C D

A B 460

268 238 171

117

71

55

41

31 kDa

Test Lubricants   Test 1: Lubricin Dose Response (n=4)

•  Lubricin (PRG4) @ 30,100,300 ug/ml in Saline •  Saline (Bausch & Lomb Sterile Plus Saline)

  Test 2: Lubricin + HA (n=5) •  CIBA Vision Aquify ~ 1mg/ml HA (AQ) •  Lubricin @ 300 ug/ml in Saline (PRG4) •  Lubricin @ 300 ug/ml in Aquify (PRG4+AQ) •  Saline

  Test 3: Lubricin + HA vs. Systane (n=5) •  Alcon Systane (Systane) •  Systane @ pH 8 (Systane 8) •  Saline •  Lubricin @ 300 ug/ml in Saline (PRG4) •  Lubricin @ 300 ug/ml in Aquify (PRG4+AQ)

Lubricin Presence At Ocular Surface   fresh porcine corneas   epithelial scrape, RIPA buffer extraction, and homogenization   SDS-PAGE Western Blot

LUBRICIN AS AN OCULAR SURFACE-CONTACT LENS BOUNDARY LUBRICANT: DOSE-DEPENDENT & SYNERGISTIC EFFECTS

S. Morrison1, B. Snider1, B. Sullivan2, E. Truitt III3, D. Sullivan4, T. Schmidt1 1 University of Calgary, Calgary, Canada; 2 TearLab Corp., San Diego, CA;

3 Singularis, Inc., San Diego, CA; 4 Schepens Eye Research Institute, Boston, MA.

  lubricin1 •  mucin-like glycoprotein from proteoglycan 4 (PRG4) gene2

•  secreted by chondrocytes3 and synoviocytes4 lining joint surfaces •  present in synovial fluid and on the surface of articular cartilage

•  cartilage boundary lubrication function5 •  decreases friction during surface-to-surface contact

•  synergism with hyaluronan (HA)   ocular surface

•  significant shear/friction forces from eyelid blink and contact lens wear •  lubricin expression

•  mRNA by human corneal and conjunctival epithelial cells6

•  protein in human meibomian gland secretions7 •  lubricin function – reduce friction at cornea-eyelid biointerface8

  dry eye disease •  decreased lubrication pain and discomfort during contact lens wear •  no effective therapeutic treatment

INTRODUCTION

Conclusions   developed a novel boundary lubrication test: cornea-contact lens biointerface   lubricin functions as an effective ocular surface boundary lubricant

•  dose dependent (30-300 ug/ml) •  synergism with HA superior to commercially available drops •  potential new & improved dry eye biotherapeutic

  lubricin protein exists at the ocular surface Future Studies   elucidation of lubricin + HA synergism   evaluation of other contact lens materials & coating +/- HA applications12

DISCUSSION

Dr. Heather Sheardown, Canadian Arthritis Network, NSERC, Southern Alberta Lions Eye Bank, The Arthritis Society, U Calgary Body Donation Program.

ACKNOWLEDGMENTS

1Swann+, Biochem J, 1977; 2Ikegawa+, Cytogenet Cell Genet, 2000; 3Schumacher+, ABB, 1994; 4Jay+, J Rheumatol, 2000; 5Schmidt+, A&R, 2007; 6Sullivan+, Trans ARVO, 2007; 7Tsai+, BR J Ophthalmol, 2006; 8Schmidt+, Trans ARVO, 2010; 9Schmidt+, OAC, 2008; 10Schmidt+, BBA, 2009; 11Schmidt+, OAC, 2007; 12Van Beek+, Biomat, 2008;

REFERENCES

1) assess boundary lubricating ability of lubricin at a contact lens-cornea biointerface, 2) determine if lubricin protein is present at the ocular surface

OBJECTIVE

Ocular Surface Materials   Human corneas: Southern Alberta Lions Eye Bank

•  fresh, preserved in Optisol-GS @ 4C, used within 2 weeks •  age: 24-90 years old

  Contact lens materials: polydimethylsiloxane (PMDS, obtained from Dr. Heather Sheardown, McMaster University)

