Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 387
Retinoic Acid Metabolism Blocking
Agents and the Skin
In vivo and in vitro Studies of the Effects on Normal
and Diseased Human Epidermis
ELIZABETH PAVEZ LORIÈ
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2008
ISSN 1651-6206
ISBN 978-91-554-7310-5
urn:nbn:se:uu:diva-9325
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I dedicate this work to former generations who did not have the same opportunities to a good education and could not pursue their dreams.
The important thing in science is not so much to obtain new facts as to
discover new ways of thinking about them
Sir William Bragg
List of Studies
This thesis is based on the following studies, which are referred to
by their Roman numerals:
I
Pavez Loriè E, Cools M, Borgers M, Wouters L, Shroot
B, Hagforsen E, Törmä H, Vahlquist A. Topical treatment with CYP26 inhibitor talarozole (R115866) dosedependently alters the expression of retinoid-regulated
genes in normal human epidermis. Br J Derm, In Press1
II
Pavez Loriè E, Gånemo A, Borgers M, Wouters L,
Blockhuys S, van de Plassche L, Törmä H, Vahlquist A.
Expression of retinoid-regulated genes in lamellar ichthyosis vs. healthy control epidermis: Changes after oral
treatment with liarozole. Acta Derm Venereol, In Press1
III
Pavez Loriè E, Chamcheu J-C, Vahlquist A, Törmä H.
Both all-trans retinoic acid and cytochrome (CYP) 26 inhibitors affect the expression of vitamin A metabolising
enzymes and retinoid biomarkers in human organotypic
epidermis, manuscript.
IV
Pavez Loriè E, Li H, Vahlquist A, Törmä H. Retinoid
signaling in human epidermal keratinocytes: The importance of cytochrome P450 (CYP) 26 for retinoic acid metabolism, manuscript.
1Reprints
ers.
were made with the kind permission of the publish-
Contents
FOREWORD ..........................................................................................................11
INTRODUCTION ................................................................................................13
THE SKIN ..............................................................................................................13
ICHTHYOSIS..........................................................................................................16
VITAMIN A AND RETINOIDS ................................................................................18
History ............................................................................................................18
Uptake, processing and transport in the body.................................................19
The uptake and processing of vitamin A in the skin .......................................21
CRABPI and CRABPII...................................................................................21
CYP26 and CYP2S1 .......................................................................................22
Biological effects of retinoids in skin ...............................................................24
Retinoid therapy of skin disorders...................................................................25
RETINOIC ACID METABOLISM BLOCKING AGENTS (RAMBAS) ..........................27
AIM OF THE RESEARCH ..................................................................................30
SPECIFIC AIMS: .....................................................................................................30
MATERIAL AND METHODS ...........................................................................31
SUBJECTS (STUDY I AND II)..................................................................................31
STUDY DESIGN .....................................................................................................31
Study I.............................................................................................................31
Study II ...........................................................................................................33
Study III ..........................................................................................................35
Study IV..........................................................................................................36
ANALYSIS OF SAMPLES...................................................................................38
EPIDERMAL THICKNESS AND SIGNS OF PROLIFERATION (I, II)............................38
MRNA EXPRESSION (I-IV)...................................................................................38
PROTEIN EXPRESSION (STUDY I-IV) ....................................................................39
Immunofluorescence........................................................................................39
Immunoblotting ...............................................................................................39
RESULTS AND DISCUSSION ..........................................................................40
STUDY I ................................................................................................................40
Effects of talarozole on retinoid biomarkers in healthy epidermis ...................40
Effects of talarozole on enzymes involved in the metabolism of vitamin A.....41
STUDY II ...............................................................................................................42
Comparison between normal and lamellar ichthyotic skin at baseline............42
Effects of liarozole on ichthyotic skin ..............................................................43
Effects of liarozole treatment on the expression of retinoid biomarkers...........44
STUDY III..............................................................................................................46
Characteristics of organotypic epidermis ........................................................46
Time dependent RA effects on the mRNA expression of retinoid regulated
genes and enzymes involved in vitamin A metabolism...................................46
The effects of CYP26 inhibitors on organotypic epidermis..............................47
Effects of CYP26 inhibitors on the vitamin A metabolism..............................48
STUDY IV .............................................................................................................48
Normal mRNA expression of CYPs involved in RA catabolism ....................48
Effects of differentiation and RA on the expression of enzymes involved in
vitamin A metabolism .....................................................................................50
Effects of CYP26 knock-down on CYP26 and CRABPII mRNA expression..51
Effects of CYP26 inhibitors on RA metabolism and vitamin A metabolising
enzymes ...........................................................................................................52
GENERAL DISCUSSION AND FUTURE PERSPECTIVES ........................53
CONCLUSION......................................................................................................55
ACKNOWLEDGEMENT ....................................................................................56
SUMMARY IN SWEDISH ..................................................................................60
SUMMARY IN SPANISH...................................................................................63
REFERENCES ........................................................................................................66
Abbreviations
ADH
ARAT
CRABPI
CRABPII
CRBPI
CRBPII
CYP
CYP26
CYP2S1
ER
HB-EGF
ICHYN
IL-1D
KRT
LB
LI
LRAT
NADPH
RA
RAL
RalDH2
RAMBA
RAR
RARE
RBP
RDH16
RDHE2
RE
ROH
RXR
SDR
TGase-1
TGM1
TNFD
Alcohol dehydrogenase
Acyl-CoA:retinol acyltransferase
Cellular retinoic acid binding protein I
Cellular retinoic acid binding protein II
Cellular retinol binding protein I
Cellular retinol binding protein II
Cytochrome P450
Cytochrome P450 26
Cytochrome P450 2S1
Endoplasmic reticulum
Heparin-binding EGF-like growth factor
Ichthyin gene
Interleukin-1D
Keratin
Lamellar bodies
Lamellar ichthyosis
Lecithin:retinol acyltransferase
Niconthinamide adenine oligonucleotide
phosphatase-oxidase
All-trans retinoic acid
Retinal
Retinal dehydrogenase 2
Retinoic acid metabolism blocking agent
Retinoic acid receptor
Retinoic acid response element
Retinol binding protein
Retinol dehydrogenase 16
Epidermal retinol dehydrogenase 2
Retinyl ester
Retinol
Retinoid X receptor
Short-chain dehydrogenase/reductase
Transglutaminase type 1
Transglutaminase type 1 gene
Tumour necrosis factor D
Foreword
Numerous dietary factors, including vitamin A, are essential for
the homeostasis of the skin. Based on this fundamental knowledge,
vitamin A and its natural and synthetic metabolites (retinoids) have
been used as treatment for different skin disorders. Some retinoids
have stood the test of time and are still in use for treating keratinisation disorders, such as psoriasis and ichthyosis, even though their
systemic use is associated with a risk of severe side-effects, such as
teratogenicity. This has prompted a search for new remedies which
might mimic the effects of retinoids, although with a more targeted
action and with lesser side-effects. One such approach is to block the
degradation of endogenous retinoic acid by using substances called
Retinoic Acid Metabolism Blocking Agents (RAMBA). This thesis
expands over the field of vitamin A in the skin and puts the present
knowledge of its metabolism and catabolism to the test, particularly
the effects of substances, which block the catabolising enzymes in
skin cells.
Elizabeth Pavez Loriè, Uppsala, 30 September 2008
11
12
Introduction
The skin
As the largest organ in the body and functioning as the body’s interface with the outside milieu, the skin’s most important function is
as a barrier protecting the body from the external environment. Other
important functions of the skin include water diffusion barrier, participation in the immune system, temperature regulator and involvement in the biosynthesis of vitamins D.
The skin comprises two major tissue layers, the inner layer, dermis,
and the outer layer, epidermis, making it a complex and dynamic
organ. Beneath the dermis the subcutaneous fat can be found (Fig. 1).
The dermis consists of a dense connective tissue, giving skin characteristics of durability and elasticity. It also contains blood and lymph
vessels, nerve endings and adnexal structures such as sweat glands,
sebaceous glands and hair follicles. The dermis regulates the body
temperature and supplies the epidermis with nutrients via the capillary network. Much of the body’s water supply is stored within the
dermis. A basal membrane connects the dermis with the epidermis
(1).
13
Figure 1. The skin consists of two main layers: epidermis and dermis. Underlying the dermis is the subcutis. Dermis contains not only of blood and
lymph vessels, but also nerves, sebaceous glands and hair follicles. The predominating cells in the epidermis are keratinocytes.
The epidermis is non-vascularised and represents stratified cornified epithelium, which provides protection against harmful mechanical, microbial, chemical and physical factors from the outer milieu.
This constantly renewing skin layer is thinnest on the eyelids (0.05
mm) and thickest on the palms and soles (1.5 mm). It mostly consists
of layers of cells called keratinocytes that produce keratins, a family
of structural proteins. In a process called terminal differentiation
(maturation) these skin cells move towards the surface where they
undergo desquamation, a process where the outer layers of the skin
are shed. The different stages of keratinocyte differentiation are represented in the different epidermal layers (strata) that can be seen
microscopically. These layers, from the bottom upwards, are stratum
basale, stratum spinosum, stratum granulosum and stratum corneum
(Fig. 2) (1).
14
Figure 2. Epidermis consists of four layers: stratum basale, spinosum, granulosum and corneum. Here the keratinocytes undergo a process of terminal
differentiation, which includes changing the expression of cytokeratins. This
process demands equilibrium between cell-proliferation, differentiation and
desquamation processes.
Stratum basale consists of keratinocyte stem cells and transit amplifying keratinocytes, which have a column-shaped structure. This
layer also harbours pigment-producing cells (melanocytes), which
protect the skin against UV-light. In this epidermal layer the keratinocytes express keratins (KRT) 5 and 14, which are part of the cytoskeleton. As keratinocytes divide they push already formed cells
(committed to differentiation) upwards to the next layer. Stratum
spinosum consists of keratinocytes that have limited dividing capacity and have a polyhedral shape. This layer also harbours Langerhans’ cells that are bone marrow derived and act as antigen presenting cells. From stratum spinosum and upwards, keratinocytes express keratins 1 and 10, which are regarded as markers of keratinocyte differentiation.
Other differentiation markers that are synthesised here is involucrin, a component of the cornified envelope, and the enzyme trans15
glutaminase 1 (TGase-1). Stratum granulosum consists of nondividing, flattened keratinocytes. These cells produce keratohyalin
granules that contain profilaggrin (the precursor of filaggrin) and
enzymes that degrade the cell nucleus and its organelles (1). In this
layer the cornified cell envelope is formed beneath the cell membrane, its formation is triggered by a rise in intracellular calcium levels (2). The cornified envelope is build up from the cross-linking of
different proteins such as loricrin and involucrin, a process that is
catalysed by TGase-1 and 3. Importantly, lipids stored in intracellular
structures called lamellar bodies (LB) are released into the extracellular environment and attach to the cell envelope (3). Parallel to this,
the keratinocytes’ nuclei degrade and many other intracellular structures enter an apoptotic process leading to “dead” corneocytes.
