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NEUROPHARMACOLOGY
Centre for Neuroscience, Guy's King's and St. Thomas' School of Biomedical Science, King's College London, Guy's Hospital Campus, London, United Kingdom
Received May 2, 2003; accepted May 22, 2003.
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Abstract |
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). In
addition, HIV within the CNS is linked to the development of HIV-associated
dementia, symptoms of which include behavioral disturbances, cognitive
slowness, and motor impairment. It has been shown that elevated HIV RNA levels
in CSF correlate with increasingly severe neurocognitive dysfunction in
patients with HIV dementia (Ellis et al.,
1997). Hence, to reduce the incidence and severity of these
complications, therapeutic concentrations of anti-HIV drugs need to reach the
CNS.
To enter the CNS, substances must be able to cross the blood-brain barrier
(BBB) and/or the blood-CSF barrier (choroid plexus and arachnoid membrane).
Transporters present at these barriers can facilitate the entry of essential
endogenous compounds, such as glucose and amino acids, into the CNS.
Conversely, additional transporters operate to remove substances from the
brain and CSF (Kusuhara and Sugiyama,
2001; Ghersi-Egea and
Strazielle, 2002). Members of the nucleoside reverse transcriptase
inhibitor (NRTI) family of anti-HIV drugs use influx and efflux transporters
present at the brain barriers, which consequently affects their concentration
within the CNS (Gibbs and Thomas,
2002; Gibbs et al.,
2003).
Lamivudine (3TC) is the negative enantiomer of the nucleoside analog
2'-deoxy-3'-thiacytidine and has potent activity against HIV-1 and
HIV-2 [mean 50% inhibitory concentrations (IC50) against various
strains of HIV-1 and HIV-2 in CD4+ lymphocyte cell lines ranged from 4 nM to
0.67 µM] (Coates et al.,
1992). NRTIs, such as 3TC, inhibit HIV replication by a common
mechanism; they are phosphorylated by intracellular enzymes to their active
5'-triphosphates, which inhibit HIV reverse transcriptase through
competition with endogenous 2'-deoxynucleoside-5'-triphosphates
and by acting as chain terminators. Structurally, 3TC is similar to the NRTI
2'3'-dideoxycytidine (ddC; zalcitabine), just differing in the
3' position of the ribose ring where the carbon atom is replaced by a
sulfur atom. In contrast to other NRTIs, including ddC, 3'-azido
3'-deoxythymidine (AZT; zidovudine), 2'3'-dideoxyinosine
(ddI; didanosine), and 2'3'-didehydro-3'deoxythymidine (d4T;
stavudine), 3TC has a more favorable safety profile and is unlikely to induce
hematological or hepatic adverse effects, neuropathy, or myopathy
(Johnson et al., 1999).
Our research group has recently investigated the ability of ddC to cross
the blood-brain and blood-CSF barriers using a long-duration (30 min) brain
perfusion method in the guinea pig (Gibbs
and Thomas, 2002). CNS uptake of ddC was limited due to its
removal via organic anion transporters present at the blood-brain and
blood-CSF barriers. Using short-duration studies (15-30 s), the brain volume
of distribution of [3H]3TC has been determined to be low, at less
than 0.05 ml/100 g once corrected for vascular space
(Wu et al., 1998).
Furthermore, 3TC has been detected at low to medium concentrations in the CSF
of humans and primates (Blaney et al.,
1995; van Leeuwen et al.,
1995; Mueller et al.,
1998). CSF to plasma ratios measured in adults ranged from 0 to
46% (2-4 h after dosing) and in children ranged from 4 to 8% (2 h after
dosing). In primates, CSF levels ranged from 8 to 41% of plasma levels (up to
24 h after dosing). Consequently, it has been proposed that 3TC uses active
efflux transport systems to leave the CNS
(Blaney et al., 1995;
Wu et al., 1998). To test this
hypothesis, we investigated 3TC movement across the guinea pig blood-brain and
blood-CSF barriers by means of the long-duration bilateral brain perfusion
method and isolated incubated choroid plexus model. Both methods are well
established and allow a thorough investigation of drug movement into the brain
and CSF simultaneously. Another advantage of the longer perfusion time is that
the uptake of slowly permeating molecules can be detected. 3TC is commonly
prescribed with other NRTIs, so in addition to using a variety of efflux
transport inhibitors, this study also examined the affect of additional NRTIs
on the CNS distribution of 3TC.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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).
