SUPPLEMENTARY MATERIAL
Article title: Evaluation of CYP3A4-Based Interactions between Levomilnacipran and Ketoconazole,
Carbamazepine or Alprazolam in Healthy Subjects
Journal: Clinical Drug Investigation
Authors: Laishun Chen, Ramesh Boinpally, Nayra Gad, William M. Greenberg, Julie Wangsa, Antonia
Periclou, Parviz Ghahramani
Corresponding author: Laishun Chen, Forest Research Institute, Harborside Financial Center, Plaza V,
Jersey City, NJ 07311, USA; Email: laishun.chen@frx.com
Online Resource 1. Levomilnacipran ER: ketoconazole interaction study: design of Study 1.
Sequence I
Day(s)
Treatment A
Day 1
Washout: Days 2–9
Treatment B
Days 10–14
Day 15
Days 16–18
Sequence II
Day(s)
Treatment B
Days 1–5
Day 6
Days 7–9
Washout: Days 10–14
Treatment A
Day 15
Treatment
Blood sampling (h) a
Levomilnacipran ER 2 x 40 mg as a single
dose (clinic confinement)
0 (predose)
2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72
and 96
–
No administration of investigational
products
Ketoconazole 400 mg (2 × 200 mg
tablets) (outpatient basis)
Co-administration of levomilnacipran ER
80 mg (2 × 40 mg ER capsules) plus
ketoconazole 400 mg (2 × 200 mg
tablets) (clinic confinement)
Ketoconazole 400 mg (2 × 200 mg
tablets) (clinic confinement)
–
Treatment
Blood sampling (h)
Ketoconazole 400 mg (2 × 200 mg
tablets) (outpatient basis)
Co-administration of levomilnacipran ER
80 mg (2 × 40 mg ER capsules) plus
ketoconazole 400 mg (2 × 200 mg
tablets) (clinic confinement)
Ketoconazole 400 mg (2 × 200 mg
tablets) (clinic confinement)
No administration of investigational
products
–
Levomilnacipran ER 2 x 40 mg as a single
dose (clinic confinement)
0 (predose)
2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72
and 96
0 (predose)
2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72
and 96
–
0 (predose)
2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72
and 96
–
–
a
Plasma samples were harvested, flash frozen and shipped by overnight courier to the bioanalytical
facilities of Forest Research Institute, Inc.
ER, extended release
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Online Resource 2. Levomilnacipran ER:carbamazepine XR interaction study: design of Study 2.
Day(s)
Treatment A (Period I,
Days 1–11)
Washout period
(Days 12–17)
Treatment B
(Period II, Days 18–38)
Treatment C
(Period III, Days 39–49)
Treatment D
(Period IV, Days 50–53)
Treatment
Levomilnacipran ER 20 mg for 1 day (AM
dose), followed by levomilnacipran ER
40 mg once daily for 3 days (AM dose),
followed by levomilnacipran ER 80 mg
once daily for 3 days (AM dose),
followed by levomilnacipran ER 120 mg
once daily for 4 days (AM dose)
No administration of investigational
products
Carbamazepine XR 100 mg twice daily
for 4 days, followed by carbamazepine
XR 200 mg twice daily for 17 days (AM
and PM dosesa)
Continuing carbamazepine XR 200 mg
twice-daily treatment for the entire
period (AM and PM doses) and
levomilnacipran ER 20 mg for 1 day (AM
dose), followed by levomilnacipran ER
40 mg once daily for 3 days (AM dose),
followed by levomilnacipran ER 80 mg
once daily for 3 days (AM dose),
followed by levomilnacipran ER 120 mg
once daily for 4 days (AM dose)
Carbamazepine XR 200 mg (AM dose)
and carbamazepine XR 100 mg (PM
dose) for 1 day, carbamazepine XR 100
mg twice daily (AM and PM doses) for 2
days, followed by carbamazepine XR 100
mg once a day (AM dose) for 1 day
Blood sampling (h) b,e
On Days 1, 10, 11c:
0 (pre-AM dose)
On Day 11c:
0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36,
48 and 72 post-AM dose
–
On Days 18 and 38d:
0 (pre-AM dose)
On Day 37d:
0 (pre-PM dose)
On Days 39, 48, 49c:
0 (pre-AM dose)
On Day 49c:
0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36,
48 and 72 post-AM dose
On Days 39 and 49d:
0 (pre-AM dose)
On Day 48d:
0 (pre-PM dose)
On Days 38 and 49d:
0.5, 1, 2, 3, 4, 5, 6, 8, 10 and 12 postAM dose
–
AM, morning; carbamazepine XR, carbamazepine extended release; levomilnacipran ER,
levomilnacipran extended release; PM, evening
a
For twice-daily doses of carbamazepine XR, the evening dose was administered 12 hours after the
morning dose.
b
Plasma samples were harvested, frozen and sent to Forest Research Institute, Inc., Jersey City, NJ
(levomilnacipran and metabolite) or PPD, Inc., Wilmington, NC (carbamazepine and metabolite) for
bioanalysis at the end of the study.
c
Samples for analysis of levomilnacipran and its metabolite.
d
Samples for analysis of carbamazepine and its metabolite.
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Online Resource 3. Alprazolam XR/levomilnacipran ER PK interaction study: design of Study 3.
