in vitro inhibition and induction of human liver
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DMD 28274
1
In vitro inhibition and induction of human liver cytochrome P450 (CYP)
enzymes by milnacipran
Brandy L. Paris, Brian W. Ogilvie, Julie A. Scheinkoenig, Florence Ndikum-Moffor, Remi Gibson and
Andrew Parkinson
XenoTech, LLC, Lenexa, KS 66219 USA (B.L.P., B.W.O., J.A.S., F.M., R.G., A.P.)
DMD Fast Forward. Published on July 16, 2009 as doi:10.1124/dmd.109.028274
Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title: CYP Inhibition and Induction by Milnacipran
Address correspondence to:
Andrew Parkinson
XenoTech LLC
16825 W. 116Th Street
Lenexa, Kansas 66219, USA
Tel: (913) 438-7450
Fax: (913) 227-7199
E-mail: aparkinson@xenotechllc.com
Document Summary:
Number of Text Pages 19
Number of Tables 5
Number of Figures 5
Number of References 32
Number of Words in the Abstract 245
Number of Words in the Introduction 606
Number of Words in the Discussion 2170
Abbreviations used are: amu, atomic mass units; ANOVA, analysis of variance; CYP, cytochrome P450;
DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO, dimethyl sulfoxide; EMs, extensive
metabolizers; ESI, electrospray ionization; FBS, fetal bovine serum; FDA, Food & Drug Administration;
IC50, inhibitor concentration that causes 50% inhibition; INR, international normalized ratio; ITS+,
insulin, human transferring, and selenous acid; LC/MS-MS, liquid chromatography/tandem mass
spectrometry; MCM, Modified Chee’s medium; MEM, minimum essential medium; PK,
pharmacokinetic(s); PMs, poor metabolizers; SNRI, selective serotonin-norepinephrine reuptake
inhibitor; SSRI, selective serotonin-reuptake inhibitor; TCA, tricyclic antidepressant; UGT, UDP-
glucuronosyltransferase.
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ABSTRACT:
Milnacipran (Savella™) inhibits both norepinephrine and serotonin reuptake and is distinguished
by a nearly three fold greater potency in inhibiting norepinephrine reuptake in vitro compared to
serotonin. We evaluated the ability of milnacipran to inhibit and induce human CYP enzymes in
vitro. In human liver microsomes, milnacipran did not inhibit CYP1A2, 2B6, 2C8, 2C9, 2C19 or
2D6 (IC50 ≥ 100 µM); whereas, a comparator with dual-reuptake properties (duloxetine
[Cymbalta®]) inhibited CYP2D6 (IC50 = 7 µM) and CYP2B6 (IC50 = 15 µM) with a relatively
high potency. Milnacipran inhibited CYP3A4/5 in a substrate-dependent manner (i.e.,
midazolam 1′-hydroxylation IC50 ≈30 µM; testosterone 6β-hydroxylation IC50 ≈100 µM);
whereas, duloxetine inhibited both CYP3A4/5 activities with equal potency (IC50 = 37 and
38 µM, respectively). Milnacipran produced no time-dependent inhibition (<10%) of CYP
activity, whereas duloxetine produced time-dependent inhibition of CYP1A2, 2B6, 2C19 and
3A4/5. To evaluate CYP induction, freshly isolated human hepatocytes (n = 3) were cultured
and treated once daily for three days with milnacipran (3, 10 and 30 µM), after which
microsomal CYP activities were measured. While positive controls (omeprazole, phenobarbital
and rifampin) caused anticipated CYP induction, milnacipran had minimal effect on CYP1A2,
2C8, 2C9 or 2C19 activity. The highest concentration of milnacipran (30 µM; >10x plasma
Cmax) produced a 2.6- and 2.2-fold increase in CYP2B6 and CYP3A4/5 activity (making it 26%
and 34% as effective as phenobarbital and rifampin, respectively). Given these results,
milnacipran is not expected to cause clinically significant CYP inhibition or induction.
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INTRODUCTION
Milnacipran (Savella™), which was recently approved for the treatment of fibromyalgia, is a
dual reuptake inhibitor of norepinephrine and serotonin, which is distinguished by an
approximately three fold greater potency in inhibiting norepinephrine reuptake in vitro compared
with serotonin reuptake (Vaishnavi et al., 2004). These two neurotransmitters have been shown
to exert significant modulatory effects on peripheral and central pain processing (Dubner and
Hargreaves, 1989). Selective serotonin-norepinephrine reuptake inhibitors (SNRIs) like
duloxetine and venlafaxine are more potent inhibitors of serotonin reuptake than norepinephrine
reuptake, whereas the converse is true of milnacipran (Vaishnavi et al., 2004).
Milnacipran is well absorbed (85-90%) after oral administration and has linear pharmacokinetics
(PK) over the therapeutic dose range (Delini-Stula, 2000). The terminal elimination half-life in
plasma is 6-8 h, and steady state levels can be predicted from single dose PK data indicating the
absence of auto-inhibition or auto-induction. Milnacipran is eliminated primarily by renal
excretion of the unchanged drug (50 - 60%), conjugation to form a carbamoyl glucuronide
(~20%) and N-dealkylation by cytochrome P450 (mainly CYP3A4) to N-desethyl milnacipran
(~8%) (Delini-Stula, 2000; Puozzo et al., 1996 and 2005; Tsuruta et al., 2000; Forest Research
Institute, personal communication). Metabolism by cytochrome P450 plays only a minor role in
the elimination of milnacipran (Caccia, 1998; Grzesiak et al., 2000; Sawada and Ohtani, 2001;
Puozzo et al., 2002). Consequently, genetic polymorphisms in CYP2D6 and inhibition of this
enzyme do not impact the pharmacokinetics of milnacipran (Puozzo et al., 2005), in contrast to
the situation with many SSRIs and TCAs (Bertilsson et al., 2002; Preskorn et al., 2007). The PK
profile of milnacipran is the same in both CYP2D6 poor metabolizers (PMs) and extensive
metabolizers (EMs), and the same holds for CYP2C19 PMs and EMs (Puozzo et al., 2005).
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Limited to no modification of the pharmacokinetic profile is expected when milnacipran is co-
administered with fluoxetine (a strong CYP2D6 inhibitor) or carbamazepine (an inducer of
CYP2B6, CYP3A4 and several other enzymes) (Puozzo et al., 2002, 2005, 2006).
