Quantitative Assessment of Drug Delivery to Tissues and Association

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX-XXX

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Quantitative Assessment of Drug Delivery to Tissues and Association with Phospholipidosis: A Case Study with Two Structurally Related Diamines in Development Irena Loryan,*,†,‡ Edmund Hoppe,‡,§ Klaus Hansen,§ Felix Held,∥,⊥ Achim Kless,§ Klaus Linz,§ Virginia Marossek,§,# Bert Nolte,§,# Paul Ratcliffe,§ Derek Saunders,§ Rolf Terlinden,§ Anita Wegert,§,# André Welbers,§ Olaf Will,§ and Margareta Hammarlund-Udenaes† †

Translational PKPD Group, Department of Pharmaceutical Biosciences, Associate member of SciLifeLab, Uppsala University, 751 24 Uppsala, Sweden § Grünenthal GmbH, 52099 Aachen, Germany ∥ Fraunhofer-Chalmers Centre, Chalmers Science Park, 412 88 Gothenburg, Sweden ⊥ Department of Mathematical Sciences, Chalmers University of Technology and University of Gothenburg, 412 96 Gothenburg, Sweden S Supporting Information *

ABSTRACT: Drug induced phospholipidosis (PLD) may be observed in the preclinical phase of drug development and pose strategic questions. As lysosomes have a central role in pathogenesis of PLD, assessment of lysosomal concentrations is important for understanding the pharmacokinetic basis of PLD manifestation and forecast of potential clinical appearance. Herein we present a systematic approach to provide insight into tissue-specific PLD by evaluation of unbound intracellular and lysosomal (reflecting acidic organelles) concentrations of two structurally related diprotic amines, GRT1 and GRT2. Their intratissue distribution was assessed using brain and lung slice assays. GRT1 induced PLD both in vitro and in vivo. GRT1 showed a high intracellular accumulation that was more pronounced in the lung, but did not cause cerebral PLD due to its effective efflux at the blood−brain barrier. Compared to GRT1, GRT2 revealed higher interstitial fluid concentrations in lung and brain, but more than 30-fold lower lysosomal trapping capacity. No signs of PLD were seen with GRT2. The different profile of GRT2 relative to GRT1 is due to a structural change resulting in a reduced basicity of one amino group. Hence, by distinct chemical modifications, undesired lysosomal trapping can be separated from desired drug delivery into different organs. In summary, assessment of intracellular unbound concentrations was instrumental in delineating the intercompound and intertissue differences in PLD induction in vivo and could be applied for identification of potential lysosomotropic compounds in drug development. KEYWORDS: drug induced phospholipidosis, unbound drug, intracellular concentration, brain slice, lung slice, pharmacokinetics

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INTRODUCTION Drug-induced phospholipidosis (PLD) is a reversible lipid storage disorder characterized by excessive accumulation of drug and polar phospholipids in acidic subcellular compartments such as lysosomes (Figure 1).1,2 It is described by specific histopathological changes identified using transmission electron microscopy i.e., membranous lamellar inclusions, concentric multilamellar bodies mimicking inherited lipidosis.2 Using light microscopy, appearance of foamy macrophages or cytoplasmic vacuoles is detected in various cells, mostly in hepatic and pulmonary tissues. Many marketed drugs have been reported to cause preclinical and clinical PLD.2,3 Symptomatic PLD may be observed in preclinical species in drug development, posing a strategic © XXXX American Chemical Society

question about its potential to be manifested in patients. Hence, early safety evaluation of PLD is becoming vital in the drug industry.3 Yet, up until now there has been no clear guideline on PLD management in drug development from either the FDA or EMA due to the incomplete knowledge on the subject. Several models have been developed to predict the PLD induction potential in an early drug discovery setting. These models utilize either a physicochemical parameter such as cLogP or pKa4,5 or toxicophore models (Table 1).6,7 While Received: June 10, 2017 Revised: October 17, 2017 Accepted: October 20, 2017

A

DOI: 10.1021/acs.molpharmaceut.7b00480 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

assessment of tissue partition coefficients (Kp, i.e., tissue to plasma total steady state concentration ratios) are required. Using the correction of partition coefficients for the organspecific uptake and plasma protein binding the unbound tissue to unbound plasma concentration ratio, Kp,uu can be derived.13 Kp,uu allows estimating the unbound extracellular concentration in the tissues in relation to the unbound plasma concentration and has so far mainly been used to describe the extent of CNS delivery of drugs. We here present a novel systematic pharmacokinetic and toxicokinetic/toxicodynamic approach intended for more mechanistic understanding of tissue-specific PLD development based on the principles presented above. The approach is exemplified by investigating two structurally related diamines GRT1 and GRT2 (Table 1). GRT1 induced PLD both in vitro and in vivo, when tested in HepG2 cells and in Wistar rats, whereas no signs of PLD were seen with GRT2. By characterizing these two compounds we were particularly interested in understanding the mechanisms that can differentiate between desired tissue delivery of unbound drug and higher in vivo efficacy on one hand and undesired nonspecific tissue binding and uptake into lysosomes with potential risk of PLD on the other hand. Toward this end, the compounds were subjected to thorough tissue-specific characterization including assays on organ-specific slices and homogenates.

Figure 1. Schematic illustration of potential mechanisms of drug induced phospholipidosis (PLD) in relation to drug-specific and tissue-specific characteristics. Lysosomes are the key pathophysiological targets in PLD. Vacuolar H+-adenosine triphosphatase (vATPase) is responsible for generation and maintenance of physiological pH gradient between the cytosol and lysosomes. BBB: blood−brain barrier. BAB: blood−alveolar barrier.

these models are quite powerful to deprioritize compounds with a PLD induction risk for development, they fail to explain differences in severity of in vivo PLD observed between structurally highly related compounds.7 In addition to physicochemical parameters it was recognized that drug disposition may be another factor that is important for the manifestation of PLD in vivo.3 The model of Hanumegowda includes the volume of distribution (Vd) as an in vivo pharmacokinetic parameter for drug disposition (Figure 1), but also fails to explain the differences in in vivo PLD outcomes observed with structurally highly related drugs, and can therefore not be used to support risk assessments that are needed in development. Therefore, one of the issues left with less attention is related to the lack of quantitative understanding of drug exposure in tissues vs PLD induction. According to the free drug hypothesis, the magnitude of the pharmacological/toxicological effect is a function of the concentration of the unbound (free) drug at the site of action. Hence, assessment of the unbound lysosomal concentration in the relevant tissues is crucial for understanding of the pharmacokinetic basis of PLD development. Direct measurement of intracellular and lysosomal concentrations are currently not applicable in the drug development setting.8 However, assessment of intracellular concentration can be achieved by means of the intracellular to extracellular unbound concentration ratio, expressed by the Kp,uu,cell parameter.9 Kp,uu,cell is derived by a combination of in vitro methods such as drug uptake into fresh tissue slices and drug binding to tissue homogenates, methods well established for brain and lung tissues. With this measurement, further estimations can be made on lysosomal to cytosolic concentrations using additional mathematical models based on pH partitioning theory where collective impact of acidic cellular subcompartments referred as lysosomes could be assessed (Kp,uu,lyso).9−12 In order to make in vivo relevant interpretations, information about tissue-specific unbound extracellular concentrations is also needed. Hence, in vivo tissue distribution studies with

