Challenges and opportunities for access to Advanced Therapy Medicinal Products in Brazil.

BACKGROUND AIMS: The marketing authorization of Advanced Therapy Medicinal Products (ATMPs) in Brazil is recent. The features of these therapies impose specialized regulatory action and are consequently challenging for developers. The goal of this study was to identify the industry's experience in clinical development, marketing authorization and access to ATMPs through the Unified Health System (SUS, acronym in Portuguese), from a regulatory perspective. METHODS: A survey containing structured questions was conducted among research participants who work at companies that commercialize ATMPs. A descriptive analysis was performed. RESULTS: We invited 15 foreign pharmaceutical companies, of which 10 agreed to participate. Overall, participants assessed that Brazil has a well-established regulatory system, especially the sanitary registration by the National Health Surveillance Agency (Anvisa), which ensures the quality, safety, and efficacy of the products. The Agency's good interaction with the regulated sector, the harmonization of sanitary and ethical assessment systems with other countries, and the analysis time in the biosafety assessment of Genetically Modified Organisms (GMOs) stand out as positive in industry's evaluation. On the other hand, it is important to advance the pricing regulation for these products since Brazilian regulations do not establish specific criteria for ATMP. One of the biggest challenges is the difficulty for the SUS in reimbursing these very high-cost therapies, especially using current Health Technology Assessment (HTA) methods. CONCLUSIONS: Considering the increasing number of approvals of cell and gene therapies in Brazil in the coming years, a close dialogue between the industry and the public sector is recommended to advance regulatory improvements (pricing and HTA). Additionally, the construction of policies to promote the national Health Economic-Industrial Complex, based on a mission-oriented vision that encourages innovative models of financing, especially those that consider risk-sharing and co-financing technologies, will help provide the population with universal, equitable and sustainable access to ATMP in the SUS.

Prevalence and impact of sarcopenia in individuals with heart failure with reduced ejection fraction (the SARC-HF study): A prospective observational study protocol.

Sarcopenia, a clinical syndrome primarily associated with reduced muscle mass in the elderly, has a negative impact on quality of life and survival. It can occur secondarily to other diseases such as heart failure (HF), a complex clinical syndrome with high morbidity and mortality. The simultaneous occurrence of these two conditions can worsen the prognosis of their carriers, especially in the most severe cases of HF, as in patients with reduced left ventricular ejection fraction (LVEF). However, due to the heterogeneous diagnostic criteria for sarcopenia, estimates of its prevalence present a wide variation, leading to new criteria having been recently proposed for its diagnosis, emphasizing muscle strength and function rather than skeletal muscle mass. The primary objective of this study is to evaluate the prevalence of sarcopenia and/or dynapenia in individuals with HF with reduced LVEF according to the most recent criteria, and compare the gene and protein expression of those patients with and without sarcopenia. The secondary objectives are to evaluate the association of sarcopenia and/or dynapenia with the risk of clinical events and death, quality of life, cardiorespiratory capacity, ventilatory efficiency, and respiratory muscle strength. The participants will answer questionnaires to evaluate sarcopenia and quality of life, and will undergo the following tests: handgrip strength, gait speed, dual-energy X-ray absorptiometry, respiratory muscle strength, cardiopulmonary exercise, as well as genomic and proteomic analysis, and dosage of N-terminal pro-B-type natriuretic peptide and growth differentiation factor-15. An association between sarcopenia and/or dynapenia with unfavorable clinical evolution is expected to be found, in addition to reduced quality of life, cardiorespiratory capacity, ventilatory efficiency, and respiratory muscle strength.

Additional improvement in regional myocardial ischemia after intracardiac injection of bone marrow cells during CABG surgery.

