Advanced Medicinal
Chemistry
Lecture 5:
Drug Metabolism and
Pharmokinetics - 2
Barrie Martin
AstraZeneca R&D Charnwood
Quantitative DMPK
Quantitative DMPK involves the measurement of a
number of pharmacokinetic parameters which
describe the fate of compounds in the body.
These can be used to compare compounds, to
highlight deficiencies in compounds (e.g. high
metabolism) and to predict how the potential drug will
behave in man – generate dose predictions.
The One Compartment Model
The simplest model to describe the fate of a compound in
the body is the ‘one compartment model’, which is
analogous to the metabolism of the compound in a beaker
containing an enzyme solution.
Although simplistic, many of the basic parameters of
pharmacokinetics (half-life, clearance and volume of
distribution) are well illustrated using this model.
Injection
BLOOD
Half-Life (T1/2) and the Elimination Rate Constant
(kel)
Following an iv injection, we might expect the plasma concentration of a
compound to vary over time as shown below:
Plasma
conc
ln c = ln co - kelt
ln c
x
-k t
C = Coe el
x
x
x
x
x
x
x
Time
- kel = slope
x
x
x x
Time
Under first order kinetics, rate of metabolism is proportional to the compound
concentration, so the rate (and gradient) decreases over time.
By plotting ln c vs. time, we can determine the elimination rate constant (kel) from the
slope of the line and, by extrapolation back to t = 0, the initial plasma concentration c0.
The main use of kel is to determine the half-life (t1/2) of the compound, defined as:
The time taken for the concentration of drug in the blood or plasma to decline to half of
its original value.
At t1/ ,
2
c
1
=
co
2
ln (0.5) = - kel t1/ ,
2
 t1/
2
=
0.693
kel
Volume of Distribution (VD)
ln c
Consider now the case where a compound is dissolved
in double the volume, what would happen to t1/2?
Time
When the volume doubles (red line), the initial
concentration halves and the half life doubles
In biological systems, compounds can distribute out of the plasma into tissues.
The volume of distribution (VD) is therefore:
The theoretical volume (L) that all the drug in the body would have to occupy if it
were present at the same concentration as that found in plasma.
It is a measure of how readily drug diffuses out of the plasma into the tissues and can
affect t1/2.
– Low VD: drug confined to plasma (vulnerable to the liver and metabolism)
– High VD: drug equilibrates with tissues
Generally: Acids - high PPB, low VD
Neutrals - ready equilibration, but not necessarily retained in the tissue - higher VD
Bases - high affinity for phospholipids in membranes (negatively charged) - highest VD
Clearance (Cl)
Clearance (Cl) is a measure of how readily compounds are eliminated (i.e.
metabolised or excreted)
It is defined as:
The volume of plasma (or blood) from which all drug is removed per unit time. (n.b.
units of Cl are units of flow ml/min)
Cl is a constant, characteristic of a drug in a particular species. It is a scaling factor
that relates the plasma concentration of a compound to the rate of elimination
( dD
)Rate of elimination (ng/min) = Clearance x concentration (ng/ml)
dt

Rate of elimination (ng/min)
Clearance (ml/min) =
concentration (ng/ml)
Clearance (Cl)
Over a small interval of time, dt:
Amount eliminated during interval dt = Cl x Conc x dt
Integrating this over the whole concentration-time profile gives:
Plasma
Conc
Total amount eliminated = Dose = Cl x AUC
dt
Cl = Dose/AUC
Conc
Time
kel = Conc. x Cl
Clearance is related to t1/2 and VD. If Cl halves, then the half life doubles
(because the rate of metabolism halves) and, as we have seen, doubling the
volume doubles the half-life (because the concentration at the metabolising
enzyme has halved).
The precise equation is:
t1/2 = 0.693 x VD/Cl
Clearance, Volume and HalfLife
Half-life is not predictable from clearance alone
- clearance characterises elimination of drug from plasma/blood
- half-life also depends on distribution of drug outside the plasma (systemic
circulation not a closed system)
For a rapid (bolus) iv dose; where D is the amount of drug in the body at time t;
Rate ofh elimination,
dD
= kelD = Clearance x concentration
dt
But D = VD x concentration, so kel x VD = Cl
Rem:
t1/2 =
0.693
kel
,
t1/2 =
or kel =
Cl
VD
0.693 x VD
Cl
NB: t½ is NOT a measure of how rapidly the drug is metabolised
Clearance, Extraction and Absorption
For the three main elimination processes, metabolism, renal, and
biliary,
clearances are additive, i.e.
ClT = ClM + ClR + ClB
e.g. If ClT = 20ml/min/Kg and %dose as parent
in urine = 20% Then ClR = 4ml/min/Kg
Can relate clearance to liver blood flow (Q):
Cl = E x Q (E = extraction ratio)
If Cl ~ Q (i.e. E~1), then 1st pass metabolism is a likely problem.
E = (Cin –Cout) / Cin
Fraction absorbed (fa)
can now be worked out
if know %F
Q = flow
(ml/min)
Q: Rat
~ 70 ml/min/kg
Dog
~ 40 ml/min/kg
Man
~ 20 ml/min/kg
Cin = conc. entering liver
Cout = conc. leaving liver
The Two Compartment Model
Injection
k12
BLOOD
TISSUES
k21
The two compartment model more accurately describes observed DMPK data. In this
model, the compound is viewed as being able to equilibrate with a second compartment
as, in addition to metabolism, drug is distributing into the tissues.
