Group sequential designs for pragmatic clinical trials with early outcomes: methods and guidance for planning and implementation.

BACKGROUND: Group sequential designs are one of the most widely used methodologies for adaptive design in randomized clinical trials. In settings where early outcomes are available, they offer large gains in efficiency compared to a fixed design. However, such designs are underused and used predominantly in therapeutic areas where there is expertise and experience in implementation. One barrier to their greater use is the requirement to undertake simulation studies at the planning stage that require considerable knowledge, coding experience and additional costs. Based on some modest assumptions about the likely patterns of recruitment and the covariance structure of the outcomes, some simple analytic expressions are presented that negate the need to undertake simulations. METHODS: A model for longitudinal outcomes with an assumed approximate multivariate normal distribution and three contrasting simple recruitment models are described, based on fixed, increasing and decreasing rates. For assumed uniform and exponential correlation models, analytic expressions for the variance of the treatment effect and the effects of the early outcomes on reducing this variance at the primary outcome time-point are presented. Expressions for the minimum and maximum values show how the correlations and timing of the early outcomes affect design efficiency. RESULTS: Simulations showed how patterns of information accrual varied between correlation and recruitment models, and consequentially to some general guidance for planning a trial. Using a previously reported group sequential trial as an exemplar, it is shown how the analytic expressions given here could have been used as a quick and flexible planning tool, avoiding the need for extensive simulation studies based on individual participant data. CONCLUSIONS: The analytic expressions described can be routinely used at the planning stage of a putative trial, based on some modest assumptions about the likely number of outcomes and when they might occur and the expected recruitment patterns. Numerical simulations showed that these models behaved sensibly and allowed a range of design options to be explored in a way that would have been difficult and time-consuming if the previously described method of simulating individual trial participant data had been used.

Renal Autologous Cell Therapy to Stabilize Function in Diabetes-Related Chronic Kidney Disease: Corroboration of Mechanistic Action With Cell Marker Analysis.

Introduction: Chronic kidney disease (CKD) is a worldwide disease without cure. Selected renal cells (SRCs) can augment kidney function in animal models. This study correlates the phenotypical characteristics of autologous homologous SRCs (formulated product called Renal Autologous Cell Therapy [REACT]) injected into patients' kidneys with advanced type 2 diabetes-related CKD (D-CKD) to clinical and laboratory findings. Methods: A total of 22 adults with type 2 D-CKD underwent a kidney biopsy followed by 2 subcortical injections of SRCs, 7 ± 3 months apart. There were 2 patients who had only 1 injection. We compared annualized estimated glomerular filtration rate (eGFR) slopes pre- and post-REACT injection using the 2009 CKD-EPI formula for serum creatinine (sCr) and the 2012 CKD-EPI Creatinine-Cystatin C equation and report clinical/laboratory changes. Fluorescent Activated Cell Sorting (FACS) Analysis for renal progenitor lineages in REACT and donor vascular endothelial growth factor A (VEGF-A) analysis were performed. Longitudinal parameter changes were analyzed with longitudinal linear mixed effects model. Results: At baseline, the mean diabetes duration was 18.4 ± 8.80 years, glycated hemoglobin (Hgb) was 7.0 ± 1.05, and eGFR was 40.3 ± 9.35 ml/min per 1.73 m2 using the 2012 CKD-EPI cystatin C and sCr formulas. The annualized eGFR slope (2012 CKD-EPI) was -4.63 ml/min per 1.73 m2 per year pre-injection and improved to -1.69 ml/min per 1.73 m2 per year post-injection (P = 0.015). There were 7 patients who had an eGFR slope of >0 ml/min per 1.73 m2 postinjection. SRCs were found to have cell markers of ureteric bud, mesenchyme cap, and podocyte sources and positive VEGF. There were 2 patients who had remote fatal adverse events determined as unrelated with the biopsies/injections or the REACT product. Conclusion: Our cell marker analysis suggests that SRCs may enable REACT to stabilize and improve kidney function, possibly halting type 2 D-CKD progression.

