Allogeneic adipose tissue‐derived stem cells ELIXCYTE® in chronic kidney disease: A phase I study assessing safety and clinical feasibility

Abstract The purpose of this phase I clinical trial is to assess the safety and tolerability of allogeneic adipose tissue‐derived stem cells (ADSCs) among chronic kidney disease (CKD) patients. 12 eligible CKD patients with an estimated glomerular filtration rate (eGFR) of 15–44 ml/min/1.73 m2 received one dose of intravenous allogeneic ADSCs (ELIXCYTE®), as 3 groups: 3 low dose (6.4 × 107 cells in total of 8 ml), 3 middle dose (19.2 × 107 cells in total of 24 ml) and 6 high dose (32.0 × 107 cells in total of 40 ml) of ELIXCYTE® and evaluated after 48 weeks. Primary endpoint was the safety profiles in terms of incidence of adverse events (AEs) and serious adverse event (SAE). Two subjects in high dose group experienced a total of 2 treatment‐related AEs which are Grade 1 slow speech and Grade 1 bradyphrenia after the infusion. One subject in middle dose group experienced an SAE unlikely related to treatment, grade 2 proteinuria. No fatal AE was reported in this study. An increase in eGFR was observed in 7 out of 12 subjects (58%) at Week 24 and in 6 of 12 subjects (50%) by Week 48. By Week 24, an increase in eGFR by more than 20% among all CKD patients with baseline eGFR ≧ 30 ml/min/1.73 m2 as compared to only 2 subjects in baseline eGFR < 30 ml/min/1.73 m2 group. No significant reduction in proteinuria was noted among all subjects. This phase I trial demonstrated single‐dose intravenous ELIXCYTE was well tolerated in moderate‐to‐severe CKD patients and its preliminary efficacy warrants future studies.


| INTRODUC TI ON
Chronic kidney disease (CKD) is a globally rising non-communicable disease with estimated global incidence of 11%-13% 1 and is important for its progressive nature which finally ensuing in end-stage kidney disease (ESKD). 2 Since kidney plays an important local role in several body homeostatic mechanisms including electrolyte balance, blood pressure control, mineral metabolism and haematopoiesis, the whole disease spectrum from early to late CKD associate with significant morbidity and mortality. The initiation of renal replacement therapy and/or transplantation in ESKD patients further inherent a huge burden on healthcare systems. 3 This pursues the idea of novel therapeutic interventions to retard the CKD progression to ESRD. [4][5][6] Regenerative cell-based therapy is one of the extensively studied novel treatments in CKD.
Chronic kidney disease is characterized by reduced kidney regenerative capacity in both renal repair and regrowth of partial or whole nephrons. In recent years, multipotent mesenchymal stem cells (MSCs) are of great interest for cell-based therapy related to their novel role in tissue regeneration and repair. 7 Various CKD models including diabetic nephropathy and allograft nephropathy revealed the ability of MSCs to preserve renal structure and improve renal function. [8][9][10][11][12][13] Among MSCs, adipose tissue-derived stem cells (ADSCs) have better anti-inflammatory and immunomodulating actions than bone marrow derived MSCs (BDMSCs). 14 Additionally, ADSCs are more feasible, high yield and lower immunogenicity than BDMCs. Zhu XY et al. found that intrarenal administration of ADSCs in animal renovascular disease improve the renal function. 15,16 Further, other studies also proved that intrarenal MSC delivery and revascularization improved hypoxia, inflammation, oxidative stress and subsequent renal fibrosis. 17,18 On the contrary, ADSCs have been proved to reduce the severity of ischaemic-reperfusion injury (IRI) and prevent renal fibrosis through inhibition of oxidative stress and inflammation. 14,19 Although several findings confirmed the beneficial effects of MSCs in these preclinical models of CKD, the diversity in CKD models, cell types and doses, administration route and different renal outcome parameters result in difficult judgement and further use in clinical settings. Few clinical trials have been investigated for the safety, dose and therapeutic potential of ADSCs, especially in different CKD patients. In this phase I clinical trial, we used ELIXCYTE ® , an investigational product composed of allogeneic human adipose tissue-derived stem cells (ADSCs) in low, middle and high dose to evaluate the safety and side effects among CKD patients with various underlying aetiologies.

