predicting phospholipidosis: a fluorescence noncell based in vitro assay for the determination of...

Published: July 26, 2011

r 2011 American Chemical Society 6980 dx.doi.org/10.1021/ac200683k |Anal. Chem. 2011, 83, 69806987

ARTICLE

pubs.acs.org/ac

Predicting Phospholipidosis: A Fluorescence Noncell Based in VitroAssay for the Determination of DrugPhospholipid ComplexFormation in Early Drug DiscoveryLiping Zhou,*, Gina Geraci, Sloan Hess,, Linhong Yang, Jianling Wang, and Upendra Argikar

Chemical and Pharmaceutical Proling, Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge,Massachusetts 02139, United States

Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, 250Massachusetts Avenue, Cambridge,Massachusetts02139, United States

Drug-induced phospholipidosis (PLD), though not called so,was rst reported in 1948 by Nelson and Fitzhugh with theobservation of foamy macrophages in rats under long-term treat-ment of chloroquine.1 Later studies have indicated that cationicamphiphilic drugs (CADs) are responsible for inducing this lipidaccumulation in cells and causing the presence of lamellar inclusionbodies that are primarily lysosomal in composition.25 The me-chanisms behind PLD are not fully understood. The two leadinghypotheses include (i) the binding between CAD and phospholi-pids through both hydrophobic and electrostatic interactionsresulting in druglipid complexes indigestible by lysosomal phos-pholipases and (ii) a direct inhibition of lipid digestion enzyme bytheCAD.3,69 There is no clear evidence linking drug-induced PLDto cell toxicity. This lipid storage disorder is considered to be a cellsadaptive response to CAD exposure10 rather than a toxicologicalmanifestation and is reversible upon the termination of the admini-stration.11,12 The altered lipid metabolism resulting from PLD is ofconcern as it can occur in a range of tissue types including lung, liver,brain, nervous system, and lymphatic system.13 In certain cases, itcan lead to an accumulation of drugs and/or their metabolites ashigh as millimolar concentration in lamellar bodies and cause cellinjury.2,14 Therefore, the extent and duration of the reversibility ofPLD by a compound is investigated to assess its safety margin. Indrug discovery and development, the accumulation of drugs incritical tissues such as brain, eye, liver, and heart are of majorconcerns and it is clearly a disadvantage when compared to acompetitor without PLD indication.

A diagnosis of PLD requires conrmation by transmissionelectron microscopy (TEM), which is widely accepted as thestandard approach to characterize drug-induced lipidosis.9,1518

Under TEM, the onionlike lysosomes in macrophages are char-acteristic of PLD. However, TEM is neither commonly availablenor readily amendable for the requirement in early drug discoveryand development because of its high cost, the need to sacricelaboratory animals, and the variation from study to study in termsof experimental design, dose, etc. Many tools, such as in silicoevaluation, in vitro biological screening, and in vivo biomarkershave been developed to address the needs at dierent phases ofdrug discovery and development to predict or identify phospho-lipidosis. Because of the characteristic properties of CADs, in silicoprediction models for PLD focus on screening for a candidateslipophilicity and its charge at neutral13,19 or a lower pH, represen-tative of the condition in the lysosome.20 Later models alsoincorporate detailed compound structural information13 or phar-macokinetic properties to improve predictability.10 Though applic-able as rst tier agging tools, in silico models shall be used withcaution as they may project a PLD snapshot especially for virtualmolecules rather than a full mechanistic assessment including dosedependency and time dependency. In addition, these tools cannotpredict inducers that are non-CADs, such as gentamicin which is

Received: March 17, 2011Accepted: July 26, 2011

ABSTRACT: This paper describes for the rst time, a high-throughput uores-cence noncell based assay to screen for the drugphospholipid interaction,which correlates to phospholipidosis. Anionic amphiphilic phospholipids canform complexes in aqueous solution, and its critical micelle concentration(CMC) can be determined using the uorescence probe N,N-dimethyl-6-propionyl-2-naphthylamine (Prodan). Upon interaction with drug candidates,this CMC may shift to a lower value due to the association between lipids anddrug candidates, the stronger the interaction, the greater the shift. Metabolism ofa drug can change the degree of phospholipidosis depending on the rate ofmetabolism and the nature of the metabolite(s). Our data from 45 drugs and metabolites of 10 drugs using this uorescenceapproach demonstrate a good correlation with phospholipidosis as reported with human studies, in vivo testing, and cellular assays.This assay therefore oers a fast, reliable, and cost-eective screening tool for early prediction of the phospholipidosis-inducingpotential of drug candidates.

6981 dx.doi.org/10.1021/ac200683k |Anal. Chem. 2011, 83, 69806987

Analytical Chemistry ARTICLE

highly hydrophilic.21 In most pharmaceutical companies PLDscreening strategies, the next level screening assays are cell-basedbiological assays with either uorescent lipids or lipophilic dyes insidecells.2225 A 96-well based gene-expression assay26 has been recentlyreported; however, this assay has been demonstrated to be lesssensitive.22 There has been no noncell based in vitro screening assayavailable until recently. A high-throughput langmuir-balance approachlinking the change in the critical micelle concentration (CMC) afterthe treatment with test compound to PLD observed in human, inanimal, and in cell hasbeen reported.27This approachgreatly enhancesthe quality of prediction compared to computational models andincreases throughput compared to cellular assays and animal models;however, this method requires the application of a specialized device,an eight channel surface-tensiometer (Delta 8, Kibron Inc., Helsinki,Finland), which is not readily available in most laboratories.

