Phospholipidosis (PLD) is a lipid storage disorder characterised by the accumulation of phospholipids within cells, and has been found to be induced by several drugs. Whilst PLD is seen in humans with inherited metabolic disorders such as Niemann-Pick and Gauchers disease. It is also of concern as an adverse side effect of cationic amphiphilic drugs (CADs). Herein we introduce a novel high-throughput physicochemical screen for predicting the PLD-inducing potential of drugs.

1. High-throughput screening assay for assessing phospholipidosis-inducing potential of drugs
Jon Mole1, Karl Box2, John Comer2, Tom Gravestock2, Elizabeth Frake2, Pavol Vitovič3, Paavo Kinnunen3,4, Juha-Matti Alakoskela3
1 Sirius Analytical Inc, Beverly, MA, USA; 2 Sirius Analytical Ltd, Forest Row, East Sussex, UK; 3University of Helsinki, Finland; 4Kibron Inc. Espoo, Finland
Purpose Instrument Screening for PLD-inducing potential
Phospholipidosis (PLD) is a lipid storage disorder characterised by the accumulation of phospholipids Measurements were made using an eight-channel tensiometer The screen for PLD-inducing potential is summarised below. Details of
sample preparation and methodology have been published [1] Lipid
within cells, and has been found to be induced by several drugs. Whilst PLD is seen in humans with (Delta 8, Kibron Inc.). The device determines surface tension from
1. Prepare stock solutions of drugs in DMSO. D/L 0.02:1
inherited metabolic disorders such as Niemann-Pick and Gauchers disease. It is also of concern as an the maximum weight of meniscus that adheres to 0.5mm diameter 2. Prepare lipid in buffer solution. 5.0mM in 20mM HEPES, 0.1mM D/L 0.1:1
adverse side effect of cationic amphiphilic drugs (CADs). Herein we introduce a novel high-throughput probes (called du Noüy probes) as they are pulled out of solution. EDTA, pH 7.4.
D/L 0.5:1
physicochemical screen for predicting the PLD-inducing potential of drugs. Full readout for 96 wells can be obtained in less than two minutes. 3. In a disposable 96-well plate, prepare dilution series in buffer of lipid,
lipid + drug, and drug alone (figure 3). D/L 0.5:1
Methods After the entire tray has been measured, the probes are cleaned
4. Transfer 50µL from each well to special 96-well plate. D/L 1:1
In our novel PLD-screening method we profiled the surface activity of compounds interacting with a by heating in a built-in furnace. 5. Insert special plate in instrument and measure surface tension. D/L 1:1
short chain phospholipid 1,2-dioctanoyl-sn-glycero-3-phospho-l-serine (diC8PS). In order to cause PLD, 6. Determine CMC values from discontinuities in isotherms (examples
Critical Micelle Concentration (CMC) Figure 1. Kibron Delta 8 Microtensiometer in figure 2).
Drug
a drug needs to enter the cell, leading to drug-anionic phospholipid complexation. Therefore the extent 7. Calculate CMCDL/CMCL for each Drug/Lipid ratio. (dividing CMCDL
At low concentrations, surface-active molecules form a monolayer on air/water interfaces, with the
of drug-anionic phospholipid complexation should correlate with its PLD-inducing potency. These by CMCL corrects for the effects of solvent on the CMC values).
hydrophilic parts (e.g. negatively charged) in water and the hydrophobic parts in air. If the concentration
interactions can be studied using surface tension measurements, which were performed using a Kibron 8. Plot CMCDL/CMCL vs. drug/lipid ratio, and determine R½, the Figure 3. Preparation of drug and lipid to
is increased, they pack closer together, and the surface tension decreases. If concentration increases drug/lipid ratio at which CMCDL/CMCL would be half way between its
Delta-8 microtensiometer system. The instrument consists of an 8-channel microbalance which records determine CMCL, and CMCDL at four different
still further, the hydrophobic parts move out of contact with the water and the molecules form micelles. highest and lowest value (Figure 4). ratios of Drug to Lipid, for a single drug.
the pull-force required to remove probes from samples in a 96-well microtitre plate. The surface tension 9. Plot log R½ vs. log min. CMCDL/CMCL (figure 5). In this example,
The concentration at which this happens is called the Critical Micelle Concentration (CMC). Above the Analysis of each plate takes 2 minutes (plus
of each drug compound was measured at 12 different concentrations in the presence of diC8PS to min. CMCDL/CMCL is the value of CMCDL/CMCL at the D/L ratio, 1:1. time for sample preparation and data analysis).
