ALTERNATIVE IN VITRO BIOEQUIVALENCE ASSESSMENT

OF TOPICAL APPLIED DERMATOLOGY FORMULATIONS

A DITEBA WHITEPAPER

WRITTEN BY:
DR. THEO KAPANADZE, D.SC., PH.D, CHIEF SCIENTIFIC OFFICER

WWW.DI TEBA.C OM
ALTERNATIVE IN VITRO BIOEQUIVALENCE ASSESSMENT
OF TOPICAL APPLIED DERMATOLOGY FORMULATIONS

Bioequivalence (BE) assessment of orally administered products intended for the systemic circulation is carried
out by measuring plasma drug concentrations following administration of a test and reference dosage form to
human subjects.

As stated at 21 CFR 320.24, approaches for BA/BE of topical products preference are:

1. pharmacokinetic (PK) measurements based on measurement of an active drug and/or metabolite in blood,
plasma, and/or urine
2. pharmacodynamic (PD) measurements
3. comparative clinical trials;
4. In Vitro studies.

The determination of the bioavailability of systemically absorbed products is defined as the rate and extent to
which the active ingredient or active moiety is absorbed from the drug product and becomes available at the
site of action.

However, when considering products which contain active ingredient(s) not intended for systemic absorption,
the US FDA, published the following statement in the US Federal Register (US Federal Register, 2009) where
topical products “may be assessed by (surrogate) measurements intended to reflect the rate and extent to
which the active ingredient or moiety becomes available at the site of action.”

For topical dermatological drug products, PK measurements in blood, plasma, and/or urine are usually not
feasible to document BE because topical dermatologic products generally do not produce measurable concen-
trations in extra cutaneous biological fluids.

Apart from the analytical difficulty of measuring concentrations of drugs in the systemic circulation following
topical administration where the concentrations generally reach only a fraction of the amount of drug applied
to the skin surface, systemic drug concentrations are not considered to reflect the concentration in the target
organ, namely, the skin.

IN VIVO STUDIES USING PHARMACODYNAMIC RESPONSE

Only a limited number of chemicals evoke a local quantifiable pharmacodynamic response, for example,
the concentration dependent vasoconstriction effect of corticosteroids. In these experiments, resulting skin
blanching is scored visually by one or more qualified investigators, using an ordinal data scale. The lack of
instrumentation has been criticized, because of possible subjective errors. However, approaches using the
Chromameter device, although recommended by the FDA, have failed due to obtain desired precision.

1
A DITEBA WHITEPAPER www.diteba.com
In addition to a single time-point measurement, skin blanching may be followed over a prolonged period of
time. In doing so, depot formation and kinetics of substance removal due to local metabolic or systemic clear-
ance may be observed. By plotting the magnitude of response against time and integrating the area under the
effect curve, different formulations can be compared. This is the method of choice to determine the bioequiv-
alence of generic products, as described in the FDA guidance for industry on topical dermatologic corticoste-
roids.

It is encouraged to perform a pilot in vivo study in order to define appropriate parameters for the pivotal study.
This includes defining the linear proportion of the sigmoidal dose response curve, the half maximal effective
dose, and assessing the lower limits of sensitivity and the maximum effect.

IN VIVO DERMATOPHARMACOKINETIC APPROACH

The BA/BE determination for topical products is thus often based on PD or clinical studies. An additional
approach considered in FDA guidance is to document BA/BE through reliance on measurement of the active
moiety(ies) in the stratum corneum. This approach is termed dermatopharmacokinetics (DPK). Although mea-
surement of the active moiety(ies) in blood or urine is not regarded as an acceptable measurement of BA/BE
for dermatological drug products, it may be used to measure systemic exposure.

RECOVERY STUDIES

Independent of any visible response, the extent of dermal absorption may be judged from the amount of
substance missing within the recovered formulation after finishing incubation. However, this method keeps the
researcher in the dark about the ultimate fate of the substance. In addition, recovery studies are not suitable
for infinite dosing, as the recovery will always exceed 90%.

TAPE STRIPPING METHOD

Tape stripping has been suggested as a surrogate measure for bioavailability and bioequivalence assessment
of topical products. Draft guidance on tape stripping for assessing the bioavailability/bioequivalence of top-
ical formulations was issued by the United States Food and Drug Administration in 1998 but has since been
withdrawn. This was due to problems associated with the method and also inconsistencies and variability in
the resulting data. Ultimately, inter-laboratory comparative studies on the same products were found to have
conflicting and opposite results. However, in spite of the withdrawal of the FDA’s draft guidance, tape strip-
ping remains a promising tool and is still being investigated and optimized by a number of researchers. Whilst
the tape stripping method has clearly been shown to be a viable alternative approach for BE assessment of
topical products, it is important to optimize the method to control sources of variability such as, the use of an
appropriate dose duration, careful removal of residual application prior to skin stripping, controlled systematic
stripping orientation of each site, normalization of individual skin thickness etc.