In this report, we describe an alternative approach for thedetermination of CMC and therefore a drugs phospholipogenicpotential in alignment with the langmuir-balance approach. Wemeasure druglipid complex formation via changes in CMC of ashort chain lipid with a uorescent dye probe. Some uorescent dyeprobes have long been used for the determination of CMCs,28,29

including lipids30,31 based on themicroenvironmental changes uponthe formation of micelles aecting the uorescent emission of thedye probe. The instrument required is a uorescent plate readerwhich is available in most biology and biochemistry laboratories.

Drug metabolism can aect PLD in at least two ways. When apolar metabolite of a CAD is formed, it can be rapidly excretedmaking the parent less likely to accumulate. On the other hand,the formation of a cationic amphiphilic metabolite from a non-CAD parent can also induce PLD.32 Such mechanistic insightsmay be valuable in explaining the disconnection between in vitroscreening results and in vivo animal data. For this reason, we havealso investigated the phospholipogenic potential of major meta-bolites of some of the test compounds.

METHODS

Equipments and Materials. Equipment. A 96-well fluores-cence plate reader (SepctraMax-Gemini EM,Molecular Devices,

Sunnyvale, CA) coupled with a 96-shallow well fluorescent plate(Greiner Bio-one, Kremsmuenster, Austria) was employed.

Materials. All standard compounds used to validate thismethod were purchased from Fisher Scientific/A.G. Scientific/Aldrich/Acros/Tocris/MP Biomedicals or obtained from No-vartis repository and used without further purification.

Buffers. A pH 7.2 N-2-hydroxyethylpiperazine-N0-2-ethane-sulfonic acid (HEPES) buffer, a 20 mM with 0.1 mM ethylene-diamine tetraacetic acid (EDTA) (MP biomedicals, Solon, OH),and a pH 4.8 acetate buffer, 40 mM were used.

Fluorescent Dye. N-N-Dimethyl-6-propionyl-2-naphthylamine(Prodan) was purchased from Fisher Scientific (Fair Lawn, NJ).

Anionic Lipid. 1,2-Dioctanoyl-sn-glycero-3-phospho-L-serine(sodium salt) (diC8PS) was purchased from Avanti Polar LipidsInc. (Alabaster, AL).Sample Preparation. Stock Solutions of Test Compounds.

Test compounds were dissolved in Dimethyl sulfoxide (DMSO)at concentrations of 10, 1, or 0.1 mM. For compounds such asgentamicin that were not fully soluble in DMSO at the aboveconcentrations, distilled water was used instead.

Lipid Solution. The 50 mg/mL stock solution of diC8PS inchloroform purchased from Avanti was further diluted in chloro-form/methanol (5:1 v/v) to yield a concentration of 16 mg/mLand stored at 80 C when not used. A volume of 4 mL of thissolution was transferred to a clean 20mL glass vial, and the solventwas evaporated under a gentle nitrogen stream. The dry lipid filmwas dispersed in 10 mL of 20 mMHEPES buffer, 0.1 mM EDTA,pH 7.2, to obtain a final concentration of 6 mg/mL. The solutionwas vortexed vigorously and placed in a 60 C water bath andincubated for 45 min before use. This solution was stored at 4 Cwhen not in use and then diluted with the same HEPES bufferdescribed above to obtain 4 and 1 mg/mL solutions prior to use.

Dye Solution. Prodan was dissolved in methanol to make a0.3 mg/mL stock solution. The container of the solution waswrapped in foil and stored in the dark at room temperature. Thesolution was diluted to 0.05 mg/mL using methanol beforeadding to the sample solutions.

Sample Plate Preparation and Measurement. The sampleplate was prepared by mixing 10 L of compound stock solution

Figure 1. Reproducibility of the assay illustrated with the average CMC of dierent test articles obtained on dierent assay dates. Error bars representedthe standard deviations.



(pure DMSO for control), 10 L of Prodan solution, and thecorrect amount of lipid solution to make the final lipid concen-trations of 3.3, 2, 1, 0.8, 0.5, 0.25, 0.1, 0.08, 0.06, 0.006, and0.0006 mg/mL, with a total volume of 110 L in each welladjusted by addition of the buffer solution. The final drugconcentrations were 910, 91, and 9.1 M from 10, 1, and0.1 mM DMSO stock solutions, respectively. The plate was readusing the SpectraMax-Gemini EMplate reader with an excitationwavelength of 360 nm and an emission wavelength of 430 nm.

Determination of Critical Micelle Concentration. The fluor-escence readings were plotted against the concentration of thelipid. The CMC was defined as the concentration point abovewhich the enhancement of fluorescent signal was a function ofconcentration as illustrated by Figure 1. This intercept of thebiphasic linear plots was determined automatically by an in-houseMicrosoft Excel worksheet developed by Novartis IT. TheMicrosoft Excel worksheet located the best fit for the vertical linearcurve by looking for the best R2 value from a moving window ofpoints in the data. The remaining data was used for the horizontallinear curve. The intercept point, which was the CMC value, wascalculated by subtracting the horizontal curve from the verticalcurve. The intersection in the control experiment representedthe CMC of the lipid in the matrix of the media.Assay Reproducibility.To test the reproducibility of the assay,

some compounds were tested multiple times on different assaydates. Together with the data of controls which were ran on everyassay date, the coefficient of variations (CVs) ranged from 4 to 8%,indicating good reproducibility of the current approach (Figure 1).