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EXPERIMENTAL SECTION Animals. Brain slice and lung slice experiments as well as brain and lung tissue binding studies were performed on 14 drug-naive male 300−350 g Sprague−Dawley rats (Taconic, Lille Skensved, Denmark), in accordance with guidelines from the Swedish National Board for Laboratory Animals. The study was approved by the Animal Ethics Committee of Uppsala, Sweden (ethical approval C189/14 and C49/16). Pharmacokinetic studies were carried out on 12 Sprague−Dawley rats (Charles River Laboratories, Germany). Toxicokinetic and toxicodynamic studies were performed in 216 female and male Wistar rats (Charles River Laboratories, Germany). The pharmacokinetic and toxicokinetic experiments were performed according to the German Animal Welfare Act and were approved by the local government authority. All rats were housed in groups at 18 to 22 °C under a 12 h light/dark cycle with ad libitum access to food and water.

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MATERIALS

Powders of GRT1 and GRT2 were provided by Grünenthal; the purity of all batches used in this study was evaluated to be equal to or greater than 95%. Acetonitrile and formic acid were purchased from Merck (Darmstadt, Germany). The water was purified using a Milli-Q system (Millipore, Bedford, Massachusetts). Brain Slice Assay. The volume of distribution of unbound GRT1 and GRT2 in rat brain (Vu,brain, mL·g brain−1), describing the ratio between total amount of drug per gram of brain in relation to unbound brain (buffer) concentration (eq 1), was estimated using the brain slice method according to previously published protocols.11,14 Six 300 μm brain coronal slices (from area of rostral striatum) were obtained from each rat brain (n = 3) using a vibrating blade microtome Leica VT1200 (Leica Microsystems AB, Sweden). The six slices were incubated in 15 mL of HEPES-buffered artificial extracellular B


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Molecular Pharmaceutics Table 1. Structure, Physicochemical Properties, and PLD Predictions for GRT1 and GRT2

The Ploemen model predicts a PLD risk if (cLogP)2 + (pKa − MB)2 ≥ 90, given that pKa ≥ 8 and cLogP ≥ 1. bThe Tomizawa model predicts a high PLD risk if net charge at pH 4 is >1 and ≤2. cThe Slavov model predicts a toxicophore for PLD with an amino group 3.5−7.5 Å away from the centroid of an aromatic ring as the main requirement. The presence of a second aromatic ring at a distance of 4−5 Å from the first ring and a distance between 5.5 and 7 Å from the amino group is also associated with a PLD positive score. The distance between the positively charged amino group and the first aromatic phenyl ring of GRT1 and GRT2 is 3.8 Å with a second aromatic ring separated by 6.5 Å from the first ring and at a distance of 6.1 Å from the amino group. dThe Goracci model is in good agreement with the Slavov model with some refinements with regard to pKa and steric hindrance around the basic amino group together with a more general extended hydrophobic interaction field for the second aromatic ring. e The Hanumegowda model predicts a PLD induction risk if pKa × cLogP × Vd ≥ 180, given that cLog ≥ 2. a

fluid, pH 7.4 (129 mM NaCl, 10 mM glucose, 3 mM KCl, 1.4 mM CaCl2, 1.2 mM MgSO4, 0.4 mM, K2HPO4, 25 mM HEPES, and 0.4 mM ascorbic acid) containing both GRT2 and GRT1 with initial concentrations of 200 nM. After 5 h incubation at 37 °C in an incubated shaker MaxQ4450 (Thermo Fisher Scientific, NinoLab, Sweden) with a rotation speed of 45 rpm and constant oxygen flow, the buffer and brain slices were sampled. To match the matrix of the brain slice samples, 200 μL of buffer was mixed with 200 μL of blank brain homogenate (1:4, w:v). The brain slices were individually removed, dried on filter paper, and weighed. The slices were individually homogenized in 9 volumes (w:v) of aECFbrain with an ultrasonic processor (VCX-130; Sonics, Chemical Instruments AB, Sweden). All samples were stored at −20 °C pending LC−MS/MS analysis. The viability of the brain slices was assessed using a dynamic pH measurement and detection of lactate dehydrogenase (LDH) activity using a cytotoxicity detection kit (Roche Diagnostics GmbH, Germany).14

The key assumption is that, at equilibrium, the concentration of the compounds in virtually protein free buffer is equal to the interstitial fluid concentration in the brain slice. The Vu,brain (mL·g brain−1) was estimated using eq 1 as a ratio of the amount of compound in the brain slice (Abrain, nmol·g brain−1) to the measured final buffer concentration (Cbuffer, μmol·L−1). Vu,brain =

Abrain − VC i buffer C buffer(1 − Vi )

(1)

where Vi (mL·g brain−1) is the volume of the surrounding brain slices layer of aECFbrain. A Vi of 0.094 mL·g brain−1 was used in the calculations, as obtained using the marker [14C] inulin by Fridén et al.11 Lung Slice Assay. The volume of distribution of unbound GRT1 and GRT2 in rat lungs (Vu,lung, mL·g lung−1) was assessed using the recently developed lung slice method.10 The lungs were removed after having been inflated with a 37 °C solution of 1.5% low melting point agarose in saline (Apollo C


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volumes of PBS, both using an ultrasonic processor VCX-130 (Sonics, Chemical Instruments AB, Sweden). A Teflon 96-well plate (model HTD96b, HTDialysis LLC, Gales Ferry, CT, USA) with cellulose membranes (molecular weight cutoff 12− 14 kDa) was used in the tissue binding experiments. Plasma, brain, and lung homogenates were spiked with either GRT1 or GRT2 to final concentrations of 1−5 μM. The spiked diluted tissue homogenates (tissue side) were dialyzed against equal volumes of PBS pH 7.4 (buffer side) for 6 h at 37 °C. At the end of the experiment samples were taken from both tissue and buffer sides. Compound recovery and stability in respective matrices were tested in each experiment. To minimize the occurrence of potential matrix effect interference during the LC−MS/MS analysis, 50 μL samples from the tissue side were mixed with equal volume of PBS and the samples from the buffer side were mixed with the respective tissue homogenate in the same manner. Samples were stored at −20 °C pending bioanalysis. The fraction of unbound drug in plasma (f u,plasma) was assessed as the buffer to plasma concentration ratio. Calculation of f u,brain and f u,lung was accompanied by an additional step accounting for the dilution of the tissue using eq 4.10,16

