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Cyclosporin A and Trifluoperazine, Two Resistance-

Modulating Agents, Increase Ivermectin Newotoxicity in Mice
Luis E Marques-Santos',', Robson R. Bernardo', Emilio F. de Paula' and Vivian M. Rumjanek4
'Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, 2National Institute of Cbncer, INCA,
RJ, Rio de Janeiro, 3Center for the Investigation of Natural Products, Rio de Janeiro, 4Department of Medical Biochemis-
try, Institute of Biomedical Sciences, UFRJ, Rio de Janeiro, Brazil
(Received March 10. 1998: Accepted October 19, 1998)

Ahs/racr: The P-glycoprotein expressed in the blood-brain barrier has been associated with the restricted access of many
compounds to the central nervous system. Mice lacking the /ndrla P-glycoprotein gene show an accumulation of various
drugs in brain tissues. P-glycoprotein is also correlated with the phenomenon of multidrug resistance in tumour cells. To
investigate the effects of drugs that modulate multidrug resistance in the selective permeability of the blood-brain barrier,
mice were treated with cyclosporin A or trifluoperazine plus ivermectin, a P-glycoprotein substrate, that has a limited
access to the central nervous system. When mice received an injection of cyclosporin A (50 mg/kg. intraperitoneally) or
trifluoperazine (750 pg/kg, intraperitoneally) one hour prior to the administration of ivermectin (10- 15 mgikg, intraperito-
neally) there was an increase in the acute toxicity of ivermectin. HPLC analysis of brain tissues indicated that the ivermec-
tin brain concentration was 2.5 times higher when mice were previously treated with cyclosporin A (50 mg/kg). These
results suggest that attention should be given to the side effects of drugs that interact with P-glycoprotein and are com-
monly used clinically and also to the possibility of creating a pharmacological gap in the blood-brain barrier that allows
the access of chemotherapeutic drugs to brain tumours.

P-glycoprotein is a member of the ATP-binding cassette su-
perfamily of transmembrane transporters (Childs & Ling
1994). P-glycoprotein is a product of the human MDRI and
MDR2/3 genes and the rodent riidrla (mdr3), mdrlb, and
mdr2 genes. P-glycoprotein mediates the membrane trans-
port of many hydrophobic compounds, including hor-
mones, sterols, lipids, phospholipids, oligopeptides, cyto-
kines, and anticancer agents (Bellamy 1996). Initially, P-gly-
coprotein was related to tumour cells and the multidrug
resistance phenomenon, which is characterized by cross-re-
sistance to structurally and functionally unrelated com-
pounds (Juliano & Ling 1976). However, it is well known
that P-glycoprotein has a physiological expression. The
product of mdr2 and nzdr2 genes are located in the liver.
MDRI and nzdrl P-glycoprotein are located in many tissues
and organs, such as the kidneys, adrenals, intestines, pla-
centa, and in the capillary endothelial cells of the testis and
blood-brain barrier (Cordon-Cardo et al. 1989 & 1990).
Many compounds are known to modulate the P-glyco-
protein by reducing the efflux activity of the pump, e.g. ver-
apamil, trifluoperazine, and cyclosporin A and their ana-
logues (Ford & Hait 1990). These drugs are capable of re-
versing the multidrug resistance phenomenon phenotype in
vitvo (Gottesman & Pastan 1993). The use of these resis-
tance-modulating agents may have physiological impli-
cations as P-glycoprotein is expressed in many cells and
tissues. Disruption of the mouse mdrla gene led to alter-
ation in the distribution of many drugs (Schinkel et al.
1994 & 1995). The most affected organ was the brain, where
the concentration of a drug could be raised 80-90 times. In
the case of ivermectin, the increase of this drug in the brain
produced toxic effects. Based on these results it was pro-
posed that P-glycoprotein has a crucial role in the mainten-
ance of the selective permeability of the blood-brain-barrier
(Schinkel et al. 1994; van Asperen et ul. 1997).
This work was designed to investigate the effects of cyclo-
sporin A and trifluoperazine in the permeability of the
blood-brain barrier and its toxicological involvement. To
approach this problem a study was performed on the acute
toxicity of ivermectin and its modulation by resistance-
modulating agents.
Materials and Methods
Animals. All experiments were carried out in 8-10 weeks old female
congenic BIOA/SnCg mice (C57B16 background H-2a), kept at the
National Institute of Cancer, Brazil. The number of animals used
in each experiment is indicated in the respective figures and tables.
Animals had free access to food and water and were housed and
handled according to institutional guidelines complying with Brazil-
ian legislation.

