Assignment on Toxicokinetics- Toxicokinetic evaluation in preclinical studies, saturation kinetics Importance and applications of toxicokinetic studies. Alternative methods to animal toxicity testing.

1. 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.
• 1)Rodents-e.g:-Rats, Mice.
• 2)Non-rodents-e.g:-Dog

2. GENERAL PRINCIPLES 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

3. TOXICOKINETIC STUDIES IN PRICLINICAL STAGE
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 .
This safety study is integral part in the CNS, CVS and respiratory assessments.
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.

4. Repeated-dose toxicity studies :
O To give support for phase 1 studies this study is carried out for four weeks in both
rodents as well as non-rodents.
O Help to support dose-selection for subsequent studies .
o 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 :
O Two in vitro studies and one in vivo study is essential to support development of drug.
O In vivo investigations usually use a rodent micronucleus (bone marrow or peripheral
erythrocytes) test or chromosome aberration (bone marrow cells) test.
1. 1

5. Reproduction toxicity studies :
O 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 :
O 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:
O There is a regulatory expectation for toxicokinetic data in pregnant animals, although no
specific guidance is given.
O Toxicokinetics may involve exposure assessment of dams, embryos, foetuses or newborn
at specified days.
O Secretion in milk may be assessed to define its role in the exposure of newborns.
O In some situations, additional studies may be necessary or appropriate in order to study
embryo/foetal transfer and secretion in milk.

6. Carcinogenicity studies:
o Sometimes drugs are used for longtime for curing purposes, this may lead to the
toxicity or carcinogenicity.
o So lifetime studies in the rodent are needed to support the long-term clinical use of
pharmaceuticals and non-rodents can also be used.
o Dose selection is usually determined as the maximum tolerated dose (MTD), which is a
25-fold AUC ratio (rodent to human), or by dose-limiting pharmacodynamic effects,
saturation of absorption, or a maximum feasible dose.
o Monitoring should occur occcasinally i.e more than six months is not necessary.
o However, pharmaceutical companies use various strategies for such monitoring times
(e.g. Weeks 1, 13, 26 and 52, Weeks 1 and 26, or Weeks 26 and 52).
o It should be noted that, owing to high variability in plasma concentration, toxico kinetic
data from aged rats (above one year old) are not useful for estimating exposure.
o Sampling times depend on available kinetic data but can range from full profile (up to 24
h) to limited time-points which are earlier stated.

7. TOXICOKINETICS STUDIES IN CLINICAL PHASES
• Regulatory bodies around the world outlining that toxicity studies are necessary to
support human Phase I, II and III studies, and product license application is available.
• The magnitude of the preclinical toxicokinetic evaluation for each clinical phase varies
significantly among pharmaceutical companies.
• For Phase I investigations the company might only generate toxicokinetic data from
the four-week repeat-dose toxicity studies.
• Full pharmacokinetic profile (including in vitro metabolism studies), and toxicokinetic
measurements from four-and 13-week repeat-dose toxicity studies prior to Phase I is
necessary.
• Toxicity assessments enable the No Observed Effect Level (NOEL) or No Observed
Adverse Effect Level (NOAEL) to be established for a potential new drug, based on
clinical observations, bodyweight, food consumption, clinical pathology, organ
weights, and histopathology.
• Toxicokinetic data from either NOEL or NOAEL can be used to give guidance to the
clinical investigator by providing suitable safe starting and upper doses in the initial
single-dose Phase I study.
• For further clinical studies using multiple dosing, toxicokinetic data from toxicity
studies provide information on possible increases or decreases of drug in plasma.

8. IMPORTANT AND APPLICTIONS OF
TOXICOKINETICS
• 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.
• TK studies also used to reduces the animal number.

9. CONTINUE:
•TK evaluation is very important in drug development phase in both regulatory and
scientific perspective.
•TK data are practically used for the purpose of drug discovery such as lead-optimization
and candidate-selection.
• 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 TECNIQUES TO BE USED AS AN
ALTERNATIVES TO ANIMAL TESTING….
• Full thickness skin model (invitro method).
• In silico methods.
• Cell line techniques.
• Patch clamp method.

12. IN-VITRO METHODS
• 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 an
ingredient 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.
• A familiar example is the Hen’s Egg Test, which scientists are now further developing
so that it can be used to test for mutagenic properties as well.

13. IN-SILICO METHODS
• 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 TECHNIQUE
• 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. TYPES OF CELL LINES:
• Mainly two types;
1)Finite cell line.
2)Continuous cell line.
FINITE CELL LINE:
• The cell line with limited culture life spans are referred to as Finite cell line.
• The cell normally divide 20-100 times before extinction.
• The actual number of doublings depends on the species, cell lineage differences,
culture conditions etc.

16. CONTINUOUS CELL LINE:
• The continuous cell lines are transformed, immortal and tumorigenic.
• A few cells in culture may acquire different morphology and get altered. Such cells are
capable of growing faster resulting in an independent culture.
• The progeny derived from these altered cells have unlimited life.

17. 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.

18. 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.

19. PATCH CLAMP TECHNIQUE IN KIDNEY CELLS:
PRINCIPLE:-
• In the different parts of the kidney fluid is reabsorbed and substances may be
transported either from the tubule lumen to the blood side (reabsorption) or vice versa
(secretion).
• Besides active transport and coupled transport systems, ion channels play an important
role in the function of kidney cells.
• The various modes of the patch clamp technique (cell-attached, cell-excised, whole-cell
mode) allow the investigation of ion channels.

20. 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 cell-
attached or inside-outcell –excised modal.

21. • 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.

22. 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.

23. Applications of Patch Clamp Technique:
• For the evaluation of antiarrhythmics agents.
• In kidney cells.
• Used for isolated ventricular myocytes from Guinea pig to study a cardio
selective inhibition of the ATP sensitive potassium channel.
• To identify multiple types of calcium channels.
• To measure the effect of potassium channel openers.
• Used in the molecular biology.
• Voltage clamp studies on sodium channels.
• Used to investigate a wider angel of electrophysiological cell properties.
• Measurement of cell membrane conductance.