This document discusses various toxicokinetic studies conducted in preclinical stages of drug development. It describes conducting single dose, rising dose, repeated dose toxicity studies in rodents and non-rodents to determine kinetic parameters and assess safety. The importance of toxicokinetic data in setting safe clinical doses is highlighted. Alternative methods to animal testing like in vitro, in silico, cell line techniques and patch clamp method are also summarized.

1. Participants
Shoaib Ahmad 020
Saira Fiaz 021
Muhammad Sohaib 022

2. Introduction
Toxicokinetics: - is defined as the generation of pharmacokinetics
data to design, conduct & Interpretation of drug safety evaluation
studies.
• Preclinical drug safety evaluation or toxicological studies .
• In these studies a minimum of two animal species are
employed, as per
regulation of FDA (Food and Drug administration).
• 1)Rodents.
• 2)Non-rodents.

3. General principle to be considered
A. Quantification of exposure .
B. setting of dose levels
1. Low dose levels .
2. Intermediate dose levels .
3. High dose levels .
C. Extent of exposure assessment in toxicity studies .
D. Complicating factors in exposure interpretation .
E. Route of administration.
F. Determination of metabolites .
G. Statistical evaluation of data.
H. Analytical methods .
I. Reporting

4. Toxicokinetics studies in preclinical stages
Toxicokinetic studies in Preclinical stage:
 Safety assessment:
 Generally safety of a molecule can be performed in in-vivo systems. This step is
not included in the guidelines but it is very useful for the researchers to assess the
systemic exposure .
 Single dose and rising dose studies :
 These studies are usually performed in rodents.
 Plasma samples may be taken in such studies and stored for later analysis.
 Results from single-dose kinetic studies may help in the choice of formulation
and in the prediction of rate and duration of exposure during a dosing interval.
 This may assist in the selection of appropriate dose levels for use in later studies.
 Rising-dose studies are performed in non-rodent models. Here, toxicokinetic
evaluation takes place at various time-points for each new dose level.

5. Repeated-dose toxicity studies :
This study is carried out for four weeks in both rodents as well as
non-rodents.
Help to support dose-selection for subsequent studies .
Performing further repeated dose studies in both rodent and non
rodents up to 6-12 months enable estimation of drug and its
metabolite(s) kinetic parameter assessment as well as long term
clinical exposure assessment.
Genotoxicity studies :
Two in vitro studies and one in vivo study is essential to support
development of drug.
In vivo investigations usually use a rodent micronucleus (bone
marrow or peripheral erythrocytes) test or chromosome aberration
(bone marrow cells) test.

6. Reproduction toxicity studies :
 Reproduction toxicity measurements are taken in studies of fertility (rat),
embryo-foetal development (rat and rabbit) and pre-or post-natal
development (rat).
Studies of fertility :
 Assessment of fertility toxicity has very important, because most of the
drugs used in fertility conditions so has to
strengthen at that time. Usually this can be done in rats.
In pregnant and lactating animals:
 There is a regulatory expectation for Toxicokinetics data in pregnant
animals, although no specific guidance is given.
 Toxicokinetics may involve exposure assessment of dams, embryos, fetuses
or newborn at specified days.
 Secretion in milk may be assessed to define its role in the exposure of
newborns.

7. Carcinogenicity studies:
Sometimes drugs are used for longtime for curing
purposes, this may lead to the toxicity or carcinogenicity.
So lifetime studies in the rodent are needed to support the
long-term clinical use of pharmaceuticals and non-
rodents can also be used.
Monitoring should occur occasionally i.e. more than six
months is not necessary.
However, pharmaceutical companies use various strategies
for such monitoring times (e.g. Weeks 1, 13, 26 and 52)

9. Importance and applications
Generation of kinetic data for systemic exposure and
toxicity assessment of the drug.
Simply it means if the safety/risk ratio is balanced or safety
is more then it will be used as good therapeutic agent.
Important in drug development stages especially in
preclinical stage.
TK evaluation is useful for the setting safe dose level in
clinical phases.
TK evaluation is useful in selection of dose, dosing form,
alternative dosing route, evaluation of toxicological
mechanism, and also used for the setting safe dose level in
clinical phases.

