Drugs partitioning:

quantification methodologies
and biological relevance
OUTLINE

 Why drugs partitioning matter

 The traditional determination method
 Theoretical methods
 The biological membrane complexity
 The necessity of other methods
 Features of mimetic model membranes
 The liposomes/water system as a drugs partitioning valid methodology
 Comparison of methodologies

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Drugs partitioning

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LINPINSKY RULE “OF FIVE”
States that compounds are likely to have good absorption and permeation in biological
systems and are more likely to be successful drug candidates if they meet the following
criteria:

 5 or fewer hydrogen-bond donors

Not too polar
 ten (2 x 5) or fewer hydrogen-bond acceptors

 molecular weight less than or equal to 500 Not too big

 calculated logP less than or equal to 5 Not too hydrophobic

*Compound classes that are substrates for biological transporters

are exceptions to the rule.

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LIPOPHILICITY

Lipophilicity is a property that has a major effect on absorption, distribution, metabolism, excretion, and
toxicity (ADME/Tox) properties as well as pharmacological activity. This molecular property represents the
affinity of a molecule for a lipophilic environment.

Lipophilicity can be quickly measured or calculated. It is most commonly described by the logarithm of
partition coefficient (log P) between two immiscible solvents, one is an organic apolar (e.g.octanol) and the
other an aqueous polar (buffer solution).

P
Xaqueous Xoctanol
[X]octanol
Partition coefficient P (usually expressed as log10P or logP) is defined as: P=
[X]aqueous
P is a measure of the relative affinity of a molecule for the
lipid and aqueous phases in the absence of ionisation.

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THE OCTANOL-WATER SYSTEM

1-Octanol is the most frequently used lipid phase in pharmaceutical research. This is because:

 It has a polar and non polar region (like a membrane phospholipid)

 Po/w is fairly easy to measure
 Po/w often correlates well with some biological properties
 It can be predicted fairly accurately using computational models

Log P depends on the partitioning of the neutral molecules between the two matrices.

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PH VARIATIONS THROUGH THE BODY

Fluid pH
Aqueous humour 7.2
The same compound will be ionised to
Blood 7.4
different extents in different parts of
Colon 5-8 the body.
Duodenum (fasting) 4.4-6.6
Duodenum (fed) 5.2-6.2 This means that, for example, basic
Saliva 6.4 compounds will not be so well
Small intestine 6.5 absorbed in the stomach than acidic
Stomach (fasting) 1.4-2.1 compounds since it is generally the
Stomach (fed) 3-7
unionised form of the drug which
diffuses into the blood stream.
Sweat 5.4
Urine 5.5-7.0

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IONISATION CONSTANTS
 The equilibrium between un-ionised and ionised forms is defined by the acidity constant Ka
or pKa = -log10 Ka

 For an acid:
Ka +
HA H + A

[H+][A-] 100
Ka = % ionised =
[AH] 1 + 10(pKa - pH)

 For a base:
Ka +
BH+ H + B

[H+][B] 100 When an acid or base is 50% ionised:

Ka = % ionised =
[BH+] 1 + 10(pH - pKa) pH = pKa

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EFFECT OF IONISATION ON PERMEABILITY AND SOLUBILITY

Permeability and solubility profiles for an acidic compound with a pKa of 5.

Permeability and solubility are pH dependent for ionizable compounds. The properties exhibit opposite effects
with pH because of the effects of ionization.

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 DISTRIBUTION COEFFICIENT
If a compound can ionise then the observed partitioning between water and octanol will be
pH dependent.

