Influence of P-glycoprotein on brain uptake of [18F]MPPF in rats

doi:10.1016/S0014-2999(00)00752-4

European Journal of Pharmacology

Volume 407, Issue 3, 3 November 2000, Pages 273-280

https://doi.org/10.1016/S0014-2999(00)00752-4 Get rights and content

Abstract

The aim of this study was to determine if the brain uptake of 4-(2′-methoxyphenyl)-1-[2′-(N-2″-pyridinyl)-p-[¹⁸F]fluorobenzamido]ethylpiperazine ([¹⁸F]MPPF), a radioligand for the imaging of 5-HT_1A receptors, is influenced by the action of P-glycoprotein.

Anesthetized male Wistar rats were injected i.v. with the 5-HT_1A receptor antagonist [¹⁸F]MPPF (2 MBq, S.A.>110 TBq/mmol) after treatment with saline (controls) or with the 5-HT_1A receptor antagonist 1-(2′-methoxyphenyl)-4-[4-(2-phtalimido)butyl]piperazine (NAN-190) (2.5 mg/kg i.v.). After 60 min, the animals were sacrificed and 13 areas of the brain were dissected for ex vivo gamma counting. The regional distribution of radioactivity was also assessed in brain slices using a storage phosphor system. Modulation of P-glycoprotein was achieved by injection of cyclosporin A (50 mg/kg) 30 min prior to injection of [¹⁸F]MPPF.

The distribution of ¹⁸F-derived radioactivity corresponded to regional 5-HT_1A receptor density as known from autoradiography. Modulation of P-glycoprotein with cyclosporin A caused a 5- to 10-fold increase in the uptake of [¹⁸F]MPPF. Tissue/cerebellum ratios in the brain correlated with receptor densities determined by in vitro autoradiography. Measurements of plasma radioactivity showed that the increased brain uptake of [¹⁸F]MPPF is partially due to a rise in ligand delivery after treatment with cyclosporin A (area under the curve, AUC, increased by a factor of 1.8). Biodistribution experiments in wild type and mdr1a(−/−) knockout mice confirmed that [¹⁸F]MPPF is a substrate for P-glycoprotein.

Introduction

Positron emission tomography (PET) and biodistribution studies in rats, cats, and monkeys have demonstrated that the regional distribution of 4-(2′-methoxyphenyl)-1-[2′-(N-2″-pyridinyl)-p-[¹⁸F]fluorobenzamido]ethylpiperazine ([¹⁸F]MPPF) in the brain corresponds to 5-HT_1A receptor localization Kung et al., 1996, Shiue et al., 1997, Le Bars et al., 1998, Ginovart et al., 2000. Recently, our group presented the first human data for [¹⁸F]MPPF Passchier et al., 1999, Passchier et al., 2000a, Passchier et al., 2000b. Again, the radioactivity distribution in human CNS was in good agreement with known receptor localization and with previous PET studies, which employed [carbonyl-¹¹C]WAY 100635 Farde et al., 1998, Gunn et al., 1998, Ito et al., 1999. Shortly afterwards, these results were confirmed by researchers from Liège (Plenevaux et al., 1999), indicating that [¹⁸F]MPPF is a useful radioligand for clinical application and may be employed to measure the 5-HT_1A receptor occupancy of new drugs during phase 1 and 2 clinical trials.

Comparison of data obtained for [¹⁸F]MPPF in rats with data for [carbonyl-¹¹C]WAY 100635, showed that MPPF displays a six- to seven-fold lower uptake in the brain (data not shown). This difference must arise from the p-fluoro-benzyl group in MPPF since the compounds are otherwise identical (Scheme 1).