Identification of functional pathways for regenerative bioactivity of selected renal cells.

BACKGROUND: Selected renal cells (SRC) are in Phase II clinical trials as a kidney-sourced, autologous, tubular epithelial cell-enriched cell-based therapy for chronic kidney disease (CKD). In preclinical studies with rodent models of CKD, SRC have been shown to positively modulate key renal biomarkers associated with development of the chronic disease condition. METHODS: A comparative bioinformatic analysis of transcripts specifically enriched or depleted in SRC component sub-populations relative to the initial, biopsy-derived cell source was conducted. RESULTS: Outcomes associated with therapeutically relevant bioactivity from a systematic, genome-wide transcriptomic profiling of rodent SRC are reported. Key transcriptomic networks and concomitant signaling pathways that may underlie SRC mechanism of action as manifested by reparative, restorative, and regenerative bioactivity in rodent models of chronic kidney disease are identified. These include genes and gene networks associated with cell cycle control, transcriptional control, inflammation, ECM-receptor interaction, immune response, actin polymerization, regeneration, cell adhesion, and morphogenesis. CONCLUSIONS: These data indicate that gene networks associated with development of the kidney are also leveraged for SRC regenerative bioactivity, providing evidence of potential mechanisms of action.

Self healable neuromorphic memtransistor elements for decentralized sensory signal processing in robotics.

Sensory information processing in robot skins currently rely on a centralized approach where signal transduction (on the body) is separated from centralized computation and decision-making, requiring the transfer of large amounts of data from periphery to central processors, at the cost of wiring, latency, fault tolerance and robustness. We envision a decentralized approach where intelligence is embedded in the sensing nodes, using a unique neuromorphic methodology to extract relevant information in robotic skins. Here we specifically address pain perception and the association of nociception with tactile perception to trigger the escape reflex in a sensorized robotic arm. The proposed system comprises self-healable materials and memtransistors as enabling technologies for the implementation of neuromorphic nociceptors, spiking local associative learning and communication. Configuring memtransistors as gated-threshold and -memristive switches, the demonstrated system features in-memory edge computing with minimal hardware circuitry and wiring, and enhanced fault tolerance and robustness.

Exosomes for repair, regeneration and rejuvenation.

INTRODUCTION: Application of regenerative medicine strategies for repair of organs/tissue impacted by chronic disease is an active subject for product development. Such methodologies emphasize the role of stem cells as the active biological ingredient. However, recent developments in elucidating mechanisms of action of these therapies have focused on the role of paracrine, 'action-at-a-distance' modus operandi in mediating the ability to catalyze regenerative outcomes without significant site-specific engraftment. A salient component of this secreted regenerative milieu are exosomes: 40-100 nm intraluminal vesicles that mediate transfer of proteins and nucleic acids across cellular boundaries. AREAS COVERED: Here, we synthesize recent studies from PubMed and Google Scholar highlighting how cell-based therapeutics and cosmeceutics are transitioning towards the secretome generally and exosomes specifically as a principal modulator of regenerative outcomes. EXPERT OPINION: Exosomes contribute to organ development and mediate regenerative outcomes in injury and disease that recapitulate observed bioactivity of stem cell populations. Encapsulation of the active biological ingredients of regeneration within non-living exosome carriers may offer process, manufacturing and regulatory advantages over stem cell-based therapies.

Selected renal cells modulate disease progression in rodent models of chronic kidney disease via NF-κB and TGF-ß1 pathways.

