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GLYCOTECHNOLOGIES
IN VETERINARY DERMATOLOGY:
A NEW ERA FOR TOPICALS
TABLE OF CONTENTS
FOREWORD __________________________________________________________________ 3
PART 1 FROM SUGARS TO GLYCOTECHNOLOGIES IN VETERINARY DERMATOLOGY __ 4
THE BODY LANGUAGE ____________________________________________________________ 5
Sugars: What are they? _______________________________________________________________________________ 5
Sugars: What are they for? _____________________________________________________________________________ 6
The many faces of sugars ___________________________________________________________________________ 6
The role of skin sugars in surface microbe-host interactions_________________________________________________ 7
The role of skin sugars in surface immunity______________________________________________________________ 9
AN INNOVATIVE TECHNOLOGY BASED ON SKIN SUGARS ___________________________________ 11
Introducing the glycotechnologies_______________________________________________________________________ 11
Glycotechnologies reduce microbial adherence ____________________________________________________________ 11
Principles of the anti-adhesive action of exogenous carbohydrates __________________________________________ 11
Proofs of the anti-adhesive action of exogenous carbohydrates: in vitro studies ________________________________ 12
Inhibition of Pseudomonas adherence to canine corneocytes ____________________________________________ 12
Inhibition of Staphylococcus adherence to canine corneocytes ___________________________________________ 15
Inhibition of Malassezia adherence to canine corneocytes ______________________________________________ 16
Glycotechnologies modulate skin inflammation ____________________________________________________________ 18
Principles of the immunomodulatory action of exogenous sugars____________________________________________ 18
Proofs of the immunomodulatory action of exogenous sugars: in vitro studies __________________________________ 19
APPLICATIONS OF GLYCOTECHNOLOGIES IN THE FIELD OF VETERINARY DERMATOLOGY ___________ 20
Benefits in dermatological therapy ______________________________________________________________________ 20
Glycotechnologies in veterinary topical products ___________________________________________________________ 21
Glycotechnologies are associated with another Virbac innovation, the non-ionic Spherulites® ________________________ 22
The value of non-ionic Spherulites® in topical therapy ____________________________________________________ 22
Spherulites®: what are they ? ____________________________________________________________________ 22
The structure of Spherulites®_____________________________________________________________________ 22
Characteristics of non-ionic Spherulites®____________________________________________________________ 23
Glycotechnologies & non-ionic Spherulites®: a synergistic combination ______________________________________ 24
PART 2 VIRBAC TOPICALS THAT FEATURE THE GLYCOTECHNOLOGIES ____________ 25
VIRBAC TOPICAL RANGE SEGMENTATION _____________________________________________ 26
ALLERMYL® SHAMPOO __________________________________________________________ 27
SEBOLYTIC® SHAMPOO __________________________________________________________ 29
PYODERM® SHAMPOO___________________________________________________________ 32
EPIOTIC ADVANCED® EAR CLEANSER ________________________________________________ 34
CONCLUSION: THE IMPORTANCE OF TOPICAL THERAPY IN DERMATOLOGY _________ 36
BIBLIOGRAPHY ______________________________________________________________ 38
FOREWORD
It has long been recognised that certain bacteria such as Pseudomonas spp. and some yeasts such as
Candida spp. exploit sugars to adhere to cell surfaces. Recent studies suggest that many more microbial
species may utilise sugars for cell adhesion.
While adherence is a highly significant part of microbial colonisation and infection it is now clear that sugar
molecules have important roles in a wide range of biological processes.
Sugars are acknowledged as central molecules in the communication business. Sugars acting as surface
receptors facilitate cells to communicate with each other as well as with their surrounding environment.
Skin infections and inflammation are two major areas that the veterinary practitioner experiences in their
clinic on a day to day basis. Sugars play key roles here. Microbial adherence to skin and the modulation
of inflammatory processes are influenced by sugars.
The knowledge gained through glycotechnology studies has opened potential therapeutic gateways. This is
both timely and apt as traditional antibiotic therapies come under threat by the increasing problem of
microbial resistance. Sugars as therapeutic agents against the common veterinary microbial skin
infections and control of dermatitis offer new solutions to some old problems.
Dr. Neil A McEwan BVM&S MVM DVM DVD Dip. ECVD MRCVS
Senior Lecturer in Veterinary Dermatology Small Animal Hospital
University of Liverpool
United Kingdom
PART 1
FROM SUGARS TO GLYCOTECHNOLOGIES
IN VETERINARY DERMATOLOGY
Sugars have long been limited to their role of energyyielding substrates. In recent years however, scientists
have begun to recognise their structural role on the
cell surface, uncovering their functional value in cellto-cell
communication
and
signalling.
These
molecules are now beginning to exert their right as
active components with beneficial effects on animal
health.
« The last decade, has witnessed the rapid emergence of the concept of the sugar
code of biological information. Mono-saccharides represent an alphabet of biological
information similar to amino acids and nucleic acids but with unsurpassed coding
capacity." Acta Anatomica 161, April 1998.
« Carbohydrate modifications of proteins and lipids are key factors in modulating
their structure and function within cells. In the extracellular milieu, they exert effects
on cellular recognition in infection and immune response. » Science 291, March
2001, Special issue on Carbohydrates and Glycobiology.
GLYCOTECHNOLOGIES PRODUCT PROFILE
THE BODY LANGUAGE
SUGARS: WHAT ARE THEY? (17, 39)
The sugars or carbohydrates are the most abundant class of organic compounds found in living
organisms. They are called carbohydrates because the carbon (carbo), hydrogen (hydr) and oxygen (ate)
they contain are usually in the proportion to form water with the general formula Cn(H2O)n.
Sugars can be divided into two major subfamilies:
•
the simple sugars also called oses or monosaccharides such as glucose, galactose,
mannose, fucose, etc. They are made of one single organic molecule.
Oxygen (O)
Carbon (C)
Hydrogen (H)
Glucose, 3D representation
D Galactose, Haworth projection
•
the complex sugars:
- oligosaccharides composed of 2-10 monosaccharide molecules.
- polysaccharides with more than 10 monosaccharide molecules. Many polysaccharides
consist of repeated disaccharide units. They include glycosoaminoglycans (GAGs).
