11

Novel Drug Targets for Reflux DiseaseNovel Drug Targets for Reflux Disease

Nikki Johnston, Ph.D.Nikki Johnston, Ph.D.Department of Otolaryngology and Communication SciencesDepartment of Otolaryngology and Communication Sciences

Medical College of WisconsinMedical College of WisconsinMilwaukee, WI, 53036Milwaukee, WI, 53036

Email: njohnsto@mcw.eduEmail: njohnsto@mcw.edu

Role of LPR in Injury and Disease is Role of LPR in Injury and Disease is Controversial Controversial

1.1. Efficacy of PPI therapy for LPR remains in doubtEfficacy of PPI therapy for LPR remains in doubt

�� Laryngeal mucosa is more sensitive to refluxate and thus Laryngeal mucosa is more sensitive to refluxate and thus patients with laryngeal symptoms require higher doses and patients with laryngeal symptoms require higher doses and longer trials of PPI’slonger trials of PPI’s

�� Studies evaluating efficacy of PPI’s for LPR used insufficient Studies evaluating efficacy of PPI’s for LPR used insufficient inclusion criteria?inclusion criteria?

VaeziVaezi et al., Laryngoscope 2006; 116: 254et al., Laryngoscope 2006; 116: 254--260260

ReichelReichel et al., et al., OtolaryngolOtolaryngol. Head and Neck Surg. 2008; 139:414. Head and Neck Surg. 2008; 139:414--420420

Role of LPR in Injury and Disease is Role of LPR in Injury and Disease is Controversial Continued..Controversial Continued..

2.2. Many nonMany non--specific signs and symptoms of LPRspecific signs and symptoms of LPR

�� OverOver--diagnosis of LPR has led to inappropriate PPI usediagnosis of LPR has led to inappropriate PPI use

�� This likely increases number of patients included in studies This likely increases number of patients included in studies who don’t have LPRwho don’t have LPR

3.3. Recent studies using MIIRecent studies using MII--pH report a positive symptom pH report a positive symptom association with nonassociation with non--acidic reflux, and persistent symptoms acidic reflux, and persistent symptoms on PPI therapyon PPI therapy

�� Role of Bile and Pepsin?Role of Bile and Pepsin?

�� Are PPI’s adequate to treat LPR?Are PPI’s adequate to treat LPR?

Sharma et al., Aliment Sharma et al., Aliment PharmacolPharmacol TherTher 2008; 25: 10032008; 25: 1003--1015 1015

TutuianTutuian et al., Am J et al., Am J GastroenterolGastroenterol 2008; 103: 10902008; 103: 1090--10961096

Role of Pepsin Role of Pepsin

�� Detected in laryngeal, but not esophageal, epithelia from 26/27 Detected in laryngeal, but not esophageal, epithelia from 26/27 patients with LPR patients with LPR (p<0.0001)(p<0.0001)

�� Detected in 0/19 laryngeal and esophageal specimens from Detected in 0/19 laryngeal and esophageal specimens from normal control subjects normal control subjects (p<0.001) (p<0.001)

�� Association between presence of pepsin in epithelial Association between presence of pepsin in epithelial specimens and depletion of CAIII and Sep70 proteins specimens and depletion of CAIII and Sep70 proteins (p<0.001)(p<0.001)

�� Exposure of porcine laryngeal mucosa to pepsin Exposure of porcine laryngeal mucosa to pepsin in vitroin vitro, , though not to low pH alone, causes depletion of CAIII and though not to low pH alone, causes depletion of CAIII and Sep70.Sep70.

Johnston et al. Laryngoscope 2004; 114 (12): 2129Johnston et al. Laryngoscope 2004; 114 (12): 2129--21342134Johnston et al. Ann Johnston et al. Ann OtolOtol RhinolRhinol & & LaryngolLaryngol 2006; 115 (1):472006; 115 (1):47--5858

22

MucinMucin Gene Expression in Human Gene Expression in Human Laryngeal Epithelia: Effect of LPRLaryngeal Epithelia: Effect of LPR

Samuels et al. Ann Otol Rhinol & Laryngol, accepted for publication April 2008Samuels et al. Ann Otol Rhinol & Laryngol, accepted for publication April 2008

Gene Expressed Not Expressed ▲▼ in LPRMUC1 √MUC2 √ ▼MUC3 √ ▼MUC4 √MUC5AC √ ▼MUC5B √ ▼MUC6 √MUC7 √MUC8 √MUC9 √MUC13 √MUC15 √MUC16 √MUC17 √MUC18 √MUC19 √MUC20 √

Effect of Acid and Pepsin on LPR-Sensitive Mucins In Vitro

PepsinPepsinControlControl

Electron Microscopy Analysis of Pepsin Electron Microscopy Analysis of Pepsin Uptake by Human Laryngeal Epithelial Cells Uptake by Human Laryngeal Epithelial Cells

200ng/ml human pepsin 3b200ng/ml human pepsin 3b20 20 minsmins at 4at 4 CC

200ng/ml human pepsin 3b200ng/ml human pepsin 3b20 20 minsmins at 37at 37 CC

= pepsin= pepsinBar = 0.5Bar = 0.5µµmm

Johnston et al. Ann Otol Rhinol & Laryngol 2007; 116(12); 934Johnston et al. Ann Otol Rhinol & Laryngol 2007; 116(12); 934--938.938.