| Treatment protocol
Firstly, three CKD patients were treated with low dose ELIXCYTE ® (ADSCs 6.4 × 10 7 cells) injection and would be observed for 14 days.
If there were no safety concern after reviewed by Data and Safety Monitoring Board (DSMB), second and third three CKD patients would be given moderate dose ELIXCYTE ® (ADSCs 19.2 × 10 7 cells) and high dose ELIXCYTE ® (ADSCs 32.0 × 10 7 cells) sequentially after with baseline eGFR ≧ 30 ml/min/1.73 m 2 as compared to only 2 subjects in baseline eGFR < 30 ml/min/1.73 m 2 group. No significant reduction in proteinuria was noted among all subjects. This phase I trial demonstrated single-dose intravenous ELIXCYTE was well tolerated in moderate-to-severe CKD patients and its preliminary efficacy warrants future studies.

K E Y W O R D S
allogeneic adipose tissue-derived stem cells, chronic kidney disease, estimated glomerular filtration rate safety assessments based on DSMB recommendation. Each study participant would sign the informed consent form after 14 days counted from the day that former patient receiving the administration of the investigational product. Due to development of suspected drug administration related adverse event, we recruited 3 more subjects in high dose (ADSCs 32.0 × 10 7 cells) group as DSMB suggestion. Finally, a total of 12 subjects, 3, 3 and 6 subjects, were treated with low, moderate and high dose ELIXCYTE ® separately in this study.
One dose of intravenous administration was given to each patient, who were followed up regularly for a total of 48 weeks. Changes in vital signs, physical and laboratory examination from baseline to post treatment visits were closely monitored. The adverse events (AEs) and serious adverse events (SAEs) and eGFR and urine total protein changes from baseline were evaluated at 0, 2, 4, 8, 12, 24, 36 and 48 weeks after administration. All adverse events occurring after MSC infusion were actively monitored. Any event either related to the MSC treatment or not was judged by investigating physicians and reported as serious adverse effects or adverse effects. This study was planned and conducted under the rules of good clinical practice (GCP), including the design of the clinical protocol and other process documents, as well as the archiving of essential documents.

| Preparation of allogeneic human adipose tissue-derived stem cell product ELIXCYTE ®
The donor was recruited under the supervision of an IRB in National Taiwan University Hospital and was screened and tested in compliance with eligibility determination guidance issued by Taiwan Food and Drug Administration (TFDA). The adipose tissue was collected from eligible donor via ultrasonic-assisted liposuction and was transferred to UnicoCell BioMed Company under hypothermal condition within 6 h. The stromal vascular fraction (SVF) was isolated by digesting the adipose tissue with type I collagenase, centrifuged and cryopreserved in liquid nitrogen tank for quarantine release. Following SVF met the release specification, SVF was revived, inoculated in T-flask and cultured at 37°C in a humidified carbon dioxide (CO 2 ) incubator for primary culture.
After approximately 7 days in culture, the adherent and highly proliferated cells termed ADSCs were detached and propagated to the predefined population doubling level (PDL). The ADSCs were then harvested and formulated at a density of 8 × 10 6 cells/ ml with cryoprotectant CryoStor ® CS10 (BioLife Solutions) containing 10% of DMSO (v/v). A gently pipetting was performed to obtain a single-cell suspension which was evenly filled to closure cryotubes. The cryotubes were cooled by a control rate freezer and temporarily stored in a vapour phase liquid nitrogen tank for quarantine release. Upon the criteria were met, the medicinal product named ELIXCYTE ® was transferred to qualified area for long term storage. Release testing of ELIXCYTE ® for microbiological evaluation (including mycoplasma, sterility and endotoxin tests) were employed to ensure safety. The product characteristics such as identity (including MSC markers, viable cell count and cell viability), product related impurities (immunophenotyping for CD34/CD45/CD11b/CD19/HLA-DR) and tri-lineage differentiation property were also assessed.

