In Vivo and In Vitro Bioavailability
Assessment of Topical Corticosteroid:
Applications in Pharma Industry
Muhammad Qamar- uz- Zaman
Assistant Professor,
Department of Pharmacy,
The Islamia University of Bahawalpur.
Objectives:
Topical formulations
In vivo and In vitro methods used for topical
corticosteroids formulations (ointments & Creams).
Applications of these studies in Pharma industry.
Topical Formulations?
• Prescribed to apply the drug on the skin surface
to get local or systemic therapeutic effects.
e.g: Ointments, Creams, Lotions, Transdermal
patches etc.
Need of Bioavailability studies
(Topical Corticosteroids)
Bioavailability leads to Bioequivalence studies
to rank the relative pharmacological activities
of the plethora of available commercial
corticosteroids.
Skin Structure
Human Skin (epidermis)
Bioavailability studies
Two types of studies are:
 In vitro

Modified Franz cell method
In vivo

Tape stripping method

Skin Blanching method

HPLC method
Franz Cell
Continued….. (Franz Cell)
Test Procedure (Franz Cell)
Materials:
Synthetic hydrophobic polyvinylidene difluoride
membrane (Durapore HVHP type, Millipore, Massachusetts)
Commercially available formulation of 0.05%
betamethasone dipropionate (Ointment or
Cream)
 Modified Franz Cell
Continued…..
Synthetic membrane is placed on the modified Franz cell over
30 hrs
Franz Cell has 1.76-cm2 orifice
HVHP filter with 25 mm in diameter and 140 μm
in thickness, 0.45 μm pore size & 75% porosity
A Plexiglas ring is placed on the membrane
Ring with internal diameter of 1.6 cm and a thickness of 0.5 cm
Ring is filled with 1 mL of 0.05% betamethasone dipropionate formulation
commercially available formulation (Ointment or Cream)
Continued…..
Drug is moved from donor compartment to
receiving compartment through synthetic
membrane
Collect the sample from receiving
compartment and perform the analysis by
HPLC method to calculate the drug content
Continued….
Precautions:
• The hydrophobic membranes used in the drug release
experiments are presoaked 5 minutes in either isopropyl
myristate (IPM) or a 40:60 mixture of PEG 400: water prior
to mounting on the modified Franz Cell.
• Thorough contact between the membrane and the
formulation is visually confirmed by viewing the underside
of the membrane.
• 40:60 (v/v) PEG400:water is used as vehicle as compare to
water or phosphate buffered saline (pH 7.4).
• The drug dose applied to the synthetic membrane is 0.28
mg of betamethasone dipropionate / cm2
In Vivo Methods
 Tape stripping method
 Skin Blanching method
 High Performance Liquid Chromatography
(HPLC) method
Tape Stripping Human Skin
Continued……
Tape Stripping Human Skin Method:
 This approach is based on the assumption
that effective cutaneous therapy requires
partitioning of the topically applied drug
from its vehicle formulation into the stratum
corneum
 The amount of drug partitioning into stratum
corneum can be objectively assessed by tape
stripping
the
same and
chemically
quantitating the amount of drug in the tape
strips.
Procedure:

0.05% betamethasone dipropionate is applied to the same human subject
forearm at the same time on the right and left forearms under occlusion for
24 hours.

160 mg dose is placed in a 1.2-cm diameter Hilltop chamber and affixed via
the supplied adhesive tape to a 1.13 cm2 surface area on the volar aspect of
the forearms. This dose, approximately 142 mg of formulation/ cm2
Continued….
Continued….
 A maximum of 3 chambers, equally spaced 2cm
apart, are applied to each forearm for each study, at
least 6 cm above the wrist and 6 cm below the
antecubital fossa to minimize the disturbance of the
drug treatment throughout normal daily activity.

All experimental skin site are maintained under
occlusion for 24 hours.
Continued…
 After 24 hours of treatment, the chamber is removed, and residual
formulation on the skin surface is gently removed with a Teflon spatula.
(The drug treated site is gently wiped with three dry cotton applicators
and allowed to air dry for 1-3 minutes).
 Ten individual 0.6 cm diameter disks of tape are utilized to tape strip
the center of the 1.2 cm diameter drug treated skin site.
 The sequential tape strips are combined and extracted in clean, labeled
1.5mL polypropylene microcentrifuge tubes with 200 μL of acetonitrile.
Continued…
 The tubes are capped and vortexed for 1minute at high speed and
centrifuged at 5000 rpm for 5 minutes.
 The supernatant is transferred into clean, labeled microcentrifuge
tubes, 20 μL is injected into HPLC .
 A single extraction of the tape strips containing drug and stratum
corneum is sufficient to routinely recover > 90% of the drug.
Continued…
HPLC conditions:
• Retention time: 6.45 min
• Column: C18 reverse phase column
• Temperature: 25 centigrade
• Flow rate: 1.2 mL/min
• Mobile phase: acetonitrile: distilled water
(65:35v/v)
• Detector: UV at 254 nm
Continued….
Precautions:
 Assessment of the amount of drug could be extremely variable due to
the differences in the amount of stratum corneum removed.
 One approach to normalize the drug content in the tape strips among
the individuals tested is to divide the drug content by the amount of
tissue removed, thus giving a drug concentration, milligram of drug per
milligram of stratum corneum.
 The amount of stratum corneum removed with the tape stripping
method can be determined by weighing the tape strips before and after
tape stripping.
Skin Blanching Assay
• Bioequivalence
of
topical
corticosteroids
from
commercial cream and ointment formulation is
currently assessed with Skin Blanching Assay.
• Assay is Subjective
• Skin blanching is monitored in the remaining drug
treated skin site after tape stripping.
• Skin blanching is score using 0-4 scale by a single
investigator at 1, 24, and 48 hours after drug removal.
Continued…
• The practicality has been enhanced by combining
the effects of time and skin blanching into a
composite skin blanching score.
• The skin blanching score
that is utilized in
correlation study assumes that the greater the
amount of drug remaining in the skin over time,
the more prolonged is the pharmacological effect.
Continued…
• The skin blanching score is therefore calculated
additively using the time interval of observation
(hours) multiplied by the extent of skin blanching
at that time interval.
For example: an extent of skin blanching at 1, 24,
48 hours of +4, +3, and +1, respectively, produces
a score of 124: [(1) x (+4) + (24) x (+3) + (48) x (+1)]
= 124.
Skin blanching Scoring System
Extent of Skin
Blanching
Description
0
No blanching
1
Slight, diffuse blanching with
indistinct outline
More Intense blanching with half of
the treated site perimeter outline
2
3
4
Marked blanching with a distinct
outline of the treated site
Extreme blanching with a distinct
outline of the treated site
Applications in Pharma Industry
 In developing generic product development
 To meet the regulatory requirements.
 Helpful tool to enhance the
promotion of medicines.
marketing and
Thank you