[3H]3TC (10.6 nM) and [14C]mannitol (1.4 µM) were
introduced into the inflowing artificial plasma via a slow drive syringe pump
for 2.5 to 30 min. A cisterna magna CSF sample was taken at the end of the
perfusion before the animal was decapitated and the brain removed. The choroid
plexuses were excised from the lateral ventricles of the brain, and together
with CSF, brain, and artificial plasma samples, were solubilized over 48 h
(Solvable; Packard, Berkshire, UK). Scintillation fluid (Lumasafe; Packard)
was then added in preparation for radioactive scintillation counting. A
Packard Tri-Carb 1900TR liquid scintillation counter was used, and the counts
per minute were converted to disintegrations per minute (disintegrations per
minute) by use of internally stored quench curves. The amount of
[3H]3TC and [14C]mannitol in the brain
(Cbrain; disintegrations per minute per gram of brain
tissue), CSF (CCSF; disintegrations per minute per
milliliter of CSF), and choroid plexuses (Cchoroid plexus;
disintegrations per minute per gram of choroid plexus tissue) was expressed as
a percentage of the amount of activity in the artificial plasma
(Cplasma; disintegrations per minute per milliliter of
plasma) and termed uptake (milliliters per 100 grams or milliliters per 100
milliters). [14C]Mannitol is a polar sugar alcohol that can only
enter the CNS by simple diffusion along the restricted paracellular pathways
of the cerebral capillaries and the choroid plexus
(Thomas and Segal, 1996), thus
[14C]mannitol was used in these studies to mark the vascular volume
of the brain and the vascular volume and extracellular space of the choroid
plexus. It has previously been shown that perfusion of 1.4 µM
[14C]mannitol into the carotid arteries does not alter the passive
permeability of the brain barriers (Thomas
and Segal, 1996). [3H]3TC uptake into the brain and
choroid plexus could be corrected for [14C]mannitol space by
subtracting the [14C]mannitol uptake from the [3H]3TC
uptake measured in each perfusion. Self-Inhibition Studies. Further brain perfusion experiments (20 min) were carried out in the presence of an excess (50 µM) of unlabeled 3TC dissolved in the artificial plasma. This was to determine whether 3TC transport across the brain barriers could be saturated.
Transport Inhibition Studies. Transport inhibitors were used to
investigate the role of transporters in the CNS accumulation of
[3H]3TC. The concentrations of the inhibitors used are above the
half saturation constant (Km) or half inhibition constant
(Ki) of the target transporters. It should be noted that
when target transporters are located on the CNS facing side of the brain
barriers (i.e., the apical side of the choroid plexus or the abluminal side of
the BBB), inhibitors need to be used at sufficient plasma levels to achieve
effective concentrations at the transporter site after passage across the
brain barriers. Progesterone (25 µM), to inhibit P-glycoprotein (P-gp); 350
µM probenecid, a broad inhibitor of organic anion transport; 10 µM
indomethacin, to inhibit multidrug resistance-associated protein (MRP); 200
µM taurocholic acid (TCA), to inhibit organic anion transporting
polypeptides (Oatps); 25 µM digoxin, to inhibit Oatp2 and P-gp; and 1 mM
tetraethylammonium bromide (TEA) and 30 µM trimethoprim to inhibit organic
cation transporters (OCTs) (Qian and Beck,
1990; Huai-Yun et al.,
1998; Gibbs and Thomas,
2002; Takubo et al.,
2002; Gibbs et al.,
2003) were added individually to the artificial plasma and
preperfused into the guinea pig for 10 min before and during a 10-min
[3H]3TC/[14C]mannitol perfusion. Digoxin and TCA were
freely soluble in the artificial plasma. The other transport inhibitors were
made into stock solutions in ethanol (progesterone) or dimethyl sulfoxide
(probenecid, indomethacin, TEA, and trimethoprim) and then diluted with
artificial plasma to make the required concentration. Solvent levels in the
artificial plasma did not exceed 0.2%. In each experiment,
[14C]mannitol CNS levels were monitored to ensure barrier
integrity. [3H]3TC uptake in the presence of the inhibitors was
compared with [3H]3TC uptake in the absence of the inhibitors as
measured in a series of "10 + 10 min" control experiments whereby
a 10-min preperfusion period (in the absence of
[3H]3TC/[14C]mannitol and any inhibitors) preceded a
10-min [3H]3TC/[14C]mannitol perfusion.
