Dr. Joel Aronowitz — CLINICAL PROTOCOL

Dr. Joel Aronowitz — Clinical Research

Cell Assisted Lipotransfer (CAL) Versus Non-Cell Enriched Autologous Fat Grafting (AFG) for Soft Tissue Augmentation in Non Diseased Tissues

Investigator: Dr. Joel Aronowitz

Protocol: Version: 0001

Sponsor: Dr. Joel Aronowitz

The information contained herein is confidential and proprietary in nature, and will not be disclosed to any third party without written approval of authorized designee. This document may be disclosed to the appropriate institutional review boards or to duly authorized representatives of the US Food and Drug Administration or a national regulatory authority under the condition that they maintain confidentiality.

STATEMENT OF COMPLIANCE

This study will be conducted in compliance with the protocol, International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), Good Clinical Practice (GCP), and the applicable regulatory requirements, United States Code of Federal Regulations (CFR) Title 45 CFR Part 46 and Title 21 CFR Parts 50, 56, and 312.

INVESTIGATOR’S AGREEMENT

I have read the attached protocol entitled, “Cell Assisted Lipotransfer (CAL) Versus Non-Cell Enriched Autologous Fat Grafting (AFG) for Soft Tissue Augmentation in Non Diseased Tissues” dated TBD (Version 1.0) and agree to abide by all the described protocol procedures. I agree to comply with the World Medical Association Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Subjects, the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Good Clinical Practice (GCP) guidelines for the scientific quality standard for conducting, recording and reporting trials that involve the participation of human subjects, applicable U.S. Food and Drug Administration (FDA) regulations and guidelines identified in the Code of Federal Regulations (CFR) Title 21 Parts: 11 (electronic records), 50 (protection of human subjects), 54 (financial disclosure by clinical investigators), 56 (institutional review boards), and section 314.126 (adequate and well-controlled studies), local Institutional Review Board (IRB) guidelines and policies, and the U.S. Health Insurance Portability and Accountability Act (HIPAA).

Print Site Principal Investigator Name: Joel Aronowitz, MD

PROTOCOL SYNOPSIS

STUDY TITLE

Cell Assisted Lipotransfer (CAL) Versus Non-Cell Enriched Autologous Fat Grafting (AFG) for Soft Tissue Augmentation in Non Diseased Tissues

CLINICAL PHASE

Phase II

STUDY OBJECTIVES

To assess the safety and efficacy of cell assisted lipotransfer (CAL) autologous fat grafting compared to non cell enriched autologous fat grafting (AFG) in soft tissue augmentation of non diseased tissues.

STUDY RATIONALE

Fat grafting by means of subcutaneous injection of autologous fat obtained by lipoaspiration, is an implant-free alternative for soft tissue augmentation. A variety of clinical conditions are currently being treated with this widely used technique. For example, soft tissue contour depressions caused by prior surgery or trauma as well as significant asymmetry due to natural variation are often improved with autologous fat grafting. The utility of the AFG procedure is limited however by unpredictable volume retention and inconsistent results. One promising strategy proposed to improve reliability is based on the observation that pluripotential cells which reside in peripheral fat play a key role in engraftment and adipose tissue regeneration after grafting. These cells, commonly known as adipose derived stem cells (ADSC) are depleted by the liposuction procedure used to harvest fat graft material. Enriching fat graft lipoaspirate with autologous ADSC’s isolated from additional lipoaspirate, a process known as Cell Assisted Lipotransfer (CAL) provides the rationale for the present study. CAL is a practical AFG modification that can be performed at the point of care as a single surgical procedure without storage or expansion of the autologous cells. The technique is supported by numerous preclinical studies and clinical studies that demonstrate improved long-term recipient site volume retention when compared to unenriched fat graft and a safety profile comparable to AFG alone. Many studies published in peer reviewed journals and in academic presentations utilize the Cytori Celution device to isolate adipose pluripotential cells. The Cytori device is an enclosed system that performs the full cycle of fat digestion, washing and ADSC concentration in an efficient and autonomous device. The performance of the device is well documented and supported. There is a lack of clinical data however to build a consensus regarding several important parameters in the treatment process. One important undetermined parameter is the optimal number of viable pluripotential adipose cells relative to the total fat graft volume for specific soft tissue defects in otherwise non diseased tissue. This study intends to add reliable and replicable data to the literature to support evidence based decision making for future acceptance and refinement of the AFG procedure.