Drugs. Ivermectin was purchased a s Ivomecm (Merck Sharp and

Author for correspondence: Luis Fernando Marques-Santos, Lab-
oratory of Tumour Immunology, Department of Medical Biochem-
istry, Institute of Biomedical Sciences, Federal University of Rio de
Janeiro, Saude Scientific Center, 21941-590 Rio de Janeiro - RJ,
Brazil (fax f 5 5 21 2708647: e-mail Ifmarques@bioqmed.ufrj.br).
Dohme), as an injectable solution (I"/" w/v) for veterinary use. For
experimentation, the drug was diluted in Phosphate-buffered saline
(pH 7.4), in plastic recipients. Cyclosporin A was purchased as san-
dimmun@(Sandoz, Switzerland), a solution for intravenous use (5%
wiv). For experimental use, it was diluted in tissue culture medium
 16000773a, 1999, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1600-0773.1999.tb00887.x, Wiley Online Library on [02/07/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
126 LUIS E MARQUES-SANTOS ET A L

(RPMl 1640, Sigma Chemical Co., St. Louis, USA) supplemented 100
with ImM L-glutamine and 25 mM Hepes (Sigma Chemical Co.).
Trifluoperazine (Sigma Chemical Co.) was first solubilized in foetal 90
calf serum (Fazenda Pigue, Brazil) and further diluted (1:5, v/v) in
tissue culture medium RPMI 1640. All drugs were freshly prepared. 80

Iverrnectin acute toxicity. Animals received a single intraperitoneal 70

injection of ivermectin. and were observed daily for the following
14 days. After this period, the number of deaths in each group was 8 60
used to estimate the lethal dose for 50% of the population (LD50).
The doses used were I , 5, 10, 15, 16, 18, 20, 30,40, 50 and 100 mgi
kg, and the numbers of animals tested at each dose were 65, 30, 50,
5k 50

45, 19, 18, 61, 10, 10, 40, and 23, respectively. The volume adminis- 2 40
tered was 10 plig.
30
Modulation protocol. Cyclosporin A and trifluoperazine were in-
jected as a single dose, intraperitoneally 1 h prior to ivermectin 20
injection (single dose), to allow a high plasma concentration of the
resistance-modulating agents at the moment of ivermectin adminis- 10
tration. The doses used were 10, 25 and 50 mg/kg (cyclosporin A),
and 75 and 750 pgikg (trifluoperazine). Different doses of ivermec-
tin were used in accordance with the study of its acute toxicity. The 1 10 15 100
volume of administration of the drugs was 10 pl/g. The control IMRMECTIN (mgkg, log scale)
groups were injected with ivermectin, cyclosporin A or trifluopera-
zine plus vehicle. The time course of the experiments was 14 days, Fig. 1. Ivermectin acute toxicity in mice. Mice received a single
and the results expressed as percentage of death of each population intraperitoneal injection of ivermectin and the mortality of each
(mortality). Significant differences between the treatments were group was determined during a period of 14 days. The lethal dose
evaluated using the chi-square test. for 50% of the population (LD,,) was estimated with 1415mg/kg.