The stratum corneum consists of flat skin cells in which keratin
filaments are aggregated and cross-linked under the influence of
filaggrin. Together with the cell envelope and lipids, they provide the
epidermis a mechanical and water-permeability barrier function (1).
The entire maturation process of keratinocytes takes 5-8 weeks and
at steady-state the process of desquamation, when corneocytes are
shed from the skin surface, counterbalances regeneration. The whole
process is controlled by the action of agents such as cytokines, growth
factors, calcium, vitamin A, vitamin D and steroid hormones. They
control proliferation and/or differentiation of keratinocytes and thus
maintain epidermal homeostasis (1).
An increased proliferation or decreased removal of keratinocytes
leads to a thicker horny layer, also called hyperkeratosis, which is a
hallmark of keratinisation disorders, such as ichthyosis.
Ichthyosis
The descriptive name ichthyosis is used for a heterogeneous group
of genetically derived keratinisation disorders. The word is derived
from the Greek word “ichthys” (fish) highlighting one of the main
features of this group of diseases: visible scales all over the body (4,
5). Ichthyosis can be a debilitating disease, causes social isolation,
require life-long treatment and may severely impair the patient’s
quality of life (6). On the basis of aetiology and clinical symptoms,
ichthyosis can be divided in four major groups (Table 1).
16
Table 1. The four major groups of ichthyosis (4, 5, 7, 8).
Autosomal
dominant ichthyosis vulgaris
(ADIV)
Incidence
Appearance
Inheritance
Symptoms
Aetiology
X-linked
recessive
ichthyosis
(XRI)
1/250
1/2000-6000
in the male
population
First months or
First week of
years of life
life
Autosomal domi- Recessive Xnant
linked
Hyperkeratosis on Brown scales
extremities
on the neck,
extremities
and trunk
Mutations in filag- Deficiency of
grin
steroid sulphatase
Lamellar ichBullous ichthyosis (LI) or
thyosis
(BI)
Autosomal
recessive congenital ichthyosis (ARCI)
1/100,0001/300,000
300,000
Present at birth
(collodion baby)
Autosomal
recessive
Generalised dry
thick skin, erythema, alopecia, ectropion,
lack of sweating
Mutations in
TGM1, ICHYN,
ABCA12,
ALOXE3,
ALOX12B,
CYP4F2
Present at birth
Autosomal
dominant
Blisters, erythema and
hyperkeratosis
Keratin mutations
At present the treatment of ichthyosis is mainly focused on reducing hyperkeratosis and other symptoms such as skin dryness, erythema, fissures and itching. The choice of treatment relies on the experience of the physicians and the symptoms and preferences of the
patients. Different keratolytics, such as salicylic acid and urea (5),
hydrating agents, such as propylene glycol (9) and lubricating creams
are used as treatment. Gånemo et al (10) showed in a pilot study that
a combination of lactic acid and propylene glycol in a cream base had
synergistic effects on the ichthyotic skin without causing too much
irritation.
Other drugs used to treat ichthyosis are vitamin D (11) and retinoids, i.e. vitamin A derivates (tretinoin, isotretionin, acitretin, tazarotene) (12). These substances modulate the expression of genes
involved in keratinocyte growth and differentiation.
Retinoids have been used topically to treat mild forms of the disease and orally to treat more severe manifestations. However this
treatment can cause significant skin irritation (12) and, in the case of
systemic treatment, carries a risk of teratogenicity, liver toxicity, skel17
etal malformations and hypertriglycemia (13). The history and effects of
retinoid therapy is discussed in more detail below.
A new approach is to increase the cellular levels of endogenously
produced all-trans retinoic acid. This can be achieved by blocking the
catabolism of this molecule with agents called, Retinoic Acid Metabolism Blocking Agents (RAMBA). This thesis focuses on the effects of
specific RAMBAs on the skin and compares their properties with
those of retinoids.
Vitamin A and retinoids
History
The word vitamin was originally derived from the term "vital
amine." In 1912, the pioneer Dr. Casimir Funk referred to these substances “vita amines” based on the belief that these accessory factors
were chemical amines, similar to thiamine, the vitamin involved in
the deficiency disorder, beriberi. A year after, two groups simultaneously and independently discovered vitamin A, then known as “fatsoluble-A” because it was found in lipid-rich food and gave growth
arrest in developing animals when deprived from this type of food.
Soon the term "fat-soluble A" was combined with Funk's designation
to become "vitamin A” (14). Later Karrer et al isolated and determined the chemical structure of retinol and Wald et al determined
retinol’s role in visual function. These two major breakthroughs in
vitamin research were awarded with the Nobel Prize in 1937 and
1967 respectively (14).
The importance of vitamin A in the body is illustrated by the
symptoms that follow vitamin A deficiency such as night blindness,
growth arrest, skin dryness and higher vulnerability to infections
(14). Two pioneers in the field of vitamin A research, Wolbach and
Howe, described as early as in 1925 that vitamin A-deficient mice
exhibited a hyperkeratotic epidermis (15). Frazier and Hu, in 1931,
also described the relationship of hypovitaminosis and follicular hyperkeratosis of the skin (16). In the 1940s, all-trans retinoic acid (RA)
was synthesised and it was found to be more potent than retinol
(ROH) in reversing the skin symptoms of vitamin A deficiency (17).
After these fundamental observations, several studies explored the
therapeutic usefulness of vitamin A in dermatology. A major breakthrough was the efficient treatment of acne with RA in 1962 (18).
Since then, many new synthetic retinoids have been developed and
tested in dermatology (19).
18
Uptake, processing and transport in the body
Vitamin A (retinol) is supplied via the diet as preformed retinyl esters (RE) from animal or is formed from certain vegetable-derived
carotenoids, which serve as vitamin A precursors (the most potent
being E-carotene). Ingested RE is hydrolysed to ROH in the intestine
lumen and absorbed as free ROH by the intestinal cells where it
forms complexes with cellular retinol binding protein II (CRBPII)
(19). E-carotene enters the enterocytes and is converted into two retinal (RAL) molecules, which are protected by CRBPII from oxidating
into RA. These two molecules are reduced to ROH by a retinal reductase. Whatever its source is, ROH serves as a substrate for the enzyme
lecithin:retinol acyltransferase (LRAT), which re-esterifies ROH to
RE. On leaving the intestinal wall, RE is incorporated into chylomicrons, i.e. lipotroteins, and transported via the general circulation to
the liver (19). In the liver vitamin A can be stored as RE, mainly in fat
storing cells (20). From 50 to 80% of the total body ROH concentration
is stored as RE in these cells and depending on the plasma concentration of ROH, it is released as ROH bound to its plasma carrier, retinol
binding protein (RBP) (19). RA also exists in plasma and other body
fluids at about 100-fold lower concentration than ROH (21). The
ROH-RBP complex is presented to the target cells for vitamin A in the
body (19). In addition to its many important functions such as vision,
spermatogenesis and bone growth, vitamin A is essential in the proliferation and differentiation of epidermal keratinocytes and thus
essential in the homeostasis of the skin (19, 22), where not only ROH,
but also E-carotene, RE, RAL, RA and 3,4-didehydro retinoids can be
found (23). Most of the biological activities of vitamin A in the skin
are carried out by two of its metabolites, i.e. RA and 9-cis retinoic acid
(9-cis RA). The principal ways of vitamin A metabolism in the cell are
shown below (Fig. 3).
19
Figure 3. Metabolism of vitamin A in keratinocytes.
20
The uptake and processing of vitamin A in the skin
The uptake of vitamin A by epidermal keratinocytes probably involves an RBP receptor expressed on the keratinocyte surface (24-26).
Intracellular retinol binds to cellular retinol binding protein I
(CRBPI), which functions as a transport protein as well as a stabiliser
of this lipid soluble molecule. Noy et al have suggested that the level
of free CRBPI in the cell determines the amount of ROH that is taken
into the cell (27).
CRBPI-bound retinol is either metabolised into RE, a process catalysed by LRAT or acyl-CoA:retinol acyltransferase (ARAT) (28, 29) or
converted into RAL depending on the ratio between free CRBPI and
bound CRBPI (22). The conversion to RAL is a reversible oxidation/reduction, which can be catalysed by three enzyme families: the
alcohol dehydrogenases (ADH), some cytochrome P450 (CYP) enzymes and short-chain dehydrogenases/reductases (SDRs) (22). In
the latter family, two enzymes have been identified in epidermis
RDH16 (RoDH4/hRDH-E) (30, 31) and RDHE2 (32), where RDH16 is
known to oxidate ROH or reduce RAL (33).
The next step is an irreversible conversion of RAL to RA, which is
catalysed by retinal dehydrogenases, where retinal dehydrogenase 2
(RalDH2/ALDH1A2) is known to be expressed in epidermis (34). It is
also believed that some CYPs are capable of catalysing RAL to RA
metabolism, but the physiological relevance of this metabolism is still
unclear (35).
Once formed, RA binds to another group of intracellular binding
proteins, cellular retinoic acid binding protein (CRABP) I and II, and
either enter the nucleus or is directed to degradation by oxidation
performed by cytochrome P450 dependent enzymes (CYPs), e.g. the
CYP26 family and CYP2S1 (19) (36).
CRABPI and CRABPII
Generally, retinoid binding proteins solubilise and stabilise their
hydrophobic and labile ligands. They are also believed to be part of
the transport, metabolism and action of their ligand (37).
The two CRABPs are highly homologous and bind RA with high
affinity (38); CRABPI has the lowest dissociation constant of the two
(0.4nM vs. 2nM) (39). Other natural retinoids, e.g. 4-hydroxy-RA, 4oxo-RA and 18-hydroxy-RA, also bind to these proteins (40). By contrast, CRABPs have low affinity for 9-cis-RA and no affinity for 13cis-RA (38). CRABPs also have a distinct tissue specific expression
pattern; CRABPI is expressed in more tissues than CRABPII (38), but
both are present in the cytosol and in the cell nuclei (41).
21
Although the exact functions and roles of CRABPI and CRABPII
are not completely understood (42), differences in their tissue expression and affinity for RA can give us an idea about their cellular function. It seems that CRABPI is widely expressed in adult tissue (22)
and it has also been demonstrated that CRABPI does not protect RA
from elimination (40), but encourages its degradation (43). Dong et al
(37) later described that CRABPI could regulate RA degradation. On
the other hand cells that synthesis RA express CRABPII, which seems
to be found in the same place as CRBP (22).