STUDY POPULATION

Males or females of any race, ages 21–65 who require adipose soft tissue augmentation.

MAIN INCLUSION/EXCLUSION CRITERIA

Inclusion:

All subjects may be included provided they meet the following inclusion criteria:

1. Subjects must consent in writing to participate in the study by signing and dating an informed consent document indicating that the subject has been informed of all pertinent aspects of the study prior to completing any of the screening procedures.
2. Males or females of any race, age 21–65 who require adipose soft tissue augmentation.
3. Subjects must have a need for augmentation of undiseased, non radiated soft tissue, i.e., peripheral adipose tissue paucity that is the result of senescence, trauma, surgery, natural variation or asymmetry between right and left sides and an otherwise normal physical exam with no abnormal subcutaneous masses, discharge, fibrocystic disease, adenopathy and/or history of abnormal or diseased tissue such as Romberg’s hemifacial atrophy, scleroderma and mixed connective tissue disorder.
4. Subjects be at a stable weight and not be fluctuating in their weight more than 10 lbs over the prior 6 month period, to avoid distortion of soft tissue volumetric measurements of graft and donor site volumetric size post operatively.
5. Subjects must also consent to be photographed before and after the procedure and at the end of the study.
6. All subjects in the study must also consent to undergo ultrasound imaging prior to the procedure and after the procedure to screen the treatment area preoperatively and assess and treat any oil-filled cysts that may arise due to the procedure.
7. All subjects in the study must also consent to allow their results to be included in any scientific research, scientific publication, or presentations at scientific meetings.
8. All subjects in the study must also consent to allow their photos to be used for publication in scientific journals, internet websites, and subject educational material such as brochures, before and after photos, and subject education videos.
9. Subjects must not be anemic at the time of the procedure (Hg <10). If they are anemic at the time of enrollment, they can still be included in the study if the anemia is corrected pre operatively with iron supplementation. This must be continued during the study to avoid any adverse reporting that may not be related to the procedure (i.e. anemia not caused by SVF enriched fat grafting).
10. Subjects must have normal preoperative screening lab studies such as CBC and U/A required by the facility or surgeon.

Exclusion:

Subjects presenting any of the following will be excluded from the study:

1. Subjects with an abnormal soft tissue exam systemically or diseased tissue on exam of the donor or recipient site.
2. Subjects with a bleeding disorder who are on anticoagulants.
3. Subjects who are anemic despite iron supplementation and treatment of the underlying cause of the anemia.
4. Subjects with fibromyalgia, regional pain syndrome, or chronic fatigue.
5. Subjects with a bleeding diathesis.
6. Subjects with urinalysis or symptoms indicating urinary tract infection.
7. Subjects whose diabetes is not adequately controlled (HgA1c > 7).
8. Subjects with a history of local anesthetic allergy.
9. History, diagnosis, or signs and symptoms of clinically significant psychiatric disorder, including but not limited to:

● Severe psychological disease (e.g. schizophrenia, manic-depressive disorder, severe depression, etc).

● Body dysmorphia syndromes, i.e., anorexia, bulimia, etc.