Lubricin Preparation, Purification, & Characterization   Preparation5:

•  cartilage disks from patellofemoral groove of mature bovine stifle joints •  cultured for 15 days in Dulbecco’s modified Eagle’s medium, 0.01%

BSA, 25 µg/ml ascorbic acid, 10 ng/ml recombinant TGF-β19

  Purification5: •  spent medium fractionated by DEAE anion exchange chromatography

•  DEAE-Sepharose column •  concentrate & buffer exchange PRG4 rich 0.3-0.6 M NaCl eluate

  Characterization: •  3-8% Tris Acetate SDS-PAGE Western Blot & protein stain

•  C terminal anti-peptide antibody10: LPN

•  gel protein stain •  BCA protein assay

Ocular Surface Boundary Lubrication Test   Test Samples (Fig. 1A)

•  cornea with ~3 mm sclera •  contact lens: Ro = 3.2 mm, Ri = 1.5 mm Reff = 2.4 mm

  Test Setup11 (Fig. 1B) •  cornea-contact lens biointerface, BOSE ELF3200 biomechanical tester •  rotate (ω rad/s) +/- 4 rev at linear sliding velocity: 30, 10, 1, 0.3 mm/s

•  12 s pre-sliding duration (dwell time) between rotations •  measure normal load N, torque τ

•  normal stress σ = N/[π(Ro2 - Ri

2)]: avg 15.7±1.2 kPa   Data Analysis

•  friction coefficients11µ = (τ/Reff)/N •  static µ ~ resistance to onset of motion: peak τ •  kinetic µ~ resistance to steady state motion: average steady τ

•  mean ± sem, repeated measures ANOVA

METHODS

Figure 3. Porcine cornea for epithelial scrape (A) and western blotting with or without anti-PRG4 Ab LPN (B)

RESULTS

B E

PRG4 Expression: Ocular Surface (Fig. 3)   high MW, LPN reactive PRG4 band

observed (*) in corrneal epithelial scrape

A

Figure 1. Ocular surface boundary lubrication test samples (A) and setup (B)

  Test 3: Lubricin + HA reduces friction more than Systane •  static µ: lubricant (p<0.05), vel (p<0.01), interact (p=0.07) (Fig. 2E) •  kinetic µ: lubricant (p<0.01), vel (p=0.41), interact (p<0.05) (Fig. 2F)

•  Lubricin + HA < Lubricin ~ Systane (p<0.05) < Systane @ pH 8 (p<0.01)

A

B 1° Ab: - LPN

cornea contact lens

Figure 2. Effect of lubricant and sliding velocity on static (A, C, E) and kinetic (B, D, F) friction at a human cornea-contact lens biointerface. Test 1: 30, 100, 300 ug/ml PRG4 , Saline (A, B). Test 2: Aquify, 300 ug/ml PRG4, PRG4+Aquify, Saline (C, D). Test 3: Systane, Systane @ pH 8, Saline, PRG4 @ 300 ug/ml , PRG4+Aquify (E, F)

F

*

Test Lubricants (Fig. 2)   Test 1: Lubricin lowers friction in a dose dependent manner (30-300 ug/ml)

•  static µ: lubricant (p<0.005), vel (p<0.01), interact (p<0.05) (Fig. 2A) •  Lubricin @ 300<100<30 (p<0.05), Saline ~ Lubricin @ 30 (p=0.99)

•  kinetic µ: lubricant (p<0.01), vel (p=0.74), interact (p<0.01) (Fig. 2B) •  Lubricin @ 300<100<30 (p<0.05), Saline ~ Lubricin @ 30 (p=0.33)

  Test 2: Lubricin + HA synergism (e.g. Aquify) •  static µ: lubricant (p=0.08), vel (p<0.001), interact (p=0.43) (Fig. 2C) •  kinetic µ: lubricant (p=0.06), vel (p<0.05), interact (p<0.01) (Fig. 2D)

•  Lubricin+HA< Lubricin < HA (p=0.1-0.15)