GAP ANALYSIS — DR. JOEL ARONOWITZ

Joel A. Aronowitz, MD

Background:

Chronic wounds negatively affect the quality of life of approximately 6 million people in the United States1. The term chronic wound refers to a wound that does not heal in the expected way or within three months using conventional wound treatments. A chronic wound can be recognized by a number of symptoms, including the loss of skin and/or tissue surrounding the wound, or by the amount of time it takes to heal. These chronic wounds are most often the result of the interplay of systemic factors such as diabetes and local factors such as arterial insufficiency, venous congestion and trophic changes1,2.

Diabetes is a chronic disease that affects roughly one-third of the adult population in the United States3. Patients with diabetes are especially vulnerable to the development of lower extremity chronic wounds. The current treatments available for diabetic chronic wounds usually involves systemic glucose control, ensuring adequate extremity perfusion, debridement of nonviable tissue, off-loading, control of infection, local wound care and patient education administered by a multidisciplinary team4. It has been reported that about fifteen percent of diabetic patients will develop a foot ulcer in their lifetime, and approximately fifteen to twenty percent of these ulcers will result in lower extremity amputation5. These amputations result from complications of diabetes that make it difficult for wounds to heal, such as neuropathy, impaired immunity and vascular deficits.

Wound healing is a complex multifactorial process involving the interaction of inflammation, granulation tissue formation, re-epithelialization, and angiogenesis6. Unfortunately, most of the available treatments mentioned are limited in effectiveness and are often not sufficient to guarantee adequate healing6. The standard rate of healing is low for chronic wounds, only 24% or 30% of diabetic foot ulcers will heal at weeks 12 or 20 respectively7. Untreated chronic wounds run the risk of infection, further tissue and bone damage, and pain that collectively may result in the need for amputation as mentioned earlier. As a result, there is a growing need to develop improved techniques in chronic wound management, specifically techniques based on the concept of converting a chronic wound into an acute wound.

Adipose-derived stem cells (ASCs) are of emerging interest in the application of wound healing due to their prolonged self-renewal capacity and their ability to proliferate and induce differentiation into various cell types6,8. Recent studies have suggested that human adipose-derived stem cells (ASCs) may play a supportive role in wound healing by converting chronic wounds into acute wounds through the formation of vascular structures via direct and indirect mechanisms8. ASCs promote wound healing by increasing vessel density, granulation tissue thickness and collagen deposition while simultaneously improving the cosmetic appearance of the resulting scar9. Additionally, ASCs secrete nearly all of the growth factors that are involved in normal wound healing, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), PDGF and TGFb1 which promote angiogenesis and accelerate wound healing9. ASCs are an advantageous resource because they can be found in abundant quantities, they can be harvested with a minimally invasive procedure, they can be differentiated along multiple cell lineage pathways, they are immunocompatible, and they can be safely and effectively transplanted into an autologous or allogeneic host10.

In an attempt to explore the contribution of ASCs to chronic wound healing, we will investigate the effects of injecting autologous ASCs into the periphery and debrided surfaces of chronic wounds. Our goal is to achieve healing in two months, and for the wounds to stay healed for the following two weeks.

Project Goal: Submission of an IND to support clinical investigation of a single treatment of autologous adipose derived regenerative cellular therapy in chronic wounds for diabetic ulcers. ADRCs will be delivered in multiple injections within and in the immediately surrounding the wound. Cells will be delivered using a 1cc syringe with an appropriate gauge and length needle. Each injection will have a volume of ≤ 250µL. The number of injections will be determined by the surgeon as a function of total wound volume.

Proposed Clinical Approach:

1. Adipose Derived Regenerative Cellular Therapy of Chronic Wounds

2. To compare outcomes between autologous fat grafting (AFG) versus autologous fat grafting enriched with adipose derived stromal vascular fraction (SVF) for breast augmentation.

Preclinical Data

A large number of studies have described the ability of freshly isolated adipose cells and cultured adipose stem cells to improve healing in several small and large animal preclinical models of cutaneous wound healing including normal animals, diabetic mice, irradiated skin, mitomycin-treated wounds, and ultraviolet-treated skin (Table 1). All investigators report improved wound healing parameters including increased wound closure rate, increased granulation tissue, and improved wound vascularity and perfusion.

Previous Clinical Studies

To date, approximately 5,000 patients have been treated with autologous ADRCs isolated in the Celution® System. These patients range in clinical indication from cardiovascular disease to soft tissue reconstruction needed post-breast cancer therapy. Recently, a case study was published showing the complete healing of a chronic, irradiated pressure sacral ulcer in an 89-year-old Japanese patient30.

