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Dr Philippe DeprezPoliclínica Estética & anti aging
National academy of Sciences BucurestiHonour Member
Port Grec, 40, pb17487 Empuriabrava - España
Tel +34 972 45 01 51 Fax: + 34 972 45 23 23http://www.estetik.com/centres/cmeempur.htm
BoNtA 568BoNtA 568Let’s relax Let’s relax
not paralysenot paralysemusclesmuscles
What means BoNtA 568 ?What means BoNtA 568 ?
BoNtA is the acronym of Botulinic Neurotoxin Alpha
568 represent the number of Amino acids in the BoNtA-like oligopeptides contained in the product
5- pentapeptide6- hexapeptide8- octopeptide
Aims of the treatmentAims of the treatment
Botulinic toxin and BoNtA 568 have the same target:
Interfere with Acetylcholine (Ach) synthesis and release in
order to control muscle contraction
BoNtA 568 www.aestheticdermal.com
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Is BoNtA 568 similar to
• Botulinic toxin injection ?
• Other “Botox-like” products of the market ?
BoNtA 568 www.aestheticdermal.com
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Is BoNtA 568 identical to Is BoNtA 568 identical to
a Botulinic toxin ?a Botulinic toxin ?
NOBotulinic toxin
• Destroys the axone that grows back in 3 to 4 months• Totally blocks Ach Release in the synaptic clefts• Induces a total flaccid palsy of treated area
BoNtA 568 • Does not destroy axone
• Slows down Ach release in synaptic cleft
• Induces a transitory muscle relaxation
BoNtA 568 www.aestheticdermal.com
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Is BoNtA 568 similar to Is BoNtA 568 similar to
other “botox like” products of the market ?other “botox like” products of the market ?
NOUsual Botox-like products
• Use only 1 oligopeptide (usually hexapeptide)• In a low concentration ( 5 or 6% usually)
BoNtA 568 • Uses 3 synergetic oligopeptides• 3 x 20 % concentration in Medical Care• 3 x 10 % concentration in Daily Care
BoNtA 568 www.aestheticdermal.com
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BoNtA 568
• Does not paralyse muscles like Botulinic toxin…
• Uses a synergetic combination of 3 “botox like oligopeptides” in a much more concentrated way comparing to any other product of the market.
BoNtA 568 www.aestheticdermal.com
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BoNtA 568 presentationsBoNtA 568 presentations
MEDICAL CARE Phials
• To be injected (CE pending)• No Needle mesotherapy use
DAILY CARE gel
• 2 times / day during the first 2 weeks• Once daily as daily care
Efficacy
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D 0 D 28 topical pentapeptidecontour eyes
D 0 D 28 topical SNAP 6contour eyes
In Vivo silicone imprints ( around the eyes ) after topical application
Blue = deep
Red = High
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D O D 28 topical Acétyl-hexa + Penta peptides
Contour eyes
D O D 28 topical Snap 8 contour eyes
Blue = deep
Red = High
Efficacy has been proven Efficacy has been proven clinically and experimantallyclinically and experimantally
1. Skin wrinkles silicone imprints2. Studies on glutamate neurotransmitter release in
vivo3. Electrophoreses of SNARE complex and SNAP
25 protein4. Studies on catecholamine exocytosis with and
without oligopeptides BoNtA like5. Studies on action of pentapeptide on external
axonal enkephaline-like receptors6. Monitoring of Snare complex formation
Evidences 2 to 6 need more extensed explanation
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There is no toxicity-cytotoxicity: human keratinocytes and human fibroblasts : NO
-Ames test (geno toxicity) . NO
-Eyes irritation text /HET-CAM test (Hen’s Egg Test-Chorio Allantioc Membrane) and NRU: (Mouse Fibroblast Neural Red Uptake TEst): NO EYES IRRITATION
-LD50 rats: > 2.500 mg/kg = strictly NO TOXICITY
-Occlusive patch tests on human skin : NOT IRRITATIVE
-Intraepidermal abrasion + occlusive application: NOT IRRITATIVE (0 / max score 8)
-HRIPT (Human Repeated Insult Patch Test)
-Hemolysis test : NOT HEMOLYTIC
-Subcutaneous injection (SNAP6) rabbits: Extremely low irritating power ( 0.55 on a 0 to 8 score)
-Intraperitoneal injection (snap 6): 1-100 mg/kg: NO TOXICITY
3 ways to use BoNtA 568
Classical Mesotherapy(CE as medical device pending)
“Botox” prolongator
No needle mesotherapy
BoNtA 568 Injections as a skin relaxing anti wrinkles treatment
INJECTIONS Inject more superficially compared to BtxDo 2 times more injection points
