1 Cellular, Tissue and Gene Therapies Advisory Committee Meeting Apligraf Allogeneic Cultured...

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Cellular, Tissue and Gene Therapies Advisory Committee Meeting

ApligrafAllogeneic Cultured Keratinocytes and

Fibroblasts in Bovine Collagen

BLA 125400

Proposed Indication: Treatment of surgically created gingival and

alveolar mucosal surface defects in adults

November 17, 2011

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FDA Presenters

Mark H. Lee, PhD – Product ManufactureDivision of Cellular and Gene TherapiesOffice of Cellular, Tissue and Gene Therapies, CBER

Robert Betz, DDS – Overview of Disease and TreatmentsDivision of Anesthesiology, General Hospital, Infection Control, and

Dental DevicesOffice of Device Evaluation, CDRH

Agnes Lim, MD – Clinical Study Design, Efficacy and SafetyDivision of Clinical Evaluation, Pharmacology and ToxicologyOffice of Cellular, Tissue and Gene Therapies, CBER

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Apligraf Review Team

ProductMark H. Lee, PhDEric Dollins, PhDCharles Durfor, PhD (CDRH)Andrew Steen, BSME (CDRH)

ClinicalAgnes Lim, MDBruce Schneider, MDRobert S. Betz, DDS, Captain (Ret.) USPHS (CDRH). Diplomate, Am. Board Periodontol.

StatisticsJohn Scott, PhD

Pharmacology/ToxicologyPatrick Au, PhD

Project ManagementTerrolyn Thomas, MS, MBA

LabelingLoan Nguyen, PharmD

Bioresearch MonitoringJanet White

EpidemiologyFaith Barash, MD, MPH

Manufacturing and Product QualityQiao Bobo, PhDGang Wang, PhD

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Product Information

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Product Description

Apligraf is a bi-layered tissue construct consisting of an upper layer of human keratinocytes and a supporting lower layer constructed of bovine derived collagen and human neonatal foreskin-derived dermal fibroblasts. The upper and lower layers of the product make up approximately 33% and 67% of the construct.

The proposed indication for Apligraf in this BLA is the treatment of surgically created gingival and alveolar mucosal surface defects in adults.

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Regulatory History of Apligraf®

• Identical to product submitted for BLA• Approved by the Center for Devices and Radiological

Health in 1998 “for use with standard therapeutic compression for the treatment of non-infected partial and full-thickness skin ulcers due to venous insufficiency of greater than 1 month duration and which have not adequately responded to conventional ulcer therapy.”

• Approved in 2000 by CDRH “for use with standard diabetic foot ulcer care for the treatment of full-thickness neuropathic diabetic foot ulcers of greater than three weeks duration which have not adequately responded to conventional ulcer therapy and which extend through the dermis but without tendon, muscle, capsule or bone exposure.”

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Product ManufactureApligraf is manufactured by combining viable allogeneic

human fibroblasts and keratinocytes with type I bovine collagen over a 3-4 week process until a bi-layered structure that resembles skin dermis/epidermis forms. Apligraf does not contain Langerhans cells, melanocytes, macrophages, lymphocytes, blood vessels or hair follicles.

Key manufacturing steps:• Establishment of Fibroblast and Keratinocyte Cell Banks• Production of Dermal Equivalent• Production of Epidermal Layer• Differentiation• Cornification

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Cell Banks

DE, EPI Layers

Differentiation Cornification

Maintenance

MatureProduct

STEP

Sterility/Mycoplasma/VirusCell Identity & PurityCytogenetic Stability, TumorigenicityIn vitro/In vivo Comparability

QC TESTS

Potency (Construct)

Sterility (Media)Mycoplasma (Media)Bioburden (Media)

Packaging

Sterility (Construct, Shipping Media)Bioburden (Construct, Ship Rinse Spent Media)EndotoxinVisual Inspection (Packaged Lots)

Sterility (Media)Bioburden (Media - Cornification)

Sterility (cellular, acellular cast mix, cell suspension residues)

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Cell Bank Qualification

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Cell Bank Qualification and Release

New cell banks of allogeneic fibroblasts and keratinocytes are introduced periodically for Apligraf manufacture.

