ADME Studies

ADME Studies

ADME (Absorption, Distribution, Metabolism and Excretion) studies are conducted during any stage of drug development to characterize molecules before final testing in humans. Experts at Blirt are specialized in in vitro ADME-Tox studies. Our ADME in vitro services include physicochemical properties analysis, in vitro permeability, protein binding, metabolism and toxicology (non GLP) (genotoxicity, cytotoxicity and proliferation).

Determination of the solubility of the organic compound in selected solvents provides preliminary information on the nature of the tested compound. It is crucial information during the preclinical stage of development of the molecule – a potential drug. The use of selected set of solvents provides package of valuable information. It helps to make a preliminary assessment of the processes’ speed of absorption and excretion of potential drugs from the body.

Compound requirements 2.5 mg solid (for solubility assessment) 1 mg solid (for standard preparation)
Solvents water, diethyl ether, 5% aqueous NaOH solution, 5% aqueous NaHCO3 solution, 5% aqueous HCl solution, concentrated sulfuric acid, 85% phosphoric acid, 5% glucose solution, 0.9% NaCl solution
Number of Replicates n = 2 (aliquots from filtrate)
Incubation Time Overnight
Incubation Temperature Ambient temperature
Analysis method HPLC-UV-VIS, Spectrophotometric determination
Output data Solubility [mg/ml]

The chemical stability assay enables to determinate degradation of compound in aqueous buffer by non-enzymatic processes (for example hydrolysis, oxidation or light-catalyzed degradation). Chemical stability of potential drugs is crucial because compounds that are highly unstable may not be suitable as drug candidates due to difficulties in maintenance of a therapeutically effective formulation. In addition compounds designed for oral administration must exhibit chemical stability at the low pH. A range of different pH values are available.

Test Compound Concentration 1 μM
DMSO Concentration 0.33%
Time Points 0, 5, 15, 30, 45, 120 min
Number of Replicates 2
Incubation Plate Material PTFE (all time points) and polypropylene (2 h only)
Compound Requirements 50 µL of 10 mM solution
Analysis Method LC-MS/MS
Output data % Parent compound remaining at each time point
% Parent compound to polypropylene compared to PTFE

The partition coefficient between the immiscible organic and aqueous phase (typically n-octanol and water) for a pharmaceutical composition is determined by extraction technique.

Analytical methods for measuring the concentration of the solute in both phases are selected on the basis of the physicochemical properties of an investigated molecule (usually a UV-VIS spectrometry is used). Organic compounds with very low solubility in aqueous solutions have often biological activity. For those compounds we propose an innovative method for determining the value of the partition coefficient using chromatographic techniques (without prior extraction from the water to the organic phase).

Method Miniaturised shake flask method
Partition Solvent n-Octanol
Ratio of Buffer:Octanol 50:1, 5:1, 1:2 (v/v)
Positive Control Compounds Acebutolol, Ketoconazole
Compound Requirements 1 mg solid compound
Analysis Method UV-VIS or HPLC-UV-VIS
Output data LogP, Log D7.4

The ability of the molecule to dissociate in physiological environment of living cell is very important for specificity of drug biological activity. An acid dissociation constant (also known as acidity constant, or acid-ionization constant) – Ka describes this ability of the compound. The ionization of the molecule affects the absorbance value and on the UV-VIS spectrum shape.

Method pH-metric titration or spectrophotometric methods
Compound Requirements 1 mg solid compound
Output data pKa

Drugs are metabolized by many sites in the body, including the gut wall, lungs, kidney and plasma. However, the majority of drug metabolism takes place in the liver which is the most metabolically active tissue per weight unit. We provide metabolic stability testing for small molecules including S9, microsomal and Hepatocytes stability assays.

S9 liver fraction is commonly used to support in vitro ADME studies, including phase I and phase II metabolism. S9 fraction is a mixture of microsomes and cytosol, which contains a wide variety of drug metabolizing enzymes and can be supplemented with cofactors such as NADPH and UDPGA. S9 fractions from different species are available to examine the differences in drug metabolism.

