Simplifying study designs and statistical models for new dose & dosage forms DIA 11 april 2019 r

Prof. (Dr.) Bhaswat S. Chakraborty
Emeritus Professor, Institute of Pharmacy, Nirma University
Former Sr.VP &Chair, R&D, Cadila Pharmaceuticals
Former Director, Biopharmaceutics, Biovail, Toronto
Former Sr. Efficacy & Safety Reviewer, TPD (Canadian FDA),
Ottawa
Simplifying Study Designs
and Statistical Models: for
new dose & dosage forms

Study Designs for New Dose or Dosage
form BE (Comparative Bioavailability)
Main Principle in Study Design
• PK based study – blood concentration can be measured reliably?
• Simple or complex PK?
• Modified‐release dosage forms?
• Drugs with serious toxicity within the normal dosage range? Drugs
exhibiting non‐linear pharmacokinetics?
• Drugs with a terminal elimination half‐life of more than 24 hours?
• Drugs with an important time of onset of effect or rate of
absorption?
• Critical dose drugs?
• Combination product?
• Highly variable drug products?
Research supports In-vitro BE?
© 2019 Prof. Bhaswat Chakraborty 2

Study Designs :Parallel or Crossover?
The standard study design used is a 2‐period cross‐over, in
which each subject is given the test and reference formulations
The advantage of the cross‐over design is that the
intra‐subject error is always lower than the inter‐subject
error used in a parallel design
Replicated cross‐over designs may also be used, where the
formulations are tested more than once in the same subjects
The main advantage of these designs is that fewer subjects are
required; however, they must appear for more periods
Parallel designs may be useful when studying drugs with very
long elimination half‐lives or some depot formulations. The error
term used is the inter‐subject variance

Higher Order Study Designs
In cases where more than two formulations are under study, or
are studied under different conditions, a higher order design
(that is (i.e.), more periods and sequences) should be
considered
Since the intra‐subject error term of these designs has more
degrees of freedom, smaller sample sizes are often adequate
The choice of a variance balanced design (Williams’ Design) or
separate incomplete block design should be justified.
A cross‐over design without a drug‐free period between
formulations may be employed for studies conducted in patients
in whom it would be unethical to discontinue treatment during a
washout period
• Instead of a drug‐free washout period, the study drugs are
administered long enough, prior to sampling, to allow elimination of the
previously administered formulation

Alternative Study Designs
When the proposed estimate of the intra‐subject variance
from the literature has large uncertainty, it is possible to
collect the data in stages based on the observed
intra‐subject variance from the first stage
Two strategies for collecting data in stages are Group
Sequential Designs and Adaptive Designs
For both types of designs the overall Type I error rate
should be maintained at 5% and the algorithm should be
defined a priori in the protocol
These approaches can be used for both cross‐over and
parallel designs.

Pharmacodynamic (PD) Study Designs
In cases where PK endpoints cannot be reliably measured, in-
vivo comparability using PD studies
• The use and design of PD studies should be justified
Design of PD studies should consider the underlying pathology
and natural history
If baseline conditions are not reproducible, it may be necessary
to use a parallel‐group design
Patients who are non‐ responders should be excluded from the
study by prior screening
Important placebo effects should also be considered, as
comparisons between drug products can be made only after a
priori consideration of such effects in the study design
A placebo cross‐over phase may be necessary to evaluate
placebo effects

N (Number of Subjects )
N should be estimated by considering the objectives of the
study, the study design, the drug products being compared
and the conditions under which the study is carried out
A complete literature search should be conducted in order
to understand the drug and drug product
Expected mean difference between T & R and anticipated
intra‐subject variance for the parameters & the power,
determine the N
All calculations are to be based on maintaining the overall
Type I error rate at 5%.
The minimum number of subjects in pivotal studies is 24

Crossover Fed Study Designs
A randomized, balanced, single-dose, two-treatment (fed vs.
fasting), two period, two sequence crossover design is used for
studying the effects of food on the bioavailability of IR or MR
product
The test product and the RLD should be administered under fed
conditions
An adequate washout period should separate the two treatments
Test product should be administered on an empty stomach (fasting
condition) in one period and following a test meal (fed condition) in
the other period
A similar, 2x2x2 crossover design for a fed BE study is often
recommended except that the treatment should consist of both T &
R following a test meal (fed condition
Usually the same 90% CI BE standard is used for AUC & Cmax

