Introduction to the Comparative Anatomical Factors Affecting Topical Skin Delivery

Nancy A. Monteiro-Riviere, PhD, ATSUniversity Distinguished Professor and Regents Distinguished Professor of

Toxicology EmeritusFormer Director, Nanotechnology Innovation Center of Kansas State University,

Manhattan, KS and

Professor Emeritus of Investigative Dermatology and ToxicologyCenter for Chemical Toxicology Research and Pharmacokinetics

North Carolina State University, Raleigh, NC nmonteiro@ksu.edu or monteiro@ncsu.edu

Conflict of Interest Statement

The presenter declares that there exists no real or perceived conflict of interest.

I acknowledge that all animal experimentation has been carried out in accordance with the Society’s Guiding Principles in the Use of Animals in Toxicology.

Outline/Objectives

Understanding how anatomy affects the topical absorption of chemicals, drugs, and nanoparticles

Potential pathways for absorption Species differences Regional differences Experimental model systems Disease and alteration of the barrier

Biological Functions of Skin

Physical and metabolic barrier to the environment: stratum corneum

Thermoregulation: hair and fur, apocrine and eccrine sweat glands, sebaceous glands, and blood flow shunts

Mechanical Support: collagen and water Endocrine (e.g., vitamin D) Neurosensory reception Immunologic responses: keratinocytes, Langerhans cells Metabolism, Biotransformation Uniquely exposed to UV light–sunburn

Skin: PORTAL of Entry and TARGET for Toxicity

Monteiro-Riviere NA: Comparative Anatomy, Physiology, and Biochemistry of Mammalian Skin. In Dermal and Ocular Toxicology: Fundamentals and Methods (Ed. DW Hobson). CRC Press, NY, Chapter 1, pp. 3-71, 1991.

Importance of Lipid Biochemistry/Biophysics

Removal of rate-limiting stratum corneum increases absorption Intercellular lipids are the primary pathway for drug absorption Consist primarily of ceramides, sterols, and other neutral lipids Exist in a liquid-crystalline matrix, the fluidity of which is related

to permeability of hydrophilic drugs Temperature, hydration, and chemical penetration enhancers

increase fluidity and thus permeability

Monteiro-Riviere NA. Structure and Function of Skin. In Toxicology of the Skin-Target Organ Series(Ed. NA Monteiro-Riviere). Informa Healthcare, NY, Vol. 29, Chapter 1, 1-18, 2010.

Composition and Orientation of Stratum Corneum Lipids are Important Determinants of Barrier Function

Roberts, Gierden, Riviere, Monteiro-Riviere. “Solvent and vehicle effects on the skin.” In Dermal Absorption and Toxicity Assessment (Eds. MS Roberts & KA Walters), Informa Healthcare, NY. Vol.177, Chapter 24, p.433-48, 2008.

Distribution of Enzymes and Transporter Proteins

Dancik, Thompson, Krishnan, Roberts. Cutaneous Metabolism and Active Transport in Transdermal Drug Delivery. In Toxicology of the Skin-Target Organ Series (Ed. NA Monteiro-Riviere). Informa Healthcare, NY, Vol. 29, Chapter 7, 69-82, 2010.

Cutaneous Biotransformation

● Although P450 activity is less in skin, it can have a profound effect on bioavailability

● Phase I and II occur in basal layer● Applications

– Prodrugs: conversion of lipid ester to free drug– Detoxification of pesticides (parathion)– Bioactivation of toxicants (benzo(a)pyrene)

There is ongoing research defining species differences in absorption related to biotransformation!

Absorption vs. PenetrationAbsorption

● Relates to the amount of chemical that penetrates the skin and then absorbed into the bloodstream to have a systemic effect.

● Detected by flux into the perfusate or blood.

Penetration● Relates to the amount of chemical that gets to targets within the

skin and could be available for local cutaneous activity.● Detected by confocal microscopy, transmission electron

microscopy, special stains, biopsies, and tape stripping.

TEM of the Basement Membrane of Skin

Monteiro-Riviere NA. Structure and Function of Skin. In Toxicology of the Skin-Target Organ Series. (Ed. NA Monteiro-Riviere). Informa Healthcare, NY, Vol. 29, Chapter 1, 1-18, 2010.

