The Novel Importance of Adventitial Therapy for PAD Christopher D. Owens, MD, MSc Associate Professor Division of Vascular and Endovascular Surgery University of California San Francisco 1

Christopher D. Owens, MD I have no real or apparent conflicts of interest to report. Off-Label: All drugs I use in early phase studies are off-label.

The Tunica Adventitia of a muscular artery Loosely organized collection of fibroblast, perivascular nerves and microvessels in a collagen-rich ECM Functional immune surveillance of the artery: Innate and adaptive immunity - inflammatory cells including resident macrophages, mast cells, T and B cells and dendritic cells Regulation of inflammatory cell trafficking into and out of the vessel Adventitial – medial loop signals to control lumen caliber by short-term regulation of smooth muscle contraction and long-term regulation of remodeling Contains resident vascular progenitor cells and may possess stem/progenitor cell niche-like activity

Porcine renal artery demonstrating tunica adventitia

Barotrauma → Restenosis: a Summary Fibrosis Adventitia Inflammation Recruitment Proliferation EEL Basement membrane fracture Phenotypic switch: quiescent → proliferative Media Recoil Migration Inflammation Recruitment Proliferation Fibrosis IEL Stretching Denudation Thrombus Recruitment Neointima INJURY Fibrosis Lumen Lumen loss

Therapeutic Targets SIROLIMUS DEXAMETHASONE Fibrosis Adventitia Inflammation Recruitment Proliferation EEL PRT-201 Basement membrane fracture Phenotypic switch: quiescent → proliferative Media Recoil Migration Inflammation Recruitment Proliferation Fibrosis IEL Stretching Denudation Thrombus Recruitment Neointima INJURY Fibrosis Lumen Lumen loss

Adventitial Drug delivery Injection 4 @ 9cm: 1.0 ml After 3 minutes, diffusion up, down, and around >15cm of SFA Injection 3 @ 13.5cm: 1.0 ml Injection 2 @ 17cm: 1.0 ml Success = longitudinal + circumferential distribution Injection 1 @ 21cm: 0.5 ml (not in frame)

Technical Success Markers D003 D004 D016 D014 D019

Recombinant Human Type I Pancreatic Elastase (PRT-201) 26 kd serine protease Degrades elastin fibers Does not degrade collagen fibers Localized effect Inactivated in blood by antiproteases Surgical (topical) and endovascular applications 10

PRT-201 Treatment Results in Fragmentation of Elastin Fibers Ex Vivo Pre-Treatment Post-Treatment Treatment has no effect on collagen fibers, which provide resistance against rupture 11

Elastin Fragmentation Results in Artery Dilation In Vivo Dilation Occurs Immediately Dilation Persists Before After Rabbit carotid artery treated with 8.3 mg/mL PRT-201 (n = 4 for each time point). Error bars represent standard deviation. 12

Initial Clinical Study PRT-201 injection to femoral or popliteal artery by Mercator catheter following PTA Open-label, dose-escalation Cohorts of 4 PRT-201 at 1, 3, 10, 30 mg Safety, technical feasibility Lumen diameter and stenosis by duplex Doppler US Entry criteria Intermittent claudication Stenosis of ≥ 70% of the SFA or popliteal artery Target lesion length of ≤ 10 cm At least one patent runoff vessel N=7 so far 13

Anti-inflammation Program The Post-PTA Inflammatory Cascade Balloon injury or vessel trauma Hours Days Weeks Months Endothelial damage DEX Targets  CRP, MCP-1, TNFa  Endothelial healing Further inflammation is prevented Vascular inflammation Cytokine expression (CRP, MCP-1, TNFa, IL-10, etc) Cellular recruitment (neutrophils, macrophages, etc.) Cellular proliferation (adventitial fibroblasts, smooth muscle cells, etc.) Cellular migration & fibrous protein synthesis (neointima, restenosis, occlusion)

+ = ? Clinical Hypothesis Adventitial delivery of dexamethasone at the time of peripheral artery endovascular revascularization will reduce inflammation and improve long-term patency* + = ? *NOTE: Dexamethasone is not currently indicated for reducing vascular inflammation or improving vascular patency FDA 510(k)-Cleared and CE Marked for infusion of agents into the vessel wall and perivascular area

