Peptides 127 (2020) 170296

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Peptides

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Review Glucagon-based therapy: Past, present and future T Mohan Patila, Nitin J. Deshmukha, Mahesh Patela,b, Ganesh V. Sanglea,* a Diabetes Research Lab, New Drug Discovery, Wockhardt Research Centre, Aurangabad, Maharashtra, India b New Drug Discovery, Wockhardt Research Centre, Aurangabad, Maharashtra, India

ARTICLE INFO ABSTRACT

Keywords: Diabesity and its related cardio-hepato-renal complications are of absolute concern globally. Last decade has Glucagon analoguess witnessed a growing interest in the scientific community in investigating novel pharmaco-therapies employing Hypoglycaemia the pancreatic hormone, glucagon. Canonically, this polypeptide hormone is known for its use in rescue treat- Glucagon antagonist ment for hypoglycaemic shocks owing to its involvement in the counter-regulatory feedback mechanism. GLP-1/glucagon co-agonist However, substantial studies in the recent past elucidated the pleiotropic effects of glucagon in diabesity and GLP-1R/GCGR/GIPR tri-agonist related complications like non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver disease (NAFLD). Diabesity Thus, the dual nature of this peptide has sparked the search for drugs that can modify glucagon signalling to combat hypoglycaemia or diabesity. Thus far, researchers have explored various pharmacological approaches to utilise this peptide in imminent modern therapies. The research endeavours in this segment led to explorations of stable glucagon formulations/analogues, glucagon receptor antagonism, glucagon receptor agonism, and in- cretin poly-agonism as new strategies for the management of hypoglycaemia or diabesity. This ‘three-dimen- sional’ research on glucagon resulted in the discovery of various drug candidates that proficiently modify glu- cagon signalling. Currently, several emerging glucagon-based therapies are under pre-clinical and clinical development. We sought to summarise the recent progress to comprehend glucagon-mediated pleiotropic effects, provide an overview of drug candidates currently being developed and future perspectives in this research domain.

1. Introduction urgent need for diabetes treatment. As a result, global understanding about pathophysiology of diabetes has evolved exponentially, and ex- The history of pancreatic hormone, glucagon begins with that of its tensive knowledge of β-cell-mediated insulin secretion is available. ‘dancing-partner’ insulin. It was primarily observed as an ‘impurity’ in Inversely, glucagon remained as an ‘overlooked entity’ during this ‘in- early crude insulin preparations responsible for an acute hypergly- sulino-centric’ era which dwarfed its importance. Furthermore, prac- caemia after injection. In 1923, Kimball and Murlin coined this ‘im- tical hurdles like scarcity of α-cells in islets, lack of its functional purity’ as “glucagon” due to its glucogenic properties (GLUCose identification patterns and limited availability of tools for glucagon AGONist). Two decades later, Sutherland and De Duve identified that quantification also blunted the study of α–cells and glucagon pharma- the endogenous origin of glucagon is in pancreatic α- cells [1,2]. cology [3–5]. However, during the last decade, sizable research high- Classically, insulin and glucagon are known as counter-parts, lighted the critical importance of α–cells and glucagon in health and sharing opposite metabolic effects. Islet of Langerhans regulates gly- diabetes [3,4,6, 7]. Significant research progress in α–cell biology and caemic homeostasis by co-ordinating secretion of these two hormones hitherto understanding of glucagon biology has fostered researchers to through pancreatic β and α –cells, respectively. Insulin increases glu- explore its utilisation in the next-generation therapies to combat dia- cose utilisation and glycogen accumulation in muscle, liver and adipose betes and obesity. This paradigm shift has initiated broad pharmaceu- tissue. Conversely, glucagon increases glucose concentrations in tical interest and ignited the search for new treatment options utilising bloodstream by stimulating hepatic glucose production via glycogen- glucagon signalling [5]. olysis and gluconeogenesis. It also stimulates lipolysis, lipid metabolism To date, researchers explored different pharmacological approaches and ketone body production [1–3]. to harness the benefits of using glucagon as a therapeutic target in Despite the discovery of glucagon, the field of diabetes research diabetes management. The incontestable approach was to use native remained insulin and β-cell-centred for several decades to fulfilan glucagon in the treatment of hypoglycaemia based on its inherent