NOTESMeso botox is less efficient than classical botox, except if similar doses are usedBoNtA-like 5-6-8 will have the same level of efficacy than low doses meso botoxBoNtA-like 5-6-8 are absolutely not toxic
CE for injection pending
NO PALSY will occur, the skin will be relaxed, no risk of blepharochalasis, or eyebrows ptosisNo risk of headache or other side effect linked to botulinic toxin injection
BoNtA 568 daily careas Botulic toxin prolongator
Botulinic toxin is injected as usual for blocking completely the muscles
BoNtA 568 daily care is applied every day to prolong the result (30% maximum)
BoNtA 568 www.aestheticdermal.com
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Transcutaneous penetration of BoNtA 568
• Botulinic Neurotoxin A– Skin is virtually impermeable to this macromolecule – BoNtA is sensitive to oxidation and would not survive long on the surface of the skin
• BoNtA-like 5-6-8 – Low sensibility to oxidation: survive on the surface of the skin– Low molecular weight: simple transcutaneous penetration has been proven (Franz Cell)
Franz Cell : penetration of hexapeptide
through human skin
After 2H: 36% of chamber 1 passed in chamber 2After 4H: 48%After 6H: 78%After 24H: 90%
2-3 mm thick human skin
Chamber 1
Chamber 2Ch 1Human Skin
Ch 2
Trans epidermic penetration
AD ROLL or Superficial abrasion dramatically increase skin permeability
Products penetrate into the dermis …. Is it true ?
Evidences:
-Application of adrenalinated lidocaine after brasion
-Or after AD roll
-Induce
-Anaesthesy: Nerves termination are located inside of deeper than the epidermal basal layer
-Vasoconstriction : Blood vessels are located inside of papillary and reticulary dermis.
In the same time, vasoconstriction slows down vascular resorption
BoNtA 568 No Needle mesotherapy a skin relaxing anti wrinkles treatment
3 steps no needle meso technique
1- skin permeabilization
Skin abrasion ( AD abrasive kit)
Or ROLLER ( AD ROLL)
2- Product application
Apply the product on the skin
Cover using a kling film ( Avoids Evaporation)
Let the active products penetrate into the dermis
3- Induce an Intracellular penetration for avoiding vascular fast resorption
EXCELLDERM
In short, about no needle meso
1- disinfect the skin
2- permeabilize the skin
AD roll (Aesthetic Dermal)
Or
ABRASION (Aesthetic Dermal)
3- apply the active products on the skin
BoNtA 568 as unique product
Or
More complex mixture ( see later)
4- cover with kling film …. Intradermal penetration
5- intracellular penetration: Excellderm …. Action every day
6- repeat 2 times month ( 4 sessions total) and use BoNtA daily care
How oligopeptides works
The detailed science under the cosmetic
product
BoNtA 568 www.aestheticdermal.com
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1- Action potential
3- SNARE complex formation
4- attraction of Ach vesicle to axone membrane
5- Membranes fusion
2- depolarisation
6- liberation of ACh in synaptic cleft
7- activation of muscle receptors
8- Muscle contractions=>wrinkles
BoNtA & BoNtA 568BoNtA & BoNtA 568act on membranes act on membranes fusion phenomenonsfusion phenomenons
axone
AChVesicle
Synaptic Cleft
Muscle
Simplified MechanismSimplified MechanismofofMuscle contractionMuscle contraction
BoNtA 568 www.aestheticdermal.com
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Vesicle containingNeurotransmitterPhospholipids bilayer
V-SNARE = VAMP protein = Vesicle Associated Membrane Protein
Neurotransmitter
Axon Synaptic membranePhospholipids bilayer
T-SNARE=Target SNARESyntaxin
SNAP-25SyNaptosomal Associated Protein
Actors playing a role in membrane fusion
Axone POROSOME
Goal is: neurotransmitter has to pass through porosomeThis can be done thanks to membrane fusion mechanism
2x4 proteins are needed for membrane fusion
Axone
Muscle
BoNtA 568 www.aestheticdermal.com
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Vesicle containingNeurotransmitterPhospholipids bilayer
V-SNARE = VAMP protein = Vesicle Associated Membrane Protein
Neurotransmitter
Synaptic membranePhospholipids bilayer
T-SNARE=Target SNARESyntaxin
SNAP-25SyNaptosomal Associated Protein
Nerve Action potential induces combination of proteins
4 proteins will combine in a
TORSION HELIX
Building the SNARE complex
SNARE = SNAp REceptoror
Soluble N-ethylaleimide-sensitive fusion factor Attachment proteins
REceptors
(vue “d’artiste”)
Axone
Muscle
BoNtA 568 www.aestheticdermal.com
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Nerve Action potential: combination of proteins
And calcium flux into the axon.
