PhysChem profiling

High throughput physicochemical profiling

Todays compound development, QSAR and ADME/tox require the availability of the true molecular scale properties of the compounds. In this respect computational methods are already in full use. However, the level of sophistication of the computational techniques is in striking contrast to the cumbersome and at the best descriptive experimental methods, which until now have been available for physicochemical profiling. No longer so: Kibron Delta-8 analyzer now brings a whole new dimension to your R&D. More specifically, Delta-8 makes surface activity profiling, i.e. the characterization of the amphiphile properties of compounds, a real, practical utility. Further, surface activity profiling by Delta-8 is a true HTS technique, meaning that it can be adapted in lead optimization and even in the hits-to-leads phase.

Kibron Delta-8 high throughput analyzer provides these key parameters, easily and by true measurements. The Delta-8 analyzer yields the mechanistic reason why a compound does not pass through the lipid bilayer, thus giving directions for improvements of molecular structures in QSAR.

True Surface Area

True Surface Area - unique molecular size descriptor,

Amphiphilic molecules adsorb at the air-liquid interface, decreasing the surface tension. Changes in interfacial tension (surface tension) with concentration provides the so called adsorption isotherm:

The isotherm can be described by various adsorption models to obtain the following molecular parameters:

  • Hydrophobicity (KAW)
  • True surface area (TSA)
  • Critical Micelle Concentration, CMC
  • Surface Excess

Efficiency of Adsorption

The magnitude and balance between polar and non-polar groups is decisive how the molecules orientate and fold which is reflected through the value of True Surface Area (=average area per adsorbed molecule). Surface area is derived from the adsorption isotherm through surface excess concentration at surface saturation (maximum value adsorption can attain).

ADME/tox implications

Characteristic parameters defining the amphiphilicity: the vectorial lipophilicity, true surface area, and critical micelle concentration are relevant for interactions between biological macromolecules (membranes, proteins) and contributes to the permeation process. The relative contribution of each molecular parameters are likely to vary for different in vivo barriers, such as BBB and the intestinal wall. Generally, the molecules with high surface areas are not as likely to cross the membranes without assistance (passive di usion). ie. in order for a molecule to partition into the membrane work must be done against the lateral pressure that prevails in the mebrane.

Critical Micelle Concentration

Critical Micelle Concentration

Amphiphilic molecules adsorb at the air-liquid interface, decreasing the surface tension. Changes in interfacial tension (surface tension) with concentration provides the so called adsorption isotherm:

The isotherm can be described by various adsorption models to obtain the following molecular parameters:

  • Hydrophobicity (KAW)
  • True surface area (TSA)
  • Critical Micelle Concentration, CMC
  • Surface Excess

Efficiency of Adsorption

Amphiphiles adsorb at air-water(buffer) interface and orientate by exposing the polar head groups with water, while hiding the tails in the air and forming a single layer of surfactant molecules. The main driving force is the hydrophobic effect. As individual amphiphile (monomer) solubility limit is reached they start to aggregate to form structures known as micelles. Now the non-polar parts are hidden inside the micelle core while the micelle surface consists of the polar parts. Again, this process is driven by the hydrophobic eff ect. More specifically, the partitioning of the compound into interface and micelle formation decreases the free energy of the system.

CMC is determined largely by the hydrophobic group. There is however an opposing force for micellization. For charged (ionic) amphiphiles there is additional electrical work required for micelle formation. Also loss of degree of freedom for hydrophobic residues as they get buried inside the micelle core increases the free energy of the system.

By measuring the surface tension of a series of 12 diluted samples (in Kibron Delta-8) from lowest to highest concentration the critical micelle concentration (CMC) can easily be determined.

ADME/Tox implications

Internal link to phospholipidosis assay

The single most important feature of micellization is solubilization of water insoluble drugs inside micelle core.

Hydrophobicity

Hydrophobicity - through surface tension

Amphiphilic molecules adsorb at the air-liquid interface, decreasing the surface tension. Changes in interfacial tension (surface tension) with concentration provides the so called adsorption isotherm:

 

The isotherm can be described by various adsorption models to obtain the following molecular parameters:

 

  • Hydrophobicity (KAW)
  • True surface area (TSA)
  • Critical Micelle Concentration, CMC
  • Surface Excess 

Efficiency of Adsorption

KAW is a term describing the affinity of a compound for the interface. In aqueous solution it can be considered as a measure of hydrophobicity. Higher the KAW value higher the hydrophobicity and vice versa. 