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GLYCOTECHNOLOGIES PRODUCT PROFILE
SUGARS: WHAT ARE THEY FOR? (17, 21, 39)
The many faces of sugars
Sugars are involved in various body functions.
Sugars have long been considered only for their role as major
sources of metabolic energy (glucose) and energy storage
molecules (glycogen).
They are also known to play a structural role, notably in cartilage
(glucosamine, chondroitin sulphate), and as one of the three
essential components of DNA (desoxyribose) and RNA (ribose).
Biochemical structure of glycogen
DNA double helix
Recent
advances
have
unveiled the fundamental role of sugar molecules at the level
of the cell membrane. Here, complex sugars link with proteins and fats to form glycoproteins and glycolipids
with a very broad range of properties.
SUGAR MOIETIES
Extracellular space
Lipid bilayer
Globular protein
Intracellular space
Alpha-helix protein
Structure of the cell membrane
The cell surface sugars are the molecules most widely used in cell-to-cell recognition, interaction
and communication. They play also an important role in immune system signalling and help the
organism to fight against pathogenic micro-organisms.
So ubiquitous are these cell surface sugar molecules that cells generally appear to one another as sugar
coated. Sugar receptors are of major importance in the epidermis where they are called “skin sugars”.
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GLYCOTECHNOLOGIES PRODUCT PROFILE
The role of skin sugars in surface microbe-host interactions (5, 8, 13, 17,18, 28)
Skin infection depends on the ability of micro-organisms to adhere to host skin cells, colonise and
proliferate and produce virulence factors. They do this by means of lectins, glycoproteins which
recognise and bind to skin sugars exposed on host cell membranes. Keratinocytes in particular, the main
cells of the epidermis, do harbour skin sugars on their surface.
Microbial lectins are important virulence factors located on the surface of yeast or bacteria, either on the cell
wall or on cell membrane developments (pili). Lectins are classified according to the type of sugar they
recognise specifically, such as D-galactose, D-mannose, D-fucose, sialic acid and N-acetyl-galactosamine
(17, 28). The interaction between microbial agents and the animal host cell is multivalent. The abundance of
carbohydrates in various forms at the animal cell surface is one reason why microbes to a large extent have
selected sugar receptor for colonisation and infection.
Staphylococcal adherence on hair
(Scanning electron microscopy)
Staphylococcus
Keratinocyte
Host carbohydrate receptor
Microbial lectin
Staphylococcus adherence
on keratinocyte
(Scanning electron microscopy)
Microbial adherence on skin and hair: mediated by host carbohydrate-microbial lectin interactions
In healthy skin, an integrated array of defence mechanisms, related to the physical and chemical
properties of the integument, allow to control microbial proliferation. Continual desquamation, the
secretions of the cutaneous glands as well as the release from cells within the epidermis of peptides and
lipid metabolites play an antimicrobial role and may also inhibit microbial adherence. (17) The cutaneous
microflora further helps to prevent microbial proliferation by competitive growth and chronic stimulation of
epithelial surfaces. (18)
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GLYCOTECHNOLOGIES PRODUCT PROFILE
CHEMICAL SYSTEM:
PHYSICAL SYSTEM:
● Corneocytes (brick and cement)
● Corneocytes desquamation
Anti-microbial film
BIOLOGICAL SYSTEM:
Cutaneous microbiota
Continuous
desquamation
Differentiation
Dense horny layer
Corneocytes
Keratinocytes
Basal layer
Proliferation
Dermis
The skin barrier: a forefront defence line
Thus, although the skin is constantly exposed to potential pathogens, such as bacteria
(Staphylococcus intermedius, Pseudomonas aeruginosa) and yeast (Malassezia pachydermatis),
infection only occurs when the normal epidermal protective functions are disrupted. On the
epidermis, the adherence of pathogenic bacteria and fungi is expressed on fully differentiated keratinocytes
(corneocytes) and hence, any alteration of the skin cells differentiation process (wound, inflammation,
seborrhea, etc.) can promote microbial adherence and trigger infection. (8, 17)
A “converse way” is also described in which microorganisms themselves harbour carbohydrates on their
surface that are recognised by host cell surface lectins (17).
Microbial cell surface carbohydrates promote intercellular
adhesion between micro-organisms, producing biofilm.
Microbes aggregation increase their virulence and resistance to
antimicrobial agents. Biofilm formation is dependent on
adherence to the substrate, such as host cells or inert material
eg catheters. (5, 13, 17)
Staphylococcus biofilm
(Scanning electron microscopy)
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GLYCOTECHNOLOGIES PRODUCT PROFILE
The role of skin sugars in surface immunity (7, 10, 11, 17, 42)
The skin, and particularly its outer layer (epidermis), is the interface between the internal milieu and the
external environment, usually providing an effective protective barrier. The keratinocytes, the most
numerous cell population in the epidermis, can however become key cells for the initiation and
maintenance of inflammation in skin.
In the healthy epidermis, keratinocytes are not activated. But many exogenous factors (skin exposure to
ultraviolet radiations, infections, irritants, allergens, etc.) and/or endogenous stimuli (cytokines produced by
cells of the immune system, etc.) may lead to keratinocytes activation.
Activated keratinocytes release a wide panel of cytokines (soluble factors mediating communication
between cells), such as IL1, TNFα, Chemokines and Growth Factors. These cytokines act as “alert signals”
meaning that something is going wrong in the skin or that external aggression is ongoing. The cytokines
alert fibroblasts, endothelial cells, melanocytes, Langerhans cells and contribute to lymphocytes
recruitment. Activated keratinocytes also express cytokine receptors on their surface, allowing them to
respond to their own cytokine secretion. This is the source of a cascading inflammatory response.
Staphylococcus intermedius
EXOGENOUS ALTERING FACTORS
ƒUltraviolet radiations
ƒCutaneous wound
ƒPathogens
ƒAllergens
ƒHumidity
ƒIrritants
ƒEtc.
Malassezia pachydermatis
MICROBIAL
PROLIFERATION
Epidermal barrier
function alteration
Keratinocytes activation
CYTOKINES
PRODUCTION
ƒEtc.
