Allogeneic Human Mesenchymal Stem Cell Infusions for Aging Frailty

Abstract Background Impaired endogenous stem cell repair capacity is hypothesized to be a biologic basis of frailty. Therapies that restore regenerative capacity may therefore be beneficial. This Phase 1 study evaluated the safety and potential efficacy of intravenous, allogeneic, human mesenchymal stem cell (allo-hMSC)-based therapy in patients with aging frailty. Methods In this nonrandomized, dose-escalation study, patients received a single intravenous infusion of allo-hMSCs: 20-million (n = 5), 100-million (n = 5), or 200-million cells (n = 5). The primary endpoint was incidence of any treatment-emergent serious adverse events measured at 1 month postinfusion. The secondary endpoints were functional efficacy domains and inflammatory biomarkers, measured at 3 and 6 months, respectively. Results There were no treatment-emergent serious adverse events at 1-month postinfusion or significant donor-specific immune reactions during the first 6 months. There was one death at 258 days postinfusion in the 200-million group. In all treatment groups, 6-minute walk distance increased at 3 months (p = .02) and 6 months (p = .001) and TNF-α levels decreased at 6 months (p < .0001). Overall, the 100-million dose showed the best improvement in all parameters, with the exception of TNF-α, which showed an improvement in both the 100- and 200-million groups (p = .0001 and p = .0001, respectively). The 100-million cell-dose group also showed significant improvements in the physical component of the SF-36 quality of life assessment at all time points relative to baseline. Conclusions Allo-hMSCs are safe and immunologically tolerated in aging frailty patients. Improvements in functional and immunologic status suggest that ongoing clinical development of cell-based therapy is warranted for frailty.

Frailty is a medical syndrome characterized by age-related diminished physiologic function, endurance, and strength that can be secondary to a multitude of factors and is associated with a high risk of hospitalizations, worse clinical outcomes, dependency, and mortality (1,2). The overall prevalence of frailty is estimated to be 9.9% and is more common in females as well as those with chronic disease (3). The etiology of frailty is multifaceted, with an age-related decline in either physical, psychological, or a combination of the two components at the helm of the disease process (1). As a result, patients with frailty will often experience a reduction in "life span" and a corresponding increase in dependence on others for activities of daily living. Over time, the elderly and frail individuals in particularly, begin to lack the ability to adapt to adverse environmental stressors secondary due to their lack of "physiologic reserve," which leads to impaired function and increases their vulnerability to death (4)(5)(6)(7). Frail patients can be classified based on severity, as frailty has been characterized as a phenotypic spectrum through which patients transition (8).
Therapeutic interventions for aging frailty have mainly focused on exercise, nutritional supplements, and multidisciplinary methods (9,10). The goal of any potential therapy for frailty is to extend the well-being and ability of a patient to regenerate functionality. In this regard, one of the key hypotheses of the aging frailty is an accelerated depletion of endogenous stem cells, thereby contributing to a reduced ability to regenerate or repair organs and tissues (11,12). Accordingly, a cell-based, regenerative treatment strategy could ameliorate signs and symptoms of frailty (13, [14][15][16]. Bone marrow-derived human mesenchymal stem cells (hMSCs) home to sites of injury, reduce inflammation and fibrosis, stimulate endogenous stem cells, and contribute to tissue regeneration (17). Clinical studies have demonstrated that hMSCs improve cardiac structure and function in patients with acute myocardial infarction (18) and ischemic and nonischemic heart failure (19,20). Importantly, hMSCs are immunomodulatory (21) and data from multiple clinical trials show that allogeneic hMSCs are safe (17)(18)(19)(20)22) irrespective of age (23). Increased chronic inflammation (24,25) and declines in cardiovascular reserve (26) have been identified in aging frail patients. These features of aging frailty support the hypothesis that hMSCs ameliorate or improve frailty.
The purpose of this Phase 1 pilot study, AllogeneiC Human Mesenchymal Stem Cell in Patients with Aging FRailTy via IntravenoUS Delivery (CRATUS) (27), was to evaluate the safety and tolerability of allogeneic hMSCs (allo-hMSCs) in patients with aging frailty and to explore domains of treatment efficacy of allo-hMSCs through the reduction of signs and symptoms of aging frailty. The study also sought to gain insight regarding optimal dosing of allo-hMSCs and potential mechanistic properties of intravenous allo-hMSC therapy in frailty vis-à-vis immune monitoring and measurements of changes in systemic biomarkers.
The study was approved by the Internal Review Board (IRB) at the University of Miami Miller School of Medicine (IRB approval #: 20130646) and monitored by a Data and Safety Monitoring Board. All subjects provided written informed consent before participating.