HPLCanalysis of brain tissue. Animals (n=3) were injected with 50

mgikg cyclosporin A one hour prior to the administration of 10
mg/kg ivermectin. One hour after ivermectin administration, the those receiving doses higher than 20 mg/kg died during the
mice were sacrificed and the brains removed and weighed. The ex- first 24 hr.
traction and clean-up of the samples as high-performance liquid There was an increase in ivermectin toxicity when the
chromatography procedures were adapted from Chiu et al. (1988) drug was preceded by the administration of 50 mg/kg cyclo-
Dickinson (1990) and Klotz et al. (1990) as described below. Each
brain was transferred to a homogenizer jar, acetonitrile-water (1:I ,
sporin A as a single intraperitoneal dose (table 1). A mor-
v/v) was added (8 mug tissue) and the tissue homogenized. To ex- tality of 100% was observed in the first 18 hr after the ad-
tract the proteins 0.2 M zinc sulfate (200 mlig tissue) were added to ministration of 50 mg/kg cyclosporin A plus 10 mg/kg iver-
the homogenized tissue. Centrifugation was performed (I700 rpm, mectin. HPLC analysis of brain tissues indicated that in
15 min.) and the pellet discarded. The supernatant was dialyzed and these animals the ivermectin brain concentration was 2.5
lyophilized. The powder was weighed and dissolved in the mobile
phase (30 mgil50 pl) consisted of acetonitrile-methanol-water times higher than that found in mice without cyclosporin
(49:33:18, viv). Injections of 20 pl were made from this solution. A
Detector SPD-M 10A Dioderray Shimadzu was used the analysis.
The columns used were Merck ODS C-18 (5 pm ,125 mmX4.6 mm). Table 1.
The mobile phase was used at a flow-rate of 1.O mlimin. All chroma-
tograms were monitored at 245 nm. Acetonitrile, milli-Q water and CvclosDorin A (CSAbincreased ivermectin (IVM) acute toxicitv.
methanol were purchased from Merck. Under these conditions the Treatment
retention time of ivermectin was 11 min.
First Second
drug drug
(mgikg) (mg/kg) Treated' Dead2 Mortality
Results
CSA 10 Vehicle 10 0 0
Ivermectin acute toxicity is illustrated in fig. I , and LD50 CSA 25 Vehicle 10 0 0
was estimated at 14-15 mg/kg. Animals presented many CSA 50 Vehicle 14 0 0
symptoms characteristic of ivermectin toxicity including Vehicle IVM 1 65 0 0
Vehicle IVM 10 50 7 14
loss of balance, tremors and immobilization that progressed
CSA 10 IVM 1 16 0 0
to comatose state and subsequently death (Schinkel et al. CSA 10 IVM 10 24 4 16.7
1994). The drug was well tolerated in animals treated with CSA 25 IVM 1 16 0 0
5 mglkg, and there were no symptoms of intoxication. Some CSA 25 IVM 10 26 17 65.4*
animals injected with doses between 10-16 mgikg showed CSA 50 IVM 1 26 9 34.6**
CSA 50 IVM 10 24 24 1oo*
some of the intoxication symptoms without progressing
into comatose state. These surviving animals had no se- ' Number of treated mice
Number of dead mice
quels, and after 180 days they were still alive. Mice that died Number of dead mice X 100inumber of treated mice
after treatment with 10-20 mg/kg ivermectin died during * P<O.OOl versus control
the first 24-72 hr after the drug administration, whereas ** P<0.001 versus control
 16000773a, 1999, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1600-0773.1999.tb00887.x, Wiley Online Library on [02/07/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
CYCLOSPORIN A INCREASES IVERMECTIN NEUROTOXICITY 127

A treatment (fig. 2). As can be seen in fig. 1, administration Table 2.