ƒAllergens
ƒ Inflammatory mediators
INFLAMMATORY
RESPONSE
(IFN-‫ﻻ‬, etc.)
ƒAlteration of anti-microbial film
Inflammatory infiltrate
secretion or composition
Neutrophils
ENDOGENOUS ALTERING FACTORS
Skin barrier alteration and keratinocytes activation
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Lymphocytes
Mastocytes
GLYCOTECHNOLOGIES PRODUCT PROFILE
The biological response to cytokines involves specific cellular receptors associated with other transducing
molecules transmitting the biological signal within the cell to the nucleus. It is now recognized that cytokines
are bi-functional molecules ie they harbour two binding domains on their surface. One domain is called the
receptor-binding domain (RBD) that binds to a receptor protein on the target cell membrane. The second
domain is the carbohydrate binding domain (CBD) that binds specific carbohydrate epitopes on the target
cell surface. This CBD therefore confers to cytokines a lectin-like activity. Once the CBD has
recognised and interacted with the target carbohydrate, the immune signal is activated and the
inflammatory cascade is perpetuated. Subtle changes in cell surface carbohydrates suppresses the
cytokine activity. Several authors have already described the lectin-like activities of several cytokines
including IL1α, IL1β, IL2, TNFα and TNFβ (7).
CBD
CYTOKINE
RECOGNITION AND INTERACTION
CARBOHYDRATE EPITOPE
TRANSDUCING MOLECULES
RBD
CELL MEMBRANE
RECEPTOR
PROTEIN
IMMUNE SIGNAL
PRODUCTION
Immune signal propagation: the role of cytokines – carbohydrate receptors interactions
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GLYCOTECHNOLOGIES PRODUCT PROFILE
AN INNOVATIVE TECHNOLOGY BASED ON SKIN
SUGARS
INTRODUCING THE GLYCOTECHNOLOGIES
Since sugars exposed on skin cell membranes are involved in microbial adherence and inflammatory
reactions by the host, a promising approach in dermatology is to use similar exogenous carbohydrates
to help control cutaneous infections and inflammatory disease. Glycotechnologies is the term
applied to the exploration and exploitation of this concept.
GLYCOTECHNOLOGIES REDUCE MICROBIAL ADHERENCE
Principles of the anti-adhesive action of exogenous carbohydrates
Microbial lectins interact with the corresponding sugars located on the epidermal cell surface. Similarly, on
the skin surface, microbial lectins will bind exogenous topically administered carbohydrates acting
as “traps”.
Micro-organisms
(Staphylococcus)
EXOGENOUS
SUGAR
Bacterial lectin
EXTERNAL
ENVIRONMENT
CORNEOCYTE
SURFACE
MICROBIAL LECTINS
- HOST SUGARS INTERACTION
BLOCKAGE OF LECTINS
Host sugar
receptor
BY EXOGENOUS SUGARS
Blocking of microbial lectins by exogenous sugars on the skin surface
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GLYCOTECHNOLOGIES PRODUCT PROFILE
The exogenous sugars saturate the lectin binding sites on the microbial surface, making impossible for the
pathogens to adhere to host carbohydrates (anti-adhesive effect).
Micro-organism
(Pseudomonas)
EXTERNAL ENVIRONMENT
Bacterial lectin
Exogenous sugars
Glycoproteins
CORNEOCYTE SURFACE
(HOST SKIN SURFACE)
Saturation of microbial lectins by exogenous sugars on the skin surface
Proofs of the anti-adhesive action of exogenous carbohydrates: in vitro studies
In vitro studies (15, 33) confirmed that specific saccharides, which mimic natural epidermal sugar moieties,
can effectively inhibit microbial pathogen adhesion. These anti-adhesive properties have therapeutic
potential in the management of corresponding infections.
Inhibition of Pseudomonas adherence to canine corneocytes (25, 26, 40, 41)
Pseudomonas aeruginosa is arguably the most important pathogen involved in
canine otitis and of major concern is the emergence of widespread and multiple
antibiotic resistances.
Adherence is an established prerequisite for microbial colonisation and
Pseudomonas aeruginosa is known to adhere to a variety of epithelial surfaces
using lectins that bind target sugars (40, 41).
Pseudomonas adherence
A study
was performed at The University of Liverpool Department of Veterinary Clinical
Science to evaluate the anti-adhesive properties of 3 monosaccharides (D-galactose, D-mannose and
L-rhamnose) against 3 strains of Pseudomonas aeruginosa cultured from clinical cases of canine otitis
(6 dogs). Initially the monosaccharides were tested individually, then a combination of all 3 test sugars was
evaluated.
(25, 26)
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GLYCOTECHNOLOGIES PRODUCT PROFILE
STUDY PROTOCOL ON MONOSACCHARIDE INHIBITION OF PSEUDOMONAS ADHERENCE TO CANINE CORNEOCYTES (25, 26, 27)
6 healthy dogs tested:
Canine corneocytes
collected from inner pinna
using adhesive discs
(D-Squame®)
One of the 3 strains
of Pseudomonas:
P1 P2 P3
One of the 4 sugar solutions:
2 sugar concentrations: 0,05% and 0,1%
- D-galactose
- D-mannose
- L-rhamnose
- The 3 sugars in combination
Or sugar-free solution (control test)
INCUBATION
at 38°C for 45 minutes
in moist chamber
CORNEOCYTES
WASHED AND STAINED
(crystal violet)
QUANTIFICATION
Image analysis > calculation of
corneocyte surface area
covered by bacteria
Corneocytes incubated with Pseudomonas
(x 1000 magnification, crystal violet staining)
Image courtesy of Dr NA McEwan
Pseudomonas
stained in violet
Corneocyte
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GLYCOTECHNOLOGIES PRODUCT PROFILE
The mean percentages of Pseudomonas aeruginosa adherence to canine corneocytes after incubation with
the test sugar solutions, by reference to the level of adherence in the control group, are represented below.
Percentage of Pseudomonas adherence
46.6
3 sugars
72.6
L-rhamnose
79.2
D-galactose
80.9
D-mannose
100
Control
0
20
40
60
80
100
Inhibition of Pseudomonas adherence by monosaccharides individually or in combination
D-galactose, D-mannose and L-rhamnose each decreased Pseudomonas adherence to canine
corneocytes. Furthermore, when the three sugars were used in combination, adherence was further
reduced by more than 50%.