From the perspective of drug-drug interactions, drugs can be viewed as victims (objects) or
perpetrators (precipitants) (Ogilvie et al., 2008). Milnacipran has low victim potential because
its clearance is not heavily dependent on metabolism by a single drug-metabolizing enzyme;
hence, its PK profile is not significantly impacted by the genetic polymorphisms, CYP inhibitors
or CYP inducers that impact the disposition of other antidepressant drugs. More than half the
drug (50-60%) is eliminated unchanged in urine, which indicates kidney function is the primary
determinant of milnacipran’s elimination. The in vitro studies described in this report were
designed to evaluate the perpetrator potential of milnacipran. The enzyme-inducing potential of
milnacipran was evaluated in three preparations of freshly cultured human hepatocytes, and
focused on the major inducible human CYP enzymes, namely CYP1A2, 2B6, 2C8, 2C9, 2C19
and 3A4/5. CYP2D6 was not examined because this enzyme is recognized by the FDA as being
non-inducible (US FDA, 2006). The ability of milnacipran to function as a direct-acting and
metabolism-dependent inhibitor of CYP enzymes was evaluated with human liver microsomes.
The enzymes evaluated included CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4/5 (with two
substrates), as recommended by the FDA (US FDA, 2006; Huang et al., 2008). In the CYP
inhibition study, the SNRI duloxetine (Cymbalta®) was included as a comparator. The
structures of milnacipran and duloxetine are shown in Fig. 1. The in vitro studies described
herein were conducted in accordance with the FDA’s draft guidance document on the conduct of
in vitro metabolism studies (US FDA, 2006) and the principles promulgated by Tucker et al.
(2001), Bjornsson et al. (2003) and Huang et al. (2008).
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MATERIALS AND METHODS
Chemicals and reagents
Milnacipran and duloxetine were provided by Forest Research Institute (Jersey City, NJ). Stock
solutions of milnacipran (10 and 50 mM) and duloxetine (10 mM) were prepared in high purity
water for CYP inhibition studies and in DMSO for enzyme induction studies.
The following reagents were purchased from Sigma-Aldrich (St. Louis, MO): bupropion HCl,
dextromethorphan, diclofenac, 4´-hydroxydiclofenac, (±)-4´-hydroxymephenytoin, 6β-hydroxy-
testosterone, midazolam, phenacetin and testosterone. Acetaminophen, N-desethylamodiaquine,
dextrorphan and 1´-hydroxymidazolam were purchased from Cerilliant (Round Rock, TX).
Amodiaquine was purchased from US Pharmacopeia (Rockville, MD). S-Mephenytoin was
purchased from Toronto Research Chemicals Inc. (North York, Ontario, Canada).
Hydroxybupropion, ITS+ and Matrigel were purchased from BD Biosciences (Bedford, MA).
Dulbecco’s Modified Eagle’s Medium (DMEM), GlutaMAX-1, insulin, MEM-non-essential
amino acids, modified Eagle’s Medium Dr. Chee's modification (MCM) and liquid penicillin-
streptomycin were purchased from Invitrogen (Grand Island, NY). PureCol was purchased from
Inamed BioMaterials (Fremont, CA). Fetal bovine serum (FBS) was purchased from SAFC
Biosciences (Lenexa, KS). Loctite 4013 was purchased from the Loctite Corporation (Rocky
Hill, CT). BCA Protein Assay Kit was purchased from Pierce Chemical Co. (Rockford, IL). All
other reagents were obtained from commercial sources, most of which have been described
elsewhere (Robertson et al., 2000; Madan et al., 2003; Ogilvie et al., 2006).
Test system.
Pooled human liver microsomes (n=16, mixed gender) were prepared and characterized at
XenoTech, LLC (Lenexa, KS). Human hepatocytes from non-transplantable livers were
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prepared at XenoTech from three individual donors, all male Caucasians (ages 51, 74 and 77);
initial cell viability was 83, 93 and 77%, respectively.
In vitro CYP inhibition
The ability of milnacipran and duloxetine to inhibit the major drug-metabolizing CYP enzymes
in a direct and time-dependent manner was investigated with a pool of human liver microsomes
(pool of 16 individuals), as described by Ogilvie et al., (2006, 2008). Briefly, duplicate
incubations were conducted at 37±1°C in 400-μL incubation mixtures containing potassium
phosphate buffer (50 mM, pH 7.4), MgCl2 (3 mM), EDTA (1 mM, pH 7.4), an
NADPH-generating system (consisting of 1 mM NADP, 5 mM glucose-6-phosphate and 1
Unit/ml glucose-6-phosphate dehydrogenase) and CYP marker substrate as indicated in Table 1.
Reactions were initiated by the addition of the NADPH-generating system and terminated after
5 min by an equal volume of acetonitrile (v/v) containing an appropriate internal standard, as
summarized in Table 1. Precipitated protein was removed by centrifugation (920 × g for 10 min
at 10°C). Calibration and quality control (QC) metabolite standards were prepared in zero-time
incubations. The analytical procedures are summarized in Table 1.
To evaluate milnacipran and duloxetine as direct-acting inhibitors, pooled human liver
microsomes (≤ 0.1 mg/ml) were incubated with CYP marker substrates (at concentrations
approximately equal to Km, as shown in Table 1) in the presence and absence of milnacipran or
duloxetine (at concentrations ranging from 0.1 to 100 µM) to determine the IC50 value.
To examine their ability to act as time-dependent inhibitors, milnacipran or duloxetine (at the
same concentrations used to evaluate direct inhibition) were preincubated at 37 ± 1 °C, in
duplicate, with human liver microsomes and an NADPH-generating system for 30 minutes. After
the preincubation period, the marker substrate (at a concentration approximately equal to its Km)
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was added, and the incubation continued for 5 min to measure residual CYP activity. Reactions
were terminated after 5 min by the addition of an equal volume of acetonitrile (containing the
appropriate internal standard, Table 1). Known direct-acting and metabolism-dependent
inhibitors were included as positive controls, most of which appear on the FDA’s list of
recommended or accepted in vitro inhibitors (Ogilvie et al. 2008; US FDA, 2006). Samples were
analyzed as described in the analytical methods section (see below).
In vitro CYP induction
The ability of milnacipran to induce or suppress the expression of CYP enzymes was
investigated in primary cultures of freshly isolated human hepatocytes with a Matrigel® overlay.
After a two-day adaptation period, three preparations of cultured human hepatocytes from three
separate human livers were treated once daily for three consecutive days with milnacipran (3, 10
and 30 µM) or one of three prototypical CYP inducers, namely omeprazole (100 µM),
phenobarbital (750 µM) and rifampin (10 µM), at the final concentrations indicated.
Milnacipran and the positive controls were dissolved in DMSO, and hepatocytes treated with
DMSO (final concentration 0.1%, v/v) served as negative controls. The isolation, culturing and
treatment procedures were performed essentially as described by Madan et al. (2003). Human
hepatocytes were harvested 24 h after the third treatment to prepare microsomes, as described by
Madan et al. (2003). Microsomes (0.004-0.02 mg/ml) were incubated with phenacetin (80 µM),
bupropion (500 µM), amodiaquine (20 µM), diclofenac (100 µM), S-mephenytoin (400 µM) and
testosterone (250 µM) for 10-30 min to measure CYP1A2, 2B6, 2C8, 2C9, 2C19 and 3A4/5
activity, respectively, essentially as described above (see in vitro CYP inhibition section).
