Introduction Bundelkhand University, Jhansi

CHAPTER 1: INTRODUCTION TO DERMATOPHARMACOKINETICS

1.1 Human skin anatomy and physiology

The human skin (integumentum commune) is the barrier between the organism’s internal
environment and its surroundings. With an area of 1.5-1.8 m2, it is our biggest organ. The
following vital functions are ensured by the skin: mechanical and chemical protection,
protection against UV radiation and micro-organisms, thermoregulation, and sensory
perception. Anatomically, the skin can be divided into subcutis and cutis. The subcutis
(tela subcutanea) is formed of small lobes of fat (panniculus adiposus) separated by septa
of connective tissue. The fat is responsible for thermo-insulation, and the connective
tissue incorporates lymph and blood vessels reaching into the dermis. The cutis is divided
into dermis (corium) and epidermis, which are firmly bound together in the dermo-
epidermal junction by hemidesmosomes on the epidermal side and by anchoring collagen
fibrils on the dermal side.

The dermis is about 1-3 mm thick, and consists of cells (fibroblasts, inflammatory cells)
and fibers (collagen, elastic, reticular) embedded in an amorphous matrix consisting of
mucopolysaccharides produced by the fibroblasts. Also present in the dermis are: blood
and lymph vessels, free nerves endings for the perception of temperature, itching and
pain, encapsulated nerve endings such as the Vater-Pacini corpuscles (sensitive to
pressure and vibration) and the Meissner’s corpuscles (sensitive to touch), nerves for the
vegetative innervation, and muscles (M. arrector pili, mimetic muscles). Skin appendages
(hair, sebaceous glands, sweat glands, nails) originate in the dermis or in the upper
subcutis (sweat glands). Structurally, the dermis comprises the deeper situated, thicker
stratum reticulare (few cells except fibroblasts, many fibers) and the stratum papillare
(many cells, capillaries, nerves), located just below the epidermis.1

The epidermis is the outermost skin layer and is a vessel-free, nerve-free, stratified,
squamous epithelium with a water content of 70%. It is nourished by the underlying
capillary loops of the stratum papillare. The thickness of the epidermis varies depending
on the anatomical region, with mean values of 77 µm at the forearm2, minimal values of
30 mm at the eye lid and maximal values of 1.6 mm at the plantar region. Two kinds of

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cells make up the epidermis. First, the keratinocytes (90%), which are responsible for
keratin production and are kept together by desmosomes. Second, the dendritic cells
(10%): melanocytes (pigment cells), Langerhans cells (immunocompetent cells), and
Merkel’s cells (responsible for the perception). The following layers characterize the
epidermis: the stratum basale (basal layer) with one cell layer, the stratum spinosum
(prickle cell layer) with 2-5 cell layers, the stratum granulosum (granular layer) with 1-3
cell layers, the stratum lucidum (in palmar and plantar skin only), and the stratum
corneum (corneal layer) with 10-20 cell layers. In a cycle of about 1 month, new
keratinocytes originate in the stratum basale, differentiate in the stratum spinosum,
produce keratohyalin containing granules and lipid/enzymes-containing lamellar bodies
(Odland bodies), which are then exocyted in the stratum granulosum and are finally
transformed into the stratum corneum.

Within the stratum corneum, the keratinocytes undergo complete keratinization, forming
the nucleus-devoid, flattened, hexagonal corneocytes of about 0.5-3 mm thickness and
30-40 mm width3. The bottom part of the stratum corneum (stratum compactum) is very
firmly bound together by corneo(desmo)somes and intercellular lipids and has an
important protective function. The top part is looser in its structure (stratum disjunctum)
and undergoes desquamation by enzymatic digestion of the corneo(desmo)somes4. The
thickness of the stratum corneum depends, like the thickness of epidermis and dermis, on
the anatomical region. Mean values of 15 mm (16 ± 4 cell layers) were recorded at the
flexor forearm and maximal values of 1 mm (86 ± 36 cell layers) at the heel.5, 6, 7

Stratum corneum, the skin barrier

The stratum corneum consists of 15% water, 70% proteins, and 15% lipids. According to
the brick-and-mortar model8, 9, two compartments can be discerned: keratinous, lipid-
devoid corneocytes as bricks and intercellular, continuous lamellar bilayers of lipids as
mortar. The corneocytes are built out of an insoluble protein complex consisting of
highly organized keratin macrofibrils, and they contain natural moisturizing factors
(NMF), low-molecular-weight, watersoluble compounds responsible for water retention.
The NMF are mainly derived from the protein filaggrin and are composed of amino acids
(40%), pyrrolidon carboxylic acid (12%), lactic acid (12%), and urea (7%).10 Each

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corneocyte is encapsulated in an insoluble tough protein shell of 10 nm thickness, the

cornified cell envelope, which is covalently bound to an outer lipid envelope consisting
of a layer of long-chain ceramides.11 The free intercellular lipid bilayers of the stratum
corneum have a unique composition compared to other epithelial lipid bilayers and
consist of ceramides (50%), cholesterol (25%), and fatty acids (10-20%, highly enriched
in linoleic acid). No phospholipids are present in healthy stratum corneum, and more than
one third of the lipids have chain lengths longer than 22 carbons (vs. 16-18 carbons in
other mammalian cell membranes).12 Most lipids of the lamellar bilayer are derived from
the Odland bodies, extruded as phospholipids, sphingolipids, and plasma membrane
constituents at the interface stratum granulosum / stratum corneum and then
enzymatically cleaved.13 It was not until the 1940’s that the stratum corneum clearly
emerged as the specific site of the skin barrier for both endogenous and exogenous
compounds.14, 15 In the 70’s, the intercellular lipids were recognized as the primary site of
the barrier.16 The qualitative and quantitative organization of the intercellular lipid
lamellae is determinant for the barrier function. Several models such as the domain-
mosaic,17 the sandwich18, and the single-gel-phase model19 have been proposed to explain
their molecular organization.20 An appropriate moisturization level of the stratum
corneum, regulated by the presence of NMF, is also important for the maintenance of an
effective skin barrier. The stratum corneum is very resistant to physical (mechanical,
thermic, actinic) and chemical (acids, to a lesser extent bases) damage. The barrier is
more sensitive to organic solvents which can extract the intercellular lipids and to
detergents which can damage the cell membrane. 21 Lipophilic compounds usually pass
more easily through the lipophilic stratum corneum barrier than hydrophilic compounds.
However, the passage through the hydrophilic (epi)dermis may then become the rate
limiting step for a lipophilic compound. Furthermore, hydrophilic substances may
penetrate the barrier also by follicular pathways.22

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1.2 Percutaneous absorption and topical bioavailability

The EMEA (European Agency for the Evaluation of Medicinal Products) defines the
term bioavailability as the “rate and extent to which the active substance or active moiety
is absorbed from a pharmaceutical form and becomes available at the site of action”.23
The “absence of a significant difference in the rate and extent to which the active
ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives
becomes available at the site of drug action when administered at the same molar dose
under similar conditions in an appropriate designed study” is termed bioequivalence.24
Topical dermatological drug products belong to the class of locally acting drug
products.25 In this case, the site of pharmacological action is the skin. Stratum corneum
and skin surface are considered to be the compartments of invasion, whereas the blood
system represents the compartment of excretion.26 Therefore, two different types of
bioavailability have to be distinguished for topical application. The topical bioavailability
reflects the rate and extent to which the active moiety becomes available at the site of
action, i.e. the skin. The systemic bioavailability, instead, may not properly reflect the
cutaneous bioavailability for medications intended to treat local skin disorders but
becomes important for the toxicological evaluation of the body burden and for
transdermal therapeutic systems (TTSs).27 Percutaneous absorption is the uptake of a
compound into the systemic circulation after topical application and describes the
movement through the various layers of the skin with respect to both rate and extent. The
percutaneous absorption process can be divided into the following 3 steps.28

Penetration is the entry of a substance (in vitro) into a particular layer. Permeation is the
passage (ex vivo) through one layer into another layer. Absorption is the uptake (in vivo)
of a substance into the vascular system (blood and/or lymph vessel), which acts as the
central compartment, and reflects the systemic bioavailability.

1.2.1 Routes of penetration into the skin

There are 3 potential routes of penetration from the skin surface into the epidermis29: 1)
the intercellular route, 2) the transcellular route, and 3) the transappendageal route

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through either the eccrine (sweat) glands or the hair follicles with their associated
sebaceous glands.

