Assessment of CYP enzyme and drug transporter suppression by

Assessment of CYP enzyme and drug transporter suppression by therapeutic proteins with an in vitro method incorporating incubations of PBMCs and hepatocyte-Kupffer cell co-cultures
Catherine Wiegand, Maciej Czerwinski, Faraz Kazmi, and David B. Buckley
XenoTech LLC, Lenexa KS, USA 66219
A DIVISION OF
Results
Hepatocyte cultures: Primary human hepatocytes were isolated by a two-step collagenase
perfusion method and plated on collagen-coated dishes. Previously, we demonstrated that
these human hepatocyte cultures contained ~1-2% Kupffer cells by staining with antibody
against macrophage marker CD68 (1). The hepatocytes were allowed to adapt to culture
conditions for two to three days, after which they were treated once daily for three
consecutive days either with MCM+ (negative control) IL-6 (10 ng/mL, positive control) for
evaluation of the direct effects of IL-6. Additionally, cultured hepatocytes were treated with
either saline-treated plasma (vehicle control for LPS), LPS-treated plasma, MOPC
31C-treated plasma (negative control antibody for ANC28.1) or ANC28.1-treated plasma at
10%, 20% or 50% v/v of cell culture media. After 72 hr of treatment, the cells were lysed with
TRIzol reagent for subsequent isolation of RNA.
Cytokine quantification: Cytokines were measured in treated plasma samples with the
Human Pro-inflammatory 9-Plex Kit and SECTOR® Imager 2400 instrument (Meso Scale
Discovery, Gaithersburg, MD). The cytokines measured are listed in Table 1.
Analysis of mRNA expression. Total RNA was phase extracted from TRIzol reagent followed
by purification with the RNeasy Mini Kit (Qiagen, Valencia, CA) or the MagMAX-96 for
Microarrays Kit (Ambion, Austin, Texas) with the KingFisher Flex (Thermo Scientific).
Purified RNA was reverse transcribed to cDNA with the High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Carlsbad, CA) and the Applied Biosystems 7300 or
7900HT Real-Time PCR System. Real time qPCR was performed on the 7900HT. Relative
quantification was calculated using RQ Manager (Applied Biosystems) and calibrators of the
relevant plasma concentration. The saline-and MOPC 31C-treated samples mRNA
expression served as controls (calibrators) for mRNA expression from LPS- and
ANC28.1-treated samples, respectively.
Table 1: Effects of saline, MOPC 31C, LPS and ANC28.1 on cytokine release in ex vivo
whole blood cultures
GM-CSF
IFN-γ
IL-1β
IL-2
IL-6
IL-8
IL-10
IL-12p70
TNF-α
Cytokine concentration (ng/mL)
Saline
MOPC 31C
ANC28.1
LPS
(diluent)
antibody
antibody
13.8
21
344
540
BLQ
2.11
293
109
*
6630
18.1
BLQ
0.527
*
10.9
BLQ
400
*
4.12
*
0.865
5.77
8.04
1.18
492
1.7
152
7610
9220
631
749
110
5400
16.3
*
48.2
6250
BLQ
227
2.60
5.73
Values are mean of duplicate determinations
BLQ = below level of quantification (limits of detection 0.6 – 2500 ng/mL)
* = Single determination, one of two duplicate determinations were BLQ
Table 3 shows the effects of treating cultured human hepatocytes with LPS- and
ANC28.1-treated plasma on the mRNA expression of various hepatic uptake transporters.
Figure 2 illustrates the effects of LPS- and ANC28.1-treated plasma on the mRNA
expression of OATP1B1 and OCT1 (Figure 2A and 2B, respectively). Similar to the
observation with CYPs, treatment of hepatocytes with LPS-treated plasma caused a marked
suppression of mRNA expression of all five uptake transporters examined, namely
OATP1B1, OATP1B3, OATP2B1, OCT1 and NTCP. Curiously, treatment of hepatocytes
with increasing concentrations of LPS-treated plasma (from 10 to 50% v/v) caused a
variable response in OATP1B3 mRNA expression. At 10% v/v, marked suppression was
observed; however, at 50% v/v, little to no change was observed.