Modified Release Dosage Forms (MRDFs)
For MR products, increased inter‐subject variability in bioavailability
will occur, including the possibility of dose‐dumping
There may also be an increased risk of adverse effects such as
gastrointestinal irritation, depending on the site of drug release, or
absorption, or both
Thus for all MR forms (including delayed‐release formulations), BE
should be demonstrated under both fasted and fed conditions
BE standards
• The 90% confidence interval of the relative mean area under the
concentration versus time curve to the time of the last quantifiable
concentration (AUCT) of the test to reference product should be within
80.0% ‐ 125.0% inclusive.
• The relative mean maximum concentration (Cmax) of the test to
reference product should be within 80.0% ‐ 125.0% inclusive

MRDFs

Modified Release Dosage Forms (MRDFs)
Steady‐state studies are not generally required. However, if a
steady‐state study is conducted, the following standards
should be met:
• The 90% confidence interval of the relative mean area under the
concentration versus time curve at steady state over the dosing
interval (AUCtau) of the test to reference product should be within
80.0% ‐ 125.0% inclusive.
• The relative mean Cmax at steady state of the test to reference
product should be within 80.0% ‐ 125.0% inclusive.
• The relative mean minimum concentration (Cmin) at steady state of
the test to reference product should not be less than 80.0%
inclusive.
• Modified‐release products with multiphasic plasma concentration
profiles demonstrated to be integral to their therapeutic effect will be
subject to standards on the partial area under the concentration
versus time

Multiphasic Modified Release Dosage
Forms
The requirement for pAUC assessment metrics for
multiphasic modified‐release formulations will be based on
data available from scientific literature
• The time course of changes in the rate of drug delivery
throughout the day should be reconciled with generally accepted
and clinically relevant response data generated from a
well‐designed randomized clinical trial program
• Specifically, standards based on the 90% confidence interval of
pAUC metrics should be met
• The specific pAUC time intervals to be considered will be based
on clinical data showing the therapeutic relevance of the
particular time interval (e.g. early onset, maintenance, dose
clearance, fasted versus fed state).

New Dose – Non-Linear Kinetics Consideration
For drugs with non‐linear pharmacokinetics in the single unit dose
range of approved strengths resulting in greater than proportional
increases in AUC with increasing dose
• BE study should be conducted on at least the highest strength.
For drugs with non‐linear dose-PK due to saturable absorption and
resulting in less than proportional increases in AUC with increasing
dose
• BE study should be conducted on at least the lowest strength
(single dose unit)
For drugs with non‐linear PK due to limited solubility of the
medicinal ingredient and resulting in less than proportional
increases in AUC with increasing dose
• BE be conducted on at least the lowest strength (single dose unit) in
the fasted state and the highest strength in both the fasted and fed
states

In Vitro Studies Sometimes better than In Vivo
Studies?
Human PK in vivo studies are often presumed to serve as
the “gold standard” to assess product BE of IR solid oral
dosage forms
It appears that in vitro studies are sometimes better than in
vivo studies in assessing BE of IR solid oral dosage forms
Reasons for in vitro studies to sometimes serve as the better
method are that in vitro studies: (a) reduce costs, (b) more
directly assess product performance, and (c) offer benefits in
terms of ethical considerations
Reduced costs are achieved through avoiding in vivo studies
where BE is self-evident, where biopharmaceutic data
anticipates BE, and where in vivo BE study type II error is
high

In Vitro Studies Sometimes better than In Vivo
Studies?..
In vitro studies more directly assess product performance
than do conventional human BE studies, since in vitro
studies focus on comparative drug absorption from the two
products, while in vivo BE testing can suffer from
complications due to its indirect approach
Ethics: “No unnecessary human testing should be
performed” and can result in faster development
Situations when in vitro test should be viewed as preferred
include Class I drugs with rapid dissolution, Class III drugs
with very rapid dissolution, and highly variable drugs with
rapid dissolution and that are not BE problem drugs
Sponsors of potential in vivo human PK BE testing should be
required to justify why in vitro data is insufficient