Results
Study subjects (n = 15) had a 2:1 male to female ratio and an average age of 78.4 ± 4.7 years ( Table 2). Over half of the patients (n = 8) were considered "vulnerable" with a Clinical Frailty Score (28,29) of 4, whereas the remainder scored a 5 ("mild" frailty; n = 6) or 6 ("moderate" frailty; n = 1). At baseline, the mean 6-minute walk distance (6MWD) and forced expiratory volume in 1 second (FEV1) were 382.6 ± 102.7 m and 2.3 ± 0.4 L, respectively. There was no significant difference between patients of each cell-dose group in either of these parameters. The average Mini-Mental State Examination (MMSE) score in this study cohort was 28.5 ± 1.4. Blood laboratory counts between patients were similar at baseline.
Allo-hMSCs were well tolerated in all three cell-dose groups with no treatment-related serious adverse events (SAE) observed during the 12-month postinfusion follow-up period. There were no persistent cardiopulmonary signs or symptoms observed immediately following infusion. Supplementary Table 1 and Table 3 list the adverse events (AE) and SAE's for this trial. Two subjects in the 20-million cell-dose group were hospitalized on Day 102 and 311 postinfusion for an SAE (uncontrolled hypertension and hypercalcemia, respectively); however, both events were considered unrelated to the product. Additionally, one infusion-related AE (intravenous infiltration) occurred in the 20-million cell-dose group, which was considered mild in severity and soon thereafter resolved. One subject in the 200-million cell-dose Table 1. Key Inclusion and Exclusion Criteria for Enrolled Subjects Key Inclusion Criteria • Provide written informed consent • Subjects age ≥60 and ≤95 y • Show signs of frailty apart from a concomitant condition as assessed by the Investigator with a frailty score of 4 to 7 using the Clinical Frailty Scale (29) Key Exclusion Criteria • Score of ≤24 Mini-Mental State Examination • Inability to perform any of the assessments required for endpoint analysis • Serious comorbid illness or any other condition that, in the opinion of the investigator, may compromise the safety or compliance of the subject or preclude successful completion of the study • Have a nonpulmonary condition that limits life span < 1 y • Have a clinical history of malignancy within 5 y (ie, subjects with prior malignancy must be disease free for 5 y), except curatively-treated basal cell carcinoma, squamous cell carcinoma, melanoma in situ or cervical carcinoma, if recurrence occurs.