In response to a motor action potential, the 3 proteins (VAMP- Syntaxin and SNAP 25) combine and form a four helix protein complex called SNARE
This combination captures the neurotransmitter-filled vesicle, attract it and dock them very closevery close to the synaptic membrane
But there is still water molecules [Ca(H20)n]2+ between the 2 bilayers and fusion is not possible as
it
This is SNARE complex
BoNtA 568 www.aestheticdermal.com
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Thanks to proteinic helix torsion forces, vesicle membrane and axone bilayer membranes are brought to within 2-3 Angtröms of each other.
This allows calcium – phosphate bridges between the 2 (-) surfaces Vesicle-axonal plasmatic membrane that usually repel each other
[Ca(H20)n]2+ is more than 6 Angströms => is physically explused
= water
= Ca-P bridge
BoNtA 568 www.aestheticdermal.com
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= water
= Ca-P bridge
Ca-P bridges simply expel the water still separing vesicle membrane and axon plasmatic membraneThe little space left now between the 2 bilayers also displaces water bound to the P group of phospholipids => membrane destabilization and fusion
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Synaptic cleft
Muscle membrane receptor Ach sensitive
Opening cation channel => muscle contraction
Membranes fusion
How can BoNtA and BoNtA-like oligopeptides
Interfere with this mechanism ?
BoNtA 568 www.aestheticdermal.com
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All serotypes of Botulic Neutotoxins type A (BoNtA) are known polypeptides
2 PHASES OF ACTIVATION ARE NEEDED FOR A NEUROTOXICITY
BoNtA is a folded polypeptide chain, linked by a disulfure bond.
Heavy chain and light chain
As it: Not toxic
Neurotoxicity needs 2 structural modifications
1st = cleavage between 2 amino acids448-449 ?418-419?422-423?
BoNtA 568 www.aestheticdermal.com
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V-SNARE = VAMP protein = Vesicle Associated Membrane Protein
2nd phase of BoNtA activation
This cleavage allows VERY RAPID entry of BoNtA into axon (endocytosis)
Into the axon: 2nd modification: Disulfure bond is cut
BoNtA 568 www.aestheticdermal.com
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V-SNARE = VAMP protein = Vesicle Associated Membrane Protein
BoNtA lyses SNAP 25 protein
Heavy and light chains are liberated
LIGHT CHAIN ISA METALLO PROTEASE
That lyses SNARE PROTEINS
BoNtA 568 www.aestheticdermal.com
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SNARE is SNARE is impossibleimpossible
MEMBRANE MEMBRANE FUSION is FUSION is impossibleimpossible
Ach release is Ach release is impossibleimpossible
Muscle Muscle contraction does contraction does not occurnot occur
BoNtA lyses SNAP 25 protein
Botulinic Toxins Light chain, a Zn dependant
endoprotease
lyse VAMP i.e.
BoNtA lyses SNAP 25
And “kills”the axone
X
BoNtA 568 www.aestheticdermal.com
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BoNtA destroys axon
• BoNtA destroys axon
• Axon regenerates by “sprouting”Sprouting begins within the first few days after BoNtA has been injected
• It takes 10 – 20 weeks for the muscle mobility to be completely regained thanks to the formation of new synapses
• BoNtA-like oligopeptides 5-6-8 have other modes of action on SMARE complex formation and do not destroy the synapse.
