Central theme of chapter

1) Drug concerntration in blood needs to be within therapeutic range to be useful. Conc outside
this range results in toxic or no effect. The amount of drug in blood is expressed in its
bioavailability.

2) ALL Drug formulation contains a) the main drug and b) other chemicals called excipient.
These excipient can influence absorption of drug so that drug concerntration in plasma is more
or less than normal. Phenytoin toxicity was caused by switching CaPO4 with lactose. The aim of
the chapter is to expose the methods of doing Bioavailability study, which if done could have
prevented the tragedy by alerting that phenytoin levels were higher than safe therapeutic
range.

3) Any Original drug is worth 15yrs, 800 million $, clinical trial with thousands of patients and is
very expensive while a generic does not do any clinical trial but is very cheap. Are they still the
same? This is answered by Bioequivalence study

Bioavailability (BA) is the rate and extent of absorption of unchanged drug from its dosage
form into the systemic circulation

Bioequivalence (BE) is the condition wherein the bioavailability of two drug products,
containing same amount of active drug and same route, is statistically similar. Two
bioequivalent products are therapeutically identical

Bioavailable fraction (F), refers to the fraction of administered dose that enters the systemic
circulation

F is always expressed in %

Bioavailability & Bioequivalence focus on release of drug substance from its dosage form &
subsequent absorption in circulation

Objectives of BA Studies

• Development of suitable dosage form for a New Drug Entity

• Comparison of availability of a drug substance from different form or same dosage form
produced by different manufacturers
 • Determination of influence of excipients, patient related factors & possible interactions
with other drugs

• Development of new drug formulations of existing drugs

• Control of quality of drug products, influence of → processing factors, storage &

stability

In-vivo BA study design

 Bioavailability-Absolute vs Relative
 Healthy people vs Patient
 Single dose vs multiple dose

Absolute Bioavailability
The fraction of the oral administered dose which is absorbed intact into the systemic circulation
relative to equivalent intravenous dose
F = (AUCtab/AUCIV) x 100%
For drugs administered intravenously, bioavailability is 100%
Relative Bioavailability
A measure of the fraction of a given drug that is absorbed intact into the systemic circulation
from a dosage form relative to a recognized standard dosage form of that drug.
F = (AUCtest/AUCstd,) x 100%

Why we use IV as standard/ don’t use oral solution as standard?

• Using IV solution allows comparison of BA from oral and parental routes and decide if
oral route is appropriate but oral solution doesn’t
• Oral solution limits pharmacokinetic model to one compartment model; cannot apply
two compartment model as with IV injection which is more realistic (one compartment
means drug going to blood only, two compartment means drug going to blood and then
to organs, muscles and fat too)
• IV route bypasses Absorption and 1st pass metabolism while oral don’t. Thus comparing
oral and IV allows to quantify these effects but two oral dosage doesn’t
• However comparison of oral tab and oral solution does help estimate effects of
disintegration and dissolution of absorption process. Oral tab has to disintegrate and
only then can it be effectively dissolved (since disintegration results in less particle size)
whereas oral solution is already in dissolved state and only needs to be absorbed
Human volunteer

Since the aim of BA/BE study is to only focus on effects of drug formulation, the volunteers
must be restricted on factors such as age, sex, state of health, race, diet since these can
influence drug absorption

(To do so, we need a group of people who are very similar to each other in terms of
physiology. Since absorption and thus BA can be influenced by gender and disease and we
don’t want that, female and patients are biased because including them will create
unnecessary variance in data which may be over or under analyzed. After all, if a new
formulation has influenced drug release and absorption in such a way that BA has gone to
toxic or sub-therapeutic levels it will be reflected in both male, females , children, old or
patients. The degree of change may vary between age and gender and state of health but
none the less change will or won’t be seen which is the only data we want.)

Criteria of volunteers

• Healthy, male, 20-40 years

• Female only for oral contraceptives
• Minimum number of subject (24-36) to avoid inter-subject variability
• Obtain permission from volunteers
• Pre-medical checkup to exclude people with any disease that can interfere with BA
• Volunteer must not take any other medicine for at least a week and fast overnight and 4
hr after dosing
• The volume and type of fluid and food must be specified
• If same subject if studied twice, there must be gap period of minimum 10 biological half
lives (to allow drugs to be COMPLETELY washed out since in the tail portion of graph
drug is still in body but its conc may be too low to detect)

Single dose vs Multiple dose studies

Single dose BA study

• Only a single dose is given to each volunteer. It is preferred by FDA in most cases
• Advantage
– Easy, offer less exposure to drugs to volunteers
• Disadvantage
 – Cannot account for steady state characteristic of drug
– (even with the best efforts to avoid this variability) There may be excessive
variability between volunteers
– Sufficiently long sampling periods are needed in order to get reliable estimate of
terminal half life which is needed for correct calculation of total AUC (it is talking
about the drug conc. in the tail portion of the graph which takes long time to
come to zero)