Background: Post-procedure residual ischemia is associated with worse prognosis in patients with coronary artery diasease (CAD). Objective: We evaluated whether autologous bone marrow-derived cells (BMC) contribute to additional reduction in regional stress-induced myocardial ischemia (SIMI) in patients undergoing incomplete coronary artery bypass graft surgery (CABG). Methods: In a double-blind, randomized, placebo-controlled trial, we enrolled 143 patients (82% men, 58 ± 11 years) with stable CAD and not candidates for complete CABG. They received 100 million BMC (n = 77) or placebo (n = 66) injected into ischemic non-revascularized segments during CABG. The primary outcome was improvement on SIMI quantified as the area at risk in injected segments assessed by cardiovascular magnetic resonance (CMR) 1, 6, and 12 months after CABG. Results: The reduction in global SIMI after CABG was comparable (p = 0.491) in both groups indicating sustained beneficial effects of the surgical procedure over 12 month period. In contrast, we observed additional improvement in regional SIMI in BMC treated group (p = 0.047). Baseline regional SIMI values were comparable [18.5 (16.2-21.0) vs. 18.5 (16.5-20.7)] and reached the lowest values at 1 month [9.74 (8.25; 11.49) vs. 12.69 (10.84; 14.85)] for BMC and placebo groups, respectively. The ischemia's improvement from baseline represented a 50% difference in regional SIMI in favor of the BMC transplanted group at 30 days. We found no differences in clinical and LVEF% between groups during the 12 month follow-up period. The 1 month rate of major adverse cerebral and cardiovascular events (MACCE) (p = 0.34) and all-cause mortality (p = 0.08) did not differ between groups 1 month post intervention. Conclusion: We provided evidence that BMC leads to additional reduction in regional SIMI in chronic ischemic patients when injected in segments not subjected to direct surgical revascularization. This adjuvant therapy deserves further assessment in patients with advanced CAD especially in those with microcirculation dysfunction. Clinical trial registration: https://clinicaltrials.gov/, identifier NCT01727063.

Landscape of Brazilian research and development public funding in advanced therapies: lessons learned and a roadmap for middle-income economies.

BACKGROUND AIMS: Advanced therapy medicinal products (ATMPs) have reached the forefront of biotechnological innovation, partly due to public funders' efforts in the early stages of research and development (R&D). Data on investment in R&D of ATMPs are recognized as scarce, particularly in developing countries. Because of the numerous peculiarities of the Brazilian health system and the science and technology (S&T) system, the country is a good example for the evaluation of public investments in R&D of ATMPs. The aim of this study is to analyze the evolution of investments made by the Ministry of Health (MoH) of Brazil and partners in the ATMP field between 2004 and 2020. METHODS: A descriptive analysis was performed based on secondary data. The analysis was based on S&T and innovation research and support for research infrastructure in the field. The database was stratified by year of funding, ATMP type, type of study or research infrastructure project, amount invested in the project, targeted disease for which clinical trials in ATMPs were developed and financing sector (health, education, S&T and economic). RESULTS: The investments coordinated by MoH (61.5%) in partnership with the S&T, education and economic sectors (38.5%) consisted of Int$137.35 million in 282 ATMP projects. Funding included S&T and innovation research (67% of the total amount) and projects to implement or maintain infrastructure in selected research centers (32.98%). With regard to global convergence, cell therapy was the type of ATMP that most benefited from public investment, totaling 82.23% of the total funding in the analyzed period. Cardiology (29%) and neurology (21%) were the main focus of clinical trials. Following the global trend of public sector R&D funding, the number of basic and pre-clinical research projects represented 78.06% of the total number of projects. CONCLUSIONS: Despite the need to implement improvements in ATMP R&D financing policy in Brazil, the country has made important steps in the field and can serve as a benchmark for other countries with socioeconomic similarities. Among the main lessons are the prioritization of research aligned with the health needs of the population, cross-sector articulation by the health policymaker to coordinate R&D efforts of the sector and formulation of a specific sector policy (Programa Genomas Brasil, the Brazilian National Program of Genomic and Precision Medicine) to promote knowledge translation.

In Situ Maturated Early-Stage Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Improve Cardiac Function by Enhancing Segmental Contraction in Infarcted Rats.

The scant ability of cardiomyocytes to proliferate makes heart regeneration one of the biggest challenges of science. Current therapies do not contemplate heart re-muscularization. In this scenario, stem cell-based approaches have been proposed to overcome this lack of regeneration. We hypothesize that early-stage hiPSC-derived cardiomyocytes (hiPSC-CMs) could enhance the cardiac function of rats after myocardial infarction (MI). Animals were subjected to the permanent occlusion of the left ventricle (LV) anterior descending coronary artery (LAD). Seven days after MI, early-stage hiPSC-CMs were injected intramyocardially. Rats were subjected to echocardiography pre-and post-treatment. Thirty days after the injections were administered, treated rats displayed 6.2% human cardiac grafts, which were characterized molecularly. Left ventricle ejection fraction (LVEF) was improved by 7.8% in cell-injected rats, while placebo controls showed an 18.2% deterioration. Additionally, cell-treated rats displayed a 92% and 56% increase in radial and circumferential strains, respectively. Human cardiac grafts maturate in situ, preserving proliferation with 10% Ki67 and 3% PHH3 positive nuclei. Grafts were perfused by host vasculature with no evidence for immune rejection nor ectopic tissue formations. Our findings support the use of early-stage hiPSC-CMs as an alternative therapy to treat MI. The next steps of preclinical development include efficacy studies in large animals on the path to clinical-grade regenerative therapy targeting human patients.

