SATURATION KINETICS

The rate process of a drugs ADME are dependent upon carrier or enzymes that are
substrate-specific, have definite capacities, and susceptible to saturation at high drug
concentration.In such cases, an essentially first-order kinetics transform into mixture of
first and zero order rate processes and pharmacokinetic parameters changes with size of
administered dose.The pharmacokinetics of such drugs are said to be dose-dependent.
The other terms synonymous with it are mixed-order, nonlinear, and capacity-limited
kinetics.
There are several test to detect non linearity in pharmacokinetics,
1. Determination of steady state plasma concentration at different doses. If the steady state
concentrations are directly proportional to the dose, then linearity in the kinetics exists.
Such proportionality is not observable when there is no nonlinearity.
2. Determination of some of the important pharmacokinetic parameters such as fraction
bioavailable, elimination half life or total systemic clearance at different doses of the
drug. Any change in these parameters which are usually constant, is indicative of
nonlinearity.
Drugs showing saturation kinetics have these following features
 Elimination of drug does not follow simple first order kinetics (non linear)
 Elimination half life changes as dose is increased.
 AUC is not proportional to the amount of bioavailable drug. The saturation my be
effected by other drugs that require the same enzyme or carrier mediated system.
 Ratio of metabolites of a drug may be affected by a change in dose
Causes of Non-linearity
1. Drug Absorption
2. Drug Distribution
3. Drug Metabolism
4. Drug Excretion

Drug Absorption
Non-linearity in drug absorption arises from 3 important sources
1. When absorption is solubility or dissolution rate limited
e.g griseofulvin`. At higher doses, a saturated solution of the drug is formed in the GIT or
at any other extra vascular site and the rate of absorption attains a constant value.
 2. When absorption involves carrier mediated transport system e.g
absorption of riboflavin, ascorbic acid, cyanocobalamin etc. saturation of the transport
system at higher doses of these vitamins results in nonlinearity.
3. When pre systemic gut wall or hepatic metabolism attains saturation e.g
propranolol, hydralazine and verapamil. Saturation of presystemic metabolism of these
drug at at high doses leads to increased bioavailability
The parameters affected will be F, Ka, Cmax ana AUC. A decrease in these parameter is
observed in the former two cases and increase in latter case. Other causes of non-linearity
in drug absorption are changes in gastric emptying and GI blood flow and other
physiological factors.
Drug Distribution
Non-linearity in distribution of drugs administered at high doses may be due to,
1. Saturation of binding sites on plasma proteins e.g phenylbutazone and naproxen. There is
finite number of binding site for a drug on plasma proteins and, theoretically, as the
concentration is raised, so too is the fraction unbound.
2. Saturation of tissue binding sites e.g thiopental and fentanyl. With large single bolus
doses or multiple dosing, saturation of tissue storage site can occur.
In both cases, the free plasma drug concentration increases but Vd increases only in the
former case where it decreases in the latter. Clearance is also altered depending upon the
extraction ratio of the drug
Drug Metabolism

The nonlinear kinetics of most clinical importance is capacity-limited metabolism since small
changes in dose administered can produce large variation in plasma concentration at steady-state.
Two important causes of non-linearity in metabolism are,
 Capacity-limited metabolism due to enzyme and/or cofactor saturation. E.g
phenytoin, alcohol, theophylline etc
 Enzyme induction e.g carbamazepine, where a decrease in peak plasma concentration
has been observed on repetitive administration over a period of time.
Other causes of non-linearity in biotransformation include saturation of binding
sites, inhibitory effect of the metabolite on enzyme and pathological situation such as
hepatotoxicity and changes in hepatic blood flow
 Drug Excretion

The two active process in renal excretion of a drug that are saturable are-
1. Active tubular secretion e.g Penicillin G . After saturation of the carrier system, a
decrease in renal clearance occurs.
2. Active tubular reabsorption e.g Water soluble vitamins and glucose. After saturation of
the carrier-system, an increase in renal clearance occurs
Other sources of non-linearity in renal excretion include forced diuresis, changes in urine
PH, nephrotoxicity and saturation of binding sites.