ADHERENCE
REDUCED
BY > 50%
Corneocytes incubated
with Pseudomonas strain 2
and the 3 monosaccharides combination.
(x 1000 magnification, crystal violet staining)
Image courtesy of Dr NA McEwan
Corneocytes incubated
with Pseudomonas strain 2 and without sugar.
(x 1000 magnification, crystal violet staining)
Image courtesy of Dr NA McEwan
In conclusion, the monosaccharides studied presented a marked anti-adhesive effect. The use of the
combination of the test sugars is anticipated to play a significant role in the management of
Pseudomonas infections in dogs.
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GLYCOTECHNOLOGIES PRODUCT PROFILE
Inhibition of Staphylococcus adherence to canine corneocytes (23, 24)
Staphylococcus intermedius are bacteria of the normal canine
skin flora but they can proliferate under favourable conditions to
cause skin infection, particularly pyoderma secondary to various
underlying dermatoses. Pyoderma is one of the most frequent skin
disease in dogs.
Adherence is an established prerequisite for microbial colonisation
and Staphylococcus intermedius is known to adhere to corneocytes
using lectins that bind target sugars. (24)
Staphylococcus intermedius on keratinocytes
A preliminary study was performed at The University of Liverpool Department of Veterinary Clinical
Science to evaluate the anti-adhesive properties of 3 monosaccharides (D-galactose, D-mannose and
L-rhamnose) against 3 strains of Staphylococcus intermedius cultured from clinical cases of canine
pyoderma. The protocol used in this study was similar to that described before for Pseudomonas. The
monosaccharides were tested individually, then all 3 test sugars in combination were evaluated.
Unfortunately the results were not those expected. The level of Staphylococcus intermedius
adherence remained unchanged after incubation with the test sugars.
Additional
more complex sugars were tested then (23). The
polysaccharides
fructooligosaccharide
(FOS)
and
alkypolyglucoside (APG) at 1% concentration were evaluated
under the same protocol.
Alkylpolyglucoside (APG)
The mean percentages of Staphylococcus intermedius adherence
to canine corneocytes after incubation with the polysaccharide solutions, by reference to the level of
adherence in the sugar-free control group, are represented below.
Percentage of Staphylococcus adherence
47.7
APG
96
FOS
100
Control
0
20
40
60
80
Inhibition of Staphylococcus intermedius adherence by polysaccharides
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100
GLYCOTECHNOLOGIES PRODUCT PROFILE
The results showed that, while the fructooligosaccharide (FOS) failed to block cocci adhesion, the
alkylpolyglucoside (APG) exhibited significant anti-adhesive properties. The APG solution was able to
reduce Staphylococcus intermedius adherence to canine corneocytes by 50%.
ADHERENCE
REDUCED
BY 50%
Corneocytes incubated
with Staphylococcus intermedius strain 2
and without sugar.
(x 1000 magnification, crystal violet staining)
Image courtesy of Dr NA McEwan
Corneocytes incubated
with Staphylococcus intermedius strain 2
and APG solution.
(x 1000 magnification, crystal violet staining)
Image courtesy of Dr NA McEwan
In conclusion, this study suggested that APG might have therapeutic potential in the treatment of
staphylococcal infections in dogs.
Inhibition of Malassezia adherence to canine corneocytes (3)
Malassezia pachydermatis are yeasts of the normal skin flora of dogs.
The factors that predispose to yeast proliferation are not completely
understood but it is involved in many dermatological problems, such as
Malassezia dermatitis and otitis in dogs, frequent complications of canine
atopic dermatitis. These represent awkward inflammatory, pruritic
conditions for affected dogs. Malassezia infections have also been
associated with the development of chronic lesional signs of atopic
dermatitis and may act as perpetuating allergens. Topical and systemic
anti-fungal medications are widely used to control Malassezia infections.
Malassezia are, however, commensal organisms, and re-colonisation and
infection from mucosal reservoirs is common. Preventing colonisation is
therefore an important goal in the long-term control of yeast complications.
Malassezia pachydermatis
Cytologic picture
(x 1000, Courtesy of Dr D Pin)
Colonisation requires adherence to the corneocytes of the superficial stratum corneum and Malassezia
pachydermatis is known to adhere to a variety of epithelial surfaces using lectins that bind target sugars. (3)
A recent study
was performed at The University of Liverpool Department of Veterinary Clinical
Science to evaluate the potential of 3 monosaccharides (D-galactose, D-mannose and L-rhamnose
each at 0.1%) and 1 polysaccharide (the alkylpolyglucoside APG at 0.5%) to inhibit the adherence of
Malassezia pachydermatis to canine corneocytes. The protocol used was similar to previous studies on
bacteria.
(45)
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GLYCOTECHNOLOGIES PRODUCT PROFILE
The mean percentages of Malassezia pachydermatis adherence to canine corneocytes after incubation
with the sugar solutions, by reference to the level of adherence in the sugar-free control group, are
represented below.
Percentage of Malassezia adherence
58.1
APG
74.6
R&M&G
79.4
L-Rhamnose
90.7
D-Mannose
100
Control
0
20
40
60
80
100
Inhibition of Malassezia pachydermatis adherence by mono and polysaccharides
(R & M & G = Rhamnose + Mannose + Galactose)
The results showed a moderate inhibitory effect of D-galactose, D-mannose and L-rhamnose taken
altogether in combination on Malassezia adherence (25% reduction vs control). The best efficacy was
achieved by the polysaccharide APG that reduced Malassezia adherence to canine corneocytes by
42%.
ADHERENCE
REDUCED
BY 42%
Corneocytes incubated
with Malassezia pachydermatis strain 3
and without sugar.
(x 1000 magnification, crystal violet staining)
Image courtesy of Dr NA McEwan
Corneocytes incubated
with Malassezia pachydermatis strain 3
and APG solution.
(x 1000 magnification, crystal violet staining)
Image courtesy of Dr NA McEwan
In conclusion, this study suggested that APG plus the 3 monosaccharides tested might have a
therapeutical interest in Malassezia infections in dogs.