Scientific, Manchester, U.K.) via the trachea. After removal, the lungs were immediately immersed in ice-cold HEPES-buffered artificial extracellular fluid, pH 7.4 (125 mM NaCl, 10 mM glucose, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, and 25 mM HEPES). Five hundred micrometer thick lung slices were obtained by using a vibrating blade microtome Leica VT1200 (Leica Microsystems AB, Sweden). Three slices (n = 3 biological replicates per compound) were incubated in 15 mL of buffer containing either GRT1 or GRT2 with an initial concentration of 100 nM. After 5 h incubation at 37 °C in an incubated shaker (MaxQ4450 Thermo Fisher Scientific, NinoLab, Sweden) with a rotation speed of 45 rpm, the buffer and lung slices were sampled. To match the matrix of the lung slice samples, 200 μL of buffer was mixed with 200 μL of blank agarose filled lung homogenate (1:4, w:v). The lung slices were individually removed, dried on filter paper, and weighed, and thereafter homogenized in 9 volumes (w:v) of buffer with an ultrasonic processor (VCX-130; Sonics, Chemical Instruments AB, Sweden). All samples were stored at −20 °C pending LC− MS/MS analysis. The viability of the lung slices was assessed using a dynamic pH measurement and evaluation of the activity released LDH. Assuming that at equilibrium the concentration of the compounds in virtually protein free buffer is equal to the interstitial fluid concentration in the lung slice, the Vu,lung (mL·g lung−1) was estimated using eq 2 as a ratio of the amount of compound in the lung slice (Alung, nmol·g lung−1) to the measured final buffer concentration (Cbuffer, μmol·L−1). Vu,lung =

fu,brain or lung =

where Vi (mL·g lung−1) is the volume of the surrounding lung slices layer of buffer. A Vi volume of 0.73 mL·g lung−1 was used.10 In Vitro Modulation of Drug Distribution in Brain and Lung Slices by Monensin. In order to investigate the contribution of pH partitioning on distribution of the compounds in the brain and lungs, the slices were incubated for 5 h with monensin. To study the pH partitioning effect on distribution of GRT1 and GRT2 in brain slices, only 50 nM monensin was studied, as higher concentrations caused a dramatic decrease in viability.15 Due to the less cytotoxic effect of monensin on lung tissue, the selected concentration range was 0.05−500 nM for the lung slices.10 Thereafter, Vu,brain and V u,lung of GRT1 and GRT2 were assessed using the experimental procedures described above. The difference between Vu,brain or Vu,lung assessed in physiological conditions and in the presence of monensin allowed an indirect evaluation of the contribution of acidic subcompartments referred as lysosomes to the distribution of compounds (eqs 3a and 3b). (3a)

Vu,lung,lyso = Vu,lung − Vu,lung,monensin

(3b)

1

− 1) +

1 D

(4)

where f u,brain or lung,D is the fraction of unbound drug in 10- or 5fold diluted (D) homogenate of brain or lung, respectively. Pharmacokinetic Studies. Systemic PK parameters were determined after intravenous or oral administration of GRT1 and GRT2 to Sprague−Dawley rats (n = 12) using 10% DMSO/5% Cremophor EL in 5% dextrose as a vehicle (Table 2). Plasma (100 μL) was sampled at 0.25, 0.5, 1, 2, and 4 h after

(2)

Vu,brain,lyso = Vu,brain − Vu,brain,monensin

(f

u,brain or lung, D

Alung − VC i buffer C buffer(1 − Vi )

1 D

Table 2. Histological Observations in (a) Lung and (b) Brain Tissues Obtained from Wistar Rats after 14 Day Repeated Oral Administration of 100 mg/kg of GRT1 or GRT2 and the Respective Controlsa GRT1

GRT2

in vivo finding

control group

treated group

control group

treated group

flocculent material foamy macrophages inflammatory infiltrate alveolar wall hypertrophy

1 0 0 0

(a) Lung 7 10 7 6

0 0 0 0

0 0 0 0

flocculent material foamy macrophages inflammatory infiltrate alveolar wall hypertrophy

0 0 0 0

(b) Brain 0 0 0 0

0 0 0 0

0 0 0 0

a

Group size was 5 males and 5 females. Severity ranged from 0 to 5 (not shown).

Drug Tissue Binding Assay. Equilibrium dialysis was used to assess the fraction of unbound GRT1 and GRT2 in plasma ( f u,plasma ), in brain homogenate ( f u,brain) and in lung homogenate ( f u,lung). The reusable Multi-Equilibrium dialysis system (Harvard Apparatus UK, Cambridge, U.K.) was used for measurement of plasma protein binding. Brain tissue was homogenized in 9 volumes of phosphate buffered saline (PBS), pH 7.4, and the agarose filled lung tissue was homogenized in 4

administration. Blood samples were centrifuged and plasma samples were stored at −18 °C pending analysis using LC− MS/MS. The in vivo brain and lung distribution experiments were part of larger plasma pharmacokinetic and tissue distribution studies. Kp,brain and Kp,lung were determined as ratios of the D


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respective compartments such as ISF, cytoplasm, and lysosomes. The ratios of cytosolic to extracellular unbound drug concentrations (Kp,uu,cyto) and lysosomal to cytosolic unbound drug concentrations (Kp,uu,lyso) for dibasic compounds based on Henderson−Hasselbalch equation were calculated for brain and lung as

areas under the curve (AUC0−t) of total tissue and plasma concentrations after intravenous administration of 0.1 mg/kg GRT1 and GRT2 to Sprague−Dawley rats. Blood and brain/ lungs were sampled at 30, 90, and 180 min after administration. At the designated time points, the rats (n = 6, for each compound) were anesthetized and blood samples were immediately collected into 10 mL BD K3EDTA vacutainers (BD Biosciences, Plymouth, U.K.). Subsequently, animals were sacrificed, and the brain and lungs were rapidly removed and homogenized in buffer (1:4, w:v). Plasma, brain, and lung homogenate samples were stored at −18 °C pending bioanalysis. To describe the extent of unbound compound transport across the blood−brain barrier (BBB) into the brain interstitium and across the blood−alveolar barrier (BAB) into the lung interstitium, Kp,uu,brain and Kp,uu,lung parameters were assessed using eqs 5 and 6, respectively. K p,uu,brain =

K p,uu,lung =

(5)

K p,lung Vu,lungfu,plasma

(6)