Challenges in BE Determination of Topical
Products
BE assessment of locally acting topical dosage forms
using traditional PK endpoints is challenging.
Historically, there were limited options for alternate
approaches to PK or clinical endpoint BE studies
FDA recognized the need to find more sensitive and
efficient surrogate approaches to demonstrate BE for
topical dermatological products.
Development of new alternate BE approaches using a
collective weight of evidence from in-vitro studies (e.g.
IVRT, IVPT)

In Vitro BE Option: Acyclovir Cream
Formulation Q1/Q2 Sameness: The test and RLD products
are qualitatively and quantitatively same.
Q3 Similarity: The physicochemical properties of test and
RLD products are similar.
In Vitro Release Test (IVRT) Studies: The test and RLD
products have an equivalent rate of acyclovir release.
In Vitro Permeation Test (IVPT) Studies: The rate and extent
of acyclovir permeation through excised human skin from the
test and reference products are comparable.
There are other options e.g., In Vivo Clinical study

In Vitro Release Test (IVRT)
An IVRT) is an established method to characterize this rate of API
release and compare the underlying sameness in product quality
characteristics
An approach to validate an IVRT method may include
• qualification of the apparatus
• IVRT method development
• validation of the analytical method
• validation of critical parameters of the IVRT method
Application of IVRT – PSGs of:
• Acyclovir Ointment
• Silver Sulfadiazine Cream
• Acyclovir Cream
• Benzyl Alcohol Lotion
• …

IVRT Method Development
Justification, Qualification and Validation
• Method Parameters: Information should be provided to support the
selection of the IVRT apparatus, product dose amount, sampling times,
stirring/agitation rate, and other parameters of the test method.
• Membrane: Information on acyclovir membrane binding and chemical
compatibility with relevant receptor solutions should be provided to
support the inertness of the membrane selected, and information on the
linearity and precision of the resulting acyclovir release rate in an IVRT
should be provided to support the selection of a membrane for the test
method.
• Receptor Solution: Information on the empirical solubility and stability of
acyclovir in the receptor solution, as well as information on the linearity
and precision of the resulting acyclovir release rate in an IVRT should be
provided to support the selection of a receptor solution for the test
method

IVRT Conduct
The IVRT pivotal study
comparing the [drug]
release rates between the
test and RLD products
should be performed in a
manner compatible with
the general procedures
and statistical analysis
method specified in the
United States
Pharmacopeia (USP)
General Chapter <1724>,
Semisolid Drug Products –
Performance Tests
An IVIVC of any kind of
IVRT results and In vivo
BE study results should
not be expected
© 2019 Prof. Bhaswat Chakraborty
Image courtesy of PermeGear
21

IVRT Method Development: Membrane
Evaluation
 3 replicate membrane
incubations for the IVRT
duration (e.g. 6 hours) at
32°C ± 1°C
 Aliquots of these solutions
may be collected before
and after the duration of
incubation, to assess any
decrease in the amount of
acyclovir in solution
 The recovery of acyclovir
in solution is
recommended to be
within the range of 100%
± 5% at the end of the test
duration to qualify the
inertness of the
membrane.
22

IVRT Method Development: Receptor
Solubility
 Minimum solubility of
acyclovir in the IVRT
receptor solution should
be empirically determined
in triplicate with acyclovir
dissolved to saturation in
the receptor solution
 This conc. should exceed
the highest sample
concentration obtained in
the pivotal IVRT study,
ideally by an order of
magnitude or
demonstrably sufficient to
facilitate the linearity of
the release rate for the
duration of the study
23