Cross-Competition Studies. To examine the effect of additional NRTIs
on the CNS uptake of [3H]3TC, further perfusions were carried out
in the presence of excess and clinically relevant concentrations of abacavir
(100 and 6.8 µM), AZT (100 and 7.1 µM), d4T (100 and 3.1 µM), and ddI
(100 and 2.2 µM) (Gibbs et al.,
2003). These drugs were dissolved into the artificial plasma and
preperfused into the guinea pig for 10 min before and during a 10-min
[3H]3TC/[14C]mannitol perfusion. [3H]3TC
uptake in the presence of additional NRTIs was compared with 10-min
[3H]3TC/[14C]mannitol perfusions carried out after
10-min [3H]3TC/[14C]mannitol-free preperfusions (10 + 10
min controls).
HPLC. To confirm that [3H]3TC remained intact and
radiolabeled when presented to the brain, HPLC analysis was carried out on
samples of the inflowing perfusate and venous outflow. In preparation for
analysis, the inflowing perfusate and a supernatant prepared from the venous
outflow by centrifugation (5 min, 3,000g, 4°C) were diluted with
acetonitrile (1:1), vortexed, and then centrifuged (15 min, 13,000g,
4°C). The resultant supernatant was diluted to produce a sample suitable
for HPLC analysis (final acetonitrile concentration <10%)
(Thomas et al., 2001). A Jasco
HPLC system was used (Jacso, Great Dunmow, Essex, UK) with a Packard
radioactive detector (Packard, Pangbourne UK). All samples were eluted from a
150 x 4.6 mm, 3 µm Luna C18(2) column (Phenomenex,
Cheshire, UK) using a linear gradient, 5 to 45% acetonitrile against an
aqueous solution, over 20 min (flow rate 0.85 ml/min)
(Simon et al., 2001). The UV
absorbance of 3TC was monitored at 250 nm. After HPLC analysis, the column
outflow continued on to the radioactive detector, where it was mixed with
scintillation fluid (Ultima Flo M; Packard) and passed through a 0.5-ml flow
cell for real-time radioactive analysis.
Isolated Incubated Choroid Plexus. An isolated incubated choroid
plexus technique was used to examine [3H]3TC accumulation from an
artificial CSF into choroid plexus tissue. Choroid plexuses were isolated from
the brains of anesthetized and heparinized guinea pigs
(Gibbs and Thomas, 2002).
Isolated tissue was incubated for 20 min in ice-cold artificial CSF (130.0 mM
NaCl, 3.0 mM KCl, 26. 4 mM NaHCO3, 0.2 mM
Na2HPO4, 1.8 mM MgCl2, 2.5 mM
CaCl2 · 6H2O, and 5.4 mM D-glucose)
before a 10-min incubation in warm CSF (in the presence or absence of an
inhibitor or NRTI), followed by a second 10-min incubation where
[3H]3TC (184 nM) and [14C]mannitol (14 µM) were
present. The tissue was then removed and weighed. The choroid plexus was
solubilized over 24 h and taken with samples of the incubation medium for
radioactive scintillation counting. [3H]3TC and
[14C]mannitol choroid plexus accumulation was measured as a ratio
of the amount in the CSF.
