Abstract
The present study was designed to evaluate the safety and potential of adipose tissue-derived stem cells (ASCs) for the treatment of Crohn's fistula. In this dose escalation study, patients were sequentially enrolled into three dosing groups with at least three patients per group. The first three patients (group 1) were given 1 × 107 cells/ml. After 4 weeks, this dose was deemed safe, and so an additional four patients (group 2) were given 2 × 107 cells/ml. Four weeks later, after which this second dose was deemed safe, a third and final group of three patients were given 4 × 107 cells/ml. Each patient was followed for a minimum of 8 weeks. Patients who showed complete healing at week 8 were followed up for an additional 6 months. Efficacy endpoint was complete healing at week 8 after injection, defined as complete closure of the fistula track and internal and external openings without drainage or signs of inflammation. There were no grade 3 or 4 severity adverse events, and there were no adverse events related to the study drug. Two patients in group 2, treated with 2 × 107 ASCs/ml, showed complete healing at week 8 after injection. Of the three patients enrolled in group 3, treated with 4 × 107 ASCs/ml, one showed complete healing. Outcome in another patient was assessed as partial healing due to incomplete closure of the external opening, although the inside of fistula track was filled considerably and there was no drainage. All three patients with complete healing at week 8 showed a sustained effect without recurrence 8 months after injection. In conclusion, this study demonstrates the tolerability, safety, and potential efficacy of ASCs for the treatment of Crohn's fistula and provides support for further clinical study.
Keywords
Introduction
Perianal fistula, one of the clinical manifestations associated with Crohn's disease, develops in 13–39% of patients with Crohn's disease (1, 6, 14, 18). The ideal goal of treatment of Crohn's fistula is complete healing without recurrence and without damage to the anal sphincter complex. Medical treatments, such as infliximab administration, or surgical treatments, such as fistulotomy, fistulectomy, seton placement, and endorectal advancement flap, have been used for the treatment of Crohn's fistula. Infliximab does not provide complete healing and shows a high recurrence rate after treatment (25, 30). Although treatment with infliximab results in closure of the external opening, an internal pocket is observed, which often leads to recurrence (31). It is recommended that infliximab be combined with surgical treatment to produce a better outcome in the management of Crohn's fistula (33). Surgical treatment also has problems of poor healing and a high rate of recurrence, even after a short-term effect is achieved (34). In addition, impaired sphincter muscle function following surgery may cause postoperative anal incontinence in some patients (16, 22). Unfortunately, currently available medical and surgical treatments are not curative, and fail to provide a long-term resolution; the result is a persistent unhealed fistula that leads to a poor quality of life for patients.
Recently, autologous or allogenic adipose tissue-derived stem cells (ASCs) (7–11, 29), autologous bone marrow-derived mesenchymal stromal cells (MSCs), haematopoietic stem cells (HSCs) (3, 4, 26), and muscle transposition (19) have been demonstrated to safely and effectively treat Crohn's fistula. Moreover, it has been reported that stem cell therapy is not associated with fecal incontinence (11). Human adipose tissue is plentiful and easily accessible (20) and contains a large number of stem cells compared to other tissues in the body (23, 32). Furthermore, ASCs readily proliferate during manufacturing, and exhibit a characteristic multipotency that enables them to differentiate into myocytes, chondrocytes, osteocytes, and adipocytes (21, 28, 38).
This phase I, dose escalation study was conducted to evaluate the safety and potential efficacy of ASCs for the treatment of Crohn's fistula.
Materials and Methods
Patients
Eligible patients were those who were at least 18 years of age with a perianal fistula associated with Crohn's disease, as confirmed by biopsy. Exclusion criteria included a history of surgery for malignant tumor within the previous 5 years; allergy to bovine-derived materials or anesthetics; an autoimmune disease other than Crohn's disease; infection with hepatitis B or C virus (HBV, HCV), human immunodeficiency virus (HIV), or other infectious diseases; an inflammatory bowel disease other than Crohn's disease; a medical or family history of variant Creutzfeldt–Jakob disease or related diseases; active tuberculosis; or symptoms of septicemia. Women were excluded if breastfeeding, pregnant, or unwilling to use contraceptive methods. Patients were also excluded if sufficient adipose tissue could not be obtained for preparation of ASCs or if patients were sensitive to fibrin glue. Patients with a significant disease considered by the investigator to be capable of impacting the study were also excluded.