Kp,uu,brain or lung values closer to unity describe a mainly passive transport at the BBB/BAB or reflect similar efflux and influx clearances. Kp,uu,brain or lung values smaller than unity indicate predominantly active efflux, and values exceeding unity indicate potential active uptake.13 Tissue-specific unbound concentrations in the interstitial fluid (ISF), Cu,brainISF or lungISF were assessed using eq 7. The key assumption was that Kp,uu,brain, Kp,uu,lung, and Kp,uu,cell parameters are dose-independent. Cu,brainISF or lungISF = K p,uu,brain or lungCu,plasma

(7)

Assessment of unbound drug partitioning into the cells was performed using eq 8.9,10 K p,uu,cell,brain or lung = Vu,brain or lungfu,brain or lung

10 pKa,1 − pHcyto + 1 10 pKa,2 − pHcyto + 1 · 10 pKa,1 − pH ISF + 1 10 pKa,2 − pH ISF + 1

(10)

K p,uu,lyso =

10 pKa,1 − pH lyso + 1 10 pKa,2 − pH lyso + 1 · 10 pKa,1 − pHcyto + 1 10 pKa,2 − pHcyto + 1

(11)

For brain tissue pHcyto 7.06, pHISF 7.3, and pHlyso 5.18 were reported by Fridén and co-workers.15 For lung tissue the following physiological values obtained from the literature were used: pHcyto 7.17,17 pHISF 7.4,17 and pHlyso 5.1.18 pKa values were experimentally determined using a SiriusT310026 system at Sirius Analytical (East Sussex, U.K.). Assessment of Drug Induced Phospholipidosis. In Vitro High Content Screening for PLD. Cell health parameters, PLD and steatosis were simultaneously assessed via a multiparametric approach using high content screening (HCS).19 In the current manuscript only data on PLD is presented. The assay measures the relative accumulation of phospholipids and lipids within lysosomes and within the cell cytoplasm across treated and vehicle control treated cells. Briefly, HepG2 cells were plated on 96-well tissue culture treated black walled clear bottomed polystyrene plates at 0.6 × 104 cells in 100 μL per well. After 24 h the cells were dosed with test compound at 0.04, 0.1, 0.4, 1.0, 4.0, 10, 40, and 100 μM. A single dilution curve in DMSO was prepared and was spiked into three wells for each concentration. However, these were not truly independent data as no standard deviation from mean (STD) of the parameter was reported. R2 was used as an indicator of how close the curve fits the data for the determined MEC and EC50. Accumulation of phospholipids was investigated in the absence and presence of a mixture of polychlorinated biphenyl Aroclor-1254 rat liver S9 fraction. Sertraline, clomipramine, and desipramine were used as positive controls. During the 24 h incubation period in the presence of the two drugs, the cells were simultaneously loaded with LipidTox Red (Invitrogen, Germany) as a fluorescent probe dye to detect PLD.20 The plates were then scanned using an automated fluorescence cellular imager ArrayScanr VTI (Thermo Scientific Cellomics, Darmstadt, Germany). Minimum effective concentration (MEC) that significantly passed vehicle control threshold and the concentration at which 50% maximum effect is observed for each cell health parameter (EC50) are reported. In Vivo Toxicokinetic Studies and Histological Examination. In vivo PLD was assessed in toxicological studies with once daily oral (gavage) administration to Wistar rats for a period of 14 days, followed by a 14-day treatment-free recovery period. The animals received compound GRT1 or GRT2 at doses of 0, 25, 50, or 100 mg/kg/day and 0, 10, 30, or 100 mg/ kg/day, respectively. The vehicle control group animals received 50 mM sodium acetate buffer (for GRT1) or 5% (m/v) aqueous hydroxypropylmethylcellulose (for GRT2) without the test item, following the same administration pattern as the groups treated with the test item. In addition, there were 9 animals per sex for the toxicokinetic investigation.

K p,brain Vu,brainfu,plasma

K p,uu,cyto =

(8)

Kp,uu,cell describes the steady state relationship of unbound compound intracellular to extracellular concentrations and indicates the average concentration ratio for all cell types within the brain or lungs. Cu,brain or lungISF was further used for evaluating intracellular fluid (ICF) unbound concentrations in brain parenchymal and pulmonary alveolar cells (Cu,brainICF or lungICF) using the respective Kp,uu,cell values, as shown in eq 9. Cu,brainICF or lungICF = K p,uu,cell,brain or lungCu,brainISF or lungISF (9)

The theoretical calculation of intracytosolic and intralysosomal concentrations based on pH partitioning theory was applied.15 The ionization stage is pH-dependent and driven by a physiological pH gradient between ISF (pH ∼ 7.4), cytoplasm (pH ∼ 7.3), and acidic subcellular compartments such as lysosomes (pH ∼ 5). It is well-known that the concentration of nonionized drug is equal on both sides of a cell membrane at equilibrium, assuming that the nonionized species are efficiently crossing the membrane. A three-compartment pH-partitioning model of Kp,uu,cell was first applied for the brain tissue15 including the pKa values of the compound, physiological volumes, and the pH of the E


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Molecular Pharmaceutics At the end of the treatment period, all surviving animals were necropsied and examined post-mortem. Histological examinations were performed on a limited set of organs and tissues such as brain and lungs. Necropsies and histological preparation were performed at Grünenthal GmbH, Aachen, Germany. The resulting sections were stained with hematoxylin and eosin, sent to AnaPath GmbH Oberbuchsiten, Switzerland, and examined by light microscope. Bioanalysis of Samples. Reversed-phase liquid chromatography followed by detection with a tandem mass spectrometer (LC−MS/MS) equipped with electrospray ionization and operating in positive ion mode was used for bioanalysis. Quantification was performed using multiple reaction monitoring mode to monitor parent > product ion (m/z) transitions with appropriate mass. Instrument settings and potentials were adjusted to optimize the mass spectrometer signal for each analyte. The source-dependent parameters maintained for compounds were as follows: capillary voltage 1.5 kV, source temperature 130 °C, desolvation temperature 400 °C, cone gas flow 40 L/h, and desolvation gas flow 1000 L/h. Prior to analysis, all frozen samples, blanks, and calibration standards were thawed and allowed to equilibrate to room temperature. One hundred fifty microliters of ice-cold 0.2% formic acid in acetonitrile was added to an aliquot of 50 μL of sample. Samples were then vortexed for 20 s and centrifuged at 10000g for 3 min at room temperature. One hundred fifty microliters of the supernatant was mixed with 300 μL of mobile phase A (0.1% formic acid). After vigorous vortexing followed by centrifugation at 10000g for 1 min, 40 μL was injected into a HyPurity C18, 3 μm particle size column with a precolumn made of the same material (Thermo Scientific, Dalco Chromatech, Sweden). Gradient elution with mobile phases A and B (a mixture of 90:10:0.1 acetonitrile:water:formic acid) was used. Individual standard curves were prepared in the respective control matrices. Appropriate dynamic ranges were achieved for the assays. Analyte concentrations in unknown samples were evaluated using linear regression models. Data quantification was performed using the Masslynx 4.1 software (Micromass, Manchester, U.K.). Data Analysis. Statistical analysis of data was performed using GraphPad Prism 6.04 for Windows (GraphPad Software, San Diego, California, USA). A D’Agostino−Pearson normality test was performed, where p > 0.05 indicated that the data passed the normality test. Descriptive statistics are presented as mean and standard deviation (mean ± SD) or standard error of mean (mean ± SEM). Standard deviation of the pharmacokinetic parameters Kp,brain, Kp,lung, Kp,uu,brain, Kp,uu,lung, and Kp,uu,cell was assessed using propagation of uncertainty. Because the Kp,brain, Kp,lung, Kp,uu,brain, Kp,uu,lung, and Kp,uu,cell pharmacokinetic parameters were not results of direct single measurement but obtained in two or three steps, the assessment of uncertainty also involved those steps.21 Propagation of uncertainty was assessed for both product and quotient of two variables, A and B, using the following equations. Propagation of uncertainty of Kp,brain, Kp,lung, and Kp,uu,cell was performed using the product rule. Let A and B be variables with standard deviations σA and σB and set f = A·B