Tissue-engineered human embryonic stem cell-containing cardiac patches: evaluating recellularization of decellularized matrix.

Decellularized cardiac extracellular matrix scaffolds with preserved composition and architecture can be used in tissue engineering to reproduce the complex cardiac extracellular matrix. However, evaluating the extent of cardiomyocyte repopulation of decellularized cardiac extracellular matrix scaffolds after recellularization attempts is challenging. Here, we describe a unique combination of biochemical, biomechanical, histological, and physiological parameters for quantifying recellularization efficiency of tissue-engineered cardiac patches compared with native cardiac tissue. Human embryonic stem cell-derived cardiomyocytes were seeded into rat heart atrial and ventricular decellularized cardiac extracellular matrix patches. Confocal and atomic force microscopy showed cell integration within the extracellular matrix basement membrane that was accompanied by restoration of native cardiac tissue passive mechanical properties. Multi-electrode array and immunostaining (connexin 43) were used to determine synchronous field potentials with electrical coupling. Myoglobin content (~60%) and sarcomere length measurement (>45% vs 2D culture) were used to evaluate cardiomyocyte maturation of integrated cells. The combination of these techniques allowed us to demonstrate that as cellularization efficiency improves, cardiomyocytes mature and synchronize electrical activity, and tissue mechanical/biochemical properties improve toward those of native tissue.

Macrophage-dependent IL-1ß production induces cardiac arrhythmias in diabetic mice.

Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1ß in DM mice. IL-1ß causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1ß-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1ß axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1ß as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.

Stem Cell-Based Therapies in Chagasic Cardiomyopathy.

Chagas disease is caused by Trypanosoma cruzi and can lead to a dilated cardiomyopathy decades after the prime infection by the parasite. As with other dilated cardiomyopathies, conventional pharmacologic therapies are not always effective and as heart failure progresses patients need heart transplantation. Therefore alternative therapies are highly desirable and cell-based therapies have been investigated in preclinical and clinical studies. In this paper we review the main findings of such studies and discuss future directions for stem cell-based therapies in chronic chagasic cardiomyopathy.

99m-Technetium binding site in bone marrow mononuclear cells.

INTRODUCTION: The increasing interest in 99m-technetium ((99m)Tc)-labeled stem cells encouraged us to study the (99m)Tc binding sites in stem cell compartments. METHODS: Bone marrow mononuclear cells were collected from femurs and tibia of rats. Cells were labeled with (99m)Tc by a direct method, in which reduced molecules react with (99m)Tc with the use of chelating agents, and lysed carefully in an ultrasonic apparatus. The organelles were separated by means of differential centrifugation. At the end of this procedure, supernatants and pellets were counted, and the percentages of radioactivity (in megabecquerels) bound to the different cellular fractions were determined. Percentages were calculated by dividing the radioactivity in each fraction by total radioactivity in the sample. The pellets were separated and characterized by their morphology on electron microscopy. RESULTS: The labeling procedure did not affect viability of bone marrow mononuclear cells. Radioactivity distributions in bone marrow mononuclear cell organelles, obtained in five independent experiments, were approximately 38.5 % in the nuclei-rich fraction, 5.3 % in the mitochondria-rich fraction, 2.2 % in microsomes, and 54 % in the cytosol. Our results showed that most of the radioactivity remained in the cytosol; therefore, this is an intracellular labeling procedure that has ribosomes unbound to membrane and soluble molecules as targets. However, approximately 39 % of the radioactivity remained bound to the nuclei-rich fraction. To confirm that cell disruption and organelle separation were efficient, transmission electron microscopy assays of all pellets were performed. CONCLUSIONS: Our results showed that most of the radioactivity was present in the cytosol fraction. More studies to elucidate the mechanisms involved in the cellular uptake of (99m)Tc in bone marrow cells are ongoing.

Expression of ganglioside 9-O acetyl GD3 in undifferentiated embryonic stem cells.

Embryonic stem cells (ES cells) express a transient and heterogeneous pattern of molecules, which suggests a notable mechanism to control self-renewal avoid the differentiation into germ layers. We show that 9-O-acetyl GD3 (9OacGD3), a highly expressed b-series ganglioside in neural stem (NS) cells, is expressed in undifferentiated mouse ES cells in a heterogeneous fashion. After sorting, undifferentiated 9OacGD3(+) ES cell population had higher levels of nestin and Sox2 mRNA than the 9OacGD3(-) cells. Even with elevated expression of these neural transcription factors, 9OacGD3(+) cells did not give rise to more neural progenitors than 9OacGD3(-) cells. Expression of 9OacGD3 was recovered from 9OacGD3(-) cell population, demonstrating that expression of this ganglioside in mouse embryonic stem cells is transient, and does not reflect cell fate. Our findings show that the ganglioside 9OacGD3 is expressed heterogeneously and transiently in ES cells, and this expression corresponds to higher levels of Sox2 and Nestin transcripts.

