Targeting the GLP-2 receptor in the management of obesity

the context of obesity. Obesity is associated with reduced gut barrier function, increasing the translocation of proinflammatory gut content to the circulation. This phenomenon constitutes a strong driver of obesity-associated systemic low-grade inflammation, which in turn plays a major role in the development of most obesity-associated complications. Thus, the intestinotrophic and gut barrier-improving effect of GLP-2, which in obese rodent models shows strong anti-inflammatory potential, may, in combination with food intake-reducing strategies, e.g., GLP-1 receptor (GLP-1) agonism, be able to rectify core pathophysiological mechanism of obesity. Here, we provide an overview of GLP-2 physiology in the context of obesity pathophysiology and review the pharmacological potential of GLP-2R activation in the management of obesity and related comorbidities.


Introduction
The global prevalence of obesity has almost tripled since 1980 [1]. Obesity (body mass index (BMI) >30.0 kg/m 2 ) thus represents a bigger challenge for healthcare systems than underweighteven in developing countries [1].The global prevalence is around 14% (with some regions exceeding 40%), corresponding to ~1 billion peoplea number that is estimated to surge to 1.5 billion by 2030 [1].
E-mail address: casper.kjaersgaard.nielsen@regionh.dk(C.K. Nielsen). 1 Currently employed at Novo Nordisk A/S, Søborg, Denmark complications, including insulin resistance and glucose intolerance, metabolic dysfunction-associated steatohepatitis (MASH), hypertension, atherosclerosis, and certain malignancies [12][13][14].Obesity-associated systemic inflammation is characterised by perturbations in circulating levels of inflammatory cytokines and acute phase proteins, including tumour necrosis factor-alpha (TNF-α), C-reactive protein (CRP), interleukin (IL)-6, and IL-18 [15][16][17].Yet the mechanisms underlying the development of obesity-associated systemic inflammation remain poorly understood.Nevertheless, accumulating evidence suggests that reduced gut barrier function, the so-called 'leaky gut', constitutes an important and underappreciated contributory cause of obesity-associated systemic inflammation by precipitating pathogen and microbial-related molecules' paracellular translocation into the underlying lamina propria and further on to the portal circulation [18].This breach is associated with gut dysbiosis, aberrant immune and inflammatory reactions, and, ultimately, pathological states.Gut dysbiosis refers to an imbalance or disruption in the composition and function of the gut microbiota (i.e.bacteria, viruses, fungi, and other microbes) and has been linked to the onset and progression of atherosclerosis and, consequently, cardiovascular disease [19].Available pharmacological anti-obesity treatments do not directly target gut barrier function, which nevertheless may be an attractive strategy in mitigatigating numerous obesity-associated comorbidities.
Historically, non-surgical treatments of obesity have been ineffective or demonstrated unfavourable safety or tolerability profiles; however, recently, pharmacological therapies based on the appetite and food intake-reducing incretin hormone glucagon-like peptide 1 (GLP-1) have demonstrated weight losses approaching the efficacy of bariatric surgeries [20,21].Interestingly, tirzepatide, a single-molecule compound stimulating both the GLP-1R and the receptor for its 'sister' incretin hormone glucose-dependent insulinotropic polypeptide (GIP), the GIP receptor (GIPR), seems to provide greater weight loss as compared to GLP-1R monoagonism [20].The further development of anti-obesity medications appears to, in general, focus on the addition of further appetite and food intake-reducing components such as amylin or energy expenditure-promoting modes of action such as glucagon receptor activation and myostatin inhibition [22,23].Interestingly, one emerging strategy focuses on combining GLP-1R activation with glucagon-like peptide 2 (GLP-2) receptor (GLP-2R) activation for the treatment of obesity; a strategy which is currently in its initial clinical stages [24].Although GLP-2R activation of central proopiomelanocortin (POMC) neurons in some rodent studies may lead to reduced energy intake [25][26][27][28][29], there is no evidence that GLP-2R activation leads to reduced appetite, food intake or body weight loss in humans.Thus, the rationale for targeting the GLP-2R in obesity mainly comprises the anti-inflammatory capabilities via improved gut barrier function, potentially mitigating the development of obesity-associated low-grade systemic inflammation known to be a driver of numerous obesity-associated complications, including MASH, cardiovascular diseases and neurodegenerative disorders-leading causes of obesity-associated mortality [30][31][32].GLP-2R agonism evidently increases mesenteric perfusion and, importantly, promote proliferation of the intestinal epithelium, which in turn ameliorates low-grade systemic inflammation by improving intestinal barrier function and, thus, reducing translocation of proinflammatory matter from the gut lumen to the bloodstream [33][34][35].Besides this indirect mitigation of systemic low-grade inflammation (Fig. 1), GLP-2 also promotes anti-inflammatory adaptations in the central nervous system and adipose tissue [35][36][37][38].