σf ≈ |f |

σAB ⎛ σA ⎞2 ⎛ σB ⎞2 ⎜ ⎟ + ⎜ ⎟ + 2 ⎝A⎠ ⎝B⎠ AB

(13)

The covariance σAB was then calculated with help of the correlation r as σAB = rσAσB. Propagation of uncertainty of Kp,uu,brain and Kp,uu,lung was performed using the quotient rule. Let A and B be variables with standard deviations σA and σB and set

f=

A B

(14)

Propagated uncertainty for a quotient, i.e., the standard deviation of f, was then calculated as follows: σf ≈ |f |

σAB ⎛ σA ⎞2 ⎛ σB ⎞2 ⎜ ⎟ + ⎜ ⎟ − 2 ⎝A⎠ ⎝B⎠ AB

(15)

As above, the covariance was calculated as σAB = rσAσB. Taking into consideration the innate correlation between the variables, |r| = 0.5 was assumed in all formulas. Negative correlation, i.e., r = −0.5 is present between Vu,brain/Vu,lung and f u,plasma as well as f u in respective tissue while all other parameters were positively correlated, i.e., r = 0.5.

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RESULTS Evaluation of Drug Induced Phospholipidosis. Prediction of PLD Induction Risk by Published in Silico Models. The risk of PLD induction was determined with five well described in silico and toxicophore models (see Table 1 for model predictions for GRT1 and GRT2, together with details for the risk categories in the different models). The predictions by these in silico models all indicate similar PLD induction potential for the two compounds and cannot discriminate between GRT1 and GRT2. This failure of the current models to predict the in vivo PLD can be easily explained by the fact that the two compounds share the same first basic amino group and lipophilic aromatic ring system, but only differ slightly in the pKa value of the second basic amino group. In Vitro Findings Related to Phospholipidosis. The in vitro model of Ceccarelli is based on the distribution coefficient between aqueous phase and porcine brain polar lipid extracts.22 Due to lack of intact intracellular organelles, this model cannot detect lysosomal accumulation and was not further considered for the investigation of GRT1 and GRT2. In vitro PLD was determined by measuring the accumulation of the specific fluorescent dye LipidTox Red in HepG2 cells following treatment for 24 h in the absence and presence of rat liver S9 fraction. In the presence of GRT1, accumulation of the fluorescent dye could be observed with a MEC of 0.513 and 0.395 μmol/L in the absence and presence of metabolic activation by S9 fraction, respectively (Table S1). In contrast, GRT2 did not show any accumulation of LipidTox Red in the absence of metabolic activation. In the presence of the S9 fraction, some dye accumulation was observed with a MEC of 7.76 μmol/L, which was more than 10-fold above the corresponding value seen with GRT1. The in vitro data suggest that the risk of seeing PLD in vivo is much lower for GRT2 than for GRT1. In Vivo Findings Related to Phospholipidosis. Compound GRT1 caused lesions in the lungs and several other organs, which are consistent with phospholipidosis. In contrast to the normally appearing reactive alveolar macrophages, there were finely vacuolated macrophages in the alveoli tending to form

(12)

Propagated uncertainty for a product, i.e., the standard deviation of f, was then calculated as follows: F


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Table 3. Pharmacokinetic Parameters (Mean ± SD) of Plasma, Brain, and Lung Exposure in Rats after Intravenous Administration of 0.1 mg/kg GRT1 or GRT2 Combined with Parameters Describing Intrabrain and Intralung Tissue Distributiona

groups. These macrophages tended to be larger than their normal counterparts. The macrophages were diffusely distributed over the lung with a trend to a denser accumulation at subpleural locations (Figure 2). In most animals, this was

parameters

Figure 2. Representative micrographs from histopathological investigation of lung tissue after 14-day repeated oral administration of respective vehicle (A) and 100 mg/kg of GRT1 (B). (A) Alveolar spaces empty except for single alveolar macrophages (→), hematoxylin and eosin, lens ×40. (B) Alveolar spaces filled with increased number of foamy macrophages. Cells are enlarged with a finely vacuolated cytoplasm and arranged separately or in small nests, hematoxylin and eosin, lens ×40. No phospholipidosis was detected in the brain tissue after 14-day repeated oral administration of 100 mg/kg of GRT1 as well as in the brain and lung tissues after 14-day repeated oral administration of respective vehicles or 100 mg/kg of GRT2 in Wistar rats (micrographs are not shown).

unit

AUCplasma f u,plasma

ng·mL−1·h−1

AUCbrain Vu,brain f u,brain Kp,brainb Kp,uu,brainb Kp,uu cell,brainb Kp,uu,cytoc Kp,uu,lysoc

ng·g−1·h−1 mL·g brain−1

AUClung Vu,lung f u,lung Kp,lungb Kp,uu,lungb Kp,uu cell,lungb Kp,uu,cytoc Kp,uu,lysoc

ng·g−1·h−1 mL·g lung−1

GRT1 Plasma 52.6 ± 12.5 0.0539 ± 0.0137 Brain 276 ± 18.4 1043 ± 59.6 0.0037 ± 0.00043 5.25 ± 1.11d 0.093 ± 0.0194d 3.86 ± 0.39d 2.20 2908 Lung 2794 ± 192 2004 ± 279 0.011 ± 0.00070 53.1 ± 11.3d 0.492 ± 0.0982d 22.0 ± 2.66d 2.07 6082