Turning scar into muscle.

After the demonstration that somatic cells could be reprogrammed to a pluripotent state, exciting new prospects were opened for the cardiac regeneration field. It did not take long for the development of strategies to convert somatic cells directly into cardiomyocytes. Despite the intrinsic difficulties of cell reprogramming, such as low efficiency, the therapeutic possibilities created by the ability to turn scar into muscle are enormous. Here, we discuss some of the major advances and strategies used in direct cardiac reprogramming and examine discrepancies and concerns that still need to be resolved in the field.

Labeling stem cells with superparamagnetic iron oxide nanoparticles: analysis of the labeling efficacy by microscopy and magnetic resonance imaging.

Stem cell therapy has emerged as a potential therapeutic option for cell death-related heart diseases. Application of non-invasive cell tracking approaches is necessary to determine tissue distribution and lifetime of stem cells following their injection and will likely provide knowledge about poorly understood stem cells mechanisms of tissue repair. Magnetic resonance imaging (MRI) is a potentially excellent tool for high-resolution visualization of the fate of cells after transplantation and for evaluation of therapeutic strategies. The application of MRI for in vivo cell tracking requires contrast agents to achieve efficient cell labeling without causing any toxic cellular effects or eliciting any other side effects. For these reasons clinically approved contrast agents (e.g., ferumoxides) and incorporation facilitators (e.g., protamine) are currently the preferred materials for cell labeling and tracking. Here we describe how to use superparamagnetic iron oxide nanoparticles to label cells and to monitor cell fate in several disease models.

Acute adenosine increases cardiac vagal and reduces sympathetic efferent nerve activities in rats.

Adenosine is the first drug of choice in the treatment of supraventricular arrhythmias. While the effects of adenosine on sympathetic nerve activity (SNA) have been investigated, no information is available on the effects on cardiac vagal nerve activity (VNA). We assessed in rats the responses of cardiac VNA, SNA and cardiovascular variables to intravenous bolus administration of adenosine. In 34 urethane-anaesthetized rats, cardiac VNA or cervical preganglionic sympathetic fibres were recorded together with ECG, arterial pressure and ventilation, before and after administration of three doses of adenosine (100, 500 and 1000 µg kg(-1)). The effects of adenosine were also assessed in isolated perfused hearts (n = 5). Adenosine induced marked bradycardia and hypotension, associated with a significant dose-dependent increase in VNA (+204 ± 56%, P < 0.01; +275 ± 120%, P < 0.01; and +372 ± 78%, P < 0.01, for the three doses, respectively; n = 7). Muscarinic blockade by atropine (5 mg kg(-1), i.v.) significantly blunted the adenosine-induced bradycardia (-56.0 ± 4.5%, P < 0.05; -86.2 ± 10.5%, P < 0.01; and -34.3 ± 9.7%, P < 0.01, respectively). Likewise, adenosine-induced bradycardia was markedly less in isolated heart preparations. Previous barodenervation did not modify the effects of adenosine on VNA. On the SNA side, adenosine administration was associated with a dose-dependent biphasic response, including overactivation in the first few seconds followed by a later profound SNA reduction. Earliest sympathetic activation was abolished by barodenervation, while subsequent sympathetic withdrawal was affected neither by baro- nor by chemodenervation. This is the first demonstration that acute adenosine is able to activate cardiac VNA, possibly through a central action. This increase in vagal outflow could make an important contribution to the antiarrhythmic action of this substance.

Bone marrow-derived cell therapy in chagasic cardiac disease: a review of pre-clinical and clinical results.

Chagas disease is caused by a protozoan parasite Trypanosoma cruzi, which infects people through blood sucking insects. It is endemic in Latin America and the disease is being spread to developed countries as a result of the migration of infected individuals. In its chronic stage, Chagas disease can lead to a severe cardiomyopathy for which there is currently no cure. End-stage patients require heart transplantation, thus demanding new therapeutic modalities. Cell-based therapy has been proposed as an alternative for various forms of heart disease. Here we review the experimental evidence that led to the use of bone marrow-derived cells in putative therapy for chronic chagasic cardiomyopathy in animal models and in clinical trials, discussing the reasons for failure of the translation of results from mice to men.