Samples were analyzed as described in the analytical methods section (see below).
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Analytical methods
All analyses of CYP enzyme activities were performed with validated HPLC-MS/MS methods.
The MS equipment was either an ABI Sciex (Applied Biosystems/MDS Sciex, Foster City, CA)
API 2000, API 3000 or API 4000 mass spectrometer with Shimadzu HPLC pumps and
autosampler systems. The HPLC columns used were as follows: Waters Atlantis C18 (5-µm
particle size, 50 mm × 2.1 mm) (Waters, Milford, MA) preceded by a Phenomenex Luna C-8
guard column (4.0 mm × 2.0 mm) (Phenomenex, Torrance, CA), or Waters Atlantis (5-µm
particle size, 100 mm × 2.1 mm) (Waters, Milford, MA) preceded by a Phenomenex Luna C-8
guard column (4.0 mm × 2.0 mm) (Phenomenex, Torrance, CA). Formic acid or ammonium
acetate based mobile phases were used for all sample analyses and flow rates ranged from
approximately 0.55 ml/min to 0.90 ml/min. All columns were maintained at ambient temperature
during analysis. Metabolites were quantified by back calculation of a weighted (1/x), linear,
least-squares regression. The regression fit was based on analyte/internal standard peak-area
ratios calculated from calibration standard samples, which were prepared from authentic
metabolite standards. Peak areas were integrated with Applied Biosystems/ MDS Sciex (Foster
City, CA) Analyst data system, version 1.4.1 or 1.4.2.
Statistical analyses
CYP inhibition data were processed with a validated, custom software program (DI IC50 LCMS
Template version 2.0.3) for the computer program Microsoft Excel (Office 2000 version 9.0,
Microsoft Inc., Redmond, WA), and IC50 values were determined by nonlinear regression with
XLfit3 (Version 3.0.5, ID Business Solutions Ltd., Guildford, Surrey, UK). XLfit is an Excel
add-in program that is a component of the validated DI IC50 LCMS Template version 2.0.3. This
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software utilizes the Levenberg-Marquardt algorithm to perform non-linear regression fitting of
the data to the following 4-parameter sigmoidal-logistic IC50 equation:
( )
⎟⎟
⎠
⎞
⎜⎜
⎝
⎛⎟⎠
⎞⎜⎝
⎛+
+=slope
50IC1
background - range background fit
x
Background was set to zero with a range up to 100 to express data as percent of control. This
software has been verified for its ability to calculate IC50 values that lie within the concentration
range of inhibitor studied. When less than 50% inhibition is observed, the data are not
extrapolated, hence, IC50 values are reported as being greater than the highest concentration of
inhibitor tested.
CYP induction data were processed with a validated, custom software program (EI Interim Data
Engine, version 1.2.1) for the computer program Microsoft Excel (Office 2003 version 11.0,
Microsoft Corporation, Redmond, WA). Statistically significant differences between group
means were calculated by equal variance and normality tests to determine if the data were
parametrically distributed. For parametrically distributed data sets, a one-way repeated measures
analysis of variance (ANOVA) was carried out to determine if there were significant differences
between the group means. For non-parametrically distributed data sets, a Kruskal-Wallis
ANOVA was performed. The ANOVA was followed by a Dunnett’s post hoc test to identify the
group means that were significantly different from the controls (p < 0.05 or 5% level of
significance). This statistical test is designed for multiple comparisons with a mean, such as
comparing multiple treatment groups with a control group. Statistical analyses were performed
with Sigma Stat Statistical Analysis System (version 2.03, Systat Software, Inc., Point
Richmond, CA). The enzyme-inducing effects of milnacipran and the prototypical inducers were
compared in terms of relative effectiveness, which was calculated as follows:
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100rate) control - rate control (Positive
rate) control - rate an(Milnacipr esseffectiven relative Percent ×=
The positive control for CYP1A2 and CYP2B6 induction was omeprazole and phenobarbital,
respectively. For all other CYP enzymes the positive control was rifampin.
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RESULTS
In vitro CYP inhibition. Milnacipran and duloxetine (0.1 – 100 µM) were evaluated for their
abilities to inhibit CYP activity in pooled human liver microsomes with CYP-selective substrates
at concentrations approximately equal to Km. In the case of CYP3A4/5, two substrates were used
(testosterone and midazolam), as recommended by the US FDA (2006). Prior to measuring CYP
activity, the test articles were preincubated with NADPH-fortified human liver microsomes for
zero or 30 minutes to assess the potential for time-dependent inhibition. The results are
summarized in Figs. 2-3 and Table 2.
Milnacipran did not inhibit CYP1A2, 2B6, 2C8, 2C9, 2C19 or 2D6 (IC50 ≥ 100 µM), as shown
in Fig. 2. Duloxetine inhibited some of these enzymes more potently than milnacipran,
inhibiting CYP2D6 with an IC50 of 7 µM, and CYP2B6 with an IC50 of 15 µM. As shown in
Fig 2, preincubation of milnacipran with NADPH-fortified human liver microsomes did not
increase its inhibitory effect on CYP1A2, 2B6, 2C8, 2C9, 2C19 or 2D6. In contrast, duloxetine
produced time-dependent inhibition of CYP1A2, 2B6 and 2C19 as indicated by the leftward shift
in IC50 curves following the 30-min preincubation period. The 30-min preincubation period
slightly decreased the inhibitory effect of duloxetine on CYP2D6 (Fig. 2).
As shown in Fig. 3, milnacipran inhibited CYP3A4/5 in a substrate-dependent manner inasmuch
as it inhibited midazolam 1′-hydroxylation (IC50 = 28 µM) more potently than it inhibited
testosterone 6β-hydroxylation (IC50 = ~100 µM). Inhibition of midazolam 1´-hydroxylation was
also evaluated with a wider range of milnacipran concentrations (up to 500 µM), which indicated
an IC50 value of 31 µM, thereby confirming the original estimate of 28 µM (data not shown).
Duloxetine also directly inhibited CYP3A4/5, with IC50 values of 37 and 38 µM for the
1´-hydroxylation of midazolam and the 6β-hydroxylation of testosterone, respectively,
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suggesting the inhibition is not dependent on substrate. Milnacipran did not produce
time-dependent inhibition (<10%) of CYP3A4/5 activity, whereas duloxetine did produce time-
dependent inhibition of CYP3A4/5 activity towards testosterone and midazolam (although the
time-dependent inhibition towards midazolam was to a lesser extent) (Fig. 3).
In vitro CYP induction. To evaluate the enzyme-inducing potential of milnacipran, three
preparations of freshly isolated human hepatocytes were cultured and treated once daily for three
consecutive days with milnacipran (3, 10 or 30 µM) or one of three prototypical enzyme
inducers, namely, omeprazole (100 µM), phenobarbital (750 µM) and rifampin (10 µM).
