Download Effects of a P-Glycoprotein Inhibitor on Brain and Plasma

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DRUG METABOLISM AND DISPOSITION
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
DMD 30:479–482, 2002
Vol. 30, No. 5
595/975193
Printed in U.S.A.
Short Communication
EFFECTS OF A P-GLYCOPROTEIN INHIBITOR ON BRAIN AND PLASMA
CONCENTRATIONS OF ANTI-HUMAN IMMUNODEFICIENCY VIRUS DRUGS
ADMINISTERED IN COMBINATION IN RATS
(Received October 1, 2001; accepted January 18, 2002)
This article is available online at http://dmd.aspetjournals.org
ABSTRACT:
6-Cl-ddP and GF120918, or 6-Cl-ddP, nelfinavir, and GF120918.
Both 6-Cl-ddP and nelfinavir were administered as i.v. infusions,
whereas GF120918 was given as an i.v. bolus 2 h before sampling.
Plasma and brain tissue concentrations of 6-Cl-ddP, ddI, and nelfinavir were determined. Neither nelfinavir nor GF120918 was shown
to alter the brain/plasma ratios of 6-Cl-ddP or ddI. GF120918,
however, increased the plasma concentrations of 6-Cl-ddP and
ddI, resulting in increased brain concentrations. GF120918 increased the brain/plasma ratio of nelfinavir significantly (⬃100fold). The brain/plasma ratios of nelfinavir were reduced nearly
2-fold in rats treated with nelfinavir, 6-Cl-ddP, and GF120918 compared with rats receiving only nelfinavir and GF120918, suggesting
a modest inhibition of nelfinavir uptake by 6-Cl-ddP. Overall, combined 6-Cl-ddP, nelfinavir, and GF120918 administration enhances
the brain/plasma ratios of both ddI and nelfinavir.
Highly active antiretroviral therapy that includes both reverse transcriptase inhibitors (RTIs1) and protease inhibitors has improved the
clinical management of HIV infection dramatically (Carpenter et al.,
2000). A problem with current antiretroviral medication, however, is
the limited entry of these drugs into the central nervous system (CNS)
(Kaufmann and Cooper, 2000). HIV-1 can enter the CNS through
infected monocytes that differentiate into macrophages and microglia
in the brain (Rausch and Stover, 2001). Productive viral replication by
macrophages in the CNS leads to loss of cognitive and motor func-
tions, referred to as acquired immunodeficiency syndrome dementia
complex (ADC) (Brew, 1999). Roughly 20 to 30% of patients with
advanced HIV infection develop ADC (McArthur et al., 1993; Brew,
1999), which occurs more frequently in HIV-infected infants and
children, resulting in increased morbidity and devastating complications (Belman, 1994).
AZT, a relatively lipophilic dideoxynucleoside RTI, has been
proven somewhat effective in ADC (Tozzi et al., 1993). However,
AZT exhibits limited brain uptake, and it is usually recommended that
the highest dose of AZT be given. The long-term use of high-dose
AZT, however, is impractical due to hematological intolerance (Gill et
al., 1987). 2⬘,3⬘-Dideoxyinosine (ddI), another commonly used nucleoside analog RTI, is very hydrophilic and exhibits very low CNS
penetration. A nonsteady-state cerebrospinal fluid to plasma ratio of
0.21 (Hartman et al., 1990) and steady-state brain parenchyma to
plasma ratio of less than 0.05 have been observed for ddI in humans
and in rats, respectively (Anderson et al., 1990; Butler et al., 1991;
Morgan et al., 1992). 6-Chloro-2⬘3⬘-dideoxypurine (6-Cl-ddP), a lipophilic prodrug of ddI activated by adenosine deaminase, has been
shown to significantly increase ddI concentrations in the brain due to
its increased lipophilicity and relatively high adenosine deaminase
activity in the brain (Ho et al., 1980; Morgan et al., 1992).
The CNS entry of protease inhibitors, such as nelfinavir, is restricted in part by the P-glycoprotein (P-gp) efflux transporter (Kim et
al., 1998; Choo et al., 2000) located within the blood-brain barrier. By
inhibiting P-gp, it is possible to obtain increased brain to plasma ratios
of nelfinavir in mice (Choo et al., 2000). GF120918 is a potent P-gp
This work was supported by National Institutes of Health Grant RO1 NS3917801. Jouko Savolainen was financially supported through fellowships from the
Academy of Finland, the Ella and Georg Ehrnrooth’s Foundation, the Saastamoinen Foundation, the Pharmacal Research and Science Foundation, and the
Finnish Pharmaceutical Society. Jeffrey E. Edwards was supported by National
Institute of Environmental Health Sciences Training Grant ES07266.