AIM: Identification of mechanistic pathways for selected renal cell (SRC) therapeutic bioactivity in rodent models of chronic kidney disease. MATERIALS & METHODS: In vivo and in vitro functional bioassays applied to investigate regenerative outcomes associated with delivery of SRC to diseased rodent kidney. RESULTS: In vivo, SRC reduces chronic infiltration by monocytes/macrophages. SRC attenuates NF-κB and PAI-1 responses while simultaneously promoting host tubular cell expansion through trophic cues. In vitro, SRC-derived conditioned media attenuates TNF-α-induced NF-κB response, TGF-ß-mediated PAI-1 response and increases expression of transcripts associated with cell cycle regulation. Observed bioactive responses were from vesicle and nonvesicle-associated factors, including specific miRNAs. CONCLUSION: We identify a paracrine mechanism for SRC immunomodulatory and trophic cues on host renal tissues, catalyzing long-term functional benefits in vivo.

Fabrication of a myocardial patch with cells differentiated from human-induced pluripotent stem cells.

The incidence of cardiovascular disease represents a significant and growing health-care challenge to the developed and developing world. The ability of native heart muscle to regenerate in response to myocardial infarct is minimal. Tissue engineering and regenerative medicine approaches represent one promising response to this difficulty. Here, we present methods for the construction of a cell-seeded cardiac patch with the potential to promote regenerative outcomes in heart muscle with damage secondary to myocardial infarct. This method leverages iPS cells and a fibrin-based scaffold to create a simple and commercially viable tissue-engineered cardiac patch. Human-induced pluripotent stem cells (hiPSCs) can, in principle, be differentiated into cells of any lineage. However, most of the protocols used to generate hiPSC-derived endothelial cells (ECs) and cardiomyocytes (CMs) are unsatisfactory because the yield and phenotypic stability of the hiPSC-ECs are low, and the hiPSC-CMs are often purified via selection for expression of a promoter-reporter construct. In this chapter, we describe an hiPSC-EC differentiation protocol that generates large numbers of stable ECs and an hiPSC-CM differentiation protocol that does not require genetic manipulation, single-cell selection, or sorting with fluorescent dyes or other reagents. We also provide a simple but effective method that can be used to combine hiPSC-ECs and hiPSC-CMs with hiPSC-derived smooth muscle cells to engineer a contracting patch of cardiac cells.

Preclinical biosafety evaluation of cell-based therapies: emerging global paradigms.

Cell-based therapies have the potential to treat a diversity of disease conditions, many representing significant and long-standing unmet medical needs. Certain properties of cell-based therapies, such as differentiation potential and proliferative potential, present safety concerns uniquely distinct from those of small molecule drugs and other macromolecule biologics. These cellular products carry risks associated with localized host tissue response, long-term persistence, ectopic tissue formation, differentiation to undesirable cell and tissue types, uncontrollable biodistribution, tumorigenicity, and immunogenicity. Such risks are generally evaluated in preclinical animal model studies as part of a comprehensive safety program prior to administration in humans. However, safety assessment for these products can be challenging because of inconsistent approaches to product characterization, inadequately defined product parameters that anticipate adverse events, and the lack of standardized approaches in evaluating in vivo host responses. In this symposium, we introduced cell-based therapies as an emerging product class to the Society of Toxicologic Pathology (STP) and highlighted key challenges for consideration during product biosafety evaluation.

Cell-based therapeutic products: potency assay development and application.

Potency is a critical quality attribute of biological products, defined by the US FDA as the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result. Ideally, a potency assay will leverage the product's mechanism of action. Alternatively, the assay may focus on a therapeutically relevant biological activity. The absence of rigorous mechanistic data for the majority of cell-based therapeutics currently in the process research pipeline has impeded efforts to design and validate indices of product potency. Development of a systematic battery of parallel functional assays that, taken together, can address all potential mechanisms of action believed to be relevant for the product platform is recommended. Such an approach is especially important during preclinical development. Here, we summarize the principal and unique challenges facing the development of functionally relevant and rigorous potency assays for cell-based therapeutics. We present perspectives regarding potency assay development for these products as illustrated by our experiences in process R&D of cryopreserved hepatocytes (Incara Pharmaceuticals) and selected renal cells (Tengion).

