nanoemulsions pour la voie oculaire - collège de france · 2010. 8. 19. · • short duration of...
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Nanoemulsions pour la voie oculaire
Simon Benita. The Institute for Drug Research,
The School of Pharmacy, The Faculty of Medicine,
The Hebrew university of Jerusalem,
Dry eye syndrome Uveitis AMD Glaucoma
Four major ocular diseases
Most pharmaceutical and therapeutic efforts are concentrated on improved treatment of
Although bacterial and viral diseases probably are the most common eye diseases
Corneal & Conjunctival diseases Dry Eye, Allergy
Anterior chamber diseases Uveitis, Glaucoma
Posterior Segment diseases Retinopathies, Uveitis
Anterior Segment Posterior SegmentEye Surface
The eye is a very sensitive and critical organ
The Eye Physiology• Turnover rate of tear film: 1µl/min
• The cornea is fairly impermeable to both hydrophilic and lipophilic drugs because of its dual composition and small surface.
• The conjunctiva is 17 times larger in surface area and 2-30 times more permeable than the cornea.
• Oily drops• Lotions• Ointments• Gels• Ocular inserts
hydrophilic drugs lipophilic drugs
Dosage forms for ocular topical application
• Collyres based on aqueous solutions
> 50% of molecules are lipophilic
The low ocular bioavailability (1-3%) of topically instilled drugs is well documented and has
been extensively reviewed
The conventional ophthalmic dosage forms, eye drops, have two major disadvantages:
• Poor drug penetration • Short duration of action
Prausnitz and Noonan, 1998Vandamme, 2002
The problem
Maximizing corneal drug absorption
• Increased retention time:
increasing the viscosity of the vehicle high spreading factor
surface charge (positive)
• Decreased particle size
• Increased cell membrane permeability & loose tight junctions by enhancers:
surfactants, bile salts, chelators, organic compounds.
Ophthalmology Market• Growing market• Numerous unmet needs• Increasing number of New molecules
But …
• Current formulation limitations:
Poor Tolerability Low Bioavailability Many hurdles for lipophilic drugs Inability to reach the back of the eye
to treat retinal diseases
Packed under nitrogen atmosphere in siliconized glass bottles
Sterilization by autoclaving at 121°C for 15 min.
Emulsion Product Sample
We have been among the fi r s t s t o p r o p o s e nanoemulsions for improved ocular drug delivery
Anionic or cationic emulsions
• Can be effective topical ophthalmic delivery systems for lipophilic drugs
• Lipophilic drug-loaded o/w ocular emulsions provide a better balance between ocular bioavailability improvement and patient comfort following topical instillation into the eye
Maissa et al.,2009 (castor oil)Jamal et al.,2009 (difluprednate)Ammar et al., 2009 (dorzolamide)Liu et al., 2009 (azithromycin)Ibrahim et al.,2009 (prednisone)
Ocular Applications for nanoemulsions
• Dry eye• Moderate to severe dry eye• Glaucoma (IOP ↓)• Retinopathies
Age Related Macular Degeneration (ARMD)
Diabetic Retinopathy
Cationic Emulsion
Droplet Size: 150 nmZeta Potential: +40 mV
200 nm
Electronic Micrograph
OleylamineBrings the Positive Charges
PhospholipidsStabilize the interface
Oily Core Solubilizes the drugs
Cationic Emulsion Rationale
• Strong electrostatic attraction between cationic emulsions and negatively charged cell surface
Adherence of opposite surface charges
Enhancement of drug absorption
(Tamilvanan – Gursoy & Benita 2002)
Novagali Pharma Technology Platform
• Proprietary technology platform originated by Hebrew University, Jerusalem (Simon Benita)
• Yissum received equity against exclusive licensing of the IP to Novagali Pharma
Strategy -Start-Up Creation: Novagali Pharma
Incorporated in 2000 as a private Co. with VC funds at Genopole, Evry
1st round of financing : Euro 3.7 million• To implement a private laboratory structure,• To transfer the technology from University to Novagali• To strengthen the valuable assets of the company,• To expand the development of the technology • To prepare clinical emulsion batches for P I studies
Water phase slowly incorporated into oil phase and mixed with a magnetic stirrer and further heated to 85°C
Heat to 70°C
Poloxamer 188Glycerol
Water up to 100
Water phase
Heat to 70°C
oilα-tocopherollipoid E-80
Stearylamine(+), Deoxycholic acid(-)CsA
Oily phase
NanoEmulsion Preparation
Sterilization by autoclaving at 121°C for 15 min.Packaging under nitrogen atmosphere in siliconized glass bottles
Adjust pH of emulsion to 7 using 0.1N HClRapid cooling below 20°C
Polytron 16000 rpm for 5 min.1 Emulsification
Homogenization
9000 psi for 5 min.