Under normal circumstances, the predominant is the intercellular route, which consists of
a tortuous route along the cornified envelope-armored corneocytes through the structured
intercellular lipid bilayers.30, 31 The tortuous diffusional path length has been estimated to
be as long as 300-500 mm32, 33 in contrast to a mean stratum corneum thickness of just 20
mm. The transport process involves sequential diffusion and partitioning between the
polar head group regions and the long alkyl chains of the lipids.34 The transcellular route
is possible for small hydrophilic substances like water. Waibler observed an in vivo
intracellular distribution of hydrophilic dyes (patent blue, sodium fluoresceine) applied in
water, and a prevalent intercellular distribution of the lipophilic dye curcumin applied in
liquid paraffin. The transappendageal route was, in the past, considered to play a
subordinate role during percutaneous penetration, since the skin surface of the
appendages yields only a maximal of 0.1% of the total skin surface. The contribution of
the appendages was regarded as initial “shunt” diffusion, whereas the main “bulk”
diffusion took place through the stratum corneum. Yet, the transappendageal route can be
relevant for lipophilic compounds35 as well as for polar steroids showing a low diffusion
through the stratum corneum.36, 37
A higher impact of the transappendageal route on
percutaneous absorption has to be expected on the forehead, where the follicle density is
very high (292 follicles/cm2 vs. 14-22 /cm2 in other skin regions).38

1.2.2 Mathematical models

[Link] Fick’s laws of diffusion

After application of a topical formulation, the active compound has to be released from
the vehicle, partition between vehicle and stratum corneum, and diffuse through (and
partition between) the different layers of the skin before it can exert its pharmacological
action, finally being “excreted” into the systemic circulation. Diffusion is a passive
kinetic process taking place along a concentration gradient from a region of higher
concentration to a region of lower concentration. The diffusion through the skin can be
described by Fick’s first law:

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……………. 1

where J is the steady state flux of the compound mass (m) through the stratum corneum
per unit area (A) and unit time (t) (mg/cm2 s), D is the diffusion coefficient of the
compound in the stratum corneum (cm2/s), c is the drug concentration, and x is the
position.39 The solution of the equation with the appropriate boundary conditions gives:

……………. 2

where K is the partition coefficient of the compound between vehicle and stratum
corneum, h is the diffusional pathlength (cm), kp is the permeability coefficient, and Dc is
the concentration difference (mg/cm2) across the stratum corneum between applied
concentration (Cappl) and concentration below the stratum corneum (in vivo) or in the
receptor phase (in vitro, Crec).40 Under normal circumstances, the applied concentration
(Cappl) is much larger than the concentration in deeper skin layers, and Dc can be
replaced with Cappl. The real diffusional pathlength (h) is the tortuous pathway along the
intercellular lipids, which is longer than the mere stratum corneum thickness. However,
the stratum corneum thickness is mostly used, since it is easily measurable.

If the steady state is not attained, the diffusional flux can be explained by Fick’s second
law, which describes the concentration change over time at a definite position x within
the membrane:

……………… 3

Different solutions of this equation with appropriate boundary conditions have been
proposed.41, 42, 43

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1.2.3 Factors governing the Percutaneous Absorption:

[Link] Solubility

The concentration difference of the compound across the skin or the membrane
(concentration gradient Dc) is the driving force for diffusion. A balance has to be found
between good solubility within the vehicle and good release from the vehicle. The
maximal stable concentration is achieved at saturation solubility (thermodynamic activity
or leaving potential = 1). Particles suspended within the vehicle do not directly contribute
to the concentration gradient but constitute a reservoir maintaining saturation conditions
in the vehicle for a prolonged time.

[Link] Partition coefficient (K)

The partition coefficient is a thermodynamic, time-independent factor and displays the

relative preference of a compound to stay either within the vehicle or within the
membrane. The degree of partitioning is dependent on the relative solubility (affinity) of
the compound in both vehicle and membrane. A hostile vehicle environment, and thus a
higher affinity to the membrane, favors partitioning.

[Link] Diffusion coefficient (D)

The degree of diffusion within a specific medium (vehicle, membrane) is described by

the diffusion coefficient. The diffusion coefficient depends on: the physicochemical
properties of both the diffusing compound (size, radius) and the diffusional medium
(viscosity), temperature, and pressure. The Einstein diffusion equation points out the
dependency on these factors:

………………. 4
R = universal gas constant = kB
NA = 8.314472 J/mol K
kB = Boltzmann constant = R/NA = 1.3806505 . 10-23 J/K
NA = Avogadro’s number = 6.0221415. 1023 /mol

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h = viscosity of the medium (vehicle, membrane)

r = radius of the compound particle
T = absolute temperature

[Link] Permeability coefficient (kp)

The permeability coefficient is a heterogeneous rate constant characteristic for a

membrane and a compound and is expressed as depth of diffusion per unit time (cm/h):

………………. 5

This constant can be calculated from experimental results by dividing the flux J with the
concentration difference Dc, whereas it is more difficult to calculate the terms K and D
separately. Permeability is influenced to a greater extent by variation of the partition
coefficient than by variation of the diffusion coefficient. This can be observed in the
analysis of a homologous series (e.g., of n-alkanols), where partition coefficient and
permeability coefficient increase with increasing chain length, whereas the diffusion
coefficient remains more or less constant.

1.2.4 Other factors affecting percutaneous absorption

Factors affecting percutaneous absorption and topical bioavailability of topically applied

compounds are:
1. The physicochemical characteristics of the compound
2. The physicochemical characteristics of the vehicle
3. The application conditions
4. The skin conditions.

[Link] Physicochemical characteristics of the compound

Molecular weight (size), degree of ionization (charge), and lipophilicity are important
factors determining the partition and diffusion coefficients.44 The molecular weight is
inversely proportional to percutaneous absorption and seems to particularly influence the
diffusion coefficient. Molecules larger than 500 Daltons have usually more difficulty to

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pass through the healthy stratum corneum.45 It is a common presumption that only non-
ionized compounds are able to diffuse through the lipophilic intercellular regions of the
stratum corneum. Yet, ionized compounds have been reported to permeate through
human skin through the intracellular and transappendageal pathway, albeit at a slower
rate. Moreover, the formation of ion pairs between compound ions and ions present in the
skin can lead to neutral compounds. As most drugs are either weak acids or weak bases,
the pH of aqueous vehicles determines the ionization state.46 The best percutaneous
absorption would be achieved by an amphiphilic compound showing both high solubility
in the lipophilic stratum corneum (maximal input into the stratum corneum) and high
aqueous solubility in the hydrophilic viable epidermis (maximal output into deeper
layers). In general, compounds with a log Koctanol/water of about 1-3 have optimum partition
behavior.47 High penetration into the stratum corneum but limited penetration into the
viable epidermis (observed for very lipophilic compounds) may induce a reservoir into
the stratum corneum. The same can occur for compounds with small diffusivity in the
stratum corneum and for compounds binding to specific tissue components.

[Link] Physicochemical characteristics of the vehicle

The vehicle can both influence drug release as well as alter the stratum corneum
structure. Drug release is affected by the viscosity of the vehicle (alteration of diffusion
coefficient) and by the solubility of the compound in the vehicle. Alteration of the
stratum corneum structure includes extraction of intercellular skin lipids and occlusive
effects. The pH of the vehicle, the state of the compound in the vehicle (dissolved,
suspended), the concentration of the compound, and the presence of cosolvents (e.g.,
propylene glycol), penetration enhancers (e.g., urea, DMSO3), and surfactants are all
factors influencing percutaneous absorption. Among the penetration enhancers, two types
can be distinguished: those influencing diffusion (e.g., Azone®4, oleic acid, surfactants)
and those influencing partitioning (most solvents, e.g. propylene glycol). After
application of a topical formulation to the skin, the vehicle undergoes important structural
changes also known as “metamorphosis of the vehicle”. The partitioning between vehicle
and stratum corneum, and thus the penetration into stratum corneum, is different for each

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component of the formulation. In addition, evaporation of the different ingredients is

possible. After application of a compound in a volatile vehicle (e.g., ethanol, acetone),
the rapid evaporation of the vehicle subsequently increases the concentration and the
saturation degree of the compound, thus altering the driving force of diffusion (enhanced
thermodynamic activity). The maximum drug penetration into the skin is known to take
place when the drug is in a saturated state.48 In some cases, supersaturation can occur and
thus further enhance percutaneous penetration.49 After complete evaporation of the
solvent, the remnant drug precipitates onto the skin as “solvent deposited solid”. In this
case, percutaneous penetration becomes a dissolution rate limited process.50, 51

Percutaneous absorption following application in a volatile vehicle is quite different than

percutaneous absorption from non-volatile vehicles and is not a steady-state process.52
Akther et al. observed, after application of flurbiprofen and ibuprofen in acetone, an
initially low percutaneous penetration which then increased as soon as evaporation of the
vehicle took place. Different mathematical models of percutaneous absorption kinetics
after application of finite vehicle volumes and solvent deposited solids have been
proposed.