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GLYCOTECHNOLOGIES PRODUCT PROFILE
GLYCOTECHNOLOGIES MODULATE SKIN INFLAMMATION (1, 7, 10, 31)
Principles of the immunomodulatory action of exogenous sugars
When keratinocytes are activated by exogenous or endogenous factors, they release cytokines such as IL1
or TNFα. These cytokines are soluble factors that initiate the inflammatory cascade. The biological
response to cytokines involves two specific cellular receptors associated with transducing molecules.
Cytokines possess both a receptor binding domain (RBD) and a carbohydrate binding domain (CBD), the
later being a lectin. The CBD recognises specific sugars and this interaction is essential for the production
of the immune signal. Exogenous sugars can interfere by competitive action on the CBDcarbohydrate interaction and consequently reduce cytokine stimulation.
BLOCKING OF CBD
BY THE EXOGENOUS SUGAR
CBD
CYTOKINE
CARBOHYDRATE EPITOPE
RBD
EXOGENOUS
SUGAR
TRANSDUCING MOLECULES
CELL MEMBRANE
RECEPTOR
PROTEIN
NO IMMUNE
SIGNAL PRODUCTION
The immunomodulatory role of exogenous sugars
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GLYCOTECHNOLOGIES PRODUCT PROFILE
Proofs of the immunomodulatory action of exogenous sugars: in vitro studies
In humans, specific monosaccharides, such as L-rhamnose, have been reported to exert inhibitory effects
on cytokine activity in vitro, and are effective in suppressing in vivo manifestations of cellular immunity (1, 31).
In dogs, keratinocytes can be activated in vitro by a cytokine produced by T cells (interferon-γ).
Such stimulation is evidenced by the release of the pro-inflammatory cytokine TNF-α by activated
keratinocytes (6).
A study (12) was conduced at the National Veterinary School of Nantes, Unit of Dermatology, Clinical
Parasitology & Micology, to evaluate the modulation of canine keratinocyte activation by Lrhamnose. The quantity of TNFα produced by activated keratinocytes incubated in 3 different solutions: a
sugar-free control solution, a rhamnose solution (10 mg/mL) and a dexamethasone solution (2 x 10-5 mol/L)
was recorded. The incubation of stimulated canine keratinocytes with L-rhamnose, or with dexamethasone,
decreased TNF-α release by 75%, and 56%, respectively.
Percentage of residual TNF-α secretion
25
Rhamnose
44
Dexamethasone
100
Control
0
20
40
60
80
100
Inhibitory effect of a sugar solution (rhamnose ) and a corticosteroid solution (dexamethasone)
on pro-inflammatory cytokine release (TNF-α) by activated canine keratinocytes
A specific monosaccharide therefore demonstrated inhibitory properties on cytokine signals
mediating inflammation in canine epidermis. The role of epidermal cell surface sugar-coating in cell-tocell signalling and communication is the basis for such effects.
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GLYCOTECHNOLOGIES PRODUCT PROFILE
APPLICATIONS OF GLYCOTECHNOLOGIES IN THE FIELD
OF VETERINARY DERMATOLOGY
BENEFITS IN DERMATOLOGICAL THERAPY
The control of both microbial proliferation and inflammation is mandatory in many skin diseases. One
classical method to manage microbial proliferation is the use of antibiotics or antiseptics, while inflammation
is often addressed by systemic (oral, injectable) glucocorticoids. The prolonged use of these drugs,
however, is not harmless. Long-term use of systemic steroid therapy may affect the way the immune
system can deal with microbial challenge and the issue of antibiotic resistances is an increasing concern.
New therapeutic options, that do not depend on antibiotics, are a welcome addition to the armoury of the
treatment of skin infections. In this context, the microbial anti-adhesive and immunomodulatory effects of
glycotechnologies represent a high tech new avenue that opens promising perspectives.
SKIN DISEASES
MICROBIAL PROLIFERATION
INFLAMMATION
USUAL CONTROL METHODS
ANTIBIOTICS, ANTISEPTICS
SYSTEMIC STEROIDS
POSSIBLE DRAWBACKS
IMMUNOSUPPRESSION
ANTIBIOTIC RESISTANCE
DISTURBANCE OF THE CUTANEOUS MICROFLORA
DETRIMENTAL MICROBIAL PROLIFERATION
PROMISING COMPLEMENTARY APPROACH
GLYCOTECHNOLOGIES
Opportunities offered by Glycotechnologies in veterinary dermatology
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GLYCOTECHNOLOGIES PRODUCT PROFILE
GLYCOTECHNOLOGIES IN VETERINARY TOPICAL PRODUCTS
Inspired by advances in human dermatology research, and after patient exploration in dogs, Virbac once
again makes the innovation by introducing the high tech skin sugars in veterinary topical therapy. This
new concept, the Glycotechnologies, is in line with the company long-term commitment to provide
veterinarians with the most effective and updated dermatological products.
The choice of the sugars newly introduced in Virbac topical products was based on the results of proof-ofconcept studies as exposed before. The sugars selected are the following:
• 3 monosaccharides:
D Mannose
D Galactose
L Rhamnose
• 1 polysaccharide:
Alkylpolyglucoside (APG)
These are precisely the sugars proved to be effective against the 3 most common pathogens found in
infectious dermatological cases: Staphylococcus intermedius, Pseudomonas aeruginosa and Malassezia
pachydermatis. Virbac has consequently filed a proprietary international patent on the veterinary
dermatological applications of these compounds.
Glycotechnologies are a ground breaking innovation in veterinary topical therapy, but their target is not to
replace existing efficient molecules. Rather, they have been developed to complement and optimise current
formulas in order to improve their efficacy.
Therefore by now, the main therapeutic Virbac shampoos and ear cleansers, include this new
technology, in addition to previous components that have made the success of the range.
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GLYCOTECHNOLOGIES PRODUCT PROFILE
GLYCOTECHNOLOGIES ARE ASSOCIATED WITH ANOTHER VIRBAC
INNOVATION, THE NON-IONIC SPHERULITES®
The value of non-ionic Spherulites® in topical therapy
Spherulites®: what are they ? (16)
Spherulites® are microvesicles made of multiple layers of surfactants.