of 25 mg/kg ivermectin gives a 90-100% mortality rate, Trifluooerazine (TFP) increased ivermectin (IVM) acute toxicitv.
which is in accordance with the results obtained with 10 Treatment
mg/kg ivermectin plus 50 mg cyclosporin A. Mice receiving First Second
50 mg/kg cyclosporin A plus 1 mg/kg ivermectin presented drug drug
acute toxicity which was not observed with administration (mg/kg) (mgikg) Treated' Dead2 Mortality (9'0)~
of the same dose of ivermectin alone (34.6% of mortality TFP 75 Vehicle 18 0 0
and no mortality, respectively). The rate of mortality in this TFP 750 Vehicle 26 0 0
group of animals (50 mg/kg cyclosporin A plus 1 mgikg Vehicle IVM 1 65 0 0
Vehicle IVM 10 50 7 14.0
ivermectin) was also higher than the group treated with 10 Vehicle IVM 15 45 24 53.3
mg/kg ivermectin alone (mortality= 14%). A dose of 25 mg/ TFP 75 IVM 1 16 0 0
kg cyclosporin A raised the mortality of the group treated TFP 75 IVM 10 30 1 3.3*
with 10 mg/kg ivermectin, but did not affect the group re- TFP 75 IVM 15 16 7 43.8**
TFP 750 IVM 1 16 0 0
ceiving 1 mg/kg ivermectin. Treatment with 10 mgikg cyclo- 28 42.9*
TFP 750 IVM 10 12
sporin A had no effect in the mortality of mice, independent TFP 750 IVM 15 24 17 70.8**
of the ivermectin dose administered. The injection of cyclo-
sporin A (10, 25 or 50 mg/kg) plus vehicle did not show
' Number of treated mice
Number of dead mice
toxic effects (table l), even when the animals were treated Number of dead mice X looinumber of treated mice
for 3 days with a dose interval of 24 hr (data not shown). * P<O.OOI versus control
The ivermectin toxicity for the doses of 10 and 15 mg/kg ** P<O.OOl versus control
was increased when the animals received a previous injec-
tion of 750 pg/kg trifluoperazine (42.9% and 70.8% mor-
tality, respectively) (table 2). In this case all deaths occurred 43.8%, respectively, when the animals were previously in-
within the first 48 hr. The dose of lmg/kg ivermectin re- jected with 75 pg/kg trifluoperazine (table 2).
mained without toxic effects even when preceded by triflu- With the same modulation protocol, we performed an
operazine administration. experiment with cyclosporin A and trifluoperazine at their
When the animals were given trifluoperazine (75 pg/kg) higher doses. Thus, an injection of 750 pg/kg trifluoperazine
and one hour later, an injection of ivermectin, there was a one hour before administration of 50 mg/kg cyclosporin A
reduction in the rate of mortality compared with that of did not produce toxic effects. Similarly, administration of
animals injected with vehicle plus ivermectin. The mortality 50 mg/kg cyclosporin A preceding the dose of 750 pgikg
of the groups treated with 10 and 15mg/kg of ivermectin trifluoperazine had no toxic implications (data not shown).
was 14 and 53.3% respectively, being reduced to 3.3 and
Discussion
Ivermectin, an acaricede and anthelmintic drug of the fam-
b
a C
ily of avermectins, produced by Streptomyces avermitilis
cultures, is a well tolerated drug with no side effects in
mammals at pharmacological doses (Fisher & Mrozik
1992). Ivermectin is an agonist of y-aminobutyric acid
(GABA) receptors and of glutamate-gated C1- channels, the
later restricted to invertebrates (Bloom 1996). The low iver-
I mectin toxicity has been attributed to its restricted access
to brain tissues, especially for being a substrate of P-glyco-
protein (Schinkel et al. 1994). Our results indicated a LDso
between 14-15 mg/kg after a single intraperitoneal adminis-
+ - tration of ivermectin in murines (fig. 1). These data are in
10
0 10 0 10 0
agreement with those found by Didier & Loor (1995), who
MInuIes
suggested that the LD5,, for a single intraperitoneal iver-
Fig. 2. Chromatograms of ivermectin (IVM) in brain tissue. a - mectin injection was higher than 10 mg/kg.
control animal (treated with 50 mgikg cyclosporin A one hour prior In the present work, the administration of resistance-
to ivermectin vehicle) b - animal treated with cyclosporin A vehicle
one hour prior to 10 mg/kg ivermectin c - animal treated with 50 modulating agents to mice was able to increase ivermectin
mgikg cyclosporin A one hour prior to 10 mgikg ivermectin. All distribution into the brain as observed by HPLC. Further-
drugs were administered intraperitoneally. The retention time of iv- more, the combination of resistance-modulating agents and
ermectin was 11 min. The relative area of ivermectin peak was 2.5 ivermectin decreased the biotolerance to ivermectin. This
times higher when animals were treated with cyclosporin A one
was probably a result of reversing P-glycoprotein activity in
hour prior to ivermectin. Data are representative of three separated
experiments. For chromatographic conditions see Materials and the blood-brain barrier as opposed to a synergistic effect
Methods. resulting from the combination of the mode of action of

