Sugar-lowering drugs for type 2 diabetes mellitus and metabolic syndromestrategies for in vivo administration: Part-II

Journal of Clinical Medicine Review Sugar-Lowering Drugs for Type 2 Diabetes Mellitus and Metabolic Syndrome—Strategies for In Vivo Administration: Part-II Raquel Vieira 1, Selma B. Souto 2, Elena Sánchez-López 1,3,4 , Ana López Machado 3, Patricia Severino 5,6 , Sajan Jose 7, Antonello Santini 8, Amelia M. Silva 9,10 , Ana Fortuna 11,12, Maria Luisa García3,4 and Eliana B. Souto 1,13,* 1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), Pólo das Ciências da Saúde, 3000-548 Coimbra, Portugal 2Department of Endocrinology, Braga Hospital, Sete Fontes, 4710-243 São Victor Braga, Portugal 3Department of Pharmacy, Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, Institute of Nanoscience and Nanotechnology (IN2UB), Av. Joan XXIII, 27-31, 08028 Barcelona, Spain 4Centro de Investigación biomédica en red de enfermedades neurodegenerativas (CIBERNED), 28031 Madrid, Spain 5Laboratory of Nanotechnology and Nanomedicine (LNMED), Institute of Technology and Research (ITP), Av. Murilo Dantas, 300, Aracaju 49010-390, Brazil 6Department of Pharmacy, University of Tiradentes (UNIT), Industrial Biotechnology Program, Av. Murilo Dantas 300, Aracaju 49032-490, Brazil 7 Department of Pharmaceutical Sciences, Mahatma Gandhi University, Cheruvandoor Campus, Ettumanoor, Kerala 686631, India 8 Department of Pharmacy, University of Naples Federico II, Via Domenico Montesano, 49-80131 Naples, Italy 9Department of Biology and Environment, University of Trás-os Montes e Alto Douro (UTAD), Quinta de Prados, 5001-801 Vila Real, Portugal 10 Centre for Research and Technology of Agro-Environmental and Biological Sciences (CITAB-UTAD), Quinta de Prados, 5001-801 Vila Real, Portugal 11 Laboratory of Pharmacology, Faculty of Pharmacy, University of Coimbra (FFUC), Pólo das Ciências da Saúde, 3000-548 Coimbra, Portugal 12 CIBIT—Coimbra Institute for Biomedical Imaging and Translational Research, University of Coimbra, 3000-548 Coimbra, Portugal 13 CEB—Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal *Correspondence: [email protected] Received: 29 July 2019; Accepted: 22 August 2019; Published: 28 August 2019   Abstract: Diabetes is a complex disease characterized by hyperglycemia, together with polyuria, polydipsia, and polyphagia. While Type 1 diabetes mellitus (T1DM) results from genetic, environmental, or immune dysfunction factors leading to pancreatic β -cell destruction depriving the organism from endogenous insulin, Type 2 diabetes mellitus (T2DM) is characterized by peripheral insulin resistance. Depending on the type of diabetes mellitus and drug mechanism to study, the animal model should be carefully selected among the wide variety of the currently available ones. This review discusses the most common animal models currently employed to study T1DM and T2DM. Moreover, an overview on the administration routes that could be used is also discussed. Keywords: diabetes mellitus; animal models; in vivo; administration routes J. Clin. Med. 2019,8, 1332; doi:10.3390/jcm8091332 www.mdpi.com/journal/jcm J. Clin. Med. 2019,8, 1332 2 of 20 1. Introduction Diabetes mellitus (DM) is one of the current leading health problems worldwide. DM comprises a group of metabolic diseases characterized by hyperglycemia, as insulin production by pancreatic β -cells is either insufficient or even absent, and target cells do not respond to circulating insulin [ 1 , 2 ]. Associated with hyperglycemia, together with common symptoms, namely polyuria, polydipsia, and polyphagia [ 1 ], DM is a silently life-threatening condition that may culminate on hemodynamic and cardiovascular complications, metabolic complications, and altered genetic susceptibility [ 3 ]. Also, long-term developing diseases, as a consequence of hyperglycemia affecting the whole organism or being organ specific, as is the case of diabetic retinopathy, is in the natural course of the disease [4]. DM is classified into two main types: type 1 DM (T1DM), previously known an insulin dependent DM (IDDM), and type 2 DM (T2DM), previously known as non-insulin dependent DM (NIDDM) [ 5 ]. In addition, gestational DM is characterized by the development of DM by pregnant women with hyperglycemia during pregnancy with no previous history of the disease [ 3 ]. T1DM results from a complex disease process in which genetic, environmental factors, and/or immune dysfunction lead to an autoimmune response, resulting in