Physico-chemical characterization

Physicochemical profiling of compounds includes the following assays:

-        Solubility (thermodynamic, kinetic)

-        Lipophilicity (log P, log D, CHI)

-        pKa

-         Chemical stability in buffers and simulated media (gastric, intestinal)

-        Development of formulations for in vivo studies

In vitro assays (absorption, metabolism)

In order to reduce the use of animals, in vitro models have been developed to predict ADME parameters.

-       Intestinal permeabilitymodels (PAMPA, Caco-2, P-gp inhibition)

-       Plasma protein binding

-       Metabolic stability (plasma, liver microsomes, liver S9 fraction, hepatocytes, enzymesrecombinant, glutathione or glucuronicacidconjugation, sulphation)

-        Blood-brainbarrier (BBB)

-        Metabolite identification

-       Drug interactions (CYP 450 inhibition, CYP 450 induction)

In vivo tests (distribution, elimination)

All pharmacokinetic properties are evaluated in vivo.

-        Pharmacokinetics in rodents (determination of Cmax, Tmax, t1/2, clearance, volume of distribution, AUC)

-        Bioavailability (i.v., i.p., i.m., p.o., topical, s.c....)

-        Biodistribution (all tissues)

-        Blood-brain barrier crossing

-        Elimination in urine, bile, faeces...

Toxicity

Evaluation of compound cytoxicity is essential before undertaking animal studies. It is also important to study toxicity in vivo.

-       Safety study in rodents

-        Cell viability tests

Mouse pharmacokinetics

Introduction

Knowing the pharmacokinetic behaviour of a compound is a prerequisite for:

1.      its rational use in in vivo experimentation

2.      the interpretation of pharmacological results and

3.      the design of toxicology studies.

Following injection of a compound, its plasma concentration will change over time. Blood and/or tissue samples (depending on customer's request) are analyzed after administration.

·         Exposure to a compound is determined by measuring the area under the concentration curve = f(time) (AUC)

·         Bioavailability is defined by the ratio of AUC: AUC (i.v.) / AUC (p.o.)

Protocol

Parallel study, typical protocol:

Animals: CD-1 mouse 8 times and 2 routes of administration: i.v. and p.o.

Single dose, n=3 mice per time point

Blood sample collection: cardiac puncture (250 µL plasma/sample)

Detection: LC-MS/MS

The data obtained are used to generate time-concentration curves and to determine fundamental pharmacokinetic parameters such as T1/2, Cmax, Tmax...

i.v. administration ofantipyrine in mice

Notes:

For other conditions (animals, time, route administration) please contact us.

Reference

K. Cox et al (2002) Combinatorial Chemistry and High Throughput Screening 5, 29-37

Metabolic stability - liver S9 fraction

Introduction

The liver is the organ mainly involved in drug metabolism.

Liver metabolism consists in 2 phases: phase I corresponds to oxidation reactions catalyzed by cytochromes P450;phase II consists in conjugation reactions to glutathione, glucuronic acid, etc.

The S9 fraction of liver corresponds to the supernatant obtained by centrifugation of homogenate at 9000g. It contains the enzymes present in microsomes (cytochromes P450) as well as soluble enzymes (glutathione transferase, glucuronyl transferase...).

·         The S9 fraction provides a complete metabolism model since it contains the majority of Phase I and II enzymes

Protocol

The compound is incubated with 1 mg/mL of S9 at 37°C in the presence of one or more co-factors involved in the reactions to be studied (NADPH, glutathione, uridine glucuronic acid diphosphate).

Ex: glutathione alone to examine a direct conjugation to glutathione or glutathione + NADPH to determine if an oxidation step is necessary to obtain the conjugation.

The compound concentration is 5 µM in a magnesium chloride containing phosphate buffer.

5 incubations (or 2 incubations) are carried out in parallel and stopped at different times. 0,15, 30, 45 and 60 min (or 0 and 60 min).

The percentage of remaining compound is determined by LC-MS by measuring the area under the peak of the compound on the chromatogram.

Controls: 0 µM of compound; without co-factor; positive control with standard compound.

Number of replicates = 2

Testosterone stability in the presence of human liver S9 fraction

Notes

For other conditions, please contact us.

Reference

R. Singh et al (1996) Rapid communications in mass spectrometry 10, 1019-1026

N. Plant et al (2004) Drug Discovery Today 9, 328-336

Metabolic stability - liver microsomes

Introduction

Metabolism is the transformation of compounds in the body by enzymatic pathways

The main organ involved in this process is the liver.

Microsomes are a subcellular fraction of liver homogenate. They contain enzymes such as cytochromes P450 linked to the membrane of the endoplasmic reticulum.

They are suitable for high throughput analyses (large number of compounds)

·         60% of marketed drugs are metabolized by cytochromes P450

·         Microsomes are prepared from multiple donors to avoid the effects of individual variability

Protocol

Incubation of the compound with microsomes (0.5 mg/mL) is done at 37°C in the presence of the NADPH co-factor (1 mM).

