Christoffer Bundgaard, Christian Jes Nyberg Jensen, Mats Garmer Discovery DMPK, H ...dmd.aspetjournals.org/content/dmd/early/2011/11/23/dmd... · 2011-11-23 · H. Lundbeck A/S Discovery

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Species comparison of in vivo P-glycoprotein mediated brain efflux using mdr1a deficient rats and mice

Christoffer Bundgaard, Christian Jes Nyberg Jensen, Mats Garmer Discovery DMPK, H. Lundbeck A/S, DK-2500 Valby, Denmark

DMD Fast Forward. Published on November 23, 2011 as doi:10.1124/dmd.111.043083

Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on November 23, 2011 as DOI: 10.1124/dmd.111.043083

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Running title: Brain efflux comparison between P-gp KO rats and mice

Corresponding author:

Christoffer Bundgaard

H. Lundbeck A/S

Discovery DMPK

9 Ottiliavej

DK-2500 Valby, Denmark

Phone: +45 3630 1311

Email:[email protected]

Number of text pages: 31

Number of tables: 3

Number of figures: 2

Number of references: 35

Number of words

In Abstract: 250

In Introduction: 736

In Discussion: 1497

Nonstandard Abbreviations: ABC, ATP-binding cassette; BBB, Blood-brain barrier;

CNS, central nervous system; P-gp, P-glycoprotein (mdr, multidrug resistance); KO,

Knockout; Kp, Total brain to plasma concentration ratio; Kp,uu, Unbound brain to

unbound plasma concentration ratio; fu,plasma, Unbound fraction in plasma; fu,brain,

Unbound fraction in brain; WT, Wild-type


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Abstract

The experiments described herein compared the extent of in vivo P-gp mediated

brain efflux between rats and mice for a set of known CNS compounds. With use of

newly introduced genetically modified mdr1a deficient rats and their gene-competent

counterparts, the brain to plasma distribution was assessed and compared to the

distribution pattern in mdr1a deficient and wild-type mice. Four compounds

(aripiprazole, citalopram, risperidone and venlafaxine) were administered using a

continuous subcutaneous osmotic minipump infusion paradigm. Steady-state brain

and plasma concentrations of the compounds, including selected metabolites (9-OH

risperidone, O-desmethyl-venlafaxine and N-desmethyl-venlafaxine) were measured

in mdr1a deficient rats and mice and their wild-type counterparts along with their free

fractions to determine total and unbound brain to plasma distribution between

genotypes within and between species. The results revealed qualitative as well as

quantitative similarities between P-gp functionality in vivo at the blood-brain barrier

level in rats and mice. All tested compounds were shown to have significantly higher

brain to plasma distribution in both mdr1a deficient rats and mice compared to their

wild-type counterparts. Moreover, the relative enhancement in extent of brain

penetration between mdr1a deficient and wild-type rats could be directly correlated to

the enhancement ratios obtained in mice. From the unbound brain to unbound

plasma distributions (Kp,uu), the impact of P-gp to the overall brain penetration

capabilities showed minor differences between rats and mice for the compounds

tested. In conclusion, a comparable functional role of P-gp between rats and mice

with respect to brain efflux mediated by this transporter is suggested.


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Introduction

The physiology of the blood-brain barrier (BBB) with its endothelial cells lining the

brain capillaries connected by tight junctions limits the extent of brain penetration for

many xenobiotics. In addition to the physical barrier, numerous transport proteins are

present at the BBB acting as efflux pumps to actively exclude a wide variety of

chemically diverse compounds from the central nervous system (CNS). One of the

most studied members of this group of transporters is P-glycoprotein (P-gp, ABCB1),

a mammalian plasma membrane phospho-glycoprotein encoded by the multidrug

resistance 1 (MDR1) gene (mdr1 in rodents). P-gp belongs to the ATP-binding

cassette (ABC) transporter superfamily and is located in the brain in the luminal

membrane of the endothelial cells but is also expressed in other organs (Thiebaut et

al, 1987). For compounds designed to exert their effect in the CNS, drug-P-gp

interactions could influence the potential efficacy by reducing the brain concentration

due to this efflux mechanism. Thus, P-gp has been shown in mice to have a profound

influence on brain concentrations of many types of drug substrates including

antidepressants and antipsychotics (Ejsing et al., 2007; Linnet and Ejsing, 2008).