pancreatic β -cell destruction, depriving the organism from endogenous insulin [ 6 , 7 ]. T2DM comprises about 85% of DM cases and is characterized by peripheral insulin resistance. Compensatory insulin hypersecretion from pancreatic β -cells precedes the decline in islet secretory function. Reduced insulin sensitivity affects mainly the skeletal muscle, liver, and adipose tissue, as these tissues have particular requirements for glucose uptake and metabolism [ 3 , 7 ]. In general, DM consequences affect various systems, organs, and tissues [ 4 , 8 – 11 ], being therefore difficult to predict in the exact consequences of the disease, as genetic, nutrition, environment, and other factors are different in each individual [ 10 , 11 ]. With these constraints, several animal models have been developed, aiming at assessing the causes and consequences of DM, in order to achieve effective and safe treatments. This review describes the animal models currently available to study DM and its complications. Additionally, since the administration routes are of crucial relevance in order to study their effectivity, a comprehensive overview about the different administration routes used for pre-clinical and clinical trials has been undertaken. In this sense, the key to successful drug development relies on the suitable selection of the animal model and the drug administration route. 2. Diabetes Mellitus Animal Models The study of DM comprises the study of normal individuals, as only by knowing how a physiological organ/tissue responds to stimuli can we correlate to the altered response in compromised organ/tissues. The study of glucose regulated insulin secretion, as well as its modulation by oral antidiabetic-drugs, has been made with resource to albino mice (Charles Rivers breeding), using isolated islets of Langerhans [12,13] or isolated pancreatic β-cells [14,15]. Other sources of pancreatic β -cells from physiological donors have also been used, such as porcine, human, canine, among others [13–15]. Animal models may be developed by two principal mechanisms: disease induction (e.g., using specific drugs) or genetic manipulation. Both are of extreme relevance as they allow the study of specific disease-related mechanisms and are essential to understand the pathogenesis and progression of the diseases and extrapolate to humans. Since T1DM and T2DM are metabolic disorders, representing complex integrating bodily systems, choosingtheadequateanimalmodeltousein specific in vivo experimentsrequirescareful consideration[ 16 ]. To attain this objective, a careful analysis must be performed when choosing a DM animal model, regarding particular aspects of the disease and the specific knowledge that is aimed in each study [ 16 ]. In this context, T1DM animal models include spontaneously developing autoimmune diabetic animals, or animals that have been subjected to chemical ablation of pancreatic β -cells. On the other hand, T2DM animal models are more numerous, and may embrace obese and non-obese models, with variable insulin resistance and β -cell failure degrees [ 16 ]. In addition, transgenic and knock-out mouse models are also available, but their use in the research field is still controversial, as it will be herein focused [16]. J. Clin. Med. 2019,8, 1332 3 of 20 Table 1. Animal models most commonly used to study type 1 diabetes mellitus. Induction Mechanism Model Main Features Possible Uses Advantages Disadvantages Ref. Chemical induction High single-dose streptozotocin (STZ) * Simple model of hyperglycemia Testing drugs (new insulin formulations) or therapies (transplantation) •A more stable model •Mortality is relatively more frequent [17,18] •Comparatively cheaper, easier to develop and maintain Multiple low dose streptozotocin (STZ) *Model of induced insulitis Treatments that may prevent β-cell death •Used for longer experimental studies •May produce toxic effects on other tissues • May be applied to higher animals •STZ is relatively unstable and solution should ideally be made immediately prior to injection Alloxan *Simple model of hyperglycemia Transplantation models • Selective loss of pancreatic β -cells leaving αand δcells intact •Hyperglycemia develops primarily by direct cytotoxic action on the β-cells and insulin deficiency rather than consequence of insulin resistance [19,20] •Animals live longer without insulin treatment (since there is a residual insulin secretion) •Less stable and reversible because of the spontaneous regeneration of β-cells •Relatively less ketosis and resulting mortality •May produce toxic effects on other tissues •Comparatively cheaper, easier to develop and to maintain •High variability of results on development of hyperglycemia J. Clin. Med. 2019,8, 1332 4 of 20 Table 1. Cont. Induction Mechanism Model Main Features Possible Uses Advantages Disadvantages Ref. Spontaneous autoimmune Non-obese diabetic (NOD) mice (Spontaneous autoimmune model of choice) β-cell destruction due to an autoimmune process Understanding genetics of T1DM •Hyperlipidemia can be also studied, as lipid content increase •Polyphagia and polyuria occurrence [16,21,22] Biobreeding (BB) rats Understanding mechanism of T1DM •Hyperglycemia persists for several days •A diabetes and obesity symptom overlaps [23,24] LEW.1AR1/-iidm rats Treatments that may prevent β -cell death and/or manipulate autoimmune process •Not identical to those in human disease Genetically induced AKITA mice *β-cell destruction due to ER stress. Insulin dependent. New formulations of insulin Transplantation models Treatments to prevent ER stress •The lack of β-cell mass makes it an alternative to STZ-treated mice in transplantation studies [25,26] Virally-induced Coxsackie B virus β-cell destruction induced by viral infection of b-cells Establish potential role of viruses in the development of T1DM •Stable and irreversible diabetes can be induced •Comparatively costlier to develop [27] Encephalomyocarditis virus Kilham rat virus LCMV under insulin promoter •Technical expert is required to handle of virus Non-rodent models Pancreatectomy Hyperglycemia induction in pigs, dogs and primates Treatments that may prevent β-cell death Transplantation models •Reasonably accurate model of auto transplantation of islets in humans •Very invasive surgery [28] •In large animal models, spontaneous diabetes is relatively rare and unpredictable in onset Chemical ablation of β-cells in large animals •Some models combine a partial pancre-atectomy with STZ treatment, thus reducing the dose of STZ •Interspecies variation in the β-cell toxicity of alloxan or STZ [28] •Narrow window of efficacy. *Also used in T2DM research. J. Clin. Med. 2019,8, 1332 5 of 20 Table 2. Animal models most commonly used to study type 2 diabetes mellitus. Induction Mechanism Model Main Features Possible Uses Advantages Disadvantages Ref. Obese monogenic models Lepob/ob mice (mutated leptin gene) Obesity-induced hyperglycemia, with hyperphagic, obese, hyperinsulinaemic and hyperglycemic animals Treatments to improve insulin resistance •Pancreatic islet volume dramatically increased •Infertile mice [29] •Metabolic aberrations (hyperlipidemia disturbance in temperature regulation, lower physical activity) •Diabetes not particular severe and thus not completely representative of human T2DM Leprdb/db mice (mutated leptin receptor gene) Treatments to improve β-cell function • Ketosis after a few months of age, having a relative short lifespan [30] Zucker Diabetic Fatty (ZDF) Rats (mutated leptin receptor gene) •Diabetic complications also develop •Hypertensive rats [31] •Females do not develop overt diabetes Obese polygenic models KK mice Obesity-induced hyperglycemia Treatments to improve insulin resistance Otsuka Long-Evans Tokushima Fat (OLEFT) rat Treatments to improve β-cell function •Three stages of histological changes can be observed [32,33] New Zealand Obese (NZO) mice Some models show diabetic complications •Renal complications TallyHo/Jng mice •Adiposity, plasma triglycerides, cholesterol and free fatty acid levels are increased •Only male mice develop hyperglycemia NoncNZO10/LtJ mice •Indicated for diabetic wound healing studies •Nephropathy presence J. Clin. Med. 2019,8, 1332 6 of 20 Table 2. Cont. Induction Mechanism Model Main Features Possible Uses Advantages Disadvantages Ref. Induced obesity models High fat feeding (mice or rats) Obesity-induced hyperglycemia Treatments to improve insulin resistance •Baboons and humans are genetically, anatomically and physiologically very similar •Handling of baboon is somewhat difficult [31,33] Desert gerbil Treatments to improve β-cell function •Cardiac complications can be studied •Veterinarian is required Nile grass rat Treatments to prevent diet-induced obesity •Costly model containing Non-obese models Goto-Kakizaki (GK) rat Hyperglycemia induced by insufficient β-cell function or mass Treatments to improve β-cell function • Allow the study of β -cell function and diabetic complications •Interstrains variability of the