The effects of the ANC28.1-treated plasma on OATP mRNA expression in cultured
hepatocytes were characterized by large inter-individual variability. At concentration of 50%
v/v of media, ANC28.1-treated plasma caused anywhere from a moderate (>50%) decrease
to little or no change in the mRNA expression of the five uptake transporters examined.
Table 4 shows the effects of treating cultured human hepatocytes with LPS- and
ANC28.1-treated plasma on the mRNA expression of various hepatic efflux transporters.
Figure 3 illustrates the effects of LPS- and ANC28.1-treated plasma on the mRNA
expression of MDR1 (P-gp) and BSEP (Figure 3A and 3B, respectively). Similar to the
observation with CYPs, treatment of hepatocytes with LPS-treated plasma caused a marked
suppression of mRNA expression of all five uptake transporters examined, namely MDR1,
MRP2, BCRP and BSEP. ANC28.1-treated plasma was a less potent and efficacious,
causing only modest mRNA suppression across majority of the efflux transporters examined.
Of note, at concentration of 50% v/v, ANC28.1-treated plasma caused modest decrease in
both bile salt transporters, NTCP and BSEP; mRNA expression of these transporters was
decreased by 63% and 46%, respectively.
[Plasma]
10%
20%
50%
OATP1B1
OATP1B3
OATP2B1
1-Oct
NTCP
LPS
ANC28.1
LPS
ANC28.1
LPS
ANC28.1
LPS
ANC28.1
LPS
ANC28.1
25.5
25.3
25.8
124.6
113.5
57.3
34.6
44.2
95.9
230.9
224.1
45.1
16
15.3
7.1
50.7
44.5
36.9
19.1
19.6
15.6
95.2
85.2
45.3
17.2
19.5
6.2
81.7
105.1
63.8
Values are mean of 3 - 6 determinations
Figure 2: Effects of LPS- and ANC28.1-treated plasma on OATP1B1 and OCT1 mRNA
expression in cultured human hepatocytes
A) OATP1B1 mRNA expression
250
B) OCT1 mRNA espression
200
ANC28.1 plasma
OCT1 mRNA level, % control
LPS plasma
200
150
100
50
ANC28.1 plasma
LPS plasma
160
120
80
40
0
0
control
10%
[Plasma]
20%
control
50%
10%
[Plasma]
20%
50%
Table 4: Effects of LPS- and ANC28.1-treated plasma on efflux drug transporter mRNA
expression in cultured human hepatocytes
MDR1
[Plasma]
MRP2
BCRP
BSEP
LPS
ANC28.1
LPS
ANC28.1
LPS
ANC28.1
LPS
ANC28.1
10%
67.8
103
52.5
76.7
48.2
90.9
29.4
50.3
20%
66.7
106.5
64.9
77.9
53.6
86.9
36.7
53.7
50%
63
93.9
94.1
68
68.8
74.9
32.8
53.6
Values are mean of 3 - 6 determinations
Figure 3: Effects of LPS- and ANC28.1-treated plasma on MDR1 and BSEP mRNA
expression in cultured human hepatocytes
A) MDR1 mRNA expression
B) BSEP mRNA expression
140
ANC28.1 plasma
120
LPS plasma
100
80
60
40
20
0
control
10%
[Plasma]
20%
50%
120
BSEP mRNA level, % control
Ex-vivo stimulation of fresh whole blood cultures: Fresh whole human blood was collected
from healthy donors (with informed consent, n = 6) into 10 mL sodium heparin vacutainers
(Becton-Dickinson, Franklin Lakes, NJ), transferred into sterile 50 mL polypropylene tubes
(BD Biosciences, San Diego, CA) and aliquoted into sterile polypropylene micro tubes
(Sarstedt, Newton, NC) for incubations with various stimuli. Endotoxin was removed from
sterile saline with the Endotoxin Removal Gel (Pierce Biotechnology, Rockford, IL). A stock
solution of lipopolysaccharides (LPS) from E.coli (5 μg/mL) was prepared with purified saline.
