Safety and clinical efficacy of the secretome of stressed peripheral blood mononuclear cells in patients with diabetic foot ulcer-study protocol of the randomized, placebo-controlled, double-blind, multicenter, international phase II clinical trial MARSYAS II.

BACKGROUND: Diabetes and its sequelae such as diabetic foot ulcer are rising health hazards not only in western countries but all over the world. Effective, yet safe treatments are desperately sought for by physicians, healthcare providers, and of course patients. METHODS/DESIGN: APOSEC, a novel, innovative drug, is tested in the phase I/II study MARSYAS II, where its efficacy to promote healing of diabetic foot ulcers will be determined. To this end, the cell-free secretome of peripheral blood mononuclear cells (APOSEC) blended with a hydrogel will be applied topically three times weekly for 4 weeks. APOSEC is predominantly effective in hypoxia-induced tissue damages by modulating the immune system and enhancing angiogenesis, whereby its anti-microbial ability and neuro-regenerative capacity will exert further positive effects. In total, 132 patients will be enrolled in the multicenter, randomized, double-blind, placebo-controlled, parallel group, dose-ranging phase I/II study and treated with APOSEC at three dose levels or placebo for 4 weeks, followed by an 8-week follow-up period to evaluate safety and efficacy of the drug. Wound area reduction after 4 weeks of treatment will serve as the primary endpoint. CONCLUSION: We consider our study protocol to be suitable to test topically administered APOSEC in patients suffering from diabetic foot ulcers in a clinical phase I/II trial. TRIAL REGISTRATION: EudraCT 2018-001653-27 . Registered on 30 July 2019. ClinicalTrials.gov NCT04277598 . Registered on 20 February 2020. TITLE: "A randomized, placebo-controlled, double-blind study to evaluate safety and dose-dependent clinical efficacy of APO-2 at three different doses in patients with diabetic foot ulcer (MARSYAS II)".

Reproducibility of GMP-compliant production of therapeutic stressed peripheral blood mononuclear cell-derived secretomes, a novel class of biological medicinal products.

BACKGROUND: The recent concept of secretome-based tissue regeneration has profoundly altered the field of regenerative medicine and offers promising novel therapeutic options. In contrast to medicinal products with a single active substance, cell-derived secretomes comprise pleiotropic bioactive ingredients, representing a major obstacle for reproducible drug product efficacy and warranting patient safety. Good manufacturing practice (GMP)-compliant production guarantees high batch-to-batch consistency and reproducible efficacy of biological medicinal products, but different batches of cellular secretomes produced under GMP have not been compared yet, and suitable quality control parameters have not been established. To this end, we analyzed diverse biological and functional parameters of different batches produced under GMP of the secretome obtained from γ-irradiated peripheral blood mononuclear cells with proven tissue regenerative properties in infarcted myocardium, stroke, spinal cord injury, and skin wounds. METHODS: We quantified key secretome ingredients, including cytokines, lipids, and extracellular vesicles, and functionally assessed potency in tube formation assay, ex vivo aortic ring sprouting assay, and cell-based protein and reporter gene assays. Furthermore, we determined secretome stability in different batches after 6 months of storage at various ambient temperatures. RESULTS: We observed that inter-batch differences in the bioactive components and secretome properties were small despite considerable differences in protein concentrations and potencies between individual donor secretomes. Stability tests showed that the analytical and functional properties of the secretomes remained stable when lyophilisates were stored at temperatures up to + 5 °C for 6 months. CONCLUSIONS: We are the first to demonstrate the consistent production of cell-derived, yet cell-free secretome as a biological medicinal product. The results from this study provide the basis for selecting appropriate quality control parameters for GMP-compliant production of therapeutic cell secretomes and pave the way for future clinical trials employing secretomes in tissue regenerative medicine.

Toxicological testing of allogeneic secretome derived from peripheral mononuclear cells (APOSEC): a novel cell-free therapeutic agent in skin disease.