In addition to a wealth of preclinical data, there are also a number of clinical studies which have demonstrated the efficacy of using mesenchymal stem cells for the treatment of ulcers and chronic wounds of the lower limbs. Studies showed that treatment with cultured and uncultured ADSCs improved wound healing and promoted vascularization and reepithelialization of ulcers. Dosages ranged from 1.5 million cells to 100 million cells.

Overall, there were very few adverse events associated with the use of autologous stromal vascular fraction cells. Adverse events reported include headache, mild fever, flu­like symptoms, hematoma and injection site pain. The use of cultured adipose­derived stem cells and uncultured stromal vascular fraction cells was determined to be very low risk in terms of safety for use in human subjects.

Chemistry Manufacturing and Controls (CMC) Data

The adipose­derived stromal vascular fraction (SVF) is acquired from fat tissue harvested through tumescent liposuction and cells are isolated by treating the aspirated fat tissue, or lipoaspirate, with collagenase and dispase (proteolytic enzymes) to break up the tissue.

Adipose tissue harvest is done under IV sedation or general anesthesia in the operation room. A standard formula tumescent solution with local anesthetic (lidocaine 1% with 1/100,000 epinephrine and Marcaine 0.5% with 1/200,000 epinephrine) is injected in the harvest sites. A non­automated, collagenase­based isolation is then performed by a trained technician at the point of care using aseptic techniques within the confines of a sterile, laminar flow biohood equipped with a centrifuge and a heated shaker. Volume of lipoaspirate digested can range from 50mL to 800 mL. Lipoaspirate is first washed 3 times using Lactated Ringer’s solution (B. Braun Medical Inc. Irvine, CA) (130 mmol/L Na+, 109 mmol/L Cl­, 28 mmol/L lactate, 1.5 mmol/L Ca2+, 4 mmol/L K+) over a period of 10­15 minutes to remove contaminating blood cells, tissue debris and tumescent solution. Washed lipoaspirate is then incubated with 35 Wünsch Units (U) of collagenase per 50 mL of tissue. Lipoaspirate is incubated with CIzymeTM AS (Vitacyte, LLC, Indianapolis, Indiana), a mixture of clostridial collagenase type I and type II (60% type I,40% type II) and neutral protease from B. polymyxa resuspended in warm (37 ºC) lactated Ringer’s solution in a temperature controlled shaker at 200 rpm for 20­30 minutes at 37 ºC. SVF cells are separated from the digested lipoaspirate via centrifugation at 2,000 rpm for 10 min. Isolated SVF cells are then strained through a 100 um cell strainer to remove large tissue fragments and washed a minimum of 3 cycles using lactated ringer’s solution to remove residual enzyme. The SVF preparation is brought to a final volume of 10mL using lactated Ringer’s solution and returned to the surgeon for injection to the patient. The total manufacturing time is approximately 60 to 90 minutes.

Total Nucleated Cell Yield and Viability Assessment: A small sample of SVF cells (0.1­0.2 mL) is analyzed using a cell counting device (Chemometec NC 200, Chemometec A/S, Davis, CA) to determine the cell yield and viability. The table below summarizes the cell count data obtained from 33 patients treated who had between 100­200 mL (average=146 mL) of adipose tissue submitted for processing (see Section 7. Clinical Safety Information). For our proposed study, any isolate which presents with fewer than 10 million total nucleated cells would not be suitable for treatment. Additionally, any sample which resulted in a viability below 70% would not be used for treatment. The minimum acceptable dosage for treatment is proposed to be 10e6 nucleated SVF cells.

Sterility Testing: A sample of the final product (2mL) will be sent to a nearby microbiology lab for sterility testing. Sterility testing will include a gram stain and a long term culture for 2 weeks using a blood culture system (ie BacT/Alert 3D system, bioMerieux, Inc., Durham, NC) to test for contamination by aerobic and anaerobic microorganisms. A 1 mL sample will be inoculated into Tryptic Soy broth (TSB) for aerobic culture, and 1mL will be inoculated into Fluid Thioglycolate media for anaerobic cultures.