Consequently, screening for P-gp substrate recognition and assessment of the

potential impact hereof are therefore important parts of the profile evaluation of new

chemical entities, particularly those targeted to the CNS (Wager et al., 2011).

During lead optimization programs and drug candidate characterization, in

vivo pharmacology and pharmacokinetic studies are often performed in rats and

mice. In such studies it is important to understand the potential differences in the

functional activity of P-gp between these species with respect to drug distribution to

the brain in order to make proper comparisons and extrapolations (Kim et al., 2008;

Syvanen et al., 2009). In both rats and mice, P-gp is the most highly expressed ABC


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protein in brain endothelial capillary cells (Warren et al., 2009). Rats and mice shares

two genes encoding for P-gp, mdr1a and mdr1b, which are similarly expressed

between the two species and in both of these species, mdr1a is the predominant

form in the brain (Croop et al., 1989; Kwan et al., 2003). In addition there is a high

degree of homology in the amino acid content of P-gp between rats and mice of

around 93% (Hubbard et al., 2007). Studies investigating differences in P-gp

functionality between rats and mice have primarily been conducted in vitro applying

efflux ratios derived from bidirectional transport studies in cell systems transfected

with mdr1 from each species. These studies suggest some correlation between rat

and mouse P-gp mediated efflux but also that differences may exist depending on

the substrate (Booth-Genthe et al., 2006; Katoh et al., 2006; Takeuchi et al., 2006).

However, comparison of efflux ratios between species from in vitro transport studies

is circumvented with indecisive biological challenges such as total protein expression

and density of the functional transporter in each cell line.

With respect to in vivo studies investigating quantitative species differences in

P-gp function between rats and mice, very limited amount of data have been

published. Cutler et al. (2006) used infusion of the P-gp inhibitor GF-120918

(elacridar) and reported that similar total plasma concentrations of the inhibitor were

required in rats and mice to achieve the same relative increase in the brain to plasma

distribution ratio of an undisclosed P-gp substrate. However, species differences in

plasma protein binding of the inhibitor or substrate are confounding factors that may

influence the total plasma concentrations required to inhibit P-gp. Thus, such

chemically induced knockout studies are not readily comparable between species.

With the advent of mdr1a/b deficient mice either from genetically knockout

modifications or mutant origin (Schinkel et al., 1996), these animals has become


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widely applied in drug discovery to assess the role of P-gp in vivo on drug disposition

including BBB transport studies. These transporter deficient animal models have

suffered from only being available in mice species making quantitative interspecies

comparisons difficult. Recently however, mdr1a knockout rats became commercially

available through the application of Zinc Finger Nuclease technology enabling

targeted gene knockout (Geurts et al., 2009; Chu et al., 2011). The purpose of this

study was therefore to compare the P-gp functionality between mdr1a deficient rats

and mice and there gene-competent counterparts (wild-types) with respect to in vivo

brain distribution. A range of known CNS compounds and their selected metabolites

previously recognized to be P-gp substrates were evaluated in these animal models

using a similar experimental design across species.


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Methods

Animals

Homozygous mdr1a knockout and wild-type rats (bodyweight 350-500 g) were

obtained from SAGETM Labs, Sigma Life Science, Sigma-Aldrich Co. (SD-

Abcb1atm1sage). Mdr1a deficient mice and CF-1TM (non-Swiss) outbred background

mice (bodyweight 25-45 g) were purchased from Charles River Laboratories

(Crl:CF1-Abcb1amds mutants). All animals arrived at the animal facility at least five

days before being used in experiments. During acclimatization, animals were housed

in pairs under controlled conditions of temperature (21 ± 2 °C), relative humidity (55 ±

5%) and a 12-h light–dark cycle (lights on at 6:00 am). Food and tap water were

freely available in the home cage. Approval of all animal procedures was granted by

The Danish National Committee for Ethics in Animal Experimentation.