3
2
4
NanoEmulsion preparation
Novagali SAS / 30.10.2001
NanoEmulsion Preparation:3 Steps to reduce droplet size to nano scale
123
> 1 µm
= 1 µm
= 200 nm
Tauber et al., 1998
Dry eye is the most common eye condition causing severe
• Discomfort• Pain• Contact lens intolerance• Visual disturbances.
A disorder of tear film that affects 10%-15% of adults
Results from either decreased tear production or excessive tear evaporation
With the loss of water tear film become unstable Progressive deterioration of the ocular surface
Supplementation of tears
• Artificial tears remain the main treatment in dry eye disease or tear deficiencies.
• Major problem in the use of artificial tears: short action (30-40min)
Current treatments up to the last 5 years
Cationic Emulsions in Ophthalmology
ConventionalEye Drops
AnionicEmulsion
CationicEmulsion
washout Electrostatic Repulsion
Electrostatic Attraction
Contact Angle on Cornea
70°
50°
30°
Saline
Anionic Emulsion
Cationic Emulsion
Contact angle
Reduced by 50 %
Studied formulations
Viscosity-increasing agents:
polyvinyl alcohol 1.4%, povidone 0.6%
Studied formulations
Viscosity-increasing agents:
Polyvidone 5%
Studied formulations
Viscosity-increasing agents:
Hydroxypropyl Methylcellulose 0.3%
Refresh Endura: anionic blank emulsioncastor oil, glycerin 1%, polysorbate 1%, carbomer, mannitol, sod. hydroxide and purified water
Studied formulations
Cationorm® : cationic blank emulsion mid chain triglycerides, phospholipids ,
oleylamine, poloxamer, glycerin, purified water Novagali
Studied formulations
Contact angles were measured by FTA125 system (First Ten Angstroms, Portsmouth, Virginia, USA)
Immediately following one droplet instillation: An image of the sessile droplet on the studied
surface was captured Automatic determination of the
contact angle was performed by the FTA software
The lowest C”A On Dry CORNEA and
CONJUNCTIVA
**
Cationorm Vs. all others = P < 0.05 (Kruskal-Wallis)
Traditional therapy:Artificial Tears:In recent decade most are preservative free Punctal (drainage) occlusion
Recent advances in dry eye therapy:Topical emulsion of CsA for the remedy of immune-related dry eye disease
Therapy:
Cyclosporin A-a drug for dry eye
• The drug is a cyclic lipophilic polypeptide consisting of 11 amino acids exhibiting complex formulation problems.
• Cyclosporin A (CsA) is an immunomodulator agent widely used for organ transplant rejection prevention.
Chemistry of CsA
Cyclosporin A-a drug for dry eyeChemistry of CsA
• The aqueous solubility of CsA is 20 - 30 µg/ml.
• No adequate aqueous formulations available for ocular administration of the drug
• If CsA oil preparations are applied directly to the eyes, irritation or a blurred vision are likely to occur
•
• Laibovitz et al. (1993)
Limitations of CsA eye drops
Acheampong et al. 1999
CsA nanoemulsion
• Previous agents were strictly palliative in nature
• CsA emulsion may represent the first therapeutic agent with a mechanism of action targeted at the underlying immune-mediated inflammatory response occurring in patients with KS
CsA emulsion
• ALLERGAN Co. has developed a negatively-charged emulsion of CsA for dry eye treatment: Restasis®
• Approved by FDA in December 2002.