Bone marrow progenitor cells do not contribute to liver fibrogenic cells.

AIM: To investigate the contribution of bone marrow (BM) cells to hepatic fibrosis. METHODS: To establish a model of chimerism, C57Bl/6 female mice were subjected to full-body irradiation (7 Gy) resulting in BM myeloablation. BM mononuclear cells obtained from male transgenic mice expressing enhanced green fluorescent protein (GFP) were used for reconstitution. Engraftment was confirmed by flow cytometry. To induce liver injury, chimeric animals received carbon tetrachloride (CCl(4)) 0.5 mL/kg intraperitoneally twice a week for 30 d (CCl(4) 30 d) and age-matched controls received saline (Saline 30 d). At the end of this period, animals were sacrificed for post mortem analysis. Liver samples were stained with hematoxylin and eosin to observe liver architectural changes and with Sirius red for collagen quantification by morphometric analysis. α-smooth muscle actin (α-SMA) was analyzed by confocal microscopy to identify GFP+ cells with myofibroblast (MF) characteristics. Liver tissue, BM and peripheral blood were collected and prepared for flow cytometric analysis using specific markers for detection of hepatic stellate cells (HSCs) and precursors from the BM. RESULTS: Injury to the liver induced changes in the hepatic parenchymal architecture, as reflected by the presence of inflammatory infiltrate and an increase in collagen deposition (Saline 30 d = 11.10% ± 1.12% vs CCl(4) 30 d = 12.60% ± 0.73%, P = 0.0329). Confocal microscopy revealed increased reactivity against α-SMA in CCl(4) 30 d compared to Saline 30 d, but there was no co-localization with GFP+ cells, suggesting that cells from BM do not differentiate to MFs. Liver flow cytometric analysis showed a significant increase of CD45+/GFP+ cells in liver tissue (Saline 30 d = 3.2% ± 2.2% vs CCl(4) 30 d = 5.8% ± 1.3%, P = 0.0458), suggesting that this increase was due to inflammatory cell infiltration (neutrophils and monocytes). There was also a significant increase of common myeloid progenitor cells (CD117+/CD45+) in the livers of CCl(4)-treated animals (Saline 30 d = 2.16% ± 1.80% vs CCl(4) 30 d = 5.60% ± 1.30%, P = 0.0142). In addition the GFP-/CD38+/CD45- subpopulation was significantly increased in the CCl(4) 30 d group compared to the Saline 30 d group (17.5% ± 3.9% vs 9.3% ± 2.4%, P = 0.004), indicating that the increase in the activated HSC subpopulation was not of BM origin. CONCLUSION: BM progenitor cells do not contribute to fibrosis, but there is a high recruitment of inflammatory cells that stimulates HSCs and MFs of liver origin.

Cysteine proteases in differentiation of embryonic stem cells into neural cells.

Glycosylated mouse cystatin C (mCysC), an endogenous inhibitor of cysteine cathepsin proteases (CP), has been suggested as a cofactor of ß-FGF to induce the differentiation of mouse embryonic stem cells into neural progenitor cells (NPCs). To investigate the possible role of CP in neural differentiation, we treated embryoid bodies (EBs) with (i) E64, an inhibitor of papain-like CP and of calpains, (ii) an inhibitor of cathepsin L (iCatL), (iii) an inhibitor of calpains (iCalp), or (iv) cystatins, and their ability to differentiate into neural cells was assessed. We show that the inhibition of CP induces a significant increase in Pax6 expression in EBs, leading to an increase in the number of nestin-positive cells after 3 days. Fourteen days after E64 treatment, we observed increased numbers of ß-III-tubulin-positive cells, showing greater percentage of immature neurons, and this feature persisted up to 24 days. At this point, we encountered higher numbers of neurons with inward Na(+) current compared with untreated EBs. Further, we show that mCysC and iCatL, but not unglycosylated egg white cystatin or iCalp, increased the numbers of NPCs. In contrast to E64 and iCatL, mCysC did not inhibit CP in EBs and its neural-inducing activity required ß-FGF. We propose that the inhibition of CP induces the differentiation of mouse embryonic stem cells into NPCs and neurons through a mechanism that is distinct from CysC-induced neural differentiation.

Heart regeneration: Past, present and future.

The heart has been considered a post-mitotic organ without regenerative capacity for most of the last century. We review the evidence that led to this hypothesis in the early 1900s and how it was progressively modified, culminating with the report that we renew 50% of our cardiomyocytes during our lifetime. The future of cardiac regenerative therapies is discussed, presenting the difficulties to overcome before repair of the diseased heart can come into clinical practice.