Tyrosine-Derived Nanospheresfor Topical Skin Delivery

Larisa Sheihet, PhD

SPOTLIGHT ON SKIN RESEARCHNew Jersey Center for Biomaterials

June 25, 2007

Team:Larisa Sheihet, Prafulla Chandra, Priya Batheja

David Devore, Joachim Kohn, and Bozena Michniak

3

Outline Introduction

Topical and Transdermal Drug Delivery• Why develop T&T Delivery• Delivery Approaches• Amphiphilic block copolymers

Tyrosine-derived polymers/nanospheres Project aim

Experimental Design: Methods & Materials Results and Discussion

Size and size distribution of nanosphere formulations Nanospheres binding efficiency Nanosphere-solute and PG-solute passive permeation studies Nanosphere-Nile Red and PG-Nile red passive permeation as function

of time Conclusions Future Direction

4

Introduction

5

Drug Delivery “…In the context of drug delivery, the needs for materials can

generally be broken into two categories: the creation of newmaterials and/or better understanding of how to manipulateexisting materials

Current needs include reducing the toxicity of drugs,increasing their absorption, improving their administration andrelease profile…”

Robert S. Langer, Institute Professor, MIT C&EN, Cover Story, August 26, 2002

6

Drug Delivery

http://www.azonano.com/details.asp?ArticleID=1538

AdministrationRoutes Peroral

Pulmonary

Parenteral• Intravenous• Intramuscular• Subcutaneous

Percutaneous• Topical• Transdermal

7

Topical & Transdermal Delivery There are several categories

of pharmaceutical products,which are targeted to the skinor utilize the skin as a port ofentry into the body:

patches gels creams sunscreens ointments lotions dermal vaccinations

Skin Care ProductsSkin Care Products

8

Common Skin Diseases Skin cancer Warts Fungal infections Dermatitis Psoriasis Acne Hand dermatitis Atopic eczema (one in six of all children) Cold sores (herpes simplex, one in five persons)

http://www.dermnetnz.org/dermatologist.htmlhttp://www.skinsite.com/index_dermatology_diseases.htm

7.5 million Americans have psoriasis2% to 3% of the world is affectedNational Psoriasis Foundation, 2007

9

Main Challenges in Transporting Drugs Intothe Skin

Low diffusion rate of drugs through the the stratumcorneum (SC)

Overcoming the resistance of the skin in a reversibleand non-damaging manner

Intra-inter variability associated with permeability ofintact and diseased skin

Pre-systemic metabolism Immunological barrier The limited number of suitable drugs is reduced to:

Low molecular mass and melting point Optimal partitioning coefficient

• Sufficient lipid (SC) and aqueous solubility Moderately lipophilic

B.W. Barry Nat Biotechnol 22 (2004) 192-197H. Patel et al. Cronin Chemosphere 48 (2002) 603-613A. Nanda et al. Curr Drug Deliv 3 (2006) 233-242

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Delivery Approaches to Overcome theSkin's Formidable Barrier Function

(1) M.B. Brown et al. Drug Deliv 13 (2006) 175-187(2) A.R. Denet et al. Adv. Drug. Del. Rev. 56 (2004) 659–674(3) Alza Website. 2005; J.A. Matriano et al. Pharm. Res.19 (2002) 63–70(4) N.B. Smith et al. Ultrasound. Med. Biol. 29, (2003) 311–317; A. Sintov et al. J. Control. Rel. 89 (2003) 311–320

Ultrasound (Sonophoresis & Phonophoresis)

MagnetophoresisRadiofrequency

Laser and photochemical wavesTemperature

Miscellaneous4

Microneedle /Puncture /PerforationAbrasion

Needless InjectionSuction

Stretching

Mechanical3

Ionophoresis (low level of electric current)

Electroporation (high voltage pulses)

Electrical2

TypeTypeActive methods - - useof external energy1

11

Delivery Approaches to Overcome theSkin's Formidable Barrier Function

Passive methods penetration enhancers supersaturated systems prodrug or metabolic approach liposomes, microemulsions, colloidal polymeric suspensions*

A.C. Williams et al. Adv Drug Deliv Rev. 56, 603-618, 2004; B. Godin et al. Crit Rev Ther Drug Carrier Syst 20, 63-102, 2003; G.M. ElMaghraby et al. J Pharm Pharmacol 58, 415-429, 2006; S.L. Borgia et al. J Control Release 110, 151-163, 2005;

(*) A. Berthold et al. Eur J Pharm Biopharm 45, 23-29, 1998; E.G. de Jalon et al. Int J Pharm 226, 181-184, 2001; S. Santoyoet al. Int J Pharm 242, 107-113, 2002.