Cultures of whole blood were stimulated with purified saline, LPS (50 ng/mL), MOPC 31C (2
μg/mL) and ANC28.1 (2 μg/mL). The cultures were mixed gently and incubated for 24 hrs at
37°C. Plasma was separated by centrifugation at 600 x g for 10 min at room temperature,
aliquoted and stored at -80°C.
Consistent with the cytokine composition, treatment of hepatocytes with LPS-treated plasma
caused a marked suppression of mRNA expression of all six CYP enzymes examined,
namely CYP1A2, CYP2B6, CYP2C9, CYP2D6, CYP3A4 and CYP3A5. Treatment of
cultured hepatocytes with ANC28.1-stimulated plasma caused a decrease in CYP1A2,
CYP2B6 and CYP3A4 mRNA expression; however, there was little or no effect observed for
CYP2C9, CYP2D6 and CYP3A5 mRNA expression. Compared to LPS-treated plasma,
treatment of hepatocytes with ANC28.1-treated plasma was less efficacious in causing
mRNA suppression of all CYP enzymes examined. These effects are consistent with
significantly higher concentration of IFN-γ, IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF-α in the
LPS-treated plasma as compared with the ANC28.1-treated plasma.
Table 3: Effects of LPS- and ANC28.1-treated plasma on uptake drug transporters mRNA
expression in cultured human hepatocytes
OATP1B1 mRNA level, % control
Chemicals and Reagents. Bacterial lipopolysaccharide (LPS) was purchased from
Sigma-Aldrich (St. Louis, MO). The anti-CD28 antibody, ANC28.1 (murine IgG1kappa), and
iso-type control antibody (MOPC 31C) were purchased from Ancell Corp. (Bayport, MN).
Lyophilized Interleukin-6 (IL-6) was purchased from EMD Biosciences (La Jolla, CA). TRIzol
reagent was purchased from Invitrogen (Carlsbad, CA).
Table 2 shows the effects of treating cultured human hepatocytes with LPS- and
ANC28.1-treated plasma on the mRNA expression of various CYP enzymes. Figure 1
illustrates the effects of LPS- and ANC28.1-treated plasma on the mRNA expression of
CYP1A2 and CYP3A4 (Figure 1A and 1B, respectively). Treatment of human hepatocytes
directly with IL-6, without stimulation or addition of plasma, demonstrated that our
hepatocytes constituted an appropriate test system for evaluation of therapeutic proteins as
enzyme suppressors, which was confirmed by suppression of CYP3A4 activity and mRNA
expression [data not shown].
ANC28.1 plasma
100
LPS plasma
80
60
40
20
0
control
10%
[Plasma]
20%
50%
Table 2: Effects of LPS- and ANC28.1-treated plasma on CYP mRNA expression in cultured
human hepatocytes
[Plasma]
10%
20%
50%
[Plasma]
10%
20%
50%
CYP1A2
LPS
ANC28.1
12.9
47.6
10.5
47.8
5.4
32.6
CYP2B6
LPS
ANC28.1
56.6
63
39.8
66.7
15.9
42.3
CYP2C9
LPS
ANC28.1
38.2
66.7
51.2
72.3
26.4
64.1
CYP2D6
LPS
ANC28.1
38.6
67.3
35.7
76.6
13.4
81
CYP3A4
LPS
ANC28.1
1.8
34.7
1.5
19.8
1
9.4
CYP3A5
LPS
ANC28.1
70.6
74.9
55.3
91.9
42.6
85.3
Conclusions
• Fresh human hepatocytes responded as expected to plasma from whole blood cultures
stimulated with various biologics, namely LPS and the anti-CD28 antibody ANC28.1.