Cross-linked gelatin microspheres with continuously tunable degradation profiles for renal tissue regeneration.

Collagen and gelatin-based biomaterials are widely used in tissue engineering applications. Various methods have been reported for the cross-linking of these macromolecules for the purpose of delaying their biodegradation to prolong their in vivo residence (in tissue engineering applications) or tailoring their drug releasing capacity (when used as drug carriers). In this study, a carbodiimide-based cross-linking method, also used in the production of United States Food and Drug Administration-approved products, was employed to obtain differentially cross-linked gelatin beads. The colorimetric determination of the in vitro enzymatic susceptibility of the beads indicated that the resistance to degradation linearly correlated with the concentration of carbodiimide used for the cross-linking reaction. This result was also confirmed in vivo by the histological evaluation of the residence time of orthotopically injected cell-seeded beads. These data would indicate that the production of gelatin-based microbeads with tunable degradation profiles might be applicable toward the development of products that catalyze regeneration of kidney and other solid organs.

Regenerative medicine of the gastrointestinal tract.

Regenerative biology/tissue engineering offers potential solutions for the repair and augmentation of diseased tissues and organs. Tissue engineering technology platforms currently under development for organ regeneration may function in part by recapitulating key mechanistic and signaling pathways associated with embryonic organogenesis. Temporal observations of observed morphological outcomes from the regeneration of tubular organs provide insights into the mechanisms of action associated with the activation of regenerative pathways in preclinical animal models and humans. These include induction of a neo-blastema, regeneration of laminarily organized mural elements (i.e., lamina propria, sub-mucosa, and muscularis), and formation of context appropriate transitional junctions at the point of anastomosis with other tissue elements. These results provide the foundation for a regenerative technology applicable to hollow organs of the gastrointestinal (GI) tract including esophagus and small intestine. Factors affecting the efficacy of observed regenerative outcomes within the GI tract include the roles of vascularization, innervations, and mesenchymal signaling. These will be discussed in the context of an overall mechanism of adult regeneration potentially applicable by the tissue engineering and regenerative medicine industry for continued development of hollow neo-organ products.

A comparative study of efficacy and safety of gabapentin versus amitriptyline as coanalgesics in patients receiving opioid analgesics for neuropathic pain in malignancy.

OBJECTIVE: To assess the efficacy and safety of gabapentin and amitriptyline along with opioids in patients suffering from neuropathic pain in malignancy. MATERIALS AND METHODS: Eighty-eight adult patients between 18 and 70 years of age with neuropathic pain in stage III malignant disease, matched for baseline charactistics, were randomly assigned to two groups. Group A received oral tramadol and gabapentin and group B received oral tramadol and amitriptyline. The treatment duration of each patient was 6 months. Visual analog scale (VAS) was the primary efficacy parameter. Verbal rating scale (VRS) score, percentage of pain relief (PPR), and global pain score (GPS) were the secondary efficacy parameters. Oral morphine tablets or fentanyl transdermal patch were used as rescue medication. Data analysis was carried out in Graph Pad instat. RESULTS: There was decline in VAS pain score from baseline in both the groups in the early phase of the study though there was no statistically detectable difference between them at any study point. Similar changes were seen in the secondary efficacy parameters too. Thus both the drugs were effective in providing relief to cancer patients with neuropathic pain though there was no statistically detectable difference in efficacy between them. Six patients in group A and eight patients in group B required rescue medication. A total of 12 subjects in the gabapentin group and 15 subjects in the amitriptyline group experienced adverse events which were of mild to moderate grades. CONCLUSIONS: Amitriptyline may be a suitable alternative for management of neuropathic pain in cancer patients although gabapentin is widely used for this purpose. The lower cost of amitriptyline may favor patient compliance with lesser number of drop-outs.

Potency evaluation of tissue engineered and regenerative medicine products.