RESULTS

Shift of Lipid CMC upon Incubation with Test Compounds.The negatively charged lipid diC8PS, being amphiphilic in nature,aggregated and formed micelles in aqueous solution, generating amore lipophilic microenvironment. When Prodan interacted withthe micelles, its fluorescence emission was shifted. A discontinuitywas observed from the fluorescence concentration profile ofdiC8PS (Figure 2) with the transitioning lipid concentration beingits CMC (CMCL = 1.1 mg/mL; L-lipid). This CMC value was

similar to the one determined by Vitovic using the surface tensionapproach under similar test conditions.27 When test compoundthiorizadine was added to the matrix, the CMC of the complex(CMCDL; DL, druglipid complex) shifted to 0.042 mg/mL(Figure 2). At lipid concentrations above 0.5 mg/mL in thesample, the fluorescent signal was quenched, giving lower values;however, this did not affect the determination of CMC. The ratiobetween CMCDL and CMCL was utilized to quantify the shift.The CMC values of the druglipid complex were determined

for 45 commercial compounds and compared with PLD ob-served in humans, in animals, or in cultured cells (Table 1).These compounds were grouped into four classes, respectively,as suggested in the literature.19,27 The degree of the CMC shiftwas represented by the CMC ratio (CMCDL/CMCL) (Figure 3).With the comparison of the PLD data derived either in humans,in animals, or in cultured cells with the CMC shift detected usingthis uorescent approach (Figure 3) and using the CMC ratio at0.75 as the cuto value, most compounds demonstrated a con-sistent ranking between the in vivo and in vitro data except for two:warfarin and amlodipine. Both warfarin20 and amlodipine10 werereported as having low or no PLD in vivo, but CMC shifts wereobserved in this uorescent assay.Concentration Dependency. The degree of PLD in in vivo

studies was dose dependent.24,33 In in vitro assays, concentrationcontrol could be used to estimate dose dependency. A group ofseven reported PLD inducers were tested at three differentconcentrations: 910, 91, and 9.1 M, respectively (Figure 4).For chloroquine and ketoconazole, either they were not fullysoluble at 10 mM in DMSO or they salted out upon mixing withbuffer when using 10 mM DMSO stocks, their highest concen-tration points were taken out. All compounds demonstratedconcentration dependency toward CMC shift the PLD inducingpotential with a varying extent. Fenfluramine appeared to be aweak inducer even at 910 M, whereas thioridazine showedsignificant changes from concentration to concentration. At thelowest concentration of 9.1 M, none of the seven compoundsappeared to be able to induce PLD according to this in vitro data.Compound Self-Aggregation. Similar to phospholipids,

most of the PLD inducers were amphiphilic. Though bearingdifferent signs of charges, they could also aggregate and formmicelles. Labetalol20,34 and quinidine35 were reported as PLDinducers; however, we were not able to determine the CMC ofthe druglipid complex of either under the test conditionsapplied. The RFU values obtained were equal or lower comparedto those in the blank (compound with fluorescent probe but nolipid). Further studies with varying drug concentrations but nolipid (0 mg/mL lipid) were carried out, and CMC values of 26and 20 Mwere revealed for labetalol and quindine, respectively,in the lipid-free assay buffer. These CMC values were well belowthe test concentrations of these compounds in the PLD screen-ing, and it was suspected that the compounds self-aggregationcould interfere with the detection of the druglipid complexformation thus making the CMCDL not determinable.PLD Induced by Metabolites. To investigate the effect of

metabolism on PLD, 10 compounds with major metabolism eventswere selected and their major metabolites were screened using thereported assay, exhibiting distinct behaviors (Figure 5 and Table 2).Desethylamiodarone, the major metabolite of amiodarone showed aclose to 10 times higher lipid binding potential than its parent. Forchloroquine, clozapine, and mianserin, their metabolites fell into thePLD noninducer group, contradictory to the parent compoundswhich were inducers. Because of solubility limitations, test

Figure 2. Fluorescence emission recorded at dierent concentrations oflipid diC8PS ranging from 64 g/mL to 3.47 mg/mL. In the control,with neat DMSO added in place of test compound (O), the sharp rise at1.1 mg/mL marked the CMC of diC8PS. With 10 L of 10 mMthioridazine (9) introduced to the matrix (diC8PS in 20 mM HEPESbuer), the CMC shifted to lower concentration (0.042 mg/mL). RFU:relative uorescence unit.



concentrations of 8-hydroxymianserin and des-methyl clozapinewere0.091 mM, lower than those of their parent compounds (1 mM).

The other metabolites showed comparable PLD inducing potentialcomparing to their parent compounds while others did not.