Quality and comparability of the cells are supported by:• Testing at the Master and Working Cell Bank (MCB, WCB) levels• Combination of direct testing of cells and of the cell-scaffold

construct produced by the cells• Both in vitro and in vivo testing

Comparability of the cells used for manufacture impacts final product quality and consistency. Scientific discussion from the Advisory Committee is requested regarding the testing approach used by the applicant.

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MCB Qualification TestingTesting performed on cells• Microbiological and viral safety testing• Isoenzyme analysis• Karyology• Senescence• Tumorigenicity• Cell purity (CD45, CD14, CD1a, CD31, HLA-DR)

Testing performed on constructs made from MCB cells• Percutaneous absorption (barrier function)• Growth Factor/Cytokine profile (PDGF , TGF-1, IL-1; IL-4)• Mitochondrial tetrazolium test (MTT)• Vascular Endothelial Growth Factor quantification (supernatant)• Histological analysis of construct (potency)• Functional assessment in athymic mouse model

Used for Comparability

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WCB Qualification TestingTesting performed on cells• Microbiological and viral safety testing• Isoenzyme analysis• Cell growth• Cell viability• Collagen biosynthesis (fibroblast)• Involucrin content (keratinocyte)

Testing performed on constructs made from WCB cells• Histological analysis of construct (potency)

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Product Potency

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Product Potency

Regulatory Requirements:Potency is defined as “the specific ability or

capacity of the product, as indicated by appropriate laboratory tests… to effect a given result.” (21 CFR 600.3(s)).

The applicant proposes to measure Apligraf potency using histological parameters. Scientific discussion from the Advisory Committee is requested.

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Proposed Potency Assay

Hematoxylin and Eosin (H&E) staining to distinguish fibroblasts and collagen within the lower dermal layer as well as keratinocytes within the upper epidermal layer.

The applicant describes the functions of the two layers to be:

• Dermal – both as structural matrix for fibroblasts and substrate for development/maintenance of upper layer

• Epidermal – impart structural elements and contribute to mechanical strength, handling and barrier properties

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* Viability is determined indirectly by using histological assessment of a basophilic cytoplasm, absence of severe vacuolization/necrosis.

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Image from BLA 125400

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Other Testing of Relevance to Product Potency and Function

- used to support histology-based assay- includes in vitro and in vivo non-clinical tests performed for cell bank qualification- not performed on a lot-by-lot basis

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Other Testing Relevant to Potency

• Testing performed on constructs generated from MCB: – Percutaneous water absorption - barrier function– GF/Cytokine profile [PDGF , TGF-1, IL-1, IL-4]– Mitochondrial Tetrazolium Testing (MTT)

• Testing on culture supernatant of constructs from MCB:– Vascular Endothelial Growth Factor (VEGF) levels

* VEGF concentration is believed by the applicant to be indicative of relevant biological factors produced by keratinocytes in the construct.

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Other Testing Relevant to Potency

• An athymic mouse graft model is used to assess comparability of Apligraf performance to that of positive control (construct made with FDA approved cell strains):

– Pre-graft morphology– Graft take and integration– Graft contraction– Graft morphology (epidermal, dermal)– Graft remodeling– Immunohistochemistry (involucrin)

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Product Potency

CBER’s Guidance on “Potency Tests for Cellular and Gene Therapy Products” clarifies that all potency assays used for release testing of licensed biological products must have the following characteristics:

• Indicate potency specific to the product• Provide test results for release of product• Provide quantitative data• Meet pre-defined acceptance and/or rejection criteria• Establish and document the accuracy, sensitivity, specificity and

reproducibility of the test methods employed through validation• Measure identity and strength (activity) of all active ingredients• Provide data to establish dating period• Meet labeling requirements

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Topics for Discussion• Two product quality issues are being posed to the

Committee for discussion:– Current approach to qualify and demonstrate

comparability for new cell banks used for Apligraf manufacture

– Usage of histology as basis for product potency and role of other testing relevant to potency