Epidermal-Dermal Separation

Monteiro-Riviere NA. Indirect Immunohistochemistry and immunoelectron microscopy distribution of eight epidermal-dermal junction epitopes in skin treated with bis (2-chloroethyl) sulfide. Toxicologic Pathology 23:313-325,1995.

Potential Pathway for Drug Delivery

Experimental Model Systems

Animal Models• Practicality• Absorption

– Toxicology─Increased absorption compared to man to assess worse case scenarioRats, Mice, Rabbits

– Pharmacology─Similar absorption to manPigs, Primates, Hairless Rodents

• Toxicity – Immunological considerations– Rabbits, Guinea Pigs, Rodents

Species differences for different mechanisms are independent ∴ one species is not the best model for all endpoints in humans!

Species Differences

• Anatomical factors such as thickness, adnexial structures, etc.

• Biochemical differences in lipid composition, enzymes, etc.

• Different receptors for immunological or pharmacological agents

• Physiological differences including blood flow, etc.

Pig Skin is Similar to Human Skin

• Large surface area makes it amenableto test transdermal patches for humans

• Similar surface characteristics

I like pigs. Dogs look up to us. Cats look down on us. Pigs treat us as equals.

• Similar body masses• Similar skin to body surface

area ratio• Sparse hair coat

Differences Between Pig and Human Skin

• Pig skin has an additional interfollicularmuscle

• Young pigs’ hair follicles occur in a triad • Pigs have apocrine sweat glands over

most of the body• Immunological and drug metabolism

differences are increasingly being characterized

Monteiro-Riviere NA. Integument. In Textbook of Veterinary Histology (Eds.HDieter Dellman & J Eurell),Williams & Wilkins, 5th ed, Baltimore,MD, Chapter 16, 303-332,1998. Stromberg MW, Huang YC, Monteiro-Riviere NA. Interfollicular smooth muscle in the skin of the domesticated pig. The Anatomical Record 201:455-462, 1981.

Porcine Skin Human Skin

Monteiro-Riviere NA: The Integument. In The Biology of the Domestic Pig (Eds. WG Pond and HJ Mersmann), Cornell University Press, NY. Chapter 14, 625-652, 2001.

Monteiro-Riviere NA, et al. Animal Skin Morphology and Dermal Absorption. In Dermal Absorption and Toxicity Assessment. (Eds. MS Roberts and KA Walters). Informa Healthcare, NY, Chapter 2, 17-35, 2008.

Mouse Skin Nude Mouse Skin

Rat Skin Rabbit Skin

Regional Differences Between Species

Rodents and Domestic Animals

• Number of cell layers• Thickness• Blood flow• Hair density

Species Epidermis Stratum Corneum Number of Cell Layers(µm) (µm)

Cat 12.97 + 0.93 5.84 + 1.02 1.28 + 0.13 Cow 36.76 + 2.95 8.65 + 1.17 2.22 + 0.11 Dog 21.16 + 2.55 5.56 + 0.85 1.89 + 0.16 Horse 33.59 + 2.16 7.26 + 1.04 2.50 + 0.25 Monkey 26.87 + 3.14 2.05 + 2.30 2.67 + 0.24 Mouse 13.32 + 1.19 2.90 + 0.12 1.75 + 0.08 Pig 51.89 + 1.49 12.28 + 0.72 3.94 + 0.13 Rabbit 10.85 + 1.00 6.56 + 0.37 1.22 + 0.11 Rat 21.66 + 2.23 5.00 + 0.85 1.83 + 0.17

Comparative Epidermal Thickness and Number of Cell Layers from the Back of Nine Species

Monteiro-Riviere et al. Interspecies and interegional analysis of the comparative histological thickness & laser Doppler blood flow measurements at five cutaneous sites in nine species. Journal of Investigative Dermatology 95:582-586, 1990.