islets morphology and metabolism [31,33] Treatments to improve β-cell survival Genetically induced models of β-cell dysfunction Human islet amyloid polypeptide-expressing (hIAPP) mice Amyloid deposition in islets Treatments to prevent amyloid deposition •Express human IAPP under the insulin promoter, which can form amyloid within the islets for further study •Transgenic mice [31,33] Treatments to improve β-cell function β-cell destruction due to ER stress Treatments to prevent ER stress •β-cell adaption to increased insulin demand is restricted Treatments to improve β-cell survival Non-rodent models Cat models Amyloid deposition in islets Treatments to improve β-cell function •Islet amyloidosis study •More expensive models [31,33–37] β-cell destruction Treatments to prevent diet-induced obesity Old-world non-human primates •Similarities to human condition J. Clin. Med. 2019,8, 1332 7 of 20 The models most commonly used to study T1DM and T2DM are summarized in Tables 1and 2, respectively, together with their main advantages and disadvantages. In an idealistic perspective and aiming a reliable representation of the diversity observed among diabetic human patients, at least two or more animal models should be employed, taking into account the principles of the four Rs, i.e., replacement (preference for methods that avoid or replace animals use), reduction (use methods which minimize the number of animals per experiment), refinement (prefer methods regarding a minimal animal suffering and that promote their welfare), and responsibility [16]. 3. Diabetes Mellitus Type 1 Animal Models T1DM is mainly characterized by pancreatic β -cells autoimmune destruction, which contributes to insufficient insulin production, or even to the absence of insulin secretion [ 16 ]. In animal models of T1DM, this deficiency may be reproduced by several mechanisms, ranging from β -cells destruction either by chemical ablation using normal animal models to breeding animals (mostly rodents) that suffer from spontaneously developed autoimmune diabetes [ 16 , 38 , 39 ]. Some of the most commonly used models of T1DM are outlined in Table 1and are mainly constituted by rodent models, but some higher animals, such as pigs, dogs, and primates, have also been used (Table 1). 3.1. Chemical Induction of Diabetes Mellitus Type 1 3.1.1. Streptozotocin (STZ)-Induced Models Streptozotocin (STZ), chemically known as N-(methylnitrosocarbamoyl)- α -d-glucosamine, is a naturally occurring compound produced by Streptomycetes achromogenes with antibiotic properties that are selectively taken up by pancreatic β -cells, causing its destruction [ 16 , 40 ]. After its intraperitoneal or intravenous administration, STZ behaves as a glucose analogue and is transported mainly by the glucose transporter subtype 2 (GLUT-2) [ 41 ] into the pancreatic β -cell, where it induces toxicity, mainly by producing DNA alkylation [ 16 ]. Due to DNA strand breaks, over-activation of poly-ADP ribose polymerase (PARP) leads to NAD+depletion, cellular ATP reduction, and consequently, insulin production is compromised, as well as the cell survival, since there is a massive loss of energy [ 42 ]. As STZ enters the cell via GLUT-2, this is also expressed in other cells behind those from the pancreas, as the toxic action of STZ is not specific to pancreatic β cells, occurring also in hepatocytes and kidney cells (Figure 1) [ 43 ]. These are probably the reasons underlying the high mortality rate associated with this model. Figure 1. Streptozocin diabetes induction model (based on [ 17 ]). ( A ) Single-dose Streptozocin, ( B ) Multiple-low dose streptozocin, ( C ) Streptozocin mechanism on the β -cells nucleus and side effects in other organs with glucose transporter subtype 2 (GLUT-2) receptors. Streptozotocin (STZ) behaves as a glucose analogue and is transported into the pancreatic β -cell by GLUT-2. It produced DNA alkylation and over-activation of poly-ADP ribose polymerase (PARP) causing NAD+depletion, cellular ATP reduction, and compromising insulin. J. Clin. Med. 2019,8, 1332 8 of 20 Depending on the severity of the intended model disease, STZ-induced DM models usually result from one of the following procedures: administration of a single high STZ dose, or as multiple low STZ doses [ 16 ]. In the high-dose STZ administration procedure, a single STZ dose is administered to mice (100–200 mg·kg−1) or rats (35–65 mg·kg−1) by intravenous or intra-peritoneal routes, producing massive pancreatic β -cell destruction with