Pilot study on the pharmokinetics of p-phenylenediamine and its metabolites after application to the skin by means of Raman spectroscopy.

With an increasing number of women, but also men and children dyeing their
hair, the prevalence of sensitization and subsequent allergic contact
dermatitis (ACD) to p-phenylenediamine (PPD) is rising. [1,2] Although the
exact prevalence is not known, large research cohorts estimate the
sensitization to PPD in the general population at 0.1 to 2.3%. [3] A less
frequent, but other way to become sensitized is the application of a temporary
henna tattoo, contact with black rubber materials or even the use of mascara
where PPD has been added. [2,4,5] Sensitization is also seen within the
framework of cross reactivity with azo-dyes in clothing. [5-7] A contact
allergic dermatitis to PPD can be very severe, with erythema, oedema,
blistering and crusts. In severe cases this can lead to hospitalization.
ACD is a delayed type hypersensitivity disorder to low molecular weight
chemicals. Despite the long recognition of ACD to PPD, the immunological
pathway has not been completely elucidated. [8,9]To date, there is no human, in
vivo data on the pharmacokinetics (distribution, metabolism, excretion) of PPD
after application to the skin. In vitro experiments showed that in aqueous
solution or in contact to the skin, PPD is susceptible to sequential oxidation
and self-conjugation, resulting in formation of instable intermediates like
benzoquinonediimine, benzoquinone and the stable end product Bandrowski*s Base
(BB). [17-19] Some of these substances are considered possible haptens. [18]
However, there is much debate on which hapten is responsible in the
sensitization to PPD. In addition, several studies have shown that
(N-acetyltransferase 1) NAT1-enzymes in the human skin are able to acetylate
PPD to mono-acetyl PPD (MAPPD) and di-acetyl PPD (DAPPD). This acetylation is
considered a detoxification step, because MAPPD and DAPPD have a reduced to
absent potential to sensitize. [20]
There are several methods to get insight in the dermal absorption and kinetics
of a chemical. The most relevant method is a human in vivo absorption
measurement, as used in experimental exposure studies with volunteers.
Logically, these are often invasive experiments which are regulated tightly and
are possible to a limited extent. In addition to the developed in vitro
studies, a number of less invasive methods like plasma- and
excretion-measurements, micro dialysis and *tape stripping* exist. [10]However,
each of these methods has its specific limitations (i.e. destructiveness, lack
of accuracy and/or spatial resolution). An entirely different in vivo method,
spectroscopy, offers several advantages: it is fast, non-invasive and is able
to provide *real-time* in vivo data on the penetration of a chemical through
the skin. Unlike Infrared Spectroscopy (IS) which only reaches the outer layer
of the stratum corneum, the Confocal Raman Microspectroscopy (CRM) is suitable
to obtain information on the molecular skin structure to the depth of several
hundred micrometers below the skin. [11]
By means of this in vivo, non-invasive study on the pharmacokinetics of PPD
after application to the skin, we try to understand and contribute to research
into the aetiology of contact sensitization to PPD. In order to obtain a
reference spectrum, the kinetics of PPD will be examined in healthy subjects
first. In subsequent studies we might examine PPD sensitized subjects in order
to study possible differences. Therefore, a better understanding of the
kinetics of PPD, may in the future lead to definition of subjects with an
increased risk of sensitization to PPD and hence, to reduction of sensitization.
CRM is an elegant, non-invasive tool that provides detailed information on
molecular structure and composition of the skin. [21] In addition, it enables
the monitoring of skin molecular changes after topical application of drugs.
[21]
In CRM the sample is illuminated by monochromatic laser light. This light
interacts with the molecules in the sample, which then are able to vibrate
internally or rotate around different axes.
Interaction between the molecules and the incident light leads to scattering of
this light. Most of the scattered light is found at frequencies of this
incident light (called elastical- or Rayleigh scattering). A small fraction
however, is found at wavelengths longer than that of the incident light and is
called inelastical- or Raman scattering. This scattered light exhibits
frequency shifts with respect to the incident light, which are associated with
molecular vibrations within the molecule. Each frequency shift, which can be
recorded and translated by an optical dispersive system into a so called Raman
spectrum, is dependent on the atom masses, chemical bonds and molecular
structure and interactions and is therefore highly molecule specific.
Therefore, a Raman spectrum represents a *fingerprint* by which the molecule
can be identified. [22]
By comparing *blank* skin (without application of PPD) with spectra derived
from skin after topical application, PPD can be detected in the skin. The
depth, at which PPD is located, can be determined with so-called water
profiles. These profiles correlate the depth of the skin with the water
gradient, naturally present in skin. [22]

By means of this in vivo study, we try to non-invasively get insights into the
pharmacokinetics of PPD applied to the human skin. By studying penetration
rate, penetration depth, allocation to different skin departments (stratum
corneum, epidermis, dermis) and metabolism (converting of PPD into the trimer
Bandrowski*s Base and/or the detoxification products mono-acetyl PPD and
di-acetyl PPD) we try to understand the immunologic pathway of PPD.