However:• the amount of drug that can be delivered is still limited

• most of the enhancers cause irreversible membrane damage• just a limited number of suitable biodegradable, polymer and solid-lipid particles areavailable

12

Choice of Formulation

Solubilise/disperse both lipophilic and hydrophilic substances

Improve the transport of highly lipophilic molecules from themainly lipidic SC into the more aqueous viable epidermis

Obtain a suitable release profile and stability of the drug

Increase uptake efficiency without causing notable damage tothe skin

13

Choice of Formulation Nanotechnology may allow the development of drug

carriers that improve the generally low skin permeation andmay also induce targeting to the specific skin strata

In particular, protect labile compounds from degradation produce occlusive properties favor drug penetration into the skin provide sustained release reduce systemic adsorption reduce irritation improve tolerance

14

Polymeric Formulation The chemical composition of polymer vehicles can be

tailor-made to have desirable physico-chemicalproperties for the investigated application

• Solid-Lipid nanoparticles1

• Collagen microparticles2

• Lactic and Glycolic acid based microparticles3

(1) H. Chen et al. J Control Release 110 (2006) 296-306; R.H. Muller et al. Adv Drug Deliv Rev 54 (2002) S131-S155; S.L. Borgia et al. J Control Release 110 (2005) 151-163.(2) A. Berthold et al. Eur J Pharm Biopharm 45 (1998) 23-29.(3) S. Santoyo et al. Int J Pharm 242 (2002) 107-113; E.G. de Jalon et al. J Pharm 226 (2001) 181-184; J. Luengo etal. Skin Pharmacol Physiol. 19 (2006) 190-197.

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Amphiphilic Block Copolymers Composed of regions (hydrophobic

and hydrophilic) that have oppositeaffinities for an aqueous solvent

Self-assemble Low toxicity Solubilization of water-insoluble

drugs Protection of the drug from

premature degradation Passive or active drug targeting of

the vehicle

Core-Shell Architecture

R. Haag Angw. Chem. Int. Ed. 45, 1198-1215, 2006

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Requirements for the TherapeuticPolymeric Nanoparticles

Core-Shell architecture: Mw of macromolecules in the range of 20-50KDa1

Inner hydrophobic block serves as the reservoir for lipophiliccompounds5

• Biocompatible polymer: poly(ester)s, poly(ether)s, poly(amino acid)s2,3,4

• Low CMC in water - stable aqueous dispersions of lipophilic drugs Outer hydrophilic block: PEG or PEO6

• Modulation of cell behavior and non-fouling characteristics• Could provide superior hydration of the skin thereby increasing the skin permeation ability of

nanospheres

Particle diameter < 200 nm penetrate SC via surface furrows on the skin6

avoid entrapment by RES7

(1) H. Maeda et al. J Cont. Rel. 74 (2001) 47-61; (2) C. Allen et al. J Cont. Rel. 63 (2000) 275-286; (3) A.V. Kabanov et al.Adv. Drug Deliv. Rev. 54 (2002) 759-779; (4) D. Shenoy et al. Pharm. Res. 22 (2005) 2107-2114; (5) R. Haag Angw. Chem.Int. Ed. 45 (2006) 1198-1215; (6) G. Cevc et al. Adv Drug Deliv Rev 56 (2004) 675-711. (7) R.B. Greenwald Adv. DrugDelivery Rev. 55 (2003) 217-250

17

Tyrosine-Derived Polymers

PIVITTM new surgical mesh forhernia repairhttp://www.tyrxpharma.com/products.html

O CH2 CH2 C NH

O

CH CH2

C

O

O

OR

C (CH2)X

O

n

C

O

Bioresorbable Mounted stenthttp://www.teamreva.com/tech_bioresorbable.html

OR

O CH2 CH2 C NH

O

CH CH2

C

O

O

OR

C

O

n

desaminotyrosyl-tyrosine alkyl ester

HO CH2 CH2 C NH

O

CH CH2

C

OH

O

OR

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Tyrosine-Based Nanospheres

CH3O(CH2CH2O)n C (CH2)X C O CH2 CH2 C NH

CH

C

CH2

O

R

O C (CH2)X C (OCH2CH2)nOCH3

O O O O O

O

m

C. Nardin, D. Bolikal and J. Kohn Langmuir 20 (2004) 11721-11725L. Sheihet et al. Biomacromolecules 2005 & 2007

By changing the length of the diacid (X) or the pendent ester chain of the dipeptide (R) or the molecular weight of PEG (n) or the molecular weight of hydrophobic core (m),

a large number of inter-related polymers can be obtained

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Hydrophobic Drug Delivery by Self-AssemblingTriblock Copolymer-Derived Nanospheres

L. Sheihet, R.A. Dubin, D. Devore, J. Kohn Biomacromolecules: 6 (2005) 2726-2731L. Sheihet, K. Piotrowska, R.A. Dubin, D. Devore, J. Kohn Biomacromolecules, 8, (2007) 998-1003