Microsomes were prepared 24 h after the final treatment and assayed for CYP1A2, 2B6, 2C8,
2C9, 2C19 and 3A4/5 activity. Under the conditions examined, milnacipran caused no cell
toxicity based on light-microscopic evaluation. Throughout the treatment period, the cultured
hepatocytes were free of detectable autophagic and lipid vesicles, were cuboidal in shape and
contained intact cell membranes and granular cytoplasm with one or two centrally located nuclei.
As shown in Table 3 and Fig. 4, all three preparations of human hepatocytes responded as
expected to treatment with prototypical CYP inducers: treatment with omeprazole produced a
marked increase in CYP1A2 (~37 fold), whereas treatment with phenobarbital or rifampin
produced an increase in CYP2B6 (6-10 fold), CYP2C8 (4-5 fold), CYP2C9 (~2 fold), CYP2C19
(3-6 fold), and CYP3A4 (~4 fold).
Treatment of human hepatocytes with up to 30 µM milnacipran for three consecutive days had
little or no effect on CYP1A2, CYP2C8, CYP2C9 or CYP2C19 activity (Table 3, Fig. 4).
Milnacipran produced a concentration-dependent increase in CYP2B6 activity with the highest
concentration tested (30 µM) effecting a statistically significant increase (2.59-fold: p < 0.05) in
CYP2B6 activity. At concentrations of 1, 10 and 30 µM, milnacipran was ~1%, 12% and 26%
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as effective as phenobarbital as a CYP2B6 inducer (Fig. 5). In one of the three preparations of
hepatocytes treated with the highest concentration of milnacipran (30 µM), which is almost an
order of magnitude greater than the highest plasma Cmaxss value observed clinically (3.1 µM)
(Forest Research Institute, personal communication), the relative effectiveness for CYP2B6
induction exceeded the FDA’s cutoff value of 40% (measured value was 51.9%, individual data
not shown).
Milnacipran produced a concentration-dependent increase in CYP3A4/5 activity (Table 3,
Fig. 4). The highest concentration of milnacipran (30 µM) produced a 2.15-fold increase in
CYP3A4/5 activity, which was not statistically significant. At concentrations of 1, 10 and
30 µM, milnacipran was ~6%, 20% and 34% as effective as rifampin as a CYP3A4/5 inducer
(Fig. 5). In one of the three preparations of hepatocytes treated with the highest concentration of
milnacipran (30 µM), which is almost an order of magnitude greater than the highest plasma
Cmaxss value observed clinically (3.1 µM), the relative effectiveness for CYP3A4 induction
exceeded the FDA’s cutoff value of 40% (measured value was 42.4%, individual data not
shown).
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DISCUSSION
The FDA provides the following guidance to design and interpret in vitro studies to evaluate the
victim and perpetrator potential of a new drug: (1) identify the role of CYP1A2, 2B6, 2C8, 2C9,
2C19, 2D6 and 3A4 in the metabolism of the drug and further clarify its victim potential by
identifying any other pathways that contribute 25% or more to the drug’s clearance; (2) evaluate
the potential for direct inhibition of CYP enzymes based on the ratio of [I], the plasma Cmaxss of
total (bound and free) drug, and the inhibition constant Ki, with a cutoff value of [I]/Ki = 0.1,
below which it is reasonable to assume a drug will not cause clinically significant CYP
inhibition; (3) evaluate the potential for time-dependent inhibition of CYP enzymes and conduct
clinical studies to assess the in vivo significance of positive in vitro findings, and (4) evaluate the
potential for enzyme induction in three preparations of human hepatocytes and conduct clinical
enzyme induction studies when, at pharmacologically relevant concentrations, a drug is 40% or
more as effective as a suitable positive control (US FDA, 2006; Huang et al., 2008).
Previous studies have established that milnacipran has low victim potential with respect to
metabolism by cytochrome P450. More than half (50-60%) of the drug is eliminated unchanged
in urine, 20% is conjugated to a carbamoyl-glucuronide and 8% is metabolized by cytochrome
P450 (Delini-Stula, 2000; Puozzo et al., 2005; Tsuruta et al., 2000; Forest Research Institute,
personal communication). The latter is most likely catalyzed by CYP3A4, which converts
milnacipran to one hydroxylated and two N-dealkylated metabolites (Puozzo et al., 1996; Tsuruta
et al., 2000). N-Desethyl-milnacipran is the major circulating oxidative metabolite of
milnacipran, accounting for approximately 10% of the dose excreted in urine (Puozzo et al.,
2005). In contrast, the SNRI duloxetine has high victim potential with respect to metabolism by
cytochrome P450. Duloxetine is extensively metabolized by CYP1A2 and CYP2D6, and clinical
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studies with [14C]-duloxetine have shown that the parent drug accounts for only 3% of systemic
exposure (plasma AUC) to [14C]-duloxetine-derived radioactivity
(http://www.fda.gov/cder/foi/nda/2004/021427_s000_Cymbalta.htm and
http://www.fda.gov/cder/foi/label/2007/021427s015s017lbl.pdf). Inhibition of CYP1A2 by
fluvoxamine results in a 5- to 6-fold increase in duloxetine AUC in CYP2D6 PM subjects;
whereas, inhibition of CYP2D6 by paroxetine increases plasma AUC by 60% in EMs (Cymbalta
package insert, http://www.fda.gov/cder/foi/label/2007/021427s015s017lbl.pdf). Cigarette
smoking, which induces CYP1A2, decreases the plasma AUC of duloxetine by about one third
(http://www.fda.gov/cder/foi/nda/2004/021427_s000_Cymbalta.htm and
http://www.fda.gov/cder/foi/label/2007/021427s015s017lbl.pdf). In contrast to the situation with
duloxetine and several other antidepressants (Bertilsson et al., 2002; Preskorn et al., 2007),
genetic polymorphisms in CYP2D6 and CYP2C19 have no impact on the PK of milnacipran
(Puozzo et al., 2005), nor does inhibition of CYP2D6 and CYP3A4 by fluoxetine (DeVane et al.,
2004; Puozzo et al., 2006). Enzyme induction by carbamazepine is associated with a small
decrease (20%) in milnacipran plasma steady state concentrations (Puozzo et al., 2002).
The present in vitro study was designed to evaluate the perpetrator potential of milnacipran. At
the pharmacologically relevant concentration of 3 µM and even at 10 µM milnacipran, which is
4-5 times the mean steady-state plasma Cmaxss of 2.2 µM, milnacipran produced no significant
induction of CYP1A2, 2B6, 2C8, 2C9, 2C19 or CYP3A4 in cultured human hepatocytes under
conditions where the positive controls exerted their anticipated inductive effects. At 30 µM
(~14x Cmaxss), milnacipran was 26% as effective as phenobarbital at inducing CYP2B6 and was
34% as effective as rifampin at inducing CYP3A4. These in vitro results indicate that, at
pharmacologically relevant concentrations, milnacipran is not 40% or more as effective as
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phenobarbital or rifampin at inducing CYP enzymes, which, based on the relevant FDA
Guidance for Industry (2006) suggests that milnacipran will not produce clinically significant
enzyme induction.