Here, we review GLP-2 physiology in the context of obesity pathophysiology and outline the potential of GLP-2R agonism in the T.G.Pálsson et al. management of obesity, focusing on GLP-2's gut barrier-improving effect and its role in obesity-associated systemic low-grade inflammation.
GLP-2 actions are mediated via the GLP-2R, which is part of the subfamily of class B superfamily of G protein-coupled receptors [49].The GLP-2R is highly ligand-specific to GLP-2 and reacts weakly or not at all to peptides with structural similarities, such as glucagon, GLP-1 and GIP, at equimolar ratios.Conversely, GLP-2 is a low-potency agonist for the GLP-1R [50,51].The GLP-2R is robustly expressed in the gastric ventricle, duodenum, jejunum, ileum, colon, gallbladder, and central nervous system, while moderate expression has been shown in the pancreas, heart, liver, adipose tissue and lungs [35,44,[52][53][54][55]. Within the gut, GLP-2R-driven LacZ expression has been primarily localised to the enteric neurons and lamina propria stromal cells in mice [56].GLP-2R activation triggers the activation of adenylate cyclase, leading to an increase in cyclic adenosine monophosphate (cAMP) levels [52,57].Increased cAMP levels further increase intracellular Ca 2+ levels, initiating the signalling cascade for the expression of proteins associated with cell survival [52,57,58].The direct prosurvival actions of GLP-2, observed in transfected fibroblasts, enhanced cell survival through a protein kinase A-dependent mechanism that inhibited the activation of glycogen synthase kinase-3, further inhibiting apoptosis in these cells [57,58].The intracellular pathway from GLP-2R activation seems to differ in various brain areas; In hippocampal neurons, GLP-2R activation acutely increases intracellular levels of calcium via L-type voltage-gated Ca 2+ channels, causing excitation of the neurons.Activation of the GLP-2R in hippocampal neurons has been speculated to modulate the release of neurotransmitters such as serotonin and nitric oxide, which are involved in synaptic plasticity, gene transcription and neuronal survival [59].In other locations of the brain, GLP-2 appears to directly modulate POMC neurons via the phosphatidylinositol 3-kinase-akt signalling pathway, modulating energy balance and glucose metabolism in rodents [60].Direct GLP-2R activation in the CNS may be beneficial for maintaining synaptic plasticity and survival of neurons.Additionally, evidence supports that GLP-2R agonism induced reduction of translocation of gut contentincluding microbiotainto the bloodstream, may have indirect neuroprotective actions [61].
Both mouse and human studies suggest that prolonged pharmacological activation of the GLP-2R does not induce tachyphylaxis [34,62], but GLP-2R desensitisation has been observed in vitro, where cells both from mice and humans demonstrated reduced GLP-2R expression through lipid-sensitive clathrin-independent mechanisms [63].A recent study found that the internalisation of GLP-2R only partly relies on β-arrestin-based mechanisms as the absence of cellular β-arrestin 1 and 2 reduced but did not eliminate GLP-2R internalisation following GLP-2 binding [64].The same study also demonstrated that alanine substitution at the N-terminal position reduced β-arrestin 1 and 2 recruitment, resulting in greater GLP-2R activation (20-fold increase in cAMP accumulation).The anorexigenic effect of GLP-1 following binding to the GLP-1R in POMC neurons, which contributes to satiety regulation in the CNS, is blunted by β-arrestin recruitment following GLP-1R internationalization resulting in desensitization [65], which-given the abovementioned results-may be preventable by GLP-2R activation.Together, this indicates that GLP-2R agonism may apart from its intestinotrophic effects also potentiate some of the anorexigenic effects of GLP-1R activation.