Table 1. List of Compounds Used in the Validation of the Fluorescent Probe Approach with Their PLD Inducing PotentialDetected/Estimated by in Vivo, in Vitro, and in Silico Models

in vivo/in vitro cellular screening in silico prediction in vitro noncell based uorescent screening, CMCDL/CMCL

compounda PLD induction classb refs

Ploemen

model19Tomizawa

model20 pH 7.2 pH 4.8

acetaminophen IV 10,20,27,56 negative low 1.043 0.91

amikacin II 10,56,57 negative none 0.193

amiodarone I, II 40,5861 positive high 0.645 0.95amitryptyline II 62 positive high 0.1

amlodipine IV 10 positive high 0.191 0.06

atenolol IV 10,27 negative low 0.98 0.86

atropine IV 10,20,27 positive low 0.944 1.05

bupropion IV 10 negative high 0.762 0.63

buspirone IV 27 negative med 0.902 0.97

carbamazepine IV 10 negative low 0.843 0.99

chloroquine (0.091 mM) I, II 2,62 positive high 0.333 0.47

chlorpromazine II 62,63 positive high 0.047 0.08

cimetidine IV 20 negative low 1.033

citalopram II 64 positive high 0.272

clomipramine II 10,23 positive high 0.082 0.84

clozapine II 62 positive high 0.665 0.83

desipramine II 65 positive high 0.155 0.28

disopyramide IV 27 positive high 1.001 0.79

erythromycin II 66 negative med 0.739 0.89

famotidine IV 20,27 negative low 0.92 0.87

fenuramine II 67,68 positive high 0.702

uoxetine I, II 69 positive high 0.047

furosemide IV 20,27 negative none 0.929 0.81

gembrozil IV 10 negative none 1.039

gentamicin I,II 36,70 negative none 0.072

haloperidol II 10,62 negative high 0.125

imipramine II 62 positive high 0.201

ketoconazole (0.091 mM) II 71 negative high 0.558 0.09

ketoprofen IV 10,27 negative none 1.026

lidocaine IV 10,20,27 negative medium 1.013

maprotiline II 72 positive high 0.072 0.15

mianserin III 62 negative high 0.217 0.94

nortriptyline III 62 positive high 0.149

pentamidine I 73 positive high 0.163 0.31

perhexiline I, II 74 positive high 0.043 0.05

procaine IV 10 positive med 0.851 0.97

promazine II 63 positive high 0.056

propranolol III 35 positive high 0.076 0.29

ranitidine IV 10 negative low 0.884

sotalol IV 10,75 negative low 0.933

suldinac IV 10 negative low 0.979

tamoxifen II 62 positive high 0.308

thioridazine II 62 positive high 0.042

valproic acid IV 10,20,27 negative low 0.997 1.04

warfarin IV 10,20 negative low 0.401 0.75a Final compound concentration was 0.91 mM unless otherwise indicated. bCompounds were grouped based on in vivo and cellular assay data. Class I:PLD in humans; Class II: PLD in animals; Class III: PLD in cells but not in animals; Class IV: no PLD observed or reported.



Assay pH Effect. The lipid disorder in PLD was mainly ob-served in the lysosomal compartment which constituted an acidicenvironment. To mimic the lysosomal condition, pH 4.8 acetatebuffer was employed in the PLD screening studies with 25commercial compounds (Table 1) using the same proceduredescribed above. Most of the compounds gave similar results inboth buffers; however, amiodarone, clomipramine, mianserin,and warfarin gave very different data, shifting the front threecompounds from PLD inducers to noninducers and warfarinfrom noninducer to inducer from pH 7.2 to pH 4.8.

DISCUSSION

Here we present a novel, high-throughput, noncell based invitro screening approach predictive of PLD using a uorimetricprobe by monitoring the CMC of phospholipid with and withoutinteraction with the test article. While the Langmuir balanceapproach27 provides a reliable avenue to assess PLD via the CMCshift, current uorimetric methodology oers an unique alter-native to predict PLD risk. Such a microplate-compatible plat-form is more cost-eective, easier to operate, and has higherthroughput, amendable for PLD risk assessment in early drugdiscovery. The uorescent probe selected, Prodan, is suitable forthe determination of CMCs of both the lipid as well as testcompounds that form micelles. We clearly demonstrate in vivoversus in vitro or noncell based versus cellular assay correlationusing the current method with an overall concordance of 91%when applying a suitable cuto value (CMCDL/CMCL = 0.75 inthis assay). This is better compared to the two in silico modelsthat we also applied to our validation set whose concordancevalues were 76 and 80% for Ploemen19 and Tomizawa20 models,respectively. The sensitivity (PLD inducers identied as positive)and selectivity (non-PLD inducers identied as negative) of ourmodel were 92% and 90%, which were also better than those forthe two in silico models (Table 3). Both in silico and in vitroassays utilized the inherent physicochemical properties of the testcompounds that made them more anitive toward phospholi-pids; however, the in vitro models could also identify the eect ofdrug concentrations on druglipid interactions which was notpredictable by in silico models. Furthermore, the steric chemistryof the molecule and the spatial distribution of charge as well as itspolar and nonpolar moieties could play a role in the reactivity ofthe test compound and these factors were not captured by insilico models. In addition, this in vitro model correctly assignedthe PLD inducing potential of non-CAD drugs like gentamicin

Figure 3. Comparison between the CMC shift (represented by the ratio between CMC of the druglipid complex (DL) and CMC of lipid alone (L):CMCDL/CMCL) observed in this uorimetric assay at pH 7.2 with in vivo human, in vivo animal, or in vitro cellular PLD assays. A cuto value of 0.75 inCMCDL/CMCL separated PLD inducers (b, in vivo assay; [, cellular assay) from PLD noninducers (9) with only two outliers: amlodipine andwarfarin.