Biliary secretion, which is also an active process, is also subject to saturation e.g tetracycline and
indomethacin

MICHAELIS MENTEN EQUATION

The kinetics of capacity-limited or saturable process is best described by Michaelis-Menten
equation.

V0 = initial velocity
[S] =substrate concentration
Vmax =maximum velocity
Km =substrate concentration at half Vmax

• As enzyme –catalysed reactions are saturable, their rate of catalysis does not show a
linear response to increasing substrate
 • If the initial rate of reaction is measured over a range of substrate concentration [S], the
initial reaction rate (V0 ) increases as [S] increases.
• As [S] gets higher , the enzyme become saturated with substrate and the initial rate
reaches Vmax, the enzyme’s maximum rate.
A plot of Michaelis Menten equation

Initially the rate increases linerly (First order) with concentration , become mixed order at high
concentration and then reaches maximum (Vmax) beyond which it proceeds at constant rate
(zero order)
The Michaelis-Menten kinetic model of a single-substrate reaction is shown above
 There is an essential bimolecular reaction between the enzyme E and substrate S to
form the enzyme-substrate complex ES .
 The rate of enzymatic reaction increases with increase of substrate concentration up to a
certain level called Vmax;
 At Vmax, increase in substrate concentration does not cause any increase in reaction rate
as there no more enzyme E available for reacting with substrate S
 The rate of reaction becomes dependent on the ES complex and the reaction become
unimolecular reaction with an order of zero.
The Michaelis-Menten equation is the basis for most single substrate enzyme kinetics
Two crucial assumption underlie this equation are,
1. Quasi-steady-state assumption- that the concentration of the substrate bound enzyme
changes more slowly than those of the product and substrate
2. Total enzyme concentration does not change over time
The Michaelis-Menten constant Km is experimentally defined as the concentration at
which the rate of enzyme reaction is half Vmax.

Three situation can now be considered depending upon the values of Km and C
1. When Km=[S]
V0=Vmax/2 (first order – mixed order – zero order)
i.e the rate of process is equal to one-half its maximum rate
2. When Km>>[S]
V0 = Vmax[S]/ Km (first order)
The above equation is identical to the one that describes first order elimination of drug where
Vmax/km = KE. This means that the drug concentration in the body that results from usual
dosage regimens of most drug is well below the Km of the elimination process with certain
exceptions such as phenytoin and alcohol.
3. When Km<<[S]
V0 = Vmax (zero order)
The rate process occurs at a constant rate Vmax and is independent of drug concentration e.g
metabolism of ethanol.
 TOXICOKINETICS
TOXICOKINETICS
toxicokinetics is defined as the generation of pharmacokinetic data, either as
an integral component in the conduct of non-clinical toxicity studies or in specially designed
supportive studies, in order to assess systemic exposure. These data may be used in the
interpretation of toxicology findings and their relevance to clinical safety issues. It also helps to
understand the relationship between observed toxicity and administered dose. Toxicokinetic
evaluation is both a regulatory and scientific requirement in the drug development process. Many
regulatory guidelines recommend toxicokinetic measurement. 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.
Toxicokinetic procedures may provide a means of obtaining multiple dose
pharmacokinetic data in the test species, if appropriate parameters are monitored, thus avoiding
duplication of such studies; optimum design in gathering the data will reduce the number of
animals required.
Toxicokinetics is thus an integral part of the non-clinical testing programme; it should enhance
the value of the toxicological data generated, both in terms of understanding the toxicity tests and
in comparison with clinical data as part of the assessment of risk and safety in humans. Due to its
integration into toxicity testing and its bridging character between nonclinical and clinical
studies, the focus is primarily on the interpretation of toxicity tests and not on characterising the
basic pharmacokinetic parameters of the substance studied.
The need for toxicokinetic data and the extent of exposure
assessment in individual toxicity studies should be based on a flexible step-by-step approach
and a case-by-case decision making process to provide sufficient information for a risk and
safety assessment.