little or no insulin production [ 43 ]. Insulinemia must be recorded to ensure that the intended model is stable, since there is some evidence that pancreatic islets regeneration may occur after this single-dose treatment. On the other hand, multiple low-dose STZ administration implies that small doses (20 to 40 mg · kg −1· day −1 ) are to be administered over a period of time in order to promote insulitis [ 16 , 18 ]. During insulitis development, infiltration of macrophages in the pancreatic islet promote cytokine production-dependent T1DM development. Therefore, therapies which target cytokines and nitric oxide are highly probable to be successful in reducing diabetes development in this model. Both STZ-induced diabetes models are cheaper and easier to perform than the remaining models and they can be used in most strains of rodents, or other animals, opening the field of diabetes research to an array of genotypic and phenotypic options that would otherwise be inaccessible [18,43]. 3.1.2. Alloxan-Induced Models Alloxan (2,4,5,6(1H,3H)-pyrimidinetetrone, 2,4,5,6-tetraoxypyrimidine,5,6-dioxyuracil) is also used to chemically induce DM and two possible mechanisms have been proposed. One suggests that alloxan selectively inhibits glucose-induced insulin secretion through specific inhibition of glucokinase, the pancreatic beta cell glucose sensor [ 44 ], and it also induces reactive oxygen species (ROS) formation, creating a redox cycle generating superoxide radicals. Alloxan is reduced to dialuric acid and then re-oxidized back to alloxan, producing superoxide radicals that undergo dismutation (by superoxide dismutase) to form hydrogen peroxide; concomitantly, hydroxyl radicals may also be formed by side reactions. These highly reactive oxygen species may cause β -cell DNA fragmentation, leading to apoptosis [ 16 , 44 , 45 ]. Although alloxan is also taken up by the liver, hepatotoxicity induced by alloxan is minimal or null since the liver has more effective protection mechanisms against ROS than β -cells [ 45 ], and they also have several mechanisms for xenobiotic biotransformations and elimination. Alloxan also promotes the essential –SH groups oxidation, especially that of gluthatione (GSH), enzymes, and proteins, and also dysregulates intracellular calcium homeostasis, leading to supraphysiological calcium concentrations and, hence, cellular damage [16,44,45]. Alloxan administered doses range from 50 to 200 mg/kg (in mice) and 40 to 200 mg/kg (in rats), and they are dependent on the chosen strain and the route of administration (e.g., intraperitoneal and subcutaneous alloxan administration require doses up to three times of those administered by intravenous route) [ 16 ]. Alloxan induces DM models as a consequence of ROS mediated beta cell toxicity (Figure 2), allowing the investigation and understanding of ROS mediated mechanisms of beta cell death in both T1DM and T2DM [44]. J. Clin. Med. 2019,8, 1332 9 of 20 Figure 2. Alloxan induced diabetes mechanism (based on [ 46 ]). Alloxan is reduced to dialuric acid and re-oxidized to alloxan producing alloxan radicals and reactive oxygen species (ROS) which undergo dismutation (by superoxide dismutase, SOD) to form hydrogen peroxide (H 2 O 2 ). Hydroxyl radicals (*OH) may also be formed by side reactions. These *OH cause β -cell DNA fragmentation, leading to apoptosis. 3.2. Spontaneous or Autoimmune of Diabetes Mellitus Type 1 3.2.1. Non-Obese Diabetic Mice The non-obese diabetic (NOD) mouse model originated in the inbreeding of the Cataract Shionogi (CTS) strain in the 1980s. The NOD mouse model is an autoimmune disease model where the T1DM develops spontaneously [ 21 ]. NOD mice exhibit polyuria, glycosuria, weight loss, and lymphocytic infiltration of the islets of Langerhans within the pancreas. It has been observed in NOD mice that innate immune cells infiltrate the pancreas of NOD mice at 3 weeks of age. In the same way, these cells types, such as dendritic cells, macrophages, and neutrophils, are also found in the human islet infiltrate [ 47 ]. The infiltration of innate immune cells into the islets attract adaptive CD4 and CD8 T cell subsets, which are required for DM development, into the islets from approximately 4 to 6 weeks of age [21,46]. Additionally, the relevance of this model relays in the fact that it was able to identify key genetic and environmental risk factors, such as effects of microorganisms including the gut microbiota, and how they may contribute to T1DM [21]. 