It is known that P-glycoprotein, an ATP-driven transmembrane efflux pump, which is present with high density in brain endothelium, has a high affinity for lipophilic molecules of moderate weight and size that contain cationic centers and planar aromatic domains Abbott and Romero, 1996, Dellinger et al., 1992, Pawagi et al., 1994, Pearce et al., 1990, Schinkel et al., 1996, Van Asperen et al., 1996, Van Asperen et al., 1997. Active transport back into the blood could result in a lower brain penetration of [¹⁸F]MPPF than might be expected from its lipophilicity. The efflux action of P-glycoprotein can be inhibited by so-called modulators (e.g. cyclosporin A, verapamil, and PSC833) Begley, 1992, Hendrikse et al., 1998, Hughes et al., 1998, Schinkel et al., 1995, Van Asperen et al., 1996. These compounds reduce P-glycoprotein functionality and thus prevent a radioligand from being expelled from the endothelial membrane back into the blood.

In rodents, two P-glycoproteins are present encoded by the mdr1a and mdr1b genes. Brain P-glycoprotein is expressed by the mdr1a gene Borst and Schinkel, 1996, Schinkel et al., 1995, Schinkel et al., 1996, Van Asperen et al., 1996. The generation of mice with homozygously disrupted mdr1a genes [mdr1a(−/−) knockout mice] has made it possible to test if the brain uptake of a drug is affected by the action of P-glycoprotein Schinkel et al., 1995, Schinkel et al., 1996, Van Asperen et al., 1996.

If uptake of the radiopharmaceutical in the brain is indeed limited by the action of P-glycoprotein, inhibition of this pump by a modulator could result in improved counting statistics within the CNS. A positive result will demonstrate the importance of taking the action of P-glycoprotein into account during the development of radioligands for CNS receptors and for 5-HT_1A receptor antagonists in particular.

The aim of the present study was to investigate if the introduction of the extra aromatic ring in MPPF results in a decreased brain uptake due to the efflux action of P-glycoprotein. To that purpose, biodistribution experiments were performed in rats with modulated P-glycoprotein functionality and in mdr1a(−/−) knockout mice.

Section snippets

Materials and methods

All solvents were of analytical grade and were obtained from Merck (Darmstadt, Germany) or Rathburn (Walkerburn, Scotland). Chemicals were purchased from Aldrich (Milwaukee, USA) and ACROS Chimica (Geel, Belgium). NAN-190 Hydrobromide and (R)-(+)-8-Hydroxy-DPAT were from Sigma (St. Louis, USA) Cyclosporin A (Sandimmune) was a product of Sandoz (Basle, Switzerland). Ketamine (Ketalar®) was purchased from Parke-Davis (Hoofddorp, The Netherlands) and xylazine (Rompun®) from Bayer (Leverkusen,

[¹⁸F]MPPF uptake and distribution in rat brain

We measured a logP of 2.14±0.06 (n=5) for [¹⁸F]MPPF. On the basis of this value, a good uptake of the radioligand in the brain could be expected. However, measured uptake values in biodistribution experiments were quite low. The uptake of [¹⁸F]MPPF in the brain of saline- and NAN-190 pretreated Wistar rats is presented in Fig. 1. Standard uptake values were in the range of 0.05–0.25. A significant reduction caused by NAN-190 (2.5 mg/kg) was observed in hippocampus, amygdala/piriform-,

Discussion

Biodistribution experiments showed a five- to seven-fold lower brain uptake for the 5-HT_1A receptor antagonist MPPF compared to the prototype ligand WAY 100635 (Fig. 1A) Plenevaux et al., 1997, Shiue et al., 1997, Pike et al., 1994. This was unexpected since the lipophilicity of MPPF is sufficient for rapid diffusion through the blood–brain barrier (logP=2.14). Since biodistribution is a rather crude method that does not provide information on ligand uptake in small (greater than a few

References (35)