Intestinal barrier function and low-grade inflammation
GLP-2 administration has been shown to improve gut-lining integrity and decrease intestinal barrier permeability in both animal studies and in humans with SBS [33,66].Subcutaneous administration of human GLP-2 in mice and intravenously administered human GLP-2 in piglets result in significant changes in enterocyte morphology, with increased thickness and villus height, as well as inhibition of apoptosis, promotion of growth, and induction of repair mechanism after intestinal injury [57,67,68].In high-fat diet-fed mice, decreased barrier function is partly due to disrupted key tight-junction proteins, zonula occludens-1, occludin, and claudin [69].In prebiotic-treated obese mice, the increase of endogenous GLP-2 secretion was speculated to cause a decrease in circulating levels of plasma lipopolysaccharide (LPS) owing to enhanced intestinal barrier function and improved tight-junction integrity, leading to decreased systemic and hepatic lipid content [70].Systemic inflammation induced by LPS represents one of several mechanisms that link alterations in gut microbiota dysbiosis with low-grade inflammation observed in obesity.LPS, a component found in the outer membrane of Gram-negative bacteria, is known for being one of the most potent immunogenic substances affecting mammals [71].Hence, plasma LPS levels are used as markers of bacterial exposure systemically [71].Elevated LPS levels signify the translocation of bacterial products across the intestinal barrier that stimulates the production of pro-inflammatory cytokines, leading to endothelial injury and heightened oxidation of low-density lipoprotein particles, thereby facilitating foam cell formation and hastening the progression of atherosclerosis [72].In addition to the effects on the epithelial mucosal lining, GLP-2 has been linked to suppressing LPS-induced inflammation in peritoneal macrophages and microglial BV-2 cells.This is believed to occur through the inhibition of nuclear factor-κВ (NF-Κβ) translocation as well as the phosphorylation of extracellular signal-regulated kinases and c-Jun N-terminal kinase 1/2 in microglial cells.These actions ultimately lead to a reduction of pro-inflammatory enzymes, including inducible nitric oxide synthase, cyclooxygenase-2 and cytokines such as TNF-α, IL-1β, and IL-6 [36,37].This indicates that the anti-inflammatory effects of GLP-2R activation originate from blunted LPS-induced inflammation, which has spurred the exploration of GLP-2 as a mitigator against systemic low-grade inflammation.Nevertheless, GLP-2R expression has only been established in mesenteric lymph nodes and not in peritoneal macrophages or microglial BV2 cellss [56].Thus, the suppression of LPS-induced inflammation by GLP-2 may be indirect.Another study suggests that GLP-2 could protect neurons from excitotoxic damage in the CNS with reduced glutamate-induced cell death in cultured hippocampal cells [73][74][75].In a mouse study, [Gly2]-GLP-2, a human GLP-2 analogue, reduced high-fat diet-induced neuroinflammation, presumably via a mechanism involving inhibition of NF-κВ [38].[Gly2]-GLP-2 was also reported to decrease neuronal stress, indicated by a reduced expression of stress markers such as phosphorylated extracellular signal-regulated T.G.Pálsson et al. kinase, inducible nitric oxide synthase, heat shock protein 60, and reactive oxygen species production.Reducing NF-κВ activation through reduction of oxidative stress will consequently decrease the expression of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 [38,76,77].Furthermore, based on a terminal deoxynucleotidyl transferase assay as an index for apoptotic cell death, GLP-2 decreased neurodegeneration in various brain areas [38].Hence, compared with high-fat diet-fed mice, intracellular DNA fragmentation, a well-known indicator for impaired cell survival, was diminished in [Gly2]-GLP-2-treated mice [38].These results support the notion that GLP-2R activation exerts anti-inflammatory effects that could dampen both systemic and neuronal inflammation associated with obesity.