Methodologies for the rigorous and quantitative evaluation of biological activity or potency are an essential aspect of the developmental pathway for all biologic product candidates. Such assays typically leverage key mechanistic pathways demonstrated to mediate observed therapeutic outcomes. Tissue engineered/regenerative medicine (TE/RM) therapeutics include cell based therapies as well as engineered tissues and neo-organs for which clarity regarding the mechanism or mechanisms of action may not be forthcoming. Here, we discuss how strategies for the development of potency assays for TE/RM product candidates may harness potential mechanisms of action or other therapeutically relevant bioactivity along with cell number and viability. As the pipeline for TE/RM product candidates expands through 2014 and beyond, the establishment of a defined framework for potency assays will facilitate successful translational outcomes.

Fabrication of a multi-layer three-dimensional scaffold with controlled porous micro-architecture for application in small intestine tissue engineering.

Various methods can be employed to fabricate scaffolds with characteristics that promote cell-to-material interaction. This report examines the use of a novel technique combining compression molding with particulate leaching to create a unique multi-layered scaffold with differential porosities and pore sizes that provides a high level of control to influence cell behavior. These cell behavioral responses were primarily characterized by bridging and penetration of two cell types (epithelial and smooth muscle cells) on the scaffold in vitro. Larger pore sizes corresponded to an increase in pore penetration, and a decrease in pore bridging. In addition, smaller cells (epithelial) penetrated further into the scaffold than larger cells (smooth muscle cells). In vivo evaluation of a multi-layered scaffold was well tolerated for 75 d in a rodent model. This data shows the ability of the components of multi-layered scaffolds to influence cell behavior, and demonstrates the potential for these scaffolds to promote desired tissue outcomes in vivo.

Isolation of pulsatile cell bodies from esophageal tissue.

Pulsatile cell bodies, three-dimensional cell clusters with satellite streaming cells, can be isolated from -esophageal tissue. One of the key features of these clusters is that they pulsate at rhythmic rates and demonstrate contractility under several in vitro conditions. Their ability to pulsate appears to be due to the presence of interstitial cells of Cajal (ICC), which mediate signal transmission from nerve to muscle cells. As predicted, the cells comprising these clusters express phenotypic and genotypic markers characteristic of smooth and skeletal muscle, neuronal, and epithelial cells. Because of the critical role of ICC in gastrointestinal tract motility, loss of function in these cells can result in a variety of pathologies. Cultures of pulsatile cell bodies may have utility as an in vitro model to study tissue engineering and regenerative medicine approaches to treating defects in gastrointestinal rhythmicity due to disease or injury.

Migration assay to evaluate cellular interactions with biomaterials for tissue engineering/regenerative medicine applications.

Regenerative medicine and tissue engineering approaches for solving current medical dilemmas such as organ failure, congenital defect, or reconstruction following disease or trauma typically require specific considerations regarding biomaterial selection, identification of key cell types, and applicable surgical techniques (Lanza et al. Principles of tissue engineering, Academic, 2007; Kikuchi, Kanama., Quart Rev 24:51-67, 2007). The ability to evaluate these components in vitro under conditions which simulate relevant in vivo environments can reduce development risks including time and money costs associated with early-stage product development. Similarly, such methods can be useful in making progress in researching features of natural and synthetic biomaterial such as porosity, strength, surface topography, and functionalization, and their singular or collective effects on cell behavior (Kikuchi and Kanama., Quart Rev 24:51-67, 2007; Furth et al. Biomaterials 28:5068-5073, 2007; Mieszawska and Kaplan., BMC Biol 8:59, 2010).Adhesion, migration, and gene and protein expression are all cell behaviors that can be affected by properties of a chosen biomaterial and vary based upon organ system (Cornwell et al. J Biomater Res 71A:55-62, 2004; David et al. Tissue Eng 8(5):787-798, 2002). Understanding of these properties and their role in combination with biomaterial in remodeling is sought in order to fully harness and direct regeneration (Lanza et al. Principles of tissue engineering, Academic Press, 2007; Mieszawska and Kaplan. BMC Biol 8:59, 2010; Matragotri and Lahann J. Nat Mater 8:15-23, 2009).