Treatment is moving from increasing tears towards the idea of correcting ocular damage
Multifactorial disease involving inflammation, autoimmunity and damage to the surface of the eye
Part of the patients (severe cases) treated by Restasis® cyclosporine anionic nanoemulsion of Allergan
More than 20 products in clinical trials.
Dry Eye Syndrome
Novel formulation approaches based on anti-inflammatory drugs : cyclosporine PIII by Sirion Therapeutics and Novagali
Doxycycline -induced protease inhibition; PIII by Alacrity Bioscience
Mucin secretion stimulants; PIII by Acucela & Otsuka; PII by Ista Pharmaceuticals
Adenosine receptor agonists PIII Allergan &Inspire Phar.
Dry Eye Syndrome
Positively charged submicron emulsion has been widely investigated by our
group for ocular applications
Novel CsA nanoemulsion
Zeta potential: 35-45mVDroplet diameter: 150-250nm
Novagali continued and improved the R&D of the cationic CsA nanoemulsion for dry eye syndrome
Rabbit Ocular Studies[3H]-Radiolabeled CsA was incorporated in
either positively or negatively-charged submicron emulsion containing 0.2% CsA
Male albino rabbits
2-2.5 kg
n=18 n=18
Treated with negatively charged radiolabeled CsA SME
Treated with positively charged radiolabeled CsA SME
• One drop in the cul-de-sac of the right eye
• Three rabbits per time point: 15, 30, 60, 120, 180 and 480 min
• Right eyes were enucleated and dissected
Human eye Rabbit eye
Rabbit is the best animal model for ocular experiments
Anatomical differences III
• The rabbit mean cornea thickness is 0.37mm while that of man is 0.51mm.
• Rabbits are more susceptible to damage (alkaline) materials, because the pH of their aqueous humor is 8.2 compared to 7.1-7.3 for man.
• The cornea represents 25% of the rabbit eye surface area, but only 7% of the surface area in man
20
515
1010
1505
2000
15 30 60 120 180 480
Sclera-choroid-retinaIrisConjunctivaCornea
CsA
Conc
., ng
/g
Effect of negative surface charge on CsA concentration in extraocular tissues following one single instillation of 50µl of
CsA emulsion in the cul-de-sac of rabbit eye
Time, min Abdulrazik et al. , 2001
0
750
1500
2250
3000
15 30 60 120 180 480
Sclera-choroid-retinaIrisConjunctivaCornea
Effect of positive surface charge on CsA concentration in extraocular tissues following one single instillation of 50 µl of
CsA emulsion in the cul-de-sac of rabbit eyeCs
A Co
nc.,
ng/g
Time, min Abdulrazik et al. , 2001
0
375
750
1125
1500
Optic ner. Blood Scl&Ret Vitreous Lens Iris Aqueo.h Conjun. Cornea
Positive emulsionNegative emulsion
CsA
Conc
., ng
/g CsA concentration in different ocular tissues after 60 min from instillation of the emulsions in the treated
eye as a function of surface charge droplet
Tissues Abdulrazik et al. , 2001
Posterior Segment
*
*
* P < 0,05
Compared Retinal BioavailabilityIndomethacin and Cyclosporine AUC in retina (single topical administration)
• There are evidences that colloidal delivery systems can facilitate the penetration of drugs into extraocular tissues through an endocytic mechanism ( Calvo et al., 1996).
• The endocytic effect is probably more pronounced with the cationic than with the anionic nano emulsion.