1
Abbreviations used are: RTI, reverse transcriptase inhibitor; HIV, human
immunodeficiency virus; CNS, central nervous system; ADC, acquired immunodeficiency syndrome dementia complex; AZT, 3⬘-azido-2⬘,3⬘-dideoxythymidine;
ddI, 2⬘,3⬘-dideoxyinosine; 6-Cl-ddP, 6-chloro-2⬘3⬘-dideoxypurine; P-gp, P-glycoprotein; GF120918, N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)
ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide; MRP,
multidrug resistance-associated protein; HPLC, high-pressure liquid chromatography.
Address correspondence to: Dr. Bradley D. Anderson, University of Kentucky, Division of Pharmaceutical Sciences, 327-G, 907 Rose St., Lexington, KY
40536-0082. E-mail: [email protected]
479
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Most of the existing anti-human immunodeficiency virus agents enter
the central nervous system (CNS) inefficiently and thus may allow
slow viral replication in the brain. This may provide a sanctuary for the
virus in the CNS and contribute to the development of acquired
immunodeficiency syndrome dementia complex. This study evaluates a prodrug approach to improve the CNS delivery of the reverse
transcriptase inhibitor 2ⴕ,3ⴕ-dideoxyinosine (ddI) in combination with
inhibition of P-glycoprotein-mediated efflux to increase the CNS delivery of the protease inhibitor nelfinavir and to determine whether any
unanticipated drug interactions occur in this combination therapy.
Three rats received either 6-chloro-2ⴕ3ⴕ-dideoxypurine (6-Cl-ddP), a
prodrug of ddI activated by adenosine deaminase, nelfinavir, nelfinavir and 6-Cl-ddP, nelfinavir and N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo4-acridine carboxamide (GF120918) (a P-glycoprotein inhibitor),
480
SAVOLAINEN ET AL.
Materials and Methods
Chemicals. 2⬘,3⬘-Dideoxyinosine was provided by the National Institute of
Allergy and Infectious Diseases (Bethesda, MD). The preparation of 6-Cl-ddP
has been described previously (Murakami et al., 1991). Nelfinavir was extracted from Viracept tablets (Agouron Pharmaceuticals, Inc., a Pfizer Company, Ann Arbor, MI), yielding a white solid having a purity of 98.6% by
microtitration and exhibiting a single peak by HPLC. GF120918 was a gift
from GlaxoSmithKline (Research Triangle Park, NC).
Surgical Procedure and Preparation of Infusion Solution. Male
Sprague-Dawley rats were obtained from Harlan, Inc. (Prattville, AL) and
housed and cared for at the Division of Laboratory Animal Research facilities
at the University of Kentucky. All animal procedures conformed to the
guidelines for the care and use of laboratory animals set by the University of
Kentucky. Animals were anesthetized with 100 mg/kg ketamine and 8 mg/kg
xylazine i.p. Using aseptic technique, catheters were implanted into the jugular
and femoral veins, as described by Waynforth and Flecnell (1994). The
animals were allowed a minimum recovery period of 24 h from the surgery,
and the catheters were flushed daily with 0.3 ml of normal saline containing
500 U/ml heparin. 6-Cl-ddP, nelfinavir, and GF120918 solutions were prepared in normal saline, deionized water adjusted to pH ⬃2.4 with HCl, and
dimethyl sulfoxide, respectively.
Experimental Designs. Intravenous infusions were performed in three rats
per group receiving either 6-Cl-ddP (target dose, 134.8 mg/kg/h), nelfinavir
(target dose, 10.5 mg/kg/h), nelfinavir and 6-Cl-ddP, nelfinavir and GF120918
(10 mg/kg), 6-Cl-ddP and GF120918, or 6-Cl-ddP, nelfinavir, and GF120918.
Body weights on the day of the experiment were 292 ⫾ 41 g (mean ⫾ S.D.;
n ⫽ 18). Nelfinavir was infused for 8 h and 6-Cl-ddP for 0.5 h beginning at
7.5 h. The time of administration of GF120918 was chosen based on its plasma
distribution kinetics and elimination half-life (2.7 h) (Hyafil et al., 1993) and
was given at 6 h as an i.v. bolus. At the end of 8 h, the samples were collected
for 6-Cl-ddP and ddI analyses, as described previously by Morgan et al.
(1992). Blood samples for analysis of nelfinavir were centrifuged, and plasma
was collected and frozen before extraction. Brain tissue samples were quickfrozen until the time of sample preparation.