Distribution coefficient
D (usually expressed as
logD) is the effective
lipophilicity of a
compound at a given
[HA]octanol pH, and is a function of
For an acidic compound: HAaq H+ aq + A- aq D= both the lipophilicity of
[HA]aq + [A-]aq the
un-ionised compound
For a basic compound: and the degree of
BH+aq H+aq + Baq
[B]octanol ionisation.
D=
[BH+]aq + [B]aq

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RELATION BETWEEN LOGD, LOGP AND PH FOR AN ACIDIC DRUG
logP=4.25
5 O

4 50% neutral O
OH
10%
3
N
1%
2
O
logD

0.1%
1

0.01% Cl Indomethacin
0
0.001% neutral
-1

-2
2 3 4 5 6 7 8 9 10
pH For singly ionising acids in general:
pKa=4.50
logD = logP - log[1 + 10(pH-pKa)]

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PH DISTRIBUTION OF BASES

Cl O
4 O

O O

3 N Amlodipine
H
Cl O
O
O pKa=9.3
2
O O NH2

1 N
H
O

H
logD

0 CN
NH3+ N N

S
-1 N N N
H H

-2 H CN Cimetidine
N N
pKa=6.8
-3 S
NH+ N N
H H
-4 For singly ionising bases in general:
3 4 5 6 7 8 9 10 11
logD = logP - log[1 + 10(pKa-pH)]
pH

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PH DISTRIBUTION OF AMPHOTERIC COMPOUNDS
OH

pKa1 = 4.4 pKa2 = 9.8

0.5
NH2
0

-0.5

O
logD

-1
OH
-1.5

-2 NH2
NH3+
-2.5
2 3 4 5 6 7 8 9 10 11 12
pH

MemBiophysics - 24 July 2017 13

LIPOPHILICITY ALSO CHANGES WITH…

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IMPACT OF LOG D7.4 ON DRUG-LIKE PROPERTIES
Log D7.4 < 1: There is good solubility but low absorption and brain penetration, owing to low passive diffusion
permeability. These compounds tend to have high clearance by the kidney, owing to their polarity.
These compounds may exhibit paracellular permeation if the molecular weight is low.

1 < Log D7.4 < 3: This is an ideal range. These compounds generally have good intestinal absorption, owing to a good
balance of solubility and passive diffusion permeability. Metabolism is minimized, owing to lower
binding to metabolic enzymes.

3 < Log D7.4 < 5: These compounds have good permeability but absorption is lower, owing to lower solubility.
Metabolism is increased in this range, owing to increased binding to metabolic enzymes.

Log D7.4 > 5: Compounds in this range tend to have low absorption and bioavailability, owing to low solubility.
Metabolic clearance is high because of high affinity for metabolic enzymes. Vd and half-life are high
because compounds partition into and stay in tissues
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 Biopharmaceutical Classification
Biopharmaceutical classification system (BCS) is an advanced tool used for classifying medicines based on dissolution,
water solubility, and intestinal permeability, which affect the absorption of active pharmaceutical ingredients (API) and
consequently its bioavailability.

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IMPACT OF LOG D ON GASTRIC ABSORPTION

A general guide for optimal gastrointestinal absorption by

passive diffusion permeability after oral dosing is to have a
moderate Log P (range 0–3)

In this range, a good balance of permeability and solubility

exists.

Compounds with a lower Log P are more polar and have

poorer lipid bilayer permeability.

Compounds with a higher Log P are more nonpolar and

have poor aqueous solubility.

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TRADITIONAL METHOD: OCTANOL-WATER
Shake-flask procedure

Phase separation
Change the
equilibrium
conditions

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PREDICTION OF LOG P AND LOG D
Fragment-based logP = S fragments
Atom-based
Knowledge-based

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PREDICTION OF LOG P AND LOG D
Fragment-based
Atom-based
Knowledge-based

logP = S fragments + S corrections

MemBiophysics - 24 July 2017 20

OCTANOL-WATER METHOD DRAWBACKS

The great majority of drugs

contain ionizable groups
Most are basic, and some are
acidic.
Only 5% are not ionizable.

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MORE ASPECTS OF DRUGS INTERACTIONS
2.

1. Drugs may interact with membrane 1.

proteins or receptors in the surface of the
cell membrane producing their effect.

2. Drugs may penetrate cell membrane to Drug

reach their intracellular target.

3. Drugs may change cell membrane and

change the activity of membrane enzymes
or membrane receptors.