Examples of the differences between the two RA binding proteins
can be found in the skin. Here CRABPI can be detected in dermis and
epidermis in basal keratinocytes (44) and melanocytes (45), whereas
CRABPII is mostly expressed in epidermis in suprabasal keratinocytes (44, 46, 47). Both CRABPII and CRBPI gene transcription are up
regulated by retinoids in the skin (48, 49). As mentioned before, both
CRABPs have been found to be present in the nucleus, but only
CRABPII has shown to affect the RA-mediated gene activation (37,
50, 51). CRABPII is also induced by keratinocyte differentiation in
vitro, which leads to increased cellular concentration of RA and
probably a retention of newly synthesised RA in the cells (52). Interestingly Siegenthaler et al (53, 54) and Karlsson et al (44) have shown
that in hyperproliferative skin diseases, the pattern of CRABPs is altered.
CYP26 and CYP2S1
The cytochrome P450 dependent enzymes (CYP, P450) consist of
heme-containing proteins found in all domains of life. These enzymes
catalyse lipid-soluble substrates, a variety of xenobiotics and environmental toxins (55). The most common reaction catalysed by cytochrome P450 is a monooxygenase reaction, e.g. insertion of one atom
of oxygen into an organic substrate (XH) while the other oxygen atom
is reduced to water:
XH + O2 + 2e-
XOH + H2O
The term P450 derives from the observation that this cytochrome
has an absorption peak at 450 nm, provided that the iron atom of the
heme group is reduced and complexed to carbon monoxide (56).
Most CYPs are membrane-bound proteins found in the endoplasmic reticulum (ER). The protein that donates electrons, needed in
22
the reaction, to P450s in the ER is called NADPH cytochrome P450
reductase, which is also a membrane bound protein (56).
The CYP enzymes are generally expressed in the liver, but have
also been found in other tissues, e.g. the skin, where some of the enzymes play an important role in RA homeostasis. RA is catabolised to
18-OH-RA, 5,6-epoxy-RA or 4-OH-RA, that is further oxidised to 4oxo-RA. All these metabolites are further metabolised to more polar
metabolites (57). These metabolites have been found to be more or
less biologically active (58, 59). As early as in 1979, Roberts et al (60)
hypothesised that the oxidation of RA could depend on CYPs because
they occurred in microsomes. Later it was shown that by adding RA
to the system the 4-hydroxylation is increased, suggesting an RA
stimulation of the enzyme (s) (61).
One family of CYPs, called CYP26, is very specific towards RA.
The first member of this family to be characterised is CYP26A1
(P450RA1) (62). This enzyme is expressed in many different tissues
and is induced by RA both in vivo and in vitro (63-65), suggesting
that RA catabolism includes feed forward loops. In man, the CYP26
family is composed of not only CYP26A1, but also by two other
members, CYP26B1 and C1 (66, 67). Although the members of the
CYP26 family are similar in their sequences and have a high specificity towards RA, they show distinct expression patterns. CYP26B1 is
for example more widely expressed in the adult brain tissue as compared to CYP26A1 (66). Furthermore, CYP26B1 appears to play a role
in hair follicle development because it is found in the developing hair
follicles in mouse embryos (68). It has also been reported that
CYP26C1 oxidise 9-cis RA. So far this enzyme has only been detected
in low levels in various human tissues (67). Another member of the
CYP26 family, CYP26D1, has been characterised in zebrafish and
showed inducibility to RA, but its role in RA catabolism has yet to be
discovered (69, 70). Therefore, from here on CYP26D1 will not be included when referring to CYP26 in the text.
The mechanisms and effects of this group of enzymes are not well
characterised. Some reports have suggested that each of the CYP26
family members have individual roles in the catabolism of RA because they do not have overlapping expression in the developing
embryo (68), but other groups have shown that a certain cooperation
or collaboration between these subtypes exists in developing mouse
embryos (71, 72). Perhaps their function depends more on the amount
of RA that is to be catabolised, shifting the expression of CYP26 subtypes towards either a more individual function or collaboration between the different CYP26 members.
23
Another CYP enzyme that has been identified in human skin is
CYP2S1. This enzyme has shown the capacity to catabolise RA into 4OH-RA and 5,6-OH-RA but not into 4-oxo-RA. CYP2S1 also shows a
two-fold induction by topical exposure to RA (73). The same report
stated that other agents, such as coal tar, also induce CYP2S1. More
recently another group showed that CYP26A1 is expressed in the
basal layer of epidermis (74) and that it was not affected by coal tar,
suggesting that CYP2S1 and CYP26A1 are differently regulated in the
skin.
Biological effects of retinoids in skin
Most of the biological activities of retinoids are carried out through
binding to specific retinoid receptors, which belong to the superfamily of nuclear receptors including steroid, vitamin D (VDR), thyroid (TR), the peroxisome proliferator-activated (PPAR) and a number of other receptors (75). Retinoic acid receptors (RAR) possess high
affinity for RA, which thus serves as a potent activator of these ligand-dependent transcriptions factors. Another receptor family, retinoid X receptors (RXR), has 9-cis-RA as its natural ligand (76). Usually RAR family members( D, E, J) and the RXR family members (D, E,
J) form heterodimer complexes (76).
In the skin the predominant RAR is RARJ, whereas RARD is expressed at minimal levels (77). The levels of RXRs are higher than
those of RARs, RXRD being the most abundant retinoid receptor (77).
The most common receptor complex in the human skin appears to be
RARJ - RXRD (77). Retinoid receptors have six functional domains of
which two are important to achieve gene transcription: the ligandbinding domain and the DNA-binding domain by which the receptor
binds to the target genes (78). The retinoid receptor dimers are localised in the nucleus accompanied by co-repressor molecules where
they bind (in its apo-form) to specific DNA regulatory sequences
called retinoic acid response elements (RAREs). These elements are
usually situated in the upstream region of target genes (75). Heterodimers bind more efficiently to these response elements than
homodimers. The binding of RA to RAR induces a conformational
change in RAR, which not only dissociates the co-repressors and
permits the binding of co-activators to the complex, but also gives
RXR the opportunity to bind its ligand, which could further induce
the RAR-mediated transcription of the retinoid-regulated gene (79,
80).
Among epidermal genes that are retinoid regulated are several cytokeratins (KRT5, KRT6, KRT14 and KRT17) and CRABPII (81, 82).
Studies performed by Steijlen et al (83, 84) and later by Virtanen et al
24
(85, 86) have shown that also KRT2 and KRT4 are retinoid regulated
in human epidermis, although no RAREs have so far been identified
in these genes. Furthermore, the CYP26A1 gene, involved in retinoid
metabolism, possesses two RAREs (87). The other CYP26 subtypes
and CYP2S1 are affected by RA, but so far no RARE have been identified (66, 67, 73).
Another mechanism by which RA and its receptors regulate the
differentiation and proliferation of epidermal keratinocytes is by acting as an antagonist of activating protein-1 (AP-1) (88), which is important for keratinocyte differentiation (89). This mechanism does not
involve RAREs in the affected genes.
Retinoid therapy of skin disorders
The term “retinoid” has changed over the years to now include the
naturally occurring vitamin A metabolites and synthetic compounds
that show biological activities, which are characteristic for vitamin A
(90). They can be divided in three groups, which are outlined in Table
2.
Table 2. Three generations of retinoids in clinical use.
Group II,
monoaromatic
all-trans retinoic acid (tretinoin) etretinate
13-cis retinoic acid (isotretinoin) acitretin
Group I,
non-aromatic
Group III,
polyaromatic
tazarotene
adapalene
The first generation includes RA and 13-cis-RA. The second generation includes substances where aromatic substitution of the beta
ionone ring structure has been performed. The third generation contain substances that interact with certain retinoid receptor and have
more than one aromatic group.
Among the diseases known to respond to retinoids are various
types of genetic disorders of keratinisation, such as psoriasis (a polygenetic disorder) and ichthyosis (17, 91, 92). Of the first generation
retinoids, tretinoin (RA) and isotretinoin (13-cis-RA) are still used in
the treatment of acne, but their skin irritating effects (93) limit their
use. (94). Acitretin and tazarotene are used in the treatment of psoriasis (91). Etretinate and acitretin, a metabolite of etretinate, are equally
effective in the systemic treatment of psoriasis and their side-effects
are similar (95, 96). Due to the teratogenic effects of these drugs, EU
25
regulations demands that women of childbearing age have to continue with contraception for two years, after finishing the treatment
(97). Topical tazarotene are in clinical use in the treatment of psoriasis, where its main side-effects are skin irritation, desquamation and
dry skin (98, 99). The Food and Drug Administration (FDA) in the
U.S. has still not approved oral treatment with this substance due to
lack of safety data.
The use of retinoids in ichthyosis depends on the severity of skin
manifestations and the patients’ general health, sex, age, etc. Mild
forms of ichthyosis can be treated with topical retinoids such as RA
(tretinoin) and tazarotene. Severe forms of ichthyosis are often continuously treated with oral acitretin. Lamellar ichthyosis responds to
etretinate, acitretin and isotretinoin. Even in some severe cases of
collodion baby systemic retinoid treatment has been used to facilitate
membrane shedding (100). Although retinoid treatment dramatically
reduces hyperkeratosis it can lead to a wide range of adverse effects
(see page 18) (16, 100) .
26
Retinoic acid metabolism blocking agents
(RAMBAs)
To achieve a better response and to reduce the risk of side-effects
caused by retinoid treatment, new indirectly acting substances have
been developed. One approach is to use RAMBAs to prevent the in
vivo catabolism of endogenous RA and augmenting its tissue level by
blocking the CYP26-dependent 4-hydroxylation of RA (36). Once
treatment is stopped these agents are quickly eliminated from the
body (101). The proposed mechanism of action for these substances is
shown below (Fig. 4).
Figure 4. Retinoic acid metabolism blocking agents, proposed mechanism of
action in the cell.
The first azole with CYP inhibitory effects was the anti-fungal substance ketoconazole. This agent showed the capacity to inhibit CYP
mediated catabolism in rodents (102), but is not specific for CYP26
(103). A second generation CYP inhibitor is liarozole (R75251; liarozole fumarate R85246) (103) (for chemical structure see Fig. 5). This
imidazole derivate lacks the anti-fungal effects of ketoconazole (103),
but enhances the endogenous plasma RA levels and reduces the elimination rate of injected RA in plasma (104, 105). It is a more specific
inhibitor of CYP26 than ketoconazole (106, 107), but still inhibits
aromatase, important in sexual development and adrenal steroid metabolising enzymes (101).