Standard S9 stability protocol:

Available species Human, rat, mouse, rabbit, dog, monkey
Test Compound Concentration 3 μM
S9 Concentration 1 mg/mL
Time Points 0, 5, 15, 30, 45 min
Temperature 37°C
Cofactors NADPH, UDPGA
Controls Blank sample
Sample without cofactor (45 min only)
Postive control (2 compounds)
Number of replicates 3 samples/time-point for test compound
1 sample/time-point for controls
Analysis Method LC-MS
Output data Percent of the parent compound remaining after each incubation period

Intrinsic clearance
Half life

Liver microsomes are commonly used to support in vitro ADME studies. Liver microsomes contain a wide variety of drug metabolizing enzymes and are commonly used to examine the potential for first-pass metabolism of orally administered drugs. Microsomes are pooled form multiple donors to minimize lot-to-lot variation caused by interindividual variability. Microsomes from different species are available to examine the differences in drug metabolism.

Standard microsomal stability protocol:

Available species Human, rat, mouse, rabbit, dog, monkey
Test Compound Concentration 3 μM
Microsome Concentration 0.5 mg/mL
Time Points 0, 5, 15, 30, 45 min
Temperature 37°C
Cofactors NADPH
Controls Blank sample
Sample without cofactor (45 min only)
Postive control (2 compounds)
Number of replicates 3 samples/time-point for test compound
1 sample/time-point for controls
Analysis Method LC-MS
Output data Percent of the parent compound remaining after each incubation period

Intrinsic clearance
Half life

Stability of compounds in biological fluids is important parameter because substances which undergo rapid degradation exhibit low efficacy in vivo. Information about plasma instability is useful for other in vitro studies (e.g., plasma protein binding where data can be difficult to interpret), in vivo studies (e.g., storage and handling pre-clinical and clinical samples may be challenging) and for screening of drugs, where rapid conversion in plasma is desirable (e.g., prodrugs and antedrugs).

Standard plasma stability protocol:

Available species Human, rat, mouse, rabbit, dog, monkey, bovine
Test Compound Concentration 1 μM
Time Points 0, 15, 30, 60 and 120 min
Temperature 37°C
Controls Blank sample
Postive control (1 compounds)
Number of replicates 3 samples/time-point for test compound
1 sample/time-point for controls
Analysis Method LC-MS
Output data Percent of the parent compound remaining after each incubation period

Hepatocytes are commonly used to support in vitro ADME studies requiring intact cellular systems. Intact hepatocytes contain the major hepatic drug-metabolizing enzymes required to study the four categories of xenobiotic biotransformation: hydrolysis, reduction, oxidation and conjugation. The assay is performed on human hepatic cells Hep2G. Primary cryopreserved hepatocytes from different species are also available up on request to examine the differences in drug metabolism.

Standard hepatocyte stability assay protocol:

Test System Human Hep2G liver cells, Human, rat, mouse, dog, monkey, minipig (cryopreserved hepatocytes)
Test Compound Concentration 3 µM
Time points 0, 5, 10, 20, 40 and 60 min
Temperature 37°C
Controls Blank sample
Postive control (2 compounds)
Number of replicates 3 samples/time-point for test compound
1 sample/time-point for controls
Analysis Method LC-MS
Output data Percent of the parent compound remaining after each incubation period

Intrinsic clearance
Half life

In vitro permeability and transport studies are performed to assess the potential of oral absorption and distribution of drug candidates. We provide whole range of in vitro tests to characterize properties of small molecules subjected to passive transport – PAMPA.

The Parallel Artificial Membrane Permeation Assay (PAMPA) is used as an in vitro model of passive, transcellular permeation and absorption of drugs by a PVDF membrane filter pretreated with a solution of the lipid. Passive diffusion is an important factor in determining transport of the drug through the gastrointestinal tract, transport across cell membranes penetration of the blood brain barrier, as well as transport across cell membranes.