Other IVRT Validation Parameters
IVRT Receptor Solution Sample Analytical Method Validation: The receptor
sample HPLC analysis procedures should be validated in a manner compatible with
the current FDA Guidance for Industry on Bioanalytical Method Validation, and/or
the ICH Harmonised Tripartite Guideline on Validation of Analytical Procedures Q2
(R1). The validation of the receptor sample analytical method should include
relevant qualifications of dilution integrity as well as stability assessments with the
highest relevant temperature in the receptor solution, which may be warmer than
32°C, for the duration of the IVRT study (e.g., 34°C for 6 hours).
IVRT Environmental Control: Ambient laboratory temperature and humidity during
the study should be monitored and reported. An environmentally controlled
temperature range of 21°C ± 2°C and a humidity range of 50% ± 20% relative
humidity are recommended.
IVRT Linearity and Range: The linearity (r2 value) of the release rate (slope) may
be calculated across the range of the sampling times, which corresponds to the
IVRT study duration. Linearity may be compared within and across all IVRT runs,
and a minimum r2 value ≥ 0.90 across the IVRT study duration (time range) is
recommended.

Other IVRT Validation Parameters
IVRT Precision and Reproducibility: The intra-run and inter-run
precision and reproducibility may be compared for the release rate
(slopes) calculated for each diffusion cell.
• a minimum intra-run and inter-run %CV ≤ 15% is recommended.
IVRT Recovery, Mass Balance & Dose Depletion: The recovery of
released acyclovir in the receptor solution may be characterized in
each diffusion cell as the accumulated amount of acyclovir in the
receptor solution over the IVRT duration (%acyclovir in the applied
dose)
IVRT Discrimination Sensitivity, Specificity and Selectivity: The
IVRT method should be able to discriminate acyclovir release rates
from similar formulations
• one with a higher strength (e.g., 7.5%) and one with a lower strength
(e.g., 2.5%)

Other IVRT Validation Parameters..
IVRT Robustness: The IVRT method may be considered robust to a
variation in the test method if the average slope of that IVRT run (under
altered conditions) is within ± 15% of the average slope of the Precision &
Reproducibility IVRT runs. Robustness testing may encompass variations
in the IVRT method that are relevant to the apparatus and test method, for
example:
• Temperature variations (e.g. - 1°C and +1°C relative to 32°C ± 1°C)
• Dose volume variations (e.g. +10% and -10% in the dose volume)
• Receptor solution variations (e.g. change in composition and/or pH)
• Mixing rate variation (e.g. differences in stirring speed, or without
stirring)

Biopharmaceutics Classification
System (BCS)
The BCS concept equation:
J = Pw*Cw
where, J is the flux across the gut wall, Pw is the permeability of the
gut wall to the drug and Cw is the concentration profile at the gut
wall
For bioequivalence (BE), highly permeable & highly soluble drugs
housed in rapidly dissolving drug products will be bioequivalent
For such drugs, unless major changes are made to the formulation,
dissolution data can be used as a surrogate for pharmacokinetic
data to demonstrate BE of two drug products or an untested
strength
BCS reduces cost of scale-up and post-approval changes, multiple
strength proportional formulations to certain oral drug products
without compromising public safety interests

Permeability
Following methods are routinely used for determination
of permeability:
a) Pharmacokinetic studies in human subjects including mass
balance studies[8] and absolute bioavailability (BA) studies or
intestinal permeability methods
b) In vivo or in situ intestinal perfusion in a suitable animal model
c) In vitro permeability methods using excised intestinal tissues
d) Monolayers of suitable epithelial cells e.g. Caco-2 cells or TC-7
cells

Permeability..
In mass balance studies, unlabelled, stable isotopes or radiolabelled drug
substances are used to determine the extent of drug absorption
In absolute BA studies, oral BA is determined and compared against the
intravenous BA as reference
Intestinal perfusion models and in vitro methods are suggested for
passively transported drugs. An interesting alternative to intestinal tissue
models is the use of in vitro systems based on the human adenocarcinoma
cell line Caco-2
• These cells serve as a model of small intestinal tissue. The
differentiated cells exhibit the microvilli typical of the small intestinal
mucosa and the integral membrane proteins of the brush-border
enzymes
• They also form the fluid-filled domes typical of a permeable epithelium
• Caco-2 cell lines also have the ability to transport ions, sugars and
peptides
• These properties have established the Caco-2 cell line as a reliable in
vitro model of the small intestine