• Clinical reviewer will discuss other studies of relevance to the potency discussion– Histology, DNA Persistence, angiogenic biomarkers

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Clinical Background

Robert S. Betz, DDS, Captain (Ret.) USPHSDiplomate, American Board of Periodontology

FDA/CDRH/ODE/DAGID

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Periodontal Anatomy

(Drawing adapted from Carranza’s Clinical Periodontology, 11th Edition)

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Periodontal Probing

(Drawings adapted from Carranza’s Clinical Periodontology, 11 th Edition)

Coronal

Apical

Cementoenamel junction

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Keratinized vs. Attached Gingiva

• A – C = Free Gingiva

• A – B = Attached Gingiva

• B – C = Keratinized Gingiva

• C – A = Probing Depth


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The Problem

Mucogingival Defects are soft tissue defects that involve both the keratinized attached gingiva and nonkeratinized alveolar mucosa at the mucogingival junction

• Insufficient Zone of Attached Gingiva (vs Keratinized Gingiva)

• Gingival recession – most frequent occurrence• <1 mm Attached Gingiva• Muscle Pull • Inflammation• Bone Loss• Inflammatory component• Progressive lesion

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Possible Causes And Effects• Toothbrush abrasion• Anatomy/Heredity

• Shallow Vestibular depth• Tooth eruption patterns

• Root prominence• Tooth crowding

• Inability to keep mucogingival junction clean• Thin gingiva

• Inflammatory Factors• Faulty dental restorations (over or under contoured)• Periodontitis• Poor oral hygiene

• Orthodontic Treatment – movement of tooth away from a central location in alveolus to a more facial location.

• Combination of the Above – multiple concurrent etiologies may be present

• Effects – tooth sensitivity, cervical tooth abrasion, root caries, esthetic problems

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Gingival RecessionGingival recession is defined as the location

of the gingival margin apical to the cementoenamel junction. (AAP)

Miller Recession Classification

• Class I: Gingival recession does not extend to the mucogingival junction. Interproximal bone and soft tissues are unaffected.

• Class II: Gingival recession extends to or beyond the mucogingival junction. Interproximal bone and soft tissues are unaffected.

• Class III: Marginal tissue recession extends to or beyond the mucogingival junction. Bone or soft tissue is lost interproximally.

• Class IV: Marginal tissue recession extends to or beyond the mucogingival junction.

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Gingival Recession

(Photograph from Franscesco Cairo et.al. article, 2010 Journal of Periodontology)

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Treatment OptionsAutogenous Grafts

Free gingival GraftsSubepithelial connective tissue graftLateral and rotated pedicle graft procedureDouble papillae pedicle graftCoronally positioned flap procedure These procedures may be performed individually or sequentially. A

popular combination is the subepithelial tissue graft + CPF.

Guided Tissue Regeneration ProceduresApligrafPlatelet rich Fibrin MembraneTunneling ProceduresDental Restorations OnlyDo Nothing

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Apligraf vs. Soft Tissue Autograft

• In the clinical studies, Apligraf was compared to soft tissue autograft (FGG)

• Does not duplicate FGG biological properties.– Connective tissue of FGG survives by “ plasmatic

circulation”.– Apligraf does not “take” like soft tissue autograft.– No Apligraf DNA present after 6 months.

• Cells of Apligraf do not survive beyond 6 months.

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Apligraf Placement

Soft tissue incision at mucogingival junction (Semilunar or with vertical releasing incisions) Releasing incisions were used in the clinical studies.

Apligraf “Z” folded and adapted to fit defect and sutured into place

(Diagrams from BLA submission)

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Treatment Notes• Autogenous palatal graft material was harvested and placed

according to standard soft tissue grafting practices.

• Width of all palate grafts was 5 mm (Study 05) or 4mm (Study 06) with the length dictated by the size of the mucosal defect. Graft thickness is usually 0.75 – 1.25 mm.