[Link] Application conditions

Dosing technique, dose, and application under occlusion are the most important
application conditions affecting percutaneous absorption. Two main dosing techniques
are known - the infinite and the finite. The infinite dose technique concerns the
application of an amount of material much higher than is expected to penetrate into the
skin. The surplus of material forms a surface reservoir which appears to be “infinite”. The
steady-state flux after application of an infinite dose follows Fick’s first law of diffusion.
The finite dose technique is more closely related to clinical settings. A definite, low
amount of material is applied. With time, the amount of material on the skin surface is
depleted and the flux into the skin decreases. No steady-state flux can be observed. In this
case, the diffusion has to be explained with an appropriate solution of Fick’s second law,
and several models have been proposed.53, 54, 55 A special case at the boundary between
finite and infinite dosing is observed after finite application of poorly absorbed
compounds (e.g., corticosteroids). In this case, the amount recovered on the skin surface

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after penetration is usually high (60-95% of the dose applied) and forms a not completely
depleted (i.e. infinite) surface reservoir. After application of a single dose, the following
parameters have been shown to influence percutaneous absorption: concentration of the
dose, applied film thickness, area on which the dose is applied, and duration of the
application. Occlusion is defined as the external insulation of the skin with a water
evaporation limiting barrier56 and can be performed either by covering the skin with an
impermeable wrap (e.g. plastic film, tape or wound wrap, gloves, impermeable textiles,
diapers) or by applying topical vehicles containing fats or oils (e.g., petrolatum or
paraffin). Because of the inhibition of the trans-epidermal water loss (TEWL) from the
skin surface by occlusion, the normal water content of the stratum corneum (10-20%) can
be increased by up to 50%. Even short-time occlusion (e.g., 30 minutes) results in a
overhydration of the skin.57, 58
Water has been shown to accumulate either within the
corneocytes, inducing their swelling,59 or within the intercellular lipids, forming
intercellular water pools.60 Moreover, occlusion has an effect on: skin surface
temperature (increase from 320C to 370C), blood flow, composition of epidermal lipids,
DNA synthesis, epidermal morphology and turnover, composition of the microbial skin
flora, pH, and activity of sweat glands and Langerhans cells of skin. In general, the
barrier function of the stratum corneum is reduced by occlusion.61 The increased stratum
corneum hydration and increased temperature can positively alter the partitioning of
applied compounds between vehicle, stratum corneum, and viable epidermis, thus
enhancing percutaneous absorption in a simple manner. After occlusion, the hydrated
stratum corneum and the viable epidermis appears more similar (facilitated partitioning),
with a greater effect for lipophilic compounds.62, 63

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1.3 Assessment of topical bioavailability

1.3.1 Regulatory requirements

In the US, the following general approaches, listed in order of preference, are regarded as
acceptable by the Code of Federal Regulations (CFR) of the FDA for bioavailability
and/or bioequivalence assessment: 1) pharmacokinetic approach based on measurements
of the concentration of the active moiety and/or metabolites in blood, plasma, serum, or
other appropriate biological fluid as a function of time; 2) pharmacokinetic approach
based on the measurement of the urinary excretion of the active moiety and/or
metabolites as a function of time (only appropriate if urinary excretion is a significant
mechanism of elimination); 3) pharmacodynamic approach based on the measurement of
an appropriate acute pharmacological effect of the active moiety and/or metabolites
(particularly appropriate to dosage forms not intended to deliver the active moiety to the
bloodstream for systemic distribution); 4) comparative clinical trials; and 5) in vitro
studies (21CFR320.24). For drug products that are not intended to be absorbed into the
bloodstream, bioavailability may be assessed by measurements intended to reflect the rate
and extent to which the active moiety becomes available at the site of action
(21CFR320.23). In the EU, the “Note for guidance on investigation of bioavailability and
bioequivalence” released by the EMEA states that for products for local use (after oral,
nasal, inhalative, ocular, dermal, rectal, vaginal administration) intended to act without
systemic absorption, the approach to determine bioequivalence based on systemic
measurements is not applicable and pharmacodynamic or comparative clinical trials are
required. The determination of the systemic exposure resulting from locally applied
products is only relevant if there is a risk of systemic adverse reactions. Comparative
clinical efficacy trials are relatively insensitive, highly variable, time consuming, costly,
and need a high number of volunteers. Pharmacodynamic investigations can only be
performed if the topically applied compound produces a measurable pharmacodynamic
response, and this is not always the case. Nevertheless, there are two outstanding
examples. Corticosteroids induce a vasoconstriction, and the degree of skin blanching can
be correlated with the efficacy and potency of the steroid. A relevant guidance (“Topical
dermatological corticosteroids: in vivo bioequivalence”) has been released by the FDA.

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Furthermore, retinoids induce an enhanced TEWL, which can be used as

pharmacodynamic measure for bioavailability assessment. An explicit approach for
measurements intended to reflect the rate and extent to which the active moiety becomes
available at the site of action is still missing in the regulatory specifications. It was
against this background that the FDA proposed in 1998 draft guidance for the
bioavailability assessment of topical dermatological drug products (“Topical
dermatological drug products NDAs and ANDAs – In vivo bioavailability,
bioequivalence, in vitro release, and associated studies”).

The guidance focused on the DPK approach, which is based on the measurement of the
active moiety in the stratum corneum. The DPK approach is comparable to a blood,
plasma, or urine pharmacokinetic approach applied to the stratum corneum. Generally,
DPK studies are performed in healthy volunteers, since diseased skin is highly variable
and changes over time. Among different models, tape stripping showed the highest
potential for the DPK characterization of topically applied drugs, since the percutaneous
penetration was assessable without invasive procedures. Yet, the draft guidance was
withdrawn in 2002 because of the following two reasons. The first concern considered
the adequacy of the DPK method to assess the bioequivalence of topical dermatological
drug products, because the products are used to treat a variety of diseases in different part
of the skin, not just the stratum corneum. Yet, despite the fact that the target site for
topical dermatological drug products may not always be the stratum corneum, the drug
must still pass through the stratum corneum barrier to reach deeper sites of action. In
certain cases, the stratum corneum itself is the site of action (e.g., for antimykotika). For
antiacne drug products, the target sites are the hair follicles and the sebaceous glands, and
it has been shown that there is a positive correlation between stratum corneum and
follicular concentrations. The second concern considered the reproducibility of the DPK
method between laboratories. The detailed presentation of the tape stripping technique in
the next chapter will provide a better understanding of this second concern.

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1.3.2 Tape stripping

Tape stripping is a technique which enables the removal of the stratum corneum layer by
layer. Adhesive tapes are sequentially pressed onto the same skin region, and then
stripped off.64 Using a standardized technique, Jacobi et al. showed that 66% of the
stratum corneum is removed with 20 tapes (using Tesa® Multi-Film Crystal-Clear tape),
and nearly the complete stratum corneum (95%) is removed with 50 tapes.65 Yet,
depending on the adhesive tapes used, up to 100 tapes may be required to remove the
entire stratum corneum from one skin site.66 With the tapes, corneocytes and substances
previously applied on the skin are removed and can be quantified with an appropriate
analytical method (e.g., HPLC, spectroscopy, scintillation counting). The tape stripping
technique was introduced in the early 40’s by Wolf. He investigated the topography of
the skin by removing layers of corneocytes with transparent adhesive tapes and by
examining them microscopically.67, 68 In the 50’s, Pinkus applied the new technique to
investigate the epidermal regeneration process. By taking punch biopsies of stripped skin
sites, he observed that tape stripping stimulated the mitotic rate in the stratum basale.69, 70
The mitotic rate was maximal after 3 days, and a couple of weeks were required for
complete skin renewing. Further studies investigating the epidermal growth kinetic after
standardized injury of healthy71, 72, 73 and atopic dermatitis skin74 by tape stripping were
performed. Recently, Choi et al. showed that tape stripping induces the production of
cytokines such as interleukins, tumor necrosis factor α, and γ-interferon75, enhances the
enzymatic activity, and increases the ceramides synthesis.76 Recently the artificial
removal of the stratum corneum barrier by tape stripping has become a frequently used
model for simulating diseased skin,77 and for assessing the efficacy of skin care products
in restoring the barrier.78, 79
Moreover, a percutaneous penetration enhancement of
macromolecules like peptides and oligonucleotides, which usually do not penetrate
healthy skin, was observed after removal of the stratum corneum, thus opening the
possibility of topical vaccinations.80

In the 80’s, the methodology was standardized by Dupuis et al.,81, 82

making of tape
stripping a method widely used in dermatological research to non-invasively investigate
the topical bioavailability and percutaneous penetration of topically applied substances.

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The investigation of the topical activity of antiviralia83, 84

and antimykotika85, 86
, the
investigation of vehicle effects87, 88, barrier function and reservoir formation within the
stratum corneum89, 90, 91
, and the investigation of the percutaneous penetration and
absorption of topical corticosteroids92, 93, 94 are some important examples of application
areas. Furthermore, tape stripping has been used to investigate stratum corneum lipids95
and epidermal enzymes.96 A prerequisite for making the tape stripping technique a
capable, robust, accurate, and reproducible method to investigate the bioavailability of
topically applied drugs is the strict standardization and a clear definition of the stripping
parameters. The technique is susceptible to numerous confounding factors, both intrinsic
and extrinsic. Among the intrinsic factors, the anatomical site, the hydration status of the
skin, the cohesion of the corneocytes (influenced as well by the vehicle used),97 and the
presence of furrows have to be mentioned.98 Van der Molen observed that topically
applied titanium dioxide could still be detected after removal of 40 tapes because of an
accumulation in superficial skin furrows, thus falsifying the results.99 In addition,
interseasonal differences have been observed.100 Among the extrinsic factors, the type of
tape used (e.g., cellophane, polyester, polypropylene, polyethylene, rayon), 101, 102
the
pressure with which the tape is applied onto the skin, the duration of the pressure, and the
force of removal (slow/quick) influence the stratum corneum removal. These are the
parameters which can and must be standardized. Surber et al. outlined a standard protocol
for tape stripping experiments. The protocol implies the use of a template to delineate the
stripping area, and the exertion of a definite pressure (e.g., with a roller)103 on each tape
before removal. The use of a roller has been shown to prevent artifacts due to furrows
and wrinkles.104 Because of inter-individual differences in stratum corneum thickness, the
complete stratum corneum of one skin site should be stripped. In newer investigations,
Weigmann et al. used the tape stripping method to calculate horny layer profiles, in
which the amount of drug penetrated is correlated not only to the tape number stripped
but also to the depth of the stratum corneum. This correlation enables the localization of
the drug within the stratum corneum layers. For this purpose, the removal of the entire
stratum corneum and the quantification of both corneocytes and drug on the tapes are
required.105 Recently, Jacobi et al. reported a procedure for the estimation of the removed
stratum corneum amount on each tape, thus providing a possibility to avoid the complete