CoCo--localization of Pepsin withlocalization of Pepsin withTransferrinTransferrin--HRP HRP

Bar = 0.3Bar = 0.3µµmm = pepsin= pepsin = = transferrintransferrin--HRPHRP

200ng/ml pepsin + 100ug/ml 200ng/ml pepsin + 100ug/ml transferrintransferrin--HRP, 15 HRP, 15 minsmins @ 37@ 37 CC

Johnston et al. Ann Otol Rhinol & Laryngol 2007; 116(12); 934Johnston et al. Ann Otol Rhinol & Laryngol 2007; 116(12); 934--938.938.

33

200ng/ml human pepsin 3b200ng/ml human pepsin 3b

CoCo--localization of Pepsin with localization of Pepsin with ClathrinClathrin

Bar = 50nmBar = 50nm

Rabbit antiRabbit anti--pepsin and goat antipepsin and goat anti--rabbit conjugated to 10nm rabbit conjugated to 10nm collidolcollidol goldgoldMouse antiMouse anti--clathrinclathrin and goat antiand goat anti--mouse conjugated to 5nm mouse conjugated to 5nm collidolcollidol gold gold

Competitive Binding for PepsinCompetitive Binding for Pepsin

Pepsin (100ng/ml) 1hr @ 4Pepsin (100ng/ml) 1hr @ 4 C, C, pepsinpepsin--TRITC (10ng/ml) 1hr @ TRITC (10ng/ml) 1hr @ 44 C, 30 C, 30 minsmins @ 37@ 37 CC

PepsinPepsin--TRITC (10ng/ml) 1hr @ 4TRITC (10ng/ml) 1hr @ 4 C, C, 10 10 minsmins at 37at 37 CC

Activity/Stability of PepsinActivity/Stability of Pepsin

Piper and Fenton. Gut 1965; 6 (5): 506Piper and Fenton. Gut 1965; 6 (5): 506--508508

0

2020

4040

6060

8080

100100

11 22 33 44 55 66 77 88pHpH

Pep

tic A

ctiv

ity (

%)

pH Activity CurvepH Activity CurvepH Stability CurvepH Stability Curve

Plasma Membrane

Exocytosis

Coated Pit

Ligands

Endosome

pH 4.0

Nucleus

Cis-Golgi

pH 5.0

pH 5.0

ER

Receptors

Trans-Golgi

Golgi Coated Pit

Lysosome

Pepsin

Receptor

CA III / Sep 70

CA III / Sep 70

44

Effect of Pepsin (0.2mg/ml)Effect of Pepsin (0.2mg/ml)pH7.4, 12 Hours at 37pH7.4, 12 Hours at 37◦◦CC

Bar = 0.5umBar = 0.5um

Effect of Pepsin (0.2mg/ml)Effect of Pepsin (0.2mg/ml)pH7.4, 12 Hours at 37pH7.4, 12 Hours at 37 CC

Bar = 2umBar = 2um Bar = 0.5umBar = 0.5um

Control: pH7.4, 12 Hours at 37Control: pH7.4, 12 Hours at 37 CC

Bar = 0.5umBar = 0.5um

Stress and Toxicity SuperArray

Symbol Genep-value

(compared to control group)

Fold Change(compared to control

group)

BAX BCL2-associated X protein 0.004822 0.4454CASP10 Caspase 10, apoptosis-related cysteine peptidase 0.002392 0.4061CCND1 Cyclin D1 0.005017 0.3703CRYAB Crystallin, alpha B 0.004478 2.3511CSF2 Colony stimulating factor 2 (granulocyte-macrophage) 0.030801 3.7321CYP1A1 Cytochrome P450, family 1, subfamily A, polypeptide 1 0.010849 5.0397DNAJA1 DnaJ (Hsp40) homolog, subfamily A, member 1 0.00236 0.4665ERCC1 Excision repair cross-complementing rodent repair deficiency 0.023851 0.3703ERCC3 Excision repair cross-complementing rodent repair deficiency 0.005941 0.3536GDF15 Growth differentiation factor 15 0.023061 1.5874HSPA2 Heat shock 70kDa protein 2 0.01701 1.4142HSPA4 Heat shock 70kDa protein 4 0.001366 0.2227HSPA6 Heat shock 70kDa protein 6 (HSP70B') 0.035053 2.9622HSPA8 Heat shock 70kDa protein 8 0.001517 0.5117HSPD1 Heat shock 60kDa protein 1 (chaperonin) 0.022321 0.6447HSPH1 Heat shock 105kDa/110kDa protein 1 0.018497 0.5878IL1A Interleukin 1, alpha 0.0081 1.7013IL6 Interleukin 6 (interferon, beta 2) 0.002131 0.4253

MT2A Metallothionein 2A 0.011441 3.325NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105) 0.001352 0.63PCNA Proliferating cell nuclear antigen 0.016192 0.6015RAD23A RAD23 homolog A (S. cerevisiae) 0.031073 0.5117RAD50 RAD50 homolog (S. cerevisiae) 0.004476 0.3703TP53 Tumor protein p53 0.042571 0.5612XRCC2 X-ray repair complementing defective repair in Chinese hamster cells 2 0.001981 0.4156RPL13A Ribosomal protein L13a 0.012154 1.8234

55

Peter Peter DettmarDettmar, PhD, PhDJoseph Joseph KerschnerKerschner, MD, MD Jamie Jamie KoufmanKoufman, MD, MD

Greg Greg PostmaPostma, MD, MD

Mark Lively, PhDMark Lively, PhD

Albert Albert MeratiMerati, MD, MD

Joel Joel BluminBlumin, MD, MD

Robert Robert ToohillToohill, MD , MD

Clive Wells, PhDClive Wells, PhD

Tina Samuels, MATina Samuels, MA

Michael Michael SyringSyring, BS, BS