Oligopeptides BoNtA like
Will mimick this action mode
BoNtA 568 www.aestheticdermal.com
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What are BoNtA - like oligopeptides ?
The actual trend to use transdermal therapies without injections has led to the creation of new products derived from BoNtA: oligopeptides BoNtA like
They do not block the muscle completely as do BoNtA but, acting at the level of SNARE complex, they MODULATE muscle contraction, giving a relaxation effect
What types of peptides are used and how do they work ?
BoNtA 568 www.aestheticdermal.com
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Types of peptides
“Pentapeptide 3” (5 amino acids)
acts as an inhibitory enkephalin
“Acetyl Hexapeptide 3” (6 amino acids)
competitor for SNAP 25 protein
“Octopeptide or SNAP-8” (8 amino acids)
competitor for SNAP 25 protein
=> The name “oligopeptides BoNtA Like 5-6-8)
BoNtA 568 www.aestheticdermal.com
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Pentapeptide 3 ( 5 amino acids)
Pentapeptide 3 is a modified enkephalin that couples to enkephalin receptors on the outer membrane of axons, that are coupled to a Gi protein (inhibitory G protein).
Stimulation of Gi receptors => Ca channel are closed and K channels are opened. Membrane fusion is a process strictly Ca dependent => less ACh exocytose. => REDUCTION OF NEURON EXCITABILITY
Pentapeptide
GI
BoNtA 568 www.aestheticdermal.com
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Acetyl – Hexapeptide 3 and Octopeptide (SNAP8)
Acetyl Hexapeptide 3 (SNAP 6) is similar to the last
6 amino acids of N-terminal of SNAP 25 protein
Octopeptide (SNAP-8) is an elongation of acetyl-
hexapeptide 3
Competition in SNARE complex formation
SNARE complex destabilized by SNAP-8 or SNAP 6 reemplacing SNAP 25
Less exocytosisMuscle relaxation
Axone POROSOME
SNARESNAREcannot be stablecannot be stablewith such a short with such a short polypeptidepolypeptide
SNAP 6 or 8
BoNtA 568 www.aestheticdermal.com
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Efficacy of BoNtA like oligopeptidesGlutamate release monitoring
Glutamate is the most common brain neurotransmitter and its essay is used to estimate that of ACH (membrane fusion phenomenons are constant)
SynergyHEXA + PENTA
BoNtA 568 www.aestheticdermal.com
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Efficacy of BoNtA like oligopeptides Neuronal exocytosis and catecholamines release
Studies carried at University Miguel Hernandez, Alicante , SpainL glutamate release after depolarization of cultured neurons: Hippocampus cells of rats embryos
Acetyl Hexapeptide-3 (SNAP6) and SNAP 8
Are more effective than Pentapeptide 3
But
Pentapeptide is synergic with SNAP 6 and SNAP 8
Exocytosis of catecholamines from chromaffines cells monotor release of catecholamins (adrenalin – noradrenalin)
SNAP 8 is also more effective than Snap 6
BoNtA 568 www.aestheticdermal.com
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Efficacy of BoNtA like oligopeptidesSNARE complex formation monitoring
SNARE complex cannot be formed correctly in presence of SNAP 6 or SNAP 8 (proteins electrophoresis)
SNARE complex formation in vitro
BoNtA 568 www.aestheticdermal.com
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Efficacy of BoNtA like oligopeptidesSNARE complex formation monitoring
SNAP 8 (octopeptide) is more effective than SNAP6 (Acetyl Hexapeptide 3) in SNARE
formation inhibition in vitro
BoNtA 568 www.aestheticdermal.com
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Policlínica Estética & anti aging
Dr Philippe DeprezDr Philippe Deprez
National Academy of Sciences - BucurestiNational Academy of Sciences - Bucuresti Honour memberHonour member
Port Grec, 40, pb17487 Empuriabrava - España
Tel +34 972 45 01 51 Fax: + 34 972 45 23 23http://www.estetik.com/centres/cmeempur.htm
Thank youThank you
ForFor
Your attentionYour attention
Ed dunitz – Ed dunitz – EnglishEnglish version version (450pp)(450pp) available february 2007available february 2007KoreanKorean version version (600pp):(600pp):
available septembre 2006available septembre 2006
See on See on www.estetik.com
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