27
Liarozole is the most studied RAMBA, showing promising effects
in different cancer forms (108) and skin disorders, such as psoriasis
and ichthyosis (96, 109-115).
More recently, a highly selective and active retinoic acid 4hydroxylase inhibitor was synthesised, named talarozole (RambazoleTM, R115866) (for chemical structure see Fig. 5). Compared to
liarozole it exhibits increased efficacy both in vitro and in animal experiments (116) and has low effects on the biosynthesis of steroids
(101). Homology models of the interaction between CYP26A1, B1 and
talarozole have also added more evidence that it is a specific RAMBA
indeed (117, 118). This drug is also quickly eliminated from the body
(119). It is also worth mentioning that the plasma levels of RA have
not increased above physiological levels in patients treated with talarozole or liarozole.
To date, three clinical studies with oral talarozole have been published (120-122). Bovenschen et al (122) assessed the expression of Tcell markers and NK-cell receptors with and without treatment of
talarozole. These biomarkers tended to be reduced after treatment,
suggesting that this agent might have anti-inflammatory effects,
which could be looked at in psoriasis inflammation studies in the
future.
Figure 5. Chemical structures of liarozole and talarozole
Only two reports have been published on the biological effects of
liarozole on retinoid-regulated genes in humans (123, 124) and little
has been reported about talarozole from this perspective. New knowledge about both the CYP26 family of enzymes and CYP2S1 has en28
abled a more detailed study of the role of these enzymes in human
skin. This knowledge and further clinical trials will lead to novel
therapies for patients suffering from different skin disorders, such as
ichthyosis.
29
Aim of the research
The objective of this study was to gain a better understanding of
the biological effects of RAMBAs (CYP26 inhibitors) on normal and
diseased human skin in vivo and to further explore the mechanisms
of action of these compounds in vitro in human keratinocytes, focusing on the role of vitamin A metabolism.
Specific aims:
x To study the effects of topically administered talarozole on
the expression of biomarkers of retinoid signalling and inflammation in the skin of healthy volunteers (Study I)
x To study the effects of oral liarozole in lamellar ichthyosis
and to gain further information about the mechanisms behind these effects (Study II)
x To investigate the effects of RA and CYP26 inhibitors on the
expression of retinoid-regulated genes in organotypic epidermis and cultured keratinocytes to explore the usefulness
of these in vitro models for evaluating pharmacological efficacy of novel CYP26 inhibitors (Study III and IV)
x To compare the effects of RA and CYP26-inhibitors on the
metabolism of RA (Study III and IV)
x To study in more detail the function of enzymes involved in
the catabolism or biosynthesis of RA with particular emphasis on CYP26 and CRABPII in cultures of normal keratinocytes (Study IV)
30
Material and methods
The following is a brief summary of the materials and methods
used in this thesis. A more detailed description can be found in the
individual studies.
Subjects (Study I and II)
All tissue specimens were collected at Uppsala University Hospital
following the ethical principles from the Declaration of Helsinki. The
local ethics committee at Uppsala University approved the studies
and the volunteers signed a written informed consent at the beginning of the trials.
Study design
Study I
This randomized, double blind, left-right comparative, and placebo-controlled single centre study was performed on healthy volunteers, including both males (n=9) and females (n=7) (Caucasians),
between the ages of 18 to 55 years. The participants had to be healthy
and only women with a negative pregnancy test at screening and
who were on oral contraception were included in the study.
Topical gel (200 mg) consisting of 0.07% or 0.35% of talarozole was
applied to one designated gluteal area (40 cm2) and vehicle to the
opposite gluteus, once daily for nine days. The applications were
done by the investigators except for the weekend when the subject
applied the test gels, after careful instruction. A timeline over the major events in this study is shown in Fig. 6.
31
Figure 6. Timeline for clinical study with topical talarozole.
Punch and superficial shave biopsies (consisting of epidermis and
a minor proportion of papillary dermis (23)) were taken from both
gluteal areas after infiltrating the skin with lidocain-adrenalin. At day
0, baseline samples were taken (one punch and one shave biopsy)
and at day 9, biopsies from both placebo and talarozole treated skin
were taken. The biopsies were kept at -70qC, until continuing with the
histological, mRNA and protein expression analysis of retinoid and
pro-inflammation biomarkers (Table 3).
32
Table 3. List of analysed biomarkers in the different studies.
Biomarker
CYP26A1
CYP26B1
CYP26C1
CYP2S1
LRAT
RDH16
RalDH2
CRABPII
KRT2
KRT4
HB-EGF
IL-1D
TNFD
Function
RA
catabolism
Retinol
storage
Retinol
and
retinaldehyde
metabolism
Binds to RA
RA-regulated
keratin
genes
RA-regulated
growth
factor
Important in
inflammation
Study I
Study II
Study III
Study IV
a
X
X
a
X
X
X
X
X
X
X
X
X
Xa
X
X
Xa
a
X
a
X
X
X
X
X
X
X
X
X
X
X
a
X
a
X
a
X
Xa
a
X
X
X
X
X
X
a
X
X
a) Examined both at the mRNA and protein level.
Study II
This study, which was part of a larger multicentre trial, included
12 volunteers both males (n=4) and females (n=8) (negative pregnancy test) no younger than 14 years of age, suffering from moderate
to severe ichthyosis (judged from overall severity of scaling and/or
erythema). Of these, 7 patients had TGM-1 mutations, 4 had ICHYN
mutations and 1 had, to this date, unknown mutations causing the
disease. Three groups of patients were randomly designated, one
group received two tablets containing 75 mg of liarozole (total of 150
mg) once daily, the second group took one tablet containing 75 mg of
liarozole and one containing a placebo tablet and the third, smaller
group received two placebo tablets daily.
The schedule for the study is outlined in Figure 7 and the patient’s
characteristics can be seen in Table 4. At baseline two punch biopsies
(for different fixations, Lana’s fix and acetone) and one shave biopsy
were obtained. This procedure was repeated after four weeks of
treatment, i.e. at the first re-visit during therapy when the full clinical
effect of the 3-month study was still pending. The biopsies were ran33
domized and stored at -70qC, followed by analysis of the histology,
mRNA and protein expression of retinoid and pro-inflammation
biomarkers (see Table 3).
Figure 7. Timeline for the liarozole clinical study.
34
Table 4. Patient’s characteristics, doses of liarozole and ichthyosis scores in
the clinical trial.
No.
1
a)
2
3
4
5
6
7
b)
8
9
10
c)
11
12
Sex
Affected
gene
Liarozole
dose
(mg)
Ichthyosis Score
Baseline
1 month
F
TGM1
Placebo
35
27
F
F
M
M
M
M
F
F
F
F
F
TGM1
TGM1
TGM1
TGM1
TGM1
TGM1
Ichthyin
Ichthyin
Ichthyin
Ichthyin
unknown
150
75
150
75
150
75
150
75
75
75
34
36
35
34
35
35
20
23
28
21
22
26
27
34
20
32
34
1
1
12
NA
7
a) The baseline sample in this case was obtained first 1 mo. after stopping therapy,
i.e. when the ichthyosis score had returned to pre-therapy value.
b) No biopsies were obtained from this patient.
c) This patient was excluded from the clinical trial but a baseline biopsy was taken
and used in the comparison with healthy controls.
NA= not applicable
Study III
This study was performed using an organotypic epidermis containing normal human keratinocytes seeded on an insert (125) to assess the effects of RA and CYP26-inhibitors (RAMBAs) on retinoidregulated genes and vitamin A metabolism in an in vivo-like skin
model. One of the reasons why we chose to work with this model
was the serum-free culture condition, which could be easily altered.
This is an important factor when studying the metabolism and function of vitamin A. A disadvantage is the lack of a normal dermalepidermal interaction, which possibly leads to a disturbed epidermal
differentiation, compared to another model, where the keratinocytes
are cultured on de-epidermised dermis (85).
First, the histology of the organotypic epidermis and the retinoid
responsiveness of this model (1 μM RA for 48 h) was examined by
looking at the expression of KRT4, which is known to be induced by
RA in the skin. This was followed by a time study (8, 24, 48 or 72 h)
35
where the organotypic model was incubated with 1 μM RA or DMSO
(as vehicle). Next, the effects of the two CYP26-inhibitors liarozole
and talarozole on retinoid-regulated genes (see Table 3) were studied.
In this case attention had to be paid to the RA levels in the culture
medium because earlier experiments showed that liarozole and talarozole alone did not alter the expression of retinoid-regulated genes
probably due to the low endogenous RA levels. A pre-incubation
with 1 nM RA for 48 h was deemed necessary for the CYP26-inhbitors
(1 μM) to increase RA and to alter the expression of the retinoid biomarkers. The uptake of [3H]RA and [3H]ROH by epidermis was examined beginning with a pre-incubation with unlabelled ROH
(100nM) after 9 days of culture to build up the levels of vitamin A
and imitate the natural vitamin A metabolism. After another 4 days, 5
μM of liarozole or talarozole were added to the culture medium and
incubated for 24 h before adding 1 μCi of [3H]RA or [3H]ROH.
Finally, the organotypic epidermis was harvested and analysed by HPLC.
In addition, some of the samples treated with ROH and CYP26 inhibitors in the metabolism experiments were also used for immunofluorescence analysis of KRT4 and CRABPII, to determine
whether accumulation of RA in the organotypic epidermis affected
the protein levels of these RA-regulated biomarkers.
Study IV
Normal human keratinocytes cultured submerged in a serum-free
medium were used in this study. The retinol and calcium concentrations of the final medium were <1 nM and 0.06 mM, respectively. The
first part paid attention to how the aspect of keratinocyte differentiation (achieved by adding 1.5mM CaCl2) affects the response of genes
involved in the metabolism of vitamin A (see Table 3). To compare
proliferating and differentiating kertainocytes the cells were additionally incubated with RA. The second part of the study focused on
the effects of CYP26 inhibitors (10PM ketoconazole, liarozole or talarozole) and CYP26A1 and B1 knock down (using siRNA, final conc.
=60 nM) on [3H]RA metabolism, studied by incubation of keratinocytes with [3H]RA for 24 h prior to HPLC analysis. At the same time
the mRNA expression of CYP26A1 and B1 were studied by incubating the cells with specific siRNA probes for 48 h. To examine the effects of CYP26B1-knock down and CYP26 inhibitors have on RA activity in the cell, the CRABPII expression (44) was assessed. The keratinocytes were incubated with RA (1PM), CYP26B1 siRNA or CYP26inhibitors (1, 5 or 10PM) for 24 h. For immunofluorescence analysis
the cells were seeded on cover slips. Lastly the influence of CYP26
inhibitors (1, 5 and 10PM) on the metabolism of vitamin A was exam36
ined by observing the expression of genes coding for enzymes involved in vitamin A metabolism.