3.2.2. Biobreeding Rats The biobreeding (BB) rat model was developed in the 1970s from outbred Wistar rats [ 21 ]. The incidence of diabetes is the same in male and female BB rats, although in humans it is reported to have a slight prevalence in males [ 23 ]. The BB rats develop T cell-specific lymphopenia and an impairment of Treg cell function. At 5 weeks, the BB rats develop insulitis, followed by hyperglycemia in around 70% of the animals [ 23 ]. Once hyperglycemia occurs, BB rats manifest polyuria, leading to a severe loss of body weight despite excessive drinking. Afterwards, these rats will develop ketoacidosis within several days [ 23 ]. According to Medina and colleagues, in this model there is a deterioration of beta cell function and mass as well as intra-islet blood flow that precedes insulitis and diabetes [ 24 ]. These underlying changes in islet function may be previously unrecognized factors of importance in type 1 diabetes development [24]. 3.2.3. LEW.1AR1/-Iddm Rats The LEW.1AR1-iddm rat is an animal model T1DM, which arose through a spontaneous mutation in the Dock8 gene within the major histocompatibility complex (MHC) congenic background strain LEW.1AR1 [48]. This Dock8 mutation provides a deepened insight into the impact of genes involved in diabetes development [ 48 ]. The mutation leads to a variable T-cell frequency in peripheral blood. J. Clin. Med. 2019,8, 1332 16 of 20 applied either to local or systemic delivery of nonirritating substances, as nasal mucosa is rich in blood vessels, there is a rapid absorption of the product and a rapid systemic effect, since it has avoided the first-pass effect by the liver [ 68 ]. Intranasal administration is becoming a common method to deliver therapeutic drugs to the central nervous system (CNS), as it is non-invasive and allows large molecules that do not cross the BBB to access the CNS, with reduced systemic exposure and unwanted systemic side effects [ 90 ]. In turn, intrapulmonary delivery may be performed by (1) intratracheal instillation, an easier but not so effective technique which involves small volumes injected directly into the trachea, or (2) inhalation, a highly complex technique that typically uses vapors or aerosols of nebulized particles in solutions which are deposited by gravitational sedimentation, inertial impaction, or diffusion in small airways; the ones deposited in large airways are then incorporated into the mucus and expelled by the mucocilliary clearance. It is thus important to evaluate solvent and propellant effects, since evaporation may lead to particle size changes [ 68 , 89 ]. Among these, intranasal has shown increased relevance, due to the direct brain connection and to the development of new pharmaceutical formulations able to enhance penetration through the nasal mucosa [90,91]. 6. Conclusions Diabetes mellitus is a metabolic disorder characterized by several disturbances in carbohydrate, protein, and fat metabolism and, apart from the need to discover new and more effective anti-diabetic drugs, animal models cannot be avoided in pre-clinical research. Several animal models are available according to the type of diabetes and also with the aim of therapy to assess. In this sense, animal models should be carefully chosen in order to fully reproduce the mechanisms and pharmacokinetics of the proposed therapies. Moreover, drug administration is of extreme relevance, both in the preclinical and clinical stages. Especially during preclinical studies, administration route advantages and drawbacks should be taken into account and the route should be adequate in order to reproduce human pathology and suitable treatment for further clinical trials. Author Contributions: R.V., S.B.S., E.S.-L., and A.L.M. have contributed to the writing and editing of the manuscript; P.S., S.J. and A.S. have structured and revised the manuscript; A.M.S., A.F., M.L.G., and E.B.S. have supervised, structured and contributed with the manuscript edition. Funding: The authors acknowledge the financial support received from Portuguese Science and Technology Foundation (FCT/MCT) and from European Funds (PRODER/COMPETE) under the project reference M-ERA-NET/0004/2015-PAIRED, co-financed by FEDER, under the Partnership Agreement PT2020. The authors also acknowledge the support of the Institute of Nanoscience and Nanotechnology under the project ART (2018). Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Beverley, B.; Eschw è ge, E. 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