Blood Flow Measurements of Nine Species at Five Cutaneous Sites

Species BUT EAR HSJ TLJ VAB

Cat 1.82 + 0.59 6.46 + 2.30 1.86 + 0.70 2.39 + 0.35 6.19 + 0.94Cow 6.03 + 1.84 6.98 + 2.19 5.51 + 2.32 5.49 + 1.49 10.49 + 2.13Dog 2.21 + 0.67 5.21 + 1.53 5.52 + 1.31 1.94 + 0.27 8.78 + 1.40Horse 3.16 + 1.22 ----- 6.76 + 1.49 2.99 + 0.86 8.90 + 1.46Monkey 3.12 + 0.58 20.93 + 5.37 8.49 + 3.28 2.40 + 0.82 3.58 + 0.41Mouse 3.88 + 0.92 10.10 + 3.51 20.56 + 4.69 36.85 + 8.14Pig 3.08 + 0.48 11.70 + 3.02 6.75 + 2.09 2.97 + 0.56 10.68 + 2.14Rabbit 3.55 + 0.93 8.38 + 1.53 5.38 + 1.06 5.46 + 0.94 17.34 + 6.31Rat 4.20 + 1.05 9.13 + 4.97 6.22 + 1.47 9.56 + 2.17 11.35 + 5.53

Units=ml/min/100g (mean + SE)But=buttocks; Ear=pinnae; HSJ=humeroscapular joint; TLJ=thoracolumbar junction; VAB=ventral abdomen

Monteiro-Riviere et al. Interspecies and interegional analysis of the comparative histological thickness and laser Doppler blood flow measurements at five cutaneous sites in nine species Journal of Investigative Dermatology 95:582-586,1990.

Hair Follicle Density

Species Area of Skin Number of Hair Follicles/cm2

Human Abdomen 11 + 1Pig Back 11 + 1Rat Back 289 + 21Mouse Back 658 + 38Hairless Mouse Back 75 + 6

Bronaugh et al. Methods for in vitro percutaneous absorption studies II. Animal models for human skin. Toxicology and Applied Pharmacology 62, 481-488, 1982.

Age of Pig Mean Hair Follicle Density per cm2 (± SEM)

8 Week 68.9 ± 2.08

14 Week 29.7 ± 0.58

15 Months 8.4 ± 0.39

Hair Follicle Density

Body Site Differences The rate of penetration and absorption differs across various body

sites Scrotum > Forehead > Axilla > = Scalp > Back = Abdomen > Palm and

Plantar surfaces Seen in humans and animals making route-to-route extrapolations

“interesting.” The major reasons are due to:

– Differences in anatomy: skin thickness– Differences in physiology: blood flow and distribution of blood

vessels– Stratum corneocyte cell size ?

Ear Skin is Different than Other Areas

• Thickness is thinner compared to other body sites and is thicker on the outside (convex) surface than the inner (concave) surface

• Hair follicle and hair density is greater on the convex surface• Glandular density is different between surfaces• Cartilage is present • Blood vessels traverse cartilage and is

different than other body regions

Monteiro-Riviere et al. Immunohistochemical characterization of the basement membrane epitopes in bis (2-chloroethyl) sulfide induced toxicity in mouse ear skin. Journal Applied Toxicology 19:313-328, 1999.

Mean Concentration of Lipid Extraction

Monteiro-Riviere, Inman, Mak, Wertz, Riviere. Effect of selective lipid extraction from different body regions on epidermal barrier function. Pharmaceutical Research 18:992-998, 2001.

Extraction with Cyclohexane–1 Min–3X

Lipid Composition

• Human and porcine skin have similar biochemical composition

• Physical chemical properties of lipids are similar between these species

• Lipid extraction increases TEWL similar to tape stripping• Pig skin lipid extraction showed relative proportions of

individual lipids were similar at different body regions • Note that these studies could serve as models of

solvent-induced skin disease

In Vitro Dermal Absorption Systems

Flow Through vs. Static Diffusion Cells

Karadzovska D, Brooks JD, Riviere JE: Experimental factors affecting in vitroabsorption of six model chemicals across porcine skin. Toxicology In Vitro 26: 1191-1198, 2012.

Topical Site Differences in Piroxicam Absorption in Pigs: In Vitro vs. In Vivo

Monteiro-Riviere NA, Inman AO, Riviere JE, McNeil SC, Francoeur ML: Topical penetration of piroxicam is dependent on the distribution of the local cutaneous vasculature. Pharmaceutical Research 10:1326-1331, 1993.

Mean Penetration Profiles 3H Piroxicam

Types of Engineered 3-D Skin Models

IPPSF Surgery and Perfusion Systems

IPPSF Closely Parallels In Vivo Human Absorption

IPPSF vs In Vivo Human

R2 = 0.91

0

5

10

15

20

25

30

35

0 5 10 15 20 25 30 35 40

IPPSF (% Dose)

In V

ivo H

uman

(% D

ose)

Wester RC, Melendres J , Sedik L, Maibach H, Riviere JE: Percutaneous absorption of salicylic acid, theophylline, 2,4-dimethylamine, diethyl hexyl phthalic acid, and p-aminobenzoic acid in the isolated perfused porcine skin flap compared to man. Toxicology and Applied Pharmacology 151:159-165,1998.