Although the exact connection between obesity and chronic lowgrade inflammation is not entirely understood, compared with lean individuals, obese people tend to overexpress pro-inflammatory adipokines such as TNF-α, IL-1β, and resistin [78].This prompts both local and systemic chronic inflammation, leading to obesity-associated comorbidities such as insulin resistance and cardiovascular diseases [78][79][80].One study found that the GLP-2R agonist, teduglutide, directly impacts both visceral-and subcutaneous human adipose tissue, where teduglutide acted as a protective factor in response to overnutrition by increasing gene expression of anti-inflammatory enzymes, adiponectin and arginase 1 in the visceral adipose tissue, resulting in decreased levels of the pro-inflammatory cytokine IL-1β, when compared with non-treated visceral adipose tissue [35].Furthermore, compared with lean individuals, people living with obesity may have a higher GLP-2R expression in subcutaneous adipose tissue and treatment with teduglutide resulted in higher basal glucose incorporation and lipogenesis, independent of insulin stimulation, suggesting that teduglutide has an anabolic effect in the subcutaneous adipocytes [35].The notion of GLP-2's anabolic effects in subcutaneous adipose tissue is further supported by increased lipogenic gene expression of fatty acid synthase and peroxisome proliferator-activated receptor-γ [35].Therefore, targeting the GLP-2R in adipose tissue in people living with obesity might help to maintain the functional integrity of both visceral-and subcutaneous adipose tissue by inhibition of pro-inflammatory IL-1β expression and increased expression of adiponectin and arginase 1, improving adipose tissues glucose tolerance and plasma glucose clearance.These effects could help alleviate some of the factors contributing to obesity-associated low-grade inflammation.Still, the precise mechanism underlying the anti-inflammatory properties of GLP-2-whether it would arise from the improved intestinal barrier function, the modulation of adipose tissue, or a cumulative effect of both processes-remains unclear.

Appetite, satiety, and gastric emptying
As aforementioned, in addition to GLP-2R expression in the gastrointestinal tract, the receptor is also expressed in the hypothalamus, hippocampus, and brainstem − regions known to partake in the regulation of energy homeostasis and hedonic food intake [73,74,81,82].In rats, CNS-derived GLP-1 and GLP-2 are predominantly synthesised in the nucleus of the solitary tract located in the hindbrain.From there, neurons expressing GLP-2R project directly to various regions of the CNS, including the hypothalamus [83].The hypothalamus is a key brain region involved in the regulation of appetite, energy homeostasis, and other physiological functions.Therefore, GLP-2 signalling from the hindbrain to the hypothalamus theoretically could influence eating behaviour, energy intake, and other metabolic processes.
Albeit controversial, one proposed route of entry for circulating GLP-1 and GLP-2 is directly crossing the blood-brain barrier into the CNS [84].After entry into the CNS, GLP-1 and GLP-2 are believed to subsequently act on key regions involved in energy homeostasis and hedonic food intake [84].Crucial, in this regard, is that the hypothalamus and hindbrain both lack a blood-brain barrier.In mice, both the GLP-1R and the GLP-2R are expressed in the POMC neurons [81].The proposed mechanism of GLP-2R activation in controlling feeding behaviour in mice is through the excitation of POMC neurons situated in the nucleus of the solitary tract and the arcuate nucleus, which leads to the release of α-melanocyte-stimulating hormone, activating the melanocortin-4 receptor and, thus, ultimately increasing satiety and decreasing appetite [81].In mice, GLP2-R deletion selectively in POMC neurons increases eating behaviour and fat mass while accelerating gastric emptying rate, ultimately leading to decreased body mass [81].Also, in murine models, intracerebroventricular administration of GLP-2 leads to hypophagia and delayed gastric emptying rate [81,82,85,86].While the seemingly anorexigenic effects of GLP-2 in murine models are intriguing, they have not been reproduced in humans; hence, intravenous GLP-2 administration in lean, healthy individuals does not lead to decrease in food intake or appetite [87][88][89].Additionally, in long-term studies of adults with SBS, native GLP-2 administration did not change energy intake [90].
Compared with low-dose or placebo, high-dose intravenously infused human GLP-2 decelerated gastric emptying rate as evaluated using a paracetamol test in healthy male subjects [89].In mice, the moderation of gastric motility via GLP-2R activation is likely mediated by inhibition of acetylcholine release from enteric neurons leading to suppressed cholinergic-evoked muscle contraction [91].Acutely, an accelerated gastric emptying rate is linked to shorter intervals of satiety, subsequently leading to hyperphagia [92][93][94].In adult SBS patients, native GLP-2 and the GLP-2R agonist, glepaglutide, reduced the accelerated gastric emptying rate [40,66].