Five incubations (or 2 incubations) are carried out in parallel and stopped at different times: 0,15, 30, 45 and 60 min (or 0 and 60 min).

The percentage of remaining compound is determined by LC-MS.

Controls: 0 µM of compound; without co-factor; positive control with standard compound.

Number of replicates = 2

Verapamil stability in the presence of human liver microsomes

Notes

For other conditions, please contact us.

Reference

Obach et al (1997) The Journal of Pharmacology and Experimental Therapeutics 283, 46-58

Linget et al (1998) Journal of Pharmaceutical and Biomedical Analysis 19, 893-901

Plasma stability

Introduction

Blood and plasma contain enzymes that can hydrolyze labile groups such as esters or amides.

It is important to determine plasma stability of a new molecule because compounds that are rapidly degraded in plasma generally have low in vivo activity.

In contrast, in the case of "prodrugs", the hydrolysis of labile groups increases pharmacological activity.

·         Compounds that are locally active and rapidly degraded to inactive molecules are called "antedrugs".

·         The plasma stability of a compound can vary greatly from one species to another

Protocol

Stability is determined at 37°C.

A dilution is performed at 5 µM in plasma (or serum) from a 10 mM stock solution in DMSO.

Five incubations (or 2 incubations) are carried out in parallel and stopped at different times.0,15, 30, 45 and 60 min (or 0 and 60 min).

The percentage of remaining compound is determined by LC-MS.

Controls: 0 µM of compound; positive control with standard compound.

Number of replicates = 2.

Stability of propanthéline in murine serum at 37°C

Notes

For other conditions, please contact us.

Reference

L. Di et al (2005) Journal of Biomolecular Screening 297, 110-119

Lipophilicity: log D (shake flask)

Introduction

The partition coefficient between octanol and water is the most commonly used physicochemical parameter for measuring lipophilicity.

The lipophilicity of a molecule influences its absorption and distribution in tissues. Moreover a hydrophobic molecule presents a high risk of non-protein-specific binding and metabolism by cytochromes P450.

The partition coefficient (log P) refers to neutral molecules. It is better to determine the partition coefficient log D which takes into account ionization of molecules.

·         For optimal oral absorption, log D should be between 0.5 and 2.

·         The difference between logDoctanol/water - log Dcyclohexane/water provides a good model of permeability of the blood-brain barrier.

Protocol

The partition coefficient is measured by the so-called "shaked flask" method: balancing an aqueous buffer (PBS) and a non-miscible organic solvent (octanol). Several solvent and buffer volume ratios are evaluated to cover a lipophilic range from -1 to 3.

Incubation: 1 hour at room temperature (environ 20°C).

HPLC UV-Vis detection (diode array).

The concentration in the aqueous phase is determined by HPLC. The concentration in the organic phase is calculated using a reference (standard solution at 100 µM).

Number of replicates = 2

Log D at pH=7.4 measured at room temperature

Notes

For other pH values, please contact us.

The measurable lipophilic values are between -1.5 and 3.

The CHI (Chromatographic Hydrophobicity Index) method allows higher lipophilicity to be determined.

References

A. Avdeef (2001) Current Topic in Medicinal Chemistry 1, 277-351

C. Young (1987) Journal of Medicinal Chemistry 31, 656-671

Permeability - (Parallel Artificial Membrane Permeability Assay)

Introduction

Permeability is defined as the rate at which a compound passes through a biological barrier.

This process is involved in intestinal absorption and in distribution to organs such as the liver, brain or kidney.

The main mechanism ofcompound permeability is passive diffusion.

The PAMPA (Parallel Artificial Membrane Permeability Assay) method provides an in vitro model of passive permeability.

·         The artificial barrier consists of phospholipids solubilized in an organic solvent

·         The method is simple, inexpensive and fast

Protocol

The compound is diluted to 10 µM in a buffer (PBS)

The system consists in a standard 96 wells plate and a plate with porous bottom on the top of the first one, which allows tocreate a donor and an acceptor compartment for each well.

An artificialmembrane is generated with a 2% phosphatidylcholine solution in dodecane introduced into the porous bottom wells of the acceptor compartment.

The incubation time is 5 hours and the measurement is performed at room temperature.HPLC UV-Vis or LC-MS detection

Controls: compound recovery, membrane integrity verified with a dye

Number of replicates = 4

Permeability of standard compounds measured at 22°C

Notes

PAMPA method just allows to model the passive diffusion mechanism. All the mechanisms involved in intestinal permeability can be performed using the Caco-2 cell line permeability method.

Référence

M. Kansy et al (1998) Journal of Medicinal Chemistry 41, 1007-1010

pKa

Protocol

Titration of compound is performed using NaOHsolution. The measurement is done using a pHmeter at room temperature at a constant ionic strength.

pKa measured at 25°C

Notes

The method used can vary according to the solubility of compound in water, so it is important to know if the compound precipitates or not in water at 250 µM between pH 1.5 and 11.5.