Isolated choroid plexus experiments were carried out in the absence and
presence of the NRTIs 3TC (50 µM), abacavir (100 µM), AZT (100 µM),
d4T (100 µM), and ddI (100 and 2.2 µM) and transport inhibitors 25 µM
progesterone, to inhibit P-gp; 10 µM indomethacin, to inhibit MRP; 500
µM p-aminohippurate, to inhibit organic anion transporters (OATs);
500 µM 2,4-dichlorophenoxyacetic acid (2,4-D), to inhibit Oatps and OATs;
25 µM digoxin, to inhibit Oatp2 and P-gp; and 1 mM TEA, to inhibit OCTs
(Gibbs and Thomas, 2002;
Gibbs et al., 2003).
Progesterone, TEA, and indomethacin were made up into stock solutions in
ethanol (progesterone) or dimethyl sulfoxide (TEA and indomethacin). Final
artificial CSF solvent levels did not exceed 0.4%, and choroid plexus
[14C]mannitol (extracellular space marker) levels in the presence
of these solvents were closely monitored.
Octanol-Saline Partition Coefficient and Protein Binding.
The octanol-saline partition coefficient of 3TC was determined by diluting
[3H]3TC with a pH 7.4 phosphate-buffered saline (final
concentration 20.7 nM) and adding aliquots of this solution (750 µl) to
equal volumes of octanol. This mixture was then vortexed and centrifuged (5
min, 1,000g, 4°C). [3H]3TC levels in the octanol
(upper) and saline (lower) phases were determined using scintillation counting
and the octanol saline partition coefficient was determined as a ratio of drug
in the octanol phase to drug in the saline phase. The percentage of 3TC
binding to proteins in the artificial plasma were determined (at pH 7.4) as
described previously (Gibbs and Thomas,
2002).
Data Analysis. Data from all the experiments are presented as mean ± S.E.M. After multiple-time uptake studies, the slope (unidirectional transfer constant, Kin) and y-intercept (initial volume of distribution, Vi) of the line were determined by least-squares linear regression analysis, where appropriate, and are reported together with the correlation coefficient (r) and the level of significance that time can be used to predict the uptake value. Statistical analysis was carried out using Sigma Stat software (Jandel Scientific, San Rafael, CA) and significance taken as *p < 0.05.
Materials. [3H]3TC (16.1 Ci/mmol), D-[14C]mannitol (53 mCi/mmol), 3TC, and abacavir were purchased from Moravek Biochemicals (Brea, CA). Ethanol and acetonitrile were purchased from Merck Eurolab Ltd. (Lutterworth, UK). Unless stated all other materials were from Sigma Biochemicals (Poole, Dorset, UK).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Self-Inhibition Studies. The effects of unlabeled 3TC on [3H]3TC uptake into brain, CSF, and choroid plexus are shown on Table 1. In each case [3H]3TC uptake (corrected for [14C]mannitol) was not significantly altered in the presence of excess drug.
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Transport Inhibition Studies. Brain perfusions in the presence of transport inhibitors (Fig. 3) found that although [3H]3TC uptake into the brain and CSF was unaffected, significant changes were evident at the choroid plexus. Digoxin and trimethoprim significantly reduced choroid plexus uptake of the test drug (one-way ANOVA and Dunnett's method, p < 0.05).
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Cross-Competition Studies. Brain perfusion experiments in the presence of additional NRTIs found that brain, CSF, and choroid plexus uptake of [3H]3TC was not significantly affected by the presence of abacavir, AZT, d4T, or ddI at both high and low concentrations (Table 1).