CMC, there is no further decrease in surface tension with increased concentration of compound.
establish a surface activity profile.
Results Effect of CADs on CMC of phospholipids
Relative Critical Micelle Concentrations (CMCs) were calculated from surface tension profiles for Lipid
The surface tension of a buffer solution containing a phospholipid
different drug/lipid molar ratios and correlated with PLD-induction potency for a range of drugs. The decreases with increasing lipid concentration until the lipid forms
Drug + Lipid
results show a distinction between CADs known to cause PLD in humans, those having an effect on micelles, with the negatively-charged phosphate groups presented min. CMCDL/CMCL values
animals and cells and CADs not causing PLD. The greater the interaction of drug with the phospholipid to the aqueous phase. The graph shows surface tension for the
as shown by the surface activity profiles, the higher the risk for PLD in humans. lipid diC8PS (o) and CMCL, the CMC for the lipid. Some drugs
(e.g. CADs) bind to the lipid and lower the surface tension and
Conclusion CMCDL CMCL CMC (■). The term CMCDL denotes the CMC for the DL complex.
Direct measurement of the interaction of cationic drugs with a short-chain phospholipid is readily
As shown in figure 4, measurements are typically made at four DL
established using a high-throughput microtensiometer system. The results correlate with PLD-inducing
ratios. For the most lipophilic drugs, e.g. amiodarone, CMC
potency of many CADs and provide a ranking scale with respect to PLD risk. The method is well-suited Figure 2. Surface tension of Lipid and
drug/Lipid complexes at increasing
decreases considerably between 0.02:1 and 0.1:1 DL ratio, but at
and easily implemented for screening in the early phases of drug discovery.
O CH3 concentrations, showing the CMC for the highest ratios (0.5:1, 1:1), there is little change in CMC.
Cationic Amphiphilic Drugs O
CH3
the Lipid and the Drug/Lipid complex
R½ = drug/lipid ratio at which
CMCDL/CMCL has changed by 50%
(CADs) Amiodarone Tamoxifen
Figure 4. CMCDL/CMCL vs. DL ratio for Figure 5. Study of 53 drugs with known PLD-inducing potential.
Drugs reported to cause PLD in humans are clustered in the bottom-
Amiodarone, fluoxetine, imipramine, tamoxifen and N
Advantages of this novel screening method amiodarone (■), promazine (●), clozapine (▲)
and amantadine (), showing a decrease in left of this plot.
fenfluramine are examples of cationic amphiphilic CH3 I I
Imipramine • Fast – 8 parallel sensors and throughput of hundreds of compounds per week. CMC with increasing DL ratio. Values of R½
drugs. Each has a hydrophobic region containing a O CH3 O CH3
• Small compound consumption (<1mg). were obtained by curve fitting and Kibron and Sirius - Sirius are the exclusive distributors of Kibron’s
substituted or unsubstituted aromatic or aliphatic
N CH3 N • Can be integrated to robotics and automatic liquid handling system. interpolation. instruments for applications in drug discovery and development in the United
N
States, Canada and UK. Kibron Delta 8 systems are available for evaluation
• Automatic sensor cleaning and calibrating after each sample.
NH CH3
ring structure and a hydrophilic region consisting of F
CH3 CH3
CH3
in Sirius’ applications laboratories in Beverly, MA and Forest Row, UK.
Fenfluramine
a primary or substituted nitrogen groups that is F
• Very good reproducibility of data, correlation to PLD risk and literature data.
Sirius - Instrumentation and CRO Services for Physicochemical Profiling
F
positively charged at physiological pH. F Fluoxetine
CH3 Reference [1] P. Vitovič et al. J. Med. Chem. 51 (2008): 1842
www.sirius-analytical.com
F
O NH F
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