⁎ Corresponding author. E-mail address: [email protected] (G.V. Sangle). https://doi.org/10.1016/j.peptides.2020.170296 Received 23 October 2019; Received in revised form 5 February 2020; Accepted 4 March 2020 Available online 05 March 2020 0196-9781/ © 2020 Elsevier Inc. All rights reserved. M. Patil, et al. Peptides 127 (2020) 170296 diabetogenic property. However, due to poor solubility and physico- chemical stability, its use outside hospital remained restricted. Hence, there is a long-awaited need for stable glucagon formulations or ana- logues to address the risk of hypoglycaemia [8,9]. ‘Glucagon receptor antagonists (GRAs)’ are designed with a ratio- ling by early nale to counter high glycaemia in diabetes by inhibiting glucagon ac- fi tions [10]. Considerable research progress in this segment led to the discovery of several molecules, albeit none of the drug candidate re- ceived regulatory approval [11]. Conversely, pre-clinical and clinical Commercially available Commercially available Phase I Approved in USA Discontinued Phase III (NDA Discontinued after Phase 1 Discontinued Phase I evidence on the positive impact of ‘glucagon agonism’ in diabetes and 2020) obesity started a race to develop ‘dual/poly incretin agonist’ as an in- novative therapeutic option [1,4,5]. As a result, “three-dimensional” research on glucagon signalling led Dose (mg) Developmental Status 1 to the discovery of a large number of glucagon-based molecules/for- NA No development after Phase 1 mulations which are currently under preclinical and clinical develop- ment. The review aims to provide a summarised overview and the most recent updates on the development of drugs that modify glucagon sig- nalling with a focus on the hurdles and future perspectives.

2. Glucagon-based therapies Delivery Pattern Ready-to-use Ready-to-use-injectable NA Preclinical (T2D, Obesity) Ready-to-use-injectable 1 Injectable Ready-to-use-injectable 1 Automatic reconstitution 1 Ready-to-use-injectable 0.15 Discontinued after Phase 1 Ready-to-use-injectable 0.5, 1 Approved in USA Ready-to-use-injectable 1 Glucagon therapy is recommended in the management of severe hypoglycaemia (glucose levels < 54 mg/dL) [12]. Currently, it is available in the form of ‘emergency kits’. Other clinical applications of glucagon include its utilisation in the treatment of refractory brady- cardia and cardiogenic shocks [13,14], and as a spasmolytic agent, during gastrointestinal tract (GIT) radiological examinations [15]. This section reviews emerging glucagon-based therapies for the treatment of metabolic disorders and related complications.

