One‐year safety, healing and amputation rates of Wagner 3‐4 diabetic foot ulcers treated with cryopreserved umbilical cord (TTAX01)

Abstract An open label, multicenter 16‐week trial of cryopreserved human umbilical cord (TTAX01) was previously undertaken in 32 subjects presenting with a Wagner grade 3 or 4 diabetic foot ulcer, with 16 (50%) of these having confirmed closure following a median of one product application (previous study). All but two subjects (30/32; 94%) consented to participate in this follow‐up study to 1‐year postexposure. No restrictions were placed on treatments for open wounds. At 8‐week intervals, subjects were evaluated for adverse events (AEs) and wound status (open or closed). Average time from initial exposure to end of follow‐up was 378 days (range 343‐433), with 29 of 30 (97%) subjects completing a full year. AEs were all typical for the population under study, and none were attributed to prior exposure to TTAX01. One previously healed wound re‐opened, one previously unconfirmed closed wound remained healed, and nine new wound closures occurred, giving 25 of 29 (86.2%) healed in the ITT population. Three of the new closures followed the use of various tissue‐based products. Three subjects whose wounds were healed required subsequent minor amputations due to osteomyelitis, one of which progressed to a major amputation (1/29; 3.4%). One additional subject underwent two minor amputations prior to healing. Overall, the study found TTAX01 to be safe in long‐term follow‐up and associated with both a low rate of major amputation and a higher than expected rates of healing.

lead to the need for minor or major lower extremity amputation. 5,6 In fact, DFU is the most common single precursor of lower extremity amputations among persons with diabetes, whose age-adjusted amputation rate is nearly 30 times that of people without diabetes. 7 The short-term goals of treatment of DFU complicated by osteomyelitis, but not ischemia, are to remove devitalized bone and tissue, identify and treat infection, and achieve wound healing with the least disruption to anatomy. The longer term goals are maintainence of healing over a meaningful length of time, and preservation of the limb, each of which is challenging given historically high rates of ulcer recurrence and amputation. 8 The present study was designed as an observational extension study from a previous interventional trial 9 of TTAX01, an aseptically processed cryopreserved human umbilical cord product derived from donated human placental tissue following healthy, live, caesarian section, full-term births, after determination of donor eligibility and placenta suitability.
The regulation of birth tissue products used in the treatment of DFU is undergoing a transition from the application of a combined set of rules (US Public Health Service Act §361, US FDA regulations 21 CFR §1271) to the application of rules governing tissue based biological products. 10 The shift to regulating these products as biologics requires manufacturers to undertake well-designed clinical evaluations of safety and efficacy under an investigational new drug exemption, in order to support a biologics license application. With the expectation of needing to treat recurrent ulcers over substantial periods of time, follow-up studies of 12-months duration are mandated by the FDA for the purpose of evaluating long-term safety.

| Trial design and participants
This was a multicenter, noninterventional follow-up study conducted at healthcare facilities in the United States (11 centers), where each center had previously enrolled at least one patient into a preceding interventional trial of TTAX01 for the treatment of Wagner 3-4 DFU (NCT03230175). 9 The protocol received Institutional Review Board approval for each participating center. Most subjects from the preceding trial consented to participate and enrolled into this follow-up (30/32; 94%) based on the only inclusion criteria that they participated in the preceding trial and were exposed to at least one application of TTAX01. Subjects enrolled into this study upon completion of the preceding trial, which occurred after confirmation of healing at any point, or upon exiting that 16-week trial with an unhealed wound. No restrictions were placed on surgical interventions, offloading techniques or the use of any therapies for wounds that were still open at the time of enrollment into this study. Visits were scheduled at 8 week intervals to complete at least 52 weeks from the time of enrollment into the preceding trial. All enrolled subjects (n = 30) were evaluable for safety, while subjects with a remaining open or closed index wound at enrollment (n = 29) were evaluable for efficacy.

| Evaluations
Safety was evaluated by frequency, expectedness, and relationship of adverse events (AEs) calculated for each body system, by preferred terminology, by the preceding treatment, for number of subjects and percentage reporting the event. Wounds were evaluated using the eKare inSight measuring device (eKare Inc., Fairfax, Virginia) by capturing an image for electronic measurement via automatic tracing of area (cm 2 ), depth (cm), and volume (cm 3 ). Wound status (healed, not healed) was recorded at each visit. New wound closures were recorded based on a single observation of closure rather than the more conservative regulatory requirement of closure confirmation at two subsequent visits each 2 weeks apart, which was followed during the preceding treatment study. Subjects who achieved closure and remained closed at the final visit were considered to have healed their wound.

| Endpoints
The primary objective of the study was to identify new AEs and examine ongoing AEs not resolved in subjects who were exposed to

| Statistical Analysis
All continuous data were expressed as mean ± SD (range), while categorical variables were expressed as frequency and percentages. The

| RESULTS
Demographics and baseline characteristics for all 30 subjects are summarized in Table 1. Details on the full cohort enrolled in the previous trial have been published. 9 Twenty-nine (97%) of 30 enrolled subjects were followed for 12 or more months from initial exposure. One subject was lost to follow-up at 48 weeks, having achieved wound closure at 32 weeks. All 30 were evaluable for safety, but only 29 were evaluable for efficacy as one subject had his unhealed wound removed in a minor amputation in the previous study.
Twenty-four (80%) of 30 subjects experienced one or more AE during the study. All AEs were expected within the demographic although not necessarily expected for individual subjects. None of them was related to prior exposure to TTAX01. Nine subjects experienced one or more serious AE, defined as requiring hospitalization; none of these events was considered related to prior exposure to TTAX01. There were no deaths.   Not only were healing rates high in the previous and present follow-up study, healing times among subjects with minor baseline amputations in the previous study were much shorter than those reported by Svensson et al, who saw a median time from primary amputation to wound healing of 26 weeks (range 2-250). 11 They further reported that 21% of these patients required re-amputation above the ankle, 3.5 times higher than in the present study.
Published experience in treating DFU with a range of amnion, chorion and umbilical cord products has been universally positive, although the majority of studies have been with shallow Wagner 1 ulcers, which have no exposure of muscle, fascia, joint capsule or bone. 12,13 Significant differences in the proportion of wounds healed in comparison to standardized care were often seen as early as 4 weeks from initiation of therapy. One dehydrated amnion and chorion product gave remarkably positive but inconsistent results over a series of single center, cross-over, multicenter and multicenter extension trials, with wound closure rates of 90 to 100% seen by 6 to 12 weeks. [14][15][16][17][18][19] A cryopreserved product showed superiority over standard care in published studies of Wagner 1 DFU, with efficacy over 12 weeks in the range of 62 to 71% in both prospective and retrospective studies. [20][21][22] A large confirmatory study (NCT02571738) was abandoned however, after approximately 50% of subjects had enrolled.
One trial of a dehydrated umbilical cord product 23 showed lower healing rates than were seen with a dehydrated amnion and chorion product from the same manufacturer, although it was still superior to standardized care.
Wound recurrence rates have not been routinely reported. Publications where recurrence is mentioned give rates of 14% 24 and 18% 20 at 3 months of follow-up with two different products. A third product reported on 82% of healed wounds followed for 9 to 12 months post healing, at which time 94% remained healed. 8 Although the present study reports follow-up over 1 year from initial exposure, rather than from the time of initial healing, persistent healing was seen in over 90% of wounds that initially healed. Of course, this study is without a control arm, therefore we do not know the freedom from recurrence rates for patients who would have healed without advanced therapy.
The patients who enrolled in the prior interventional study,