10. Continue.
Toxicokinetic data is important to know the toxic response
to that of drug exposure obtained in drug development
stages.
It is used to set safe dose for clinical use of new drugs.
Useful in the understanding of differences in responses or
sensitivity between individual animals, genders, species or
life stages, and supporting the extrapolation of findings in
experimental animals to humans.
Even though toxicokinetic evaluation is only a small part of
the process of understanding the fate of a drug, it has a
vital part in drug development a role that proceeds to
advance.

11. Different techniques to be used as
an alternative to an animal testing.
• Full thickness skin model (in-vitro method).
• In silico methods.
• Cell line techniques.
• Patch clamp method

12. In vitro method
 Instead of using animals, cell and tissue cultures can be
used to test product ingredients.
 Cell culture experiments can show, for example, the lowest
concentration at which aningredient causes damage to
cells.
 The results enable conclusions to be drawn about the
ingredient’s compatibility with tissue.
 Cell cultures are now also used routinely to test substances
for mutagenic properties.
 Tissue cultures are additionally used to test substances for
compatibility with mucous membranes.

13. In- Silico method
 Substances with similar chemical structures often have similar properties.
 In these cases, therefore, a knowledge of the properties of a few representative
substances is sufficient to be able to deduce the properties of a series of similar
substances.
 By analogy, certain properties of these representative substances can also be
assumed to be properties of the other substances in the series.
 The required calculations are performed using specially developed computer
programs.
 It is anticipated that combinations of such calculations will make it possible to
narrow down the number of substances to be tested.
 Only these selected substances will then have to be tested according to the
legally prescribed test methods.

14. Cell line techniques
The term cell line refers to the propagation of culture after
the first subculture.
Once the primary culture is sub cultured, it becomes a cell
line.
A cell line derived by selection or cloning is referred to as
cell strain.
Cell strain do not have infinite life, as they die after some
divisions.
Types of cells used in cell line:
a)Precursor/ stem cells/ master cells.
b)Undifferentiated but committed precursor cells.
c)Mature differentiated cells.

15. APPLICATIONS OF CELL LINE
 Screening of anti cancer drugs
 Cell based bioassays.
 To determine the cytotoxicity.
 In vitro screening of several drugs.
 Production of anti viral vaccines
 Cancer research, which require the study of uncontrolled cell division in
culture.
 Cell fusion technique.
 Genetic manipulation.
 Gene therapy.
 Recombinant DNA therapy.
 Biotechnology.
 Molecular biology……etc.

16. PATCH CLAMP TECHNIQUE:
The Technique was developed by Erwin neher & bert
Sakman.
Patch clamp technique is a technique in
electrophysiology that allows the study of individual ion
channels in cells.
The technique is used to study excitable cells such as
neurons, muscle fibers and the beta cells of the
pancreas.

17. PROCEDURE:
 The patch clamp technique can be applied to cultured kidney cells
freshly isolated kidney cells or to cells of isolated perfused kidney
tubules.
 Segments of late superficial proximal tubules of rabbit kidney are
dissected and perfused from one end with a perfusion system.
 The non-cannulated end of the tubule is freely accessible to a patch
pipette the patch pipette can be moved through the open end into the
tubule lumen and is brought in contact with the brush border
membrane.
 After slight suction of the patch electrode, giga seals for
instantaneously and single potassium or sodium channels can be
recorded in the cellattached or inside-outcell –excised modal.

18.  In order to obtain exposed lateral cell membranes suitable
to the application of the patch clamp method , pieces of
the tubule are to off by means of a glass pipette.
 As to facilitate the tearing off , the tubules are incubated
for about 5min in 0.5g/L collagen a seat room temperature.
 After tearing off part of the cannulated tubule, clean lateral
cell membranes are exposed at the non-cannulated end.
 The patch pipette can be moved to the lateral cell
membrane and gigaseal scan be obtained.
 It was possible, to investigate potassium channels and
nonselective cations channels in these membranes.

19. EVALUATION:
 In isolated perfused renal tubules , concentration
response curves of drugs which inhibition channel
scan be obtained with the patch clamp technique.
 In isolated cells of the proximal tubule, the whole cell
mode of the patch clamp technique enables the
investigation of the sodium- alanine cotransport
system.