2.1. Glucagon kits and their limitations

Glucagon treatment is safe, tolerable and effectively restores hy- poglycaemia within minutes of injection [16,17], but this native pep- tide is not soluble in aqueous buffers at physiological pH. Even when formulated at low or high pH, it demonstrates poor physicochemical properties [18]. Thus, commercial glucagon kits are available in Native peptide prepared by recombinant DNA technology Need reconstitution 1 Formulation characteristics/technology Short acting glucagon analogues Native peptide formulation with Phospholipd & Cyclodextrin Ready-to-use-nasal spray 3 using XeriSol technology Glucagon analogue powder form, along with solvents to reconstitute before their in- BioChaperone technology tramuscular or subcutaneous use. Currently, two glucagon kits are available in the market, ‘Glucagon Emergency Kit’ (Eli Lilly, In- dianapolis, USA) and ‘GlucaGen HypoKit’ (Novo Nordisk, Copenhagen, Denmark). These products require reconstitution immediately before their use [19,20]. Involvement of multi-steps in their preparation and fi delivery process often leads to erroneous or delayed administration Xeris PharmaZealand Pharma Native peptide formulation in dimethyl Glucagon sulfoxide analogue (DMSO) Intarcia TherapeuticsSano Glucagon analogue Adocia Biotech Native peptide formulation with m-cresol using Albireo Pharma Lyophilized formulation Hanmi Pharma Long acting glucagon analogue using LAPScovery technology Ready-to-use-injectable 1 during stressful emergencies [16]. Therefore, it requires specialised Novo Nordisk Glucagon receptor agonists training and education for non-medico-professionals or caregivers [21]. Moreover, solubilised preparations of these kits are unstable due to the tendency to form aggregations and fibrillation [22]. Due to such com- plexities, conventional glucagon therapy remains as an under- appreciated and underused approach in hypoglycaemia management [16]. Dasiglucagon Gvoke (G-Pen) HM15136 ICA6150349 SAR438544 Formulation Name Innovator GlucaGen HypoKItGlucagon Emergency Kit Eli Lilly Novo Nordisk NativeBC-glucagon peptide prepared by recombinant DNA technology Need reconstitutionBIODEL-961 1 BAQSIMI MAR 531 & MAR-D28Rescue G & PumpaGon Roche Latitude Pharma Native peptide formulations NN 9030 2.2. Emerging glucagon formulations/analogues

Due to an unmet need in the management of hypoglycaemias, sev- eral pharmaceutical companies are currently aiming to develop stable and ready-to-use glucagon formulations or analogues (Table 1). These emerging therapies are expected to retain the biological profile of na- tive peptide, minimize risk of hypoglycaemic events and offer clinical advantages over conventional products. Eli Lilly is the first pharmaceutical company to commercialize na- tive glucagon for the treatment of hypoglycaemia [23]. The company recently received United States Food and Drug Administration (US- FDA) approval for first-ever nasal glucagon, Baqsimi [24]. The product analogues analogues is a portable, single-use dispenser containing ready-to-use nasal spray of Emerging glucagon formulations/ Discontinued glucagon formulations/ Regulatory approved glucagon products

3 mg native glucagon with improved stability [25]. Lilly acquired Table 1 Glucagon formulations/analogues (approved, under-development and discontinued). NA: Information not available.