In Vivo: Similar anti-tumor activity in a breast cancerxenograft model that to that of clinically used

formulation of Cremophor-paclitaxel

In Vivo: No change in vital behavior,skin irritation and sensitization weredetected when mice were inoculated

with the nanospheres

0

10

20

30

40

50

-1.93 1.91 3.64

Bin

din

g E

ffic

ien

cy

, %

Log, D

Ara CCamptothecin

Paclitaxel

An effective sink for binding lipophilic drugs

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The effects of nanospheres on the secretionof inflammatory molecules

88487 ±51841942 ± 921655 ± 1761948 ± 377MCP-1

27587 ±965246 ± 210 ± 0330 ± 31TNF - α

Lipopoly-saccharides

DTE-containing

nanospheres

DTO-containing

nanospheresControl

These results suggest a potential to utilize the tyrosine-derived nanospheres with octyl pendent chains as an anti-

TNF- α therapeutic tool for the particular inflammatorydiseases, such as psoriasis and rheumatoid arthritis

Mouse macrophage cell line RAW264.7, ELISA, N=3

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Project Aim

Evaluation of tyrosine-derived nanospheres asdelivery vehicles for highly lipophilic moleculesfor passive skin permeation

Fluorescent dyes as model agents were used todetermine the efficiency of the nanosphere approachas compared to non-particulate enhancerrepresented by propylene glycol

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Materials & Methods

23

Experimental Design: Materials

Log D values (pH 7) were obtained from ACD/Labs (© 1994-2006 ACD/Labs).

CH3O(CH2CH2O)n C (CH2)6 C O CH2 CH2 CHN CH

C

CH2

O

R

O C (CH2)6 C

O O O O O

O

m

R = DTO (Octyl)

(OCH2CH2)nOCH3

OHO O

C

OH

O

HN

C

(CH2)10

O

CH3

DAF (Log D 7.54)

O

N

Et2N O

Nile Red (Log D 3.10)

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Preparation of Nanosphere-SoluteComplexes

NSP-Solute

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Solute Binding Efficiency

Solute extraction procedure: Freeze dry predetermined aliquot of the purified

nanosphere-solute complexes suspension Accurately weight the dry residue Thoroughly dissolve in extraction solvent

Solute concentration assay (for NSP and/or PG) RP-HPLC method developed for each solute

!

BindingEfficiency(%)=massof solute inthenanospheresmassof solute inthe feed

" 100%

26

Transport Studies - Experimental Design

Franz diffusion cells: Donor compartment:

• Nanospheres alone and/or PBS alone• Nanosphere-solute formulations (NSP-solute)• Propylene Glycol-solute solutions (PG-solute)

Human cadaver skin (full thickness, ~300-500 µm)• Vertical cross-sectioning of treated skin• Fluorescent microscopy analysis

Receptor compartment:• phosphate buffer (pH 7.4)• HPLC analysis of receptor compartment after 24 hrs ofpermeation experiments

Time:• 24 hours for DAF permeation studies• 1, 3, 6 and 24 hrs in case NR permeation studies

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Results & Discussion

28

NSP Morphology - TEM

(A) Freeze-fracture (the largest spheres are 60 nm in size, magnification54000 x )

(B) Negative staining(C) Cryo-transmission - membrane thickness 6 mm

(A) (B) (C)

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Size Distribution of NSP-Solute Formulations

Neither the presence of thesolute in the nanospherespreparation nor the solute

hydrophobicity have asignificant effect on

nanosphere size and SD

() NSP alone() NSP-DAF() NSP-Nile Red

Assuming that the average width of transepidermal hydrophilic pathwaysis in order of 0.4 (water evaporation pathways) to ~ 100 nm

(intercorneocyte space), the relatively small size of tyrosine-derivednanospheres will easily allow their penetration into the stratum corneum

along the surface furrows on the skin

0

0.2

0.4

0.6

0.8

1

1.2

0 12 18 28 43 66 101 156 200

Hydrodynamic Diameter (nm)

Inte

nsit

y

30

Nanosphere Binding Efficiency

~ 0.02% (wt/v) nanosphere-or PG-solute formulations were used inpermeation studies providing sufficiently strong signal to be detected byfluorescent microscopy

DAF NILE RED

g/mL

of NSP or PG

1 8 0

200

A binding efficiency of 65% of all solutes with nanospheres was obtainedregardless of the extent of the solute hydrophobicity and solutes’ molecularweight

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HPLC Analysis Tyrosine-derived nanospheres do not facilitate

transport across the human cadaver skin• the amount of solutes in the receptor compartments was

below the limit of detection

1. E.G. de Jalon, M.J. Blanco-Prieto, P. Ygartua, S. Santoyo Int J Pharm 226, 181-184, 20012. R. Alvarez-Roman, A. Naik, Y.N. Kalia, R.H. Guy, H. Fessi Pharm Res 21, 1818-1825, 2004