Drugs and dosing

The administered test compounds included citalopram, venlafaxine, risperidone and

aripiprazole which all are extensively used CNS compounds. In addition, these

compounds and their selected metabolites represent a relative wide span in their

functional extent of substrate affinity towards P-gp at the BBB level in mice (Linnet

and Ejsing, 2008). Alzet osmotic minipumps (Durect Corporation, Cupertino, CA)

were used to deliver the test compounds in steady-state dosing regiments. Alzet

model 2ML1 (delivery rate 10 µl/h) and model 2001 (delivery rate 1 µl/h) were applied

for rats and mice respectively. Citalopram (hydrobromide, H. Lundbeck A/S),

venlafaxine (hydrochloride, Sigma) and risperidone (free base, Sigma) were

dissolved in 0.9% NaCl. Aripiprazole (free base, Sequoia Research) was dissolved in

20% hydroxylpropyl-β-cyclodextrin, pH4. Test compounds were used in the following


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concentrations (as free base) in the pumps with corresponding average doses

calculated based on body weight and pump delivery rate: Citalopram: rats 15 mg/ml

(~9 mg/kg/day), mice 6 mg/ml (~5 mg/kg/day). Venlafaxine: rats 15 mg/ml (~9

mg/kg/day), mice 20 mg/ml (~12 mg/kg/day). Risperidone: rats 10 mg/ml (~5

mg/kg/day), mice 4 mg/ml (~4 mg/kg/day). Aripiprazole: rats 12 mg/ml (~6

mg/kg/day), mice 3 mg/ml (~3 mg/kg/day). Prior to implantation, the pumps were

equilibrated by soaking them overnight in sterile saline solution at 37 ºC. Before

surgery animals received Rimadyl (carprofen) subcutaneously as analgesic

treatment. During brief isoflurane anesthesia, minipumps were implanted

subcutaneously on the back of the animals under aseptic conditions. Postoperative

treatment was provided by Temgesic (buprenorphine) on the day of surgery and

Rimadyl the following two days. 3-5 wild-type and mdr1a deficient rats and mice were

used in each treatment group. The continuous delivery was maintained for 3 days in

both mice and rats to attain steady-state after which the animals were decapitated

and the brains dissected out. Trunk blood was collected in EDTA-coated tubes and

plasma were harvested following centrifugation (5000 g for 10 min at 4 °C). Brain

homogenate was prepared by homogenizing the whole brain with 70% acetonitrile

(1:4 v/v) followed by centrifugation and collection of the supernatant. Plasma and

brain supernatant samples were frozen at -80°C until analysis.

Bioanalysis

Rat and mice plasma and brain homogenate samples were prepared by precipitation

and addition of generic internal standard. Plasma and brain concentrations of dosed

test compounds and selected metabolites (O-desmethyl venlafaxine, N-desmethyl

venlafaxine and 9-OH-risperidone) were determined using UltraPerformance LC


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chromatography (Acquity UPLC, Waters, Milford, MA, USA) followed by MS/MS

detection using a Sciex-API 4000 MS (AB Sciex, Foster City, CA, USA) (Erichsen et

al., 2010). The lower limit of quantification was typically 0.5 ng/ml in plasma and 1

ng/g in brain (peak S/N > 5).

Free fraction determinations

Plasma protein binding and non-specific binding in brain homogenates were

determined in naïve male rats (Sprague-Dawley) and mice (Crl:NMRI(Han)) by use of

equilibrium dialysis. The procedure was modified from Kalvass and Maurer (2002).

Dialysis membranes (96 well formats, cut-off 12-14 kDa, HTDialysis) was soaked in

PBS/ethanol 80:20 and rinsed in deionized water before use. Brain homogenate or

plasma was added to the donor side and spiked with test compound to a final

concentration of 1 µM. The receiver side contained phosphate buffer (pH 7.4).