Advantages of In Vitro Models

• Ease of screening large number of compounds• Can screen potential toxic agents that would be unethical to

study in animals• Alternative animal models help in the 3 “Rs”

- Reduction- Refinement- Replacement

Disadvantages of In Vitro

• Difficulty of reproducing results and standardizing between laboratories

• Intralaboratory variability and Intra/Interlot variability• Subjectivity of recording techniques• Absorption of compounds is much higher than normal• Mathematical modeling greatly increases the ability to use in

vitro models to study mechanisms and predict human absorption (Baynes and Kasting will discuss this)

Nanoparticles and Skin Interactions: Safety Evaluation

Nanomaterials

Materials having a physicochemical structure on a scale greater than atomic/molecular dimensions but less than 100 nm, which exhibit physical, chemical, and/or biological characteristics associated with its nanostructure

International Association of Nanotechnology’s Nomenclature and Terminology Subcommittee and American National Standards Institute Nanotechnology Standards Panel (ANSI-NSP)

Physicochemical Properties• Size distribution• Agglomeration state• Shape• Crystal structure• Chemical composition• Surface area• Surface chemistry• Surface charge• Porosity

Physicochemical Parameters

Modify cellular uptakeProtein binding

Translocation Interactions

CellsBody fluidsProteins

Ability to distribute throughout the body

TEM of Human Epidermal Keratinocytes

Monteiro-Riviere et al., Science, 306, p. 2164, 2004Monteiro-Riviere NA, Nemanich RJ, Inman AO, Wang YY, Riviere JE: Multi-walled carbon nanotube interactions with human epidermal keratinocytes. Toxicology Letters 155:377-384, 2005

Featured in an exhibition on Nanotechnology–London Science Museum–2005 for one year

Quantum Dots1µM-QD621-PEG 10µM-QD621-PEG

Zhang LW, Yu WW, Colvin VL, Monteiro-Riviere NA: Biological interactions of quantum dot nanoparticles in skin and in human epidermal keratinocytes. Toxicology Applied Pharmacology 228:200-211, 2008.

Dry TEM – QD- 621-Drs. Vicki Colvin/ William Yu

Flow-Through Diffusion Cell-10mM QD-PEG621- 24hr

Zhang L, Yu WW, Colvin VL, Monteiro-Riviere NA: Biological interactions of quantum dot nanoparticles in skin and in human epidermal keratinocytes. Toxicology Applied Pharmacology228:200-211, 2008

Flexing of Baa-Lys(FITC)-NLS over 24hr

Rat Skin-Flow-Through 8hr 1mM QD655-COOH

Human Skin─QD565-NH2 8h

Sunscreen Formulations

CM 630 10% rutile, crystallite TiO2 (T-Lite SF) oil/water TiO2 coated with hydrated silica and dimethicone/methicone copolymer and aluminum hydroxide for a mean size of 10 x 50nm, surface area of 100m2/g

CM 634 10% rutile, crystallite TiO2 (T-Lite SF) in water/oilTiO2 coated with hydrated silica and dimethicone/methicone copolymer (and) aluminum hydroxide for a mean size of 10 x 50nm, surface area of 100m2/g

CM 643 5% ZnO (Z-COTE HP1) in oil/waterZn coated /triethoxycaprylylsilane, mean size of 140nm, surface area of 12-24m2/g

CM 544 5% ZnO (Z-COTE) in oil/water Uncoated zinc oxide with a mean size of 140nm,surface area of 12-24m2/g

Sunscreen Formulations

TEM–In Vivo Porcine Skin-TiO2 CM 630

TEM–In Vivo Porcine Skin–ZnO CM 643

a: No UVB-treated with CM643 ZnO. Zn was seen on surface-3-4 cell layers 48hrb: UVB-exposed CM643 ZnO. Zn was seen in the superficial layers 48hr

Schematic Illustrating NP Penetration into SC Layers of Skin

a, b, and c represent different sections through skin as imaged by TEM. (a) NP found only in deeper layers of the SC, (b) NP in the upper and lower layers of the SC, and (c) NP in all layers of the SC. Arrows denote presence of NP in the intercellular spaces between SC cells.