GRT2 2.36 ± 0.250 0.247 ± 0.0952 30.9 ± 1.91 50.3 ± 2.58 0.052 ± 0.021 13.1 ± 1.21d 1.05 ± 0.361d 2.62 ± 0.996d 1.73 78 193 ± 16.1 97.6 ± 21.6 0.127 ± 0.031 81.8 ± 7.91d 3.39 ± 1.11d 12.4 ± 2.89d 1.69 122

Data presented as a mean ± standard deviation of the mean obtained minimum in three independent experiments. bParameters calculated on the basis of the mean values using combinatory mapping approach.12 cPredicted parameters. dStandard deviation is calculated on the basis of the propagation of uncertainty (see Experimental Section). a

associated with inflammatory cell infiltrate. Furthermore, there were reactive changes mainly at the terminal sacs that took the form of alveolar wall hyperplasia/hypertrophy, indicating the recruitment of macrophages. In addition, there was an increased incidence and severity of a flocculent material within the alveolar spaces that is considered partly released from ruptured macrophages, representing remnants of aspirated material, of which the exact source was not possible to be established by light microscopy (Tables 2, S2). There was a trend toward recovery during the treatment-free period. In contrast to the findings described above, the histopathological evaluation of the same tissues of the animals treated with compound GRT2 revealed no morphological lesions that were different from the vehicle control animals, and, therefore, it was considered not related to phospholipidosis or to the compound. Assessment of Lysosomal Contribution to in Vitro Intracerebral and Intrapulmonary Intracellular Distribution. The overall uptake into brain and lung tissue slices, incorporating possible active uptake, binding, and pH partitioning, as described with Vu,brain or lung (eq 1 and eq 2), was 1043 and 2004 mL·g−1 for GRT1 and 50.3 and 97.6 mL· g−1 for GRT2, respectively (Table 3). Comparing lung and brain uptake, the lung uptake was almost 2-fold higher than the uptake into brain tissue for both drugs. Comparing the two compounds, the uptake of GRT1 was 21-fold higher than that of GRT2 in both brain and lung. Estimation of cellular uptake (eq 8) showed that both GRT1 and GRT2 accumulate intracellularly (Table 3). GRT1 exhibited very high intracellular accumulation, which was revealed by a Kp,uu,cell value of 3.9 in brain and an even higher value of 22 in lungs. Decreasing the basicity of the second amino group in GRT2 was associated with reduced lysosomal trapping, demonstrated by Kp,uu,cell values of 2.6 in brain and 12.4 in lungs. Thus, despite the high similarity in structure

GRT1 revealed higher binding and cellular accumulation compared to GRT2. Using a modification of the Henderson−Hasselbach equation (eqs 10 and 11) it was possible to predict the extent of accumulation of the dibasic GRT1 and GRT2 in the cytosolic and lysosomal compartments (Table 1). The ratio of cytosolic to extracellular unbound drug concentrations, Kp,uu,cyto (eq 10) was similar between the compounds and tissues (Table 2). However, the ratio of lysosomal to cytosolic unbound drug concentrations, Kp,uu,lyso (eq 11), was for GRT1 2908 and 6082, and for GRT2 78 and 122 in brain and lung, respectively (Table 3). To understand the role of lysosomal trapping for the tissue distribution of drugs, bafilomycin A1, which is a specific inhibitor of V-ATPase, and the less selective monensin, which uncouples the proton gradient present in lysosomes, have been used by several groups.23−25 Incubation of tissue slices with monensin showed that lysosomal trapping contributes the most to the intracellular accumulation of GRT1 (Figure 3). The Vu,lung of GRT1 decreased 6-fold with increasing concentrations of monensin to 325 mL·g lung−1 at 500 nM monensin, while the Vu,lung of GRT2 dropped 4.5-fold. The contribution of lysosomal accumulation of GRT1 in pulmonary alveoli, presented as Vu,lung,lyso (eq 3b), was 1678 mL·g lung−1, i.e., contributing with 84% to the distribution, while that of GRT2 was 22-fold lower, 76 mL·g lung−1, contributing with 78% to the distribution. The impact of monensin on Vu,brain was smaller for both compounds (Figure 3B). Vu,brain,lyso (eq 3a) was 290 G


Article


Figure 3. Modulation of the unbound volume of distribution of GRT1 (blue circles) and GRT2 (red squares) in lungs (Vu,lung) (A) and brain (Vu,brain) (B) with increasing concentrations of monensin. The half-filled symbols represent Vu recalculated from lung and brain homogenates (1/ f u,lung or 1/f u,brain), where 1/f u,lung or 1/f u,brain reflects only tissue binding properties of compounds in the absence of active intracellular uptake. As the homogenates lose cellular integrity and physiological pH gradients, these Vu numbers represent the lowest possible values. Due to its cytotoxic effect, only concentrations up to 500 nM (lung) and 50 nM (brain) were used. It can be observed that 1/f u,lung is somewhat smaller than Vu,lung in the presence of 500 nM monensin, and 1/f u,brain is smaller than Vu,brain at 50 nM monensin. This indicates that monensin at the concentrations applied was not able to fully inhibit uptake into acidic subcompartments such as lysosomes and/or that other possible active uptake processes are present. NB: Observe the differences in the scales of the Y-axes for lung and brain.

mL·g brain−1 (28%) and 11.5 mL·g brain−1 (23%) for GRT1 and GRT2, respectively. Thus, the lysosomal accumulation in brain was less profound than that in the lung. Evaluation of Unbound Intracellular Concentrations in Vivo. The total maximal plasma concentrations (Cmax) assessed at day 14 after daily oral administration of 100 mg/kg of GRT1 or GRT2 (Table 4) were used to estimate the achieved unbound intracellular concentrations in brain and lungs. After correction for plasma protein binding, the Cmax values were sequentially used for calculation of unbound interstitial concentrations (eq 7).