If this pilot-study provides us insights into the pharmacokinetics of PPD in
the skin of human volunteers, in the future we might be able to detect possible
pharmacokinetic differences between healthy volunteers and sensitized subjects.
Eventually, this may lead to definition of subjects with increased risk or
reduction or avoidance of sensitization

Furthermore, several concepts exist on the mechanism of T-cell activation by
PPD. The hapten-protein concept suggests that PPD represents a pro-hapten which
will be modified after penetration through the skin, in order to subsequently
being presented by antigen presenting cells to naïve T-cells.[17,23] Another
concept states that PPD is able to directly - by means of a reversible
non-covalent binding to MHC-class II molecule without further modification -
stimulate T-cells. [18,23] This PPD-protein complex seems to represent an
additional antigenic signal for T-cells of allergic patients. Advanced
techniques in Raman microscopy may allow visualizing binding of PPD to
residential skin proteins. This is a possible follow-up experiment for the
future.

Experiment 1: PPD penetration and clearance
a. Stratum corneum (SC) penetration as a function of application and clearance
time
b. Deep penetration (epidermis (ED)/dermis) for 2 application times and 2
clearance times

Product : 1% PPD petrolatum
Application : covered, preferably Finn-chamber
Site : inner forearm (we can define 3 areas per arm and use left and right)
Volunteers : at least 2

Required : measurement of SC thickness (water profile)
measurement of PPD profile (fingerprint profile) across entire SC thickness

Experiment 1a:

Measurement times:
Each spot requires both a water profile (SC thickness) and a PPD profile.
Because of biological variation, take 10 repeat measurements per area.
Water profiles are measured (required) -4 to 40 micrometer in 4 micrometer
steps at 1s/frame (appr. 24s / profile).
Fingerprint profiles can be measured 0 to 24 micrometer in 2 micrometer steps
at 6 s/frame (appr. 91s / profile). Alternative settings for range and step
size are possible (e.g. larger steps to save time at the cost of spatial
resolution).
Total time for 10 repeats (including overhead times): 26 min.

Time scheduling
Based on a total time of little under 30 min per area, a time schedule is
prepared to best fill all the desired time points, at highest efficiency.
(Based on an explorative experiment, it is not likely that we will see much PPD
after 6 hrs or later. If necessary, we can verify one if PPD is still visible
6 hrs after 30 min application. If it is, we can then expand.)

Measurements
Application time T = 0 T = 1 hr T = 2 hrs T= 4 hrs T= 6 hrs T = 12 hrs T = 24
hrs
5 min. X X X X
30 min. X X X X
2 hrs. X X X X X X X
48 hrs. X

With the application and time points given above, a full series can be measured
in 3 days for 2 volunteers. This should be done 3x to obtain a more reasonable
number of 6 volunteers.

Experiment 1b:
Based on an exploratory experiment, a small amount of PPD is expected in the
viable epidermis after 2 hrs or longer application. In order to determine the
depth of penetration beyond the SC, the required signal collection time is
considerably longer than in exp. 1a. This should be explored first, which can
be done in about half a day.

Explorative experiment:
Time: 0.5 day
Volunteers: 2 volunteers)
After 48 hrs application (deepest penetration; moreover, this is the
applicationtime used in diagnostic patch testing)
- measuring water profiles to determine SC thickness
- measuring deep fingerprint profiles (0-40 micrometer and deeper in 4
micrometer steps), with long exposure time of 20s per point. This results in
~3.5 minutes per profile.
- Repeat only a few times. Based on the outcome a detailed experiment can be
designed.


Experiment 2: formation of MAPPD, DAPPD, BB
- Raman spectra of MAPPD, DAPPD and BB must be established first.
- These experiment requires high quality spectra (= long exposure times) in
order to identify and quantify the downstream products.
- Exploratory experiments are required to determine if downstream products can
be detected at all, and how long we should measure in order to quantify the
amounts.