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removal of the stratum corneum. After a review of all the influencing and confounding
factors affecting the tape stripping technique, the withdrawal of the DPK draft guideline
for the bioavailability assessment of topical dermatological drug products becomes more
comprehensible. The concern of inadequate reproducibility between laboratories is not at
all amazing. The FDA draft guidance recommended the application of the test
formulation on ≥ 8 skin sites on each forearm, with tape stripping performed at
incremental times after application. Only 12 tapes, without specification of the type of
tape to be used, had to be stripped. Moreover, the first 2 tapes were discarded from
evaluation and the remaining 10 tapes pooled and extracted together. The protocol
described in the draft guidance was not yet mature and did not satisfy the regulatory
requirements for bioavailability/bioequivalence assessment at the time of withdrawal. In
the future, the proposal of a more realistic and elaborated protocol could ensure a
successful regulatory basis for a promising and already widely used DPK method like
tape stripping.

In the determination of BE of topical products, the method of choice is the tape-stripping

technique. The method consists of the standardized protocol of repeated applications and
removal of adhesive tape on the skin surface, whereby consecutive layers of stratum
corneum cells are sampled. As discussed by Jacobi U and et al, Tape stripping is a
standard measuring method for the investigation of the dermatopharmacokinetics of
topically applied substances using adhesive films. These tape strips are successively
applied and removed from the skin after application and penetration of topically applied
substances; thus, the layers of the corneocytes and certain amount of topically applied
substances are removed. The amount of the substances and the amount of stratum
corneum removed with the single tape strip is to be determined for calculation of the
penetration profile. The topically applied substances removed from the skin can be thus
determined by various analytical methods like UV, HPLC, Mass Spectroscopy and other
spectroscopic measurements.97

The weakness of the DPK approach is the end-point nature of the data obtained and the
lack of studies confirming the correlation between DPK data and clinical efficacy. The

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objective of this study is to demonstrate that DPK is a valid means of comparing a test
and reference product for their ability to deliver drug to the deeper layers of the skin.106

To investigate this method for its reliability we have taken two marketed Erythromycin
topical preparations and performed the DPK clinical study using human subjects. All the
formulations are already available in the market, hence one of the formulation from
similar dosage form category i.e. Erythromycin ACNECLIN gel was considered as
reference or standard product, where as remaining formulation, Erythromycin Clid Gel
was taken as test product for the Pharmacokinetic comparison.

1.3.3 Further techniques

Other approaches for the assessment of the drug concentration profile in the skin
following topical application do exist. The most invasive method is represented by the
direct excision of skin tissue. The punch biopsy is performed with round disposable
knives (diameter 2-10 mm), and the biopsy contains epidermis, dermis, and part of the
subcutis. The shave biopsy is more superficial, contains epidermis and part of the dermis,
and is usually performed with a razor blade.107 The skin scraping (curettage) technique
usually removes the superficial stratum corneum with a dermal curette, but deeper parts
of the epidermis can be sampled as well with a more vigorous scraping.108 The skin
surface biopsy uses quick drying cyanoacrylate glue to remove the stratum corneum in
few samplings. The glue is applied to a glass slide, pressed onto the skin surface, and
then removed after polymerization of the glue.109 Cutaneous microdialysis enables the
real-time assessment of cutaneous drug delivery in a minimally invasive manner. A
probe, consisting of a semipermeable dialysis membrane tube and simulating a blood
vessel, is inserted into the dermis and perfused by a physiological solution, which
equilibrates with the extracellular fluid of the surrounding tissue. Topically applied
compounds can thus be determined in the dermal extracellular water.110

The suction blister technique is usually performed to assess drug levels in the skin after
systemic administration. Intra-epidermal blisters are artificially produced by placing a
chamber with several holes onto the skin and applying a vacuum. The suction blister fluid
displays approximately the same composition of the interstitial fluid.111 In addition to

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these invasive techniques, several imaging methods have been applied to the skin in the
last years. The most promising optical methods seem to be the confocal scanning laser
microscopy112 and the confocal Raman microspectroscopy.113, 114 These new techniques
allow to focus a beam to a given depth within the skin and to determine the concentration
of the topically applied compound at this level in an absolutely non-invasive manner.

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1.4 Review of Literature

The tape stripping technique was introduced in the early 40’s by Wolf, who investigated
the topography of the skin by removing layers of corneocytes with transparent adhesive
tapes and by examining them microscopically.115 In the 50’s, Pinkus applied the new
technique to investigate the epidermal regeneration process. By taking punch biopsies of
stripped skin sites, he observed that tape stripping stimulated the mitotic rate in the
stratum basale.69 The mitotic rate was maximal after 3 days, and a couple of weeks were
required for complete skin renewing. Further studies investigating the epidermal growth
kinetic after standardized injury of healthy and atopic dermatitis skin by tape stripping
were performed.75

Recently, Choi et al. showed that tape stripping induces the production of cytokines such
as interleukins, tumor necrosis factor α, and γ-interferon, enhances the enzymatic activity,
and increases the ceramides synthesis.76 The artificial removal of the stratum corneum
barrier by tape stripping has become a frequently used model for simulating diseased skin
and for assessing the efficacy of skin care products in restoring the barrier. 79 Moreover, a
Percutaneous penetration enhancement of macromolecules like peptides and
oligonucleotides, which usually do not penetrate healthy skin, was observed after
removal of the stratum corneum, thus opening the possibility of topical vaccinations.

In the 80’s, the methodology was standardized by Dupuis et al, making of tape stripping
a method widely used in dermatological research to non-invasively investigate the topical
bioavailability and percutaneous penetration of topically applied substances.82 The
investigation of the topical activity of antimykotic88 and antiviral89, the investigation of
vehicle effects97, barrier function and reservoir formation within the stratum corneum84,
and the investigation of the percutaneous penetration and absorption of topical
corticosteroids116 are some important examples of application areas. Furthermore, tape
stripping has been used to investigate stratum corneum lipids and epidermal enzymes. 117
A prerequisite for making the tape stripping technique a capable, robust, accurate, and
reproducible method to investigate the bioavailability of topically applied drugs is the
strict standardization and a clear definition of the stripping parameters. The technique is
susceptible to numerous confounding factors, both intrinsic and extrinsic. Among the

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intrinsic factors, the anatomical site, the hydration status of the skin, the cohesion of the
corneocytes influenced as well by the vehicle used, and the presence of furrows have to
be mentioned.

Van der Molen observed that topically applied titanium dioxide could still be detected
after removal of 40 tapes because of an accumulation in superficial skin furrows, thus
falsifying the results.99 In addition, interseasonal differences have been observed.102
Among the extrinsic factors, the type of tape used (e.g., cellophane, polyester,
polypropylene, polyethylene, rayon), the pressure with which the tape is applied onto the
skin, the duration of the ressure, and the force of removal (slow/quick) influence the
stratum corneum removal. These are the parameters which can and must be standardized.
Surber et al. outlined a standard protocol for tape stripping experiments. The protocol
implies the use of a template to delineate the stripping area, and the exertion of a definite
pressure e.g., with a roller on each tape before removal. The use of a roller has been
shown to prevent artifacts due to furrows and wrinkles. Because of inter-individual
differences in stratum corneum thickness, the complete stratum corneum of one skin site
should be stripped.118

In newer investigations, Weigmann et al. used the tape stripping method to calculate
horny layer profiles, in which the amount of drug penetrated is correlated not only to the
tape number stripped but also to the depth of the stratum corneum. This correlation
enables the localization of the drug within the stratum corneum layers. For this purpose,
the removal of the entire stratum corneum and the quantification of both corneocytes and
drug on the tapes are required.104 Recently, Jacobi et al. reported a procedure for the
estimation of the removed stratum corneum amount on each tape, thus providing a
possibility to avoid the complete removal of the stratum corneum.