N.J. Abbott et al.
Transporting therapeutics across the blood–brain barrier
Mol. Med. Today
(1996)
D.J. Begley
The interaction of some centrally active drugs with the blood–brain barrier and circumventricular organs
Prog. Brain Res.
(1992)
P. Borst et al.
What have we learnt thus far from mice with disrupted P-glycoprotein genes?
Eur. J. Cancer
(1996)
P. Doze et al.
Validation of S-1′-[¹⁸F]fluorocarazolol for in vivo imaging and quantification of cerebral ß-adrenoceptors
Eur. J. Pharmacol.
(1998)
R.A. Glennon et al.
NAN-190: an arylpiperazine analog that antagonizes the stimulus effects of the 5-HT_1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)
Eur. J. Pharmacol.
(1988)
R.N. Gunn et al.
Tracer kinetic modeling of the 5-HT_1A receptor ligand [carbonyl-¹¹C]WAY-100635 for PET
Neuroimage
(1998)
S.P. Hume et al.
Evaluation of [O-methyl-³H]WAY-100635 as an in vivo radioligand for 5-HT_1A receptors in rat brain
Eur. J. Pharmacol.
(1994)
X. Khawaja
Quantitative autoradiographic characterisation of the binding of [³H]WAY-100635, a selective 5-HT_1A receptor antagonist
Brain Res.
(1995)
D. Le Bars et al.
High-yield radiosynthesis and preliminary in vivo evaluation of p-[¹⁸F]MPPF, a fluoro analog of WAY-100635
Nucl. Med. Biol.
(1998)
A.B. Pawagi et al.
Transmembrane aromatic amino acid distribution in P-glycoprotein. A functional role in broad substrate specificity
J. Mol. Biol.
(1994)

H.L. Pearce et al.

Structural characteristics of compounds that modulate P-glycoprotein-associated multidrug resistance

Adv. Enzyme Regul.

(1990)

L. Rydelek-Fitzgerald et al.

NAN-190: agonist and antagonist interactions with brain 5-HT_1A receptors

Brain Res.

(1990)

J. Van Asperen et al.

The functional role of P-glycoprotein in the blood–brain barrier

J. Pharm. Sci.

(1997)

L.E. Arvidsson et al.

8-Hydroxy-2-(di-n-propylamino)tetralin, a new centrally acting 5-hydroxytryptamine receptor agonist

J. Med. Chem.

(1981)

M. Dellinger et al.

Structural requirements of simple organic cations for recognition by multidrug-resistant cells

Cancer Res.

(1992)

L. Farde et al.

Quantitative analyses of carbonyl-carbon-11-WAY-100635 binding to central 5-hydroxytryptamine-1A receptors in man

J. Nucl. Med.

(1998)

N. Ginovart et al.

In vivo characterization of p-[(18)F]MPPF, a fluoro analog of WAY-100635 for visualization of 5-HT(1a) receptors

Synapse

(2000)

Cited by (60)