First, tissue distributional studies revealed high brain and lung total concentrations in relation to the total plasma concentrations (Table 3). GRT1 had 10-fold higher drug partitioning coefficient into the lungs than into the brain. Similarly, GRT2 showed 6.2-fold higher Kp,lung compared to Kp,brain. Second, assessment of Kp,uu,brain revealed that GRT1 is efficiently effluxed at the BBB (Kp,uu,brain of 0.093) while GRT2 has Kp,uu,brain of 1.05, indicating predominant passive diffusion across the BBB. Kp,uu,lung was 0.49 and 3.39 for GRT1 and GRT2, respectively, which will have a propagating influence on the concentrations achieved also intracellularly. Estimation of unbound concentrations in the intracellular fluid (eq 9) showed that GRT1 achieved 1.2 μM and 0.05 μM, while GRT2 reached 30 μM and 2 μM in the lungs compared to the brain (Figure 4). Assessment of unbound lysosomal concentrations based on Kp,uu,cyto and Kp,uu,lyso revealed extensive accumulation of both compounds in the lysosomes, that was more pronounced in the lung tissue. Remarkably, the lysosomal concentration of GRT1 in the lung tissue was 5872fold higher than its unbound plasma concentration, while GRT2 had a 698-fold higher lysosomal concentration in relation to its unbound plasma concentration (Figure 4).

Table 4. Pharmacokinetic Parameters (Mean ± SEM) Obtained after Oral Administration of GRT1 and GRT2 to Male and Female Wistar Rats GRT1 day

parameters

25 mg/kg

50 mg/kg

100 mg/kg

1

Cmax [ng/mL] Tmax [h] AUC0−t [ng/mL × h] Cmax [ng/mL] Tmax [h] AUC0−t [ng/mL × h]

135 ± 37.9 1.00 782 ± 156

175 ± 45.5 1.00 1560 ± 244

336 ± 201 1.50 4180 ± 1820

240 ± 122 1.00 1560 ± 451

289 ± 64.8 1.50 3810 ± 660

707 ± 3.35 3.50 14200 ± 768

14

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DISCUSSION In a preclinical CNS drug development program focused on identifying compounds with a sufficient CNS exposure and a safe profile, two structurally related diamines GRT1 and GRT2 were explored. GRT1 induced severe tissue-specific side effects in a 2-week rat toxicity study, primarily in the lungs and liver, which were attributed to PLD. At similar doses of GRT2, no toxicity findings related to PLD were observed. None of the compounds induced PLD in the CNS. By assessing in vivo intracellular and lysosomal unbound concentrations of GRT1 and GRT2 in brain and lung tissues with the novel combinatory mapping approach,12 a pharmacokinetic link in the development of PLD was established.

GRT2 day 1

14

param Cmax [ng/mL] Tmax [h] AUC0−t [ng/mL × h] Cmax [ng/mL] Tmax [h] AUC0−t [ng/mL × h]

10 mg/kg

30 mg/kg

100 mg/kg

39.6 ± 9.61 1.00 62.2 ± 9.74

306 ± 58.5 1.00 633 ± 82.7

839 ± 153 1.50 3764 ± 345

59.0 ± 11.7 1.00 121 ± 18.7

505 ± 95.8 1.00 1697 ± 180

1168 ± 118 1.00 7254 ± 316

H


Article


Figure 4. Comparison of achieved unbound concentrations of GRT1 and GRT2 in plasma, interstitial fluid, intracellular fluid, and acidic subcompartments referred as lysosomes (predicted) in brain parenchyma and pulmonary alveoli after 14-day repeated oral administration of 100 mg/kg of GRT1 and GRT2 in Wistar rats. The interfaces between blood and interstitial fluid in brain and lung are the blood−brain barrier (BBB) and the blood−alveolar barrier (BAB). The cellular barriers (CB) represent the cellular membrane of average brain parenchymal cells (neurons, astrocytes, pericytes, microglia, etc.) and lung parenchymal cells (type I pneumocytes, type II pneumocytes, interstitial and alveolar macrophages, pericytes, fibroblasts, etc.). Mean maximal total plasma concentrations of GRT1 and GRT2 were 707 ng/mL (1939 nM) and 1168 ng/mL (2865 nM), n = 18. Lysosomal concentrations are calculated based on pH partitioning theory using eqs 10 and 11 and indicated in red. For comparison, the detected minimum effective concentration in in vitro induced PLD model for GRT1 was 0.513 μM, while GRT2 was classified as a nonresponder (Table S1).

Drug Disposition in Tissues and Impact of TissueSpecific Characteristics on PLD. The integration of PLD induction potential and drug disposition in tissues can help to understand the risk of in vivo PLD manifestation (Figure 1). Lysosomes have a central role in PLD pathogenesis that occurs in lysosome-rich organs such as the lungs, liver, and kidneys and in the brain.26 Susceptibility to PLD may exist in the organspecific structural and functional differences, as exemplified below by a comparison of lysosomal trapping capacity and protective barrier involvement in lungs and brain. In lungs, alveolar and pulmonary interstitial macrophages belong to the lysosome-rich cell types.27 Both types of macrophages have similar lysosomal pH around 5, and both can contribute to the lysosomal accumulation of substances.18,28 Compared to lung tissue, brain is considered to have less lysosomal capacity. This was reflected in considerably lower intrabrain distribution (Vu,brain) and cellular uptake (Kp,uu,cell) for both compounds (Table 3). The estimation of density and fractional volume of lysosomes in the entire pulmonary tissue is so far complicated. However, in analogy with the liver one can assume that lysosomes contribute to about 1% of average lung cells.29 In spite of such a small physiological volume, more than 86% of the estimated total amount of GRT1 in rat lung tissue was associated with lysosomes (Figure 4), taking into account that compartmental volume of interstitial space is only 11.6% in alveolar septal lung tissue.27 In contrary, only 16% of estimated total amount of GRT2 in lungs was accumulated into acidic subcompartments. However, the information on tissue-specific lysosomal content and lysosomal pH is not sufficient to understand organ-specific differences in PLD induction. The barrier role of membranes must be taken into account for the evaluation of unbound concentrations in the interstitium in respect to plasma. It is well acknowledged that the brain is protected by the BBB, limiting drug transport into the interstitial fluid.9,13,15 The defensive role of the BBB is also reflected in the fact that

PLD is quite rarely described for CNS. For instance, PLD in the brain of rats treated with chloroquine and quinacrine was only documented in circumventricular areas where the BBB is leaky, suggesting that neural cells might not have an intrinsic resistance to PLD, but rather that they do not obtain sufficient concentrations to induce PLD.26 In this regard the assessment of Kp,uu,brain (eq 5) is very instrumental.12,13 In spite of a very high total brain to plasma concentration ratio (Table 3), GRT1 had very low extent of BBB transport reflected in a Kp,uu,brain of 0.0985, i.e., only 10% equilibrating into the brain across the BBB as unbound drug. In contrast, GRT2 equilibrated with a BBB ratio of unity. Together with the assessment of unbound intracellular exposure, this suggests that the concentration of GRT1 in the brain was not sufficient to induce PLD. Drug distribution into the pulmonary interstitium and alveolar cells is less studied compared to into the brain. Blood and pulmonary interstitium are separated by the endothelium. It is well established that pulmonary endothelium and epithelium separated by a thin basement membrane form BAB.30 The lung endothelium consists of nonfenestrated continuous endothelial cells.31 Barrier function of this monolayer is regulated by cell−cell and cell−extracellular matrix adhesion.32,33 The alveolar and capillary walls delimit the pulmonary interstitium, a thin compartment where fluid is freely moving within the fibrous extracellular matrix drained by lymphatics. In this state the thin side of the BAB remains almost “dry”.34 Except for the thin side of BAB, the pulmonary cellular interstitium also contains pericytes, fibroblasts, pulmonary macrophages, etc. Deguchi et al. were the first ones to assess unbound interstitial concentrations by using pulmonary microdialysis, studying β-lactams and comparing them to the unbound plasma concentration, proving the validity of the free drug hypothesis for lung drug transport.35 Later, additional pulmonary microdialysis studies have shown evidence on asymmetry in the BAB regarding unbound drug transport of various drugs.36 Herein, assessment of Kp,uu,lung was I