128 LUIS E MARQUES-SANTOS ET A L .
ivermectin and resistance-modulating agents a t the GABA
receptors level. Although it has been reported that cyclo-
sporin A and trifluoperazine can interact with these recep-
tors, it is still not completely clear how this interaction takes
place. It is believed that while cyclosporin A and its ligand
cyclophilin A are able to increase the recovery from desens-
itization of GABA receptor in hipocampal neurones (Mar-
tina et. al. 1996), trifluoperazine is a blocker of GABA-
gated chloride currents (Zorumski & Yang 1988; Yang &
Zorumski 1989). In our experiments, however, both drugs
were capable of potentiating the toxic effect of ivermectin,
and the involvement of targets other than GABA receptors
cannot be discarded.
In favour of the increase of bioavailability of ivermectin
as a result of disruption of the blood-brain barrier is the
fact that the administration of 50 mg/kg cyclosporin A one
hour prior to the injection of 10 mg/kg ivermectin produced
the same toxic effects of a ivermectin dose, at least three
times higher (30 mg/kg ivermectin). The toxicological par-
ameters are in agreement with HPLC analysis of brain
tissues indicating that the ivermectin brain concentration
was 2.5 times higher when animals were previously treated
with 50 mg/kg cyclosporin A. The toxicological symptoms
observed loss of balance, inability to right themselves, and
~ decreased. Nevertheless, interaction at the level of GABA
immobilization, leading to a comatose state and subse-
quently death - are in agreement with an enhancement of
GABA action mediated by an increase of ivermectin con-
centration in the brain. This resulted from cyclosporin A
administration and its effect on P-glycoprotein expressed in
the endothelial cells that constitute the blood-brain-barrier.
In accordance to our results, Wang et al. (1995) demon-
strated that the concentration of Rhodaminel23 - a fluor-
escent dye - in the brain was 3 times higher when rats were
treated with cyclosporin A. Didier & Loor (1995), using
an experimental design similar to that used in this work,
demonstrated that SDZ PSC 833 or SDZ 28W46, two re-
sistance-modulating agents and cyclosporin A analogue, in-
creased ivermectin toxicity. Nevertheless, in their work,
cyclosporin A administered orally at 50 mg/kg, was not able
to increase cyclosporin A toxicity. Cyclosporin A exhibits a
low availability when administered orally (22-36%) (Fahr
1993), whereas the cyclosporin A dose and route of admin-
istration used here (50 mg/kg, intraperitoneally) was cap-
able of producing an immunossupressive effect in rodents
(Rumjanek et al. 1985). This dosage had no toxicological
implications even when administered for 3 consecutive days
at 24 hr intervals (data not shown).
Pouliot et al. (1997) using in vitro models demonstrated
that ivermectin increased the vinblastine and doxorubicin
toxicity in cells expressing high levels of P-glycoprotein. Iv-
ermectin could compete with the chemotherapeutic drugs
as a P-glycoprotein substrate. Similarly, the toxicological ef-
fects found in the present experiments could result from
an ivermectin reversing effect, inducing cyclosporin A or
trifluoperazine neurotoxicity. However, trifluoperazine and
others phenothiazines have free access to the CNS (Baldes-
sarini 1996) what rules out a modulatory effect of ivermec-
16000773a, 1999, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1600-0773.1999.tb00887.x, Wiley Online Library on [02/07/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