Equilibrium dialysis was performed by incubating at 37°C for 5 h. Subsequently, the

compound concentration was measured in the buffer phase using liquid

chromatography-mass spectrometry (LC-MS). The free fraction in plasma (fu,plasma)

and intact brain tissue (fu,brain) was calculated according to Kalvass and Maurer

(2002) with their dilution factor in brain taken into account. For all compounds,

recovery following incubation were in the range of 91-109 %.

Data analysis and statistics

Brain to plasma distribution ratios (Kp) were calculated from the measured total drug

levels in the respective matrices (Cbrain,total and Cplasma,total). To compare brain to

plasma distribution in mdr1a deficient and wild-type animals between species, Kp

values were normalized and expressed relative to wild-types (100%) for each


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species. In addition, the unbound brain to unbound plasma partition coefficients,

designated Kp,uu, were calculated by associating free fractions with total brain to

plasma distribution at steady-state for each species as Cbrain,total × fu,brain / Cplasma,total ×

fu,plasma (Hammarlund-Udenaes et al., 1997; Gupta et al., 2006). Student’s t-test was

used to compare brain to plasma distribution ratios data between mdr1a deficient and

wild-type animals. Statistical and regression analyses were performed using

GraphPad Prism® (Vers. 4.02, GraphPad Software, USA).


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Results

Following 3 days of continuous treatment using minipumps, the four test compounds

and three selected formed metabolites were measured in plasma and brains of wild-

type and mdr1a deficient rats and mice. The observed total plasma and brain

concentrations and corresponding Kp values are summarized in Table 1 for rats and

Table 2 for mice. For all compounds except 9-OH risperidone there were no

statistically significant differences between the systemic plasma exposure in mdr1a

deficient animals and their wild-type counterparts. For 9-OH risperidone, plasma

concentrations were significantly higher in mdr1a deficient mice as compared to wild-

type mice (p<0.05). This trend was also observed for 9-OH risperidone in mdr1a

deficient rats versus wild-type rats although not to a statistically significant degree.

From the derived Kp values, all the tested compounds tended to display higher brain

distribution in mice as compared to rats when comparing the species within in each

genotype. Thus, risperidone, 9-OH risperidone and aripiprazole all had Kp values

below 0.22 in wild-type rats. Among wild-type mice, 9-OH risperidone and

aripiprazole had the lowest Kp values of 0.28 and 0.49. Of the compounds evaluated,

citalopram exhibited the highest Kp value in both species.

All the tested compounds in the data set were identified as being substrates

for P-gp at the BBB level in both rats and mice. Thus, from the Kp values, all the

tested compounds were shown to have significantly higher brain distribution in both

mdr1a deficient rats and mice compared to their wild-type counterparts. To

quantitatively compare the impact of P-gp on brain distribution capabilities of the

compounds between rats and mice, Kp values were normalized to account for

differences in absolute total plasma and brain exposure. Figure 1 shows the

normalized Kp values for the test compounds in mdr1a deficient animals relative to


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their wild-type controls (set as 100%) for each species. All the compounds exhibited

significant enhancement in their normalized Kp value in mdr1a deficient animals. In

both rats and mice, the most pronounced effect of P-gp mediated brain efflux was

observed for risperidone (835% ±197 enhancement in mdr1a deficient rats; 737%

±221 in mice) and its metabolite 9-OH risperidone (1100% ±274 enhancement in

mdr1a deficient rats; 1200% ±155 in mice). The weakest effect of P-gp on the relative

brain distribution was seen with venlafaxine (192% ±18 enhancement in mdr1a

deficient rats; 176% ±20 in mice) and citalopram (330% ±27 enhancement in mdr1a

deficient rats; 201% ±8 in mice). As for the most pronounced P-gp substrates, the

weaker relative effects of P-gp on brain distribution of venlafaxine and citalopram

were also shown to be present in both species. When ranking the impact of P-gp on