ADME/Tox properties

For passive dissusion the compounds lacking or with only few functional groups capable of hydrogen bonding with water(buffer) adsorb

at the interface. Too lipophilic compounds may be retained by the cell membrane.

Early in vitro ADMEtox

An in vitro screen for early ADME prediction

Amphiphilicity is known to be an important characteristic for drugs. Amphiphiles are kind of drugs that have polar and non-polar moieties (acts as surfactants) and thus have the affinity for both aqueous and non-aqueous solvents/ media. For the same reason they have the tendency to adsorb into biological membranes with no requirement of energy. This passive di usion of drugs into membranes is dependent on the size, shape and lipophilicity of the compounds.

Kibron ADMEscreenTM is based on on characterizing amphiphilic properties of the drug compounds. It is ideal ADME related compound profiling at the early stages of drug discovery enabling researchs to gain better understanding on their molecules for analysis of phospholipidosis, blood brain barrier permeation and fraction absorbed.

Blood Brain Barrier Permeation

Blood-brain barrier permeation through amphilicity profiling

Poor ADME/Tox properties including (bioavailability, blood-brain barrier permeability) are the major reason for the failure of NCEs in pharmaceutical R&D. This can be extremely costly, in particular, if adverse effects such as serious CNS side effects manifest just in clinical trials. Accordingly, there is an increasing pressure to screen compound libraries and NCEs for their ADME/Tox properties in vitro, preferably already before lead optimization.

Kibron ADMEscreenTM delivers comprehensive physicochemical profiles of compounds, by providing the amphiphilicity parameters: KAW (hydrophobicity)TSA (true surface area)CMC (critical micelle concentration) further correlating with ADME properties such as blood-brain barrier (BBB) permeation.


Selected publications:

"Prediction of blood-brain barrier penetration of poorly soluble drug candidates using surface activity profiling"

Dressman et. al. Eur J Pharm Biopharm. 2010 Aug;75(3):405-10. Epub 2010 Mar 21.

 

"Surface activity profiling of drugs. Correlation to passive blood-brain barrier permeation"

Suomalainen, P., Johans, C., Söderlund, T., Kinnunen, P.K.J., J.Med.Chem. 47, 1783-1788 (2004).

 

"Blood-Brain Barrier Permeation: Molecular Parameters Governing Passive Diffusion."

Fischer, H., Gottschlich, R., Seelig, A., J. Membr. Biol. 165, 201-211 (1998).

 

"A method to determine the ability of drugs to diffuse through the blood-brain barrier"

Seelig, A., Gottschlich, R., Devant, R. M., . Proc. Natl. Acad. Sci. USA. 91, 68-72 (1994).

Fraction Absorbed

INTESTINAL PERMEABILITY THROUGH AMPHIPHILICITY PROFILING

Poor ADME/Tox properties including (bioavailability, blood-brain barrier permeability) are the major reason for the failure of NCEs in pharmaceutical R&D. This can be extremely costly, in particular, if adverse effects such as serious CNS side effects manifest just in clinical trials. Accordingly, there is an increasing pressure to screen compound libraries and NCEs for their ADME/Tox properties in vitro, preferably already before lead optimization.

Kibron ADMEscreenTM delivers comprehensive physicochemical profiles of compounds, by providing the amphiphilicity parameters: KAW (hydrophobicity), TSA (true surface area), CMC (critical micelle concentration) further correlating with ADME properties such as blood-brain barrier (BBB) permeation or intestinal permeability.

Selected publications

Kiehm, K., (2009), Development of a novel screening tool for the prediction of oral drug absorption based on surface activity profiling, Diss., Frankfurt am Main, Univ.

