üUnderstanding of in vitro-in vivo correlation and frontloading PK/PD is crucial for the
compound selection even at an early stage.
ü Obviously, an excellent PD data
can save a poor PK as long as the PD effect is notable.
In Vivo ADME studies are mostly custom-designed. Often these studies are performed in multiple
species by administering the test compounds by variety of routes (includes
oral, intravenous, dermal, intraperitoneal, inhalation to name a few). These in-life studies provide information
for:
·
mass balance
·
metabolite isolation and
characterization
·
dosing frequency
·
routes of administration
·
exposure
·
interspecies comparisons
·
PK/TK
·
tissue distribution
studies to determine clearance routes and rates
·
tissue half-life
·
Potential sites of
toxicity after systemic exposure.
The basic assumption in all pre-clinical ADME studies is that the
administered NCEs and dose levels are not toxic and do not cause any harm to
the welfare of the animals.
In
general rats of similar age and weight are chosen for the experiments. Experimental procedures are kept strictly in
accordance with the related ethics regulations.
In many studies, all the subjects are kept in an environmentally controlled
breeding room (temperature maintained at about 25 ± 2 oC and with a 12 h light
/ 12 h dark cycle) for at least once week before starting the experiments and
fed with standard laboratory food and water.
Prior to each experiment, the rats are fasted for 12 h with free access
to water.
RANK ORDER OF COMPOUND
EXPOSURE: A small cohort of animals (selected number),
is administered compound orally (p.o.) or by intraperitoneal injection (i.p.)
at a single dose (5 - 50 mg/kg). Blood samples are collected at 20 and 120
minutes. The plasma samples are analyzed which provides a snapshot of compound
exposure as the area under the curve (AUC(20-120 min)), providing a rank order
of estimated AUC values to prioritize compounds for further investigation.
Based on the concentration vs. time data, pharmacokinetic
parameters such as body clearance, volume of distribution and bioavailability
can be calculated, information which is used to refine in vivo experiments with
the same series of drugs in the development.
MATERIAL BALANCE STUDY:
- Groups of male and female rats at a single dose level (equal
number of male and female rats are used)
- Daily collection of urine and feces for 7 days
- Collect blood and major internal organs, and retain carcass (corpse) upon necropsy
- Total radioactivity level in each sample is assayed and the
recovered dose is compared with the administered dose. The percentage of
distribution of the administered dose in urine, feces, and each major
internal organ and the half-time for excretion are calculated.
A
necropsy
is a surgical examination of a dead body, most commonly a dead animal, in order
to learn why the animal died. A more common word for necropsy is autopsy. Either way, it's the
dissection of a corpse performed to learn something about the cause of death or
about a particular disease.
PLASMA LEVEL AND BIO-AVAILABLITY
STUDY:
The
objective of this study is to examine the pharmacokinetic profiles of the
chemicals under test. These studies can be conducted by using unlabeled
chemicals provided that appropriate analytical procedures (e.g., HPLC) are
available.
·
Groups
of 5 male and 5 female rats per route of dosing
·
One
group is dosed intravenously and the second group is dosed by an extra-vascular
route (e.g., oral, percutaneous)
·
The
dosages for the 2 groups need not be identical
·
Blood
is sampled at frequent intervals (e.g., 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, and
48 hr), and plasma levels of the chemical are assayed.
·
The
plasma- concentration-time data are analyzed to obtain the pharmacokinetic
profile, including the area under the curve (AUC). Comparison of the AUCs after
extra-vascular and intravenous dosing, after correcting for the dosage,
provides a measure of bioavailability.
Alternatively, if a radioactive chemical is used, comparison of the
administered doses excreted in urine can be used to calculate the approximate
bioavailability of the orally dosed chemical.