• Marked suppression of CYP and drug transporter mRNA expression were observed,
which were consistent with known modulation of drug metabolism by pro-inflammatory
cytokines elevated in infection, inflammation and upon exposure to certain therapeutic
proteins.
• The method applied in this study, which consisted of ex vivo stimulation of whole blood,
collection of the stimulated plasma, and in vitro co-culture of human hepatocytes and
liver macrophages with the plasma, was previously presented. The results of this study
further supported the method’s utility to identify therapeutic proteins with the potential to
cause immune system-mediated suppression of drug metabolism and disposition and
consequently to precipitate DDI with small molecule drugs.
Values are mean of 3 - 6 determinations
Figure 1: Effects of LPS- and ANC28.1-treated plasma on CYP1A2 and CYP3A4 mRNA
expression in cultured human hepatocytes
A) CYP1A2 mRNA expression
B) CYP3A4 mRNA expression
120
ANC28.1 plasma
100
LPS plasma
80
60
40
20
0
control
10%
[Plasma]
20%
50%
120
CYP3A4 mRNA level, % control
Materials and Methods
Table 1 shows the concentrations of key inflammatory cytokines in plasma treated with LPS
and ANC28.1 and their controls, saline and MOP 31C, respectively. The concentrations of
cytokines in saline-treated blood were very low, similar to those found in untreated blood.
Similarly, plasma from the MOPC 31C-treated blood cultures (control antibody) caused only
minimal increases in IFN-γ, IL-6 and IL-8 compared to the saline-treated controls. As
expected, stimulation of whole human blood with LPS resulted in a marked increase of all
cytokines tested. Similarly, stimulation of whole blood cultures with the ANC28.1 antibody
caused an increase in the concentration of all cytokines examined as compared with MOPC
31C-treated controls. Of note, IL-2 was elevated to a higher extent in ANC28.1-treated
plasma than in the LPS-treated plasma (Table 1, highlighted fields), consistent with
stimulatory effects that antibodies against CD28 receptor have on helper T cells (e.g.
TGN1412).
CYP 1A2 mRNA level, % control
Suppression of drug metabolizing enzymes by certain therapeutic proteins (TPs) and
co-administration of TPs with small molecule drugs (SMD) create a potential for drug-drug
interactions (DDI). The suppression of drug metabolizing enzymes (DME), which constitutes
a mechanism of these interactions, is mediated by pro-inflammatory cytokines released by
peripheral blood mononuclear cells (PBMCs). In a previous study, we proposed an in vitro
method to evaluate immune system-mediated and direct effects of TPs on DME in human
hepatocytes.1 The method involves treating whole human blood with a biologic drug ex vivo,
after which plasma is prepared and added to primary human hepatocyte-Kupffer cell
co-cultures, to evaluate the effects of the drug on the expression of cytochrome P450 (CYP)
enzymes. In the aforementioned study, we demonstrated that this proposed method was
capable of determining suppression of CYP1A2, 2B6 and 3A4 activity and mRNA
expression in cultured human hepatocytes following treatment with LPS- and an anti-CD28
antibody-stimulated plasma. In the current study, we demonstrate that this method is
suitable to evaluate the suppression of other drug-metabolizing CYPs and several hepatic
drug transporters by TP drugs in vitro.
MDRI mRNA level,% control
Introduction
Cytokine
P80
1
ANC28.1 plasma
LPS plasma
100
80
60
40
20
0
control
10%
[Plasma]
20%
References
50%
Maciej Czerwinski, Christina Renneke, Joanne Parker, Chad Pope, Kevin Lyon,
Catherine Wiegand, Jason Neat, Faraz Kazmi, David Buckley, and Andrew Parkinson;
An in vitro test system to evaluate drug-drug interactions with biologics. Drug Metabolism
Reviews 43, #112, 2011

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