Effect

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DRUGS TRANSPORT

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THE BIOLOGIC MEMBRANE

Cellular membranes are highly complex systems

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MEMBRANES BASIC COMPOSITION

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MEMBRANE LIPIDS
Alcohol
H
Phosphatidylcholine (PC)
Choline

Phosphatidylethanolamine (PE)
Ethanolamine

Phosphatidylserine (PS)
Serine

Phosphatidylinositol (PI)
Inositol

Phosphatidylglicerol (PG)
Glicerol
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MEMBRANE LIPIDS

- They can be saturated or insaturated and have 1 to 4 double bonds.

They can have several sizes:
 C12 – Lauric
 C14 – Myristic
 C16 – Palmitic
 C18 – Stearic
 C20 Araquidic

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PHOSPHOLIPIDS NOMENCLATURE

DPPC Dipalmitoylphosphatidylcholine
Fatty acids
DMPC Dimyristoylphosphatidylcholine
Headgroup
DMPE Dimyristoylphosphatidylethanolamine

POPC Palmitoyl-2-oleoyl-glycero-3-phosphorylcholine
POPG Palmitoyl-2-oleoyl-glycero-3-phosphorylglicerol

EPC Egg phosphatidylcholine

MemBiophysics - 24 July 2017 28

CHOLESTEROL

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MEMBRANE FASHION
Brain Cells
EPC:CHOL:SMYEL (1:1:1)
Mycobacterium tuberculosis

DMPC/DMPG
Pulmonary Surfactant (negative charge)
Mycolic acids
DPPC
Curosurf®

Gastric mucosa
Helicobacter pylori
DPPC (gel phase)
DMPE:DMPG:CL
(1:1:1)
Brush Border membrane

DPPC:DMPE:SMYEL (3:3:1)

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PHOSPHOLIPID ASYMMETRY
Normal cell membrane Cancer cell membrane

pHe 7.3–7.4 pHe 6.2–6.9

-
PC PS PE S Cho
M l

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ORGANELLES LIPID COMPOSITION

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LIPIDS-WATER SYSTEM TO MEASURE LOG P AND LOG D

Controlled variables: Ionic strength, pH, temperature…

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PHOSPHOLIPIDS GEOMETRY
Geometry of Structure of
monomers agregates Agregate / Lipid phase

Lysophospholipids –
lead to membrane pore
formation
Micelle

DOPE - induce
membrane fusion
Hexagonal phase II (HII)

phosphatidylcholines – Lamelar phase

lipid bilayers (L)

bilayer Liposomes
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MEMBRANE MODEL SYSTEMS
Advantages membrane model systems:

control the composition choosing the type and concentration of the

phospholipids;
used more than one phospholipids or use cholesterol.
introduce membrane proteins during the liposome preparation.

The complexity of the model can be increased in order to obtain a better

mimetic cell membrane model system.

Mimic
•cell membranes
•physiological barrier

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PARTITION COEFFICIENT METHODOLOGIES

Drug [Drug]octanol
P
[Drug]water
Aqueous Phase

Organic/lipidic [Drug]lipid
Kp 
Phase
Octanol/Water Micelle/Water Liposome/Water Membrane/Water
[Drug]water

Parallel artificial membrane permeability assay

(PAMPA)

Other examples:
Partition Chromatography

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TYPES OF LIPOSOMES

Lipid
bilayer

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DERIVATIVE SPECTROSCOPY – EXPERIMENTAL CONDITIONS

Increasing concentrations of
lipid (in buffer)
Kp determination

or
Derivative
Spectroscopy
Liposomes Micelles

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Incubation

Spectroscopic
Drug + lípid
Analysis
suspensions
Fluorimetry
Addition of drug UV-VIS
Fixed concentration

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SCATTERING ELIMINATION

Scattering

Scattering elimination

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KP DETERMINATION (MICROPLATE PROTOCOL)

Second derivative

2nd derivative
NSAID (M) Third derivative

3rd derivative
3rd derivative

Dm  Da K p LV
D  Da 
1  K p LV

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KP DETERMINATION (MICROPLATE PROTOCOL)
b K p [L]
Dt  a 
1  K p [L]

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