IN VIVO PHARMACOKINETIC STUDY
(PLASMA PHARMACOKINETICS):
Depending up on the study, rats were
randomly assigned for pharmacokinetic investigation. Test compound is administered by i.v.
injection via the lateral tail vein and orally at the dose of for example, 10
and 20 mg/kg, respectively. At the time points of 0 (pre-dose), 5, 15, 30, 45,
60, 90, 120, 240 and 480 min post injection / administration, blood samples
(0.5 mL) are collected in heparinized (add heparin to (blood or a container about to be filled with
blood) to prevent it from coagulating) tubes
from the orbital vein, and then centrifuged to obtain plasma. The plasma is
stored at −70 °C prior to analysis by HPLC.
METABOLISM STUDY IN RAT URINE
AND FECES:
Similar to above study, but here feces
and urine samples are separately collected at 24 h after administration of test
compound. All the samples were stored at
-70 oC prior to analysis.
TISSUE DISTRIBUTION STUDIES:
For tissue distribution study, number
of rats is first divided into several groups randomly and test compound is
administered intravenously through the tail vein at a particular dose, say 20
mg/kg. After injection, the rats are
sacrificed at 0.5, 1.0, 2.0, 4.0 and 8.0 h following administration and the
tissue specimens including lung, liver, heart, spleen, stomach, small
intestine, brain, thymus, muscle, fat and kidney are collected. Tissue samples are
then rinsed in saline and blotted dry with filter paper, and then weighed for
wet weight and homogenized in ice-cold physiological saline solution (500
mg/mL). The obtained tissue homogenates are stored at −70 °C until analysis
performed.
ALTERNATIVE ROUTES OF ADMINISTRATION:
Alternative routes of administration,
typically intraperitoneal (i.p.) or subcutaneous (s.c.), may be used depending
on the targeted type of treatment and indication or to avoid first-pass
metabolism in the liver occurring after absorption from the gut wall when oral
dosing is used.
REPEATED DOSE STUDY:
Other typical experimental set-ups
include a repeated dose study, where the drug is administered once a day for up
to two weeks. This experimental design can be used to find out not only the
drug pharmacokinetics but also pharmacokinetic changes in the drug, for
example, in a situation where the drug metabolism is either induced or
saturated.
____________________________________________________
When the
bioanalysis has been completed and the results processed to gain plasma
concentrations, plasma concentration vs. time data is then input into a
software program for the calculation of PK parameters.
Pharmacokinetic parameters calculated
include:
The area under the plasma concentration–time curve (AUC) is first calculated.
Cmax: After the drug administration, the highest
concentration obtained is considered the peak plasma concentration.
Tmax: Time
take for peak plasma concentration is Tmax
C0: Concentration at time zero
Vd: Volume of distribution. The apparent volume to which the mass of compound
is distributed in the body at any given time.
CL: Clearance. This value describes
the tendency of the NCE to disappear from plasma.
T1/2: Half-life. Time taken for the plasma
concentration of an NCE to halve.
BA: Bioavailability. This is the
fraction of the NCE that reaches the systemic circulation unchanged.
____________________________________________________
WHAT IS A GOOD PK PROFILE OF A PRECLINICAL CANDIDATE THAT IS
BEING CONSIDERED FOR TAKING INTO DEVELOPMENT?
Ideally, preclinical candidate should,
have acceptable solubility for development;
be completely absorbed, preferably via passive absorption;
have high bioavailability (e.g., F>50%) for oral drug;
have a low plasma clearance CL (e.g, <30% blood flow), long half‐life (t1/2) (e.g., >6 hrs), and
acceptable distribution volume;
have a linear kinetics, i.e., exposure proportional to dose and a clear PK/PD correlation;
be eliminated by several pathways, i.e., renal excretion and hepatic metabolism, also metabolised preferably by more than one enzyme for de‐risking DDI;
have a simple metabolite profile, with no reactive metabolite;
have no obvious CYP and major transporters like P‐gp inhibition or induction or low DDI potential;
and
have a sufficient or at least acceptable safety margin (safety margin >10x, depending on different
therapeutic targets).