Figure 4. The CMC shift (represented by the ratio between CMC ofthe druglipid complex (DL) and CMC of lipid alone (L): CMCDL/CMCL) of test compounds with a dierent nal concentration in thesample wells at pH 7.2.



and erythromycin because in vitro models evaluated the overallinteractions between the test compound and lipid thus weremore advanced than in silicomodels. Conversely, for compoundswith solubility issues at desired test concentrations, an alternativesolvent should be identied or the data could be misleading. Insilico models did not have this barrier and could be applied to anycompounds with dened structures.

Pharmacokinetics was reported to have an impact on PLD asthe dose,24,33,36 the metabolism,32,3739 and the disposition10 ofthe drug all aected the severity of PLD in vivo. Creation of asuccessful candidate required clear understanding of the cause of

PLD and thus redesigning the chemical structure or assessing anappropriate therapeutic window.

The minimum dose required for PLD diered from com-pound to compound and sometimes was gender dependent.24

Directly linking the in vivo dose with the in vitro test concentra-tion could be challenging as the plasma concentration of the drugcould be well aected by its permeability, solubility, metabolism,excipient utilized, route of administration, frequency of drugintake, and so on. However, by monitoring the concentrationdependency of the test molecule toward PLD capability, whichwas unlikely to be linear as shown in Figure 4 and would be hardto estimate, we could identify the potential relationship betweendose and lipid binding, thus assess the safety window with alimited number of in vivo tests.

There have been a number of publications describing PLDcaused by drug metabolites. Among them, the primary metabo-lite of amiodarone, desethylamiodarone, had been discussedextensively.14,3840 Preferential accumulation of the metaboliteover amiodarone had been observed especially for short-termtreatments.39 This oered a possible explanation for our assay datawhere desethylamiodarone, amajormetabolite of amiodarone,41,42

demonstrated much higher PLD inducing potential. Similarly,the CMC shift dierence between 8-hydroxymianserin, themajor oxidative metabolite of mianserin,43,44 and mianserinwas more signicant than the dierence between the other twopairs of parents andmetabolites. Because of solubility limitations,dose concentrations were dierent for the parents and metabo-lites for clozapine and mianserin. It was possible that this mightlead to remarkable dierences in the CMC shift observedbetween parent and metabolite. When tested at a lower concen-tration, clozapine switched from a low potential inducer to anoninducer, whereas mianserin remained as an inducer but withhigher CMCDL/CMCL value. It was worth noting most CADs inFigure 5. The CMC shift (represented by ratio between CMC of the

druglipid complex (DL) and CMC of lipid alone (L): CMCDL/CMCL) of test compounds (black) and their major metabolites (gray,primary metabolite; white, secondary metabolite) analyzed in pH 7.2HEPES buer. The drug concentrations used were 1 mM unlessotherwise indicated in Table2. Error bars were assigned to thosecompounds with n g 3 tests. For clozapine and mianserin, both 0.91and 0.091 mM were used in the tests but only data from the 1 mM testswere plotted.

Table 2. List of Compounds Used in the Validation of Metabolite Eects on PLD

parent

compound

PLD

class

CMC shift of parent

compound metabolite 1

CMC shift of

metabolite 1 metabolite 2

CMC shift of

metabolite 2 refs

amiodarone I, II 0.604 desethylamiodarone 0.075 41,42

amitryptyline II 0.175 nortryptyline 0.065 amitriptalline N-glucuronide N/D 4850amlodipine IV 0.045 dehydro amlodipine oxalate 0.180 76,77

chloroquine

(0.091 mM)

I, II 0.657 desethyl chloroquine 0.980 bidesethyl chloroquine N/D 51,52

chlorpromazine II 0.047 N-desmethyl chlorpromazine 0.056 chlorpromazine-5-sulfoxide N/D 4547clomipramine II 0.083 N-des methyl clomipramine 0.060 clomipramine-5S-oxide

clomipraimne n-oxide

0.877 53,54

clozapine II 0.712 des-methyl clozapine (0.1 mM) 0.917 clozapine-N-oxide (0.1 mM) 1.015 78,79

clozapine

(0.091 mM)

0.924

desipramine II 0.161 N-desmethyl desipramine 0.080 desipramine N-hydroxy 1.040 43,50

imipramine II 0.235 desipramine 0.155 imipramine-N-glucuronide N/D 43,50

mianserin III 0.225 8-hydroxymianserin (0.1 mM) 0.877 43,44

mianserin (0.091 mM) 0.675

Table 3. Predictability of Various Models

model Ploemen19 Tomizawa20 Novartis

sensitivity (%) 72 88 92

selectivity (%) 80 70 90

concordance (%) 76 80 91



the current test set were reported to be metabolized oxidativelyto their corresponding des-alkyl primary metabolites. In order tounderstand the PLD potential of the corresponding primarymetabolites, we tested the additional metabolites. Among themetabolites tested were primary des-methyl metabolites chlor-promazine,4547 amitryptyline,4850 imipramine,43,50 and pri-mary and secondary des-ethyl metabolites of chloroquine.51,52

In addition, oxidative metabolites formed by heteroatom oxida-tion and des-alkyl metabolites of desipramine43,50 and clomin-pramine53,54 were evaluated. Metabolites, like other CADs, couldinduce PLD if they had suitable physicochemical properties.32

Conversely, a metabolite would lose the capability of bindingwith lipid if CAD charactersistics are lost, such as observed withclomipramine N-oxide and desipramine N-hydroxy (Figure 4and Table 2).