brain distribution across all tested compounds, considerable overlap between rats in

mice was observed. In rats this was (low to high) venlafaxine, O-desmethyl

venlafaxine, citalopram, aripiprazole, N-desmethyl venlafaxine, risperidone, 9-OH

risperidone. The corresponding ranking for mice was venlafaxine, citalopram,

aripiprazole, O-desmethyl venlafaxine, N-desmethyl venlafaxine, risperidone, 9-OH

risperidone. The overall relationship between the enhancements in the normalized Kp

values in mdr1a deficient rats and mice is visualized in Figure 2. Regression analysis

revealed a linear correlation between the two species with a slope close to unity

(y=1.08x - 102; r2=0.95, p=0.0002).

To assess the impact of P-gp deficiency on the brain to plasma distribution of

the compounds independent of binding to plasma proteins and brain tissue, the

plasma and brain free fractions determined in vitro were combined with measured

total brain and plasma concentrations at steady-state to calculate Kp,uu for each

compound in wild-type and mdr1a deficient animals (Table 3). In wild-type rats all


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compounds had Kp,uu values well below unity consistent with active efflux dominating

the BBB distribution of these compounds. The Kp,uu values in mdr1a deficient rats

were increased to around unity for the majority of the compounds. In mice, all the

compounds except for citalopram exhibited Kp,uu values below unity. Citalopram was

shown to have a Kp,uu value of 2.4 in wild-type mice which was increased to 4.8 in

mdr1a deficient mice whereas the other compounds were increased to values closer

to unity as compared to respective values in wild-type animals.


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Discussion

The experiments described herein compared the prevalence and magnitude of in

vivo P-gp mediated brain efflux between rats and mice for a set of known CNS

compounds. In order to compare the extent of brain distribution between rats and

mice and the impact of P-gp, all experiments were performed at steady-state. This

design allowed for calculations of Kp and Kp,uu between brain and plasma with

species dependent pharmacokinetics taken into account.

From the Kp values obtained in mdr1a deficient rats and mice and their wild-

type counterparts, the experiments revealed qualitative as well as quantitative

species similarities between P-gp functionality at the BBB level. Thus, all compounds

evaluated were shown to be substrates in both species. Moreover, as shown in

Figure 2, the extent of increase in the normalized Kp values between mdr1a deficient

animals and there wild-type counterparts could be directly correlated between rats

and mice. The indication of a comparable quantitative role of in vivo P-gp

functionality at the BBB between rats and mice observed in this study corroborate

with expression levels and protein homology of mdr1 between these species (Kim et

al., 2008). Also in vitro studies in mdr1a transfected cells indicate a comparable

functional role of P-gp between these species although some substrate dependency

has been reported (Booth-Genthe et al., 2006; Katoh et al., 2006; Takeuchi et al.,

2006).

The tested compounds have previously been reported to be substrates for P-

gp in vivo at the BBB in mice as shown by an increased brain to plasma distribution

in P-gp knockout versus to wild-type mice. Citalopram has been shown to have 2-3

fold higher Kp value in P-gp knockout mice (Uhr and Grauer, 2003; Doran et al.,


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2005; Uhr et al., 2008) in line with the present mice data (201% ±8 increase). In

mdr1a deficient rats, citalopram showed an enhancement ratio of 330% ±27 which

could indicate that this compound has a slightly higher propensity for P-gp mediated

brain efflux in rats as compared to mice. Assessment of bidirectional transport

properties across the BBB including impact of P-gp can be based on the ratio

between unbound concentrations in brain and plasma at equilibrium conditions,

expressed as Kp,uu. If Kp,uu is close to unity, the BBB transport is predominantly

mediated by passive diffusion, or the impact of influx and efflux transport is equal. If

Kp,uu is below unity, efflux transport may explain this whereas a Kp,uu above unity is an

indication that active uptake processes are involved (Hammarlund-Udenaes et al.,

1997; Hammarlund-Udenaes et al., 2008). Interestingly, citalopram displayed a Kp,uu

value in wild-type mice of 2.4 which was increased to 4.8 in mdr1a deficient mice,

indicating that, despite clear P-gp mediated efflux, some involvement of uptake

transporters is also suggested to contribute to the overall BBB distribution of

citalopram. This pattern was also observed for citalopram in rats although to a lesser

extent. A putative involvement of active uptake is supported by in vitro transport

studies across bovine brain endothelial cells, where citalopram has been reported to

be transported by means of a carrier-mediated mechanism (Rochat et al., 1999).

