3.2.4 Increased expression and activity of P-gp and BCRP
A selective increase in microvascular expression of P-gp and BCRP during
disease progression in three ALS mouse models
(SOD1G93A, SOD1G86R and
TDP43A315T) has been reported in the cerebral cortex
and spinal cord (Jablonski et al., 2012; Milane et al., 2010). mRNA
levels were also significantly increased in lumbar spinal cord
homogenates 2.13-fold for P-gp and 1.72-fold for BCRP in mice at
symptomatic stages compared to those at presymptomatic stages (Jablonski
et al., 2012). In addition, Western blot was employed to assess P-gp and
BCRP abundance in the whole spinal cord and cerebral cortex of
SOD1G93A mice. Increased expression of P-gp
(1.96-fold) and BCRP (1.69-fold) was observed in the spinal cord of
symptomatic mice compared to presymptomatic SOD1G93Amice and elevated levels of P-gp (1.35-fold) and BCRP (1.28-fold) were
reported in the cerebral cortex of symptomatic mice compared to
presymptomatic SOD1G93A mice (Jablonski et al., 2012).
The increased expression of P-gp and BCRP was demonstrated in another
mouse model of ALS by Jablonski et al. comparing P-gp and BCRP
levels in the spinal cord and cerebral cortex of symptomatic
TDP43A315T mice with presymptomatic mice. An
independent group (Milane et al., 2010) demonstrated increased P-gp
abundance (1.5-fold) in microvessels isolated from the brains of
presymptomatic SOD1G86R mice compared to age-matched
wildtype controls. However, no alteration of BCRP abundance was detected
in these SOD1G86R mice.
In SOD1G93A rats, microvessels from brain and spinal
cord sections were assessed immunohistochemically and microvessels were
also isolated for protein quantification using Western blot (Chan,
Evans, Banks, Mesev, Miller & Cannon, 2017). In this study Chanet al . demonstrated immunohistochemically that P-gp protein
expression was significantly increased in the cerebral cortex
(1.88-fold) and the spinal cord (1.46-fold) from symptomatic
SOD1G93A rats compared to age-matched wildtype rats.
In addition, the expression of BCRP was not affected when assessed
immunohistochemically. Given immunohistochemistry is semi-quantitative,
protein expression in isolated microvessels was reassessed using Western
blot, and a 1.5-fold increase in P-gp expression was demonstrated in
isolated microvessels from brain and spinal cord of symptomatic
SOD1G93A rats. It was also noted that microvascular
BCRP expression increased 1.15- and 1.25-fold in brain and spinal cord,
respectively, although statistical significance was not achieved. These
studies clearly demonstrate increased expression of P-gp and sometimes
BCRP in the brain and spinal cord in models of ALS. Some differences in
the results were observed. For example, whole brain and spinal cord
homogenates were used by Jablonski et al . and the samples
obtained from the microvascular isolation techniques employed by Chanet al. and Milane et al. often contain astrocytes, which
express P-gp (Golden & Pardridge, 1999).
In addition to assessing expression of P-gp and BCRP, Jablonski et
al. assessed their activity using a confocal microscopy-based transport
assay where microvessles were incubated with fluorescent substrates
(NBD-cyclosporin A for P-gp; bodipy-prazosin for BCRP) (Jablonski et
al., 2012). Luminal substrate accumulation was measured in the absence
and presence of specific transport inhibitors (PSC833 for P-gp, Ko143
for BCRP). This assay successfully demonstrated that the transport
activity of P-gp was increased 1.8-fold and 2-fold in brain and spinal
cord microvessels, respectively, from symptomatic
SOD1G93A mice relative to age-matched wildtype mice
and presymptomatic SOD1G93A mice. In a follow up
study, the in vivo accumulation of a specific P-gp substrate,
LD800, in the spinal cord of symptomatic SOD1G93A mice
was assessed and compared to wildtype mice following intraperitoneal
administration (Jablonski et al., 2014). Spinal cord levels of LD800
levels were significantly lower in SOD1G93A mice
comparing to wildtype mice, suggesting increased P-gp activity at the
BSCB, in line with increased P-gp expression. A similar study was
performed, where a P-gp substrate [3H]-digoxin and
a BCRP substrate [3H]-prazosin were dosed
intraperitoneally to both SOD1G86R mice and wildtype
mice (Milane et al., 2010). A reduced disposition of
[3H]-digoxin (1.5-fold) but not
[3H]-prazosin was observed in the brain of
presymptomatic SOD1G86R mice compared to wildtype
controls, suggesting increased function of P-gp but not BCRP in this
mouse model. This alteration is in line with reduced P-gp protein
abundance (1.5-fold) and unchanged BCRP abundance observed in this mouse
model.
Chan et al. demonstrated a 2-fold increase in P-gp activity in
microvessels isolated from the brain and spinal cord of symptomatic
SOD1G93A rats compared to those from presymptomatic
rats using the same confocal microscopy-based transport assay mentioned
above (Chan, Evans, Banks, Mesev, Miller & Cannon, 2017). Overall,
these studies demonstrate increased P-gp and BCRP abundance and
activities at the CNS barriers in ALS rodent models, suggesting that the
barriers may be more restrictive to substrates of these transporters.
This is of particular concern given that riluzole, which is effective in
early ALS and less effective in late stage ALS, is a substrate of P-gp
and BCRP (Zoccolella et al., 2007). The reduced effectiveness of
riluzole in SOD1G93A rats may result from P-gp/BCRP
overactivity, as less riluzole may be able to enter the CNS. In fact, a
1.7-fold reduction in riluzole disposition into the brain of
SOD1G86R has been reported compared to wildtype mice
following intraperitoneal administration (Milane et al., 2010).
Therefore, approaches to overcome increased P-gp and BCRP activities in
ALS may improve the effectiveness of riluzole. This has been confirmed
in SOD1G93A mice that received riluzole with or
without elacridar (an inhibitor of P-gp and BCRP) (Jablonski et al.,
2014). The efficacy of riluzole improved when co-administered with
elacridar and this was associated with increased riluzole delivery to
the brain. However, such generic inhibition of P-gp/BCRP comes with side
effects, and therefore, an approach to restore P-gp/BCRP to levels
similar to the healthy CNS barriers is more likely to be a more
effective clinical strategy. In addition, while much has been reported
on P-gp and BCRP in ALS, our knowledge on other transporters at the CNS
barriers is limited. Profiling the expression at the CNS barriers could
potentially identify efflux transporters that should be avoided for
effective CNS delivery or influx transporters, which could be
effectively targeted to increase CNS exposure of drugs intended to reach
the CNS.