Arima et al. investigated the effect of hydroxypropyl-P-cyclodextrin (HP-P-CD) on the

cutaneous penetration and activation of ethyl 4- biphenylyl acetate (EBA), a prodrug of
the nonsteroidal anti-inflammatory drug 4-biphenylylacetic acid (BPAA), from
hydrophilic ointment, using hairless mouse skin in vitro. When the hydrophilic ointment
containing a complex of EBA with HP-P-CD was applied to full-thickness skin, HP-P-
CD facilitated the penetration of EBA into the skin. When the ointment containing the

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EBA:HP-P-CD complex was applied to the skin, the BPAA flux through the tape-
stripped skin was greater than that through full-thickness skin, while the activation of the
prodrug in the skin was slowed by TS. Their results suggest that the enhancing effect of
HP-P-CD on the cutaneous penetration of EBA would be largely attributed to an increase
in the effective concentration of EBA in the ointment.119

Curdy et al. administered piroxicam from a commercially available gel to human

volunteers, both passively and under the application of an iontophoretic current. After
treatment, the SC at the site of application was progressively tape-stripped and piroxicam
transport into the membrane was assessed by UV analysis of drug extracted from the
tape-strips. Current application enhanced drug uptake into the SC, as indicated by both
increased piroxicam concentrations in the horny layer and detectable concentrations at
greater depths in the membrane. The total amount of drug recovered in the SC post-
iontophoresis was significantly higher than that found following passive diffusion for
each application time.120

Escobar-Chávez et al. determined the penetration of sodium naproxen, formulated in

Pluronic F-127 gels ontaining Azone® and Transcutol as penetration enhancers, through
human skin in vivo by using the TS technique. It was found that the combination of
Azone® and Transcutol in PF-127 gels enhanced sodium naproxen penetration, with up to
two-fold enhancement ratios compared with the formulation containing Transcutol only.
These results were confirmed by TEWL and ATR-FTIR spectroscopy, suggesting a
synergistic action for Azone® and Transcutol®.121

Esposito et al. produced and characterized monoleine (MO) dispersions as drug delivery
systems for indomethacin. An in vitro diffusion study was conducted using Franz cells
associated to SC epidermal membrane on cubosome dispersions viscosized by carbomer.
In vivo studies based on skin reflectance spectrophotometry and TS were performed to
better investigate the performance of cubosome as an indomethacin delivery system.
Indomethacin incorporated in viscosized MO dispersions exhibited a lower flux with
respect to the analogous formulation containing the free drug in the aqueous phase and to
the control formulation based on carbomer gel. Reflectance spectroscopy demonstrated
that indomethacin incorporated into MO dispersions can be released in a prolonged

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fashion. TS experiments corroborated this finding. MO dispersions can be proposed as

nanoparticulate systems able to control the percutaneous absorption of indomethacin. 122

Ganem-Quintanar et al. used naproxen-loaded nanoparticles to prepare, in a one-step

process, unilaminar films of Eudragit E- 100. Nanoparticle films and conventional films
were characterized in vitro by drug release studies through a cellulose membrane using
Franz-type cells, and in vivo by penetration experiments with the TS technique.
Concerning in vivo penetration studies, no statistical differences were found for the
amount of naproxen penetrated across the SC and the depth of penetration for the two
films.123

Herkenne et al. investigated pig ear skin as a surrogate for human skin in the assessment
of topical drug bioavailability by sequential TS of the SC. Ex vivo experiments on
isolated pig ears were compared with in vivo studies in human volunteers. Four
formulations including ibuprofen in different propylene glycol (PG)-water mixtures were
compared. Derived DPK parameters characterizing the diffusion and partitioning of the
drug in the SC ex vivo were consistent with those in vivo following a 30 minute
application period. Furthermore, non-steady- state ex vivo results could be used to predict
the in vivo concentration profile of the drug across the SC when a formulation was
administered for 3 h (i.e., close to steady-state). Taken together, the results obtained
suggest that pig ear skin ex vivo is promising as a tool for topical formulation evaluation
and optimization.124

Hostýnek et al. sheded light on the long-standing controversy on whether wearing copper
bangles benefits patients suffering from inflammatory conditions such as arthritis.
Sequential TS was performed on healthy volunteers to examine the diffusion of copper
through human SC in vivo, following application of the metal as powder on the volar
forearm for periods of up to 72 h. Exposure sites were stripped 20 times, and the strips
were analyzed for metal content by inductively coupled plasma mass spectroscopy. The
results indicate that, in contact with skin, copper will oxidize and may penetrate the SC
after forming an ion pair with skin exudates. The rate of reaction seems to depend on
contact time and oxygen availability. A marked inter-individual difference was observed
in baseline values and amounts of copper absorbed.125

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Lodén et al. compared the bioavailability of ketoprofen in a photostabilised gel

formulation without photoprotection using a new DPK tape stripping model and an
established ex vivo penetration method using human skin. Analyses of the SC showed
that during the first 45 minutes, about 12 µg/cm2 of ketoprofen were absorbed into the
skin from the formulations. The area under the ketoprofen concentration–time curve
(AUC0–6) for the photo-stabilised gel/transparent gel ratio was 73%. The rate of
penetration of ketoprofen through isolated skin was approximately 0.2 µg/cm2 h for both
formulations. The ratio’s AUC0–36 was 84%. Thus, the two methods did not disagree in
terms of the relative efficacy of the two gels. The comparison of the amount of
ketoprofen in the skin after 45 minutes with the amount penetrated through the excised
skin during 36 h, suggests a change in the thermodynamic activity of ketoprofen during
exposure. A supersaturated formulation may have been formed initially due to
evaporation of ethanol.126

Wagner Heike et al. carried out penetration experiments to investigate several incubation
times with three different skin flaps using the Saarbruecken penetration model and the
lipophilic model drug flufenamic acid. Drug distribution within SC was obtained by the
TS technique, while the drug present in deep skin layers was determined by
cryosectioning. In addition, for the lipophilic drug flufenamic acid, a direct linear
correlation was found between SC/water partition coefficients and the drug amounts
penetrated into the SC for all the time intervals tested. The authors concluded that
SC/water-partition coefficients offer the possibility to predict drug amounts within the SC
of different donor skin flaps, without a time consuming determination of the lipid
composition of the SC.127

The aim of Pellanda et al. was to investigate the effect of i) dose and ii) application
frequency on the penetration of triamcinolone acetonide (TACA) into human SC in vivo.
The experiments were conducted on the forearms of 15 healthy volunteers, with i), single
TACA doses (300 µg/cm2 and 100 µg/cm2), and ii) single (1 x 300 µg/cm2) and multiple
(3 x 100 µg/cm2) TACA doses. SC samples were collected by TS after 0.5, 4 and 24 h (i)
and after 4, 8 and 24 h (ii). In Experiment 1, TACA amounts within SC after application
of 1 x 300 µg/cm2 compared to 1 x 100 µg/cm2 were only significantly different

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immediately after application, and were similar at multiple applications of 3 x 100

µg/cm2 yielded higher TACA amounts compared to a single application of 1 x 300
µg/cm2 at 4 and 8 h. At 24 h, no difference was observed. In conclusion, by using this
simple vehicle, considerable TACA amounts were retained within the SC, independently
of dose and application frequency.128

Lboutounne et al. investigated the sustained bactericidal activity of chlorhexidine base

loaded poly (Є-caprolactone) nanocapsules against Staphylococcus epidermidis
inoculated onto porcine ear skin. The antimicrobial activity of these colloidal carriers was
evaluated (i) in vitro against eight strains of bacteria, and (ii) ex vivo against
Staphylococcus epidermidis inoculated for 12 h onto porcine ear skin surface treated for
3 minutes either with 0.6% chlorhexidine base loaded or unloaded nanocapsules
suspended in hydrogel, or 1% chlorhexidine digluconate aqueous solution. Chlorhexidine
absorption into the SC was evaluated by the TS technique. The results showed that
chlorhexidine nanocapsules in aqueous suspension with a 200–300 nm size and a positive
charge exhibited similar minimum inhibitory concentrations against several bacteria,
compared with chlorhexidine digluconate aqueous solution. Ex vivo, there was a
significant reduction in the number of colony forming units from skin treated with
chlorhexidine nanocapsules suspension for 3 minutes compared to chlorhexidine
digluconate solution after an 8 h artificial contamination. Interestingly, nanocapsules
were present in porcine hair follicles. Topical application of chlorhexidine base-loaded
positively charged nanocapsules in an aqueous gel achieved a sustained release of
bactericide against Staphylococcus epidermidis for at least 8 h.129

Fresno-Contreras et al. designed an all-trans retinoic acid (RA) topical release system
that modifies drug diffusion parameters in the vehicle and the skin in order to reduce
systemic absorption and side-effects associated with the topical application of the drug to
the skin. RA, either in free form or encapsulated in SC lipid liposomes, was included in
hydrogels prepared with Carbopol® UltrezTM 10 and hyaluronic acid. In vitro
permeability experiments with [3H]- t-RA were carried out using a Franz-type diffusion
cell in abdominal rat skin samples. Accumulation of the drug in the surface and skin
layers was evaluated by both the TS technique and a dissection technique. The results

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show that RA encapsulation not only prolongs drug release, but also promotes drug
retention in viable skin. At the same time, interaction between RA and hyaluronic acid
has an obstructive effect on diffusion, which contributes to the formation of a
reservoir.130

Padula et al. studied the behavior of a skin bioadhesive film containing lidocaine, in vitro
and in vivo. Film characterization included in vitro and in vivo drug transport studies with
and without iontophoresis. The release rate was compared with a lidocaine commercial
gel. The permeation kinetics across the skin was not linear but the patch acted as a matrix
controlling drug delivery. Additionally, permeation rate increased with drug loading. The
in vivo experiments with TS indicated that the presence of water during film application
is essential to achieve not only the proper adhesion, but also an effective accumulation.
The application of an electric current to the patch can further increase the amount of drug
accumulated in the SC.131