Sequence I
Day(s)
1
Treatment
Alprazolam XR 1 mga
2–9
10
11–13
14–16
17–20
Washout period
Levomilnacipran ER 20 mg
Levomilnacipran ER 40 mg ODb
Levomilnacipran ER 80 mg ODb
Levomilnacipran ER 120 mg ODb,c
21
Levomilnacipran ER 120 mg ODb plus
alprazolam XR 1 mga
21
As above
22–23
Levomilnacipran ER 120 mg OD
Sequence II
Day(s)
1
2–4
5–7
8–11
Treatment
Levomilnacipran ER 20 mg
Levomilnacipran ER 40 mg OD
Levomilnacipran ER 80 mg ODb
Levomilnacipran ER 120 mg ODb,c
12
Levomilnacipran ER 120 mg ODb plus
alprazolam XR 1 mga
12
As above
13–14
15–20
21
Levomilnacipran ER 120 mg ODb,c
Washout period
Alprazolam XR 1 mga
Blood sampling (h)f
0 (predose)
1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72
and 96
–
0 (predose)
–
–
On Day 20:
0 (predose)
1, 3, 5, 6, 8 and 12
0 (predose)
1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72
and 96d
0 (predose)
1, 3, 5, 6, 8, 12 and 24e
–
Blood sampling (h)f
0 (predose)
–
–
On Day 11: 0 (predose)
1, 3, 5, 6, 8 and 12
0 (predose)
1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72
and 96d
0 (predose)
1, 3, 5, 6, 8, 12 and 24e
–
–
0 (predose)
1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72
and 96
ER, extended release; OD, once daily; PK, pharmacokinetic; XR, extended release; –, no samples
taken
a
Fasted state
b
Given as single or multiple 40 mg capsules
c
Fasted state on last day
d
Samples for analysis of alprazolam XR pharmacokinetics
e
Samples for analysis of levomilnacipran ER pharmacokinetics
f
Plasma was harvested and frozen and sent to PPD, Inc., Wilmington, NC (alprazolam) or Forest
Research Institute, Inc., Jersey City, NJ (levomilnacipran) for bioanalysis at the end of the study.
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Online Resource 4
Bioanalytical methods
Levomilnacipran and N-desethyl levomilnacipran
Concentrations of levomilnacipran and N-desethyl levomilnacipran free bases were determined in
plasma using a published liquid chromatography–tandem mass spectrometry (LC-MS/MS) method
that had been validated for accuracy, precision, linearity and reproducibility [1]. Briefly, plasma
samples were mixed with a solution containing internal standards for levomilnacipran and Ndesethyl levomilnacipran. The analytes were extracted using methyl t-butyl ether and the organic
phase was evaporated. The dried residue was reconstituted in 2% formic acid in water–methanol
(70:30, v/v). The final extract was analysed via high-performance liquid chromatography (HPLC) with
MS/MS detection. The instrument response, which is the ratio of levomilnacipran free base product
ion peak area to that of its internal standard, was the response used for quantification of
levomilnacipran free base. Similarly, peak area ratio of N-desethyl levomilnacipran free base was the
response used for quantification of N-desethyl levomilnacipran free base.
For the ketoconazole study, the method used to determine the concentrations of levomilnacipran
and N-desethyl levomilnacipran free bases was linear over the concentration range of 1 to 200
ng/mL, with a lower limit of quantification (LLOQ) of 1 ng/mL in 100 μL of plasma with dipotassium
EDTA as the anticoagulant. The precision (%CV) and accuracy (%bias) for the plasma levomilnacipran
standards were 2.8 and ± 1.6 % respectively. The precision and accuracy for the plasma
levomilnacipran quality control samples were 6.0 and ± 0.9 respectively. Similarly, the plasma Ndesethyl levomilnacipran standards had a precision and accuracy of 4.2 and ± 2.7 % respectively,
while the plasma quality control samples had a precision and accuracy of 7.0 and ± 4.3 %
respectively.
For the carbamazepine and alprazolam studies, the method used was linear over a concentration
range of 1 to 500 ng/mL for both levomilnacipran and N-desethyl levomilnacipran, with an LLOQ of 1
ng/mL in 100 μL of plasma with dipotassium EDTA as the anticoagulant.
Carbamazepine and carbamazepine-10,11-epoxide
Plasma carbamazepine and carbamazepine-10,11-epoxide concentrations were determined at PPD
Bioanalytical (Richmond, VA, USA) using a sensitive LC-MS/MS method that had been validated for
accuracy, precision, linearity and reproducibility, and that is proprietary to PPD.
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The method was linear over a concentration range of 0.0500 to 50.0 μg/mL for both analytes, with
LLOQ at 0.0500 μg/mL in 50 μL of plasma with sodium heparin as the anticoagulant.
Alprazolam
Plasma concentrations of alprazolam were also determined at PPD Bioanalytical (Richmond, VA,
USA) using a sensitive LC-MS/MS method that had been validated for accuracy, precision, linearity
and reproducibility, and that is proprietary to PPD.
A 500 μL sample aliquot was fortified with 50 μL of internal standard working solution. Analytes
were isolated through liquid–liquid extraction. The organic phase was evaporated and the remaining
residue was reconstituted with 400 μL of reconstitution solution. The extract was further purified
through a hexane wash. The final extract was injected and analysed via HPLC with MS/MS detection.
The instrument response, which is the ratio of alprazolam product ion peak area to that of its
internal standard ([2H5] alprazolam), was used for quantification. The method was linear over the
concentration range of 0.25 to 40 ng/mL with an LLOQ at 0.25 ng/mL using 500 μL of human plasma
with dipotassium EDTA as the anticoagulant. The precision (%CV) and accuracy (%bias) for the
plasma alprazolam standards were 5.0 and ± 3.1% respectively. The precision and accuracy for the
plasma alprazolam quality control samples were 4.0 and ± 9.4% respectively.
Reference
1. Chen L, Boinpally R, Greenberg WM, et al. Effect of hepatic impairment on the pharmacokinetics
of levomilnacipran following a single oral dose of a levomilnacipran extended-release capsule in
human participants. Clin Drug Invest. 2014;34:351–9.
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