Drug accumulates in the tissues because the plasma concentration is initially greater
than the tissue concentration and so k12>k21. However, eventually the plasma
concentration falls to such an extent that the net drug movement is from tissues back
into blood. At this point in time, plasma concentration begins to fall far more slowly as
diffusion from tissues back into blood becomes more and more significant.
Plasma
conc
ln c
x
Distribution phase
x
cp = c1e-k1t + c2e-k2t
x
x
x
Elimination phase
x
Time
x
x
x x
x
x
Time
Oral Dosing - Bioavailability
Plasma
conc
Upon oral dosing of a drug, there is an
initial increase in the systemic
concentration of the drug, as it is absorbed
from the gut.
iv
oral
As absorption is completed and the
compound is eliminated from the body, the
concentration of drug decreases over time.
Time
Absorption phase
Elimination phase
Oral Bioavailability (F%) is defined as:
The fraction of the dose which makes it to the systemic circulation (i.e. survives 1st pass metabolism).
F=
AUC after an oral dose
AUC after an equivalent iv dose
Limiting factors include:
Chemical instability, eg acid sensitive compound in the stomach
Incomplete absorption - solubility, formulation
Gut wall metabolism, 1st pass metabolism - labile functional groups
Predicting in vivo DMPK using in vitro Measurements
A number of DMPK parameters may be predicted from in vitro assays to
build up understanding of compound properties – predict behaviour in
man
• Absorption – Pampa, Caco-2
• Clearance - Microsomes, Hepatocytes
• Distribution - Plasma protein binding,
• Cytochrome P450 inhibition (5 major isozymes)
• Physical parameters – log D, pKa, solubility
These assays are used extensively to profile compounds and filter out
those that do not possess the required properties to be drugs.
Permeability in vitro
Pampa (Parallel Artificial Membrane
Caco-2 Human colon adenocarcinoma
Permeability Assay)
Models transcellular (passive) absorption only
Human colon carcinoma cell line which grow as
monolayers, similar to small intestine enterocytes
All mechanisms modelled - express key transporter
proteins (e.g. PGP)
No tissue culture
Culturing over several days
Assay 96 cpd
(2 days experimental and analysis)
Assay 96 cpds AB or 48 cpd BA
Artificial membrane separates 2 compartments
drug
Can investigate different directions (A-B) and (B-A)
Apical chamber
(gut lumen)
drug
Absorptive Flux (A-B)
Basolateral
Chamber (blood)
Secretory flux (B-A) drug
Apparent permeability (Papp) measurements calculated (units are cm/sec x 1E-6)
Typically use PAMPA assay as primary assay for absorption followed by oral
data in two species
Both can be related to human fraction absorbed
Cell
monolayer
In vitro Measurement of
Metabolism
In vivo Cl can also be predicted using in vitro assays:
Microsomes (species – rat, dog, human)
A subcellular fraction obtained by centrifugation of liver cells. Mainly composed of
vesicles containing CYP450 enzymes formed from fragmented endoplasmic
reticulum. Perform Phase I reactions.
Hepatocytes (species – rat, dog, human)
Isolated whole liver cells. Capable of performing both Phase I and II reactions.
Rates of metabolism are reported as intrinsic clearance - Clint (ul/min/106 cells)
Typically: Rat Hepatocytes Clint Low (< 10), Moderate (10-20), High (> 20)
Human Microsomes Clint Low (< 15), Moderate (15-30), High (> 30)
In vitro – In vivo scaling
Cannot measure in vivo human PK until phase I trials. If we can predict consistently how a
compound will behave in other species, then we can have greater confidence that it will behave
predictably in man. In vitro - in vivo scaling is the prediction of in vivo Cl from Clint measured in
hepatocytes.
The in vitro assay gives the maximum possible metabolic rate – but need to factor in drug
delivery i.e. liver size, PPB, liver blood flow etc, extrahepatic clearance.
Clint: ml/min/106 cells
120 x 106 hu heps
per gram liver
Clint*: ml/min/kg
Liver
ml/min/g liver
g liver per kg body weight
ml to ml
Species 1
Species 2
Human
In vitro
Heps low
Heps low
Hu heps low
In vivo
Compound scales
Compound scales
FTIM
DMPK well understood, predictable from hepatocytes, PPB etc.
Dose Prediction to Man
DMPK measurements enable prediction of human PK parameters. Incorporation
of potency and safety data enables Dose to Man (DtM) and safety margin
predictions.
Predicted human PK appropriate for once a day oral dosing:
Therapeutic Dose
Pred. Human DMPK
Plasma
conc
< 5mg/kg uid
t1/2 6-12h, F > 30%
Toxic
Plasma
conc
Therapeutic
Safety
Margin
Cssmax
Cssmin
(typically 3 x potency)
Time
Ineffective
Time
Summary
Definitions and qualitative aspects of absorption, distribution and elimination.
Quantitative PK studies allowing the determination of:
Absorption
Distribution
Elimination
Permeability Efflux Aqueous
Renal Metabolic Biliary
solubility excretion stability excretion
fa
Protein Tissue
binding binding
Cl
%F
(poor/med/high)
VD
t1/2
(UID/BID/>3-4x)
Knowledge of these parameters allows identification of where improvements
need to be made to end up with a pharmacokinetically optimized drug.