According to the USA FDA and most regulatory authorities, the declaration of bioequivalence between a test
and reference product using the 2 one-sided t-test, requires that the 90% confidence interval (CI) should fall
within the range of 80-125%. Thus, the same approaches , these criteria have been adopted to assess BE for
dermatology products The first tape strip analyzed but not included in the data analysis since the first tape
strip may still contain some formulation residue not removed from the skin by swabbing.

2
www.diteba.com A DITEBA WHITEPAPER
IN VIVO DERMAL MICRODIALYSIS

Dermal Microdialysis (DMD) is a relatively new In vivo application of Microdialysis (MD) which allows continu-
ous monitoring of endogenous and/or exogenous solutes in the interstitial fluid of dermal tissue with minimal
tissue trauma and involves the placement of small perfused membrane systems at given depths within the
dermis.

When a topical formulation is applied onto the skin and perfusate is pumped through the implanted mem-
brane system, drug molecules from the topical formulation present in the dermal fluid diffuse (driven by the
concentration gradient) into the lumen of the membrane, resulting in the presence of drug in the perfusion
medium collected as dialysate. The dialysate is sampled at various intervals of time and the drug concentra-
tion in the dialysate can be determined quantitatively. It is believed that DMD has the potential for use for BE
assessment of some topical products.

An increasing number of studies are using microdialysis of a wide range of drugs in animals and humans, sup-
porting the potential of this technique for bioavailability and bioequivalence studies.

PERFUSED SKIN MODELS

Perfused skin models are considered the missing link between In vivo and In Vitro methods. Circumventing
the potential harm of testing hazardous compounds in living humans or animals, as well as inconveniences
connected to repeated sampling from the venous system or extensive biopsies, perfused skin models offer the
benefits of living tissue with fully active microcirculation and metabolism.

Ears of different animal origin, such as hairless mice, rabbits, or pigs, have been used. However, the exposed
location of the ears and their involvement in thermoregulation implement a high degree of vascularization.
Because of their unusually high permeability, perfused ear models have been proposed to be predictive mod-
els of premature neonate skin.

Basically, these models comprise a surgically prepared portion of animal skin including a continuous vascular
circulation which can be cannulized, perfused with tissue culture medium, and sampled for cutaneously ap-
plied chemicals or their metabolites. The easy sampling of substance levels within the vascular system itself al-
lows analysis of systemically available drugs. Methods known from in vivo investigations, such as mass balance,
including perfusate levels, surface washings, stratum corneum tape stripping, and deeper skin layer biopsies,
are easily transferable to the perfused skin. Concerns about using animal skin as a surrogate for human dermal
drug absorption are associated with differences in stratum corneum thickness, number of corneocyte layers,
hair density, water content, lipid profile, and morphology. When comparing results of several species, human
skin finds its closest match in porcine tissue.

However, in the case of topical preparations that are intended for local action, the general BE procedure can-
not be used. Thus, BE assessment of topical products usually requires clinical studies in patients to compare a
new formulation (test) versus an approved product (reference). However, clinical efficacy trials are time con-
suming and expensive.

3
A DITEBA WHITEPAPER www.diteba.com
For an NDA preapproval, or for an NDA or ANDA postapproval, FDA recommends guidance for industry,
“SUPAC-SS Nonsterile Semisolid Dosage Forms, Scale-up and Postapproval Changes” In Vitro Release Testing
and In vivo Bioequivalence Documentation (May 1997)

If alternative approaches are not possible, then BE data based on comparative clinical trials may be crucial.
However, as stated comparative clinical trials are generally difficult to perform, highly variable, and insensitive.

Currently, the human skin blanching assay is the only acceptable BE method approved by the USA Food and
Drug Administration and also by many other international regulatory bodies for the BE assessment of topical
products.

More specifically, bioavailability of products refers to the release of steroids from the preparation followed by
its penetration of the epidermis to produce the characteristic blanching response. Skin blanching assay by col-
orimetry is a promising technique, its measure of color difference. The advantages of the blanching assay are
that it is noninvasive, inexpensive, reproducible, and safe. In clinical circumstances, when the degree of sen-
sitivity of the skin to blanching is required, one can simply estimate the degree of blanching while comparing
it to normal neighboring skin as the value of color difference. The instrument records color reflectance three
dimensionally and compare differences in color between treated site and the control sites.

This method was originally, but it is only applicable for assessing topical corticosteroid products which produce
skin blanching following application to the skin. Therefore, alternative methods for the BE assessment of other
topical dosage forms are recommended.

Consequently, up to date, the only “surrogate” measure for the assessment of bioequivalence of topical prod-
ucts “officially” recognized is the human skin blanching assay for topical corticosteroids. Although an instru-
mental method of assessing blanching is generally recommended, recent data have shown that visual assess-
ment is as good as the instrumental data for BE of topical corticosteroid products. Hence tape stripping has
been shown to be a viable alternative BE method for the assessment of topical preparations.

