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DECONTRACTOR BoNT-L-peptide Effective Botox-like Therapy

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Page 1: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... BoNT-L-peptide

DECONTRACTOR BoNT-L-peptide

Effective Botox-like

Therapy

Page 2: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... BoNT-L-peptide

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

Page 3: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... BoNT-L-peptide

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

Page 4: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... BoNT-L-peptide

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

Page 5: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... BoNT-L-peptide

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

Page 6: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... BoNT-L-peptide

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)

Page 7: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... BoNT-L-peptide

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

Page 8: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... BoNT-L-peptide

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

Page 9: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... BoNT-L-peptide

BoNT-L-peptide advantages

• Cost

• Effective

•Cosmetically satisfactory

• Non-invasive treatment

• Without side effects

• Painless

Page 10: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... BoNT-L-peptide

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

Page 11: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... 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

Page 12: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... 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

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

Bo

NT-

L-P

epti

de-

2

Bo

NT-

L-P

epti

de

Co

ntr

ol

Bo

NT-

L-P

epti

de-

3

Bo

NT-

L-P

epti

de-

4

Bo

NT-

L-P

epti

de-

5

SNARE Complex

Den

sito

met

ry d

atas

Page 13: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... BoNT-L-peptide

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%

Page 14: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... BoNT-L-peptide

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

Page 15: BoNT-L-peptide - Magical Ariya · PDF fileneuromuscular disorders characterized by involuntary muscle contractions ... BoNT into the extraocular muscles, ... BoNT-L-peptide

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