2 M. Patil, et al. Peptides 127 (2020) 170296

Baqsimi (formerly AMG504-1) from Locemia solutions (Montreal, Ca- increased peroxisome proliferator-activated receptor gamma co-acti- nada) in 2015 [26]. In this formulation, cyclodextrin is a bulking agent; vator 1-alpha (PGC-1 α) and uncoupling protein-1 (UCP-1) [48]. As- whereas phospholipid facilitates glucagon absorption into the nasal tonishingly, HM15136 also improved insulin resistance, lipotoxicity, mucosa to preclude its delivery to the lung. Pharmacokinetic (PK), and inflammation in DIO mice by increasing fibroblast growth factor 21 pharmacodynamic (PD) and safety profile of Baqsimi in humans is (FGF-21) levels, reducing expression of F4/80 and pro-inflammatory comparable to existing conventional glucagon products [27]. cytokines in WAT [49]. Moreover, when given in combination with Similarly, Xeris pharma (Chicago, USA) also received US-FDA ap- other oral anti-diabetic drugs like sitagliptin and empagliflozin, it ex- proval for first-ever, ready-to-use stable liquid glucagon, Gvoke (G- hibited enhanced weight reduction as compared to respective mono- Pen). The product will be available in two separate dosing regimens for therapies. It suggests the additive weight-loss potential of glucagon in paediatric (0.5 mg/0.1 ml) and adult (1 mg/0.2 ml) patients as a pre- combination with oral anti-diabetic drugs [50]. Adocia is also focusing filled syringe and auto-injector. This premade glucagon formulation is on utilising BC-glucagon as a treatment option for obesity by devel- likely to reduce the time of preparation and administration errors oping its fully-aqueous co-formulation with exenatide (BC-Glu-Exe). Its compared to conventional glucagon kits [28]. The company confirmed fourteen days of treatment in DIO mice, achieved superior (25 %) its efficacy, safety and tolerability in a Phase 3 trial conducted in 161 weight-reduction compared to exenatide alone (15 %) [51]. adult type-1 diabetes (T1D) patients [29]. The product is designed by Intarcia Therapeutics (Boston, USA) is developing ICA 6150349, a using XeriSol technology. In this formulation, dimethyl sulfoxide peptidase resistant, 38-amino acid glucagon analogue, with high se- (DMSO) is used as a solvent to dissolve native human glucagon. In in- lectivity for glucagon receptors (GCGR). It is found safe and tolerable in itial animal and human studies, the product demonstrated improved rodents and non-human primate models [52]. Its treatment exhibited physicochemical properties and promising efficacy with few cases of weight loss potential in Long Evans (LE) DIO rats and Zucker diabetic injection site discomfort [30–32]. fatty (ZDF) rats when given alone and in combination with exenatide. Zealand Pharma (Glostrup, Denmark) is developing Dasiglucagon Additionally, combination treatment also restored glycated hae- (ZP4207), a stable glucagon analogue, which is currently under Phase 3 moglobin (HbA1c), glucose, and food intake in obese and T2D rat development. It is a ready-to-use glucagon analogue with seven sub- model [53]. Novo Nordisk initiated the development of a glucagon stitute amino acids in native peptide and is stable in an aqueous solu- analogue, NN 9030 as mono or in combination with liraglutide for tion at neutral pH. The product exhibited better stability and compel- obesity. Two separate studies are planned in obese or overweight sub- ling results in pre-clinical and clinical studies. Since it is a novel jects to evaluate its potential as monotherapy (NCT02835235) [54] and analogue prepared by modifying the native glucagon peptide sequence, in combination with liraglutide (NCT02870231) [55]. However, no immunogenicity remains a concern and needs to be evaluate further. recent updates are available on these studies [56]. However, initial clinical studies did not report any signs of treatment- related anti-drug antibodies or neutralizing antibodies formation 2.2.1. Future perspective on glucagon formulations/analogues [33,34]. Currently, two separate clinical studies are underway for The clinical management of hypoglycaemia has not changed for congenital hyperinsulinism (CHI) (NCT04172441) and bariatric sur- decades. Poor stability and physicochemical properties of conventional gery-induced post-prandial hypoglycaemia (NCT03984370) [35,36]. glucagon products restrict their use. The emerging