IN VITRO SKIN PERMEATION STUDIES AS AN EXTENSION OF THE METHODOLOGY

The human cadaver skin (ex vivo) model is a powerful and sensitive tool by which to accurately quantitate a
drug’s rate of percutaneousabsorption, and it has application within multiple areas of the drug development
process. The area of greatest application historically has been preclinical development, where it is used princi-
pally to screen and select the optimum formulation for further development.

Most publications in the field of skin permeation research are carried out using a large variety of setups and
experimental protocols varying from laboratory to laboratory. This raises questions of standardization and reg-
ularization.

For all In Vitro skin experiments, human skin is considered to be the “gold-standard.” Supply is usually provid-
ed from plastic surgery, amputations, or cadavers. While for In vivo studies the mid-forearm (volar and dorsal
sides) is used, In Vitro skin sources are mostly abdominal, back, leg, or breast skin. Much attention has been
turned to assessing the degree and sources of variability.

4
www.diteba.com A DITEBA WHITEPAPER
Nonetheless, large intra and inter individual variations of up to 45% in vivo are documented. These may be due
to differences in lipid composition, skin thickness, or hydration, which depend on body site and sex or are a
consequence of skin diseases.

Current scientific consensus is that In Vitro release is an acceptable regulatory measure to signal inequivalence
in the presence of certain formulation and manufacturing changes. With suitable validation, In Vitro release
may be used to assess batch-to-batch quality, replacing a series of tests that in the aggregate assess product
quality and drug release (e.g., particle size determination, viscosity, and rheology). Because topical dosage
forms are complex dosage forms, manufacturers should optimize the In Vitro release test procedure for their
product in a manner analogous to the use of In Vitro dissolution to assess the quality of extended release
products from batch to batch. In addition, In Vitro release might be used in a sponsor specific comparability
protocol to allow more extensive postapproval changes in formulation and/or manufacturing, provided that BE
between two products representing the extremes of the formulation and manufacturing changes have been
shown to be bioequivalent, using approaches recommended earlier in this document.

However, as new data continue to emerge to support the validity of the model as a surrogate for In vivo mea-
surements of bioavailability and bioequivalence, its application within other phases of the drug development
process have become evident.

These include:

• Reformulation during Phases I–III,

• Development of line extensions and products with enhanced delivery,
• Scale-up and post-approval changes,
• Generic drug development,
• Use as a surrogate for bioequivalence.

Greater recognitions by FDA of its standing as a scientifically sound test method and acceptance of its use as
a valid surrogate for more costly and time consuming human/animal tests would help to simplify the develop-
ment of both new and generic topical/ transdermal drug products.

The prior examples illustrating the advantage of the cadaver skin model in generic drug development raise a
question about its potential use as a bioequivalence testing method. Indeed, this has been a subject of discus-
sion in the past at meetings jointly sponsored by AAPS and FDA. Although insufficient data were available at
that time to make a compelling argument for its use as a bioequivalence test method, and regulatory interest
in other bioequivalence methods has subsequently sidetracked further consideration, new data continue to
emerge and strongly support use of the cadaver skin model as a BE method.

In conclusion: Two, methodologies, have been given consideration by the FDA as potentially suitable surro-
gates for clinical tests:

1) In vivo dermatopharmacokinetic (DPK) method in which drug levels in the stratum corneum are assessed
through the use of tape stripping,
2) In Vitro DPK method in which the rate and extent of permeation through ex vivo human skin is measured.

5
A DITEBA WHITEPAPER www.diteba.com
The first method is based on the fact that the stratum corneum is the compartment through which drugs must
pass to move from the skin surface to their site of action in the deeper layers of the skin, epidermis and der-
mis. Therefore, analysis of the kinetics of drug entry and exit from this compartment could be used to assess
bioequivalence, a situation analogous to the use of the blood stream to assess the bioequivalence of oral
drugs.

An in vitro DPK method using human skin was actually considered by FDA much earlier than the In vivo DPK
method. However, the lack of a sufficient compelling data at that time stopped further consideration. The sit-
uation that has now changed, new data have been obtained last 10 years offer substantial evidence to support
the belief that the in vitro assessment of pharmacokinetics in excised human skin is a viable alternative to
clinical testing.

Review of the In Vitro data exists in both the topical and transdermal drug fields of the past 10 years reveals
the great extent to support the proposition that the use of excised human skin is a valid model by which to
assess the BE/BA of various drugs and other chemicals which come in contact with the skin.

Combination of our expertise and harmonized Diteba’s protocol/report with FDA, OECD requirements, made
alternative In Vitro methodology, successful, for submission and approval numerous topical formulations, not
only as supportive data, but as waiver of clinical In vivo bioequivalence.

6
www.diteba.com A DITEBA WHITEPAPER