Applications, Advances, and Limitations of Molecular Imaging of Brain Receptors
2021, Molecular Imaging: Principles and Practice
The molecular imaging of brain receptors by positron emission tomography (PET) and single photon emission computed tomography (SPECT) has evolved into a mature technology through parallel developments in radiopharmaceutical chemistry, instrumentation, image reconstruction, and kinetic modeling. PET researchers had early success in studies of dopamine D₂-like receptors using spiperone derivates labeled with carbon-11 or fluorine-18. However, quantitation of these ligands is difficult due to their slow approach to equilibrium binding, and due to offtarget binding at serotonin receptors. These problems were resolved with the discovery of [¹¹C]raclopride, a benzamide antagonist ligand with rapid kinetics, near absence of brain-penetrating metabolites, and admirable selectivity for dopamine D_2/3 receptors. The displaceability of its specific binding by endogenous dopamine enabled a new field of research aiming to measure indirectly the competition from dopamine in the living brain. Furthermore, higher affinity benzamide ligands can detect the less abundant ex-striatal D_2/3 receptors, albeit with the requirement of hours-long PET recordings for the striatum. Meanwhile, there emerged a series of PET ligands with selectivity for serotonin receptor subtypes, notably the 5HT_2A receptors of the cerebral cortex, the 5HT_1A receptors found in the hippocampus and serotonin neurons. Opioid receptors are the next best documented pharmacological class, with the preponderance of PET studies focusing on the μ-subtype. The opioid receptor literature is matched in scope by studies of acetylcholine receptors, notably the muscarinic receptors and the nicotinic receptors, now extending to the α₇-subtype. Overall, the literature is heavily weighted to this short list of receptor types, and important receptor types such as the NMDA-type glutamate receptors remain elusive targets for molecular imaging. PET technology is now widely used for measuring target engagement in the living human brain. However, there are few instances where molecular imaging of brain receptors is pathognomonic of brain disease.
PET imaging to study the functional impact of P-glycoprotein in neuropharmacokinetics
2020, Therapies
La P-glycoprotéine (P-gp) est un transporteur d’efflux exprimé au niveau de la barrière hématoencéphalique (BHE) qui joue un rôle déterminant dans le passage cérébral de nombreux médicaments. La translation clinique des données obtenues chez l’animal par des méthodes invasives est complexe et il est difficile de prédire les répercussions neuropharmacocinétiques de la P-gp chez l’Homme. La tomographie par émission de positons (TEP) est une méthode d’imagerie permettant de mesurer la cinétique des concentrations tissulaires d’une molécule radiomarquée injectée en microdose. À ce jour, trois radioligands substrats de la P-gp ont fait l’objet d’un développement clinique : le [¹¹C]vérapamil, [¹¹C]-N-desmethyl-lopéramide et le [¹¹C]métoclopramide. Cette approche innovante a permis de montrer une régulation fonctionnelle de la P-gp au niveau de la BHE chez l’Homme au cours de certaines pathologies neurologiques. Cette approche a également permis d’évaluer le risque d’interactions médicamenteuses impliquant des inhibiteurs de la P-gp au niveau de la BHE et d’en mesurer les conséquences sur le passage cérébral de médicaments substrats de ce transporteur.
P-glycoprotein (P-gp) is a major efflux transporter at the blood-brain barrier (BBB) where it controls the brain distribution of many drugs. Clinical translation of preclinical data obtained using invasive methods in animals is difficult due to species differences in P-gp expression and function. Positron emission tomography (PET) is an imaging technique allowing for the determination of the tissue kinetics of microdose of radiolabeled compounds. Three radiolabeled substrates of the P-gp have been developed for clinical use: [¹¹C]verapamil, [¹¹C]-N-desmethyl-loperamide and [¹¹C]metoclopramide. These innovative tools enabled the study of P-gp in humans in various patho-physiological conditions including neurological diseases. This approach is also useful to predict the risk for drug-drug interaction caused by P-gp inhibitors at the BBB and address its impact for neuropharmacokinetics in vivo in humans.