Isolated Choroid Plexus. In initial isolated choroid plexus experiments, [3H]3TC tissue accumulation after 10 min was 1.13 ± 0.14 ml/g. Accumulation of the extracellular marker, [14C]mannitol, was significantly lower at 0.51 ± 0.11 ml/g (paired t test, p < 0.005). [3H]3TC accumulation was not significantly altered in the presence of 100 µM unlabeled 3TC, abacavir, AZT, or d4T; however, 100 µM ddI (but not 2.2 µM) significantly increased [3H]3TC accumulation from the incubation medium (Fig. 4). Further isolated incubated choroid plexus experiments in the presence of transport inhibitors show that 2,4-D significantly increased the choroid plexus accumulation of [3H]3TC (Fig. 4). The integrity of the choroid plexus cells was confirmed by comparison of the uptake of the extracellular space marker in each experimental group against the control (one-way ANOVA).
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Octanol-Saline Partition Coefficient and Protein Binding. The octanol-saline partition coefficient for 3TC was 0.108 ± 0.006. Protein binding analysis revealed that 3TC binding to proteins in the artificial plasma was negligible (<1%).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Banks et al.,
2001). Thus, substantial 3TC accumulation in the choroid plexus
may have beneficial effects on the treatment of HIV infection within the CNS.
Despite significant [3H]3TC accumulation in the choroid plexus from
the blood, the subsequent passage of [3H]3TC into the CSF was more
restricted. Nevertheless, drug uptake into the CSF was still significantly
greater than [14C]mannitol uptake. [3H]3TC CSF uptake
after 30 min (1.9 ± 0.3 ml/100 g) is comparable with [3H]ddC
uptake (1.4 ± 0.5 ml/100 g) measured by the perfusion method at the
same time point (Gibbs and Thomas,
2002). Clinical studies have reported CSF/plasma ratios as 6% in
adults and 0 to 46% (median, 10%) in children
(van Leeuwen et al., 1995;
Mueller et al., 1998).
Interestingly, in primates, mean 3TC CSF/plasma ratios are higher in lumbar
CSF (41 ± 23%) than ventricular CSF (7.9 ± 4.7%)
(Blaney et al., 1995). This
could be explained by the presence of an active efflux mechanism for 3TC
localized to the ventricles.
[3H]3TC was detected in the brain at very low levels after
carotid artery perfusion. This reflects findings in the rat where 3TC brain
uptake after 15-s unilateral carotid artery perfusion was less than 0.05
ml/100 g (Wu et al., 1998).
The rate of [3H]3TC entry (Kin) into the guinea
pig brain was no greater than the rate of [14C]mannitol entry,
implying that 3TC passage across the BBB, like mannitol, is restricted to
diffusion. Considering the low lipophilicity of mannitol (octanol-saline
partition coefficient 0.002 ± 0.0004;
Gibbs and Thomas, 2002)
compared with 3TC (0.108 ± 0.006), it would be expected that 3TC uptake
across the BBB be greater than mannitol uptake. Because this was not the case,
the influence of transporter systems which limit CNS uptake is implicated.
Only 3TC that is not bound to plasma proteins is free to cross membranes and
distribute in anatomical compartments. [3H]3TC passage across the
guinea pig BBB was not limited by protein binding within the plasma, because
the majority of the drug (99%) was unbound. 3TC binding to protein in human
plasma is, however, very substantial (36%, Epivir prescribing information;
GSK, September 2001) and may impact on the ability of 3TC to move from blood
to CNS. However, the low levels of [3H]3TC protein binding in these
studies allows us to determine the ability of the unbound drug to cross the
brain barriers.
