05-BoNtA 568

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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

[email protected]

BoNtA 568BoNtA 568Let’s relax Let’s relax

not paralysenot paralysemusclesmuscles

http://www.estetik.com/centres/cmeempur.htm

mailto:[email protected]

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 ?


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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


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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


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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.


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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)


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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


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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


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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


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Vesicle containingNeurotransmitterPhospholipids bilayer


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


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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


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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


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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 ?


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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?


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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


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BoNtA lyses SNAP 25 protein

Heavy and light chains are liberated

LIGHT CHAIN ISA METALLO PROTEASE

That lyses SNARE PROTEINS


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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


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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


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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 ?


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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)


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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


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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


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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


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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


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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


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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


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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

[email protected]

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

http://www.estetik.com/centres/cmeempur.htm

mailto:[email protected]

http://www.estetik.com/

05-BoNtA 568

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