A cell-free approach using secretomes derived from stem cells or peripheral blood mononuclear cells is an active area of regenerative medicine that holds promise for therapies. Regulatory authorities classify these secretomes as biological medicinal products, and non- clinical safety assessment thus falls under the scope of ICH S6. A secretome of stressed peripheral blood mononuclear cells (APOSEC) was successfully tested in a toxicology program, supporting clinical use of the new drug candidate. Here, to allow for topical, dermal treatment of patients with diabetic foot ulcer, several non-clinical safety studies were performed. Acute toxicity (single dose) and neuropharmacological screening were tested intravenously in a rat model. Risk for skin sensitisation was tested in mice. A 4-week intravenous toxicity study in mice and a 4-week subcutaneous toxicity study in minipigs were conducted to cover the clinical setting and application in a rodent and a non-rodent model. Acute and repeated-dose toxicity studies show that APOSEC administered intravenously and subcutaneously does not involve major toxicities or signs of local intolerance at levels above the intended total human maximal dose of 3.3 U/kg/treatment, 200 U/wound/treatment, and 100 U/cm2/treatment. The non-clinical data support the safe topical use of APOSEC in skin diseases related to deficient wound healing.

Safety and tolerability of topically administered autologous, apoptotic PBMC secretome (APOSEC) in dermal wounds: a randomized Phase 1 trial (MARSYAS I).

Developing effective therapies against chronic wound healing deficiencies is a global priority. Thus we evaluated the safety of two different doses of topically administered autologous APOSEC, the secretome of apoptotic peripheral blood mononuclear cells (PBMCs), in healthy male volunteers with artificial dermal wounds. Ten healthy men were enrolled in a single-center, randomized, double-blinded, placebo-controlled phase 1 trial. Two artificial wounds at the upper arm were generated using a 4-mm punch biopsy. Each participant was treated with both topically applied APOSEC and placebo in NuGel for 7 consecutive days. The volunteers were randomized into two groups: a low-dose group (A) receiving the supernatant of 12.5 × 106 PBMCs and a high-dose group (B) receiving an equivalent of 25 × 106 PBMCs resuspended in NuGel Hydrogel. Irradiated medium served as placebo. The primary outcome was the tolerability of the topical application of APOSEC. All adverse events were recorded until 17 days after the biopsy. Local tolerability assessment was measured on a 4-point scale. Secondary outcomes were wound closure and epithelization at day 7. No therapy-related serious adverse events occurred in any of the participants, and both low- and high-dose treatments were well tolerated. Wound closure was not affected by APOSEC therapy.

Paracrine Factors from Irradiated Peripheral Blood Mononuclear Cells Improve Skin Regeneration and Angiogenesis in a Porcine Burn Model.

Burn wounds pose a serious threat to patients and often require surgical treatment. Skin grafting aims to achieve wound closure but requires a well-vascularized wound bed. The secretome of peripheral blood mononuclear cells (PBMCs) has been shown to improve wound healing and angiogenesis. We hypothesized that topical application of the PBMC secretome would improve the quality of regenerating skin, increase angiogenesis, and reduce scar formation after burn injury and skin grafting in a porcine model. Full-thickness burn injuries were created on the back of female pigs. Necrotic areas were excised and the wounds were covered with split-thickness mesh skin grafts. Wounds were treated repeatedly with either the secretome of cultured PBMCs (Sec(PBMC)), apoptotic PBMCs (Apo-Sec(PBMC)), or controls. The wounds treated with Apo-Sec(PBMC) had an increased epidermal thickness, higher number of rete ridges, and more advanced epidermal differentiation than controls. The samples treated with Apo-Sec(PBMC) had a two-fold increase in CD31+ cells, indicating more angiogenesis. These data suggest that the repeated application of Apo-Sec(PBMC) significantly improves epidermal thickness, angiogenesis, and skin quality in a porcine model of burn injury and skin grafting.

Circulating heat shock protein 27 as a biomarker for the differentiation of patients with lung cancer and healthy controls--a clinical comparison of different enzyme linked immunosorbent assays.