[3H]3TC CNS uptake was unaffected by 50 µM 3TC
(Table 1); however, a
low-affinity (i.e., only significantly affected at 3TC concentrations greater
than 50 µM) transport system for 3TC at the brain barriers could possibly
exist. Specific transport inhibitors assessed the involvement of OATs, Oatps,
OCTs, and ATP-binding cassette transporters (P-gp and MRP) on
[3H]3TC CNS distribution. These studies failed to detect any BBB
transport systems that may function to limit [3H]3TC brain uptake
(Fig. 3). However, they did
provide evidence for transporters at the blood-CSF barrier. Digoxin
significantly reduced [3H]3TC choroid plexus uptake from blood.
Digoxin has a high affinity for Oatp2 (Km 0.24-1.07
µM), a transporter found on the choroid plexus basolateral membrane
(Noe et al., 1997;
Gao et al., 1999).
Additionally, digoxin inhibits P-gp
(Schinkel et al., 1996), which
is also expressed at the choroid plexus. However, P-gp is believed to function
as an apically directed efflux transporter
(Fricker and Miller, 2002) and
so the observed effect of digoxin on [3H]3TC choroid plexus
accumulation in unlikely to be via this transporter. Instead, an Oatp2-like
transporter at the blood-facing side of the choroid plexus, which facilitates
[3H]3TC tissue entry is implicated [although digoxin effected
[3H]3TC choroid plexus accumulation, TCA and probenecid, which
could also be expected to inhibit Oatp2, did not. This may be explained by the
inhibition of other transporters, which remove 3TC from the choroid plexus, by
TCA (i.e., Oatp) and probenecid (Oatp, Oat, and MRP)]. The notion of an
Oatp2-like transporter facilitating 3TC choroid plexus influx is supported by
previous work that identified the involvement of an Oatp2-like transporter in
ddI choroid plexus influx (Gibbs et al.,
2003). This study indicated a significant interaction between
[3H]ddI and 3TC at the basolateral membrane of the perfused guinea
pig choroid plexus. Although [3H]3TC choroid plexus uptake in this
study was reduced in the presence of 100 µM ddI (from 36.5 ± 5.8% to
19.6 ± 3.3%; Table 1),
this did not attain statistical significance.
Evidence that 3TC interacts with a further choroid plexus transporter comes
from incubated choroid plexus experiments
(Fig. 4). [3H]3TC
choroid plexus accumulation from CSF was significantly increased by ddI (100
µM) and 2,4-D, signifying the presence of a transporter-facilitating drug
efflux from the tissue. The 2,4-D sensitivity suggests that it is a
transporter for organic anions (OAT or Oatp)
(Gibbs and Thomas, 2002).
OAT1-3 are expressed in the mouse and rat choroid plexus
(Sweet et al., 2002) and rat
OAT1 can transport 3TC (Wada et al.,
2000). However, we found no further evidence for the involvement
of an OAT transporter, because p-aminohippurate and salicylate
(OAT1/OAT3 and OAT2 inhibitors, respectively
(Sweet et al., 1997;
Kusuhara et al., 1999;
Morita et al., 2001), did not
effect [3H]3TC choroid plexus accumulation. Oatp1-3 are present in
rat choroid plexus (Ohtsuki et al.,
2003). However, Oatp1 and Oatp3 influx organic anions from CSF to
choroid plexus, so are unlikely to be involved in the efflux system reported
here. Although we have evidence for a 2,4-D-sensitive system, which removes
3TC from choroid plexus, the nature of this transporter remains unclear.
3TC is a weak organic anion that exhibits weak organic cation properties. A
TEA and trimethoprim sensitive OCT system for 3TC uptake
(Km of 2.28 ± 0.387 mM) has been seen in rat renal
cortex brush-border membrane vesicles
(Takubo et al., 2002). Because
several OCTs are present at the CNS barriers
(Sawada et al., 1999;
Kido et al., 2001;
Sweet et al., 2001), their
involvement in [3H]3TC CNS distribution was investigated. Results
demonstrated (Figs. 3 and
4) that [3H]3TC CNS
uptake was unaffected by TEA, a broad OCT inhibitor. Interestingly,
trimethoprim significantly attenuated [3H]3TC uptake from blood to
choroid plexus. Although trimethoprim can inhibit OCT transporters
(Takubo et al., 2002), our
data, from experiments in the presence of TEA, suggest that this trimethoprim
sensitivity is not via an OCT. Subsequently, we conclude that the OCTs do not
facilitate [3H]3TC movement across the guinea pig CNS barriers.