Article


this type of investigation represents a new frontier in drug delivery research, where much can be learned and applied to improve drug efficacy. In this regard, the brain/lung slice assays combined with the brain/lung homogenate method could provide experimental evidence on cellular transport by means of Kp,uu,cell, supporting identification of potential lysosomotropic compounds.9,10,12,15 To our knowledge, the only method for direct evaluation of the accumulation of the compound in the lysosomes is difficult to perform and has never been applied to in vivo studies.8 Hence, only compilation of in vitro and in vivo organ-specific pharmacokinetic parameters, predicted lysosomal concentrations with in vitro and in vivo evaluation of findings indicating PLD induction, allows systematic evaluation of PLD development and its management in individual cases. In summary, the results obtained in the present study highlight the importance of multidirectional methodical investigation of the safety profile of drug candidates. Estimation of extra- and intracellular unbound concentrations by defining the extent of unbound compound transport across the BBB, BAB, and cellular barriers was instrumental in delineating the pharmacokinetic basis of tissue-specific and intercompound differences resulting in better tissue delivery for high in vivo efficacy on one hand and reduced nonspecific lysosomal accumulation with lower risk of in vivo PLD on the other hand.

essential for estimation of the average pulmonary interstitial fluid unbound concentration of GRT1 and GRT2, which indicated possible active uptake of GRT2 across the endothelial cell layer. PLD Induction Potential and Impact of Drug-Specific Characteristics on PLD. Most PLD positive drugs are cationic amphiphilic substances containing hydrophobic ring structures and hydrophilic side chains with cationic amine groups that enable binding to phospholipids, lysosomotropism, etc.3 How lysosomotropism can cause drug-induced toxicity is not well understood. Hypothetically, compounds can inhibit the synthesis of various lysosomal enzymes and/or increase the synthesis of phospholipids.37,38 Numerous in silico and in vitro assays for identification of lysosomotropic compounds have been developed and can be implemented in early preclinical safety evaluations to help identify compound-induced lysosomal dysfunction or lysosomal accumulation.6,8,38,39 Interestingly the common models fail to explain the observed differences in in vivo PLD observed with GRT1 and GRT2 (see Table 1). As was shown in the current study, the use of in vitro HCS using LipidTox Red was very supportive in elucidating the observed paradoxes. Thus, despite a high phospholipidogenic profile of GRT1 (Table 1 and Table S1), the 14-day administration of 100 mg/kg of GRT1 did not cause cerebral PLD. This is likely due to its very effective efflux at the BBB resulting in the interstitial brain concentration being far below the MEC value detected in the LipidTox assay. Yet, PLD was clearly shown in the periphery. Another paradox was that GRT2 administered at the same dose, 100 mg/kg, reached high intracellular and lysosomal concentrations in vivo. This was due to its more efficient brain and lung uptake properties, counteracting the lower lysosomal uptake and resulting in comparable lysosomal concentrations to GRT1 (Figure 4). However, the chronic administration of GRT2 was not associated with PLD, which was supported in the HCS assay identifying the dibasic GRT2 as a safe compound regarding PLD. Hence, the readouts of in vitro PLD assays should be interpreted in relation to the achieved in vivo intracellular concentrations in the respective tissues. Pharmacokinetic Considerations in Management of PLD. Recently, many investigators have begun to realize that, besides intrinsic PLD induction potential, also drug disposition and intracellular distribution of a drug are extremely important determinants of in vivo drug PLD toxicity.3 The volume of distribution Vd was introduced as an in vivo disposition parameter to improve the correlation between in vitro and in vivo PLD data. However, incorporation of Vd did not help to explain the difference between GRT1 and GRT2. Consequently, we were looking deeper into the tissue disposition by calculating absolute in vivo unbound drug concentrations in the target tissues and compartments by the combinatory mapping approach.12 The combinatory mapping approach reveals that Vd like total tissue/plasma ratios Kp are “lump” parameters that do not discriminate between productive unbound tissue delivery resulting in higher efficacy and nonspecific mechanisms, for instance, uptake into lysosomes, increasing the risk of PLD. The combinatory mapping approach helped to dissect the differences between high nonspecific accumulation in lysosomes and PLD as seen with GRT1 and high tissue delivery of unbound drug as seen with GRT2. Despite the fundamental importance, it is not experimentally evaluated in most instances. It is the opinion of the authors that

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00480. Quantitative Assessment of Drug Delivery to Tissues and Association with Phospholipidosis: A Case Study with Two Structurally Related Diamines in Development (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Translational PKPD Group, Department of Pharmaceutical Biosciences, Associate member of SciLifeLab, Box 591, 751 24 Uppsala, Sweden. Tel: +46 18 471 4995; +46 73 891 4982 (mobile). E-mail: [email protected]. ORCID

Irena Loryan: 0000-0002-1557-4416 Achim Kless: 0000-0001-8301-5934 Present Address #

V. Marossek: Bayer AG, Wuppertal, Germany. B. Nolte: Peter Greven Physioderm GmbH, Euskirchen, Germany. A. Wegert: Mercachem, Nijmegen, Netherlands. Author Contributions ‡

I. Loryan and E. Hoppe shared first authorship.

Notes

The authors declare the following competing financial interest(s): E. Hoppe, K. Hansen, A. Kless, K. Linz, V. Marossek, B. Nolte, P. Ratcliffe, D. Saunders, R. Terlinden, A. Wegert, A. Welbers, and O. Will are/were employed by Grunenthal GmbH, Germany. F. Held is employed at the Fraunhofer-Chalmers Centre in Gothenburg, Sweden.

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ACKNOWLEDGMENTS We express our sincere gratitude to Anke Heuser (Grunenthal GmbH) for support with preparation of histological microJ


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Quantitative Assessment of Drug Delivery to Tissues and Association

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