| Statistical analysis
The variables were reported as mean ± standard deviation or median (interquartile). The differences in changes between the groups were analysed by the unpaired t-test. The differences in changes within groups were analysed by the paired t-test. For non-normal distributed variable, Mann-Whitney U-test was used to analyse the difference between the two groups. The statistical analyses were performed by using the SPSS version 16.0 statistical software program.

| Disposition of subjects
Disposition of subjects participating in this clinical study is presented in Table 1. Enrolment status by study site is also summarized in Figure 1. Twelve subjects were screened for the study at Taipei Medical University Shuang Ho Hospital, and all subjects were eligible to be enrolled to the study. Patients were administered with one of three dose levels of investigational product based on the enrolled order. All the eligible subjects completed the study and were included in efficacy and safety analyses.

| Demography
The demographic data of all treated subjects (n = 12) are summarized in Table 2. Result showed that mean (±SD) age of all enrolled subjects was 53.4 ± 10.85. Their mean (±SD) BMI was 25.0 ± 4.61.
Ten males and 2 females were enrolled in the study.

| Adverse events
Incidence of AEs and SAEs are the primary endpoint in this phaseⅠstudy. Forty-one of AEs were reported by 11 subjects ( Table 3.
All AEs were coded using medical dictionary for regulatory ac-    Phase II study for all 3 dose levels.

| Change of estimated glomerular filtration rate
As for preliminary efficacy data, the change from baseline to Week 24 visit in estimated glomerular filtration rate was evaluated. The eGFR changes from baseline versus visit over 48 weeks in all treated subjects were shown in Figure 2A. An increase in eGFR was observed in 7 out of 12 subjects (58%) at Week 24 and in 6 of 12 subjects (50%) at Week 48. The increased percentages of eGFR at Week 24 by more than 20% were noted in all subjects with baseline eGFR ≧ 30 ml/min/1.73 m 2 ( Figure 2C). On the contrary, only 2 subjects with baseline eGFR < 30 ml/min/1.73 m 2 acquired increased eGFR more than 20% by 24 weeks ( Figure 2B). However, no significant reduction in proteinuria was noted in all subjects.

TA B L E 3 Summary of adverse events
No. of subjects with study treatment-related AEs 0 (0.0%) 0 (0.0%) 2 (33.3%) 2 (16.7%) No. of subjects with withdrawn of study treatment due to AEs 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) No. of subjects with SAEs 0 (0.0%) 1 (33.3%) 0 (0.0%) 1 (8.3%) No. of subjects with fatal AEs 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) Abbreviations: AE, adverse event; No., number; SAE, serious adverse event.  Papazova,D. A. et al. 9 also found that there is no dose dependency in stem cell therapy, which is supposed by the fact that such therapy acts by switching on the endogenous repair not by supplying persistent source of exogenous cells. Indeed, ADSCs secrete cytokines and growth factors in a paracrine or autocrine manner to promote endogenous repair. 24 Studies also demonstrated that ADSCs suppress T-cell activation, induce reg-T cells, inhibit apoptotic factors and further promote recovery of renal function. [25][26][27][28] This study aimed to evaluate safety and tolerability in CKD patients. Being a single-centre dose-escalation study, shorter observation period, limited number of subjects and variable study subject disposition became major limitations. We gave one intravenous infusion of ADSCs to patients; however, the frequency and dose of ADSCs in CKD are still controversial. Multiple administration compared with single dose might be beneficial for chronic conditions, since paracrine actions could reduce with time. Preclinical animal studies confirmed that weekly MSC administration significantly improve the kidney function than single dose. 20 Thus, whether second booster dose is required is to be consider in future clinical trials.
In conclusion, we proved the safety and feasibility of allogeneic ADSCs in CKD patients. Whether underlying aetiology and stage of CKD, dose and frequency of ADSCs, and comorbid condition influence the efficacy of ADSCs in these patients is still needed to be considered by future larger clinical trials.

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was reported by the authors.