Sample Preparation. Plasma samples for 6-Cl-ddP and ddI analysis. The
plasma samples were treated as described previously by Morgan et al. (1992).
Dried extracts were stored at ⫺20°C before analysis.
Plasma samples for nelfinavir analysis. Samples were thawed, and 0.5 ml of
acetonitrile was added to 0.1 ml of plasma. Then the samples were vortexed for
4 min and centrifuged at 3500 rpm for 5 min. Supernatants were removed and
dried under a nitrogen stream. Dried extracts were resuspended in mobile
phase and analyzed by HPLC.
Brain samples for 6-Cl-ddP and ddI analysis. Brain tissue samples were
treated as described previously by Morgan et al. (1992). Dried extracts were
stored at ⫺20°C before analysis.
Brain samples for nelfinavir analysis. Brain homogenates were prepared in
the same manner as above (Morgan et al., 1992). The prepared homogenates
were vortexed with 12.5 ml of acetonitrile and centrifuged (400g for 10 min).
The supernatants were collected in 20-ml glass scintillation vials and evaporated to dryness under a nitrogen stream. Dried extracts were stored at ⫺20°C
before analysis. Frozen spiked plasma and brain controls that were prepared
and analyzed simultaneously with actual samples indicated no degradation of
analytes over the time period of storage in the freezer.
HPLC Analyses. Dry frozen samples were thawed and redissolved either in
phosphate buffer (6-Cl-ddP and ddI) or in mobile phase (nelfinavir). Plasma and
brain concentrations were determined by reversed-phase HPLC with UV detection
at 254 nm. The separations were achieved on a Supelcosil LC-18S column
(Bellefonte, PA; 6-Cl-ddP and ddI) and Supelcosil ABZ⫹ Plus column (nelfinavir). Assays for the spiked tissue samples showed good linearity (r2 between
0.9953 and 0.9999) over the concentration ranges studied. Recoveries (mean ⫾
S.D.) of 6-Cl-ddP, ddI, and nelfinavir from spiked plasma samples were 83.0 ⫾
4.3% (n ⫽ 10), 94.3 ⫾ 3.3% (n ⫽ 10), and 81.3 ⫾ 7.3% (n ⫽ 5), respectively.
Recoveries of 6-Cl-ddP, ddI, and nelfinavir from spiked brain samples were
96.8 ⫾ 2.9% (n ⫽ 10), 105.5 ⫾ 6.0% (n ⫽ 11), and 86.3 ⫾ 2.8% (n ⫽ 8),
respectively.
Statistical Analysis. Two-way analysis of variance with Tukey’s post hoc
test was used to determine the statistical difference between the dose combinations, and p ⬍ 0.05 was considered to be significant.
Results and Discussion
The infusion times for nelfinavir and 6-Cl-ddP were sufficient to reach
steady state in plasma. Previous pharmacokinetic studies have shown that
plasma and brain steady-state levels for 6-Cl-ddP are reached within 30
min at the dose used in this study (Anderson et al., 1990; Morgan et al.,
1992). Nelfinavir’s elimination half-life is ⬃1.3 h in rats (Shetty et al.,
1996), and plasma steady state is obtained with an 8 h infusion (data not
shown). The time necessary for nelfinavir concentrations to reach steady
state in the brain during an i.v. infusion has not been established. Parenchymal brain concentrations were obtained by correcting the measured
brain sample concentrations for the vascular space component, which
was set at 2% based on our previous findings (Anderson et al., 1990;
Morgan et al., 1992). The concentrations in brain were calculated according to the equation: Ci ⫽ Cm ⫺ Vp Cp, where Ci is the brain concentration
(extracellular ⫹ intracellular), Cm is the drug concentration in the brain
sample, Vp is the fraction of brain tissue space occupied by plasma, and
Cp is the drug concentration in plasma. The specific gravity of brain tissue
was assumed to be 1.0. The brain parenchyma to plasma ratio (mean ⫾
S.D.) of nelfinavir was 0.022 ⫾ 0.015 and 0.011 ⫾ 0.013 in the absence
and presence of 6-Cl-ddP, respectively (Fig. 1A). Similar low brain
penetration of nelfinavir has been reported in mice (Choo et al., 2000).
GF120918 increased the brain/plasma ratio of nelfinavir significantly
(⬃100-fold; Fig. 1A). This increase exceeds those reported in the literature when using other P-gp inhibitors (Choo et al., 2000). However,
Choo et al. (2000) used mice, and nelfinavir was given as an injection
rather than as an infusion to steady-state concentrations in plasma, making it difficult to interpret the differences in apparent P-gp inhibitor
potencies observed.