DANCE Pilot Design 20 patients enrolled between February 2011 and September 2012 at San Francisco VA Medical Center DESIGN: Open-label, non-randomized, single-arm, single-center clinical feasibility evaluation of adventitial DEX after PAD intervention OBJECTIVE: To evaluate technical success, safety and effectiveness outcomes of adventitial DEX delivered by Bullfrog® Catheter PRINCIPAL INVESTIGATOR Christopher D. Owens, MD University of California San Francisco Baseline blood draw for hsCRP levels Revascularization Procedure Adventitial DEX Treatment 24-hour blood draw for  hsCRP Clinical, Hemodynamic and Duplex U/S follow-up at 6 months Clinical, Hemodynamic and Duplex U/S follow-up at 12 months

DANCE Pilot Objectives Technical success: Fluoroscopic blush of dex/contrast delivery into adventitia of SFA and popliteal artery with Bullfrog® Catheter. Dex/contrast delivered to the adventitia Safety: vascular injury, thrombosis, embolization, dissection, death, and amputation Effectiveness: C-reactive protein rise from baseline to 24 hours Assessments at 1, 3, 6, 9, 12 mo: Lack of binary restenosis (Duplex U/S PSVR<2.5) ABI improvement relative to baseline Rutherford improvement relative to baseline

DANCE Pilot Demographics (N=20) Age (mean ± SD) 66 ± 10 Gender (male,%) 20 (100%) Race (caucasian, %) 10 (50%) *Diabetes (positive, %) 11 (55%) Hypertension (positive, %) 19 (95%) Hyperlipidemia (positive, %) Preoperative Rutherford classification 9=Rutherford 2 3=Rutherford 4 7=Rutherford 3 1=Rutherford 5 TASC II classification A=3 B=9 C=7 D=1 *Occlusions (%) Mean % occlusion (mean ± SD) 88 ± 12 *Mean lesion length cm (mean ± SD) 8.9 ± 5.3 Revascularization method PTA in 20 patients (100%) + atherectomy in 3 patients (20%) + provisional stent in 6 patient (30%)

DANCE Pilot Treated Segment Proximal SFA: 10% Mid SFA: 14% Note: 1 patient had treated segments in all three areas Distal SFA: 38% Popliteal: 48%

Safety and Feasibility (Technical) Endpoints Safety: no adverse events. No dissection, hemorrhage, thrombosis, or pseudoaneurysm Feasibility: Drug was delivered to the target vessel in 100% of cases.

DANCE Pilot Results Hemodynamic and Clinical Improvement, P<.01 21

DANCE Pilot Efficacy Endpoint Adventitial DEX Inhibits Inflammatory Biomarker of Restenosis Rise in C-reactive protein (CRP) predicts 6-month restenosis No statistical rise from baseline to 24-hours in DANCE trial C-Reactive Protein levels Schillinger , Radiology 2002

6-month Patency Rates after Drug-Enhanced Fem-Pop Angioplasty 23

Conclusions Adventitial may have theoretical advantages for local drug deliver over intimal-based methodologies Need to exploit more than proliferative pathways in lower extremity occlusive disease Chemical stenting promising but need to establish dose and safety parameters Dexamethasone infusion is safe, feasible, and demonstrates efficacy through reduced inflammation. A larger registry is set to begin and will be sponsored by Mercator MedSystems MIC has a favorable profile in the SFA and popliteal arteries. Drug titration, scalable delivery, and visual confirmation of treatment offers theoretical advantages over DCB

Acknowledgements Kirk Seward, Warren Gasper, Hugh Alley, Michael Conte, Karen Chong, Diana Kim Funding: NIH NHLBI HL-92163, & R43HL102998, American Vascular Association, and the Department of Surgery University of California San Francisco, Proteon Therapeutics Bullfrog Micro-Infusion Catheters were a gift from Mercator MedSystems, Inc. (San Leandro, Calif.)