Interestingly, clinical evidence suggests an interaction between digoxin and
trimethoprim: increased digoxin serum levels are reported in patients who
received trimethoprim with digoxin
(Petersen et al., 1985).
Hence, it may be possible that trimethorprim interacts with digoxin at the
Oatp2 transporter.
MRP4 and MRP8, have also been implicated in 3TC transport
(Schuetz et al., 1999;
Turriziani et al., 2002). MRP4
and MRP8 are known to be expressed in rat and human brain, respectively
(Bera et al., 2001;
Hirrlinger et al., 2002) and
MRP4 is present at the bovine BBB (Zhang
et al., 2000). To date, MRP1 is the only MRP to be localized to
the choroid plexus (Rao et al.,
1999). The MRP inhibitor indomethacin did not effect
[3H]3TC CNS uptake (Figs.
3 and
4); thus, we conclude that MRP
is not involved in [3H]3TC transport across the guinea pig CNS
barriers. Experiments carried out in the presence of the P-gp modulator
progesterone showed that P-gp does not effect the CNS distribution of
[3H]3TC.
An interaction between 3TC and ddI at the choroid plexus has been implicated by this work. In contrast, there was no evidence for interactions between 3TC and abacavir, AZT, or d4T at the choroid plexus (Fig. 4). Given that the choroid plexus is considered a target for treating HIV within the CNS, this finding could impact on drug selections for combination therapy intended to tackle HIV within the CNS. Importantly, this study demonstrated that none of the NRTIs significantly effected [3H]3TC brain or CSF uptake, indicating that their inclusion in a treatment regime containing 3TC does not have detrimental effects on the 3TC accumulation within these regions.
In conclusion, this study demonstrates that 3TC has a limited ability to cross the brain barriers and accumulate in the brain and CSF. Although we found no evidence for an efflux transporter restricting 3TC brain entry, our results indicate the involvement of a digoxin-sensitive transporter in [3H]3TC uptake from blood to choroid plexus. The involvement of a further transporter acting to remove 3TC from the choroid plexus is indicated. We have demonstrated that ddI may interact with these two transporters to affect 3TC choroid plexus accumulation. However, it may be important clinically to note that none of the NRTIs acted to alter [3H]3TC uptake into brain or CSF.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: HIV, human immunodeficiency virus; CNS, central nervous system; BBB, blood-brain barrier; NRTI, nucleoside reverse transcriptase inhibitor; 3TC, (-)-2'-deoxy-3'-thiacytidine; ddC, 2'3'-dideoxycytidine; AZT, 3'-azido 3'-deoxythymidine; ddI, 2'3'-dideoxyinosine; d4T, 2'3'-didehydro-3'deoxythymidine; CSF, cerebrospinal fluid; P-gp, P-glycoprotein; MRP, multidrug resistance-associated protein; TCA, taurocholic acid; Oatp, organic anion transporting polypeptide; TEA, tetraethylammonium bromide; OCT, organic cation transporter; HPLC, high-performance liquid chromatography; OAT, organic anion transporter; 2,4-D, 2,4-dichlorophenoxyacetic acid; ANOVA, analysis of variance; Ki, half inhibitory constant; Kin, unidirectional transfer constant; Km, half saturation constant; Vi, initial volume of distribution.
Address correspondence to: Dr. Sarah A. Thomas, Centre for Neuroscience, Guy's King's and St. Thomas' School of Biomedical Science, King's College London, Guy's Hospital Campus, London Bridge, London SE1 1UL, UK. E-mail: sarah.thomas{at}kcl.ac.uk
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