Bashir et al. studied the keratolytic efficacy of topical preparations containing salicylic
acid (SA) in humans by the TS technique, quantifying SC removal by protein analysis. In
combination with TS, squamometry was used to evaluate the influence of SA on skin
surface scaliness and desquamation. Furthermore, skin barrier perturbation and skin
irritability were recorded and related to the dermatopharmacological effect of the
preparations. In contrast to squamometry, TS combined with protein analysis was
sensitive in detecting the keratolytic effect of SA within hours of application.
Importantly, whereas the pH of the preparations had only a minimal influence on
efficacy, local dermatotoxicity was significantly increased at an acidic pH. This indicates
that the intent to increase the amount of free, non-dissociated SA is, in fact,
counterproductive, as more acidic preparations resulted in skin irritation and barrier
disruption.132

Abdulmajed et al. used a novel synthetic technique to synthesize the co-drug retinyl
ascorbate (RA-AsA) ester from all-trans-retinyl chloride (RA) and l-ascorbic acid (AsA)
suspended in ethanol at low temperature. The flux and permeation coefficient were
determined using heat separated human skin membrane, and skin penetration was
determined by TS using full thickness human skin. All experiments were performed in

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parallel with retinyl palmitate and ascorbyl palmitate. Overall, the data suggest the
potential value of RA-AsA co-drug for treating damage to skin resulting from UV-
induced production of free radicals Aquaporine-3. Hara et al showed that glycerol
replacement corrects each of the defects in aquaporine-3 (AQP3)-null mice. SC water
content, measured by skin conductance and H2 O accumulation, was 3-fold lower in
AQP3-null vs. wild-type mice, but was similar after topical or systemic administration of
glycerol in amounts that normalized glycerol content in the SC. Orally administered
glycerol fully corrected reduced skin elasticity in AQP3-null mice, as measured by the
kinetics of skin displacement after suction, and the delayed barrier recovery, as measured
by TEWL after TS. The analysis of [14C] glycerol kinetics indicated a reduced blood-to-
SC transport of glycerol in AQP3-null mice, resulting in slowed lipid biosynthesis. These
data provide functional evidence for a physiological role of glycerol transport by
aquaglyceroporin, and indicate that glycerol is a major determinant of SC water retention
and of mechanical and biosynthetic functions. Their findings establish a scientific basis
for the >200 year old empirical practice of including glycerol in cosmetic and medicinal
skin formulations.133

Morgan et al. measured the contribution of SC barrier and microvascular perfusion in

determining dermal tissue levels of two hydrophilic drugs (acyclovir and pencyclovir) in
vivo. Removal of the SC by TS resulted in a 1300-fold increase in pencyclovir absorption
and a 440-fold increase in acyclovir absorption, confirming that SC is the major barrier to
hydrophilic drug absorption.134

Jarvis et al. determined the in vitro dermal delivery of a new class of lipophilic, highly
potent and uniquely selective anti-Varicella Zoster virus nucleoside (VZV) analogue
compared with acyclovir. Three test compounds (Cf1698, Cf1743 and Cf1712) and
acyclovir were formulated in propylene glycol/aqueous cream, and finite doses were
applied to full-thickness pig ear skin for 48 hours in vertical Franz-type diffusion cells.
Depth profiles were constructed following TS and membrane separation. All three test
compounds reached the target basal epidermis in concentrations suggesting they would
be highly efficacious in reducing viral load. Furthermore, the data showed that each of
the test compounds would have a far superior performance than acyclovir. The

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Introduction Bundelkhand University, Jhansi

dermatomal site of viral replication during secondary infection the basal epidermis was
successfully targeted.135

Inoue et al. examined the effect of CpG-oligodeoxynucleotide (CpG-ODN) on the

immune response to an antigen applied to tapestripped mouse skin, by evaluating the
production of cytokines and Ig isotypes. Confocal laser scanning microscopy revealed
that the OVA (model antigen) and CpG-ODN easily penetrated the tape-stripped skin.
Co-administration of CpGODN and OVA to the disrupted skin elicited an antigen-
specific, Th1-predominant immune response, and enhanced the production of Th1- type
cytokines, IL-12 and IFN-γ. On the other hand, the production of a Th2-type cytokine,
IL-4, was drastically suppressed. In terms of antigenspecific antibody production, the
IgG2a level, which is regulated by IFN-γ, was increased by CpG-ODN, but IgE
production regulated by IL-4 was suppressed. Furthermore, the administration of CpG-
ODN through the skin drastically attenuated the production of IgE in mice experiencing
IgE-type immune response. Administration of CpG-ODN through the skin may shift the
immune response from a Th2 to a Th1-like response.136

Alvárez-Román et al. determined whether encapsulation of lipophilic compounds in

polymeric nanoparticles is able to improve topical delivery to the skin. The penetration of
octyl methoxycinnamate (OMC) encapsulated in poly (Є-caprolactone) nanoparticles,
into and across porcine ear skin in vitro, was investigated using TS. Quantification of
OMC in the skin using TS demonstrated that nanoparticulate encapsulation produced a
3.4-fold increase in the level of OMC within the SC. Nanoparticulate encapsulation of
OMC increased its “availability” within the SC.137

Olvera-Martínez et al. prepared polymeric nanocapsules (NCs) containing OMC and

their in vivo distribution profile through the SC were determined by the TS technique.
The penetration degree of OMC formulated in NCs was compared with that obtained for
a nanoemulsion (NE) and a conventional oil-in-water (o/w) emulsion (EM). In vivo
Percutaneous penetration, evaluated by the TS technique, demonstrated that NE increased
the extent of OMC penetration relative to the penetration achieved by NCs or EM.
Likewise, OMC accumulation in the skin was significantly greater with NE than with EM
or NCs.138

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Sarveiya et al. developed a reverse HPLC assay to quantify four common sunscreen
agents, namely, 2-hydroxy-4- methoxybenzophenone, 2-ethylhexyl-pmethoxycinnamate,
octylsalicylate and salicylic acid 3,3,5-trimethcyclohexyl ester in a range of biological
matrices. This assay was further applied to study skin penetration and systemic
absorption of sunscreen filters after topical application to human volunteers. The assay
allows the analysis of sunscreen agents in biological fluids, including bovine serum
albumin solution, plasma and urine, and in human epidermis by using the TS technique.
The results from the preliminary clinical study demonstrate a significant penetration of all
sunscreen agents into the skin.139

In vitro human skin permeation and distribution of geranyl nitrile (GN) were determined
by Brian et al. using epidermal membranes, following application in 70% ethanol, under
non-occlusive conditions, at maximum in-use concentration (1%). Levels of GN in the
epidermis (plus any remaining in SC after TS), filter paper membrane support, and
receptor fluid were combined to produce a total absorbed dose value of 4.72±0.32%. The
systemic exposure resulting from the use of GN as a fragrance ingredient, under
unoccluded conditions, would be low based on the currently reported use levels. 140

Alberti et al. evaluated, using attenuated total reflectance Fourier transform infrared
spectroscopy, the SC bioavailability of terbinafine (TBF) following topical treatment
with four different formulations, based on a vehicle consisting of 50% ethanol and 50%
isopropyl myristate. Three of these formulations included a percutaneous penetration
enhancer: either 5% oleic acid, 10% 2-pyrrolidone or 1% urea. The SC concentration
profile of TBF was measured by repeated infrared spectroscopic measurements while
sequentially stripping off the layers of this barrier membrane with adhesive tape. TEWL
measurements were also performed, to permit facile estimation of SC thickness. The SC
concentration profiles of TBF were fitted to the appropriate solution of Fick's second law
of diffusion. This analysis enabled the efficacies of the different formulations tested to be
compared to the non-enhancer control. While it was found that the formulation
containing 5% oleic acid significantly enhanced the SC availability of TBF, the other
formulations did not improve the apparent drug delivery.141

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Kalia et al. determined whether a structurally heterogeneous biomembrane, human SC,

behaved as a homogeneous barrier to water transport. Impedance spectra (IS) of the skin
and measurements of the rate of TEWL were recorded sequentially in vivo in human
subjects as layers of the SC were progressively removed by the serial application of
adhesive tape strips. The low frequency impedance of skin was much more significantly
affected by TS than the higher frequency values; removal of the outermost SC layer had
the largest effect. In contrast, TEWL changed little as the outer SC layers were stripped
off, but increased dramatically when 6-8 microns of the tissue had been removed. It
follows that the two noninvasive techniques probe SC barrier integrity in somewhat
different ways. After SC removal, recovery of barrier function, as assessed by increasing
values of the low-frequency impedance, apparently proceeded faster than TEWL
decreased to the pre-stripping control. The variation of TEWL as a function of SC
removal behaved in a manner entirely consistent with a homogeneous barrier, thereby
permitting the apparent SC diffusivity of water to be found. Skin impedance (low
frequency) was correlated with the relative concentration of water within the SC, thus
providing an in vivo probe for skin hydration. Finally, the SC permeability coefficient to
water, as a function of SC thickness, was calculated and correlated with the
corresponding values of skin admittance derived from IS.142

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1.5 Research Envisage

Bioequivalence studies in India, though the major break-though, in the Indian Pharma-
growth and about a decade old, has not yet penetrated equally as far as the dermal
products are concerned. Like many other dosage forms, those getting absorbed through
various routes, the dermal preparations which are absorbed from skin, also contributes the
respectable market performance. But unlike for other dosage forms the bioavailability or
bioequivalence studies, for dermal preparations are very less studied.