glucagon formula- Dasiglucagon received “Orphan drug” and “Rare Paediatric Disease” tions are stable and ready-to-use injections, which do not require pre- designation both by US-FDA and European Medicines Agency (EMA) in parative steps, in contrast to traditional products. Gvoke is now avail- the treatment of CHI [37]. able in the USA market, while, Zealand is aiming to file a New Drug Adocia Biotech (Lyon, France) is currently developing Application (NDA) for Dasiglucagon by early 2020 [57]. Hopefully, BioChaperone (BC) glucagon, a stable, ready-to-inject aqueous solution both these novel glucagon formulations will overcome the current ob- of human glucagon using BC technology. BC-glucagon showed com- stacles encountered in the management of hypoglycaemia. parable PK/PD profile with GlucaGen hypo kit in the minipig model Nasal glucagon, Baqsimi is the only product that is likely to address [38,39]. A Phase 1 trial in T1D patients reported that BC-glucagon the unmet need for needle-free delivery of glucagon in critical emer- treatment is safe, effective and has comparable PK with GlucaGen gencies [58]. The product is yet to establish the feasibility of its usage in (NCT03176524) [40]. Hanmi Pharma (Seoul, South Korea) has dis- the real-world-setting, but its approval is encouraging for future pro- covered long-acting glucagon analogue, HM15136, employing its ducts focusing on intranasal delivery of glucagon. Transdermal glu- LAPScovery (Long Acting Protein/Peptide Discovery) technology plat- cagon-patches could be another alternative delivery approach for stable form. Conjugation of native glucagon to the human glycosylated fc glucagon formulations. In the past, an attempt was made by Zosano fragment through PEG linker attributed extended duration to Pharma (Fremont, USA) to develop ZP-glucagon by using a microneedle HM15136. Its preliminary efficacy evaluated in a rodent model of CHI patch system technology [59]. Moreover, there is long-awaited interest [ 41]. The company research pipeline indicates a plan to consider from medical device manufacturers to utilise stable glucagon in artifi- HM15136 for further clinical development [42], including its potential cial pancreas systems to mimic the bi-hormonal condition [60,61]. Beta in other clinical indications such as obesity [43], [44]. Bionics (Boston, USA) recently announced positive results on Phase 2, In contrast to such progressive developments, several companies home-use trial of iLet™ (a pocket-sized, wearable, autonomous bionic discontinued development of their glucagon-based products at different pancreas device) with Dasiglucagon [62]. Future availability of stable stages. The discontinued products include SAR438544 (Sanofi, Paris, glucagon formulations may ease explorations on such new delivery France) [45], BIODEL-961 (Albireo Pharma, previously Biodel, Boston, systems and expand usage of glucagon therapy. USA) [46], MAR-D28 and MAR531 (Marcadia Biotech Carmel, USA) ‘Mini-dose glucagon (MDG)’ is proposed as an alternative sub- and Rescue G, PumpaGon (Latitude Pharma, San Diego, USA) [46]. cutaneous glucagon treatment to oral carbohydrate intake during mild- Besides hypoglycaemia, glucagon products are also gaining im- to-moderate hypoglycaemia. T1D patients/children with inadequate portance for their usage in obesity and type-2 diabetes (T2D) due to carbohydrate intake or prone to oral carbohydrate-induced nausea, pleiotropic effects of native glucagon peptide on food intake, lipid vomiting may benefit from MDG. Also, it can reduce the risk of exercise- metabolism and energy expenditure. Intriguingly, Hanmi and Zealand induced hypoglycaemia in T1D patients [32,63–66]. Future availability Pharma separately reported body weight loss in diet-induced obese of new stable glucagon formulations as MDG therapy may improve the (DIO) mice upon chronic administration of stable glucagon formula- quality of life in diabetic patients. However, additional clinical studies tions [43,47]. Chronic treatment of HM15136 resulted in body weight are needed to calculate the risk associated with the chronic use of MDG. reduction in DIO mice [44] possibly due to pleiotropic mechanisms on Another unique application of glucagon therapy could be to mini- energy expenditure via white-adipose tissue (WAT) browning, mize the insulin requirement and balance the insulin-induced weight