It has been known that the levels and time in circulation mayvary for dierent metabolites and from one drug to another;however, the amounts (in moles) or dose equivalents of circulat-ing metabolites may never be greater than those of the adminis-tered drug. In the present study, the metabolites were tested atthe same or lower concentrations compared to their parentsbased on the allowance of their solubility. It should be noted thatthis assay was developed as a prospective screening tool; there-fore, the underlying premise is the testing metabolites at thesame concentration as that of the parent. In cases where thePLD potential of the metabolites is of interest, a concentrationdependent study may be conducted with the concerned meta-bolite. However, such a study was beyond the scope of thepresent investigation. The identication of lamellar bodies viatransmission electron microscopy (TEM) was the rst indicationof lysosome involvement in PLD9 and indicated that lysosomalpH may be a critical factor;20 however, initially there was no directevidence of the origin and function of lysosomal involvement.3 Astudy carried out by Maxeld and MacGraw showed evidence ofrapid sequestration of endosomes formed in the presence ofphospholipogenic drugs that became lysosomes;55 however, itwas possible that the acidic pH of lysosomes could change theseverity of interaction between the lipid and drug of interest withthe formation of lysosomal inclusion bodies. For these reasons, webelieved it valuable to study both pH conditions and thereforeobtain amore comprehensive risk assessment. In our validation set,most compounds gave similar CMC shifts under both pH 7.4 and4.8 except for mianserin, amiodarone, and warfarin, and all threecompounds were proven to be PLD inducers by varied in vivo andcellular assays.

This assay showed high reproducibility and predictability.Similar to other in vitro assays which could not replace in vivomeasurements for a conrmative result, this less expensive, lesstime-consuming uorimetric approach could provide usefulinformation on druglipid interaction early on. Furthermore,such an approach could be used for rank ordering candidates,aiding drug design, and estimating potential risk.

AUTHOR INFORMATION

Corresponding Author*Phone: 617-871-7143. E-mail: [email protected].

Present AddressesDepartment of Chemistry, University of Colorado-Denver,Denver, CO 80205.

ACKNOWLEDGMENT

We would like to thank Dr. Pavel Landsman for his insight onuorescent dye probe selections, design of the experiments, andhelpful discussions. Our thanks also go to Mr. Alan Bushey fromNovartis IT for development of the data processing MicrosoftExcel worksheet.

REFERENCES

(1) Nelson, A. A.; Fitzhugh, O. G. Arch. Pathol. 1948, 45 (4),454462.

(2) Hostetler, K. Y.; Reasor, M.; Yazaki, P. J. J. Biol. Chem. 1985, 260(1), 215219.

(3) Kodavanti, U. P.; Mehendale, H. M. Pharmacol. Rev. 1990, 42327354.

(4) Vanhaelst, U. J. G. M.; Elving, L.; Hoefnagels, W. Ultramicro-scopy 1986, 19 (1), 107108.

(5) Schneider, P. Arch. Toxicol. 1992, 66 (1), 2333.(6) Halliwell, W. H. Toxicol. Pathol. 1997, 25 (1), 5360.(7) Reasor,M. J.; Hastings, K. L.; Ulrich, R. G. Expert Opin. Drug Saf.

2006, 5 (4), 567583.(8) Martin, W. J.; Kachel, D. L.; Vilen, T.; Natarajan, V. J. Pharmacol.

Exp. Ther. 1989, 251 (1), 272278.(9) Anderson, N.; Borlak, J. FEBS Lett. 2006, 580 (23), 55335540.(10) Hanumegowda, U. M.; Wenke, G.; Regueiro-Ren, A.; Yorda-

nova, R.; Corradi, J. P.; Adams, S. P. Chem. Res. Toxicol. 2010, 23 (4),749755.

(11) Reasor, M. J.; Castranova, V. Exp. Mol. Pathol. 1981, 35 (3),359369.

(12) Reasor, M. J.; Walker, E. R. Exp. Mol. Pathol. 1981, 35 (3),370379.

(13) Pelletier, D. J.; Gehlhaar, D.; Tilloy-Ellul, A.; Johnson, T. O.;Greene, N. J. Chem. Inf. Model. 2007, 47 (3), 11961205.

(14) Sun, E. L.; Petrella, D. K.; McCloud, C. M.; Cramer, C. T.;Reasor, M. J.; Ulrich, R. G. In Vitro Toxicol. 1997, 10 (4), 459470.

(15) Watanabe, Y.; Watanabe, K.; Tashiro, I.; Enomoto, Y.J. Electron Microsc. 1971, 20 (3), 255.

(16) Tashiro, Y.; Watanabe, Y.; Enomoto, Y. Acta Pathol. Jpn. 1983,33 (5), 929942.

(17) Monteith, D. K.; Morgan, R. E.; Halstead, B. Expert Opin. DrugMetab. Toxicol. 2006, 2 (5), 687696.