New Trends and Current Status of Positron-Emission Tomography and Single-Photon-Emission Computerized Tomography Radioligands for Neuronal Serotonin Receptors and Serotonin Transporter
2017, Bioconjugate Chemistry
The critical role of serotonin (5-hydroxytryptamine; 5-HT) and its receptors (5-HTRs) in the pathophysiology of diverse neuropsychiatric and neurodegenerative disorders render them attractive diagnostic and therapeutic targets for brain disorders. Therefore, the in vivo assessment of binding of 5-HT receptor ligands under a multitude of physiologic and pathologic scenarios may support more-accurate identification of disease and its progression and the patient’s response to therapy as well as the screening of novel therapeutic strategies. The present Review aims to focus on the current status of radioligands used for positron-emission tomography (PET) and single-photon-emission computerized tomography (SPECT) imaging of human brain serotonin receptors. We further elaborate upon and emphasize the attributes that qualify a radioligand for theranostics on the basis of its frequency of use in clinics, its benefit to risk assessment in humans, and its continuous evolution, along with the major limitations.
18F-FCWAY, a serotonin 1A receptor radioligand, is a substrate for efflux transport at the human blood-brain barrier
2016, NeuroImage
Citation Excerpt :
The authors interpreted the results to mean that 5-HT1A receptors were decreased in the temporal lobe of patients, but the findings could also reflect increased removal of the radioligand from brain. In fact, other 5-HT1A radioligands with a structure similar to that of FCWAY (Liow et al. 2007; Passchier et al. 2000) have been reported to be substrates for permeability-glycoprotein (P-gp), one of the two most prevalent efflux transporters at the blood–brain barrier. In addition, most patients in that study were drug-resistant.
Efflux transporters at the blood–brain barrier can decrease the entry of drugs and increase the removal of those molecules able to bypass the transporter. We previously hypothesized that ¹⁸F-FCWAY, a radioligand for the serotonin 5-HT_1A receptor, is a weak substrate for permeability glycoprotein (P-gp) based on its very early peak and rapid washout from human brain. To determine whether ¹⁸F-FCWAY is a substrate for P-gp, breast cancer resistance protein (BCRP), and multidrug resistance protein (MRP1) — the three most prevalent efflux transporters at the blood–brain barrier — we performed three sets of experiments. In vitro, we conducted fluorescence-activated cell sorting (FACS) flow cytometry studies in cells over-expressing P-gp, BCRP, and MRP1 treated with inhibitors specific to each transporter and with FCWAY. Ex vivo, we measured ¹⁸F-FCWAY concentration in plasma and brain homogenate of transporter knockout mice using γ-counter and radio-HPLC. In vivo, we conducted positron emission tomography (PET) studies to assess changes in humans who received ¹⁸F-FCWAY during an infusion of tariquidar (2–4 mg/kg iv), a potent and selective P-gp inhibitor. In vitro studies showed that FCWAY allowed fluorescent substrates to get into the cell by competitive inhibition of all three transporters at the cell membrane. Ex vivo measurements in knockout mice indicate that ¹⁸F-FCWAY is a substrate only for P-gp and not BCRP. In vivo, tariquidar increased ¹⁸F-FCWAY brain uptake in seven of eight subjects by 60–100% compared to each person's baseline. Tariquidar did not increase brain uptake via some peripheral mechanism, given that it did not significantly alter concentrations in plasma of the parent radioligand ¹⁸F-FCWAY or its brain-penetrant radiometabolite ¹⁸F-FC. These results show that ¹⁸F-FCWAY is a weak substrate for efflux transport at the blood–brain barrier; some radioligand can enter brain, but its removal is hastened by P-gp. Although ¹⁸F-FCWAY is not ideal for measuring 5-HT_1A receptors, it demonstrates that weak substrate radioligands can be useful for measuring both increased and decreased function of efflux transporters, which is not possible with currently available radioligands such as ¹¹C-loperamide and ¹¹C-verapamil that are avid substrates for transporters.
5-HT2A receptor SPECT imaging with [123I]R91150 under P-gp inhibition with tariquidar: More is better?
2016, Nuclear Medicine and Biology
Citation Excerpt :