● Somatoform disorders

1. Potential research participants with a substance abuse (e.g. alcoholism, medical narcotics (morphine, Vicodin, codeine, Percodan, etc.) illicit drug, prescription medications without a valid prescription, etc.) within 2 years of screening, and tobacco habit.
2. Research participants who are or have been receiving immunosuppressants such as Cyclosporin A or azathioprine within the past six weeks.
3. Research participants on anticoagulants that cannot be stopped or corrected.
4. Use of biologics, e.g. TNF-inhibitors, such as adalimumab, etanercept, infliximab, including any live vaccines within 3 months of the initial pain assessment period.
5. Research participants who are taking anticoagulants such as coumadin, fixed dose, non fractionated heparin or low molecular weight heparin (Lovenox).
6. Oral or parenterally administered systemic corticosteroids within 30 days prior to the initial pain assessment period.
7. Individuals largely or wholly incapacitated, e.g. bedridden or confined to a wheelchair, permitting little or no self-care.
8. Pregnant women, lactating mothers, women suspected of being pregnant, subjects with a positive urine or blood pregnancy test, women who wish to be pregnant during the course of the clinical study.
9. Research participants who are presently or have been enrolled in other clinical trials within the past four weeks.
10. Subjects with prior radiation or direct chemotherapeutic infusion to the donor or recipient site may not be included in the study.

ENDPOINT (S)

1. Primary Endpoint: Improvement in long-term volume retention. i.e. minimum 6 months post-op, when using cell assisted lipotransfer technique compared to regular fat grafting, as evaluated by post-op volumetric measurement of the donor site using a Vectra 3D photo system with a volumetric algorithm. The quantitative volume tracking over time is based on comparison of the CAL versus unenriched AFG treatment areas assessed over time to compensate for changes in body weight and fluid balance.
2. Secondary Endpoint: Safety, as evaluated by Adverse Events and Serious Adverse Events

STUDY DESIGN

A Phase II, single center, unblinded, safety and efficacy study comparing results of the unmodified AFG versus the grafting process using CAL. Subjects meeting all Eligibility Criteria and providing Informed Consent and will be given the option of participating in Group A or Group B until either group has met their enrollment number. At that point, subjects would be placed in the remaining group.

1. Group A will be composed of subjects with bilateral, i.e., right and left, fat grafting recipient areas. Subjects will receive enriched fat graft material in one of two dosages (high: 1.0 x 106 cells/grams of fat, Group Ah and low: 0.5 x 106 cells/grams of fat, Group Al) to one soft tissue site and opposite bilateral side will be treated with AFG only, Group A control and the other soft tissue site with CAL, Group Ah or Al. The AFG tissue will be the control and those receiving CAL grafting will be considered the experimental side. N = 80 subjects.
2. Group B subjects will compare non cell enriched AFG to CAL in different subjects with unilateral recipient sites. In this study, the subject can choose to have CAL soft tissue augmentation. The two subject groups will be compared. Here the group with AFG soft tissue augmentation sites will be the control and subjects with CAL soft tissue augmentation sites will be the experimental group. N = 60 subjects.

SUBJECT NUMBER

N = 140 total subjects

N = 80 in Group A (bilateral soft tissue augmentation with self control)

N = 60 in Group B (unilateral soft tissue augmentation: SvF-enriched AFG experimental with AFG control)

TREATMENT DURATION

Treatment occurs in a single outpatient surgical setting with the operation duration expected to be 3 to 4 hours.

DURATION OF FOLLOW-UP

Systematic clinical assessments will be performed at intervals over 12 months post-operatively.

DOSE LEVEL (S) AND DOSE JUSTIFICATION

The total volume of CAL injected will be based on the subject’s desired size of soft tissue augmentation, on the clinical requirement as quantified by preoperative Vectra 3-D contour analysis and determined by the operating surgeon. Graft volumes are anticipated to be 50 to 400mL. Group Ah high dosage CAL and Group B subjects will undergo CAL with a graft containing at least 1.0 x 106 cells/grams of fat, isolated from up to 400mL of autologous lipoaspirate obtained at the same surgery using the Cytori Celution Device. Subjects in Group Al receiving a low dosage of CAL will receive a graft containing between 0.5 x 106 cells/grams and 0.7 x 106 cells/grams of fat, isolated from up to 400mL of autologous lipoaspirate obtained at the same surgery using the Cytori Celution Device.