Standard PAMPA assay protocol:

Test Compound Concentration 10 μM
Number of Replicates 3
Membrane Composition Lecithin in dodecane (1% v/v)
Incubation Time 16 hours
Temperature Room temperature
Compound Requirements 100 µL of 10 mM DMSO solution
Integrity Marker Lucifer Yellow
Analysis Method LC-MS/MS or HPLC-UV-VIS
Data Delivery Log Pe

To understand the distribution potential of tested compound a fraction of unbound in plasma compound is determined with a usage of equilibrium dialysis. The measure of binding to plasma affects the way of potential drug distribution into tissues in the body. Extensive plasma protein binding also limits the amount of free compound available to access sites of action in the cell, this may cause slower metabolism and elimination. Equilibrium dialysis is the most widely accepted method for determination of plasma protein binding due to minimized non-specific binding effects, when compared to other methods, for example ultrafiltration.

Standard plasma protein binding assay protocol:

Method Equilibrium Dialysis (at 10 %, 50 % or 100 % plasma)
Test Compound Concentration 5 μM (different concentrations available)
Number of Replicates 2
Compound Requirements 150 μL of 10 mM solution
Analysis Method LC-MS/MS quantification (both plasma and buffer standards prepared)
Data Delivery Fraction unbound in 100% plasma Recovery

Protein binding has a very important effect on drug dynamics since only the free (unbound) fraction of drug interacts with receptors and may cause pharmacological effects. It is more common to determine the concentration of a drug in plasma rather than whole blood. On the other hand it is crucial for the interpretation of pharmacokinetic data if differential binding to a specific component in the blood occurs.

Standard whole blood protein binding assay protocol:

Test method Rapid equilibrium dialysis (RED, Pierce)
(8K MWCO, distribution: 100% blood-buffer)
Available species Human, rat, mouse, rabbit, dog, monkey
Test Compound Concentration 5 µM
Incubation time 4h
Temperature 37°C
Controls Blank sample
Positive control (2 compounds: Metoprolol; Chlorthalidone)
Number of replicates 3 samples for test compound
1 sample for controls
Analysis Method LC-MS
Output data Fraction unbound in blood
Recovery

Differences between plasma and brain composition are significant. Plasma contains twice as much protein and brain contains 20 fold more lipids.

Standard brain tissue binding assay protocol:

Test method Rapid equilibrium dialysis (RED, Pierce)
(8K MWCO, distribution: 100% brain tissue homogenate-buffer)
Available species Human, rat, mouse, rabbit, dog, monkey
Test Compound Concentration 5 µM
Incubation time 4h
Temperature 37°C
Controls Blank sample
Positive control (2 compounds: Metoprolol; Chlorthalidone)
Number of replicates 3 samples for test compound
1 sample for controls
Analysis Method LC-MS
Output data Fraction unbound in brain tissue homogenate
Recovery

It is proved that observed kinetics of metabolism in the in vitro assays can be influenced by nonspecific liposomal binding causing problems with accurate prediction of clearance. Several examples proved that knowledge of microsomal binding leads to more precise understanding of connections between in vitro metabolism and in vivo pharmacokinetics.

Standard microsomal binding assay protocol:

Test method Rapid equilibrium dialysis (RED, Pierce)
(8K MWCO, distribution: 100% microsomes-buffer)
Available species Human, rat, mouse, rabbit, dog, monkey
Test Compound Concentration 5 µM
Incubation time 4h
Temperature 37°C
Controls Blank sample
Positive control (2 compounds: Atenolol, Propranolol)
Number of replicates 3 samples for test compound
1 sample for controls
Analysis Method LC-MS
Output data Fraction unbound in microsome
Recovery

Blood to plasma distribution is calculated using plasma protein binding assay and whole blood protein binding assay.