37
Analysis of samples
In all four studies the effects of retinoids and CYP26 inhibition
were analysed at the mRNA and protein level. In addition the epidermal thickness and signs of skin inflammation were evaluated in
Study I and II. Here is a brief summary on the methods used.
Epidermal thickness and signs of proliferation (I,
II)
In both studies the viable epidermis was measured and its thickness calculated from Haematoxylin stained sections from the biopsies. Also, in Study II the stratum corneum thickness was calculated.
In both Study I and II, a dermatologist looked for any signs of adverse events such as erythema on each visit.
In Study I the expression of Ki67 in the basal layer was examined
in sections where the number of Ki67 positive cells per length of basal
membrane was calculated.
mRNA expression (I-IV)
Total RNA was extracted from shave biopsies (I and II), organotypic epidermis (III) or monolayer cultures (IV). cDNA was synthesised and the mRNA expression of the genes of interest (see Table 3)
was analysed by using quantitative real-time PCR. For the two in
vivo studies (I and II) the housekeeping genes cyclophilin and E-actin
were analysed in parallel. A normalisation factor for each sample was
calculated from the expression of the two housekeeping genes, using
the geNorm software (126). All values for each gene were normalized
using this factor. For the two in vitro studies (III and IV) cyclophilin
was used as housekeeping gene.
38
Protein expression (Study I-IV)
Immunofluorescence
In both in vivo studies (I and II) the expression of KRT 2 and 4, and
of the enzymes CYP26A1 and B1 were examined. In Study II, one
additional punch biopsy from each patient was placed in Lana’s fix
and sucrose for examination of CRABPII (44). The expressions were
studied by immunofluorescence (IF) and the intensity evaluated by
means of scoring. In Study III two punch biopsies were taken from
the centre of each organotypic epidermis. One of the biopsies was
fixed in 4% formaldehyde followed by methanol for 10 min at room
temperature to be used in the protein expression analysis of KRT4.
The other was placed in Lana’s fix for 2 h followed by incubation in
10% sucrose for 24 h and later sectioned and stained for CRABPII. In
all four studies the samples were stored at -70°C. The monolayer cultures in Study IV were fixed with 2% paraformaldehyde for 10 min,
before staining for CRABPII (44).
Immunoblotting
In Study I the protein expression of CYP26A1 and B1 was also examined by Western blot. Because no separate samples were taken for
this analysis, the tissue extracts were obtained from sections of frozen
punch biopsies in which dermis was microscopically removed and
the remaining epidermis was lysed.
The cells in Study IV were homogenised and lysed using a common cell-lysing buffer, RIPA. In the two studies the lysed samples
were centrifuged and the supernatant was stored at -70qC. The total
protein extracts were then fractioned in SDS-PAGE followed by blotting and visualisation of CYP26A1, B1 or CRABPII protein by the use
of specific antibodies.
39
Results and discussion
Study I
Effects of talarozole on retinoid biomarkers in healthy
epidermis
In this study we examined whether two different doses (0.07% or
0.35%) of topically applied talarozole could alter retinoid-regulated
genes, thus serving as a proof-of-principle for the drug.
Talarozole increased the mRNA expressions of CRABPII and
KRT4, whereas KRT2 expression was reduced. The changes were
similar to those seen in skin treated topically with RA (83, 85). The
magnitude of the changes was concentration-dependent; the highest
concentration (0.35%) of talarozole being most effective. KRT4 protein
expression was also affected by talarozole application, showing the
appearance of a distinct immunostaining in stratum granulosum.
KRT2, on the other hand, was only marginally down regulated by
talarozole (Fig. 8).
Only two subjects showed mild signs of skin irritation after 9 days
of exposure to the drug and analysis of epidermal thickness (and Ki67
staining), showed no major induction of epidermal hyperplasia.
Figure 8. Gene and protein expression of KRT2 and 4, in normal untreated
human skin and after topical application of vehicle or 0.35% talarozole.
40
Effects of talarozole on enzymes involved in the
metabolism of vitamin A
The CYP26 family and CYP2S1 are enzymes known to control the
levels of RA in the cells and are also induced by RA. CYP26 enzymes
are not only both primary targets for RAMBAs, but also positive
biomarkers of increased RA activity (for review see (36)). Our results
for CYP26 A1 and B1 mRNA expression are in total agreement with
these concepts, the effects being dose-dependent and most pronounced for CYP26A1. However, the results of immunofluorescence
staining and Western blot analysis showed no increased expression of
the two proteins after exposure to RAMBAs, but neither did RAexposed control skin. In theory the results could be explained by a
rapid turnover of protein or by the fact that a large amount of mRNA
is not translated into protein (Fig. 9). CYP2S1 had a lower expression
and CYP26C1 was only marginally present in epidermis.
Figure 9. Gene and protein expression of CYP26A1 and B1 in normal untreated human skin and after topical application of vehicle or 0.35% talarozole.
LRAT and RalDH2 are two enzymes that have a rate limiting function in retinol esterificaton and retinal oxidation respectively (127).
They play an important role in the regulation of vitamin A metabolism and are also putative retinoid biomarkers. For example, it is
known that exposure of normal skin to topical RA leads to increased
retinol esterification (28), which may serve as an autoregulatory loop
diverting retinol from oxidation to RA. In our analysis we detected a
slight decrease of RalDH2 mRNA expression when applying the
higher dose, while no alteration was detected in LRAT expression.
41
Although retinoid-like effects were observed at the mRNA and
protein levels, only two subjects experienced signs of skin irritation,
which is a common side-effect of RA and other retinoids (13). Even
more surprisingly, IL-1D, a biomarker of early inflammatory processes (128) and known to be up regulated after RA application (129),
was down regulated at the mRNA level. The expression of TNFD,
another pro-inflammatory cytokine, was not significantly affected.
Although the protein expression of IL-1D did not detect any reduction of this cytokine, it would be of interest in future clinical studies
of talarozole treatment to be able to confirm or reject these findings.
Study II
In comparison with the talarozole study (I), the liarozole study on
lamellar ichthyosis patients showed a less uniform response pattern
of the retinoid biomarkers. One reason could be that out of the nine
biopsied patients, only two had received the higher dose of liarozole
(150 mg/day).
Comparison between normal and lamellar ichthyotic skin
at baseline
One of the retinoid biomarkers that has been described before in LI
epidermis and psoriasis lesions is CRABPII. The gene and protein
expressions were both higher in the diseased epidermis compared to
healthy skin, and expression was seen in all suprabasal layers instead
of normally only in stratum granulosum (44, 54). These observations
could be observed in our study as well (Fig. 10). Therefore, increased
CRABPII expression may be secondary to epidermal hyperplasia in
LI and not a reliable indicator of RA stimulation during liarozole
therapy.
42
Figure 10. Gene and protein expression of CRABPII in normal (left) and
lamellar ichthyotic (right) skin.
Effects of liarozole on ichthyotic skin
No systemic toxicity was noted in any of the patients that participated in this study and a comparison between baseline and treatment
samples showed no significant difference in epidermal thickness.
However, a tendency to decrease str. corneum thickness was observed after liarozole treatment consistent with an anti-keratinising
effect of the drug.
The clinical results after 4 weeks of liarozole therapy were particularly good in the patients with ICHYN or other non-TGM1 mutations.
Ichthyin is believed to act as a lipid transporter to the lamellar bodies
(LB) essential for the formation of a skin water barrier (130). Interestingly, the absence of Ichthyin in ichthyosis patients (131) and retinoic
acid receptor J in knockout mice (132), both lead to similar structural
abnormalities in LBs. This implies a connection between retinoid signalling and the transport of lipids by Ichthyin, possibly explaining
why patients with ICHYN mutations responded more promptly to
increased endogenous RA levels during liarozole therapy than did
patients with TGM1 mutations.
43
Effects of liarozole treatment on the expression of retinoid
biomarkers
Five subjects that demonstrated a strong induction of KRT4 expression during liarozole treatment also had predominant changes in
the mRNA levels of other genes, i.e. KRT2 and CYP26A1 indicating a
retinoid-like response (Fig. 11).
Figure 11. KRT4, 2 and CYP26A1 gene expression in LI patients treated
orally with liarozole. Five patients had a response in all three genes (black).
KRT4 expression
In contrast to the findings of Lucker et al (112), our KRT4 mRNA
data showed only a mild induction, which could be explained by differences in the treatment duration and applied drug concentration in
the two studies. To be sure that the effects of treatment were primary
and not secondary to improvement of ichthyosis, we waited the
shortest possible period of treatment before taking the biopsies. We
also used lower liarozole concentrations (75 mg vs. 150 mg) since it
has been reported that liarozole given at high doses elicits similar
side-effects as seen with retinoid treatment (109, 112).
KRT2 expression
The gene expression of KRT2 is known to be down-regulated with
RA treatment (85). This was also the case after liarozole treatment
although no dose-related (75 mg and 150 mg) response could be observed and the overall change in keratin gene expression was small
compared to the changes seen with topical RA application in normal
human skin (85). Lucker et al (112) described the protein expression
of keratins in ichthyosis patients treated with oral liarozole although
KRT2 was not included in their study. In our study the KRT2 protein
expression seems to be lower than in normal skin already at baseline,
which could explain why no further reduction occurred after oral
44
liarozole treatment. Instead a small increase occurred, which suggests
that the effects of liarozole tend to normalise the protein expression of
this gene in epidermis (Fig. 12).
CYP26A1
One of the main targets of liarozole is believed to be CYP26A1. The
mRNA of this enzyme tended to dose-dependently increase during
liarozole therapy. Several LI patients displayed strong protein staining of CYP26A1, which was distributed all over epidermis before
therapy in comparison to the specific basal layer staining in normal
human skin (74, 133). The differences in CYP26A1 and CRABPII expressions (see above) suggest that the RA signalling is disturbed in LI
patients.
After therapy the changes in CYP26A1 (and KRT2) expression indicate that ichthyotic epidermis strives to normalise the protein levels
(Fig. 12).
Figure 12. Protein expression of KRT2 and CYP26A1 in normal human skin
(NS) and in lamellar ichthyotic skin, before and after liarozole treatment.
On the other hand the mRNA expression of CYP26A1 does not
correlate with its protein expression after liarozole treatment. This
might be due to methodological problems, such as obtaining skin
samples from a heterogeneous group of patients. However, the poor
correlation between mRNA and protein levels is not exclusive to LI
but was also observed in normal skin treated with talarozole or RA.