THE OBJECTIVES OF TOXICOKINETICS AND THE PARAMETERS WHICH MAY

BE DETERMINED
The primary objective of toxicokinetics is:
• to describe the systemic exposure achieved in animals and its relationship to dose level and the
time course of the toxicity study.
Secondary objectives are:
• to relate the exposure achieved in toxicity studies to toxicological findings and
contribute to the assessment of the relevance of these findings to clinical safety.
• to support the choice of species and treatment regimen in non-clinical toxicity studies.
• to provide information which, in conjunction with the toxicity findings, contributes to the
design of subsequent non-clinical toxicity studies.
These objectives may be achieved by the derivation of one or more pharmacokinetic parameters
from measurements made at appropriate time points during the course of the individual studies.
These measurements usually consist of plasma (or whole blood or serum) concentrations for the
parent compound and/or metabolite(s) and should be selected on a case-by-case basis

• Following parameters are usually measured

– maximum plasma concentration (C)
– area under curve (AUC) of plasma concentration
(exposure) versus time
– time to reach maximum plasma concentration (Tmax).
Toxicity studies which may be usefully supported by toxicokinetic information include single
and repeated-dose toxicity studies, reproductive, genotoxicity and carcinogenicity studies.

PRINCIPLES INVOLVED IN TOXICOKINETICS

1.QUANTIFICATION OF EXPOSURE
The quantification of systemic exposure provides an assessment of the burden on the test
species and assists in the interpretation of similarities and differences in toxicity across
species, dose groups and sexes. The exposure might be represented by plasma (serum or
blood) concentrations or the AUCs of parent compound and/or metabolite(s). In some
circumstances, studies may be designed to investigate tissue concentrations. When designing
the toxicity studies, the exposure and dose-dependence in humans at therapeutic dose levels,
should be considered in order to achieve relevant exposure at various dose levels in the animal
toxicity studies
Pharmacodynamic effects or toxicity might also give supporting evidence of exposure or even
replace pharmacokinetic parameters in some circumstances.

Toxicokinetic monitoring or profiling of toxicity studies should establish what level of exposure
has been achieved during the course of the study and may also serve to alert the toxicologist to
non-linear, dose-related changes in exposure (Note 3) which may have occurred. Toxicokinetic
information may allow better interspecies comparisons than simple dose/body weight (or surface
area) comparisons.

2.EXTEND OF EXPOSURE
In toxicity studies, systemic exposure should be estimated in an appropriate number of animals
and dose groups to provide a basis for risk assessment.
Concomitant toxicokinetics may be performed either in all or a representative proportion of the
animals used in the main study or in special satellite groups. Normally, samples for the
generation of toxicokinetic data may be collected from main study animals, where large animals
are involved, but satellite groups may be required for the smaller (rodent) species.
The number of animals to be used should be the minimum consistent with generating adequate
toxicokinetic data. Where both male and female animals are utilised in the main study it is
normal to estimate exposure in animals of both sexes unless some justification can be made for
not so doing.

3.Contribution to the setting of dose levels in order to produce adequate exposure

The setting of dose levels in toxicity studies is largely governed by the toxicology findings and
the pharmacodynamic responses of the test species. The following toxicokinetic principles may
contribute to the setting of the dose levels.
1. Low dose levels
At the low dose, preferably a no-toxic-effect dose level, the exposure in the
animals of any toxicity study should ideally equal or just exceed the maximum
expected in patients
2. Intermediate dose levels
Exposure at intermediate dose levels should normally represent an appropriate multiple
(or fraction) of the exposure at lower (or higher) dose levels dependent upon the
objectives of the toxicity study.
3.Higher dose level
The high dose levels in toxicity studies will normally be determined by toxicological
considerations.
Where toxicokinetic data indicate that absorption of a compound limits exposure to
parent compound and/or metabolite(s) , the lowest dose level of the substance producing the
maximum exposure should be accepted as the top dose level to be used.

