bont-l-peptide - magical ariya · pdf fileneuromuscular disorders characterized by involuntary...
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DECONTRACTOR BoNT-L-peptide
Effective Botox-like
Therapy
Wrinkles Why ?
WRINKLES
Natural Factors
Chronologic aging Enviroment
Secondary Factors
Mimic Facial
Wrinkles Gravity
Pressure on
the skin
Contraction of
muscles due to
facial
expressions
e.g. during sleep The constant
pull of gravity
Clostridia a Rich Source of Toxins
Definitions :
Poison: any substance that is harmful or lethal to a
biological (living) system
Toxin: poison derived from a biological source
Clostridia: among the eubacteria, clostridia
produces more protein toxins than any other bacterial
genus
Botulinum NT (BoNT) :
Is the most poisonous biological substance known
The extraordinary potency of BoNT derives from :
its remarkable neurospecificity
its catalytic cleavage at exceedingly low
concentration
Backbone trace of the BoNA/A
molecule
Neurospecificity
BoNT Therapeutic agent
Applications:
Used for treatment of a myriad of human
neuromuscular disorders characterized by involuntary
muscle contractions
FDA approval in 1989 for 3 disorders:
Strabismus
Blepharospasm
Hemifacial spasm
New treatments :
Dystonias
Spasticity
Tremors
Migraine
Cosmetic applications
Representative treatment of strabismus (cross eyed) by injection of
BoNT into the extraocular muscles, weakening them and allowing
compensatory alignment by the agonist muscle. Top, before
treatment; bottom, after injection
Problems:
Formation of antibodies and obliteration of response
to type-A toxin
Diffusion of BoNT to neighboring muscles with
transient and sometimes debilitating ptosis
Need for repeated injection of toxin in chronic
disorders
Lack of alternate botulinum serotypes with the
potency and active duration of type A
Certain toxin preparations lack consistency and
show low specific activities
BoNT Problems with therapy
(a) Ptosis of the left upper lid
(b) Spock´s sign
BoNT-L-peptide Areas to be treated
Glabellar frown lines
Periorbital lateral
line folds
(crow´s feet) Chin and nasal
folds
(bunny lines)
Upper lip wrinkles
Platysmal folds
(turkey neck) Décolleté folds
Horizontal forehead
area
(thinker´s wrinkles)
The SNARE proteins are :
Synaptobrevin/VAMP
SNAP 25 (Synaptosomal Associated Protein)
Syntaxin (STNX)
They form a complex with the ATPase NSF
The SNARE complex is involved in trafficking the
synaptic vesicle containing neurotransmitters to the
presynaptic membrane
Fusion and exocytosis of the neurotransmitters
occurs
SNARE complex mechanism of action
BoNT-L-peptide mechanism of action
BoNT-L-peptide is split off in the cell
BoNT-L-peptide inactivates SNAP-25 from the
SNARE complex
Prevents the fusion of the acetylcholine filled vesicle
with the plasma membrane
The consequence is prevention of the release of
acetylcholine into the synaptic cleft
VAMP VAMP
STNX
SNAP-25
VAMP VAMP
STNX
BoTN-L-peptide BoNT-L-peptide
MUSCLE CONTRACTED
SNARE complex activated
MUSCLE RELAXED
SNARE complex inactivated
BoNT-L-peptide advantages
• Cost
• Effective
•Cosmetically satisfactory
• Non-invasive treatment
• Without side effects
• Painless
efficacy test
In vitro test :
Inhibition of the SNARE complex formation
In vivo test :
Macro-relief of human skin (silicon replica)
Evaluation of the photographs of the
wrinkles
BoNT-L-peptide
BoNT-L-Peptide Inhibition of the formation of SNARE complex
• Objective: To assess the capability of BoNT-L-Peptide to
inhibit the SNARE complex formation.
• Methodology:
• Incubation of SNARE proteins’ soluble fragments
(primary sintaxin, SNAP-25 and sinaptobrevin/VAMP-2).
Visualization through electrophoresis in a poliacrylamide
gel (SDS-PAGE).
•Addition of labelled SNAP-25 with 35Met in order to
detect and quantify the quantity of complex
• BoNT-L-Peptide is added to the mixture
•Gels are fixed, dried and exposed to the
Phospholggamer (Biorad) screen for time periods
comprised between 3 -12hrs.
•The images are scanned and the bands are quantified
through Quantity-One software from Bio-Rad.
CONTROL BoNT-L-Peptide
BoNT-L-Peptide Inhibition of the formation of SNARE complex
• Results :
• BoNT-L-Peptide inhibits 30% of the formation of
SNARE complex
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SNARE Complex
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BoNT-L-Peptide Macro-relief of human skin
N=15
Ra RMS
-37,15 -39,23
• Objective : evaluate the capability of BoNT-L-Peptide at
5% concentration to reduce wrinkles
• Methodology :
• Assessing the macro-relief of silicon replica from
15 volunteers obtained from the area surrounding
the eyes.
• 4 weeks treatment, analysis of samples at time 0,
before the beginning of treatment and at 4 weeks
(28 days of application)
• The rugosity average has been assesed through
confocal profilometry with a Profilemeter Pl and
stereoscopic microscopy Optech ST3
• Results :
• Reduction of wrinkles of 38%
BoNT-L-Peptide photographs of the wrinkles
• Objective : evaluate the capability of BoNT-L-Peptide at
5% to reduce wrinkles
• Methodology :
The evaluation of the wrinkles is based on
photographs taken by qualified, trained judges. The
evaluation criteria that the experts must take into
account is the depth of the wrinkles.
The visual evaluation of these descriptive
parameters and the response to the application of
anti-wrinkle products by the skin itself implies a
close follow-up training of the evaluation personnel.
The judges are regularly trained using a portfolio of 50
photographs of crow´s feet. The variability of a
measurement method is the criteria which resumes the
narrowness of the agreement between a series of
measurements carried out on the subject. The repeatability
expresses variability under the same conditions. The
accuracy expresses the distribution of the difference
between notation of the subject and notation of the
reference.
T0
T28
BoNT-L-Peptide dosage and presentations
Presentations
Cod. AA0001 BoNT-L-Peptide Powder 3-5% of 1 g of AA0001 in 1 L of water water
Cod. AA0002 BoNT-L-Peptide Solution Aqueous solution 3-5%
Dosage