Thus, a possible species dependent affinity to uptake transporters could in theory

influence the functional transport activity at the BBB differently between mdr1a

deficient rats and mice although this remains to be explored.

Aripiprazole has previously been shown to be a substrate for P-gp in mice with

a 2.7-fold increase in Kp in knockout mice following sub-chronic minipump treatment

(Kirschbaum et al., 2010). In the present study a similar increase in Kp was observed

in mdr1a deficient mice (269% ±39 increase). Like for citalopram, a slightly higher


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increase in Kp for aripiprazole was attained in mdr1a deficient rats compared to wild-

type rats (338% ±38 increase) which cannot rule out some species difference of P-gp

functionality for this compound. The Kp,uu values of aripiprazole in mdr1a deficient

rats and mice were increased to reach similar values and did not reached unity.

However, some uncertainty in these derived values is to be expected as the free

fractions of aripiprazole were extremely low in plasma as well as in brain.

Risperidone and its metabolite 9-OH risperidone are well studied P-gp

substrates in mice with profound P-gp effects on extent of brain penetration shown by

at least 10 times higher Kp values in knockout mice in a range of different studies

(Wang et al., 2004; Doran et al., 2005; Ejsing et al., 2005). These reports aligned well

with the present results where these compounds were the most pronounced

substrates in the test set with increases in Kp values in similar ranges in both mice

and rats. In both mdr1a deficient rats and mice, Kp,uu values for risperidone and 9-OH

risperidone were increased to values very close to unity, suggesting P-gp to be the

main gatekeeper for brain penetration of these substances in both species.

Brain entry of venlafaxine and its demethylated metabolites O-desmethyl-

venlafaxine and N-desmethyl-venlafaxine have previously been shown to be limited

by P-gp using knockout mice with brain penetration enhancement ratios between 2

and 4 in mice lacking P-gp following sub-chronic minipump treatment (Uhr et al.,

2008; Karlsson et al., 2011). In the current experiments similar enhancement ratios

were observed in mice. Venlafaxine and O-desmethyl-venlafaxine displayed equal

relative enhancement ratios between mice and rats lacking P-gp. N-desmethyl-

venlafaxine had a slightly higher enhancement ratio in rats (634% ±117) as

compared to mice (461% ±13). However, relatively high variability in the Kp data of

formed N-desmethyl-venlafaxine was observed in rats. In regards to this, dosing of


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this metabolite could be considered as means to a more accurate determination of

potential species differences in the P-gp mediated effects of this compound. Kp,uu

estimates of venlafaxine and its metabolites in mdr1a deficient and wild-type animals

indicated that passive diffusion was the dominating factor for BBB distribution without

the presence of P-gp across the parent-metabolite system. Kp,uu values of N-

desmethyl-venlafaxine in rats and O-desmethyl-venlafaxine in mice increased to

values above 2 in the mdr1a deficient species but the aforementioned variability in N-

desmethyl-venlafaxine exposure and the very high free fraction of O-desmethyl-

venlafaxine in mice plasma suggests that these values should be interpreted with

caution.