4.SAMPLING POINT
The time points for collecting body fluids in concomitant toxicokinetic studies should be as
frequent as is necessary, but not so frequent as to interfere with the normal conduct of the study
or to cause undue physiological stress to the animals. In each study, the number of time points
should be justified on the basis that they are adequate to estimate
exposure . Sample size is typically 0.25–0.50 ml /day in rodents and up to1ml /day in non-
rodents.
5. ROUTE OF ADMINISTRATION
The toxicokinetic strategy to be adopted for the use of alternative routes of administration, for
example by inhalation, topical or parenteral delivery, should be based on the pharmacokinetic
properties of the substance administered by the intended route. If the drug is intended to
administer through oral route then oral toxicity should be checked
If a new clinical route of administration is established, then it is necessary assess the effect of
changing the clinical route. (effect on safety margin).
This process may include a comparison of the systemic exposure to the compound and/or its
relevant metabolite(s) (AUC and Cmax) in humans generated by the existing and proposed
routes of administration. If the new route results in increased AUC and/or Cmax, or a change in
metabolic route, the continuing assurance of safety from animal toxicology and kinetics should
be reconsidered.

6.METABOLITE DETERMINATION
A primary objective of toxicokinetics is to describe the systemic exposure to the administered
compound achieved in the toxicology species. However, there may be circumstances when
measurement of metabolite concentrations in plasma or other body fluids is especially important
in the conduct of toxicokinetics

 Measurement of metabolite concentrations in plasma or other body fluids is also

important in the conduct of toxicokinetics.
 When the administered compound acts as a 'pro-drug‘
 compound is metabolized to one or more pharmacologically or toxicologically active
metabolites
 drugs which are extensively metabolized and the measurement of the metabolites is only
the quantifiable factor
7.STATISTICAL EVALUATION OF DATA
The data should allow a representative assessment of the exposure. However, because large
intra- and inter-individual variation of kinetic parameters may occur and small numbers of
animals are involved in generating toxicokinetic data, a high level of precision in terms of
statistics is not normally needed. Consideration should be given to the calculation of mean or
median values and estimates of variability

8.ANALYTICAL METHODS
The analytical methods to be used in toxicokinetic studies should be specific for the entity to be
measured and of an adequate accuracy and precision. The limit of quantification should be
adequate for the measurement of the range of concentrations anticipated to occur in the
generation of the toxicokinetic data.
The choice of analyte and the matrix to be assayed (biological fluids or tissue) should be
stated and possible interference by endogenous components in each type of sample (from
each species) should be investigated. Plasma, serum or whole blood are normally the matrices
of choice for toxicokinetic studies.
The methods used in analytical purposes are Gas chromatography, HPLC (UV or fluorescence),
LC, LC–MS, LC-MS-MS, and capillary electrophoresis

9.REPORTING
A comprehensive account of the toxicokinetic data generated, together with an evaluation of the
results and of the implications for the interpretation of the toxicology findings should be given.
An outline of the analytical method should be reported or referenced. In addition, a rationale
for the choice of the matrix analysed and the analyte measured should be given.
The positioning of the report within the application will depend upon whether the data are
specific to any one toxicity study or are supportive of all toxicity testing.

TOXICOKINETICS EVALUATION IN PRECLINICAL STUDIES

 Safety pharmacology studies
 Single-dose toxicity studies
 Repeat-dose toxicity studies
 Reproduction toxicity studies
 Genotoxicity studies
 Carcinogenicity studies