Positively charged
emulsion 1:60
SalineNegatively charged emulsion 1:60
Cow stroma P5 cell cultures after 24hrs incubation
• This hypothesis has been partially verified in an independent cell-culture study which revealed that the cationic o i l d r o p l e t s o f t h e emulsion are more easily internalized within the cells than the anionic oil droplets
Novagali Pharma Expansion • Private Company Founded in 2000 1st Round in 2000 : 3.78 M€ 2nd Round in 2004 : 14 M€ 3rd Round in 2006/7: 26 M€ 4th Round in 2008: 15 M€
• Expertise in low soluble drugs Formulation Pre-clinical & Clinical
development in Ophthalmology• Integrated Pharmaceutical company
Development activities GMP Pilot production
Emulsion scaling up completed
> 1 µm
= 1 µm
= 150 nm
1. Phases Mix
2. Ultraturrax
3. Homogeneization
4. Autoclavage
5. Bottlepack / Monodoses
Cationorm®
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The unique & patented milky emulsion preservative free ►For the treatment of dry eye symptoms ►Dose regimen: 1 to 4 drops per day►Compatible with contact lens ►Medical device Eu ( OTC in the US)
Presentation►2 sizes: 30 or 10 single sterilized vials ►Storage at Room temperature
Commercialization►Promotion to ophthalmologists (9 reps)● Sampling, Conference (booth & symposia), Gift, Mailing
►Sold in pharmacies (via wholesalers), PR
CsA Cationic Emulsion –Current Status
• Phase III clinical studies in Europe completed by Novagali Pharma
Cationic emulsions: potential intraocular nanocarriers for antisense oligonucleotides
T. Hagigit1, M. Abdulrazik1, F. Valamanesh2 M.Hagedorn2 , G. Lambert3 F. Behar-Cohen2 and S. Benita1.
1Pharmaceutics Dept., The Hebrew University of Jerusalem, Israel, 2 INSERM, U598, University Paris-Descartes, Paris France,
3Novagali Pharma, Evry, France
EU CRAFT program (FP VI)
Disease ReviewARMD – Age Related Macular Degeneration
‣A degenerative disease that focuses on a specific part of the retina - the macula.
‣Main cause of blindness to elderly people in the industrialized world.
‣Over 30 million people suffer from it.
‣The onset age of the disease is 50 and above.
The most impressive therapeutic progress was done in this important and serious ocular disease
The recent introduction of VEGF inhibitors has revolutionized the management of wet AMD.
Macugen ( pegaptanib, OSI Pharma.), Avastin, & Lucentis ( bevacizumab & ranibizumab
Genentech) Avastin for systemic administration & Lucentis
designed for intravitreal injection
AMD – Age Related Macular Degeneration
Antisense oligonucleotide delivery for the treatment of age related macular degeneration
The aim of antisense strategy is to interfere with gene expression by preventing the translation of proteins from mRNA.
A New Gene Therapy Approach for Treating AMD
‣Rapid degradation in biological fluids.
‣Inability to efficiently cross cell membranes
‣hydrophilic polyanionic character
‣large molecular structure
‣Difficulty to reach the target tissue (retina)
Obstacles facing ODN
‣ 17 bases single stranded DNA oligonucleotide.
‣ Sequence: 5’GsCsAsTsCTCCTTTTCsTsGsAsC3’.
‣ Partially phosphorothioated oligonucleotide with a MW of 5217 Daltons.
‣Anti-angiogenic activity by specific down-regulation of VEGFR-2.
‣ Specific inhibitor of endothelial cell proliferation.
Antisense oligonucleotide (ODN17)
Separation of free ODN by a porous membrane
Incubation of ODN with emulsion
ODN association
‣ MCT
‣ Cationic lipid agent
‣ Vit-E (antioxidant)
‣ Lipoid E-80
‣ (mixture of phospholipids)
‣ Poloxamer 188 (nonionic surfactant)
‣ Glycerol (osmotic agent) Distilled water
Oily core
Phospholipids Stabilize the interface
Positive charge/ association site
Emulsion by TEM
Emulsion formulation characterization
Cationic lipids
0
20
40
60
80
100
0.05 0.1 0.2 0.4
OD
N a
ssoc
iati
on, %
Cationic lipid (AOA), %
60
70
80
90
100
Without cationic lipid AOA 0.2 DOTAP 0.33
OD
N a
ssoc
iati
on, %
Emulsion type
Oleylamine (OA), Arginine octadecyl amide (AOA) DOTAP Equimolar concentrations of 4.67mM
ODN association extent of various nanoemulsion formulations
Hagigit et al., 2008
Nanoemulsion
ODN associated cationic nanoemulsion
Before accelerated test After accelerated test
Zeta potential mV
Particle sizenm
Zeta potentialmV
Particle size nm
Oleylamine 0.125 26.7±1 116.7±6 14.9±0.6 177.5±10
DOTAP 0.33 38.2±2.3 122.8±5 50.2±1.7 126.4±7
AOA 0.1 22.1±0.8 160.8±17 21.8±0.7 209.2±21
Shaking at constant rate of 100rpm over 48h at 37°C.