Development of the genetically mdr1 deficient rat model, provides a simple

tool for evaluating the functional consequence of P-gp efflux at the BBB by the

measurement of drug concentrations in brain and plasma at steady-state. However,

some drug and/or species dependent limitations in this model and methodology likely

exist that may affect interpretation of the obtained Kp and Kp,uu estimates. Firstly, it

has been shown that knockout of P-gp in mice can result in alterations in the mRNA

expression levels of other transporter proteins (Cisternino et al., 2004). Thus, if the

compound under investigation has substrate affinity for other efflux or uptake

transporters besides P-gp, this could potentially affect the brain to plasma distribution

obtained in mdr1 deficient models. A very recent characterization of the mdr1a

deficient rat model suggest that only minor compensatory changes in expression of

other transporter related genes occur at the BBB level in male rats lacking P-gp (Chu

et al., 2011). Secondly, continuous drug administration may increase P-gp

functionality at the BBB as a result of mdr1 induction. This has been suggested for

venlafaxine in vitro in human cell systems (Ehret et al., 2007; Bachmeier et al., 2011)


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and in vivo in rats following 3 weeks of continuous treatment (de Klerk et al., 2010)

and. Thus, if species differences exist in terms of liability and extent of mdr1

induction following sub-chronic treatment, this could theoretically hamper a

quantitative comparison of P-gp mediated efflux between rats and mice. Thirdly,

intrabrain drug distribution governed by other processes than nonspecific binding

may potentially impact the estimates of Kp,uu in the present study. Hence, determining

brain free fractions using the homogenate methodology only measures nonspecific

binding and does therefore not account for other intrabrain distributional aspects

such as sequestration into lysosomes which might affect cellular retention for basic

compounds. Also, The homogenization process may alter the binding properties by

unmasking binding sites that are not accessible to a drug in intact brain tissue.

However, recent studies suggest that a reasonable correlation can be established

between nonspecific binding from homogenates and binding obtained using brain

slices with a preserved cellular structure (Friden et al., 2011).

By use of the same experimental methodology in rats in mice, the validity of

the mdr1a deficient rat model was confirmed in the present study where all selected

P-gp substrates identified in mice was also recognized in rats. This model thus allows

for a direct means to assess the potential influence of P-gp on pharmacokinetic and

pharmacodynamic parameters in rats without the need for chemical P-gp inhibitors.

Besides qualitative similarities in the P-gp functionality between rats and mice, the

data obtained here also indicates substantial quantitative similarities in the extent of

P-gp mediated efflux at the BBB between these species. This information might

prove useful when extrapolating existing knowledge on P-gp mediated efflux

obtained in mice to the rat species during pharmacokinetic and in vivo

pharmacological evaluations.


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Acknowledgements

Mona Elster is acknowledged for technical assistance.


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Authorship Contributions

Participated in research design: Bundgaard, Jensen

Conducted experiments: Jensen, Garmer

Performed data analysis: Garmer, Bundgaard

Wrote or contributed to the writing of the manuscript: Bundgaard


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Footnotes

Reprint requests:

Christoffer Bundgaard, PhD

H. Lundbeck A/S

Discovery DMPK

DK-2500 Valby, Denmark

Phone: +45 3630 1311

Email:[email protected]


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Legends for figures Fig. 1. Relative brain to plasma distribution for the 4 tested compounds and their

selected metabolites in mdr1a deficient rats and mice and their wild-type

counterparts following 3-day continuous minipump treatment. Data are displayed as

relative Kp values expressed as average percentage of Kp values in wild-types

(100%) ± relative SEM). Statistical difference were assessed between mdr1a

deficient and wild-type animals within each species (*p<0.05; **p<0.01; ***p<0.001).

Fig. 2. Relationship between the enhancements in the normalized Kp values in mdr1a

deficient rats and mice (designated P-gp defrat/WTrat and P-gp defmice/WTmice

respectively). Dotted line represents the line of unity and the solid line the resulting fit

of the linear regression. Kp values are shown as average ± relative SEM obtained in

each species.


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Table 1. Total plasma and brain concentrations and derived Kp values of

administered test compounds and formed metabolites in wild-type and mdr1a

deficient rats following 3-day continuous minipump treatment. Values are shown as

mean ± SEM (n=3-5).