• Incisions made to alveolar bone were used to “tack down” both Apligraf and FGG

• Apligraf membranes and FGGs were free of movement as evaluated by muscle traction after placement

• Root coverage was not performed (not studied in either 05 or 06 Study)

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Keratinized vs. Attached GingivaDiscussion Issues

If probing depth is significant, there may be a measurable difference between attached and keratinized gingiva measurements.

Could the change from measuring attached gingiva (05 Study) to keratinized gingiva (06 Study) be clinically significant?

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FIN

Photograph from R. Betz, D.D.S.

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ApligrafBLA 125400

Office of Cellular, Tissue and Gene TherapiesCenter for Biologics Evaluation and Research

Advisory CommitteeCTGTAC Meeting #54November 17, 2011

Agnes Lim, M.D.Bruce Schneider, M.D. (Team Leader)

Clinical Evaluation BranchCenter for Biologics Evaluation and Research (CBER)

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Presentation Outline

• BLA Supported by Two Studies;Overview of study plans• Study 05-PER-001 (05)• Study 06-PER-002-CTX (06)

• Efficacy Results from Studies 05 and 06• Safety Results

• Studies 05 and 06• Additional safety experience from Apligraf for

chronic cutaneous wounds

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Study 05-PER-001 (05)

Study Design

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Study 05-PER-001 (05)

Title

“A pilot Clinical Trial to Assess the Safety and Efficacy of Apligraf in Establishing A Functional Zone of Attached Gingiva”

• Objectives To assess safety and efficacy in establishing a functional zone of attached gingiva

• Study Design

Study 05 was a randomized, single-center, within-subject controlled (graft sites matched for teeth and gingival condition) study; n=25 subjects

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Study 05

Efficacy EndpointsPrimary Endpoint 1) The change in the amount of attached gingiva with Apligraf

compared to control treatment at 6 months

Secondary Endpoints1) Inflammation score 2) Color and texture match of the graft to the adjacent tissue3) Resistance to oral muscle pull4) Probing depth 5) Clinical attachment level 6) Subject preference or satisfaction (including pain

experience) 7) Change in recession depth 8) Width of keratinized tissue

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Study 05

Major Eligibility Criteria• Included

• Adults with an insufficient zone of attached gingiva that required soft tissue grafting

• At least two non-adjacent teeth in contralateral quadrants of the same jaw

• Root coverage was not desired at the time of grafting• Excluded

• Treatment of exposed root is recommended• Acute infection in the areas intended for surgery• Health conditions/medications that could compromise

wound healing --- such as, diabetes, cancer; receiving systemic corticosteroids, immunosuppressive agents

• Current smokers

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Study 05

Study Plan• 25 subjects; Single center • Apligraf vs. soft tissue palatal autograft (FGG)• Within-subject control• 1st three subjects participated as training subjects;

excluded from efficacy analysis, included in safety analysis

• Treatment site and order of treatment were randomized; following randomization, subjects received both a palatal graft and Apligraf

• Neither subject nor Investigator could be blinded• Primary efficacy evaluation at Month 6, with interim visits

at Week 1, Month 1, and Month 3 • 2 adjunct laboratory studies: Histology Study and DNA

Persistence Study

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Study 05

Assessment Method for

Primary Endpoint


• The amount of attached gingiva was evaluated using a calibrated periodontal probe, measured to the nearest 0.5 mm

Attached gingiva

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Study 05

Assessment Methods for Secondary Endpoints

• Color compared to surrounding tissue• More/ Less/ Equally Red; at 1 week, 3 months, and 6 months

• Texture compared to surrounding tissue• More/ Less/ Equally Firm; at 4 weeks, 3 months, 6 months

• Inflammation • Scored by an examiner on a scale of 0 (absence of inflammation) to 4; at Week

1, Months 1, 3, and 6• Probing Depth and KT width measured in mm• Clinical Attachment Level calculated from probing measurements• Resistance to Muscle Pull

• Free gingiva movement when cheek/lip retracted; assessed at 6 months• Patient Satisfaction was based on responses to 2 Questionnaires

• Subject Aesthetics Questionnaire – marked response on a line between “Disappointed” and “Fully Satisfied”

• Subject Discomfort Questionnaire - queried subjects for perceptions of the severity of pain, bleeding, swelling, and sensitivity for the Apligraf site, control site, and palate site. These were rated as None, Mild, Moderate, or Severe.

