Newsletter - September 30, 2009
In this issue
SOLVO
Biotechnology introduces the MRP4 and the rat Mrp2 Vesicular Transport Assays
and N-methyl-quinidine (NMQ).
__________________________________________________________________________________
MRP4 Vesicular Transport Assay
SOLVO's MRP4 vesicular transport assay uses membrane vesicles
isolated from transfectant LLC-PK1 cells stably expressing human MRP4, and
employs 3H-dehydroepiandrosterone-sulfate (DHEAS) as the probe
substrate. Human MRP4 transports DHEAS efficiently with negligible
background transport in control vesicles (Figure 1).
Figure 1. DHEAS transport (cpm
values) into human MRP4 containing and control vesicles.
IC50
values were determined for a set of known MRP4 interactors using DHEAS as the
probe substrate. Inhibition curves are displayed in Figure 2.
The IC50 values calculated are in good agreement with those
obtained from the literature.
Figure 2 - Effect
of known MRP4 interactors on the MRP4-mediated DHEAS transport.
For more details on the MRP4 transporter and assay validation
data please click here.
rat Mrp2 Vesicular Transport Assay
A simple method for measuring rat Mrp2-mediated transport is
the vesicular transport (VT) assay. SOLVO's rat Mrp2 vesicular
transport assay uses membrane vesicles (ratMrp2-HEK293) isolated from
transfected HEK293 cells stably expressing rat Mrp2 and control membrane
vesicles (HEK293-CTRL) isolated from parental HEK293 cells, and employs
estradiol 3H-estradiol-17-β-D-glucuronide (E217βG) as
the probe substrate. E217βG is a widely used probe substrate
of human MRP2 and rat Mrp2. The interaction is detected as the
modulation of the initial rate of E217βG transport by rat Mrp2
into inside-out vesicles containing the transporter.
All assay validation steps were executed both at low (1 µM)
and high (100 µM) E217βG concentrations based on the human MRP2
studies of Zelcer et al (JBC, 2003). According to their observation
many compounds stimulate the MRP2-mediated transport of E217βG at
low substrate concentrations. Such drug interactions could potentially
affect the pharmacokinetic properties (e.g. oral bioavailability, biliary
elimination) of drugs transported by MRP2. Similar stimulation was
observed in the case of rat Mrp2-mediated E217βG transport shown
in this study (Figure 4).
Rat Mrp2 transports E217βG efficiently with
negligible background transport in control vesicles (Figure 3).
The transport was tested at 1 and 100 µM concentrations of E217βG.
Figure 3 - E217βG
transport (cpm values) into rat Mrp2 containing and control HEK293 vesicles.
The experiment was performed in the presence of 1 and 100 µM E217βG,
at 37°C and 50 µg protein/well for 5 minutes.
The
inhibition curves determined both at low (1 µM) and high (100 µM) E217βG
concentrations are displayed in Figure 4.
Figure
4 - Effect of known MRP2 interactors
on the rat Mrp2-mediated E217βG transport.
For more details on the rat Mrp2 transporter and assay
validation data please click here.
The unlabeled probe substrate of SOLVO's MDR1 and rat Mdr1b
PREDIVEZ Kits is now available from SOLVO Biotechnology.
N-methyl-quinidine (NMQ, Fig.5.),
a low permeabily amphipathic monoquaternary molecule is an excellent cationic
model compound. NMQ was shown to be actively transported by human
MDR1/P-gp and rat Mdr1b efflux transporters (Hooiveld et al, 2002) as well as
by the human OATP-A (OATP1A2) uptake transporter (van Montfoort et al,
1999). The apparent Km values were 15 µM, 20 µM and 26 µM
for the MDR1, Mdr1b and OATP-A transporters, respectively.
Figure 5: N-methyl-quinidine
Kinetic parameters of human MDR1- and
rat Mdr1b-mediated NMQ transport into transporter containing and control
membrane vesicles have been validated using SOLVO membrane preparations
(SB-MDR1-K-VT, SB-K-CTRL, SB-ratMdr1b-Sf9-VT, SB-defPgp-Sf9-VT).
The optimized assays are available as
SOLVO's MDR1-PREDIVEZTM and ratMdr1b-PREDIVEZTM Kits.
For more details on the NMQ validation data please click here.
CONTACT INFORMATION:
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E-mail: sales@solvo.com
web: www.solvo.com
Phone: +36-23/503-940
Fax: +36-23/503-941
SOLVO distributors:
in US and Canada: XenoTech LLC www.xenotechllc.com,
in Europe: SOLVO and tebu-bio www.tebu-bio.com,
in Japan: Nosan www.nosan.co.jp,
in India: Krishgen www.krishgen.com
Contact us
Tel.: +36-23-503-940
Fax.: +36-23-503-941
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