Compound Wild-type rats Mdr1a deficient rats

Plasma (ng/ml)

Brain (ng/g) Kp

Plasma (ng/ml)

Brain (ng/g) Kp

Citalopram 27 ± 2.4 265 ± 16 10 ± 0.78 35 ± 5.8 1198 ± 289 33 ± 2.7

Venlafaxine 50 ± 3.7 158 ± 4.6 3.3 ± 0.29 63 ± 11 389 ± 33 6.3 ± 0.60

O-Des-venlafaxine 4.5 ± 1.4 4.4 ± 0.89 1.0 ± 0.10 11 ± 3.3 33 ± 7.8 3.3 ± 0.38

N-Des-venlafaxine 6.8 ± 1.5 9.3 ± 1.1 1.4 ± 0.14 10 ± 4.2 79 ± 21 8.9 ± 1.7

Risperidone 194 ± 34 41 ± 0.88 0.22 ± 0.04 168 ± 25 295 ± 36 1.9 ± 0.44

9-OH Risperidone 153 ± 24 22 ± 1.6 0.15 ± 0.03 192 ± 41 289 ± 14 1.7 ± 0.41

Aripiprazole 18 ± 5.3 3.0 ± 0.60 0.19 ± 0.04 22 ± 1.0 14 ± 2.1 0.64 ± 0.07


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Table 2. Total plasma and brain concentrations and derived Kp values of

administered test compounds and formed metabolites in wild-type and mdr1a

deficient mice following 3-day continuous minipump treatment. Values are shown as

mean ± SEM (n=3-5).

Compound Wild-type mice Mdr1a deficient mice

Plasma (ng/ml)

Brain (ng/g)

Kp Plasma (ng/ml)

Brain (ng/g)

Kp

Citalopram 8.8 ± 0.40 191± 19 22 ± 2.2 8.2 ± 0.7 361 ± 33 44 ± 1.6

Venlafaxine 85 ± 9.8 403 ± 63 4.7 ± 0.40 105 ± 16 828 ± 70 8.3 ± 1.0

O-Des-venlafaxine 5.7 ± 0.35 10 ± 0.87 1.8 ± 1.13 7.5 ± 1.6 40 ± 6.9 5.7 ± 0.58

N-Des-venlafaxine 17 ± 2.8 25 ± 5.1 1.5 ± 0.12 11 ± 1.7 77 ± 11 7.0 ± 0.20

Risperidone 43 ± 10 21 ± 2.3 0.52 ± 0.08 40 ± 10 149 ± 16 3.8 ± 1.1

9-OH Risperidone 45 ± 8.4 13 ± 1.7 0.28 ± 0.01 110 ± 26 366 ± 91 3.4 ± 0.44

Aripiprazole 51 ± 8.7 25 ± 4.8 0.49 ± 0.04 32 ± 6.2 41 ± 7.2 1.3 ± 0.17


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Table 3. Free fraction values obtained in plasma (fu,plasma) and brain homogenate

(fu,brain) and calculated unbound brain to plasma partition coefficients (Kp,uu) in wild-

type (WT) and mdr1a deficient (P-gp def.) rats and mice. fu-values are shown as

average of two independent determinations.

Compound

Free fraction, rats (%) Kp,uu, rats

Free fraction, mice (%) Kp,uu, mice

fu,plasma fu,brain WT P-gp def.

fu,plasma fu,brain WT

P-gp def.

Citalopram 50 3.1 0.61 2.1 32 3.5 2.4 4.8

Venlafaxine 64 9.2 0.45 0.89 >99* 18 0.86 1.4

O-Des-venlafaxine 89 35 0.38 1.2 >99* 45 0.80 2.4

N-Des-venlafaxine 32 8.0 0.34 2.0 80 16 0.29 1.4

Risperidone 11 9.0 0.17 1.4 27 5.8 0.11 0.81

9-OH Risperidone 20 15 0.11 1.1 54 18 0.10 1.1

Aripiprazole 0.10 0.10 0.19 0.64 0.20 0.10 0.25 0.64

*fu,plasma values of 99% was used to estimate Kp,uu for venlafaxine and O-Des-venlafaxine.


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