Similar observations were previously reported:• PLGA-fluorescent microparticles were clearly visualized withinthe skin layers but were not able to reach the receptorcompartment of the diffusion cells1

• use of particulate drug carriers appeared to improve the drugresidence in skin without increasing transdermal transport2

32

Histology of Cryo-Sectioned Skin UsingH&E Staining

Schematic representation of cryo-sectioned skin (Left) and Hematoxylin & Eosinstaining (Right)

33

Cross-Sectional Fluorescent Images of UntreatedSkin and Skin Treated with NSP alone

485 and 520 nm for DAF

Skin sections were analyzed in the green band atexcitation and emission wavelengths of:

546 and 585 nm forNile Red

Untreated

Nanospheresalone

34

Cross-SectionalFluorescent Imagesof NSP-Solute vs.PG-Solute - 24 hrs

(A) PG-DAF vs. (B) NSP-DAF(C) PG-NR vs. (D) NSP-NR

*** Average of 30-40replicates of skin sections

35

Quantitative Analysis of Penetration Effect of NSP-Solute vs. PG-Solute

Penetration effect (PE ± standard error, *p < 0.01) of NSP with respect to PG after 24 hrs of passive penetrationwas quantified using Image J software (v1.36, NIH)SD: superficial dermis; LD: lower dermis

() PG and () NSP

DAF

Nile RedX 2

X 10

36

Cross-SectionalFluorescent Imagesof NSP-NR vs. PG-NR PassivePermeation as aFunction of Time

1: 1 hour3: 3 hrs6: 6 hrs

(A) PG-NR vs. (B) NSP-NR

37

Cross-Sectional Fluorescent Images of NSP-NR vs.PG-NR Passive Permeation as Function of Time

Penetration effect (PE ± standard error, *p < 0.01)

of NSP with respect to PGSD: superficial dermis; LD: lower dermis

() PG and () NSP

x 1.8

x 3

38

Proposed Explanations forNanospheres Enhanced Permeability

Vehicle nature Neat PG ⇒ dehydrating effect ⇒ less flux PEG in the nanosphere corona ⇒ superior hydration of the skin -

permeation enhancing effect An individual nanosphere could be considered as a supersaturated

system ⇒ higher thermodynamic activity ⇒ increase the partitioning Size

Given the small size, size distribution and dynamic structure ofnanosphere-dye complexes, it is conceivable that they may traversethrough the intercorneocyte spaces

Mechanism of penetration Alteration in the barrier properties and/or a greater degree of partitioning

of the nanospheres into the stratum corneum It is possible that the furrows between the corneocyte islands provide a

place into which nanospheres may accumulate within the skin

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Conclusions Tyrosine-derived nanospheres significantly enhanced skin penetration of

highly lipophilic model agents (DAF and Nile Red) in human cadaver skinas compared to a non-particulate formulation

No detectable transdermal permeation was observed even after 24 hoursapplication, suggesting that these nanospheres can be used in topicaldrug delivery

An increase in rate and extent of dyes penetration to deeper skin layerscould be due to the higher thermodynamic activity of the dye (relative tothat in propylene glycol), small size and hydration properties of thenanospheres

No acute toxicity, skin irritation or sensitization was detected when micewere injected with nanospheres

Hence, these nanospheres can offer a promising tool for the topical skindelivery of lipophilic drugs and personal care agents such as Vitamins Aand D, sunscreens, glucocorticoids, or retinoids

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Future Directions

Deliver solutes tothe lower skinstrata avoidingtransdermalpenetration

Tunablestructure,biocompatible,biodegradable,non- toxic

Encapsulatelipophilic agents:paclitaxel,vitamin D3,

DTO-containingnanospheresinactivate thecytokine TNF-α

PSORIASISchronic scaling and

inflammatorydisease

41

Future Directions

PSORIASIS

Targets of therapeuticintervention:

1. Hyperproliferation andabnormal differentiationkeratinocytes

• Increased release of TNF-α

2. Vascular changes withelongated and dilated bloodvessels

• An angiogenic phenotype3. Dermal inflammation with

prominent presence of Tlymphocytes

• Type 1 T-cell cytokines ⇒elevated levels of TNF-αand IL-2

Therapies1. Calcipotriol (Vitamin D3

analogue, Log D ~ 3)• “Normalization” of

keratinocytes• Antiangiogenic activity

2. Anti-TNF- α biologicals

3. Anti-cancer drugs (paclitaxel!)

• Antiangiogenic,antiproliferative andantiflammatory

√

√

√

42

Acknowledgements The TEAM Dr. Gleb Shumyatsky Mr. Matt Treiser NJCBM and CEMBR (W81XWH-04-2-003) for

funding

Thank You!Thank You!