3 M. Patil, et al. Peptides 127 (2020) 170296 gain in diabetic patients. Animal experiments revealed that co-admin- hurdle and confers selectivity as a critical criterion for the drugs that act istration of long-acting glucagon analogue and insulin at a fixed-dose through glucagon receptor antagonism [73]. As a collective con- ratio not only increases the therapeutic window and duration of action sequence of such hurdles, further developmental efforts are impeded in of insulin but also attenuates insulin-induced body weight gain [67,68]. this area, thus dampening the research interest. However, another study reports defective β-cell function resulting from Future studies on GRAs are needed to elucidate the exact mechan- chronic GCGR activation in DIO mice [69]. Indeed, further investiga- isms of untoward effects to propose possible approaches to mask them. tions are needed to evaluate the long-term efficacy and safety of long- Altogether, it is evident that the road for regulatory approval of GRAs as acting glucagon analogues, especially their impact on β-cell function. monotherapy will be difficult unless clinical studies demonstrate a To date, no formulation/analogue reported hepatic glucagon resistance positive risk-benefit profile. Thus, instead of monotherapy, a combi- or desensitization, howbeit it remains a concern for future development nation approach may ease the success path for GRAs development. It is [70]. theoretically convincing that partial blockage of glucagon receptors by So far, available pre-clinical findings on sustained GCGR activation low-dose GRAs in combination with other anti-hyperglycaemic agents by stable glucagon products suggest their potential as mono or in (like GLP-1RA) may reduce the severity of unwanted effects and offer combination therapy for the management of diabesity. However, clin- favourable clinical outcome. However, further experimental and clin- ical translatability of this approach is yet to be established and needs ical investigations are required to speculate the long-term risk-benefit further mechanistic evaluation. The favourable bench-to-bedside profile of such combination therapies. translation of this approach may start a new race to hunt for long-acting glucagon analogues which may completely change the fate of glucagon 2.4. Dual and triple -incretin agonists utilising glucagon therapy in the future. 2.4.1. GLP-1 receptor (GLP-1R)/GCGR dual-agonist 2.3. Glucagon receptor antagonists (GRAs) as a treatment for T2D The complementary weight-loss mechanisms of GLP-1 and glucagon founded the basis of their pharmacological integration in a unim- Hyperglucagonemia is widely evident in diabetes [4,5,71]. The ra- olecular dual-agonist. Their high sequence homology eases this en- tionale for designing GRAs was to control hyperglycaemia in diabetes gineering and the insulinotropic property of GLP-1 balances diabeto- by suppressing or blocking glucagon action [10]. The glucagon re- genic liability of glucagon further, utilising its pleiotropic effects such ceptors ablation or treatment with the glucagon receptor antibody as lipolysis and thermogenesis to amplify GLP-1 induced-centrally improved the glycaemic index in diabetic animals [72–74]. So far, mediated satiety. Moreover, the non-overlapping expression of their several GRAs have been evaluated in the preclinical and clinical sce- receptors at many cell types provides broader effects in multiple tissues nario with expectations to provide the evolutionary treatment option [1,92,93]. for T2D patients (Table 2). In 1994, the first peptide was discovered, which demonstrated a Based on chemical nature, GRAs can be categorized into three dif- capability to activate both the receptors [94]. During mid-2009, a study ferent types i) small molecule ii) monoclonal antibody, and iii) anti- employing PEGlylated peptide built on glucagon sequence was first to sense oligonucleotide. To date, various GRAs demonstrated their effi- report the therapeutic benefits of GLP-1R/GCGR dual co-agonism in an cacy in different stages of clinical trials, although none of the molecules animal model of metabolic disease. In this study, dual agonist treatment succeeded as therapeutic entities. Most of the trials evaluating the long- showed improvement in lipid metabolism and liver steatosis along with term efficacy of GRAs raised safety concerns due to the concomitant positive primary outcomes on body weight, glycaemic index and energy occurrence of unwanted side effects despite their ability to reduce expenditure. Furthermore, the study suggested a possibility of incretin HbA1c and glycaemia. The concerns with GRAs include body weight poly-agonism in a single molecule for further enhancement of efficacy gain, elevation in hepatic enzymes and low-density-lipoprotein-C (LDL- by closely mimicking physiological regulations [95]. During the same C) levels, liver fat content, systolic blood pressure and α-cell hyper- period, a research group at Merck (New Jersey, USA) designed a pep- plasia [75,76]. tide using the sequence of a known endogenous GLP-1R/GCGR dual- It appears that the GRAs-induced increase in hepatic transaminase agonist, oxyntomodulin (OXM). This long-acting, dipeptidyl peptidase- attributes to an elevated hepatic fat content [75]. The plausible reason IV (DPP-IV)-resistant analogue demonstrated reversal of obesity in DIO for an abnormal increase in LDL-C could be increased plasma levels of mice model [96]. Native OXM peptide is known to promote the re- glucagon-like peptide-2 (GLP-2), phytosterols and bile acids resulting in duction in food intake and body weight. However, its weak affinity at enhanced cholesterol absorption; leading to cardiovascular (CV) safety each receptor and a short half-life (approximately 10−12 min), pre- concern [77]. Moreover, the blockage of glucagon receptor stimulates cludes its utility as a drug [97]. compensatory over-secretion of endogenous glucagon. This repulsive The first clinical study supporting GLP-1R/GCGR dual agonism increase in glucagon concentration makes the way difficult for the de- delineated increased energy expenditure and abolished hyperglycaemia velopment of drugs that down-regulates glucagon signalling. Further- in healthy humans after co-infusion of both native peptides [98]. A year more, the resemblance of GLP-1 and glucagon receptors poses another later, another human study endorsed a reduction in food intake after