tin on trifluoperazine distribution to the brain. In addition,
the highest dose of trifluoperazine used (750 pg/kg) is much
smaller than its LDS0(29 mg/kg) for intravenous adminis-
tration. The transport of cyclosporin A across the blood-
brain barrier is restricted (Sakata et al. 1994), however, the
administration of 750 pg/kg trifluoperazine one hour prior
to 50 mg/kg cyclosporin A injection produced no toxic fea-
tures. This indicate that even if cyclosporin A distribution
to CNS was increased it was not toxic. The same dose of
trifluoperazine was able to increase the acute toxicity of iv-
ermectin (10 and 15 mg/kg).
The administration of the lower dose of trifluoperazine
(75 pg/kg) had a protective effect against the ivermectin
toxicity, decreasing it significantly (table 2). This dose is
very close to that used in the treatment of psychiatric dis-
orders (Baldessarini 1996). It is possible that at the lower
dose trifluoperazine was not able to modulate the P-glyco-
protein expressed in the blood-brain barrier to the extent of
raising the ivermectin access to the CNS. However, this
same dose may affect the active transport mediated by P-
glycoprotein in other organs (i.e., kidneys or liver), modify-
ing the pharmacokinetics of ivermectin. Therefore, the vol-
ume of distribution of the drug into the brain would be
receptor antagonizing the action of ivermectin can not be
ruled out. Despite the dose of 750 pgikg trifluoperazine
have decreased the biotolerance to ivermectin.
The studies performed in mice lacking P-glycoprotein en-
coded by the mdrla gene, as well as studies using resistance-
modulating agents in wild type mice, demonstrated the im-
portance of this protein in the pharmacokinetic parameters
of many drugs, including vinblastine (Schinkel et al. 1994;
Arboix et al. 1997), dexamethasone, digoxin, cyclosporin A
(Schinkel et al. 1995), and doxorubicin (Colombo et al.
1994). Our results demonstrated that cyclosporin A and tri-
fluoperazine were able to raise the neurotoxicity of ivermec-
tin. The possibility that resistance-modulating agents alters
in a distinct manner the pharmacokinetics of the drugs
should be considered. The pattern of distribution of the
drugs under a resistance-modulating agents treatment, may
not have a typical or generalized model, it is necessary,
therefore, to amplify the investigation of the resistance-
modulating agents effects, including its actions in the
homeostasis of the organism. The utilization of the resis-
tance-modulating agents may have serious and important
clinical implications. The possibility of reversing the re-
stricted access of chemotherapeutic drugs to the CNS - cre-
ating a pharmacological gap in the blood-brain barrier - to
treat brain tumours can be a question of finding the ideal
modulation protocol, i.e. the right resistance-modulating
agents plus the right chemotherapeutic agent.
Acknowledgements
The authors are thankful to Dr. Elimar Teresa Brand
from the Analytical Center, Nucleo de Pesquisa de Produtos
Naturais, UFRJ, for her assistance in the HPLC analysis.

CYCLOSPORIN A INCREASES IVERMECTIN NEUROTOXICITY
Luis E Marques-Santos is recipient of a FundaGBo Capacit-
a@o e AperfeiGoamento de Pessoal de Ensino Superior
(CAPES) M.Sc. fellowship. This work was supported by
grants from PRONEX, Conselho Nacional de Desenvolvi-
mento Cientifico e Tecnologico ( C N P q ) and Financiadora
de Estudos e Projetos de Pesquisa ( F I N E P ) .
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