Study Design
The study was conducted according to the principles of the Declaration of Helsinki and was consistent with Good Clinical Practice guidelines. The protocol was approved by institutional review boards at each of three study sites and by the Korean Food and Drug Administration. Written informed consent was obtained from all patients before enrollment.
This was an open-label, multicenter, phase I, dose escalation study designed to evaluate the safety and preliminary efficacy of ASCs in the treatment of Crohn's fistula. Patients were sequentially enrolled into three dosing groups with at least three patients per group. The first three patients (group 1) were given 1 × 107 cells/ml. After 4 weeks, this dose was deemed safe and an additional four patients (group 2) were given 2 × 107 cells/ml. After an additional 4 weeks, at which point the second dose was deemed safe, a third and final group of three patients were given 4 × 107 cells/ml. Each patient was followed up for a minimum of 8 weeks. The volume of injected cells was proportional to fistula size. Patients who showed complete healing at week 8 were examined at 4, 6, and 8 months to monitor the sustained effectiveness and safety of ASC treatment.
Patients received physical examinations and were clinically assessed for adverse events. Blood samples were also collected for measurement of laboratory parameters. Immunological tests were performed on patients in group 2 as part of the safety evaluation.
Preparation of ASCs
Subcutaneous adipose tissue was collected by liposuction or extraction of fat tissue from the abdomen or thigh of patients under local anesthesia. Fat tissue was digested in phosphate-buffered saline (PBS; Hyclone) containing 1% bovine serum albumin (BSA) and 0.025% collagenase for 80 min at 37°C with intermittent shaking. Isolated ASCs were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1 ng/ml basic fibroblast growth factor (bFGF) to obtain the required number of cells for injection (Anterogen Co., Ltd., Seoul, Korea). ASCs at passages 3–4 were harvested by trypsin treatment, suspended in DMEM, and packaged in single-use vials containing 4 × 107 cells/ml. All procedures were performed in a Good Manufacturing Practice facility authorized by the Korean Food and Drug Administration. Fibrin glue (Tissucol Duo Quick, Baxter AG, Wien, Austria) and diluents (DMEM) in separate vials were also provided to achieve the required concentration for each group. Characteristically, ASCs expressed the stromal cell-associated markers, CD10, CD13, CD29, CD44, and CD90, and did not express the hematopoietic stem cell-associated markers, CD34 and CD45, or the bone marrow-derived stem cell-associated marker, STRO-1 (15). When analyzed for lot release testing, cell population both positive for CD90 and negative for CD45 was more than 90%. Cell viability was determined more than 90%. To confirm safety from contamination of adventitious agent, bacteria, fungi, mycoplasma, and virus tests were performed.
Injection
Before injection with ASCs, investigators performed a throughout tract curettage and then closed the internal opening by multiple suturing with 2-0 vicryl. Injected cell volume was proportional to the size of the fistula track, which was measured prior to injection using a probe. The volume of ASCs required in each group was evenly injected into the fistula tract wall and the mucosa surrounding the internal opening. The fistula track was filled with a mixing solution of thrombin and fibrinogen using a Duploject injection system (Tissucol Duo Quick, Baxter AG, Wien, Austria).
Assessments
In this study, safety evaluations included analysis of adverse events reported after injection with ASCs, serious adverse events during study period, and laboratory toxicity observed after injection with ASCs. Toxicity grade was determined using WHO toxicity scale and CTCAE version 3.0. Immunological responses were also examined in group 2 patients by performing a lymphocyte analysis and comparing CD4/CD8 ratios on day 1 (i.e., prior to injection) and week 8. CD4/CD8 T-cell ratio was determined with immunofluorescent reagent kit (Simultest IMK-Lymphocyte, BD) by analyzing flow-cytometry. The efficacy endpoint was complete healing at week 8 after injection of ASCs, defined as complete closure of the fistula track, and internal and external openings without drainage or signs of inflammation.