Tissue engineering of esophagus and small intestine in rodent injury models.

Regenerative constructs composed of synthetically sourced, biodegradable biomaterials seeded with smooth muscle-like cells have been leveraged to mediate regeneration of bladder and bladder-like neo-organs. Here, we describe how such constructs may be applied to catalyze regeneration of esophagus and small intestine in preclinical rodent models.

Molecular characterization of the regenerative response induced by intrarenal transplantation of selected renal cells in a rodent model of chronic kidney disease.

Dedifferentiation and proliferation of resident tubular epithelial cells is a mechanism of action potentially contributing to repair and regeneration in kidneys presenting with ischemic or chronic disease. To more efficiently develop cell and tissue engineering technologies for the kidney, we have developed molecular assays to evaluate the acquisition of a pluripotent state associated with stem/progenitor cell phenotype during induction of a regenerative response within the kidneys of rats with chronic kidney disease (CKD) following therapeutic intervention. Intrarenal delivery of selected bioactive renal cells leads to significant upregulation of pluripotency-associated SOX2 mRNA within the diseased kidney tissue from 1 to 24 weeks after treatment. The overall regenerative response index was assessed by quantitative composite expression of CD24, NODAL and LEFTY1 proteins, which were induced within 1 week of cell treatment and peaked at 12 weeks after treatment, reaching statistical significance (p < 0.05) compared to untreated CKD controls. Molecular assays that incorporate the assessment of SOX2 and the regenerative response index may prove to be valuable tools for the detection and monitoring of the tissue response after the delivery of regenerative treatments for CKD, thereby significantly shortening the developmental timelines associated with such therapies.

Developmental engineering the kidney: leveraging principles of morphogenesis for renal regeneration.

Multiple methodological approaches are currently under active development for application in tissue engineering and regenerative medicine of tubular and solid organs. Most recently, developmental engineering (TE/RM), or the leveraging of embryonic and morphological paradigms to recapitulate aspects of organ development, has been proposed as a strategy for the sequential, iterative de novo assembly of tissues and organs as discrete developmental modules ex vivo, prior to implantation in vivo. In this article, we focus on the kidney to highlight in detail how principles of developmental biology are impacting approaches to TE of this complex solid organ. Ultimately, such methodologies may facilitate the establishment of clinically relevant therapeutic strategies for regeneration of renal structure and function, greatly impacting treatment regimens for chronic kidney disease.

Construction of a tubular scaffold that mimics J-shaped stress/strain mechanics using an innovative electrospinning technique.

Soft tissues such as blood vessel, lung, ureter, skin, etc., possess mechanical behavior characterized by a "J"-shaped curve on a stress-strain diagram with a low-stiffness highly elastic zone giving rise to a high-stiffness zone. This mechanical behavior may be adaptive and protective against aneurysm formation in tissues whose primary loading is pressure-based. "J"-shaped behavior arises from the synergistic interplay of two main structural proteins: collagen and elastin. An innovative electrospinning technique has been utilized to form tubular scaffold composites with structural features reminiscent of the corrugated laminae seen in blood vessels. In doing so, tubular scaffolds have been fabricated with complex "J"-shaped behavior through the use of elastic polyurethane and reinforcing poly-glycolic acid (PGA) woven mesh. In these studies, corrugated laminae were formed on the 175 µm and 1.5 mm scale. Initial moduli were 0.5±0.17 MPa (mean±standard deviation) giving rise to stiffer moduli of 36.09±6.72 MPa at a strain of 1.31±0.15. Burst pressures were physiologically relevant at 3095±1016 mmHg. The toughness of these prototypes was 6.3±1.9 MJ/m(3). The ability to employ different materials and different formation parameters utilizing this technique promises the ability to match complex stress-strain behaviors in soft tissues with a high degree of fidelity.