Many regulatory bodies including US FDA feels it necessary to set up the guidelines for
the BA BE studies of the dermal preparations. Accordingly, FDA had already published
the draft guideline for this purpose. Though this guideline is not for the commercial
purpose, but hold the greatest interest for academic research, where in, the use and
importance of Dermatopharmacokinetic studies, could be verified.

Transdermal drug delivery offers many important advantages. For instance, it is easy and
painless, it protects the active compound from gastric enzymes, and it avoids the hepatic
first-pass effect. Also, it is simple to terminate the therapy if any adverse or undesired
effect occurs. But skin is a natural barrier, and only a few drugs can penetrate the skin
easily and in sufficient quantities to be effective. Therefore, in recent years, numerous
studies have been conducted to study the various factors governing the penetration of
drugs through skin.143, 144

Human cadaver skin is the first choice as a skin model for a study of a final product to be
used in humans.145 Al-Saidan et al showed that in vitro permeation studies using rat skin
could also provide information useful for manipulating the design of transdermal
therapeutic system (TTS) patches so that the desired permeation of the drug across
human skin would be achieved.146

Through this research proposal, the standardization of the Dermatopharmacokinetic

procedures, through the skin stripping method and thereby, studying the possible
bioequivalence between the marketed dermal products of Erythromycin is proposed. The
in vitro penetration characteristics of these formulations and the in vivo absorption profile
will be compared to establish the in vitro- in vivo correlation for these Erythromycin

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dermal formulations. The drug Erythromycin is selected for the reason of wide variety of
marketed preparations available and also the faster and reliable quantitative analysis of
Erythromycin is reported.

The proposed project is designed to investigate into whether any two marketed dermal
preparations when compared for the bioequivalence shows the significant difference
when other parameters are kept constant like subject’s age, race and the dose of the drug
and to investigate if we could establish the in vitro- in vivo correlation for these
Erythromycin dermal formulations.

In the marketed dermal dosage forms of the same drug, the only difference lies in the
recipe of the formulation and therefore the consistency of the dosage form (cream / lotion
/ spray etc). These formulations therefore mostly differ in their drug releasing pattern.
The topical dosage forms are tested for the drug release pattern during their formulation
development process, the one which is as well applicable to the other dosage forms
except for those intended for the administration into systemic circulation. But like
practiced for other dosage forms including those intended for the administration into
systemic circulation, the dermal formulations do not undergo through Clinical in vivo
analysis before they are marketed.

The clinical research is also required to be completed on various populations for most of
the drugs and their dosage forms because it is so frequently observed during their
bioavailability studies that the inter-individual, inter-racial differences do exist,
necessitating their BA BE studies on variable population.

The widely used skin stripping method for Dermatopharmacokinetic analysis should be
tested using various population samples, to cross verify its race / location dependency. It
is also a known fact that even the skin type varies per population, and there lies the
possibility that the pharmacokinetic parameters of the drug could vary in population.

Thus this project is a way to determine whether if the Dermatopharmacokinetic studies if

done in the given population results in reliable outcome and if they are reproducible. And
finally, whether the dermal products are in need of the strict regulatory requirement of

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going through the BA BE determination or not, and it could only be verified through the
clubbed results of such various local studies globally.

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Introduction Bundelkhand University, Jhansi

1.6 Research Objective

Assessment of bioequivalence of topical dermatological formulations is a challenge. A

new dermatopharmacokinetic (DPK) approach has been proposed for bioequivalence
determination of topical drug products by comparing the drug content kinetics in stratum
corneum. Various in vivo methods have been studied for this purpose, out of which the
skin stripping or tape stripping is faster and reliable technique. Tape stripping method
measures the drug concentration in stratum corneum at the site of application. Various
studies have shown the DPK to be reliable and reproducible; however, confidence in this
methodology needs to be established, particularly by developing the DPK methods for
various topical drug formulations. Simultaneously the impact of factors like regional and
genetic variations in skin type on DPK, the reproducibility of DPK results for a particular
drug in particular population must be studied. The DPK method is not yet a regulatory
compulsion in India or even in other countries. Though its need is recognized, which is
evident by USFDA’s draft guideline publication for DPK.

Erythromycin topical formulations are widely available in India. Indian topical generic
market of Erythromycin is also of respectable size. These generic Erythromycin topical
products find their way in market through various in vitro analytical checks.

Hence we considered its worth to develop the analytical and clinical method for
Erythromycin DPK analysis using the Indian Human skin and to validate it.

The Objectives could thus be summarised as below:

1. To develop and validate simple, specific and sensitive analytical method in vitro
for the estimation of Erythromycin in human Stratum corneum.

2. To assess the in vivo pharmacokinetics of two Erythromycin Marketed

formulations by Dermatopharmacokinetic methods using Indian male subjects to
meet the following sub-objectives:
a. Efficacy: To assess the pharmacokinetic profile of Erythromycin
Marketed formulations by Dermatopharmacokinetic methods.
[Link]: To monitor the safety of the study formulations in subjects.

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Introduction Bundelkhand University, Jhansi

3. To investigate the in vitro penetration characteristics of the two Erythromycin

formulations and to check if any in vitro-in vivo correlation could be established.

As Erythromycin is one of the most frequently used transdermal antibacterial drug, many
of the analytical methods have been established. We have validated one of such
established methods to check whether the quantity of drug that has penetrated through the
skin could be estimated or not and whether it could be done precisely and accurately.

The in vitro penetration kinetics through the human cadaver skin was then studied
extensively. Then the penetration and absorption of the same drugs were clinically
checked using human subjects. The kinetics of the absorption were also studied
extensively

As this in vivo, drug quantity absorbed when compared with in vitro, drug quantity
penetrated, it was found that the in vivo drug absorption is at very small quantity, hence a
in house sensitive HPLC method was developed to estimate the drug absorbed through
the skin accurately. The samples collected from the subjects were analyzed by HPLC
method.

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Introduction Bundelkhand University, Jhansi

1.7 DRUG PROFILE:

Figure 1.1: Structure of Erythromycin

Erythromycin, a macrolide antibiotic is effective in vitro against Mycoplasma, Gram

positive Coci, Neisseria, some strains of Haemophilus, Corynebacterium, Listeria,
Pasteurella mutocida, Brucella, and Treponemes. Proteus, Pseudomonas and [Link] are
relatively resistant to the drug. In veterinary medicine, this compound is used for the
treatment of clinical and subclinical mastitis in lactating cows, for the treatment of
infectious diseases due to erythromycin sensitive bacteria (cattle, sheep, swine, poultry)
and for the treatment of chronic diseases due to mycoplasma in poultry (EMEA, 2000). In
Europe, for broiler chickens and laying hens, the most often recommended dose (as
erythromycin base) is 20 mg/kg/day.

Absorption

Erythromycin base is destroyed by gastric acid, except if administered with a protective

enteric coating. Acidic media degrades erythromycin rapidly to form derivates with little
antimicrobial activity. Erythromycin stearate is more stable, however in vitro studies
have demonstrated that erythromycin stearate dissolves in gastric acid, retains only 2%
antibiotic activity and is rapidly destroyed (DiSanto and Chodos, 1981; Periti et al., 1989;
Martindale, 1989). The major site of absorption in rat, dogs and humans is the small

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Introduction Bundelkhand University, Jhansi

intestine. Erythromycin is only slightly absorbed from the stomach. In man, absorption
occurs mainly in the duodenum (Anderson et al., 1959; Huber, 1977).

In humans, erythromycin is rather slowly absorbed after oral administration. Peak serum
concentrations occur 1 to 6.3 hours after dosing and vary from 0.1 to 4.8 µg/ml,
depending on the formulation, and the coating of erythromycin administered. The oral
absorption is less than 50% and erythromycin is degraded by gastric acid. It is absorbed
in the small intestine as erythromycin base (DiSanto and Chodos, 1981; Griffith and
Black, 1970; Burrows, 1980). The oral bioavailability of unprotected erythromycin base
and salts is less than 50 % of the dose. Food reduces the absorption of erythromycin
(Griffith and Black, 1970; Burrows, 1980). Wilson and Van Boxtel (1978) observed that
erythromycin propionate and stearate were better absorbed before rather than after
breakfast.

Distribution

Plasma protein binding

In humans, erythromycin is highly bound to plasma proteins. The extent of protein

binding is >74% in vitro and >90% in vivo (Wilson and Van Boxtel, 1978). Erythromycin
undergoes a relatively low extent of binding to bovine serum proteins (37-43%) (Baggot
and Gingerich, 1976).

Milk protein binding

Studies show that antibiotics are bound only to a minor extent to milk proteins. However,
the unbound fraction of erythromycin may be decreased because it may be bound to milk
casein. Erythromycin is <25% bound to dry udder secretion and to dry udder tissue
homogenates (Ziv, 1980).