The burden for the subjects in this pilot study will consist of:

Voorafgaand aan deelname: anamnestisch uitsluiten van allergie voor PPD,
actieve huidziekte onderarmen en mogelijk zwangerschap, d.m.v. vragenlijst
meegestuurd met informatiebrief. Tevens anamnese tijdens een eerste afspraak
(30 min.) en inspectie van de onderarmen naar evt. actieve huidziekte en uitleg
zelf bereiden en aanbrengen PPD pleister.


- Prior to participation: excluding sensitization to PPD and pregnancy by
taking history, excluding active skin disease by questionnairre attached to the
information letter. In addition taking a history during a first visit (30 min.)
together with physical examination of the lower arms and instruction of PPD
patch test preparation and application.
- Three consecutive measuring days, according to a schedule as optimal as
possible. Day 1 :6.5 hrs, day 2: 5.5 hrs and day 3: 4.5hrs (including breaks).
- Measurements will take place at the Erasmsu Medical Centre in Rotterdam
- True burden exists of: application of several patchtest (plaster containing
1%PPD in petrolatum) during 5min, 30min, 2 hrs and 48hrs. After removal of the
patchtest, the skin will be wiped clean by a tissue, where after the CRM
measurements can start.
- For one measurement (application 2hrs, measurement T=12), the patch test has
to be applied and removed at home, by the subjects themselves. They will be
carefully instructed.
- During (especially the 48hrs) patch test, the patient will be asked to keep
the lower-arm dry
- During the measurements, the subject places his/her arm on measurement window
of glass, placed above the objective of the Raman spectrometer, which can be
seen as an inverted microscope, while resting their hand and elbow on specially
equipped armrests. During the 10 repeated measurements of approximately 90sec,
the subjects will be asked to keep their arm as still as possible. Between the
measurements the subjects will be able to move their arm and relax. The
measurements are pain- and harmless.
- For exploratory experiment 1b measurements will be conducted on the
researcher and spectroscopist after 48hrs application. Timeframe: 0.5 day.
- For exploratory experiment 2 attempts will be made to detect the formed
PPD-metabolites in the skin of the researcher, if spectra of these metabolites
will be obtained successfully. Timeframe: 0.5 day

The risks:
- Mild irritation on the spot of the patchtest. To minimize these complaints,
hypoallergenic patches will be used. Possible irritation/itch will disappear
spontaneously after removal of the patch and can be compared to itch while
wearing a normal patch.
- Extremely small chance (<= 0,3 %, in 10-years review) on active sensitization
to PPD.
Based on the history we assume that participating subjects don*t have an
allergy to PPD.
This means that they have never been exposed to PPD and subsequent never could
have been sensititized or that they have been exposed (very likely f.e. through
dye in clothing), but are tolerant to PPD and thus do not develop allergic
contact dermatitis.

Raman spectroscopie is a unique method to non-invasively study pharmacokinetics
of PPD in vivo.[21] Knowledge regarding penetration, distribution and
metabolism is necessary to unravel the to date unknown mechanism of
sensitization to PPD. When more knowledge on the pathway of sensitization in
present, we may react to the field of prevention as well as the field of
treatment. For the future, this may contribute to decreasing the prevalence of
allergic contact dermatitis to PPD in the general population. In terms of these
interests, we consider the negligible risks acceptable. Furthermore,
application of the 1%PPD patchtest, needed for this experiment, is being used
as a general diagnostic tool on a considerable amount of subjects of the
dermatology department.[12] PPD has been included in the European Standard
Series (a series with several, common contact allergens) which has been
selected after extensive consideration and repeated evaluation by the European
Society of Contact Dermatitis. [13] An allergen is being tested in a
concentration that maximizes the number of sensitized subjects being detected,
while the risk of active sensitization is as low as possible.[14] Scientific
studies show that patch testing with PPD on subjects which have not been
sensitized prior to the test, or which only showed one positive reaction, do
not cause active sensitization and can be can be performed with minimal risk.
[14,15] Furthermore, the dose to which subjects will be exposed (in regular
patch tests as well in this pilot study) is much lower than the dose in f.e.
hair dyeing. [16]


The objective of these experiments is to study the pharmacokinetics of PPD in
the skin. Using six healthy volunteers, allows us to get a clear image. For the
future, this may help to distract a protocol (optimal application times and
follow-up measurements) for experiments on other healthy volunteers and/or
sensitized patients, which then will be less time consuming.