Impact on glucose homeostasis
GLP-2 administration has not demonstrated any noticeable impact on plasma insulin, glucose or C-peptide levels in humans [25,95].However, during a liquid mixed meal test in healthy male subjects, compared with placebo, high-dose intravenously infused synthetic human GLP-2(1− 33) decreased postprandial glucose peak while drastically increasing glucagon levels [89,95].This leads to the hypothesis that supraphysiological levels of GLP-2 might be glucagonotropic, which is supported by the expression of GLP-2 receptors on the α-cell in humans (shown by means of immunohistochemistry and real-time PCR) [54].However, in mice, GLP-2 did not elevate glucagon plasma levels during hypoglycemic and hyperglycemic conditions [44].The discrepancy in the physiological actions of GLP-2 regarding glucagonotropic effects might be ascribed to the natural variance within different species, doses, or other methodological considerations.Currently, there is no evidence of prolonged GLP-2 administration exerting direct glycemic effects in humans.In a recent study involving persons living with type 2 diabetes, subcutaneously administered GLP-2 did not acutely affect insulin, C-peptide or glucose levels in the fasting state (circulating glucagon was not reported) [96].However, since GLP-2R activation improves gut-barrier function, mitigating microbiota dysbiosis and lowering systemic low-grade inflammation, prolonged administration could be speculated to improve obesity-associated insulin resistance indirectly [31,90].

Nutrient absorption and hepatic influence
In SBS patients, an increase in mesenteric blood flow following GLP-2 administration has repeatedly been demonstrated [97,98].GLP-2 has been shown to upregulate intestinal endothelial nitric oxide synthase activity, suggesting that GLP-2-induced increased blood flow may depend on the formation of vascular nitrous oxide [99].The increase in mesenteric blood flow contributes to improved gastrointestinal uptake of nutrients via expansion of the absorptive mucosal epithelium with increased crypt cell proliferation and reduced apoptosis [33,100].In animal studies, GLP-2 also stimulates the absorption of nutrients by increasing the expression or activity of hexose and transmembrane transporters such as glucose transporter 2, sodium-glucose transporter 1, and peptide transporter 1 in enterocytes [101][102][103][104][105]. Other studies, both in GLP-2R knockout mice and in humans, suggest that GLP-2 action is important for intestinal amino acid absorption rather than lipid and glucose absorption [25,106].Postprandially, in mice, GLP-2 rapidly augments the absorption of lipids and increases chylomicron secretion from the gut mucosa through a functional cluster of differentiation 36 (CD36; a fatty acid translocase) [95,107].Importantly, similar observations have been made in humans using the GLP-2R agonist teduglutide and native GLP-2, where GLP-2R activation seemed to increase plasma triacylglyceride levels approximately 7 hours after consuming a high-fat meal [108][109][110].Since GLP-2R is not expressed in enterocytes (site of chylomicron synthesis), this increase was attributed to the mobilisation of chylomicrons, presumably from the lamina propria and mesenteric lymph nodes through lymphatic circulation.This increase in postprandial triacylglycerides has raised concerns that GLP-2 could play a pro-atherogenic role [108][109][110].Conversely, endogenous GLP-2 is believed to act as a beneficial factor for dysmetabolic conditions in the liver [111]; Obesity is a major risk factor for developing metabolic dysfunction-associated steatotic liver disease (MASLD).The prevalence of MASLD in obese populations is approximately 75% compared with 15-30% in the general population [112].High-fat diet-fed mice receiving intra-peritoneal administration of the GLP-2R antagonist, GLP-2(3− 33), for four weeks experienced dyslipidemia and exacerbated morphological alterations in hepatocytes, including macrovascular steatosis in the liver when compared with untreated counterparts [111].These findings concur with another mouse study, identifying the expression of GLP-2R in hepatic stellate cells, where genetic ablation of the GLP-2R worsened hepatic steatosis and upregulated hepatic cytokine expression [113].Taken together, these studies point to the importance of GLP-2 in lipid homeostasis and mitigating liver steatosis, albeit the findings are yet to be reproduced in humans [114,115].