Descriptive Statistical Analysis
All patients who received ASC treatment were included in the safety analysis (intention-to-treat analysis). Efficacy evaluations were analyzed on the basis of a modified PP (per protocol) analysis, which included patients who received an ASC treatment and completed more than the three scheduled visits, including a postinjection week 8 visit. Any development of a new fistula related to the target fistula during the study period was regarded as an inefficacious outcome, whereas any newly developed fistulas unrelated to the target fistula were not considered in the efficacy analysis.
Results
Study Population
A total of 10 patients with more than one fistula were enrolled between November 26, 2008, and June 9, 2009, at three sites. Of these, one patient enrolled in group 2 dropped out after injection due to an insufficient number of ASCs. Therefore, 10 patients were included in the safety analysis and nine patients (three patients in each group) who met the criteria for the modified PP analysis were included in the efficacy analysis (Fig. 1). Five patients had previously experienced a surgical operation for fistula, such as fistulectomy and/or the use of setons.
Patient disposition. ASC, adipose tissue-derived stem cells.
Overall, 40% (4/10) of patients were male and 60% (6/10) were female; their average age was 26.5 ± 6.0 years, average weight was 52.1 ± 11.9 kg, and average body mass index (BMI) was 19.7 ± 3.0 (Table 1). There were five patients with transsphincteric, four patients with suprasphincteric, and one patient with extrasphincteric fistula. The average diameter and length of fistulas was 1.53 ± 1.32 cm and 4.45 ± 1.58 cm, respectively. The injected volume of ASCs was 3–20 ml, determined based on the fistula size; the total number of injected ASCs in this study ranged from 3 × 107 to 40 × 107 cells.
Baseline Characteristics
Safety and Tolerability
The safety analysis was performed on 10 patients who received injections of ASCs. There were no discontinuations due to adverse events. During the study period, 13 adverse events were reported in seven patients (70%). The adverse events, which were mild or moderate in severity, were not related to study drug. There were no grade 3 or 4 adverse events and no laboratory toxicity greater than grade 3 in this study. Adverse events reported in two or more patients included pain (n = 3) and diarrhea (n = 2). During the study period, two patients reported three serious adverse events (enterocolitis, seton application, and infliximab administration for new fistulas unrelated to the target fistula) requiring hospitalization (hospitalization should be reported as a serious adverse event regardless of toxicity grade). The reported serious adverse events, which were resolved during the study period, were toxicity grade 2. Immunological tests performed on blood samples to evaluate CD4/CD8 ratio showed no clinically significant change at week 8 (CD4/CD8 ratio of 0.63) compared to day 1 (before injection) (CD4/CD8 ratio of 0.59).
Healing
Three patients in group 1 showed partial closure with decreased drainage. Two patients in group 2, injected with 2 × 107 ASCs/ml in proportion to fistula size, showed complete healing at week 8 after injection. Of the three patients in group 3, injected with 4 × 107 ASCs/ml in proportion to fistula size, one showed complete healing. Outcome in another patient was assessed as partial healing due to incomplete closure of the external opening, although the inside of the fistula track was considerably filled and there was no drainage. Outcome in the third patient in group 3 was also assessed as partial healing due to a small amount of drainage.
Long-Term Follow-Up (Up to 8 Months) After Injection
The three patients who showed complete healing at week 8 were followed up for an additional 6 months (8 months after injection) to evaluate the safety and sustained effect of ASC injection. All patients tolerated the treatment well without safety concerns and the healing effect was sustained without recurrence at postinjection month 8 (Figs. 2 and 3).
Patient A. Treatment with ASCs. (a) Before injection. (b) At week 4 after injection. (c) At week 8 after injection, showing healed fistula with complete epithelialization of the external opening. (d) At month 8 after injection. Short white arrow, opening before injection; open-headed arrow, injection site.