PLDscreen

PLDscreen - a fast screen for unparalleled prediction of risk for drug-induced phospholipidosis

Phospholipidosis (PLD), the accumulation of phospholipids in tissues such as lungs, kidneys and liver, has been recognized to represent a potential causative process for a number of serious side effects, particularly for drugs intended for long term usage by the patients. Consequently, it is desirable to identify potential PLD inducing compounds at an early drug development stage, such as lead optimization.

The reason for PLD is believed to be the formation of drug-phospholipid complexes, which cannot be metabolized by phospholipid-degrading enzymes, phospholipases.

Kibron PLDscreen is a robust in vitro HTS assay which measures directly the strength of drug-phospholipid interaction. More specifically, it detects the drug-induced shift in the critical micelle concentration (CMC) for a phospholipid, measured using our Delta-8 surface tension plate reader. PLDscreen predicts with unparalleled accuracy the ability of compounds to induce PLD. This proprietary assay allows for high throughput screening of molecules with minimal compound consumption, hundreds of compounds in a single working day!

The PLDscreen comprises of the Kibron Delta-8 surface tension plate reader and software, PLDnanosol reagent, and DynePlate microtiter plates.

Read more:

Vitovic et al. Assessment of drug-lipid complex formation by a high throughput Langmuir balance and correlation to phospholipidosis. J. Medicinal Chemistry 51(6), 1842-1848 (2008).

Features & Benefits

Kibron PLDscreen - features & benefits

Kibron PLDscreen is based on quantitative surface activity profiling of compounds by the high throughput Kibron Delta-8 analyzer, PLDnanosol reagent, software for data acquisition & analysis, and Kibron microtiter plates.

Basic features and benefits of the Kibron PLDscreen are:

  • compound classification by PLD index
  • compound consumption < 1 mg
  • throughput up to 1400 compounds / week, in full HTS mode, with automatic liquid handling system > 2000 compounds / week
  • easy integration to robotics and automatic liquid handling systems
  • comprehensive and intuitive software, immediate data read-out
  • standard 96-well platform
  • automatic sensor cleaning after each assay

The Kibron PLDscreen offers high throughput PLD screening with minimal compound consumption, resulting in a fast and cost-efficient, yet highly accurate prediction of PLD.

Kibron vs. cell-based assays

Kibron PLDscreen vs. cell-based assays

Kibron PLDscreen requires minimal manual labor and working space, and greatly diminishes the need for costly and labor intensive cell-based assays for phospholipidosis. The results from PLDscreen are obtained in real time, providing immediate feedback to the drug development team.

Comparing the results from Kibron PLDscreen and a cell-based assay (see below) reveals an excellent correlation between these two sets of data. However, unlike the cell-based assay, PLDscreen does not give false negatives. Accordingly, the phospholipidosis-inducing potential of gentamicin and ketoconazole is readily revealed by PLDscreen, while these compounds produce no effect in the cell-based assay (gentamicin) or an uncalculable result (ketoconazole). This is of particular concern, since gentamicin is one of the few drugs known to cause phospholipidosis in humans.


 

For more detailed comparison with other assays, please read:

Vitovic et al. Assessment of drug-lipid complex formation by a high throughput Langmuir balance and correlation to phospholipidosis. J. Medicinal Chemistry 51(6), 1842-1848 (2008).

PLDscreen assay

Kibron PLDscreen assay

PLDscreen is a simple assay, involving first making serial dilutions of the drug, Nanosol, and drug-Nanosol mixture. Subsequently, the values of surface tension are measured by the Kibron Delta-8 plate reader. One 96-well plate is read in approxim. 3 minutes, your answers come in real time!

PLDscreen principle

PLDscreen principle

The Kibron PLDscreen assesses phospholipidosis by measuring drug-phospholipid complex formation.

The binding affinity is reflected in changes in CMC for PLDnanosol and drug-PLDnanosol mixture, monitored by surface tension.

Hydrophobic effect and head group interactions (e.g. electrostatics) are the driving forces in the formation of micelles, allowing for detailed interpretation of data and the molecular mechanism(s) involved.

Unlike e.g. cell based and toxocogenomics assays PLDscreen does not give false positives or false negatives!

How it works

Recommended instruments:

Delta-8

Delta-8 is a unique high throughput surface tension plate reader, for e.g. HTS screening of critical micelle concentration (CMC) values of surface active materials.