Monteiro-Riviere, Wiench, Landsiedel, Schulte, Inman, Riviere. Safety evaluations of sunscreen formulations containing titanium dioxide nanoparticles in UVB sunburned skin: An in vitro and in vivo study. Toxicological Sciences 123 :264-280, 2011.

Important Considerations in Assessing Absorption/ Penetration Penetration is a function of NP type, species, and skin treatment (flexion, mechanical

action, tape stripping, abrasion)

Nanoparticle size, size distribution, shape, surface coatings, pH, and vehicle

Current literature suggests minimal penetration of nanoparticles with intact skin

Several factors can influence the extent of chemical and nanoparticle uptake in the skin: barrier integrity (UV, chemicals, flexion, tape stripping, abrasion), contaminated surface, anatomical structures (blood flow, thickness, appendageal structures), presence of skin diseases such as allergic and irritant contact dermatitis, atopic eczema, psoriasis, hydration, detergents, metabolism, and drugs may all increase skin absorption.

Schematic Depicting the Anatomical Factors That Can Influence Absorption

Monteiro-Riviere NA: Anatomical Factors that Affect Barrier Function. In Dermatotoxicology. (Eds. H. Zhai, KP Wilhelm, HI Maibach), 7th ed, CRC Press, Chapter 4, pp. 39-50, 2008

THANK YOU

Monteiro-Riviere NA: Comparative Anatomy, Physiology, and Biochemistry of Mammalian Skin. In Dermal and Ocular Toxicology: Fundamentals and Methods (Ed DW Hobson). CRC Press, Inc., NY, Chapter 1, pp. 3-71, 1991.Monteiro-Riviere NA (Ed). Toxicology of the Skin-Target Organ Series. Informa Healthcare, NY, Vol. 29, 2010.Monteiro-Riviere NA. The Integument. In The Biology of the Domestic Pig (Eds. WG Pond and HJ Mersmann), Cornell University Press, NY. Chapter 14, 625-652, 2001. Monteiro-Riviere et al. Interspecies and interegional analysis of the comparative histological thickness & laser Doppler blood flow measurements at five cutaneous sites in nine species. Journal of Investigative Dermatology 95:582-586, 1990.Karadzovska D, Brooks JD, Riviere JE: Experimental factors affecting in vitro absorption of six model chemicals across porcine skin. Toxicology In Vitro 26: 1191-1198, 2012. Monteiro-Riviere NA, Inman AO, Riviere JE, McNeil SC, Francoeur ML: Topical penetration of piroxicam is dependent on the distribution of the local cutaneous vasculature. Pharmaceutical Research 10:1326-1331, 1993. Samberg ME, Oldenburg SJ, Monteiro-Riviere NA. Evaluation of silver nanoparticle toxicity in skin in vivo and keratinocytes in vitro. Environmental Health Perspectives 118: 407-413, 2010.Rouse JG, Yang J, Ryman-Rasmussen JP, Barron AR, Monteiro-Riviere NA: Effects of mechanical flexion on the penetration of fullerene amino acid-derivatized peptide nanoparticles through skin. Nano Letters 7:155-160, 2007.Zhang and Monteiro-Riviere. Assessment of quantum dot penetration into intact, tape stripped, abraded, and flexed skin. Skin Pharmacology and Physiology 21:166-180,2008.Monteiro-Riviere, Wiench, Landsiedel, Schulte, Inman, Riviere. Safety evaluations of sunscreen formulations containing titanium dioxide nanoparticles in UVB sunburned skin: An in vitro and in vivo study. Toxicological Sciences 123:264-280, 2011.Riviere JE, Monteiro-Riviere NA: The isolated perfused porcine skin flap as an in vitro model for percutaneous absorption and cutaneous toxicology. CRC Critical Reviews in Toxicology 21:329-344, 1991. Monteiro-Riviere NA and Tran CL (Ed). 2nd Edition. Nanotoxicology: Progress toward Nanomedicine, CRC Press/ Taylor& Francis, Boca Raton, Fl. 2014.Riviere JE (Ed). Dermal Absorption Models in Toxicology and Pharmacology. CRC Press/Taylor & Francis, Boca Raton, Fl. 2006.

References