To our knowledge, this is the first randomized, double – blinded, placebo controlled multicenter phase I/II trial to assess primarily the safety and also the potential effects of intramuscular allogeneic BM-MSC administration in patients with critical limb ischemia. Our results highlight the safety of using allogeneic bone marrow derived MSCs in patients with CLI and showed positive trends towards improvement as evidenced by the increase in parameters such as ankle pressure and ABPI, consistent with previous reports on cell based therapies in CLI [20
]. However, patients with impending amputation did not derive any benefit from BM-MSC administration. This group of patients had a poor prognosis, due to the advanced disease process and the clinical outcome was predetermined. For this study, CLI was determined by an ankle pressure of
70 mmHg along with that of ABPI of
0.6. Both these parameters showed significant improvement in patients included in the BM-MSC arm while this was not the case in patients treated with placebo (p
Pooled analyses from different studies conducted using stem cells in CLI have shown that ABPI increased between 0.1 and 0.2 points and a TcPO2 increase between 10 and 20 mmHg [24
]. The randomized, double – blind, placebo – controlled PROVASA (Intra-arterial progenitor cell transplantation of bone marrow mononuclear cells for induction of neovascularization in patients with peripheral arterial occlusive disease) study showed no significant difference in the primary outcome of improvement in ABPI [25
]. However, there were significant improvements in other secondary end points, including ulcer healing and rest pain reduction in the BM-MSC arm group. The authors debated that ABPI was a poor selection as a primary end point. In a recently completed Investigator led trial, we have shown that intra-arterial administration of allogeneic BM-MSCs resulted in significant decrease in pain score (VAS score), ABPI and TcPO2 parameters [26
]. It also showed complete ulcer healing to 70% reduction in ulcer area as compared to the baseline values. The TACT trial [23
] (therapeutic angiogenesis by cell transplantation) also reported that autologous BM-MNC administration did not alter the ABPI and TcPO2 in patients with atherosclerotic PAD or patients with Buerger’s disease over a period of 3 years, however led to improvement of other efficacy parameters such as extension of amputation free interval and improvement of ischemic rest pain. They concluded that ABPI value is not a useful predictor for evaluating the long – term efficiency of the angiogenic therapy using bone marrow cells. In a previous TACT trial [19
] and in our published study [26
] ABPI and TcPO2 values were significantly improved in patients with atherosclerotic PAD at 4 and 24 weeks. In another study, Idei et al. [27
] reported an increase in ABPI and TcPO2 after BM-MNC implantation in patients with atherosclerotic PAD and Buerger’s disease. In Buerger’s disease, ABPI and TcPO2 were significantly increased after 1 month and remained high during the 3 – year follow – up period. However, in patients with PAD, ABPI and TcPO2 pressure increased significantly after 1 month and gradually decreased during the follow – up, and, returned to the base line values at the end of 3 – year follow – up period. The differences in the severity of PAD may in part explain the observed differences in changes in perfusion between these studies.
We observed decrease in subjective rest pain in all patients irrespective of the arms they belonged to. This may partially be explained by the sufficient concomitant analgesics administered; however, the intake of analgesics was not analyzed objectively. We included patients with diabetes mellitus type 2, which might have caused similar outcomes of rest pain due to the decreased sensitivity of pain with diabetic peripheral neuropathy which was also reported in a study published by Lu et al. [28
]. Furthermore, other published studies which showed improvements in rest pain in the BM-MSC arm measured the rest pain score on the Visual Analogue Scale (VAS) of 0 to 10 [20
] which was not followed in our study.
The adverse events in the BM-MSC arm were less as compared to the placebo. Most of the adverse events were abnormal clinical laboratory values and or symptoms related to the progression of the disease. One patient in the BM-MSC arm died suddenly within one week of IMP administration. DSMB and the Indian FDA did an audit of the patient records, his past history and concluded that the sudden death was not related to IMP and the likely cause of death was a sudden cardiac event, which is not uncommon in patients with CLI [29
]. Most studies have shown that cell therapy is promising for angiogenesis and has no severe adverse effects in patients afflicted with Buerger’s disease [19
]. However, patients with atherosclerotic PAD may have increased mortality due to associated cardiovascular risk factors.
Ulcer healing and amputation rates were similar in both arms in our study. This may be due to the fact that these critically ill patients with impending amputation due to the advanced nature of the disease did not derive much benefit from allogeneic BM-MSC administration. In a study by Walter et al. [25
] all 4 patients with extensive gangrene with impending amputation (Rutherford class 6) at inclusion in the study had to undergo amputation above the ankle during the initial 3 month period.
There are few published reports on clinical trial results using autologous or allogeneic BM-MSCs in CLI since majority of the reported trials used autologous BM-MNCs. The mechanism through which MSCs exerts angiogenesis is mainly by secreting angiogenic growth factors or cytokines and also through differentiation into endothelial cells [33
]. The pro-angiogenic effect of MSC has been demonstrated in several studies both in vitro
and in vivo
]. MSCs have been shown to express and secrete stromal cell-derived factors - 1 (SDF-1), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF); matrix metalloproteinases (MMPs), all of which are important for triggering and maintaining angiogenesis [35
]. However, apart from their angiogenic activity, MSCs obtained from bone marrow and other tissues have also been shown to mediate anti-inflammatory, anti-apoptotic, anti-fibrotic, mitogenic and wound healing properties [36
Many clinical trials are using autologous BMMNCs for evaluating the efficacy in PAD patients but it has many limitations. Firstly, the active cellular constituent of bone marrow that is the agent of repair is not well characterized. Secondly, it is widely accepted that therapeutically active bone marrow constituents likely represents only 1 in 10,000 bone marrow cells [37
]. Thirdly, aspiration of the bone marrow is an invasive process and lastly, concerns exist that patients most likely to be affected by atherosclerosis are also likely to have impaired marrow function [38
]. The use of allogeneic BM-MSCs has important advantages. They are likely to represent an enriched population of cells with therapeutic angiogenic capacity. They are readily prepared from healthy donors and may be used as an allogeneic, “off – the - shelf” cryopreserved product [39
] without HLA matching because of their hypoimmunogenic, immunosuppressive and immunomodulatory properties.
Although this is a small study performed with a small number of patients (20), it offers some potential clinical insights. As the primary goal of the study, potential safety concerns are alleviated by our findings. Delivery of the BM-MSC via the IM route (in this study) or our earlier published study by IA route [26
] did not compromise, rather appeared to have improved the hemodynamic parameters in the lower limbs of the treated patients. This work also forms the basis for future clinical trials aimed at establishing the therapeutic possibility of using allogeneic BM-MSCs in CLI patients to improve angiogenesis and increasing amputation – free survival in these patients.