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Study 05

Statistical Analysis Plan• Primary Efficacy Endpoint:

The absolute change in the amount of attached gingiva over 6 months between Apligraf and Control using a non-inferiority comparison

• 5% significance level• n=22 subjects• Non-inferiority margin was a 1.0 mm difference in

change• Analysis population: Number of subjects enrolled minus

those who participated as training subjects• Secondary Efficacy Endpoints: No detailed statistical

analysis plan for secondary endpoints and no adjustments were made for multiplicity

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Study 05-PER-001 (05)

Efficacy Results

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Study 05-PER-001

• Subject Disposition • 25 enrolled and treated; all 25 completed all

visits

• Demographics• Mean age was 49 years• 17 female (68%); 8 male (32%)• 22 Caucasian (88%); 1 Hispanic, 1 Asian, 1

Middle Eastern

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Study 05

Primary Efficacy Endpoint ResultsAttached Gingiva (mm), n=22

• At Apligraf-treated sites at 6 months, 14/22 subjects (64%) showed an increase in attached gingiva; average increase from 0.30 mm at baseline to 1.14 mm ± 0.37 mm at 6 months

• At Control sites at 6 months, 21/22 subjects (95%) showed an increase in attached gingiva; average increase from 0.27 mm at baseline to 2.71 mm at 6 months

Thus, Apligraf failed to demonstrate non-inferiority to Control for the Primary Efficacy Endpoint

BaselineMean (95% CI)

6 MonthsMean (95% CI)

ChangeMean (95% CI)

Attached Gingiva

Apligraf sites

Control sites

0.30 (0.13, 0.46)

0.27 (0.11, 0.44)

1.14 (0.77, 1.50)

2.71 (2.34, 3.07)

[p<0.001]

0.85 (0.48, 1.21)

2.43 (2.06, 2.79)

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Study 05 Secondary Efficacy Endpoint Results

• Secondary Endpoints1) Inflammation score 2) Color and texture match of the graft to the adjacent tissue3) Resistance to oral muscle pull4) Probing depth 5) Clinical attachment level 6) Subject preference or satisfaction (including pain experience) 7) Change in recession depth 8) Width of keratinized tissue

Results of selected secondary endpoints will be highlighted

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Study 05

Secondary Efficacy Endpoint Results (cont.)

• The Applicant found Apligraf to be superior to Control for the following secondary endpoints

• Tissue color

• Texture matching

• Patient Satisfaction

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Study 05


• There was no difference between the 2 groups in

• Probing Depth • Recession• Clinical Attachment

• There was also no clinically meaningful difference in

• Resistance to muscle pull • Inflammation • Bleeding on probing

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Study 05


Width of Keratinized Tissue (mm), n=22

• Larger increase from baseline to 6 months in the width of KT in Control sites (22/22; 100%) compared to Apligraf sites (21/22; 95%)

• Additionally, at 6 months• At least 2 mm of keratinized tissue width was established in 18/22 (82%)

of Apligraf sites; mean increase was 1.37 mm (0.97, 1.77).• At control sites, 22/22 (100%) established at least 2 mm of keratinized

tissue width; mean increase was 3.33 mm (2.93, 3.74).

BaselineMean (95% CI)

6 MonthsMean (95% CI)

ChangeMean (95% CI)

Width of Keratinized Tissue (KT) Apligraf site

Control site

1.13 (0.92, 1.33)1.24 (1.03, 1.44)

2.50 (2.18, 2.82)4.57 (4.25, 4.89)

1.37 (0.97, 1.77)3.33 (2.93, 3.74)

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Study 05


• The perception of pain at Week 1 was one of the variables in the Patient Satisfaction assessment, as evaluated by the “Subject Discomfort Questionnaire.”