They represent a new generation of delivery systems for the
encapsulation of active ingredients.
The Spherulites® were developed by the CNRS (Centre National de la
Recherche Scientifique, the equivalent of the NIH in France) and
veterinary dermatological applications are protected by a VIRBAC patent.
Internal structure of Spherulites®
(Scanning electron microscopy)
The structure of Spherulites® (16)
Spherulites® are composed of tensioactive molecules (surfactants). These molecules feature two opposite
poles: one hydrophilic (the head) and one lipophilic (the tail). Consequently, surfactants tend to locate at
the interface between substances with different polarities, such as oil and water.
LIPOPHILIC COMPARTMENT
HYDROPHILIC COMPARTMENT
Lipophilic tail
Lipophilic active agents
Hydrophilic head
Hydrophilic active agents
The structure of Spherulites®
Spherulites® diameter may vary between 0.2 and 20 µm, depending on the number of concentric layers,
ranging from 10 to 1000.
22/39
GLYCOTECHNOLOGIES PRODUCT PROFILE
Characteristics of non-ionic Spherulites® (2, 16)
Encapsulation of multiple active ingredients
Spherulites® are multilamellar structures allowing the encapsulation of different molecules into the
same microvesicle. The surfactant constituents allow the incorporation of hydrophilic, as well as lipophilic
ingredients. This is not the case of alternative encapsulation technologies (nanocapsules or liposomes).
Penetration in skin deeper layers
The surfactants used in non-ionic Spherulites® do not contain electronic charge so that the resulting
microvesicles do not bind to negatively charged hair and skin. Non-ionic Spherulites® therefore are able
to penetrate into deep skin structures and this favours the flow of active ingredients to their site of
action. This property was demonstrated using radioactive non-ionic Spherulites® on dog skin biopsies (2).
Non-ionic Spherulites®
(appear as small violet grains)
Distribution of 14C-labelled non-ionic Spherulites® in canine skin biopsies.
Histological section of a hair follicle and visualisation of radioactive grains
showing Spherulites® distribution.
Image courtesy of N Barthe
Penetration of non-ionic
Spherulites® in skin
Progressive release of active ingredients
Each layer of the multilamellar structure acts as a barrier limiting the diffusion of active molecules outside
the microvesicle. The breakdown of the first, or even the first few, outer layers does not lead to complete
structure destruction. Therefore, as opposed to liposomes, Spherulites® display great intrinsic stability. As
the layers slowly breakdown, progressive release of active ingredients occurs into the skin.
Consequently, their efficacy is prolonged.
23/39
GLYCOTECHNOLOGIES PRODUCT PROFILE
GLYCOTECHNOLOGIES & Non-ionic SPHERULITES®-: a synergistic combination
Some of the sugars encapsulated in Non-ionic Spherulites®
are conveyed in the skin deeper layers, where they can
exert their immunomodulatory properties.
Encapsulated
sugars
The other non-encapsulated sugars and the remaining
encapsulated sugars on the skin surface provide quick and
prolonged microbial anti-adhesive effects.
Glycotechnologies and Spherulites® therefore work
together in synergy.
Non-ionic Spherulites® encapsulating skin sugars
24/39
PART 2
VIRBAC TOPICALS THAT FEATURE THE
GLYCOTECHNOLOGIES
As an ongoing effort to improve its products, Virbac
introduces the latest innovation in topical therapy.
The “Glycotechnologies included” label is the
privilege for veterinarians of prescribing the most
advanced solutions.
VIRBAC TOPICAL RANGE SEGMENTATION
Virbac developed a large medicated topical range, organised around the 4 main therapeutical challenges
in veterinary dermatology:
•
•
•
•
Allergies: Class A
Keratoseborrheic disorders: Class K
Cutaneous Infections: Class I
Otitis: Class O
Unlicensed topicals, shampoos or ear cleansers, provide a valuable aid in the management of the above
skin disorders.
Products in each Class that benefit from the glycotechnologies innovation include:
Class
A
K
Brand name
Allermyl® shampoo
I
Sebolytic® shampoo
Pyoderm® shampoo
O
Epiotic® Advanced ear cleanser
26/39
ALLERMYL® SHAMPOO
PROPERTIES:
A
APPLICATIONS:
• Soothing
• Restructuring
Cleansing shampoo as an aid for the management of
allergic skin conditions in dogs and cats
• Antibacterial
• Antifungal
What goes wrong in the skin of atopic dogs?
ATOPIC DOG STRATUM CORNEUM
NORMAL DOG STRATUM CORNEUM
Keratinocyte
Intercellular space
(cement)
Courtesy of: T. Olivry, NC State University, USA.
Courtesy of: T. Olivry, NC State University, USA.
Electron microscopic observations of the stratum corneum intercellular lipids in normal and atopic dogs (30)
The results of this study (30) revealed structural abnormalities in intercellular lipid deposits
within the stratum corneum of atopic dogs.
These defects can explain the IMPAIRED SKIN BARRIER FUNCTION OF ATOPIC DOGS.
●
ALLERGENS
PENETRATION
Staphylococcus
intermedius
Malassezia
pachydermatis
Dermatophagoides farinae
(House dust mite)
MICROBIAL
PROLIFERATION
EPIDERMAL BARRIER
FUNCTION ALTERATION
CHRONIC INFLAMMATION AND PRURITUS
Inflammatory infiltrate
Neutrophils
Lymphocytes
27/39
Mastocytes
Allermyl® response: the triple action
TARGET ACTIONS
ACTIVE INGREDIENTS
PROPERTIES
BENEFIT
1. Reinforcement of the
cutaneous barrier
Skin lipid complex (32)
= Ceramides
+ EFAs + cholesterol
Multi-lamellar system which mimics
the structure & composition of
intercellular lipids in the stratum
corneum
Restore skin integrity
Excellent activity against
Staphylococcus intermedius
& Malassezia pachydermatis
Antimicrobial activity
High affinity for keratines
(skin and hair)
Targeted action in skin and hairs
Efficient at low concentrations
Completely safe
Microbial anti-adhesive effect
Antimicrobial activity
Natural sugars present in epidermal
cell membranes
Respect of the skin / microflora
Reduction of cytokine signalling
Immunomodulatory properties
reduction of inflammation
Piroctone olamine
(44)
2. Control of microbial
proliferation
Glycotechnologies
(11,23,26,45)
3. Reduction of skin
inflammation
An optimised formula
A formulation for optimal tolerance
ƒ
ƒ
Physiological pH.