SAFETY PHARMACOLOGY STUDIES

 Core studies in safety pharmacology comprise in vivo CNS,
cardiovascular and respiratory assessments.
 Toxicokinetic assessment is not specifically mentioned in
the guidelines
 It enables researchers to correlate any observed effects with
systemic level of the drug.
 Cross-reference dose level with exposure in toxicity studies.
SINGLE DOSE TOXICITY STUDIES
 Single-dose studies are usually performed in rodents.
 Toxicokinetic evaluation is not routinely included in such studies
 performed in a very early phase of drug development
  Bioanalytical method has been developed and toxicokinetic monitoring of these studies is
therefore not normally possible.
 Plasma samples may be taken in such studies and stored for later analysis
REPEATED –DOSE TOXICITY STUDIES
 Both rodents and non rodents and are usually of four-week duration
 The treatment regimen and species should be selected whenever possible with regard to
pharmacodynamic and pharmacokinetic principles.
 information on exposure, dose proportionality, sex- and species-difference, and potential
accumulation and inhibition, and help to support dose-selection for subsequent studies.
GENOTOXICITY STUDIES
 Drug development usually needs to be supported by two in vitro studies and one in vivo
study.
 In vivo investigations usually use a rodent micronucleus (bone marrow or peripheral
erythrocytes) test or chromosome aberration (bone marrow cells) test
CARCINOGENICITY STUDIES
Sometimes drugs are used for longtime for curing purposes, this may lead to the toxicity or
carcinogenicity
Dose selection is usually determined as the maximum tolerated dose
Sighting or dose ranging studies
 Appropriate monitoring and profiling should be undertaken to generate toxicokinetic data
 Special attention have to be paid to the species and strains which have not been included
in earlier toxicity studies
 The ideal study design would ensure that dose level in oncogenicity studies generate a
range of systemic exposure value that exceed the maximum therapeutic exposure for
humans by varying multiples.
The Main studies
 The treatment regimen, species and strain selection should, as far as is feasible, be
determined with available PK and TK information.
 Vast majority of these studies are conducted in rat and mouse
REPRODUCTIVE STUDIES
 The information on pharmacokinetics is preferable to carryout reproduction studies, this
may suggest the need to adjust the choice of species, study design and dosing schedules.
  The limitation of exposure in reproductive study is usually governed by maternal
toxicity.
 Data from non-pregnant animals is useful to set dose levels, and the limitation of
exposure.0
 A satellite group of female animals may be used to collect the toxicokinetic data
Fertility studies
 The general principles for repeated-dose toxicity studies apply. The need to monitor these
studies will depend on the dosing regimen used and the information already available
from earlier studies in the selected species

Studies in pregnant and lactating animals

 The treatment regimen during the exposure period should be selected on the basis of the
toxicological findings and on pharmacokinetic and toxicokinetic principles.
 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
APPLICATIONS
 More precise scenario of drug kinetics and metabolism
 Improved assessment strategy with greater efficiency
 Use fewer animals and provide better data for risk assessment purposes
 Rescue- at risk program in preclinical/ early clinical development
 Screen/ evaluate leads at early stages using predictive tool for toxicity and MOA
 Develop pre-clinical biomarkers of drug response and toxicity
 Adoption of toxicity management approaches to improve the therapeutic outcome
 It helps to understand the molecular basis of drug toxicities
 ALTERNATIVE METHODS TO ANIMAL TESTING
 The term alternative is used to refer to those techniques that replace the use of laboratory
animals altogether.
 Reduce the number of animals required
 Refine an existing procedure or technique to minimise the level of stress endured by the
animals.

Some alternative to the use of animal in testing include,

• In vitro test method and model based on human cell and tissue culture .
• In silico methods
• Cell line techniques
• Patch clamp technique
• Computer aid drug designing methods
• Molecular biology techniques
• Computerized-patient drug data bases and virtual drug trial
• Stem cell and genetic testing method
• Non- invasive imaging techniques such as MRI and CT scan
BENEFITS OF NON-ANIMAL TESTING
• Alternative scientific test are often more reliable than animal test
(an in vitro test derived from cultured human skin cells, was found to be more accurate in
identifying chemical skin irritant than traditional animal test)
• The use of human tissue in toxicity testing is more accurate than animal model
• More cost effective, practical and expedient
• Cruelty-free products are more environment friendly .(cruelty free testing is less harmful
to the environment or create less waste
IN-VITRO TESTING
• In vitro, tissue-based models are common and widely used for screening and ranking
chemicals, especially in testing of drugs at the pre clinical stages.
• The toxic effect include general cytotoxicity, genotoxicity, mutagenisis and
carcinogenesis.
• The possibility of using cell or tissue cultures as suitable material for testing agent in
pharmacology has often been suggested.
• The culture methods include organ culture, sphere culture, suspension culture, clone
culture on soft agar and monolayer cell culture