(18) Nonoyama, T.; Fukuda, R. Toxicol. Pathol. 2008, 21 (1), 924.(19) Ploemen, J. P. H. T.; Kelder, J.; Hafmans, T.; Van De Sandt, H.;

van Burgsteden, J. A.; Salemink, P. J. M.; Van Esch, E. Exp. Toxicol.Pathol. 2004, 55 (5), 347355.

(20) Tomizawa, K.; Sugano, K.; Yamada, H.; Horii, I. J. Toxicol. Sci.2006, 31 (4), 315324.

(21) Laurent, G.; Carlier, M. B.; Rollman, B.; Vanhoof, F.; Tulkens,P. Biochem. Pharmacol. 1982, 31 (23), 38613870.

(22) Nioi, P.; Perry, B. K.; Wang, E. J.; Gu, Y. Z.; Snyder, R. D.Toxicol. Sci. 2007, 99 (1), 162173.

(23) Morelli, J. K.; Buehrle, M.; Pognan, F.; Barone, L. R.; Fieles, W.;Ciaccio, P. J. Cell Biol. Toxicol. 2006, 22 (1), 1527.

(24) Miyamoto, S.; Matsumoto, A.; Mori, I.; Horinouchi, A. Toxicol.Mech. Methods 2009, 19 (8), 477485.

(25) Natalie, M.; Margino, S.; Erik, H.; Annelieke, P.; Geert, V.;Philippe, V. Toxicol. in Vitro 2009, 23 (2), 217226.

(26) Sawada, H.; Taniguchi, K.; Takami, K. Toxicol. in Vitro 2006, 20(8), 15061513.

(27) Vitovic, P.; Alakoskela, J. M.; Kinnunen, P. K. J. J. Med. Chem.2008, 51 (6), 18421848.

(28) De Vendittis, E.; Palumbo, G.; Parlato, G.; Bocchini, V. Anal.Biochem. 1981, 115, 278286.

(29) Deb, N.; Shannigrahi, M.; Bagchi, S. J. Phys. Chem. B 2008, 11228682873.

(30) Moyano, F.; Silber, J. J.; Correa, N. M. J. Colloid Interface Sci.2008, 317, 332345.



(31) Moyano, F.; Biasutti, M. A.; Silber, J. J.; Correa, N. M. J. Phys.Chem. B 2006, 110, 1183811846.

(32) Gum, R. J.; Hickman, D.; Fagerland, J. A.; Heindel, M. A.;Gagne, G. D.; Schmidt, J. M.;Michaelides, M. R.; Davidsen, S. K.; Ulrich,R. G. Biochem. Pharmacol. 2001, 62 (12), 16611673.

(33) Wilson, B. D.; Clarkson, C. E.; Lippmann, M. N. Am. Rev.Respir. Dis. 1991, 143, 11101114.

(34) Lahoti, S.; Lee, W. M. Gastroenterol. Clin. North Am. 1995, 24(4), 907922.

(35) Kita, N.; Sugihara, N.; Furuno, K. J. Pharmacobiodyn. 1992, 15(4), 181189.

(36) Giuliano, R. A.; Paulus, G. J.; Verpooten, G. A.; Pattijn, V. M.;Pollet, D. E.; Nouwen, E. J.; Carlier, M. B.; Maldague, P.; Tulkens, P. M.;Debroe, M. E. Hum. Toxicol. 1984, 3 (5), 432.

(37) Kacew, S.; Narbaitz, R.; Ruddick, J. A.; Villeneuve, D. C. Exp.Mol. Pathol. 1981, 35 (1), 98107.

(38) Kodavanti, U. P.; Mehendale, H.M. Am. J. Respir. Cell Mol. Biol.1991, 4 (4), 369378.

(39) Antonini, J. M.; Reasor, M. J. Biochem. Pharmacol. 1991, 42S151S156.

(40) Kannan, R.; Sarma, J. S. M.; Guha, M.; Venkataraman, K. Eur.J. Pharmacol. 1990, 183 (6), 24522453.

(41) Nattel, S. J. Cardiovasc. Pharmacol. 1986, 8 (4), 771777.(42) Latini, R.; Tognoni, G.; Kates, R. E. Clin. Pharmacokinet. 1984,

9 (2), 136156.(43) Riley, R. J.; Roberts, P.; Kitteringham, N. R.; Park, B. K.

Biochem. Pharmacol. 1990, 39 (12), 19511958.(44) Lambert, C.; Park, B. K.; Kitteringham, N. R. Biochem.

Pharmacol. 1989, 38 (17), 28532858.(45) Wojcikowski, J.; Boksa, J.; Daniel, W. A. Biochem. Pharmacol.

2010, 80 (8), 12521259.(46) Stevens, J. C.; Shipley, L. A.; Cashman, J. R.; Vandenbranden,

M.; Wrighton, S. A. Drug Metab. Dispos. 1993, 21 (5), 753760.(47) Hartmann, F.; Gruenke, L. D.; Craig, J. C.; Bissell, D. M. Drug

Metab. Dispos. 1983, 11 (3), 244248.(48) Gupta, R. N.; Molnar, G.; Hill, R. E.; Gupta, M. L. Clin.

Biochem. 1976, 9 (5), 247251.(49) Prox, R.; Breyer-Pfa, U. Drug Metab. Dispos. 1987, 15 (6),

890896.(50) Vandemark, F. L.; Adams, R. F.; Schmidt, G. J. Clin. Chem.