The potential for milnacipran to inhibit CYP enzymes in human liver microsomes was compared
with that of duloxetine. The only CYP enzyme potentially inhibited by milnacipran was
CYP3A4, the enzyme implicated in its N-dealkylation and hydroxylation, which are minor
pathways of milnacipran clearance (Tsuruta, 2000). Milnacipran inhibited the 1′-hydroxylation
of midazolam with an IC50 of approximately 30 µM. The concentration of midazolam was 4 µM,
which is approximately equal to its Km; hence, the Ki value would be ~15 µM or ~30 µM
depending on whether the inhibition of CYP3A4 by milnacipran was competitive or non-
competitive, respectively (Ogilvie et al., 2008). Following a dosage of 100 mg b.i.d., the mean
Cmax at steady state is 2.2 µM, and ranges from 1.3 to 3.1 µM (Forest Research Institute,
personal communication). Based on a conservative estimate of Ki (~15 µM) and the mean
Cmaxss of 2.2 µM, the value of [I]/Ki for CYP3A4 inhibition by milnacipran is ~0.15, which
slightly exceeds the FDA’s cut-off of 0.1. When testosterone was used to measure CYP3A4
activity, the Ki for milnacipran was conservatively estimated to be 51 µM (based on an IC50 of
~102 µM). Accordingly, [I]/Ki is 0.043 (based on mean Cmaxss of 2.2 µM), which falls below
the FDA’s cut-off value of 0.1.
Milnacipran has been reported to have no effect on the urinary excretion of 6β-hydroxycortisol
or the PK of carbamazepine, which, despite being imperfect markers of CYP3A4 activity,
provides some evidence that milnacipran does not cause clinically significant inhibition of
CYP3A4 (Table 4) (Puozzo et al., 2005). For all other CYP enzymes, estimates of [I]/Ki are less
than 0.1, and there was no evidence of metabolism-dependent inhibition. Based on this cut-off,
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the lack of time-dependent inhibition and the rank order approach to extrapolating in vitro
findings to the in vivo situation (Obach et al., 2005; Obach et al., 2006), milnacipran would not
be expected to cause clinically significant inhibition of CYP1A2, 2B6, 2C8, 2C9, 2C19 or 2D6.
In clinical drug-drug interaction studies, (summarized in Table 4), milnacipran has been shown
to cause no inhibition (or induction) of CYP1A2 (with caffeine as the in vivo probe substrate),
CYP2C19 (racemic mephenytoin) and CYP2D6 (sparteine) (Puozzo et al., 2005). Overall, there
is good correspondence between the in vitro results and the available in vivo clinical findings.
Compared with milnacipran, duloxetine was a more potent inhibitor of all the CYP enzymes
examined, and showed evidence of time-dependent inhibition of CYP1A2, 2B6, 2C19 and
CYP3A4/5 (Figs. 2-3). This study did not establish the effects of NADPH, or whether the
metabolism-dependent inhibition of CYP enzymes by duloxetine was due to the formation of
metabolites that are more potent reversible inhibitors or are irreversible inhibitors of CYP1A2,
2B6, 2C19 and 3A4. Based on experiments with recombinant human CYP enzymes, Lobo et al.
(2008) reported that the hydroxylation of the naphthyl ring of duloxetine is catalyzed by
CYP1A2 (in the 4-, 5- and 6-positions) and by CYP2D6 (in the 4- and 5-positions). These
hydroxylated metabolites (as well as di-hydroxylated metabolites and conjugates) are major
duloxetine metabolites in human plasma and urine, whereas N-demethylated and O-dealkylated
metabolites are minor in vivo metabolites (Lantz et al., 2003). N-Demethylation of duloxetine
would be expected to produce a more potent direct-acting CYP inhibitor than the parent drug,
whereas di-hydroxylation of duloxetine to a catechol metabolite on the naphthyl ring may
potentially lead to irreversible inhibition of one or more CYP enzymes. In this regard, it is
interesting that CYP1A2 (which showed evidence of time-dependent inhibition) catalyzes the 5-
and 6-hydroxylation of duloxetine. Consequently, hydroxylation at both these sites (which
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appear on the same ring and are adjacent to each other) would lead to catechol formation. In
contrast, CYP2D6 (which showed no evidence of time-dependent inhibition) catalyzes the 4- and
5-hydroxylation of duloxetine. Hydroxylation at both these sites would not produce a catechol
because these two sites are not adjacent to each other but appear on different rings of the
naphthyl moiety (Lantz et al., 2003; Lobo et al., 2008). Duloxetine also contains a thiophene
ring. Although metabolites involving thiophene oxidation have not been reported for duloxetine,
this particular functional group is associated with several cases of irreversible CYP inhibition, as
in the case of tienilic acid, ticlopidine and clopidogrel (Fontana et al., 2005; Ogilvie et al., 2008;
Parkinson and Ogilvie, 2008).
Duloxetine inhibited both of the enzymes implicated in its metabolism, namely CYP1A2 and
CYP2D6. Duloxetine inhibited CYP1A2 with an IC50 of 50 µM without any preincubation and
an IC50 of 18 µM with a 30-min preincubation. Lobo et al. (2008) also evaluated duloxetine as a
direct-acting inhibitor of CYP1A2 and reported that duloxetine causes competitive inhibition of
CYP1A2 with a Ki value of 18 µM. When incubated with marker substrate at a concentration
equal to Km, the Ki value for a competitive inhibitor is half its IC50 value, hence, the Ki of
18 µM reported by Lobo et al. (2008) translates to an IC50 value of 36 µM, which is comparable
to our value of 50 µM (determined without a preincubation period) and 18 µM (determined with
a preincubation period). Lobo et al. (2008) do not specifically report having evaluated duloxetine
as a time-dependent inhibitor of CYP1A2. Clinical interaction studies with theophylline, a
CYP1A2 substrate, established that duloxetine is a weak inhibitor of CYP1A2 in vivo; it
increased the plasma AUC of theophylline by 7% (1-15%) in one study and 20% (13-27%) in
another (Table 5). With no pre-incubation, the enzyme most potently inhibited by duloxetine was
CYP2D6 (IC50 = 7 µM). With a preincubation, the enzyme most potently inhibited by
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duloxetine was CYP2B6 (IC50 = ~5 µM). Duloxetine produces clinically significant inhibition
of CYP2D6 based on its ability to cause up to a 3-fold increase in the plasma AUC of
desipramine, a sensitive CYP2D6 in vivo probe drug (Table 5). The enzyme most potently
inhibited by duloxetine in vitro was CYP2D6, and the most pronounced clinical drug-drug
interaction reported for duloxetine is its interaction with desipramine, a drug whose clearance is
largely dependent (80-90%) on metabolism by CYP2D6. However, the inhibition of CYP2D6
observed in vivo would not be predicted from the in vitro inhibition data based on [I]/ Ki. The
average maximum plasma concentrations of duloxetine at steady state (Cmaxss) is 20.7 ng/ml or
~0.07 µM (Skinner et al., 2003), and a conservative estimate of Ki for the inhibition of CYP2D6
by duloxetine is 3.5 µM (assuming the inhibition is competitive, such that Ki is half the IC50
value). Accordingly, the [I]/ Ki value for the direct inhibition of CYP2D6 by duloxetine (0.02) is
well below the FDA’s cut-off of 0.1. It is not clear why the extrapolation of the in vitro data to
the in vivo situation based on the [I]/ Ki value underestimates the clinical inhibition of CYP2D6
by duloxetine. Duloxetine undergoes rapid and extensive first-pass metabolism in the liver and
gut, for which reason parent drug accounts for only 3% or 9% of drug-related material in plasma
based on AUC and Cmax, respectively (Lantz et al., 2003). Accordingly, hepatic levels of
duloxetine may be considerably greater than those in plasma, hence, the underestimation may be
the result of basing the [I]/ Ki value on too low a value of [I].