The possibility that part of the increase in brain tissue concentration
of nelfinavir following GF120918 treatment may be due to displacement of protein-bound nelfinavir merits further consideration because
nelfinavir has been shown to be ⬃98.5% protein bound in human
serum (Zhang et al., 2001). However, there are several observations
suggesting that protein-binding effects were not the source of the
increased brain tissue concentrations caused by GF120918. First, as
shown in Table 1, the nelfinavir concentration in plasma was 12.5 ␮g/ml
in the absence of GF120918 and 12.3 ␮g/ml 2 h after a dose of
GF120918. Significant displacement of protein-bound nelfinavir might
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inhibitor in humans (Witherspoon et al., 1996) and has been shown to
inhibit P-gp in the blood-brain barrier, increasing the entry of P-gp
substrates into the CNS in rats (Letrent et al., 1999) and in mice (Polli
et al., 1999). The observation that probenecid, an inhibitor of multidrug resistance-associated protein (MRP) in brain endothelial cells
(Huai-Yun et al., 1998), also increases brain concentrations of certain
dideoxynucleosides (Galinsky et al., 1991) implicates MRPs in the
brain uptake of anti-HIV agents. Since MRP and P-gp have many
substrates in common (Hollo et al., 1998), including small hydrophobic peptides (De Jong et al., 2001) and the HIV protease inhibitors
(Srinivas et al., 1998), it is reasonable to speculate that there may be
interactions between nelfinavir and 6-Cl-ddP/ddI when given in combination. Our goal is to introduce a different approach in the possible
treatment of HIV infection in general and ADC in particular. Prodrug
technology (6-Cl-ddP) is used to deliver an RTI (ddI) in sufficient
amounts to the CNS, and a P-gp inhibitor (GF120918) is used to
obtain higher concentrations of a protease inhibitor (nelfinavir) in the
CNS. The aim of this study is to examine whether coadministration of
6-Cl-ddP, nelfinavir, and GF120918 enhances the brain delivery of
ddI and nelfinavir in rats and whether significant interactions in CNS
delivery occur in the combination therapy.
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EFFECTS OF A P-gp INHIBITOR ON BRAIN ENTRY OF ANTI-HIV DRUGS
be expected to have altered the steady-state plasma concentration. This
may not have occurred, however, due to the likelihood that the plasma
concentration of GF120918 was much lower than the steady-state concentration of nelfinavir. Although the steady-state nelfinavir concentration in plasma was found to be ⬃12 ␮g/ml, Hyafil et al. (1993) determined the pharmacokinetics of GF120918 in mice following a 10 mg/kg
i.v. bolus (the same dose administered in this study) and found a blood
concentration at 2 h after i.v. administration of ⬃0.1 ␮g/ml. Although we
did not monitor GF120918 concentrations in this study, it is likely that
they were substantially below the nelfinavir concentrations in plasma.
Additional evidence for a minimal effect of GF120918 on protein binding
comes from a study of the tissue distribution of amprenavir, another HIV
protease inhibitor that is also extensively protein bound (Polli et al.,
1999). These authors demonstrated that the distribution of amprenavir in
blood, brain, cerebrospinal fluid, testes, and muscle was similar in mdr
1a/1b genetic double-knockout mice and after GF120918 pretreatment,
TABLE 1
The plasma and brain parenchyma concentrations of 6-Cl-ddP, ddI, and nelfinavir after 6-Cl-ddP, nelfinavir, and GF120918 administrations (mean ⫾ S.D.; n ⫽ 3)
Values in parenthesis are from Morgan et al. (1992).
Nelf in
Plasma
Nelf in Brain
Parenchyma
␮g/g
␮g/ml
␮g/g
␮g/ml
␮g/g
⫹ Nelf
⫹ 918
14.7 ⫾ 6.0
(17.2 ⫾ 3.6)
18.8 ⫾ 10.4
22.6 ⫾ 9.8
5.5 ⫾ 2.3
(11.6 ⫾ 4.6)
7.3 ⫾ 2.2
10.9 ⫾ 0.8
11.6 ⫾ 0.8
(7.9 ⫾ 1.8)
12.3 ⫾ 1.2
13.9 ⫾ 3.1
6.2 ⫾ 0.2
(6.4 ⫾ 1.7)
5.3 ⫾ 0.8
7.9 ⫾ 0.2a,b
25.4 ⫾ 10.3
11.0 ⫾ 4.5
20.2 ⫾ 3.0
12.2 ⫾ 0.3
⫹ 918
⫹ Nelf ⫹ 918
ddI in Plasma
ddI in Brain
Parenchyma
6-Cl in Plasma
6-Cl
6-Cl
6-Cl
Nelf
Nelf
6-Cl
6-Cl in Brain
Parenchyma
Dose
␮g/ml
a,b,c
6-Cl, 6-Cl-ddP; Nelf, nelfinavir; 918, GF120918.