1978, 24 (1), 8791.(51) Kim, K. A.; Park, J. Y.; Lee, J. S.; Lim, S. Arch. Pharm. Res. 2003,

26 (8), 631637.(52) Projean, D.; Baune, B.; Farinotti, R.; Flinois, J. P.; Beaune, P.;

Taburet, A. M.; Ducharme, J. Drug Metab. Dispos. 2003, 31 (6),748754.

(53) Wu, Z. L.; Huang, S. L.; Ou-Yang, D. S.; Xu, Z. H.; Xie, H. G.;Zhou, H. H. Zhongguo Yao Li Xue Bao 1998, 19 (5), 433436.

(54) Nielsen, K. K.; Flinois, J. P.; Beaune, P.; Brosen, K. J. Pharmacol.Exp. Ther. 1996, 277 (3), 16591664.

(55) Maxeld, F.; McGraw, T. Nat. Rev. Mol. Cell Biol. 2004, 5121132.

(56) Sawada, H.; Takami, K.; Asahi, S. Toxicol. Sci. 2005, 83 (2),282292.

(57) Toubeau, G.; Nonclercq, D.; Zanen, J.; Lambricht, P.; Tulkens,P. M.; Heuson Stiennon, J. A.; Laurent, G. Kidney Int. 1991, 40 (4),691699.

(58) Poucell, S.; Valencia, P.; Ireton, J.; Downar, E.; Patterson, J.;Blendis, L.; Phillips, M. J. Hepatology 1983, 3 (5), 805.

(59) Poucell, S.; Ireton, J.; Valenciamayoral, P.; Downar, E.; Larratt,L.; Patterson, J.; Blendis, L.; Phillips, M. J. Gastroenterology 1984, 86 (5),926936.

(60) Goldman, I. S.; Winkler, M. L.; Raper, S. E.; Barker, M. E.;Keung, E.; Goldberg, H. I.; Boyer, T. D. Am. J. Roentgenol. 1985, 144 (3),541546.

(61) Riva, E.;Marchi, S.; Pesenti, A.; Bizzi, A.; Cini,M.; Veneroni, E.;Tavbani, E.; Boeri, R.; Bertani, T.; Latini, R. Biochem. Pharmacol. 1987,36 (19), 32093214.

(62) Hruban, Z. Environ. Health Perspect. 1984, 55, 5376.(63) Kodavanti, U. P.; Lockard, V. G.; Mehendale, H. M. J. Biochem.

Toxicol. 1990, 5 (4), 245251.(64) Lullmann-Rauch, R.; Nassberger, L. Acta Pharmacol. Toxicol.

1983, 52, 161167.(65) Yano, T.; Ohmori, S.; Igarashi, T.; Ueno, K.; Kitagawa, H. Jpn.

J. Pharmacol. 1987, 43, 207.(66) Gray, J. E.; Weaver, R. N.; Stern, K. F.; Phillips, W. A. Toxicol.

Appl. Pharmacol. 1978, 45 (3), 701711.(67) Lullmann-Rauch, R.; Reil, G. H. Arch. Pharmacol. 1974,

285, 175184.(68) Valodia, P. N.; Syce, J. A. Med. Sci. Res. 1998, 26 (7), 491493.(69) Gonzalezrothi, R. J.; Zander, D. S.; Ros, P. R. Chest 1995, 107

(6), 17631765.(70) Auberttulkens, G.; Vanhoof, F. Arch. Int. Physiol. Biochim.

Biophys. 1978, 86 (2), 403405.(71) Pakuts, A. P.; Parks, R. J.; Paul, C. J.; Bujaki, S. J.; Mueller, R. W.

Res. Commun. Chem. Pathol. Pharmacol. 1990, 67 (1), 5562.(72) Staubli, W.; Schweizer, W.; Suter, J. Exp. Mol. Pathol. 1978,

28, 177195.(73) Glaumann, H.; Bronner, U.; Ericsson, O.; Gustason, L. L.;

Rombo, L. Pharmacol. Toxicol. 1994, 74, 1722.(74) Lullmann, H.; Lullmann-Rauch, R. Klin. Wochenschr. 1978,

56, 309310.(75) Kasahara, T.; Tomita, K.; Murano, H.; Harada, T.; Tsubakimoto,

K.; Ogihara, T.; Ohnishi, S.; Kakinuma, C. Toxicol. Sci. 2006, 90 (1),133141.

(76) Beresford, A. P.; McGibney, D.; Humphrey, M. J.; Macrae,P. V.; Stopher, D. A. Xenobiotica 1988, 18 (2), 245254.

(77) Stopher, D. A.; Beresford, A. P.; Macrae, P. V.; Humphrey, M. J.J. Cardiovasc. Pharmacol. 1988, 12, S55S59.

(78) Pirmohamed, M.; Williams, D.; Madden, S.; Templeton, E.;Park, B. K. J. Pharmacol. Exp. Ther. 1995, 272 (3), 984990.

(79) Tugnait, M.; Hawes, E. M.;McKay, G.; Eichelbaum,M.;Midha,K. K. Chem. Biol. Interact. 1999, 118 (2), 171189.

predicting phospholipidosis: a fluorescence noncell based in vitro assay for the determination of...

Documents