Glycotechnologies complement the antimicrobial activity of piroctone olamine while respecting the cutaneous
ecosystem.
A formulation for optimal distribution and penetration in skin
MICRO-EMULSIONED
SHAMPOO
• Very small droplets:
high power of solubilisation & diffusion
• Oily phase:
introduction of liposoluble ingredients (fatty acids)
A formulation for more satisfaction
Allermyl® new formula improved cosmetic properties:
• Better lathering power
• Optimal viscosity
28/39
SEBOLYTIC® SHAMPOO
PROPERTIES:
K
APPLICATIONS:
• Antiseborrheic
Cleansing shampoo as an aid for the control of
kerratoseborrheic disorders in dogs and cats
• Keratolytic
• Keratomodulating
K
• Antibacterial
• Antifungal
What goes wrong in keratoseborrheic disorders (KSD)?
S h a m p o o
Staphylococcus
intermedius
HYPERSEBORRHEA
Alteration in sebum
and epidermal lipids
secretion / composition
Malassezia
pachydermatis
MICROBIAL
PROLIFERATION
KERATINIZATION DEFECTS
Abnormal
differentiation
Epidermal barrier
deficiency
Excessive scale formation
= Hyperkeratosis
INFLAMMATION AND PRURITUS
Basal cells
hyperproliferation
Inflammatory infiltrate
Neutrophils
29/39
Lymphocytes
Mastocytes
MALODOR
Sebolytic® response: a multi-site action
TARGET ACTIONS
Remove
excess
scale
ACTIVE
INGREDIENTS
Keratoplastic effects on the
intercellular cement
Exfoliant action
Decrease horny layer thickness
Mildly bacteriostatic
Help to control bacterial
proliferation
Incorporation in the intercellular
cement
Restore the epidermal barrier
‫ ﻻ‬linolenic acid
Major structural component of cell
membrane phospholipids
Maintain functional integrity of
keratinocytes and promote their
normal maturation
Zinc gluconate
Inhibits the enzyme 5α reductase
which stimulates sebaceous
secretion
Reduce sebum secretion
Salicylic acid
Linoleic acid
2. Regulate sebum production
Vitamin B6
Piroctone olamine
3. Control microbial proliferation
Glycotechnologies
4. Reduction of skin inflammation
5. Control malodor
BENEFITS
Solubilises intercellular cement
1. Correct keratinisation defects
Regulate
the keratinisation
process
PROPERTIES
Tea tree oil
Synergistic inhibition of 5α reductase Reduce sebum secretion
Large antimicrobial spectrum
(Gram + & Gram – bacteria, yeasts)
Antimicrobial activity
High affinity for keratines
High persistence on skin and
hairs
Microbial anti-adhesive effect
Antimicrobial activity
Natural sugars present in epidermal
cell membranes
Respect the skin / microflora
Reduction of cytokine signalling
Immunomodulatory properties
reduction of inflammation
Natural pleasant odor
Reduce malodor
Interact with microbe membranes
Antimicrobial activity
Reduction of histamine and
inflammatory mediators production
Reduction of skin inflammation
and itching
30/39
An optimised formula
A formulation for optimal tolerance
ƒ
ƒ
ƒ
ƒ
ƒ
Mild cleansing agents
Physiological pH.
No coal tar, therefore :
o No skin irritating, drying and staining effect
o No unpleasant shampoo odor, appearance and color
o Can be used in cats
Tea Tree Oil provides a natural pleasant herbal scent therefore no perfume was added to the shampoo, reducing
hypersensitization hazards.
Glycotechnologies complement the antimicrobial activity of piroctone olamine while respecting the cutaneous ecosystem
A formulation for deep targeted action in skin
To exert their inhibitory action on sebaceous gland secretion, topical zinc and vitamin B6 should penetrate and diffuse
into deep skin structures. For this purpose they were incorporated, together with monosaccharides, in Non-ionic
Spherulites®, that favour the flow of active ingredients to their sites of action.
Zinc gluconate
Vitamin B6
Sebaceous
glands
Hair
follicle
Non-ionic Spherulites® delivering zinc gluconate and vitamin B6 in deep cutaneous structures
31/39
I
PYODERM® SHAMPOO
PROPERTIES:
APPLICATIONS:
Cleansing shampoo as an aid for the control of
bacterial and fungal infections in dogs and cats
• Antibacterial
• Antifungal
P y o d e r m ®
S h a m p o o
What happens in skin infections?
MICROBIAL
PROLIFERATION
●
Staphylococcus
intermedius
Surface proliferation (BOG)
Folliculite
Malassezia
pachydermatis
Furunculosis
Cellulitis
CHRONIC INFLAMMATION AND PRURITUS
Inflammatory infiltrate
Neutrophils
Lymphocytes
Mastocytes
Pyoderm® response: the dual action
TARGET ACTIONS
1. Control of
proliferation
ACTIVE INGREDIENTS
PROPERTIES
Large antimicrobial spectrum
Chlorhexidine gluconate (Gram + & Gram –, yeasts)
(3%)
microbial
Adapted and efficient formulation
Glycotechnologies
2. Reduction of skin
inflammation
BENEFITS
Antimicrobial activity
Excellent tolerance
Microbial anti-adhesive effects
Antimicrobial activity
Reduction of cytokine signalling
Immunomodulatory properties
reduction of inflammation
Natural sugars present in
epidermal cell membranes
Respect the skin / microflora
32/39
An optimised formula
A formulation for optimal tolerance
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ
Mild cleansing agents.
Physiological pH.
Moisturising agents
Glycotechnologies strenghten the antimicrobial activity of chlorhexidine while respecting the cutaneous ecosystem
balance.
Chitosanide is a filming agent that has healing properties.
The quality of chlorhexidine formulation (9) provides efficiency on bacteria and yeast at 3% concentration (20), with
excellent dermal tolerance.