BACKGROUND: Increased heat shock protein 27 (HSP27) has been described in patients with non-small cell lung cancer (NSCLC). The aim of this study was to evaluate five commercially available assays for HSP27 measurement with respect to their capabilities to differentiate NSCLC patients from healthy controls. METHODS: We measured HSP27 serum concentrations in 40 NSCLC cases and 40 healthy controls by different assays (i.e., R&D, Enzo Life Sciences, Invitrogen, Abcam, and MyBioSource). We analyzed receiver operating characteristic plots and calculated areas under the curve (AUCs) for the five HSP27 assays with the case-control status as the classification variable. RESULTS: The following AUCs were obtained: R&D, 0.834 (95% CI, 0.734 - 0.908); Enzo Life Sciences, 0.823 (95% CI, 0.722 - 0.899); Invitrogen, 0.780 (95% CI, 0.674 - 0.856); Abcam, 0.642 (95% CI, 0.528 - 0.747); and MyBioSource 0.523 (95% CI, 0.408 - 0.636). An explorative comparison of the AUCs revealed that the R&D, Enzo Life Sciences, and Invitrogen assays perform better than the Abcam and MyBioSource assays in the setting evaluated. Results obtained by different HSP27 assays had up to 10-fold difference of serum concentrations, and correlation coefficients of pairwise assay comparisons ranged from 0.184 - 0.938. CONCLUSIONS: The results of our clinical method comparison study revealed that commercially available HSP27 assays are not equally useful to differentiate NSCLC patients from healthy controls. Our study suggests that certain HSP27 methods cannot be applied for diagnostic purposes in lung cancer and probably also not in other diseases.

Increased serum levels of HSP27 as a marker for incipient chronic obstructive pulmonary disease in young smokers.

BACKGROUND: Although chronic obstructive pulmonary disease (COPD) is amongst the leading causes of morbidity and mortality, no biomarkers for its early detection are known. We have recently demonstrated that COPD is accompanied by elevated serum heat shock protein (HSP) 27 levels as compared to a control population. OBJECTIVES: In an open prospective study, we investigated whether elevated HSP27 levels are associated with the early radiological signs of COPD, i.e., air trapping (AT), emphysema (E) and impaired lung function. METHODS: In total, 120 apparently healthy smokers underwent lung function testing and serum sampling. Serum levels of HSP27, phospho-HSP27, CXCR2 chemokines and proteins related to inflammation, tissue remodeling and apoptosis were evaluated by ELISA. Of these 120 subjects, 94 voluntarily underwent a high-resolution computed tomography scan. RESULTS: AT or AT and E were detected in 57.45%. Subjects with AT and E (n = 23) showed significantly higher HSP27 levels than those without any pathology [i.e., nothing abnormal detected (NAD)] (4,618 +/- 1,677 vs. 3,282 +/- 1,607 pg/ml; p = 0.0081). In a univariate logistic regression model including NAD and AT and E, the area under the curve of HSP27 in the receiver-operating-characteristic curve was 0.724, (0.594­0.854, 95% CI; p = 0.0033). Interestingly, proinflammatory IL-8 was elevated in those subjects with evidence of AT and E compared to those with AT and NAD. Lung function did not correlate with increased HSP27 levels or pathological radiological findings. CONCLUSIONS: HSP27 serum levels correlated with the early radiological signs of COPD, whereas lung function did not match with radiological findings or HSP27 serum levels. Serum HSP27 levels may serve as a potential marker to identify the early signs of COPD independent of lung function in young smokers.

Discrimination of clinical stages in non-small cell lung cancer patients by serum HSP27 and HSP70: a multi-institutional case-control study.

INTRODUCTION: Lung cancer represents a major healthcare problem. Accordingly, there is an urgent need to identify serum biomarkers for early diagnosis of lung pathology. We have recently described that patients with manifest COPD evidence elevated levels of heat shock proteins (HSPs). Based on these data, we speculated whether HSPs are also increased in patients with diagnosed lung cancer. METHODS: Serum levels of HSP27, phospho-HSP27 (pHSP27) and HSP70 in patients with non-small cell lung cancer (NSCLC) diagnosed at an early (stages I-II, n=37) or advanced (stages IIIA-IV, n=72) stage were determined by using ELISA. Healthy smokers (n=24), healthy never-smoker volunteers (n=33) and COPD patients (n=34) according to GOLD classification served as control population. RESULTS: Serum levels of HSP27 were elevated in patients with NSCLC diagnosed at an early or advanced stage when compared with both healthy control groups (P<0.005 and P<0.0001 respectively). Statistically significant differences were furthermore found between the groups of patients with early vs. advanced stage NSCLC (P=0.0021). Serum levels of HSP70 were also significantly elevated in patients with NSCLC diagnosed at an early or at an advanced stage when compared with either healthy control groups (P=0.0028 and P<0.0001 respectively). In univariate logistic regression models including healthy subjects and patients with NSCLC, HSP70 had an area under the curve (AUC) of 0.779 (P<0.0001) and HSP27 showed an AUC of 0.870 (P<0.0001). CONCLUSION: Our data suggest that serum HSP27 levels might serve as a possible tool to discriminate between early and advanced stages NSCLC.