Physico-chemical properties of
compounds
MBDD 22.2.2018
ADME :
Absorption – Distribution – Metabolism –
Excretion
Exploration of ADME is at least of same
importance as exploration of activity of the
compound.
Large pharmaceutical companies are able to
screen over 3 000 000 of new molecules for
biological activity per year.
Some 30 000 hits may be found.
Most of them, however potent they are, have
not suitable physical, metabolic and safety
properties.
Some 30 molecules pass for pharmacological
evaluation.
0 - 3 molecules are introduced into market.
About 30% of molecules in pharmacological
evaluation are rejected due to ADME problems.
„A“ from ADME (Absorption)
properties affecting passive absorption:
acid-base character
lipophilicity
solubility
membrane permeability
Transport model
permeability – solubility - charge state –
pH-partition hypothesis
Passive diffusion: product of diffusivity and
concentration gradient
For ionizable molecules to permeate, molecule
needs to be uncharged
The amount of uncharged form is pH-dependent
Cm
0, Cm
h : concentrations of uncharged forms in
membrane (hard to estimate)
h : thickness of membrane
logK : partition coefficient lipid/water
CD, CA : water concentrations (easy to estimate
by HPLC)
Dm : diffusivity
Pm : permeability
Physiological properties of the GIT
Jejunum + ileum > 99% of absorbable surface
Time needed to pass through:
Empty stomach : water solution up to 0.4 hour
solids 0.5 - 3 hours
fatty food up to 13 hours
Jejunum + ileum : 3 – 5 hours
Colon: 7 – 20 hours (depends of sleeping period)
pH decrease in colon may be caused by short
fatty acids produced by bacteria
Intestine
-glycocalyx slows absorption of lipophillic
molecules
-tight junctions allows to come through small
molecules (< 200 Da)
-positively charged drugs has better permeability
through basal membrane
-acid pH microclimate prefer weak acids for
permeation
Intracellular pH environment:
Structure of octanol.
Octanol serves for years as a model.
Water saturated octanol:
water-octanol clusters allows to enter
hydrophyllic compounds, partially charged
compounds, as well
Biopharmaceutics classification system
Four BCS classes due to solubility and
permeability
For this classification:
Solubility is amount of watter needed to
dissolve highest single dose at pH (1-8) with
worst solubility: low s > 250 mL > high s
Permeability in human jejunum in vivo
high > 10-4 > low
Biopharmaceutics classification system
Charge state
Weak acids and bases ionize in solutions to
varying extent, dependent on pH of environment.
This affects amount of uncharged molecules ready
for absorption.
Thermodynamic parameter of this process is the
ionization constant KA (pKA)
Knowlwdge of compound´s pKA is very imortant –
it can predict absorption, distribution and
excretion of the compound.
Charge state Example
Urine pH (normal 5.7-5.8) can be altered by oral
doses of NH4Cl or NaHCO3 to ease excretion of
ionized compounds in toxicological emergencies.
Weak acids are excreted in alkaline urine, weak
bases in acidic urine.
Henderson-Hasselbach equation
thermodynamic equations for acid, base and
diprotic ampholyte:
negative logarithm of these equations give
Henderson-Hasselbach equations:
Henderson-Hasselbach equation
pH = pKA : concentration of ionized and uncharged
form is equal
pH = pKA – 2 : ratio (1:100) 99.9% uncharged
pH = pKA + 2 : ratio (100:1) 99.9% charged
Constant ionic medium reference state
Ionic strenght of the solution is involved in
dissociation rates.
Measurements of pKA has to be performed in
standard ionic conditions:
0.15 M KCl or NaCl solutions are used
(physiological ionic strenght)
Potentiometric measurement
Titration of water solutions of substance with
addition of 0.15 M KCl or NaCl
by HCl or KOH/NaOH
Potentiometric titration curve is obtained
In the case of multiprotic compounds, simple
curve can be misleading!
Bjerrum plot must be constructed:
Bjerrum plot construction:
Bjerrum plot construction:
1. Subtract titration curve with no compound
(blank) from a titration curve with sample (b)
2. x and y axis are rotated (c)
3. volume difference is turned to number of
ionizable hydrogens ratio (known from structure):
Difference between total ionizable hydrogens and
actually ionized hydrogens
Equilibrium states indicates pKA values (d)
Solubility problems:
Most of bioactive compounds are poorly soluble
in water.