Associated ODN emulsion stability
Hagigit et al., 2008
Pharmacological Model
Induction of corneal neovascularization using a silver nitrate cauterization technique.
8 treatment groups and 6 rats in each group.
Mahoney and coll. Curr. Eye Res. (1985).
Peyman and coll. Graefes Arch. Clin. Exp. Ophthalmol.(2007).
Topical application immediately following cauterization. Corneal photographs were taken on the 7th day and quantitatively analyzed using NIH imageJ software.
Treatment groups: Saline Blank DOTAP nanoemulsion ODN17 aqueous solution Scrambled ODN sequence solution ODN17 DOTAP nanoemulsion (topical & sub conj.) Scrambled ODN DOTAP nanoemulsion Avastin®
Dose: 50µl 10µM immediately following cauterization.
Fluorescein sodium I.V. injection to the tail vein prior to photography
Time
Healthy cornea
Fluorescein
Fluorescein
Corneal neovascularization
Calculations
Area of neovascularization measured in pixels
Entire corneal areaX 100
Image analysis was performed on each cornea using an image processing and analysis software program.
Percentage of corneal neovascularization:
Effect of ODN17 Nanoemulsion In-Vivo
Effect of various ODN formulations on the cornea vascularization following induction of corneal neovascularization using a silver nitrate cauterization technique.
Topical application of 50μl from various formulations immediately after cauterization. Corneal photographs were taken on the 7th day and quantitatively analyzed using NIH imageJ software (N=6 rats).
Conclusion
‣DOTAP cationic emulsion can be considered a potential intra ocular delivery system for the sensitive active ODN17
Novagali Pharma Company-Update
• French Société Anonyme à directoire et conseil de surveillance– Founded in 2000 – ~ 45 employees including 13 doctors of science, medicine or pharmacy– Located in Evry (Paris suburb – Genopôle)– 4 rounds of financing for a total amount of € 59m
Formulation Analytical & QC Scale Up Pilot Manufacturing
pre-clinical Studies Clinical Development Regulatory Commercialization
44
The innovative cationic emulsion for the treatment of dry eye symptoms
Simon Benita
IDR
Novagali Pharma received in 2009 a 91 d’or, a distinction from the French Business Confederation (MEDEF) and the Siemens“Health Award”
Frost & Sullivan Recognizes Novagali Pharma for Innovation in Ophthalmic Therapies and grants the 2009 Best Practices Award
Collaborators-Acknowledgments
Pharmaceutics Dept. School of PharmacyThe Hebrew University of Jerusalem
Dr. M. Abdulrazik
Dr. S. TamilvananDr. S. Klang
T. Hagigit
K. KhouryD. Gilhar
M. Yarkoni
Hadassah University Medical Center
Pediatric Ophthalmology UnitProf. D. BenEzra G. Maftzir
Department of OphthalmologyProf. J. Frucht-PeryDr. M. AbdulrazikDr. S.C. SiganosDr. F. Orucov
INSERM U 598, Paris, France Prof. F. Behar-Cohen
Collaborators- Novagali Pharma
First CEO: Mr Ben van Der Kooij
Present CEO: Mr Jerome Martinez
First R & D Dir. : Dr. Gregory Lambert
Present R & D Dir. : Mr Jean Sebastien Garrigue
Acknowledgments: Betty Philips, Celine Caria and Dr. Frederic Lallemand
Thank You!