Sample Characterization: The stromal fraction of the cell population will be characterized using flow cytometry. Surface antigens tested will include CD31, CD34 and CD45. The population of interest is the fraction of cells which display a CD31­/CD34+/CD45­ phenotype. In 2013, the International Federation of Adipose Therapeutics and Sciences (IFATS) and the International Society of Cellular Therapy (ISCT) issued a joint position paper in which they described the resident cell populations found in the stromal vascular fraction from adipose tissue (Table 3) [41]. For our method, this is between 5% and 15% of the total nucleated cell population is stromal cells; however the exact number varies based on a variety of factors including, tissue harvest method, method of isolation, and amount of patient bleeding (blood contamination). The SVF contains the progenitor cells of interest (adipose­derived stem cells) and a variety of other cells including white blood cells, macrophages, pericytes, vascular endothelial cells and a few others. It has been characterized, but it tends to vary in the frequency of each cell type from patient to patient. The population of adipose­derived stem cells can vary from about 1% to 10% of the final cell population.

The StemSource System is an automated, closed processing system that can isolate a heterogeneous population of regenerative cells from adipose tissue in real-time at the patient’s bedside for autologous therapy. This is an approved device and is covered by a 510(k).

Regulatory Assessment

Per the FDA guidance, “Regulatory Considerations for Human Cells, Tissues, and Cellular and Tissue-Based Products: Minimal Manipulation and Homologous Use — Guidance for Industry and Food and Drug Administration Staff” presents the following flow-chart to determine the regulatory path:

Two important points:

· Degree of manipulation of the SVF cells exceeds that outlined for minimal manipulation, and

· This product is not homologous as defined by FDA, “Homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor (21 CFR 1271.3(c)), including when such cells or tissues are for autologous use.”

Cedars-Sinai Biomanufacturing Center (CBC) Summary and Recommendations:

After review of the presented data (FDA Briefing book and the clinical protocols), the CBC Team has the following recommendations on how to move the project forward. Given that we do not meet the criteria for exemption under 21 CFR 1271 we will need to pursue and IND to formalize the clinical study.

We recommend approaching FDA with a more concise plan to get the clinic and with a more detailed CMC section. To communicate with FDA we will need to address the following:

Clinical

Build a defined and focused clinical protocol. We suggest targeting wound healing for diabetic foot ulcers as the first clinical indication.

• In the clinical protocol we should pick a quantifiable criterion for improvement of the chronic wounds.
• What is the patient population — data presented was in diabetic patients?
• Number of patients.
• Do we need to control what type of diabetes?
• Duration of diabetes
• Wound size — smaller vs. larger
• Duration of wound?
• Specify the dosage: Determine the dosage range — how many cells per 0.25 mL.
• How many injections will be administrated per wound (a determination method of injection number per wound should be established based on the size of wound)

Manufacturing — Process Refinement

Regardless of whether enzymatic (current procedures) or automated mechanical methods for isolating SVF (Cytori Celution device). We would prefer to move forward with process refinement utilizing the Cytori cell separation device to isolate SVF in this protocol. This is the best choice for isolating SVF as this device is already cleared by FDA for such use.

We need more clarity on the delivery methodology of the SVF cell product to patients — consider collagen patch vs. direct injection. Are their Cedars-Sinai proprietary methods that can be leveraged versus other competitors who use similar SVF cell population and cell separation devices?

I. If the cells are expected to have metabolic activity to actively help the wound to heal, then we will need to file and IND with the FDA and proceed with that regulatory oversight. Note, even with an IND in place we will still need to have Cedars IRB approval.

In addition to the refined protocol we will need to pull together a more robust manufacturing (CMC — Chemistry, Manufacturing and Controls) process and documentation.

We will need to

• better characterize the final product and
• understand the mechanism of improvement of wound healing imparted by the autologous adipose derived stromal vascular fraction (SVF) cell product.

Develop a more detailed manufacturing process — additional process development may be needed in the future and can be used to address some safety concerns by using the product generated in this testing in an appropriate animal model

• Qualified procedures including all input materials used and any devices
• The process should demonstrate the consistency and reproducibility of the final product.
• All materials/reagents used in the process should be qualified for use of GMP grade production.
• Preparation of standard operating protocols (SOPs) for isolation of SVF
• Detailed collection of the raw material — adipose tissue and cleaning process.
• Need qualified procedures
• Testing to be done against specification
• Stability testing at each stage of the process needs to be performed.

Generate Release specifications for the cells
A procedure should be in place for the production to prevent any of cross contamination and labeling mix up if patients will be staggering during the procedures.

• Release specifications and testing to demonstrate
• Bioactivity vs. viability — which cells and how much
• Measurement of growth factors and cytokines

Provide at a minimum a qualification of the final product shipping method if the final product will not be on site.