This has led to the development of strategies to overcome the impact of P-gp activity on PET and SPECT preclinical and clinical imaging with pharmacological inhibition of P-gp. In this context, previous studies have demonstrated the potential of two P-gp inhibitors, cyclosporine A (CsA) [2,6–8] and tariquidar (TQD) [5,9,10] to augment brain uptake of radiotracers and signal-to-noise ratio. However, translating the aforementioned effects to an amelioration of quantitative measures is not straightforward.
Pharmacological P-glycoprotein (P-gp) inhibition with tariquidar (TQD) is considered a promising strategy for the augmentation of radiotracer brain uptake. However, a region-dependent effect may compromise the robustness of quantitative studies. For this reason, we studied the effect of a TQD pretreatment on 5-HT2A imaging with [¹²³I]R91150 and compared results with those obtained in Mdr1a knock-out (KO) rats.
Ex vivo autoradiography was performed in TQD (15 mg/kg) pretreated wild-type (WT-TQD), Mdr1a knock-out (KO) and untreated WT rats for Specific Binding Ratio (SBR) estimation. In vivo dynamic SPECT imaging with serial arterial blood sampling was performed in the former two groups of rats and kinetic analysis was performed with a one tissue-compartment (1TC) model and the Specific Uptake Ratio (SUR). Results were analyzed statistically using repeated measures ANOVA.
SBR values differed between WT-TQD, Mdr1a KO and WT rats in a region-dependent manner (p < 0.0001). In vivo brain uptake of radiotracer did not differ between groups. Similarly, kinetic analysis provided distribution volume (V_T) values that did not differ significantly between groups. SUR binding potential (BP_ND) values from both groups highly correlated with corresponding V_T (r = 0.970, p < 0.0001 and r = 0.962, p < 0.0001, respectively). However, SUR measured over averaged images between 100 and 120 min, using cerebellum as reference region, demonstrated values that were, by average, 2.99 ± 0.53 times higher in the WT-TQD group, with the difference between groups being region-dependent (p < 0.001). In addition, coefficient of variation of the SUR BP_ND values across brain regions was significantly higher in the WT-TQD rats (41.25% ± 9.63% versus 11.13% ± 5.59%, p < 0.0001).
P-gp inhibition with TQD leads to region-dependent effect in the rat brain, with probably sub-optimal effect in cerebellum. This warrants attention when it is used as a reference region for quantitative studies.
N-(4-[18F]-fluoropyridin-2-yl)-N-{2-[4-(2-methoxyphenyl) piperazin-1-yl]ethyl}carboxamides as analogs of WAY100635. New PET tracers of serotonin 5-HT<inf>1A</inf> receptors
2014, European Journal of Medicinal Chemistry
N-(4-[¹⁸F]-Fluoropyridin-2-yl)-N-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-carboxamides were prepared by labeling their 4-nitropyridin-2-yl precursors through nitro substitution by the ¹⁸F anion. In vitro and in vivo tests showed that the cyclohexanecarboxamide derivative is a reversible, selective and high affinity 5-HT_1A receptor antagonist (IC₅₀ = 0.29 nM, k_i = 0.18 nM) with high brain uptake, slow brain clearance and stability to defluorination when compared with conventional standards. This PET radioligand is a promising candidate for an improved in vivo quantification of 5-HT_1A receptors in neuropsychiatric disorders.

View all citing articles on Scopus

View full text

Influence of P-glycoprotein on brain uptake of [18F]MPPF in rats

Abstract

Introduction

Section snippets

Materials and methods

[18F]MPPF uptake and distribution in rat brain

Discussion

Mol. Med. Today

Prog. Brain Res.

Eur. J. Cancer

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Neuroimage

Eur. J. Pharmacol.

Brain Res.

Nucl. Med. Biol.

J. Mol. Biol.

Adv. Enzyme Regul.

Brain Res.

J. Pharm. Sci.

8-Hydroxy-2-(di-n-propylamino)tetralin, a new centrally acting 5-hydroxytryptamine receptor agonist

J. Med. Chem.

Structural requirements of simple organic cations for recognition by multidrug-resistant cells

Cancer Res.

Quantitative analyses of carbonyl-carbon-11-WAY-100635 binding to central 5-hydroxytryptamine-1A receptors in man

J. Nucl. Med.

In vivo characterization of p-[(18)F]MPPF, a fluoro analog of WAY-100635 for visualization of 5-HT(1a) receptors

Synapse

Influence of P-glycoprotein on brain uptake of [¹⁸F]MPPF in rats

[¹⁸F]MPPF uptake and distribution in rat brain