Therefore, we cannot exclude the possibility that the influence of
45
CYP26 inhibitors, and their metabolites, or RA fundamentally altered
the translation of CYP26A1 mRNA. Nevertheless, the fact that
CYP26A1 reacted most strongly to the higher dose of CYP26 inhibitors suggests that this gene in particular has capacity to respond to
excessive RA accumulation in epidermis and thereby “protect” the
cell from cytotoxic levels of RA.
LRAT and RalDH2
In the present study we found no significant changes in the expression of
two enzymes, LRAT and RalDH2 in LI skin, which implies that the RA
levels during liarozole therapy remained below the threshold value needed to
activate any autoregulatory mechanisms in the epidermal retinol metabolism.
Pro-inflammatory cytokines
The pro-inflammatory cytokine, TNF-D, showed a significant
down regulation, whereas another cytokine, IL-1D, was more variably
affected. Although a decreased IL-1D expression occurred in most
patients on therapy, those with a poor clinical response or a high dose
level of liarozole (150 mg per day) occasionally showed increased expression of this cytokine. At the moment it is not known if this effect
is related to a mild retinoid-like dermatitis, which is sometimes observed as a side-effect in liarozole-treated patients (111, 112).
Study III
Characteristics of organotypic epidermis
A validation of the organotypic model showed typical features of
epidermis on histology and a specific KRT4 staining in stratum
granulosum after addition of RA to the culture medium, also observed in vivo (85). Other retinoid-regulated genes such as KRT2,
CRABPII and HB-EGF were also affected after exposure to RA, in the
same manner as reported after topical treatment in vivo (44, 85, 134).
Time dependent RA effects on the mRNA expression of
retinoid regulated genes and enzymes involved in vitamin
A metabolism
A time study showed that of the four retinoid-regulated genes,
CRABPII had the weakest response to RA addition. In the same study
genes encoding for enzymes involved in RA biosynthesis and catabolism, i.e. LRAT, RDH16, RalDH2, CYP26A1 and CYP26B1 were also
46
analysed. The two CYP26 enzymes had a rapid response to RA,
which is in line with CYP26 being involved in the degradation of RA
(135). The increased mRNA expression of CYP26A1, which has two
RAREs in the promoter region (87), could not be confirmed at the
protein level for unknown reasons.
Increased LRAT gene expression was a late event, suggesting a
feedback mechanism where keratinocytes react to increased cellular
levels of RA by increased retinol esterification and thus deviating
retinol from oxidation to RA (28). A feedback regulation is further
supported by the down regulation of RDH16 and RalDH2 (Fig. 13).
Figure 13. Time study - the gene expression of CYP26 enzymes (left) and the
enzymes involved in the vitamin A metabolism (right).
The effects of CYP26 inhibitors on organotypic epidermis
Due to the low levels of RA in the culture medium a preincubation with 1nM RA had to be included to build up the amount
of retinoid in the tissue before adding the inhibitors. Unexpectedly,
the expression of CRABPII and the keratin biomarkers was maximally stimulated already at this low level of RA. This is probably
because both the nuclear retinoid receptors have dissociation constants (Kd) in the nanomolar range (136) and the CRABPII gene has a
RARE (82). However it could also be because in a virtually retinoiddeficient organotypic epidermis, the response to even minor increases
in the tissue retinoid concentration is strong. Only the CYP26A1 expression was synergistically induced by a combination of RA and
talarozole, which can be interpreted as a feed-forward control (137) of
this gene mediated by its RARE sites (87). Paradoxically, RA or
CYP26 inhibitors did not increase the CYP26A1 and B1 protein expressions in organotypic epidermis, an observation that confirmed
the findings from Study I.
47
Effects of CYP26 inhibitors on the vitamin A metabolism
These effects were assessed by adding ROH and inhibitors to the
culture medium followed by addition of [3H]RA or [3H]ROH, which
showed that talarozole more effectively accumulated [3H]RA in the
tissue than liarozole. Similar results could be seen with [3H]ROH (Fig.
14). In the same series of experiments the ROH- and talarozoletreated samples exhibited the strongest KRT4 and CRABPII protein
staining.
Figure 14. Effects of RAMBAs on the cellular uptake of [3H]RA (left) and
[3H]ROH (right).
Study IV
This last study explored the importance of the individual CYP26
members and how they are regulated in human keratinocytes. This
was achieved by examining the mRNA expression of three CYP26
family members and other enzymes, which play a role in the vitamin
A homeostasis (127) during keratinocyte differentiation. Furthermore
their regulation by RA and CYP26 inhibitors was studied.
Normal mRNA expression of CYPs involved in RA
catabolism
Our results show that CYP26A1 mRNA levels are low in normal
keratinocytes, which supports previous reports on this subject (74,
138). We also found CYP26B1 to be expressed at a higher magnitude
48
than both CYP26A1 and CYP26C1. In fact CYP26A1 expression was
barely detectable and no expression of CYP26C1 at all was found. We
also examined the CYP2S1 expression, which showed much higher
mRNA level than CYP26B1 (Fig. 15). This is not the same relationship
we detected in vivo, where CYP26B1 was more expressed than
CYP2S1.
Figure 15. Gene expression of CYP26A1, B1 and CYP2S1 in cultured human
keratinocytes at resting state.
Smith et al (73) argued that CYP2S1 is mostly responsible for RA
being catabolised into 4-OH-RA and 5,6-epoxy-RA and not at all for
the oxidation into 4-oxo-RA. White et al (62) showed that CYP26 produces a broad range of hydroxylated forms of RA including 4-OH-RA
and 4-oxo-RA although the work was performed in COS-1 cells transfected with human CYP26. This suggests that perhaps the main
source of catabolism in vivo fluctuates with different epidermal conditions leading to various spectra of metabolites. During in vitro culture, on the other hand, a preference for 4-OH-RA, 5,6-epoxy-RA
driven catabolism, mainly accomplished by higher levels of CYP2S1,
may exist rather than 4-oxo-RA catabolism. These different metabolites have been shown to be more or less biologically active (58, 59);
for example 4-OH-RA and 5,6-epoxy-RA are more active than 4-oxoRA (58).
49
Effects of differentiation and RA on the expression of
enzymes involved in vitamin A metabolism
In differentiated keratinocytes the mRNA levels of RDH16 increased, corroborating a previous report by Jurukovski et al (31). The
expression of genes encoding other enzymes involved in vitamin A
metabolism e.g. LRAT and RalDH2, also increased. This was however
not the case for the CYP26B1, which was reduced, whereas CYP26A1
and CYP2S1 were unaffected by differentiation. On the other hand,
CYP26A1 and B1 responded rapidly to RA addition, both showing
the highest response in differentiated keratinocytes.
Interestingly our results in proliferating keratinocytes show a tendency of RA to early induce RDH16 levels. No corresponding induction was seen in differentiating cells. RalDH2 is marginally reduced
in both cell types after RA exposure, implying that RA also controls
its own biosynthesis. The increased LRAT mRNA levels were similar
to previous described results (28) (Fig. 16). Thus, in supra-basal keratinocytes the whole machinery for RA synthesis seems to operate
(high expression of RDH16 and RalDH2) together with degradation
mechanisms in case the RA level is too high (RA-inducible CYP26
enzymes).
Figure 16. Time study of RA effects on the gene expression of CYPs (above)
and enzymes involved in vitamin A metabolism (below), in proliferating
(left) and differentiating (right) keratinocytes.
50
Effects of CYP26 knock-down on CYP26 and CRABPII
mRNA expression
Targeting siRNA against CYP26A1 and B1 demonstrated that cellular accumulation of RA was slightly increased when inhibiting the
B1 subtype, but not the A1 subtype. The reasons why CYP26A1
siRNA had no effect could be that the mRNA levels of CYP26A1 are
low at resting state, and thus no further inhibition can be expected. It
is also possible that the CYP26A1 protein, mainly confined to keratinocytes in the basal layer (both in vitro and in vivo) (74, 133), has a
long turn-over time, leading to no changes in the amount of RA being
catabolised.
The importance of CYP26B1 in controlling the cellular RA level
was examined by studying the expression of CRABPII, a wellcharacterised biomarker of retinoid activity (49), in CYP26B1-silenced
keratinocytes. The results show increased CRABPII mRNA and protein levels. Similar effects were also detected in cells exposed to talarozole. Interestingly, the cellular localisation of CRABPII protein was
altered and a specific dotted staining pattern was observed after either CYP26B1-siRNA or talarozole treatment, but not in RA-exposed
cells (Fig.17).
Figure 17. CRABPII expression in normal human keratinocytes (far left) and
after incubation with RA, CYP26B1-siRNA and talarozole (far right). Scale
bar: 20Pm.
As mentioned above the CYP26 family will be part of catabolising
RA to a wide range of metabolites including 4-oxo-RA (73). This proposes that inhibiting the action of CYP26B1 in keratinocytes by either
siRNA technique or talarozole would increase the amount of both RA
and probably also other metabolites like 4-OH-RA and 5,6-epoxy-RA,
but greatly reduce 4-oxo-RA. Reynolds et al (58) showed in mouse
skin that CRABPII is mostly induced by RA followed, in order of potency, by 4-OH-RA, 5,6-epoxy-RA and 4-oxo-RA. Combining these
data with our results concerning CRABPII expression (Study IV) may
51
suggest that the altered CRABPII expression in talarozole and siRNA
exposed cells, not seen with RA, is a response to elevated levels of
other more active metabolites than 4-oxo-RA. To clarify this matter,
experiments should be performed where CRAPBII expression in
keratinocytes is studied by culturing the cells with RA, talarozole,
CYP26siRNA or RA together with different metabolites. Another interesting experiment to perform would be to examine the effects of
CYP26B1-silencing on the uptake and metabolism of [3H]RA, determining the amount of RA, 4-OH-RA and 4-oxo-RA at different times.
Effects of CYP26 inhibitors on RA metabolism and vitamin
A metabolising enzymes
Three different CYP26 inhibitors, ketoconazole, liarozole and talarozole, were examined by assessing [3H]RA metabolism and the
changes in mRNA of enzymes involved in vitamin A metabolism.
Once again talarozole was found to be the most potent RAMBA.
As seen in the studies mentioned above (I and III) CYP26A1 was
highly induced by talarozole. This effect was not seen in the expression for CYP26B1, which was reduced in differentiated cells. This
observation and the fact that both RDH16 and RalDH2 were induced
by talarozole together suggest that the effects of talarozole are similar, but not identical to those seen with RA.