Fig: Single dose vs Multiple Dose

Multiple dose study
• Same dose is given repeatedly to same volunteer for a fixed time interval until a steady
state condition is reached. Single dose study is sufficient in most cases. But multiple
dose study is required if:
– There is a difference in the rate of absorption but not in the extent of absorption.
– There is excessive variability in bioavailability from subject to subject which can
be reduced at steady state
– The concentration of the active drug in the blood resulting from a single dose is
too low for accurate determination by the analytical method.
– The drug product is an extended-release dosage form.
• Advantages
– More accurately reflects the manner in which drug will be used clinically
– Allows blood levels to be measured at same concerntration encountered
therapeutically
– Requires collection of fewer blood samples
– Higher drug blood concerntration is observed which makes its determination
possible even by less analytical methods
– If fewer subjects are taken, then inter-subject variability is small
– Better evaluation of controlled release formulation
– Nonliner pharmacokinetics can be easily detected
– Eliminates needs for long wash out period between doses
 • Disadvantage
– Tedious, requires more time to complete
– More costly to conduct (prolonged dosing to subjects)
– Poor compliance by subjects
– Greater exposure of subjects to test drug which increases chances for adverse
reactions

Measurement of Bioavailability

We need such measurement that can be related to no effect, therapeutic effect or toxic
effect of drug.

• Pharmacokinetic Methods
– Plasma level-time studies
– Urinary excretion studies
• Pharmacodynamic Methods
– Acute pharmacological response
– Therapeutic response

I) Pharmacokinetic Methods

Pharmacokinetic deals with studying drug concerntration in plasma as a effect of absorption,

distribution, metabolism and elimination ie What body does to drug?

The assumption is that unchanged drug concerntration in plasma or sometime in urine

correlates to drug concerntration at site of action inside body which then correlates to the
response produced.

1) Plasma Level-Time Studies

• Principle: Drug’s response can be measured as a function of drug concerntration in
blood
• Drug dosing can be single or multiple
A) Single dose study

Process involves collecting blood samples periodically and make plot of plasma drug conc.
versus corresponding time. Parameters of interest are Cmax, Tmax and AUC0-t,AUC∞

Period and Frequency of blood sampling:

Blood sample is drawn for period of about 3 drug half-lives to capture at least 80% of AUC.

– Sampling frequency during absorption phase

• At least 3 sampling
– Sampling frequency during elimination phase
• if one compartment model à 3 sampling
• and if two compartment model à 5-6 sampling
• For IV dosage sampling must start from 5 mins after dosing and then for
each 15 min interval for 3 half live period

The extent of Bioavailability for single dose can be measured as

Relative bioavailability
B) Multiple dose study

Process involves steady state condition which can be achieved by a fixed drug dose
administration for at least 5 biological t1/2 with dosing interval equal to or greater than t1/2.
Parameters of interest are Css,max , τ , AUCss where ss means steady state, τ is the dosing
interval.

Period and Frequency of blood sampling:

• Blood sample should be taken at the end of previous dosing interval and

• 8-10 times after the administration of the next dose

(There is no defined time interval to draw blood in both Single and multiple doses. The ideal
time intervals is found by first doing small scale trial study with few volunteers to know the
optimum times at which to draw blood so that the graph comes out very smooth and proper)

The extent of bioavailability is given by

For single dose

For multiple dose

2) Urinary Excretion Studies

The principle is that urinary excretion of drug is directly proportional to the plasma
concerntration of drug. To use this method a minimum of 20% of administered drug has to be
excreted unchanged from kidney. Although Plasma study is preferred; however, it is useful for
Drugs that are extensively unchanged in the urine eg thiazide diuretics and sulphonamides or
Drugs that have urine as site of action eg urinary antiseptics such as nitrofurantoin and
hexamine. Parameter of interest is

 (dXu/dt)max à Max urinary excretion rateà like Cmax

 (tu)maxà time for max excretion rateà like Tmax

 Xu∞ àcumulative amount of drug excreted in the urine

The process involves

• Water-loading 400ml after fasting overnight and 200 ml with drug and 200ml hourly for
next 4 hours

• collection of urine at regular intervals, not specific time (ideally less than one drug t1/2)
for a time-span equal to 7 biological half-lives which ensure 99% drug collection

• analysis of unchanged drug in the collected samples

• Determination of the amount of drug excreted in each interval and cumulative amount
excreted

For valid results, following criteria must be met too

• At each sample collection , total emptying of the bladder is needed to avoid addition of
residual amount to the next urine sample