Table 2 Glucagon Receptor Antagonists (GRAs).

Nature of Drug Drug Name Inventor/Developer Development Status References

Small Molecules Bay 27-9955 Bayer Discontinued [78] MK 0893 Merck & Co Discontinued [11] MK 3577 [79] PF 6291874 Pfizer Discontinued [80], [81][75], RVT 1502 (LGD 6972) Metavant Sciences and Ligand Pharmaceuticals Phase 2 [82][83], Adomeglivant (LY-2409021) Eli Lilly Discontinued [76], [84], [85][86], NNC 252504 Novo Nordisk Discontinued [87] Monoclonal Antibodies PF 6293620 (PF-06293620; PF-6293620; RN909) Pfizer Phase 1 [88] Volagidemab (AMG477; REMD 477) REMD Biotherapeutics Phase 2 [89] Antisense Oligonucleotide IONIS-GCGRRx (ISIS-449884; ISIS-GCGRRx) Isis Pharmaceuticals and Ionis Pharmaceuticals Phase 2 [90] ISIS 325568 Isis Pharmaceuticals Discontinued [91]

4 M. Patil, et al. Peptides 127 (2020) 170296

Table 3 Emerging GLP-1R/GCGR Dual Agonists.

Drug Name Inventor/Developer company Indications Development Phase

Cotadutide (MEDI0382) MedImmune (AstraZeneca) T2D, Obesity Phase 2 Pegapamodutide (LY2944876/TT-401/OPK 88003) Transition Therapeutics (OPKO Health) T2D Phase 2 Efinopegdutide (JNJ-64565111,HM12525A) Hanmi Pharmaceutical T2D, Obesity Phase 2 MK-8521 Merck T2D Discontinued NN9277 (NNC9204-1177) Novo Nordisk Obesity Phase 1 ZP2929 Zealand Pharma T2D, Obesity Phase 1 PSA-oxyntomodulin Xenetic Biosciences T2D, Obesity Phase 1 MOD-6031 OPKO Biologics T2D Phase 1 BI 456906 Boehringer Ingelheim Pharmaceuticals T2D Phase 1 SAR425899 Sanofi-Aventis T2D, Obesity Phase 1 JNJ-54728518 Janssen T2D, Obesity Preclinical VPD-107/ SP-1373 / ALT-801 Spitfire Pharma T2D, Obesity, NASH Preclinical glucagon and GLP-1 co-infusion [99]. A concrete proof-of-concept in in unimolecular dual-agonist is an intricate task. It appears that optimal animal models and highly positive clinical translation ignited a broad specificities for GLP-1R and GCGR in Cotadutide were achieved by interest to focus on GLP-1R/GCGR co-agonism as an innovative ap- clinical modelling and animal studies. Cotadutide claims to have a fi- proach to fight diabesity and related co-morbidities. Consequently, ve‐fold higher affinity towards GLP‐1R versus GCGR [118]. However, several unimolecular GLP-1/GCGR dual agonists are currently under- no clinical study so far compared PK-matched GLP-1R mono-agonist development (Table 3). with Cotadutide depicting GCGR-agonism specific contribution in its Mostly, native peptides (glucagon/GLP-1/OXM/exendin) are used efficacy. In Phase 2a study, the magnitude of increase in the heart rate as a starting template to design the new dual co-agonists. To date, and numerical reduction in blood pressure observed in Cotadutide various modifications were explored on these native peptides to achieve treated patients was similar to liraglutide [117]. These effects are maximum metabolic benefits in resultant novel unimolecular dual- possibly due to its higher affinity towards GLP-1R and thus may retain agonist. Metabolic outcomes of such modifications are