ROUTE OF DELIVERY

Injection using a blunt fat grafting injection cannula introduced in the subcutaneous plane.

STOPPING RULES

1. Subject mortality or a critical medical event such as stroke, sepsis, or pulmonary thrombosis either related or unlikely related to investigational treatment

CLINICAL ASSESSMENTS

Physical examination of the soft tissue depression via volumetric assessment using Vectra 3 D photo documentation, reported in cm3.

CAL PREPARATION

After induction of anesthesia

1. Surgical site is prepped and draped in sterile fashion
2. Infusion of 1000cc of tumescent solution to areas to be harvested using a small infiltration cannula
3. 3.5 mm blunt tip cannula is introduce through a stab incision and the liposuction is performed with a low vacuum less than 28 mmHg
4. Lipoaspirate is inserted into the Tissue Collection Container of the Cytori 800/CRS system where the tissue is then washed with lactated ringer solution. The appropriate amount of Celase is added to begin the process. Cytori 800/CRS system completes two full centrifugation cycles of pellet wash. A 10 mL syringe with an 18 gauge x 3–1/2” needle is used to slowly aspirate the solution into and out of the syringe three times, in order to re-suspend the nucleated cell output.
5. Count the cell output from Celution device and adjust dose if required
6. Nucleated cells are added to fresh lipoaspirate fat graft and injected to the soft tissue recipient site in a careful grid like pattern with small ribbons of graft to assure maximum graft take, using a blunt tipped injection cannula of appropriate size for the injection site.
7. Surgical incisions are closed with a small absorbable suture

AFG PREPARATION

After induction of anesthesia

1. Surgical site is prepped and draped in sterile fashion
2. Infusion of 1000cc of tumescent solution to areas to be harvested using a small infiltration cannula
3. 3.5 mm blunt tip cannula is introduce through a stab incision and the liposuction is performed with a low vacuum less than 28 mmHg
4. Lipoaspirate is decanted into a sterile container and quantified
5. Lipoaspirate is injected to the soft tissue recipient site in a careful grid like pattern with small ribbons of graft to assure maximum graft take, using a blunt tipped injection cannula of appropriate size for the injection site.
6. Surgical incisions are closed with a small absorbable suture

STATISTICAL ANALYSIS PLAN

The collected data for CAL grafting will be compared with contra-lateral AFG soft tissue augmentation of the same individual for Group A study. For the Group B study, the data for CAL fat grafting will be compared with that of the AFG alone control group. All statistical analyses will be performed using Student’s t test and P<0.05 will be defined as significant.

OUTCOME CRITERIA

1. Volumetric analysis of soft tissue
2. Postoperative sequelae and complications

RISKS

The usual risks associated with liposuction and autologous fat grafting under local or IV sedation anesthesia, including donor site contour irregularity, recipient site oil cysts, fat nodules and infection. Side effects of medications used in AFG surgery:

● Pain medications: sleepiness, nausea, constipation, difficulty breathing

● Nausea medications: abnormal movements, headache, dizziness, diarrhea

● Medications used to treat constipation: abdominal cramps, diarrhea

● Muscle relaxants: difficulty breathing, slowing of heartbeat

● Blood clot prevention medications: bleeding including mild or serious blood from the surgery and drug delivery system used during surgery:

● Pain around the operative site

● Nausea and vomiting

● Headache, which may be severe and last up to 1 week post-operatively

● Temporary slowing or stopping of normal intestinal movement associated with postoperative narcotic pain medication

● Pain or irritation or infection of the urethra due to indwelling Foley (bladder) catheter

● Infection of skin, near the site of the surgery

● Damage to soft tissues surrounding the surgery site

● Separation of the edges of the 2–3 mm surgical injection wound that might require minor surgery

● Infection in the blood (bacteremia)

● Bleeding which cannot be controlled

SCHEDULE OF EVENTS

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