a
Values are significantly different from 6-Cl.
b
Values are significantly different from 6-Cl ⫹ Nelf.
c
Values are significantly different from 6-Cl ⫹ 918.
d
Values are significantly different from Nelf.
e
Values are significantly different from Nelf ⫹ 918 ( p ⬍ 0.05, Two-way analysis of variance and Tukey’s post hoc test).
a,b,c
15.9 ⫾ 4.4
12.5 ⫾ 1.2
12.3 ⫾ 2.5
14.4 ⫾ 5.8
0.2 ⫾ 0.2
0.3 ⫾ 0.2
23.2 ⫾ 2.6b,d
16.0 ⫾ 4.9b,d,e
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FIG. 1. Brain parenchyma/plasma ratios (mean ⫾ S.D.; n ⫽ 3) of nelfinavir (A) and ddI (B) after different drug combinations.
The ddI brain parenchyma/plasma ratio after ddI treatment in 1B is from Morgan et al. (1992). a, values are significantly different from 6-Cl-ddP ⫹ Nelf; b, values are
significantly different from Nelf; c, values are significantly different from Nelf ⫹ 918 (p ⬍ 0.05; two-way analysis of variance and Tukey’s post hoc test). 918, GF120918;
Nelf, nelfinavir.
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SAVOLAINEN ET AL.
Division of Pharmaceutical Sciences,
University of Kentucky, Lexington,
Kentucky (J.S., M.E.M., P.J.M., B.D.A.);
Department of Pharmaceutical Chemistry,
University of Kuopio, Kuopio,
Finland (J.S.);
Graduate Center for Toxicology,
University of Kentucky, Lexington,
Kentucky (J.E.E.)
JOUKO SAVOLAINEN
JEFFREY E. EDWARDS
MICHAEL E. MORGAN
PATRICK J. MCNAMARA
BRADLEY D. ANDERSON
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producing “chemical knockouts”, a highly unlikely outcome if protein
binding of nelfinavir had been substantially altered after GF120918
pretreatment.
In rats administered GF120918, the brain/plasma ratios of nelfinavir were significantly reduced (nearly 2-fold) with 6-Cl-ddP treatment
(1.95 ⫾ 0.48 versus 1.14 ⫾ 0.13) (Table 1; Fig. 1A), suggesting an
effect of 6-Cl-ddP on nelfinavir uptake into the brain. A reduction
upon 6-Cl-ddP treatment was also seen in brain/plasma ratios of
nelfinavir in the absence of GF120918, but the significance of this
difference was not established, perhaps due to the limited number of
animals used in each group. Although ddI and other reverse transcriptase inhibitors do not affect the transport of nelfinavir in an LLC-PK1 cell
line (Shiraki et al., 2000), the reduced uptake of nelfinavir upon 6-Cl-ddP
treatment is reminiscent of the observed reduction of nelfinavir brain/
plasma ratios in mdr1a(⫺/⫺) mice caused by valspodar, another P-gp
inhibitor, which was assumed to reflect inhibition of one or more drug
uptake transport systems (Choo et al., 2000). Coadministration of nelfinavir with 6-Cl-ddP did not change the distribution of 6-Cl-ddP and ddI
between plasma and brain (Table 1; Fig. 1B). The actual brain and plasma
concentrations are similar to those found earlier after 6-Cl-ddP infusion
alone (Morgan et al., 1992). After 6-Cl-ddP infusion, the brain/plasma
ratio of ddI increases significantly compared with an infusion of ddI
(Morgan et al., 1992; Fig. 1B). GF120918 increased the brain concentrations of both 6-Cl-ddP and ddI (up to 2-fold). The magnitude of the
increase, however, parallels that in plasma; thus, GF120918 did not seem
to affect the brain/plasma ratios of 6-Cl-ddP or ddI (Table 1). The
elevated brain and plasma levels of 6-Cl-ddP and ddI in the presence of
GF120918 may be due to a reduction in their systemic elimination. These
results indicate that coadministration of 6-Cl-ddP, nelfinavir, and
GF120918 significantly enhances the brain concentrations of ddI and
nelfinavir in comparison with the administration of ddI and nelfinavir
alone. Thus, the combination of a prodrug-approach and P-gp inhibition
may provide a new alternative to treat HIV infection in general and ADC
in particular.