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Study 05


Perception of Pain at Week 1, n=22

At Week 1, more subjects reported severe pain at the Apligraf graft site than at either the Control graft or palate Donor site

Pain at Specified Sites

Grading: Apligraf Control Palate (Donor)

None 3 (13.6%) 2 (9.1%) 8 (36.4%)

Mild 5 (22.7%) 9 (40.9%) 9 (40.9%)

Moderate 9 (40.9%) 10 (45.5%) 4 (18.2%)

Severe 5 (22.7%) 1 (4.5%) 1 (4.5%)

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Study 05-PER-001 Summary of Efficacy Results

• In summary, Apligraf failed to demonstrate success in the non-inferiority primary endpoint of establishing a zone of attached gingiva comparable to that of a control palatal graft.

• There was some indication of success in three (tissue color matching, texture matching, and patient satisfaction) of the eight secondary endpoints. However,• Greater increase in KT width at Control sites• More severe pain at Apligraf sites at Week 1

Results from Study 05 were used to guide the study design for Study 06.

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Study 05 Adjunct Laboratory Studies

• Biopsy/Histology • Tissue specimens from 7 subjects at baseline and at

6 months, taken from both Apligraf and Control sites; histologic evaluation (H&E) to examine the cellular composition and tissue architecture

• 6-month biopsies generally demonstrated the presence of both gingival and alveolar mucosal phenotypes with a transition between these two types at the biopsied sites

• The origins of these cell types were not determined in this study

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Study 05 Adjunct Laboratory Studies

(cont.)• DNA Persistence

• Frozen biopsy specimens obtained at 6 months from 2 of the 7 subjects who participated in the histology study

• The Apligraf specimens, as well as the subjects’ buccal samples were analysed.

• Evaluated by PCR analysis of DNA; samples amplified (Applied Biosystems, AmpFISTR Identiflier™)

• At 6 months, no evidence of allograft DNA persistence at the Apligraf sites

• Control sites were also negative for allograft DNA.

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Study 06-PER-002-CTX (06)

Study Design

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Study 06-PER-002 (06)• Title: “A Clinical Trial to Evaluate CelTx (Apligraf) as an Alternative

to Tissue from the Palate to Enhance Oral Soft Tissue Regeneration and Wound Healing”

• Objective To assess the ability of Apligraf to achieve a “clinically acceptable threshold for keratinized tissue (KT) at 6 months (≥ 2 mm KT)”

• Study Design Study 06 was a randomized, multi-center (4 sites), within-subject controlled (graft sites matched for teeth and gingival condition) study; n=96 subjects

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Study 06

Major Eligibility Criteria• Included

• Adults with an insufficient zone (≤ 1 mm) of attached gingiva that required soft tissue grafting

• At least two non-adjacent teeth in contralateral quadrants of the same jaw

• Root coverage was not desired at the time of grafting• Excluded

• Class III recession in the presence of a shallow vestibule, or class IV recession

• Vestibule depth of less than 7 mm from base of recession• Teeth that have Miller Grade 2 or higher mobility • Acute infection in the areas intended for surgery • Health conditions/medications that could compromise wound

healing --- such as diabetes, cancer; receiving systemic corticosteroids, immunosuppressive agents

• Current smokers

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Study 06

Efficacy EndpointsPrimary Endpoint 1) Proportion of subjects with KT ≥ 2mm at the Apligraf-treated

sites vs. 50% success rate (single arm comparison)

Secondary Endpoints1) Color same as adjacent tissues 2) Texture same as adjacent tissues 3) KT ≥1 mm for Apligraf after 6 months (compared to an 80%

success standard)4) Patient Preference5) Surgical Site Sensitivity mild or absent after 1 week6) Pain absent after 3 days

5/6 secondary endpoints were Apligraf compared to Control

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Study 06

Study Plan• 96 subjects; four investigational sites • Apligraf vs. soft tissue autograft (FGG)• Within-subject (split-mouth) control• 1st two subjects per Investigator participated as training

subjects; excluded from efficacy analysis, included in safety analysis

• Treatment site and order of treatment were randomized; following randomization, subjects received both a palatal graft and Apligraf

• Neither subject nor Investigator could be blinded• Primary efficacy evaluation at Month 6, with interim visits

at 48 and 72 hrs, 1 and 2 weeks, Month 1, and Month 3 • Adjunct laboratory study: Angiogenic biomarkers

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Study 06 Study Plan Assessment of Primary Endpoint

• Width of keratinized tissue (KT) was measured by examiner • at Months 3 and 6 using the "roll technique."• Schiller's iodine was applied to the oral

mucosa to aid in visual differentiation between keratinized and non-keratinized areas.