Duloxetine produced metabolism-dependent inhibition of CYP2B6 and CYP2C19 in vitro;
however, the effect of duloxetine on the in vivo disposition of probe drugs for these enzymes has
not been investigated. Duloxetine did not produce direct or time-dependent inhibition of
CYP2C9, however, in a single case report, duloxetine was found to be the likely cause of an
increased INR in a patient who had been on a stable dose of warfarin for a year before starting
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duloxetine (Glueck et al., 2006). Milnacipran did not inhibit CYP2C9 in vitro, and did not alter
the pharmacokinetics of S-warfarin (Forest Research Institute, personal communication).
Furthermore, milnacipran did not affect the pharmacodynamics of warfarin as indicated by the
International Normalized Ratio (INR) (Forest Research Institute, personal communication). At
steady state, the plasma Cmax of duloxetine ranges from 15-35 ng/ml (about 0.05 to 0.1 µM).
Based on a conservative estimate of 3.5 µM for the Ki value for inhibition of CYP2D6 (i.e., half
the IC50 value with [S] = Km) and a Cmaxss value of 0.1 µM, the [I]/Ki value is well below the
FDA cut-off of 0.1, and yet duloxetine causes clinically significant inhibition of CYP2D6.
Duloxetine is metabolized so extensively that parent drug accounts for only 3% of systemic
exposure to [14C]-duloxetine-derived radioactivity. Although metabolites of duloxetine might
account for the greater degree of CYP2D6 inhibition observed in vivo compared with that
predicted from in vitro studies, it is interesting that CYP2D6 was not among the enzymes that
duloxetine inhibited in a metabolism-dependent manner.
In summary, the results of this in vitro study established that duloxetine inhibits CYP2D6 and
other CYP enzymes, and has been shown to cause clinically significant inhibition of CYP2D6.
In contrast, the only human CYP enzyme inhibited by milnacipran is CYP3A4, which
milnacipran inhibited weakly and in a substrate-dependent manner (midazolam, but not
testosterone). Milnacipran would not be expected to produce clinically significant inhibition of
CYP enzymes, which is consistent with clinical data demonstrating a lack of interaction between
milnacipran and drugs metabolized by CYP1A2, 2C9, 2C19, 2D6 or 3A4. In addition, the
results of the present study suggest that milnacipran will not produce clinically significant
induction of CYP enzymes.
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Footnotes:
This study was sponsored by Forest Research Institute, Jersey City, NJ; however, the views
expressed herein are those of the publishing authors alone.
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FIGURE LEGENDS
Fig. 1: Structures of the dual reuptake inhibitors milnacipran (Savella™) and duloxetine
(Cymbalta®).
Fig. 2: Effects of milnacipran and duloxetine on selected CYP activities with and without a 30-
min preincubation with NADPH-fortified human liver microsomes. For each CYP enzyme
assayed, the substrate concentration was approximately equal to Km (see Table 1 for details).
Fig. 3: Effects of milnacipran and duloxetine on CYP3A4/5 activity towards testosterone and
midazolam with and without a 30-min preincubation with NADPH-fortified human liver
microsomes. The concentration of testosterone and midazolam was approximately equal to Km
(see Table 1 for details).
Fig. 4: Effects of treating cultured human hepatocytes with milnacipran or prototypical inducers
on microsomal CYP activity. Three preparations of human hepatocytes were treated once daily
for three consecutive days with milnacipran or one of three prototypical enzyme inducers.
Microsomes were prepared 24 h after the last treatment and assayed for CYP activity as
described in Materials and Methods. Values are presented as fold increase over negative control
(microsomes from DMSO-treated hepatoctyes) based on the absolute values shown in Table 3. *,
significantly different from vehicle control (DMSO) p<0.05 when the positive control groups
were excluded from the statistical analysis; †, statistically significance found among treatment
groups according to Kruskal-Wallis One Way Analysis of Variance on ranks (p<0.05) and
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Dunnett’s Method when the positive control groups (omeprazole, phenobarbital and rifampin)
were included in the statistical analysis; ††, significantly different from the vehicle control
(DMSO) according to the Dunnett’s Method (p<0.05) when the positive control groups
(omeprazole, phenobarbital and rifampin) were included in the statistical analysis.
Fig. 5: Comparison of the effectiveness of milnacipran as an enzyme inducer in human
hepatocytes relative to prototypical inducers. Percent relative effectiveness was calculated as
described in Materials and Methods, based on the CYP activities in Table 3. For CYP1A2 and
CYP2B6, the positive control was omeprazole and phenobarbital, respectively. For all other
CYP enzymes, the positive control was rifampin. ND, not detected; OME, omeprazole; PB,
phenobarbital; RIF, rifampin.