Caco-2 cell monolayers from a) Bock et al.(14) in a 12-well, 21-day
assay, b) Lentz et al. in a six-well, 4-day assay (53), and c) Withington
(54) in a 24-well, 3-day assay. Triangle HP drugs, Circle LP drugs,
Dashed line LP/HP boundary, Dotted line 90% absorption
33

The BCS Classification
According to BCS, drug substances or APIs are divided
into high/ low solubility and permeability classes:
Class I : High Solubility - High Permeability
Class II : Low Solubility - High Permeability
Class III : High Solubility - Low Permeability
Class IV : Low Solubility - Low Permeability
In combination with the dissolution, the BCS takes into
account the three major factors governing BA, viz.
dissolution, solubility and permeability
The BCS in accordance with WHO guideline is shown in
next slide

The BCS Classification

Additional Considerations by WHO
BCS classification is associated with drug dissolution
and absorption model, which identifies the key
parameters controlling drug absorption as a set of
dimensionless numbers
• Absorption number, An = mean residence time/mean absorption
time
• Dissolution number, Dn = mean residence time/mean dissolution
time
• Dose number, Do = (maximum dose strength/250)/solubility
Class I drugs exhibit a high absorption number and a
high dissolution number.
• The rate-limiting step is drug dissolution and if dissolution is very
rapid then gastric emptying rate becomes the rate-determining
step
36

Additional Considerations by WHO..
Class II drugs have a high absorption number but a low
dissolution number
• In vivo drug dissolution is then a rate-limiting step for absorption
except at a very high dose number
• The absorption for Class II drugs is usually slower than Class I and
occurs over a longer period of time
For Class III drugs, permeability is a rate-limiting step for
drug absorption
• These drugs exhibit a high variation in the rate and extent of drug
absorption
• Because the dissolution is rapid, the variation is attributable to
alteration of physiology and membrane permeability rather than the
dosage form factors
Generally, Class IV drugs exhibit problems for effective
oral administration
Examples of drugs for different classes are given in
Table in next slide
37

FITC-insulin transport across Caco-2 monolayers. (a) Time-course study of FITC-insulin
transport (mg) at different loading concentrations. FITC-insulin was loaded in apical
chambers at 0.05 (open circles), 0.15 (filled circles), 0.3 (squares), and 0.6 (triangles)
mg/well respectively; and permeation was measured by measuring the fluorescence in
samples collected from basolateral chamber at different time-points up to 5 hrs. (b) % FITC-
insulin transport across Caco-2 monolayers. Data represent mean ± SD (n = 3).
39

Sulforhodamine-B transport across Caco-2 monolayers. (a) Time-course study of
sulforhodamine-B transport (mg) at different loading concentrations. Sulforhodamine-B was
loaded in apical chambers at 0.05 (open circles), 0.15 (filled circles), 0.3 (squares), and 0.6
(triangles) mg/well respectively; and apical-to-basolateral permeation was measured by
measuring the fluorescence in samples collected from basolateral chamber at different time-
points up to 5 hrs. (b) % Sulforhodamine-B transport across Caco-2 monolayers over of 5
hrs of incubation. Data represent mean ± SD (n = 3).
40

Innovative Model for Future ANDAs
Source: Robert Lionberger. Application of PBPK models
in assessment of bioequivalence (AAPS Annual
Meeting 2014)
Source: Robert Lionberger. Application of PBPK models in assessment of bioequivalence (AAPS Annual Meeting 2014)
41

References
Canadian bioavailability and bioequivalence guidelines
https://www.canada.ca/en/health-canada/services/drugs-health-
products/drug-products/applications-submissions/guidance-
documents/bioavailability-bioequivalence.html
https://www.fda.gov/downloads/Drugs/NewsEvents/UCM591918.pd
f
Kregar et al, Int J Pharm. 2015 May 15;485(1-2):202-14. doi:
10.1016/j.ijpharm.2015.03.018. Epub 2015 Mar 12.
Gupta et al. PLOS One
https://doi.org/10.1371/journal.pone.0057136

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