52
General Discussion and Future
perspectives
Both talarozole and liarozole have already shown to be good candidates in the treatment of different keratinisation disorders, such as
ichthyosis and psoriasis (101) and our results both confirm these findings in lamellar ichthyosis (liarozole) and show a good tolerance at
topical treatment of normal skin (talarozole). Also by using an indirect approach, i.e. measurement of retinoid biomarkers, we can confirm the drugs’ ability to accumulate RA in human epidermis. Unfortunately direct analysis of the very low levels of RA in skin tissue is
not possible with current technologies.
RA controls its own biosynthesis from retinol via an autoregulatory enzymatic system in keratinocytes (28), which balances the endogenous RA levels and counter acts excessive RA effects. In vivo
(Study I) the higher dose of talarozole caused a minor down regulation in the gene expression of one of the enzymes involved in RA synthesis, but the retinol esterification was not altered. However, in vitro
(Study III and IV) we found induction of retinol storage capacity and
of other enzymes involved in RA synthesis in keratinocytes exposed
to talarozole, which was not seen after RA exposure. The effects could
be concluded as being talarozole derived instead of being a purely
RA-derived response. Therefore future studies of long-term treatment
should include analysis of a broader spectrum of enzymes involved
in RA biosynthesis.
Adding to this understanding, results from our two in vivo studies
(I and II), showed that both CYP26 inhibitors reduced the expression
of certain pro-inflammatory mediators (IL-1D and TNFD) in both
normal and ichthyotic epidermis. These findings are interesting because patients undergoing retinoid treatment often report skin irritation as side-effect, which could be circumvented by RAMBA treatment. Therefore larger studies of RAMBAs in patients with inflammatory skin conditions, i.e. psoriasis, where their effects are compared with those of retinoids, need to be performed to verify our
findings.
53
One of the questions considered important to address in this project was that of CYP26 and CYP2S1 expression in epidermis. Our results show that CYP26A1, B1 and CYP2S1 are expressed both in vivo
and in vitro in epidermis and that CYP26B1 plays an important role
in RA catabolism in keratinocytes under physiological conditions. For
many of these RA-degrading enzymes the expression was altered
during keratinocyte differentiation (Study IV). This suggests that they
have different roles during keratinisation aimed at keeping RA levels
at steady state.
From our results we also conclude that the differences between the
effects of RA and CYP26 inhibitors are not restricted to proinflammatory and RA metabolism auto-regulatory mechanisms, but
also involve expression of retinoid-regulated biomarkers, e.g. CYP26
and CYP2S1. This difference was most dramatically seen in the protein expression of CRABPII, where talarozole completely altered its
localisation and pattern compared to RA treated cells (Study IV). Unexpectedly, the same pattern was seen in CYP26B1 knock-down cells.
This suggests that i) talarozole is specific to CYP26B1, ii) the outcome
of talarozole and CYP26B1 knock-down on RA catabolism is similar,
iii) talarozole and CYP26 knock-down affect the regulation of
CRABPPII protein in a similar manner.
All these findings point to the fact that talarozole and liarozole
have potential as novel drugs. Therefore it is of interest to understand
more about their mechanism of action and effects to be able to understand their full potential.
It is also worth mentioning that for many patients suffering from
ichthyosis it is important to find a treatment that allows them to live
more normal and functional lives. It was therefore exciting to see the
positive clinical response to the liarozole study (II), where many of
the patients seemed content and happy with the results and wanted
to continue with the treatment.
54
Conclusion
In this project both talarozole and liarozole have been shown to
have the ability to increase the RA levels in keratinocytes and elicit
effects on retinoid-regulated biomarkers, e.g. CRABPII and KRT4 that
are similar but not identical to those obtained with RA. For instance,
the intracellular distribution of increased CRABPII protein expression
differs between RA-treated and talarozole-treated keratinocytes. The
RAMBAs also alter the expression of CYP26 enzymes (their main
target) and other vitamin A metabolising enzymes in a way that is
both similar to RA, e.g. induction of CYP26 mRNA, and different, e.g.
conditions under which CYP26 induction occurs compared to RA.
Our overall results demonstrate that talarozole and liarozole are
promising drugs that might in the near future complement retinoid
therapy in different skin disorders. However, they should also be
considered as substances that have their own effects unrelated to RA,
which may yield unpredicted clinical and biological effects.
55
Acknowledgement
Now that “the long and winding road that leads to…” my dissertation
is almost over I would like to express my sincere gratitude to all of
you (you know who you are!) who made my journey easier and contributed to my scientific and personal advancements. I would especially like to express my gratitude to the following:
Anders Vahquist, my supervisor for introducing me to the field of
dermatology, especially genodermatoses. I’m immensely thankful
that you gave me the opportunity to be part of clinical research. To
meet patients and volunteers has made my sometimes hectic lab
work worth a million times more. It is fulfilling to know that I am
doing something worth the effort.
Hans Törmä, my co-supervisor for being a great support and
teacher in the daily lab work and having a never-ending amount of
patience. Also for always having time for me and my thoughts about
the project.
Marie Virtanen my co-supervisor for the social and emotional
support along with practical advice.
All co-authors of my papers/manuscripts for contributing to my
research.
Agneta Gånemo and Christina Hymnelius for their help with the
in vivo studies.
The volunteers in the two in vivo studies for making this thesis
possible.
Inger Pihl-Lundin the best research engineer by far. Your help in
matters of research and life has been unconditional and never-ending.
You are the soul of our research group! I consider you my friend and
I hope that I have been a good roommate over the years.
56
Eva Hagforsen for her advice in the field of immunohistochemistry and help in both in vivo studies. Also for knowing when I needed
to talk and a cheer up. It has been very easy to laugh (and cry) with
you.
Hao Li, the “junior” in the lab. Your joy and positive thinking is
the best cure in any situation. Thank you for being a good co-worker
in the last part of my thesis. Your sense of humour and childfulness is
very similar to mine and I appreciate you a lot for this.
Jean-Christopher Chamcheu my “Cameroonian prince” for all scientific talks and making me believe in myself and in my ideas that
concerned the project. Thank you for making me “auntie Elizabeth”
to little Sven. I’ve always wanted to be called “auntie”.
Sofi “Soffan” Forsberg for being a good listener in issues concerning my thesis, my lab work and life.
Izabela “Iza” Buraczewska for being a good listener and friend.
To Gerd Michaëlsson our former dermatology professor for her
enthusiasm and curiosity in my project and life.
Teresa Karlsson my first roommate for introducing me to the life
of a PhD student.
The rest of the dermatology group (e.g. Mats, Håkan, Calle S.) for
good chats and laughs.
The late professor Lennart Juhlin whom I had the privilege to talk
to several times. He made everything seem simple and made me start
believing in my capabilities.
Former and present members of the SAD group (e.g. Anne, Maija,
Doreen, Tanja, Jessica, Gunnar A, Katharina and Olle) our neighbours
for making "fika" and other daily life moments joyful.
All friends and colleagues at CKMF (e.g. Tijana, Sara, Fredrik C,
Anders L, Anders N, Calle R, Majd, etc) for the nice talks and laughter.
Former and present CKMF personnel (e.g. Gunnar, Rezza, Pernilla, Gun, Siw, Lena, Eva, Susan etc) for creating a good work environment.
57
My extensive family abroad for your support over the years
The Åkesson and Sandberg families, especially Jan-Åke and Rigmor, for being supportive and understanding.
My beloved brother Diego for always believing in my capacity and
for being right to the point.
My parents have always - no matter what - expressed their love
and support for each aspect of my life. Being this the acknowledgment of my Doctor’s thesis I would like to thank them separately and
point out parts in their support that have meant so much through out
my PhD student years.
To my dear mother for teaching me the art of work discipline and effectiveness. Also for always telling me to be content with my
achievements and to see the mistakes more as a learning experience.
To my dear father for your never-ending belief in my capabilities
and for introducing me to the interesting field of science and biology.
You have taught me to question and reason. I would like to thank
you for the wonderful sketches of the skin, which are included in this
thesis.
…I love you both to the moon (stopping in a far off galaxy) and back!
Signe Hässler you have been an excellent moral support over the
years and a truly good friend.
My friend Julia Wilson who has been like a mentor to me and has
a never-ending belief in me.
My other friends who have become a part of my family (Emma,
Magnus, Stina, Mattias etc). Thank you for making me forget about
work and enjoy the little things in life.
Helena Reuterwall for her support and enthusiasm.
My upper secondary school biology teacher, Malin Fagerås for introducing me to PCR and being an exemplary teacher.
Pascal “mi tesorito chico” I could not have hoped for a more understanding and patient son. Thank you for your joy and openness.
The flower is for you as I promised. I love you!
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My “heart” and best friend Henrik. Thank you for always giving
the best advice and for helping me with everything that concerns this
thesis other than the writing itself. Thank you for sharing me with my
work. You are the love of my life.
This doctoral research was carried out at the Department of Medical Sciences, Uppsala University and was financially supported by
the Swedish Research Council, Barrier Therapeutics and Edvard Welander and Finsen Foundation.
59
Summary in Swedish
Vitamin A och linknade molekyler (retinoider) har stor inverkan
på cellers delning (proliferation) och utmognad (differentiering). I
kroppens största organ, huden påverkar naturligt förekommande
retinoider bl.a. differentieringsprocessen av de vanligaste hudcellerna
i epidermis (keratinocyter). Med anledning av detta introducerades
för ca 35 år sedan syntetiska retinoider i behandlingen av hudsjukdomar som kännetecknas av störd differentiering, tex medfödd iktyos. Denna sjukdom beror på specifika mutationer i keratinocyterna,
som förorsakar kraftig fjällbildning. Nackdelen med retinoidbehandling är dess biverkningar. Kvinnor med iktyos som behandlas med
retinoider måste undvika att bli gravida och bör fortsätta ta preventivmedel två år efter det att behandlingen avslutats.
Ett sätt att minska dessa problem är att istället för retinoider tillföra läkemedel som blockerar nedbrytningen av kroppens “eget” vitamin A och på så sätt få retinoidnivåer som är högre som förhoppningsvis ger mindre biverkningar. Detta kan uppnås genom att
blockera det enzym (CYP26) i cellerna som bryter ner (katabolism)
det hormonliknande vitamin A metabolit i cellen som heter retinsyra
(RA). I djurförsök och kliniska studier på bl.a. psoriasis och iktyos
patienter har man visat att denna form av blockering ger retinoidliknande effekter utan biverkningar. Denna doktorsavhandling har haft
som mål att studera verkningsmekanismen av sådana substanser
(ketoconazole, liarozole, talarozole) som blockerar CYP26 funktionen
i epidermis, samt att undersöka funktionen av CYP26 i epidermis.
Studierna på människa (delarbete I och II) var granskade och godkännda av etisk kommitté.