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Table 1: Experimental conditions for measuring microsomal CYP activity for enzyme inhibition studies
Enzyme CYP Activity Substrate
concentration (µM)
Protein (µg/ml)
Ionization mode a
Mass transition monitored
(amu b)
Internal standard
CYP1A2 Phenacetin O-dealkylation
40 100 ESI+ 152 � 110 d4-Acetaminophen
CYP2B6 Bupropion
hydroxylation 50 100 ESI+ 256 � 238 d6-Hydroxybupropion
CYP2C8 Amodiaquine N-
dealkylation 7 100 ESI+ 328 � 283 d5-N-Desethylamodiaquine
CYP2C9 Diclofenac 4´-hydroxylation
6 100 ESI– 310 � 266 d4-4´-Hydroxydiclofenac
CYP2C19 S-Mephenytoin 4´-
hydroxylation 40 100 ESI– 233 � 190 d3-4´-Hydroxymephenytoin
CYP2D6 Dextromethorphan O-demethylation
7.5 100 ESI+ 258 � 157 d3-Dextrorphan
CYP3A4/5 Testosterone 6β-
hydroxylation 100 100 ESI+ 305 � 269 d3-6β-Hydroxytestosterone
CYP3A4/5 Midazolam 1´-hydroxylation
4 50 ESI+ 342 � 324 d3-1´-Hydroxymidazolam
a Indicates the type of ionization (i.e., electrospray ionization [ESI]) and the polarity (+ or −).
b Atomic mass units
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Table 2: Comparison of milnacipran and duloxetine as inhibitors of selected CYP enzymes with and without a 30-min preincubation with NADPH-fortified human liver microsomes
Enzyme CYP Activity
Direct inhibition (Zero-minute preincubation)
Time-dependent inhibition (30-minute preincubation)
Milnacipran IC50 (µM)
Duloxetine IC50 (µM)
Milnacipran IC50 (µM)
Duloxetine IC50 (µM)
CYP1A2 Phenacetin O-dealkylation >100 50 >100 18
CYP2B6 Bupropion hydroxylation 98 15 >100 4.7
CYP2C8 Amodiaquine N-dealkylation >100 60 >100 49
CYP2C9 Diclofenac 4´-hydroxylation >100 >100 >100 >100
CYP2C19 S-Mephenytoin 4´-hydroxylation >100 27 >100 8.6
CYP2D6 Dextromethorphan O-demethylation >100 7.0 >100 13
CYP3A4/5 Testosterone 6β-hydroxylation >100 a 38 >100 17
CYP3A4/5 Midazolam 1´-hydroxylation 28 37 24 26
a The IC50 value calculated was 102 µM, but is reported as >100 µM since concentrations beyond the concentration range studied should be considered estimates.
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Table 3: Effects of treating cultured human hepatocytes with DMSO, milnacipran or prototypical inducers on microsomal CYP activity
Treatment Concentration Enzyme activity (pmol/mg microsomal protein/min) a
CYP1A2 CYP2B6 CYP2C8 CYP2C9 CYP2C19 CYP3A4/5
DMSO 0.1% (v/v) 63.5 ± 50.8 65.9 ± 34.4 236 ± 73 928 ± 320 6.41 ± 4.45 2980 ± 1040
Milnacipran 3 µM 64.6 ± 50.1 76.1 ± 48.9 259 ± 124 979 ± 348 6.25 ± 3.71 3470 ± 480
Milnacipran 10 µM 72.1 ± 56.1 111 ± 61 276 ± 81 997 ± 305 6.45 ± 3.44 4700 ± 540
Milnacipran 30 µM 73.4 ± 59.4 168 ± 90* 285 ± 74 1010 ± 280 5.80 ± 3.08 5840 ± 580
Omeprazole 100 µM 1630 ± 750† 419 ± 214† 904 ± 483 1320 ± 650 8.27 ± 6.30 4100 ± 2280
Phenobarbital 750 µM 124 ± 69 666 ± 399† 959 ± 257† 1680 ± 650† 20.8 ± 17.0 11400 ± 1300††
Rifampin 10 µM 114 ± 86 388 ± 262† 1130 ± 250† 1930 ± 570† 43.7 ± 33.1†† 11500 ± 1000††
a Values (rounded to three significant figures) are the mean ± standard deviation (rounded to the same degree of accuracy) of three determinations (i.e., three human hepatocyte preparations).
* Significantly different from the vehicle control (DMSO) according to the Dunnett’s Method (p<0.05) when the positive control groups (omeprazole, phenobarbital and rifampin) were excluded from the statistical analysis.
† Statistical significance found among the treatment groups according to Kruskal-Wallis One Way Analysis on ranks (p<0.05) and Dunnett’s Method when the positive control groups (omeprazole, phenobarbital and rifampin) were included in the statistical analysis.
†† Significantly different from the vehicle control (DMSO) according to the Dunnett’s Method (p<0.05) when the positive control groups (omeprazole, phenobarbital and rifampin) were included in the statistical analysis.
CYP1A2: Phenacetin O-dealkylation
CYP2B6: Bupropion hydroxylation
CYP2C8: Amodiaquine N-dealkylation
CYP2C9: Diclofenac 4´-hydroxylation
CYP2C19: S-Mephenytoin 4´-hydroxylation
CYP3A4/5: Testosterone 6β-hydroxylation
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Table 4: Summary of clinical drug-drug interactions studies to evaluate the effect of milnacipran on the disposition of coadministered drugs.
Victim drug Na Enzymeb Treatment Change in
pharmacokinetics of the victim drug
Reference
Sparteine 25 CYP2D6
Milnacipran: 50 mg single dose (Day 1), then 50 mg b.i.d. (Days 2-8) Sparteine: 100 mg q.d. (Days -2, 1, 8, 20)
19.5% increase in sparteine/metabolite ratio
in CYP2C19 EMsc
Puozzo et al., 2005
Mephenytoin 25 CYP2C19
Milnacipran: 50 mg single dose (Day 1), then 50 mg b.i.d. (Days 2-8) Mephenytoin: 100 mg q.d. (Days -2, 1, 8, 20)
No increase in S/R-mephenytoin ratiod
Puozzo et al., 2005
Caffeine 25 CYP1A2
Milnacipran: 50 mg single dose (Day 1), then 50 mg b.i.d. (Days 2-8) Caffeine: 200 mg q.d., (Days -2, 1, 8, 20)
No increase in the caffeine/paraxanthine
AUC0-12 ratioe
Puozzo et al., 2005
Warfarin 25
CYP2C9 for S-warfarin and CYP1A2/3A4 for R-warfarinf
Milnacipran: 25 mg bid (Days 1-3), 50 mg bid (Days 4-6), and 100 mg bid (Days 7-11) Warfarin: 25 mg single dose on Day 11 and Day 25 Or Milnacipran: 25 mg bid (Days 15-17), 50 mg bid (Days 18-20), and 100 mg bid (Days 21-25) Warfarin: 25 mg single dose on Day 1 and Day 25
No change in S- or R-warfarin pharmaco-
kinetics or pharmaco-dynamics (INR)
FDA drug label and FRI personal communication