> 100 µM: no problems in potentiometry
10 – 100 µM: measurable after carefull electrode
calibration
< 10 µM: mixed solvent environment have to be
used
Co-solvent mixtures:
alcohol - water (methanol, ethanol, propanol)
dimethylsulfoxide (DMSO) – water
dioxane – water
Measured values in the mixture can be
extrapolated to poor water using standard sets
Spectroscopic measurement (UV-VIS):
pH-dependent chromophore necessary
construction of molar absorbance to pH curves
at various vawelenghts
searching for suitable vawelenghts
specific method development for each compound
needed
Capillary electrophoresis mesurements
mobility of ionizable compounds depends on pKA
apparent mobility to pH curves are constructed
sigmoideal shape, midpoint pH equals to pKA
Macroconstants / microconstants
Certain type of multiprotic molecules posses
different tautomeric arrangement
measured pKA are average constants for more
complex equilibria – macroconstants
Microconstants can be elucidated by multiple
series of measurements with cosolvents shifting
pKa values in combination with UV-VIS detection
of chromophore changing
Macroconstants / microconstants (cetirizine)
Partitioning into octanol
P – partition coefficient
D – apparent partition coefficient (pH-dependent)
Partitioning equilibria:
non-ionizable molecules can be directly measured
Partitioning into octanol
ionizable molecules are partitioned too, but to
much lesser extent:
Partitioning into
octanol:
Propranolol
Partitioning into octanol
Difference between ionized and non-ionized
forms:
Difference between partition coefficients is equal
to difference between pKas
logD
Distribution ratio D is used at ionizable molecules
refers to a collection of all species:
lipophilicity profiles:
Shake-flask method
concentration is determined in aqueous phase
(HPLC)
HPLC methods
retention times at hydrophobic column/aqueous
buffer system are hydrophobicity indices
logP values can be calculated from retention
times using standards (structurally similar
compounds with known logP)
pH metric logP method
dual phase titration of compounds
Bjerrum plots are constructed to potentiometric
curves for octanol and water separately
Difference between pKa and apparent pKa is
equal to partition coefficient
pH metric logP
method
Partitioning into liposomes
liposomes are more „biologic-like“
distribution between membrane and aqueous
phase
Partitioning
into
liposomes
Partitioning into
liposomes
Changing of
dielectric
properties of
a layer
Partitioning into liposomes
Liposomes are added to defined solution of the
compound
after equilibria is established, liposomes are
separated by:
dialysis
ultrafiltration
centrifugation
Amount of unabsorbed substance in water
solution is determined
Partitioning into liposomes
liposome partitioning can be estimated from logP
valuaes by complex computational process
Partitioning into liposomes
complex non-linear
similarity
Solubility
solubility of ionizable molecules depends on pKa
monoprotic molecules:
diprotic ampholyte:
Solubility
many experimental complications:
crystalline/amorphous form
amorphism
polymorphism
solvates of solids
crystalline cosolvent
self-associates formation
micelles formation
Shake-flask method
thermostated saturated solution is shaken
between two phases (solid/liquid)
long equlibrium times (12hours – 7 days)
concentration in water phase is determined by
HPLC after microfiltration and centrifugation
Membrane permeability
In simple model, permeability can be linearly
related to the membrane-water partition
coefficient
In practice, unlinearity often occures:
-unstirred water layer
-aqueous pores in membranes
-membrane retention of lipophilic solute
-precipitation of solute
-transmembrane pH gradients
-hydrogen-bonding, electrostatic, hydrophobic
interactions with membrane constituents
-membrane surface charge
Membrane permeability
in vivo additional problems:
-different composition of inner and outer surface
-active transporters
-efflux system P-gp
-metabolism in membrane
Artificial membrane models
Paralell artificial-membrane permeability assay
(PAMPA)
-sendwich microplates covered by phospholipide
bilayer
-composition near to cell membrane
-allows high-throughoutput screening
Cell monolayer models
permeability through epitelial cell monolayer
e. g. caco-2 cell line