Avhandlingen är indelad i fyra delarbeten (I-IV):
I
I detta arbete studeras effekten av lokalbehandling med
talarozole på 16 friska frivilliga försökpersoner. Två olika
koncentrationer av talarozole jämfördes med kontroll. Ett
hudområde behandlades med kräm med aktiv substans
och ett annat med enbart krämbas. Hudbiopsier togs på
bägge områdena och analyserades med hjälp av mikroskopi, protein- och genuttrycksmetoder av olika retinoid biomarkörer. Efter nio dagars behandling kunde man iaktta
60
signifikanta och i vissa fall dos-beroende förändringar i
genuttrycket av keratiner (cytoskelettproteiner), CRABPII
(protein som binder RA), CYP26A1 (medlem i CYP26 familjen) och andra retinoid markörer. Effekterna var inte
förenade med den hudirritation man ser i samband med
retinoidbehandling.
II
I detta arbete studeras effekten av liarozole, i tablettform,
på iktyosepidermis . Två olika doser av liarozole jämfördes
med placebo i behandlingen av lamellär iktyos, vilket är en
svår form av iktyos, som ofta behandlas med syntetiska retinoider. Studien var en del av en större klinisk multicenterstudie, där de kliniska effekterna av liarozole stod i fokus. Elva patienter (20-65 år) slumpades i tre olika grupper, som fick tabletter motsvarade hög dos, låg dos och
placebo. Hudbiopsier togs efter fyra veckors behandling. I
princip samma biomarkörer som i föregående arbete analyserades. Resultaten visade på milda retinoid-liknande effekter hos liarozole-behandlade patienter. Effekterna varierade från individ till individ, vilket kan bero på att hudbiopsierna visade en varierande grad av förhorning och att
utgångsproverna i vissa anseenden var kraftigt avvikande
jämfört med normalt epidermis.
III
I denna in vitro studie undersöktes effekterna av liarozole
och talarozole. Effekten av substanserna studerades med
avssende på uttrycket av flera retinoid reglerade gener och
vitamin A metaboliserande enzymer i organotypiskt epidermis (bestående av keratinocyter i olika mognadsstadier)
i kombination med RA. Resultaten visar att talarozole är
mer effektiv än liarozole på att höja vitamin A och RA nivåerna i epidermis in vitro. Studien visar också att
CYP26A1 är den gen som är mest känslig för förändringar i
vitamin A nivåerna.
IV
I denna studie analyserades uttrycket av retinoidmetaboliserande enzymer i prolifererande eller differentierande keratinocyter. CYP26A1 och CYP26B1 (en annan medlem av
CYP26 familjen) bildningen förhindrades med hjälp av
siRNA (korta RNA strängar), för att undersöka deras betydelse i RA katabolismen och RA signaleringen i cellen.
Därutöver undersöktes inverkan av ketoconazole, liarozole
och talarozole på genuttrycket av retinoid-metaboliserande
61
enzymer. Studien visar att CYP26 familjen och andra CYPenzymer noggrant kontrollerar RA katabolismen i såväl
prolifererande
som
differentierande
keratinocyter.
CYP26B1 är det enzym som verkar ha störst roll i keratinocyterna under mer fysiologiska omständigheter. CYP26A1
däremot verkar ha större roll vid höga doser av RA. Liksom i arbete III var talarozole den substans som mest effektivt påverkade RA metabolismen. Den påvisade en hög
specificitet mot CYP26 och kraftigt inducerade CYP26A1.
En annan slutsats är att substanserna har olika effekter i
prolifererande och differentierande keratinocyter och att
dessa effekter skiljer sig från retinoid (RA) effekterna.
Sammanfattningsvis har de fyra studierna visat att liarozole och talarozole kan vara goda ersättare till retinoider i behandlingen av
hudsjukdomar. Vidare att deras effekter varierar i styrka och enbart
delvis är identiska med retinoidernas. Dock måste fler studier genomföras där man fokuserar mer på RA katabolismen, för att tillfullo förstå dessa substansers verkningsmekanism och effekter i huden.
62
Summary in Spanish
La vitamina A y las moléculas similares a esta (retinoides) tienen
una gran influencia en la división celular (proliferación) y desarrollo
(diferenciación). En el órgano más grande del cuerpo, la piel, van los
retinoides a tener, entre otras cosas, influencia en el proceso de diferenciación de las células más comunes de la epidermis (keratinocitos).
Es por ello que se utilizan retinoides sintéticos en el tratamiento de
enfermedades de la piel que se caracterizan por la alteración en la
diferenciación celular, por ejemplo la ictiosis. Esta enfermedad depende de mutaciones específicas en los keratinocitos. Dando, entre
otras cosas, una gran escamocidad. La parte negativa del tratamiento
con retinoides son sus secuelas. Mujeres que sufren de ictiosis y son
tratadas con retinoides deben evitar quedar embarazadas y usar medios preventivos hasta lo menos dos años después de haber terminado su tratamiento con retinoides.
Una forma de disminuir esos problemas es que a cambio de administrar retinoides al cuerpo se bloquee la degradación de la ”propia”
vitamina A y de ésta manera se obtengan niveles retinoidales más
altos que causen menos daños. Esto se puede lograr a través de bloquear la enzima (CYP26) en la célula que cataboliza el acido retinoico
(AR). Con la ayuda de ensayos con animales y estudios clínicos, entre
otros, pacientes con soriasis e ictiosis se ha podido comprobar que
ésta forma de bloqueo da los resultados esperados sin efectos colaterales.
Esta tesis ha tenido como objetivo estudiar los efectos y mecanismos de esas substancias (ketoconazole, liarozole, talarozole) que bloquean la función de CYP26 en la epidermis, al mismo tiempo que se
investigan las funciones de CYP26 en la epidermis. Los estudios hechos en personas (parte I y II) están aprobados por el comité ético.
63
La tesis está dividida en cuatro partes (I – IV)
I. En este trabajo se estudió la influencia de talarozole como
tratamiento local en una piel normal. Dos concentraciones
diferentes de talarozole se compararon con el control. Allí, a
una parte de la piel se le administró una crema con la substancia y a la otra con sólo una crema base. Las biopsias se
tomaron en ambas zonas y se hicieron análisis histológicos,
protenicos y de expresión genética de biomarcadores retinoidales. Luego de nueve dias de tratamiento se pudo observar
significativos resultados y en algunos casos, dependientes de
la dosis en la expresión genetica de los keratinos (proteinas de
la extructura celular), CRABPII (proteina que acopla AR),
CYP26A1 (miembro en la familia CYP26) y otros marcadores
retinoidales. El efecto no estaba asociado con la irritación de la
piel que se puede ver en relación con el tratamiento retinoidal.
II. En éste trabajo se estudió la implicancia de liarozole en la
epidermis de pacientes con ictios. Dos dosis diferentes de
liarozole se compararon con placebo en el tratamiento de
ictiosis laminar, la cual es una forma severa de ictiosis que a
menudo es tratada con retinoides sintéticos. El trabajo fué
parte de un estudio de multicentro-clinico donde los efectos
clinicos de liarozole era el objetivo. Once pacientes (20-65
años) se escogieron al azar y se agruparon en tres diferentes
grupos, allí se les administraron tabletas que equivalian dosis
alta, baja y placebo. Las biopsias se obtenieron despues de
cuatro semanas de tratamiento. En principio los mismos
biomarcadores que se analizaron en el trabajo anterior se
analizaron tambien en este estudio. Los resultados mostraron
moderados efectos de parecido retinoidal en aquellos que
obtuvieron liarozole, y tambien los efectos variaban entre
individuos. Esto puede ser causa de que las biopsias tenian
diferentes grados de piel callosa. Ademas, las muestras de
referencias de los pacientes fueron en algunos casos, anormal
en comparación con la epidermis normal.
III. En este item se ha estudiado los efectos de liarozole y
talarozole en la expreción de varios genes regulados por los
retinoides y enzimas metabolizadoras de vitamina A en un
organo tipo de epidermis (compuesta de keratinocitos en
diversas etapas de madurez ) en combinación con AR. Los
resultados muestran que talarozole es más eficaz que liarozole
para aumentar la vitamina A y los niveles de AR en un organo
64
tipo de epidermis in vitro. El estudio muestra tambien que
CYP26A1 es el gen más sensible a cambios de niveles de la
vitamina A.
IV. En éste item se ha estudiado como las diferentes enzimas del
metabolismo retinoico se expresaron en los cultivos de
keratinocitos que se hayaban en face de proliferacion o
diferenciacion. CYP26A1 y CYP26B1 (otro miembro de la
familia CYP26) fueron disminuidos con la ayuda del ARN de
silenciamiento (fragmentos cortos de ARN), para investigar su
importancia en el catabolismo de AR y la señalización del AR
en la celula. Ademas fué examinada la influencia de
ketaconazole, liarozole y talarozole en la expresión genetica
de enzimas del metabolismo enzimatico retinoidal. El estudio
muestra que la familia CYP26 y otras enzimas CYP parecen
controlar el catabolismo de AR debido a que ambos tienen
funciones en la proliferación y diferenciación de los
keratinocitos. CYP26B1 es el que muestra tener un rol más
activo en los keratinocitos en circumtancias mas fisilogicas.
CYP26A1 por otra parte parece tener un rol más de
emergencia. Al igual que en item III talarazole fué la
substancia más efectiva. Tambien muestra una especificación
contra CYP26 y gran inducción de CYP26A1. Además quedó
claro que las subtancias tenian diferentes funciones en la
proliferación y diferenciación de los keratinocitos los cuales
difieren del efecto retinoidal (AR).
En resumen, los cuatro estudios mostraron que liarozole y
talarozole podrían ser buenos reemplazantes para los retinoides en el
tratamiento de enfermedades a la piel. Adicionalmente que los
efectos de estas substancias varian en efectividad y son parcialmente
iguales a los retinoidos. Sin embargo, más estudios deben llevarse a
cabo donde se enfoque el catabolismo del AR, con el fin de
comprender el mecanismo de efecto de estas substancias y los efectos
en la piel.
65
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Acta Universitatis Upsaliensis
Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 387
Editor: The Dean of the Faculty of Medicine
A doctoral dissertation from the Faculty of Medicine, Uppsala
University, is usually a summary of a number of papers. A few
copies of the complete dissertation are kept at major Swedish
research libraries, while the summary alone is distributed
internationally through the series Digital Comprehensive
Summaries of Uppsala Dissertations from the Faculty of
Medicine. (Prior to January, 2005, the series was published
under the title “Comprehensive Summaries of Uppsala
Dissertations from the Faculty of Medicine”.)
Distribution: publications.uu.se
urn:nbn:se:uu:diva-9325
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2008