Human trials with GLP-2 receptor agonism
Most clinical trials involving the administration of GLP-2R agonists have focused on its potential in treating SBS; a chronic disease characterised by malabsorption due to surgical loss of large portions of the intestine [66,116,117].In SBS, GLP-2's intestinotrophic and pro-adaptive effects are clearly beneficial, illustrated by the reduced need for parenteral support [66].The intestinotrophic effects from these studies would also be advantageous for people living with obesity since maintaining intestinal barrier function and ameliorating gut microbiota dysbiosis perhaps would reduce systemic low-grade inflammation otherwise linked with the development of obesity-associated comorbidities [8].Currently, teduglutide (Revestive®, Takeda Pharmaceuticals, Japan) is the only GLP-2 analogue approved for the treatment of SBS [39].Nevertheless, upcoming GLP-2R agonists such as glepaglutide (Zealand Pharma, Søborg, Denmark) and apraglutide (Ironwood Pharma, Boston, Massachusetts, USA) are currently in phase III development for the treatment of SBS.Both glepaglutide and apraglutide promote long-lasting GLP-2R activations; while apraglutide only retains potency and selectivity to the GLP-2R, glepaglutide also elicits a rather high affinity and potency to the GLP-1R [40,41,118].Dapiglutide (Zealand Pharma, Søborg, Denmark), a GLP-1R/GLP-2R dual agonist, reportedly induced dosage-dependent weight loss of up to 4% after four weeks of treatment in lean, healthy men and is currently being evaluated for its weight-loss potential in persons living with obesity in a phase IIa proof-of-concept study [24].

Safety of GLP-2 Receptor Agonism
Some safety concerns have been raised regarding prolonged GLP-2 treatment.This mainly pertains to the intestinotrophic effects of GLP-2, which has been speculated to increase the risk of intestinal neoplasia [119].Evidence from non-sensitized preclinical murine models exposed to stable GLP-2 analogues does not support an increased cancer risk, but mice pretreated with a carcinogen, 1, 2-dimethylhydrazine and GLP-2 indicates an increased risk of intestinal polyps [120].For this reason, human subjects in trials with GLP-2R agonists have been screened for previous or current conditions that increase the likelihood of neoplastic formation in the gut [121][122][123].Based on current data, the risk of neoplastic formation from teduglutide treatment is minimal [119], although long-term (i.e.>10 years) safety data are lacking and highly warranted.In a 2-year follow-up study involving patients with SBS treated with teduglutide for a median of 4.5 years, 10 out of the 35 included patients had polypoid lesions without the manifestation of clinical symptoms (i.e.abdominal pain or intestinal occlusion) [121].Importantly, no morphological dysplasia was observed, and the highly selected population and sample size should be kept in mind when interpreting these results.
Another concern has been raised due to GLP-2's ability to reduce smooth muscle contractility in the gallbladder [53,89].This could potentially increase the risk of gallbladder-related diseases such as bile duct and pancreatic duct obstruction with GLP-2R agonist treatment [89,124].The short-term side effects that can occur and are known to be related to the treatment of the GLP-2R agonist, teduglutide, include nausea, vomiting, bloating, dyspepsia and abdominal pain, respiratory infections, and injection site reaction [125].

Conclusion
GLP-2R agonism improves intestinal barrier function, which may ameliorate obesity-associated low-grade systemic inflammation, hepatic inflammation, neuroinflammation, and adipose tissue inflammation.However, no evidence from human trials indicates that GLP-2R activation directly leads to decreased food intake, appetite modulation or increased energy expenditure; hence, body weight loss from GLP-2R monotherapy seems unlikely.Consequently, the current literature does not provide strong evidence for GLP-2-based monoagonism as an antiobesity treatment; however, combining pharmacological GLP-2R agonism with weight-loss-inducing drugs like GLP-1R, GIPR, glucagon receptor and/or amylin receptor-activating properties, may be beneficial in targeting body weight reduction, gut barrier function and systemic low-grade inflammation.
Antag Therapeutics; and is currently emplyed by Novo Nordisk.P.B.J. has received consultancy fees from Albumedix A/S; Protara Therapeutics; Baxter; Coloplast; Ferring Pharmaceuticals; Fresenius Kabi; GLyPharma Therapeutic; Naia Pharmaceuticals; Novo Nordisk Foundation; Shire, a Takeda company; Therachon; Takeda; VectivBio AG; and Zealand Pharma.A.B.L. has received consultancy fees from Novo Nordisk and Sanofi.B.A.H.J. has served as scientific advisor and/or received consultancy fees from Novozymes A/S and Defensin Therapeutics ApS; is co-founder and shareholder in Aesculus Bio ApS, and minority shareholder in MycoBiotix Inc.