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…..

Continued…..

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 SystemExtent of Skin Blanching Description

0 No blanching

1 Slight, diffuse blanching with indistinct outline

2 More Intense blanching with half of the treated site perimeter outline

3 Marked blanching with a distinct outline of the treated site

4 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 marketing and

promotion of medicines.

Thank you