A formulation for deep targeted action in skin
Hydrating agents
To exert its inhibitory activity on deep microbial infections, some
chlorhexidine should penetrate and diffuse into deep skin
structures. For this purpose, chlorhexidine molecules are also
incorporated, together with monosaccharides, in Non-ionic
Spherulites® that favour the flow of active ingredients to
their sites of action.
Chlorhexidine
A formulation for long lasting action
ƒ
ƒ
Spherulites provide a progressive release of chlorhexidine and Glycotechnologies onto the skin.
Chitosanide exerts it filming action on the skin and hair surface, thanks to its positive charges.This film keeps active
ingredients in contact with target sites.
Captive
active ingredient
Chitosanide
cationic group
Skin negatively loaded
33/39
Chitosanide
Glucosamine unit
O
EPIOTIC® ADVANCED
PROPERTIES:
APPLICATIONS:
Ear cleanser as an aid for the
management of bacterial and yeast otitis
• Cleansing
• Antibacterial
• Antifungal
What happens in otitis externa?
E p i o t i c ®
N e w
G e n e r a t i o n
Accumulation of cerumen,
hairs, foreign bodies, etc.
Dermatologic disorders
(allergies, seborrhoea, parasites,
etc.)
MICROBIAL
PROLIFERATION
Cerumen, debris
Dead cells
Cocci (Staphylococcus)
Rod (Pseudomonas)
INFLAMMATION AND PRURITUS
Neutrophils
Yeast (Malassezia)
Inflammation
Lymphocytes
Mastocytes
Pus
Epiotic® Advanced response: the triple action
TARGET ACTIONS
ACTIVE INGREDIENTS
Dioctyl sodium
sulfosuccinate (DSS)
1. Emulsification and removal
of cerumen, debris, pus,
exsudates, etc.
PROPERTIES
Emulsify and disperse wax and
debris
Makes greasy dirt easily soluble
Solubilise intercellular cement
Salicylic acid
Exfoliant action
Astringent agent
Mildly bacteriostatic
2. Control of microbial
proliferation
Remove cerumen, debris, etc.
Keratoplastic effects on the
intercellular cement
Decrease horny layer thickness
Dry the ear canal surface and prevent
maceration
Help to control bacterial proliferation
Parachlorometaxylenol
Bactericidal
Antimicrobial properties
(PCMX)
Permeabilise the outer membrane of
EDTA Sodium
Potentiate PCMX activity
Gram-negative bacteria
Glycotechnologies
3. Reduction of inflammation
BENEFITS
Microbial anti-adhesive effect
Antimicrobial activity
Reduction of cytokine signalling
Immunomodulatory properties
Reduce inflammation
Natural sugars present in epidermal
cell membranes
Respect of skin / microflora
34/39
PCMX
Antiseptic
EDTA
Permeabilizer
Glycotechnologies
Anti-adhesive
Epiotic® Advanced 3-way road to antimicrobial success
An optimized formula
A formulation for optimal tolerance
ƒ
ƒ
ƒ
ƒ
ƒ
Mild cleansing agents.
Physiological pH.
Glycotechnologies strengthen the antimicrobial activity of PCMX and EDTA while respecting the cutaneous ecosystem
balance.
Non-irritant formula: in tolerance tests conducted in compliance with the most stringent legal requirements for topical
products, the Primary Cutaneous Irritation Index (CPI) of Epiotic Advanced was 0. Products are classified according
to the following CPI scale:
- 0 to 0.5: non-irritant
- 0.5 to 2: slightly irritant
- 2 to 5: irritant
- 5 to 8: very irritant
Good preservation: repeated microbial challenge of Epiotic Advanced bottles was performed according to the
procedures of the 4th European Pharmacopea (2002). All tests qualified for the best criteria (A) of antimicrobial
preservation recommended for topical formulations.
A formulation for enhanced satisfaction
Improved product perception.
Includes a patented anti-odor technology for neutralizing unpleasant smells, based on the reactivity of aldehyde
compounds with volatile molecules (neutralisation).
35/39
CONCLUSION:
THE IMPORTANCE OF TOPICAL THERAPY
IN DERMATOLOGY
Why topicals matter ?
TOPICAL THERAPY
SYSTEMIC TREATMENT
Topical therapy plays an important role in veterinary dermatology for many reasons:
• Unlike many organs, the skin is readily accessible to medications. Topicals convey active
ingredients in direct contact with the skin, without prior dilution, to produce immediate
action on target sites.
• As the outer barrier of the body, the skin surface accumulates material (allergens, dust, foreign
bodies,..), secretions and organisms that may become harmful. Topical products, such as
shampoos, have a mechanical cleansing effect that removes detrimental material (scales,
crusts, debris, exudate..) or organisms (invading pathogens) from the skin surface. (27)
• The cleansing effect of shampoos (first application) moreover enhances the ability of
active ingredients to get in direct contact with deeper structures or sites (second
application).
• The water content and moisturising agents in the shampoo have a great beneficial effect on
skin hydration, an important parameter to maintain an effective epidermal barrier.
• Topicals act locally, minimising the risk of general adverse events.
• Topicals are unique in improving skin and coat aspect, resulting in increased owner
satisfaction.
• Topicals act in synergy with systemic treatments, helping to reduce the dose or frequency of
the later, or providing a quicker or stronger response. (22)
• On the long term, topical therapy is often helpful to prevent relapses and control chronic skin
diseases.
Therefore topical use should be the MAINSTAY of ANY dermatological treatment.
It can be associated with a systemic medication to increase overall efficacy and safety.
36/39
RECOMMANDATIONS FOR THE
BEST USE OF SHAMPOOS
(27)
PROCEDURE:
1. 1st application = cleaning
2. Rinse
3. 2nd application = treatment
4. WAIT (at least for 5 minutes, better if 10 minutes)
5. Final rinse (thorough)
SHAMPOO FREQUENCY (general rule):
1. Initially 2-3 times a week for 2 weeks
2. Then reduce gradually to longest interval over
which treatment is still effective (usually 1 to 2
weeks) for maintenance
To be adjusted according to the severity of the skin
problem, response to treatment & owner's possibilities
(compliance)
37/39
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