Références 

X. Gong et al. (2008) Chromatographia. 68, 219-225 -

Kinetic solubility

Introduction

Theshaked flask method allowsthe determination of thermodynamic solubility, i.e. to characterize the balance between a compound in solid form and the buffer in which it has been dissolved.

It is not always necessary to have such information. The measurement of an apparent solubility can be enough.

Biological screening assays are generally performed using chemical libraries (in DMSO buffer).

It is possible to determine an apparent solubility from these stock solutions by diluting them in an aqueous buffer.

·         This method essentially identifies compounds with very low water solubility.

·         It is faster and cheaper than a thermodynamic solubility measurement.

Protocol

A 200 µM dilution is prepared from a 10 mM stock solution in DMSO inHepes buffer at pH=7.4. This mixture is equilibrated at room temperature for 24 hours.

After centrifugation, the supernatant is injected into HPLC (UV detection).

A reference solution made of  an identical dilution in a CH3CN/water 1/1 v/v mixture is prepared. A reference point at 100 µM is used to check the linearity of the UV response.

The comparison of chromatograms allowstodetermine the apparent solubility of the compounds (if it is less than 200 µM)

Number of replicates = 2

Apparent solubilities at pH=7.4 measured at room temperature

Notes: For different buffer or incubation temperature, please contact us.

For a solubility threshold other than 200 µM, please contact us.

In any case, the final proportion of DMSO will not exceed 2%.

Thermodynamic solubility

Introduction

Modern techniques used to discover new therapeutic agents give rise to more and more compounds with low water solubility.

Low solubility limits the intestinal absorption of compounds and therefore their bioavailability when administered orally. It is also a concern for the formulation of intravenous injectable solutions.

Poorly soluble molecules will also affect the results of analyses carried out during the "discovery" phase (e.g. precipitation problem during serial dilutions)

Therefore, low solubility can lead to a waste of time and high development costs.

·         In the case of ionizable compounds, solubility is highly dependent on pH

·         High solubility does not necessarily mean good intestinal absorption

Protocol

Solubility is measured by the so-called "shaked flask" method: saturation of the Hepes buffer pH=7.4 then ultracentrifugation and determination of compound concentration in the saturated solution.

Incubation: 24 hours at room temperature (around 22°C).

HPLC UV-Vis detection (diode array).

Determination of solubility using 3-point calibration (from a reference solution in the DMSO).

Number of replicates = 2

Solubilities at pH=7.4 measured at room temperature

Notes

For different buffer or incubation temperatures, please contact us.

The minimum measurable solubility limit depends on the detection of the compound.

For highly soluble compounds, the maximum limit depends on the quantity of product supplied.

References

A. Avdeef (2007) Advanced Drug Delivery Reviews 59, 568-590

N. Patel (2006) Predictive ADME and Toxicology Strategies

Chemical stability

Introduction

A compound may be chemically degraded by non-enzymatic processes.

Instability of a compound in solution can lead to erroneous data in in vitro tests.

Compounds with high chemical instability cannot become drug candidates as it is difficult to find a formulation to prevent this degradation.

·         Orally administered compounds must be stable in acidic medium

·         Some compounds may decompose in organic storage solvents (DMSOs)

Protocol

Stability is generally determined at room temperature.

A dilution is performed at 20 µM in a buffer or biological fluid (plasma, serum) from a 10 mM stock solution in DMSO. 5 samples are taken at different time points depending on the medium (or 2 samples).

Buffer: 0, 2, 4, 6 and 24 hours (or 0 and 24 hours).

The percentage of remaining compound is determined by HPLC or LC-MS by measuring the area under the peak of the compound on the chromatogram.

Number of replicates = 2

Stability of erythromycin at pH 2

Notes

For other conditions, please contact us.

Reference

CE. Kibbey et al (2001) Journal of  Pharmaceutical Sciences 90, 1164-1175

Plasma protein binding

Introduction

Plasma contains between 6 and 8% protein including serum albumin and alpha -1-acidic glycoprotein.

The binding of a compound to plasma proteins influences its distribution in the body. Protein-bound compounds are retained in the blood and cannot cross biological membranes.

Plasma protein binding can be measured by ultrafitration using a semi-permeable membrane.

·         The ultrafiltration technique is easy, fast and inexpensive.

Protocol

The compound is diluted to 10 µM in plasma.

After centrifugation unbound molecules pass through the membrane while the protein-bound molecules are retained in the upper part of the tube.

HPLC UV-Vis or LC-MS detection

The unbound percentage is calculated from the initial concentration and the concentration measured in the lower compartment.

Controls: Material balance

Number of replicates = 2

Plasma protein binding measured at 22°C (mouse plasma)

Notes

For other conditions, please contact us.

Reference

J. Oracova et al (1996) Journal of Chromatography B: Biomedical Applications, 677, 1-28