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®

61

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!