Discussion
The major new findings of CRATUS are that intravenous allo-hMSC infusions are safe and well tolerated in elderly individuals with early signs and symptoms of frailty. Importantly, there were improvements in a constellation of parameters that are important predictors of morbidity and mortality in patients with aging frailty.
With no current standard of care for frailty, allo-hMSCs may hold great promise as a cell therapy agent for patients with this syndrome. The underlying basis for positive effects of allo-MSCs are likely due, at least in part, to anti-inflammatory and proregenerative effects (21). In this regard, frailty is characterized by systemic inflammation (17) and low "reserve capacity" of organ systems thought due to diminished endogenous stem cell production (16). Though baseline stem cell levels are difficult to detect, aging in itself is likely associated with reduced stem cells (11,12) and of those which remain viable, there is a loss in their differentiation capacity types (15), which could contribute to a decline in multiorgan physiologic reserve. Replenishment of the body's stem cell "factory" and/or revitalization of stem cell niches via intravenous infusion of allo-hMSCs may help treat the morbidities associated with aging frailty. Additionally, the decline in function of aged stem cells (12) can be circumvented by administration of young donor allo-hMSCs that do not exhibit senescence or functional impairments that are a characteristic of older donor MSCs (13, 16).
The safety of allogeneic MSC therapy has been demonstrated in numerous studies (18)(19)(20)22). Additionally, cPRA testing indicated that infusion of allo-hMSCs does not induce a clinically relevant immune response. This may be related to the immunoregulatory properties of MSCs (21), specifically on T-helper and T-regulatory cells (30). Furthermore, our data demonstrate that the allo-hMSCs had an immunomodulatory effect suppressing early and late/chronic T cell activation markers in all treatment groups.
At 6 months postinfusion, we observed a significant decrease in the inflammatory biomarker TNF-α in all cell-dose groups, with the greatest change in the 100-and 200-million groups. These findings corroborate the association between elevated cytokines and systemic inflammation, as well as the role of this marker in the accelerated aging process (24). Indeed, systemic inflammatory biomarkers have been implicated in augmenting cell mitochondrial dysfunction, altered cell-to-cell interactions, and stem cell reserve exhaustion (4). Moreover, elevated inflammatory cytokines like TNF-α have been linked to the disruption of physiological organ systems and their homeostatic properties (24). Other biomarkers, including IL-6 and CRP, did not show any significant decrease following cell therapy in this Phase 1 study, although these cytokines have been previously found to be associated with aging frailty (24,31).
The 6MWD is a validated field test, widely used to assess functional exercise capacity, treatment effectiveness, and prognosis since it is accurate, reproducible, easy to administer, and well-tolerated by patients (32)(33)(34)(35). It has been commonly used as a prognostic instrument to assess the functional status of patients with cardiovascular (32,36), pulmonary (33), and more recently, muscular diseases (34). Using the 6MWD to evaluate physical functioning after allo-hMSC intravenous infusion therapy in patients with frailty, we observed significant improvements in distance walked in the 20-million and 100-million cell-dose groups, with the greatest change seen in the 100-million group. Interestingly, we did not find a significant difference in the 200-million cell-dose group. Although safety and tolerability was demonstrated at this cell-dose, significantly valid conclusions can only be made regarding efficacy with additional studies (37). Similarly, in regards to efficacy, it is important to note that there may be an effect in regards to practice with the 6MWD over time. Importantly, the 6MWD test is particularly applicable to frailty as it is an integrated global assessment of patients' cardiac, respiratory, circulatory, as well as muscular capacities. Age-related skeletal muscle loss, or sarcopenia, can lead to diminished strength, lack of endurance, and low tolerance for physical exertion; all common core clinical presentations of frailty (7). Indeed, the strong association between sarcopenia and functional impairment in older patients has been reported (38). As such, the 6MWD can be used as a meaningful reflection of a frail patient's ability to perform basic activities of daily living and given the improvement noted in this study, future clinical testing of intravenous allo-hMSC infusion in frail subjects is warranted.
The minimal clinically important difference MCID is defined as the smallest difference in score in the domain of interest, which patients perceive as beneficial and which would mandate a change in patient management (39). In regard to the 6MWD, Perera and colleagues have suggested that an improvement by more than 49 m is considered a "substantial change" (40) and this threshold was indeed exceeded in the 100-million group. However, it is important to consider that although small changes may be statistically significant, they may not be clinically relevant. The MCID is a dynamic and subjective concept, and its derivations are usually estimated for a specific population at a particular stage of recovery (32). Therefore, the fact that 6MWD improves after cell-therapy in this study warrants future clinical testing of intravenous allo-MSC infusion therapy on efficacy outcomes.
Other outcome measurements for efficacy in this Phase 1 trial, including pulmonary function and cognitive function, showed variable results or suggested trends. In regards to the MMSE, the analysis of all cell-dose groups combined showed significant improvement from baseline raising an intriguing possibility that MSCs may improve cognition, a prediction that will require further testing.
Taken together, the dose-finding portion of this study supports the idea that 100-million cells represent the optimal dose level, with no added benefit or loss of effect at the 200-million dose. Interestingly, TNF-α levels appear to be a useful biomarker of efficacy in this population, as they decline in response to all doses of allo-hMSCs. Studies in patients with ischemic cardiomyopathy also suggest a dose threshold effect. However, the small sample size of this Phase 1 and inadequate powering of the secondary outcomes likely contributed to the mixed results noted in regards to pulmonary function and cognition. Phase 2 clinical trials in aging frailty patients with larger sample sizes and blinded, randomized study designs are underway that are designed to confirm or refute the provocative efficacy results shown here. Another limitation of this study was the patient demographics. All subjects were of white race, with no inclusion of African Americans or Hispanics, which would be reasonable to attain in a larger, Phase 2 study sample.
In the United States, the population of individuals 65 years and older is projected to increase to almost 30 times the number recorded in 1990 (3,080,498 persons) (NP2008-T12, U.S. Administration on Aging). Although the overall prevalence of aging frailty has been reported to be near 10%, studies have shown a wide range in the actual prevalence numbers, from less than 5% to 59% (3). This phenomenon is at least in part due to the lack of consensus on the syndrome's true defining criteria in addition to the array of models used to describe it (41). The various sets of diagnostic models for the syndrome are related to the fact that frailty involves social and psychological components in addition to physical or clinical symptoms (5-7). Therefore, no single model exists that can comprehensively encompass all the essential aspects of the syndrome. As patients with frailty are a major source of medical resource expenditures, early interventions and/or treatments for this disease can improve patient quality of life in addition to lowering healthcare costs (42).
In conclusion, intravenous administration of allo-hMSCs was safe, well tolerated, and free from any concerning adverse events. Furthermore, given the promising results of this study, larger, doubleblinded, placebo-controlled clinical trials examining the potential benefits of allo-hMSCs are warranted to further bolster understanding of the efficacy profile of allo-MSCs in individuals with aging frailty.

Supplementary Material
Supplementary data is available at The Journals of Gerontology, Series A: Biological Sciences and Medical Sciences online.

Funding
This work was supported by the Starr Foundation and the Soffer Family Foundation.