• Measurements taken with a UNC-15 probe and rounded to the nearest 0.5 mm.

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Study 06 Study Plan Assessment of Secondary Endpoints• Assessments of color and texture performed at Months 1, 3 and 6

and were rated as either• less red/firm than adjacent mucosa• the same color/firmness, or • more red/firm than adjacent mucosa

• Patient Preference was assessed at Month 6 by asking subjects to respond to the question “Taking into account all aspects of treatment (surgery, recovery, and appearance) which treatment is preferred?” ;

• Surgical site sensitivity (none, mild, moderate, severe) assessed at Week 1 using a 3-second puff of air and the subject was asked (none, mild, moderate, severe) to report the amount of sensitivity at each site (Apligraf-treated, palatal-harvest, and control-treated)

• Pain and general sensitivity were recorded on a study-provided diary each day from Day 1 to Day 14.

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Study 06

Statistical Analysis Plan

• Primary Endpoint: The superiority of Apligraf relative to a pre-defined standard (50% success) for a 2 mm KT threshold after 6 months

• 5% level of significance• Analysis Population for effectiveness (n=85)

• All 96 subjects enrolled completed all required visits.• 11 subjects participated as training subjects – not

included in efficacy analysis.

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Study 06-PER-002-CTX (06)

Efficacy Results

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Study 06-PER-002 (06) • Subject Disposition

• 119 screened; 96 enrolled and treated. • All 96 completed all required visits.

• Demographics • Mean age was 47 years • 52 female (54%); 44 male (46%)• 87 Caucasian (91%); 1 African American, 5 Asian, 3

Others

(Note: Of the 96 subjects, 11 participated as training subjects and were not included in the efficacy analysis cohort; n=85 for efficacy analysis)

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Study 06

Primary Efficacy Endpoint Results• 81/85 subjects (95.3%) met success criterion

of ≥ 2mm KT at the Apligraf site at 6 months in at least 50% of subjects

• Exact binomial 95% CI interval was 88.4 - 98.7%.

• All 11 subjects in the training cohort also met the primary endpoint at the Apligraf-treated site.

• All 96 subjects met the primary endpoint at the Control site.

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Study 06

Secondary Efficacy Endpoint ResultsSequential order of testing pre-specified for secondary endpoints

• Apligraf was statistically superior to Control for 3 of the 4 secondary endpoints and met criteria for the fourth (KT ≥ 1mm success rate):1) Color matching 2) Texture matching3) KT ≥ 1 mm success rate (greater than the 80%

success standard) 4) Patient Preference

• No significant difference between Apligraf and Control in surgical site sensitivity (the 5th secondary endpoint)

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Study 06 (cont.)

Secondary Efficacy Endpoint Results• Therefore, the 6th secondary endpoint, the absence of pain

after 3 days between Apligraf and control sites, was not tested. Although it was not tested statistically, results showed no important differences between Apligraf and Control sites

Pain at Day 3 (n=84)

Pain assessment at Control reflects the most pain at either donor or control site; Pain = moderate or severe, No pain = mild or none

Apligraf

Control No Pain Pain

No pain 54 7 Pain 5 18

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Study 06

“Other Effectiveness Endpoints”• At FDA’s request, the applicant also provided summaries

and post-hoc statistical analyses of other effectiveness endpoints; Changes from baseline to Month 6 were assessed in each of these endpoints.