ADVANTAGES
 Controlled testing conditions
 A high level of standardization
 Reduction in variability between experiments
 The absence of systemic effects
 Low cost testing
 Small amount of material needed
 Limited amount of toxic waste, cells, and human tissues used
 Reduced animal testing.
DISADVANTAGES
 In vivo- dose response not available
 No systemic effect could be studied
 Organ specificity lacking
 Chronic & long term effects could not be studied
 Possible change of properties
 More difficult extrapolation
 pK values cannot be evaluated
Types of in vitro models
 Replacement
i) Use of living system
ii) Use of non- living system
iii) Physical & mechanical system
 Reduction
i) Animal sharing
 ii) Improved statistical design
iii) Phylogenetic reduction
iv) Better quality animals
 Refinement
i) Decreased invasiveness
ii) Improved instrumentation
iii) Improved control of pain
iv) Improved control of techniques

IN SILICO METHODS
In silico is an expression used to mean “performed on a computer or via computer
simulation .in silico study in medicine is thought to have the potential to speed the rate of
discovery while reducing the need for expensive lab work and clinical trials.
CELL LINE TECHNIQUES
Cell line refers to propagation of culture after the first sub culture OR Once the primary
culture is sub cultured it becomes cell line
• Such a cell line derived by selection or cloning is referred to as cell strain
• Cell strain does not have infinite life, as they die after some divisions
Types of cells used in cell line
 Precurser/stem cells/master cells
 Undifferentiated but committed precurser cells
 Mature differentiated cells
Characteristics of cell culture
• Cells can be isolated by grading the tissue & subsequent treatment with trypsin
• Cell line can be obtained by culturing isolated cell
• Such culture consisting of differentiated cell types are known as primary culture
• A secondary culture can be established from primary culture by culturing & repeated sub
culture.
PATCH CLAMP TECHNIQUE
• It is a laboratory technique in electrophysiology that allows the study of single or
multiple ion channels in cells
• Especially useful in the study of excitable cells such as neurons, cardiomyocites, muscle
fibers & pancreatic beta cells.
• It uses, an electrode ---a glass micropipette--- open tip diameter of about 1 μm, a size
enclosing membrane S.A / patch that often contains just 1/ a few ion channel molecules
• In some experiments the micropipette tip is heated in a micro forge --- produce a smooth
surface ---assist in forming high resistant seal with cell membrane
• Interior of the pipette filled with a solution matching ionic composition of bath solution
• Chloride silver curve is paced in contact with these solution & conduct electric current to
the amplifier
Variations in patch clamp technique
• “Excised patch” à patch is excised from main body of the cell
• One cell patch or cell attached
• Whole cell patch
• Out side out patch
• Inside out patch
• Perforated patch
• Loose patch
MOLECULAR BIOLOGY TECHNIQUES
• Study of biology at a molecular level
• The field overlap with other areas of biology & chemistry, particularly genetics &
biochemistry
• It concerns with understanding interaction between various system of a cell, including the
interrelationship of DNA,RNA & protein synthesis
 Expression cloning
• To study protein function
• DNA coding for a protein of interest is cloned (using PCR/restriction enzymes) in to a
plasmid (expression vector) or viruses or pathogenic bacterias as carriers
• Introducing DNA in to eukaryotic cells is called transfection
 Polymerase chain reaction( PCR )
• Extremely versatile technique for copying DNA
• PCR allows a single DNA sequence to be copied (millions of times) & altered in
predetermined ways
• Via enzymatic replication

APPLICATIONS
• Detection of hereditary disease
• Identification of genetic finger prints
• Diagnosis of infectious diseases
• Cloning of genes
• Paternity testing
• DNA computing
 Southern blotting
• used for probing for presence of specific DNA sequence within DNA sample
 Northern blotting
• Used to expression patterns of a specific type of RNA molecule as relative comparison
among a set of different sample of RNA
 Western blotting
• Here, proteins are first separated by size by SDS-PAGE
 Gel electrophoresis
• DNA, RNA, and protein can all be separated using an electric field