Patient B. Treatment with ASCs. (a) Before injection. (b) At week 4 after injection. (c) At week 8 after injection, showing healed fistula with complete epithelialization of the external opening. (d) At month 8 after injection. Short white arrow, opening before injection; open-headed arrow, injection site.
Discussion
The present study showed that treatment with ASCs is safe and well tolerable at all dosing levels tested. The primary purpose of this study was to evaluate safety. Therefore, we should start with low dose although we speculated the first dose of 1 × 107 cells/ml could not produce the optimal efficacy. In this study, there were no drug-related adverse events and there were no clinically significant laboratory changes at all dosing levels.
Although this study has a limitation on efficacy evaluation due to small number of patients, ASCs may be effective in the treatment of Crohn's fistula when ASCs are administered at a dose of at least 2 × 107 cells/ml in proportion to fistula size.
In our study, healing was observed in 50% of the patients treated with at least 2 × 107 cells/ml after a single one time injection, while in Spanish group study, healing was observed in 46% of patients after first injection, which is similar (11). Literature reports have shown that fistula healing is observed with fibrin glue alone for 16% of patients with complex perianal fistula. In line with the Spanish study in which the second injection improved total efficacy, we plan on using a second injection in a future phase II study.
Both the Spanish study group and our group used ASCs, while the cell number is different. The Spanish study group used 2 × 107 cells/ml of ASCs regardless fistula size, while our group used ASCs in proportion to fistula size. We believe that cell number in proportion to fistula size may improve the efficacy because cells can be injected evenly in the fistula.
Most of the patients who had previously experienced recurrence after other surgical operations before participating in this study showed a decrease in drainage after injection with ASCs. These results suggest that ASCs may be effective for the treatment of fistula in patients who were unable to achieve complete healing by conventional medical or surgical interventions.
The patients who had completely healed at week 8 showed sustained complete healing up to 8 months after injection, a result that may predict the long-term efficacy of ASCs in clinical use. While, infliximab, which is an approved agent for the treatment of Crohn's fistula, is not curative in many patients and shows a high recurrence rate (43%) (25). Currently available surgical options also have a high recurrence rate (50% recurrence after advanced flap procedures in patients with fistula related to Crohn's disease) (27) and fecal incontinence caused by surgical damage to the anal sphincter (35% after advanced flap procedures for the repair of transsphincteric fistulas) (24). However, treatment with ASCs provided long-term efficacy without recurrence in the patients who showed complete healing at week 8 after injection. In addition, injection with ASCs did not induce fecal incontinence because the treatment does not involve any procedures that cause damage to the anal sphincter.
The precise mechanism underlying the long-term efficacy of ASCs in the treatment of Crohn's fistula is not yet known but may reflect the characteristics of MSCs, including their potential for differentiation (5, 12, 13, 37), and anti-inflammatory and immunomodulatory effects (17, 35, 36). Injected ASCs may differentiate into myocytes, leading to replenishment of tissue defects caused by chronic inflammation under the surrounding environment. In a preclinical study, expanded ASCs injected into the femoral muscle of a mouse or the sphincter muscle of a rat, were shown to exhibit myogenic differentiation (data not shown, manuscript in preparation). The immunomodulatory effects of MSCs may also encourage healing of fistulas caused by autoimmune diseases such as Crohn's disease (36). Indeed, ASCs obtained from healthy donors or patients with Crohn's disease exhibited immunosuppressive activity in vitro (2). These experiments showed that proliferation of activated peripheral blood mononuclear cells and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α or interferon (IFN)-γ, were markedly inhibited by ASCs.
We conclude that ASCs were well tolerated and demonstrated a potential for the treatment of Crohn's fistula. Although the number of patients was insufficient for a statistical evaluation of efficacy, complete healing was sustained in several patients for 8 months after treatment. Moreover, patients with complete healing in this phase I study have shown no recurrence 2 years after treatment (data not shown). An additional large-scale study is ongoing to clarify the safety and efficacy of ASCs.
Footnotes
Acknowledgments
This study was supported by grants from the Health Technology R&D Project, Ministry of Health and welfare, Republic of Korea (A100041), and Anterogen Co. Ltd., Seoul. The authors declare no conflict of interest.