• Recession Depth• Recession (%) • Probing Pocket Depth• Clinical Attachment Level • Keratinized Tissue Width• Attached Gingiva • Bleeding on Angulated Probing • Muscle Pull Resistance • Plaque scores• Inflammation Score • Bleeding and Swelling

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Study 06

“Other Effectiveness Endpoints”Recession Depth• Recession (%) • Probing Pocket Depth• Clinical Attachment Level Keratinized Tissue (KT) WidthAttached Gingiva • Bleeding on Angulated Probing • Muscle Pull Resistance • Plaque scores• Inflammation Score • Bleeding and Swelling

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Study 06

“Other Effectiveness Endpoints”• For the Apligraf-treated site, there were

improvements from baseline to Month 6 in • Recession depth • KT width • Attached gingiva width– No other endpoint yielded clinically important changes

from baseline to Month 6. • For the Control site, there were improvements

from baseline to Month 6 in • KT width• Attached gingival width. – There was no clinically important improvement in

recession depth.

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Study 06 (cont.)

“Other Effectiveness Endpoints”Results comparing Apligraf and Control sites for KT width and attach gingiva

• The Control site had greater KT width and attached gingival width than the Apligraf site at 6 months

• There was no other difference between Apligraf and Control for the ‘other effectiveness endpoints’ at 6 months.

KT Width (mm)Mean (SD)

Attached Gingiva (mm)Mean (SD)

Apligraf site

Baseline

6 months

1.41 (0.72)

3.21 (1.14)

0.02 (0.76)

1.77 (1.32)

Control site

Baseline

6 months

1.43 (0.69)

4.57 (1.00)

0.08 (0.79)

3.17 (1.17)

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Study 06-PER-002 Summary of Efficacy Results

• In summary, the study met its primary endpoint, KT ≥ 2 mm at 6 months in at least 50% of subjects.• 81/85 subjects (95%) met success criterion at the

Apligraf site.

• Apligraf met its secondary endpoints for color matching, texture matching, KT ≥ 1mm, and patient preference; but it did not meet the 5th secondary endpoint (sensitivity), and pain was not tested due to prespecified fixed testing sequence.

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Study 06 Adjunct Laboratory Study

• Angiogenic biomarkers • Wound fluid samples from Apligraf- or FGG-treated

sites at 1, 2, 3, and 4 weeks post-treatment from 29-44 subjects

• Analyzed with human angiogenesis array kit for an array of proteins, such as angiogenin (ANG), fibroblast growth factor-2 (FGF-2), etc.

• No biomarker correlations to the quality of healing results (i.e., color, texture, pain and inflammation) in either treatment group

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Safety Analysis of

Apligraf

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Safety Database• Safety database derived from three clinical

studies • 05-PER-001 (n=25)• 06-PER-002-CTX (n=96)

(07-PER-004-CTX; “submerged” study; n=15)

• Pre-marketing pivotal studies• Venous Leg Ulcers• Diabetic Foot Ulcers

• Post-marketing data• FDA MAUDE database

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Safety Results Studies 05, 06, and 07

• Study 05 (n=25): No SAEs, deaths; no related AEs; no infection at wound sites; no evidence of clinical immune response

• Study 06 (n=96):• 3 SAEs

- pneumonia, chest pain (unrelated)- metastatic malignant fibrous histiocytoma (unlikely)

• Additional non-oral neoplasm (follicular thyroid neoplasm; Hurthle cell lesion) not reported as SAE (unrelated)

• Local AEs by Apligraf, Control, and Donor sites unremarkable

• Study 07 (n=15): No important AEs identified

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Apligraf for Cutaneous WoundsPre- and Post-market Data

• Pre-market Studies Total of 273 subjects treated with Apligraf for both studies:• Venous Leg Ulcers (VLU)• Diabetic Foot Ulcers (DFU)

• Post-market Data (MAUDE)• Since approval in 1998 for VLU, > 400,000 units

shipped for patient treatment• Total 9 medical device reports (MDRs) submitted to

FDA MAUDE safety database• No allergy or acute rejection of Apligraf• No malignancy attributed to Apligraf

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Topics for Discussion

• Effectiveness

• Patient population

• Safety

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Thank you

1 Cellular, Tissue and Gene Therapies Advisory Committee Meeting Apligraf Allogeneic Cultured...

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