• Frequent sampling of urine is also essential in the beginning in order to compute

correctly the rate of absorption

• The fraction excreted unchanged in the urine must remain constant

Advantages
 • Urine has a small volume and can show high concerntration for same amount of drug as
compared to plasma that has very high volume. Thus this method requires less sensitive
analytical method while plasma requires more sensitive analytical techniques which may
not present

• Convenient and Non-invasive and thus better subject compliance

• When coupled with plasma level-time study, it can estimate renal clearance

• Direct measurement of BA, both absolute and relative without need for fitting data to a
mathematical model

• Derive other information such as 1st order elimination and metabolism, excretion and
absorption rate constant

Limitations

• Can’t compute Vd (volume of distribution) and Clt (systemic clearence at time t)

• Difficult to apply in case where drug is slowly released or has long half-life cause
concerntration in urine will be very low
II) Pharmacodynamic methods

Pharmacodynamic studies physiological changes caused by drug ie What drug does to body?
The principle of this method is that magnitude of response and dose given is correlated

1) Acute Pharmacological Response Method

Acute Pharmacological Response means any physiologic changes that occur shortly after
administration of drug. It is not necessarily a curative response eg changes in ECG, blood
pressure, pupil diameter etc. A dose-response graph or pharmacological effect-time curve is
made.This method is used when PK methods is difficult, inaccurate or non-reproducible

Measurement of response for at least three half lives is done

Disadvantage
• Pharmacological response is naturally more variable among persons, which prevents
proper correlation between measured response and drug available from formulation
• The observed effect may be due to an active metabolite

2) Therapeutic Response Method

This method is based on observing clinical response to a drug formulation given to patients who
are suffering from the associated disease the drug was meant to be used. Here also dose
response curve is used.

It has many drawbacks such as

 • In the second round of crossover BE studies, in which each of the two group of subjects
receive both standard and test drug products, the two groups might be more healthy
than at beginning of study which defeats the purpose of selecting patients
• Unless multiple dose study is done, a patient won’t be able to take his medicine multiple
times a day because single dose study requires a washout period between two doses
which can be very long. This might worsen the patient's health
• Many patients are on more than one drug which can interact which the study drug and
make BA study difficult to understand
• Difficult to judge relative BA because the measured response from two drug products is
not the same

Application is limited to drugs that only act at site of administration and are not meant to be
absorbed into the blood such as topical antifungal creams and sucralfate used in ulcer therapy
or drugs that can harm healthy people such as anticancer drug etoposide and anti-s
schizophrenic clozapine

Study of paracetamol 1-g oral solution bioavailability.

Abstract
The aim of this work was to assess paracetamol bioavailability after administering 1 g
in oral solution. Eighteen healthy volunteers were selected for this open-label study. A
total of 15.4 ml of Gelocatil Oral Solution (Laboratorios Gelos, S.L.), corresponding to 1
g of paracetamol, were administered to fasting
Up to now summarysubjects. Blood samples were collected
at 0 min, 10 min, 20 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h and 12
We are studying BA because:
h. Paracetamol plasma concentrations were determined by reverse-phase high-
• performance liquidofchromatography.
to evaluate effect formulation on BA The study
to avoid waslike
cases conducted
phenytoinwithout deviations
toxicity where we came
to understand that change in formulation can place BA outside the therapeutic
from protocol. Pharmacokinetic data from 18 subjects were allowed for estimating range

• fast
BAand
datahigh-paracetamol
is needed to do BE,bioavailability: t(max)paracetamol
ie to compare generic 20 min (10-45)
(Niko)[median (range)],
and proprietary
C(max) 24. 3 mg/l
paracetamol (6.5) [mean (standard deviation)], AUC(0-t) 64.0 mg h/l (16.1) and
(tylenol)
AUC(0-00) 68.1 mg h/l (17.9). These results are comparable to those described for
4 methods to do In-vivo (within living being) BA study.
Gelocatil Oral Solution given at a 650 mg dose and for immediate release Gelocatil 650
• mgPlasma – plasma
tablets. drug conc
Absorption vs time
speed graph,fast,
was very method of choice
similar to that described for other oral-
• solution
Urine –formulations,
plasma drug concwhich provides
vs time graph, an immediate
drugs onset
that act on of pain and fever relief. The
bladder
results of this study show suitable bioavailability for 1 g Gelocatil Oral Solution, with
• Acute –dose/log[dose] vs response graph,
fast-absorption speed that provides an immediate onset of pain and fever relief.
• Therapeutic - dose/log[dose] vs response graph, done on patients for locally acting drugs such
as antifungal cream, BE study problem

For every method, three things to consider

• Volunteer type and size (healthy 20-40 aged male, same race)

• Single or multiple dose ( single mostly preferred by FDA)

• Absolute or relative (absolute gives data about effect of 1 st pass metabolism and rate of
absorption)