Carbamazepine 25 CYP3A4
Milnacipran: 50 mg bid (Days 1-4) and Days (29 – 35) Carbamazepine: 100 mg bid (Days 8-11); 200 mg bid (Days 12 - 35)
No change in the pharmacokinetics of carbamazepine or its epoxide metabolite
FDA drug label and FRI personal communication
a Number of subjects completing each study b Principal metabolizing enzyme c Prior to milnacipran treatment, the sparteine/metabolite ratio was 0.51 and 14.6 in CYP2C19 extensive
metabolizers (EMs) and poor metabolizers (PMs), respectively
d Prior to milnacipran treatment, the S/R-mephenytoin ratio was 0.066 and 1.56 in CYP2C19 extensive metabolizers (EMs) and poor metabolizers (PMs), respectively
e Prior to milnacipran treatment, the caffeine/paraxanthine AUC ratio was 2.3 f Two-sequence crossover design used
FDA, Food & Drug Administration; FRI, Forest Research Institute; INR, international normalized ratio (a measure of prothrombin time)
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Table 5 Summary of clinical drug-drug interaction studies to evaluate the effect of duloxetine on the disposition of coadministered drugs
Victim drug Na Enzymeb Treatmentc
Arithmetic mean AUC
change (%)
AUC Geometric mean ratio
Reference Mean 90% CI
Theophylline 28 CYP1A2
Duloxetine: 60 mg b.i.d. (Days 1-4), then 60 mg q.d. (Day 5) Theophylline: 197.5 mg (Day 5)
1.13 1.07 – 1.18d Lobo et al., 2008
Temazepam Unk. UGT
Duloxetine: 20 mg q.d. (Days 1-6) Temazepam: 30 mg q.d. (Days 1-6)
8.6 1.11 1.02-1.21 Duloxetine NDA (21-427)e
Lorazepam 16 UGT
Duloxetine: 60 mg b.i.d. (Days 1-6) Lorazepam: 2 mg b.i.d. (Days 4-6)
8.2 1.08 1.00-1.16 Duloxetine NDA (21-427)e
Desipramine 17 CYP2D6
Duloxetine: 30 mg b.i.d. (Days 6-15) Desipramine: 50 mg (Day 11)
107 2.22 1.95-2.51 Patroneva et al., 2008
Desipramine 13 CYP2D6
Duloxetine: 40 mg b.i.d. (Days 8-13), then 60 mg b.i.d. (Days 14-27) Desipramine: 50 mg (Day 21)
168 2.92 2.55-3.34 Skinner et al., 2003
Metoprolol 16 CYP2D6
Duloxetine: 30 mg q.d. (Day 1), then 60 mg q.d. (Days 2-17) Metoprolol: 100 mg q.d. (Day 17)
180f
94.5g
Preskorn et al., 2007
Tolterodine 16 CYP2D6,
3A4
Duloxetine: 40 mg b.i.d. (Days 1–4, then 40 mg q.d. (Day 5) Tolterodine: 2 mg b.i.d. (Days 1-4, then 2 mg q.d. (Day 5)
1.71 1.31-2.23h Hua et al., 2004
a Number of subjects completing each study b Principal metabolizing enzyme c q.d. and b.i.d. refer to once-a-day and twice-a-day dosing, respectively d Theophylline AUC increased 20% in women (n=18; statistically significant) but only 7% in men (n=10;
statistically insignificant). e Duloxetine NDA (21-427): http://www.fda.gov/cder/foi/nda/2004/021427_s000_Cymbalta.htm f Original value reported in Table 5 in Preskorn et al., 2007 g Recalculated value based on AUC values reported in Table 4 in Preskorn et al., 2007. The recalculated value
agrees with that reported in the University of Washington Metabolism and Transport Drug Interaction Database (MTDI database: http://www.druginteractioninfo.org).
h Value represents 95% CI (all other values are 90% CI) CI, Confidence interval; IV, intravenous; Unk, Unknown; UGT, UDP-glucuronosyltransferase
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N
O
NH2
O
S
NH
1R(S), 2S(R)-Milnacipran (S)-Duloxetine
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CYP1A2
[Drug concentration] (µM)0.1 1 10 100 1000
Phe
nac
etin
O-d
ealk
ylat
ion
(Per
cent
con
trol
)0
20
40
60
80
100
120
>100 µM
>100 µM
50 µM
18 µM
IC50 values
CYP1A2
[Drug concentration] (µM)0.1 1 10 100 1000
Bu
pro
pio
n h
ydro
xyla
tio
n(P
erce
nt c
ontr
ol)
0
20
40
60
80
100
120
>100 µM
>100 µM
15 µM
4.7 µM
IC50 values
CYP2B6
[Drug concentration] (µM)0.1 1 10 100 1000
Am
od
iaq
uin
e N
-dea
lkyl
atio
n
(Per
cent
con
trol
)
0
20
40
60
80
100
120
>100 µM
>100 µM
60 µM
49 µM
IC50 values
CYP2C8
[Drug concentration] (µM)0.1 1 10 100 1000
Dic
lofe
nac
4´-
hyd
roxy
lati
on
(P
erce
nt c
ontr
ol)
0
20
40
60
80
100
120
>100 µM
>100 µM
>100 µM
>100 µM
IC50 values
CYP2C9
[Drug concentration] (µM)0.1 1 10 100 1000
S-M
ephe
nyt
oin
4´-
hyd
roxy
latio
n
(Per
cent
con
trol
)
0
20
40
60
80
100
120
>100 µM
>100 µM
27 µM
8.6 µM
IC50 values
CYP2C19
[Drug concentration] (µM)0.1 1 10 100 1000
Dex
tro
met
ho
rph
an O
-dem
eth
ylat
ion
(Per
cent
con
trol
)
0
20
40
60
80
100
120
>100 µM
>100 µM
7.0 µM
13 µM
IC50 values
CYP2D6
Milnacipran zero preincubation
Milnacipran 30-min preincubation
Duloxetine zero preincubation
Duloxetine 30-min preincubation
Figure 2
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Milnacipran Duloxetine
Milnacipran zero preincubation
Milnacipran 30-min preincubation
Duloxetine zero preincubation
Duloxetine 30-min preincubation
[Milnacipran concentration] (µM)0.1 1 10 100 1000
Tes
tost
ero
ne
6 β-h
ydro
xyla
tio
n
(Per
cent
con
trol
)
0
20
40
60
80
100
120
>100 µM
>100 µM
IC50 values
CYP3A4/5: Testosterone
[Duloxetine concentration] (µM)0.1 1 10 100 1000
Tes
tost
eron
e 6 β
-hyd
roxy
latio
n (P
erce
nt c
ontr
ol)
0
20
40
60
80
100
120
38 µM
21 µM
IC50 values
CYP3A4/5: Testosterone
[Milnacipran concentration] (µM)0.1 1 10 100 1000
Mid
azo
lam
1´-
hydr
oxy
latio
n (P
erce
nt c
ontr
ol)
0
20
40
60
80
100
120
28 µM
24 µM
IC50 values
CYP3A4/5: Midazolam
[Duloxetine concentration] (µM)0.1 1 10 100 1000
Mid
azo
lam
1´-
hyd
roxy
latio
n
(Per
cent
con
trol
)
0
20
40
60
80
100
120
37 µM
26 µM
IC50 values
CYP3A4/5: Midazolam
Figure 3
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