Serum levels

According to Wilson and Van Boxtel (1978), dose levels of 250 mg of erythromycin base
or erythromycin stearate in adults produce similar peak serum concentrations (0.4 µg/ml)
within 2-4 hours after oral administration. In cattle, after intramuscular administration of

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Introduction Bundelkhand University, Jhansi

erythromycin in cattle, peak serum concentrations are maintained for several hours and
then decline slowly. The l2-hour levels are about 25% of peak concentration (Burrows,
1980).

Tissue distribution

Animal studies indicate that erythromycin is well distributed in the body and tissue levels
(e.g. liver, spleen, kidneys, and lungs) are general1y higher than serum levels and persist
longer (Wilson and Van Boxtel, 1978).

In humans, erythromycin is distributed to various tissues and fluids. About 10% of

erythromycin is estimated to cross the placenta and fetal blood levels are no higher than
10% (usually closer to 2%) of those present in normal circulation. An estimated 0.1% of
a daily dose appears in breast milk in pregnant women (Wilson and Van Boxtel, 1978).

Metabolism

The metabolism of erythromycin has been studied in different animal species and in
humans. (Lee et al, 1956a; Lee et al, 1956b; Wilson and Van Boxtel, 1978; Pineau et el.,
1990; Tsubaki and Ichikawa, 1985). These studies show that erythromycin is rapidly
metabolized in the liver, mainly through an Ndemethylation process in both rats and dogs
and in the liver microsomal system of rabbits. Collectively, these studies strongly suggest
that the metabolism of erythromycin by N-demethylation occurs in all species tested.
Des-N-methyl-erythromycin is the major metabolite and the only microbiologically
active metabolite of erythromycin. However, the antimicrobial activity is presumably low
and the only form of erythromycin known to be active in vivo is the free base. It is
excreted in the bile and eliminated through the faeces. Only erythromycin was found in
the liver and the absence of des-N-methyl-erythromycin indicates that it is excreted in the
bile immediately after erythromycin demethylation. It is absorbed from the intestinal tract
but the very minute amount of des- N-methyl-erythromycin available in the body may
explain its absence from urine. The hepatic cytochrome P-450 isozymes that catalyse
erythromycin demethylation in rat are highly similar to the form of liver cytochrome P-
450 present in rabbit, hamster, gerbil and mouse and this may also extend to humans
since human liver contains a protein equivalent to the rat cytochrome P- 450. Similarly, a

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Introduction Bundelkhand University, Jhansi

high degree of similarity was found between the ovine cytochrome P-450 involved in N-
demethylation of erythromycin and the form isolated in rabbits. This suggests that an
equivalent form of these liver cytochrome P-450 isozymes, with similar catalytic
activities, is present in the species tested. In cattle a form of cytochrome P-450 isozyme
exhibiting a high catalytic activity for Ndemethylation was found; this activity was not
measured for erythromycin but for other substrates having a N-methyl group structure.

Excretion

Renal excretion

In humans, the portion of an erythromycin dose excreted in the urine varies from 0.02 to
20% and the elimination half-live may be prolonged in renal disease. However, except
complete renal failure, renal impairment has only a minor impact on the
pharmacokinetics of erythromycin (Wilson and Van Boxtel, 1978).

Urinary excretion of erythromycin accounts for approximately 10% of an administered

oral or IM dose (Burrows, 1980). Twenty hours after administration of isotopic
erythromycin in rats, 27 to 36% of the radioactivity was recovered in the urine (Lee et al.,
1956a).

Faecal Excretion

In humans, 15% of an administered dose was excreted in the bile (Griffith and Black,
1970).

In rats, erythromycin and its metabolites are excreted mainly by way of bile, but in part,
also by direct passage through the intestinal wall (Baggot and Gingerich, 1976). Two
hours following intravenous injection of isotopic erythromycin, 15.1% of the dose was
excreted in the bi1e (Lee et al, 1956b). Twenty hours following intravenous injection of
isotopic erythromycin, 37-43% of the radioactivity is recovered in the intestinal tract plus
faeces (Lee et al. 1956a). An enterohepatic recirculation may also contribute to the high
concentrations of erythromycin in faecal samples (Kroboth et al., 1982).

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Introduction Bundelkhand University, Jhansi

Antimicrobial Activity:

Gram-positive bacteria, mycoplasma pneumoniae, chlamydia trachomatis, chlamydia

pneumoniae, chlamydia psittaci, ureaplasma urealyticum, legionella pneumophila,
campylobacter jejuni, bordatella pertussis

Mechanism of Action:

Macrolides are inhibitors of protein synthesis. They impair the elongation cycle of the
peptidyl chain by specifically binding to the 50 S subunit of the ribosome. Specificity
towards prokaryotes relies upon the absence of 50S ribosomes in eukaryotes.

Pharmacodynamics:

Macrolides are considered time-dependent antibiotics, which means that their efficacy
will be related to the time interval during which their concentration at the infected site
remains above the MIC of the offending organism.

Pharmacokinetics: (500mg P.O. dose)

Cmax: 3mg/L; Half-life: 2 hours; Volume of distribution: 0.64L/kg; Bioavailability: 25-

60%;

Adverse Effects:

 Gastrointestinal: abdominal pain, nausea, vomiting, diarrhea

 Cardiovascular System: prolongation of QT interval, ventricular fibrillation
 Hepatic: hepatotoxicity
 Otic: auditory and vestibular dysfunction
 Hematologic: eosinophilia
 Dermatologic: skin rashes, pain at injection site, thrombophlebitis

Dosage:

 Capsule: 250mg
 Topical gel: 2%

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Introduction Bundelkhand University, Jhansi

 Granules for oral suspension: 200mg/5ml

 Injection, powder for reconstitution: 500mg, 1g
 Ophthalmic ointment: 2%
 Topical ointment: 2%
 Powder for oral suspension: 200mg/5ml, 400mg/5ml, 100mg/2.5ml
 Topical solution: 1.5%, 2%
 Oral suspension: 125mg/5ml, 250mg/5ml, 200mg/5ml, 400mg/5ml
 Swab: 2%
 Chewable tablet: 200mg
 Delayed release tablet: 250mg, 333mg, 500mg
 Film coated tablet: 250mg, 333mg, 400mg, 500mg

Susceptible infections: 250 mg PO every 6 hr or 500 mg PO every 12 hr; Max = 4 g/day

Susceptible infections: (delayed-release base) 250 mg PO every 6 hr or 333 mg PO every
8 hr or 500 mg PO every 12 hr; Max = 4 g/day

Amebiasis, intestinal: 250 mg PO every 6 hr or 500 mg PO every 12 hr for 10-14 days;

(delayed-release base) 333 mg PO every 8 hr for 10-14 days

Chancroid: 500 mg PO 3 times a day for 7 days

Chlamydia: 500 mg PO 4 times a day for 7 days; (delayed-release base) two 333 mg
tablets PO every 8 hr for 7 days

Intraocular infections: 1 cm ribbon ophthalmic ointment applied up to 6 times daily

(depending on severity of infection)

Nongonococcal urethritis: 500 mg PO 4 times a day for at least 7 days; (delayed-release

base) two 333 mg tablets PO every 8 hr for at least 7 days

Pelvic inflammatory disease (N. gonorrheae): 500 mg IV (lactobionate) every 6 hrs for
3 days, then 500 mg PO every 12 hr or 333 mg (delayed-release base) every 8 hr for 7
days

Rheumatic fever prophylaxis: 250 mg PO twice daily

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Introduction Bundelkhand University, Jhansi

DISEASE STATE BASED DOSING

Hepatic failure: drug may accumulate in patients with severe liver disease; no specific
dosing recommendations available.

Contraindications/Warnings/Precautions:

Contraindications: concomitant therapy with astemizole, cisapride, pimozide,

terfenadine

Precautions: concomitant therapy with lovastatin, history of myasthenia gravis (risk for
exacerbation), impaired hepatic function

Drug Interactions:

Antipsychotics (major severity):

MOA: additive effects on QT prolongation

Management: Caution is advised if erythromycin and antipsychotics are used

concomitantly. Monitor QT interval at baseline and periodically during treatment.

Arsenic Trioxide (major severity):

MOA: additive effects on QT prolongation

Management: Caution is advised if erythromycin and arsenic trioxide are used

concomitantly. Monitor QT interval at baseline and periodically during treatment.

Astemizole (major severity):

MOA: additive effects on QT prolongation

Management: The concurrent administration of erythromycin and astemizole is

contraindicated.

Atorvastatin (major severity):

MOA: inhibition by erythromycin of atorvastatin metabolism

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Introduction Bundelkhand University, Jhansi

Management: If concurrent therapy is required, monitor the patient for signs and
symptoms of myopathy or rhabdomyolysis (muscle pain, tenderness, or
weakness). Monitor creatine kinase (CK) levels and discontinue use if CK levels
show a marked increase, or if myopathy or rhabdomyolysis is diagnosed or
suspected.

Bepedril (major severity):

MOA: additive effects on QT prolongation

Management: The concurrent administration of erythromycin and bepedril is

contraindicated.

Pregnancy:

Category B: No evidence of risk in humans but studies inadequate.

Monitoring Requirements:

Therapeutic: Periodic WBC, chest X-ray if pneumonia, cultures, temperature

Toxicity: EKG, drug serum level

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