well docu- CV effects of native GLP-1. Similarly, in Phase 1 study, few incidents of mented and reviewed elsewhere, mostly by Richard DiMarchi and ‘vomiting’ occurred, which may be due to elevated glucagon portion in Matthias Tschop group [1,93,100–116]. In this section, we focused on higher dose regimen (300 μg). However, future clinical studies on a providing an overview of drug candidates currently being developed as large number of patient populations will unravel the exact magnitude of GLP-1R/GCGR dual agonist. CV and gastrointestinal (GI) related safety aspects of Cotadutide. MedImmune, a subsidiary of AstraZeneca (Cambridge, United Transition Therapeutics, a subsidiary of OPKO Health Miami, USA, Kingdom) is developing Cotadutide (MEDI0382), as once-daily, sub- recently announced results on Phase 2b study NCT03406377 conducted cutaneous therapy for T2D and obesity. The company lately reported its in T2D and obese patients for its subcutaneous, once-weekly, phase 2a, multiple-ascending dose (MAD) study in obese or overweight PEGlylated-OXM-peptide-analogue, Pegapamodutide LY2944876/TT- T2D patients [117]. Design of this synthetic, OXM-related, degradation- 401/OPK 88,003. The 30 weeks treatment of Pegapamodutide ex- resistant peptide is in such a way that it exerts optimally-balanced dual hibited a significant reduction in HbA1c (1.30 % from baseline) and agonism. The extended half-life is achieved by the insertion of a pal- body weights (4.4 kg from baseline). Future Phase 3 trial will evaluate mitic acid side chain at Lys10 to promote its reversal albumin binding. this candidate in T2D and NASH patients [124]. Earlier, Eli Lilly (ori- Its preclinical studies on DIO mice and cynomolgus monkeys elicited ginator) conducted Phase 2 study in T2D patients demonstrating su- sustained weight loss, improvement in the glycaemic state, and reduc- perior HbA1c reduction at 30 and 50 mg dose over placebo (1.15 % and tion in hepatic fat content. Four-week treatment in DIO mice at 30 1.04 %, respectively) and non-inferior (0.4 % margin) with extended nmol/kg dose demonstrated a 30 % weight reduction compared to lir- released (ER) exenatide (0.02 % and 0.09 %, respectively) [125]. OPKO aglutide (21 %), whereas the weight-loss in monkeys was in the range was also developing MOD6031, a long-acting, reversible-PEGylated- of 5–13 % accompanied by a decrease in food intake after 29 days of OXM peptide, consisting of native human OXM conjugated to poly- dosing (Doses 8−27 nmol/kg) [118]. These pre-clinical outcomes are ethylene glycol using a hydrolysable linker. Preliminary evaluation in supported by Phase 1, single ascending dose (SAD) study in healthy ob/ob mice revealed its positive impact on body weight, glucose, in- individuals, wherein MEDI0382 was found safe and tolerable (up to 150 sulin, and cholesterol levels [126]. Though the company completed the μg) with no signs of immunogenicity. The PK profile was linear at doses Phase 1 clinical study (NCT02692781) in healthy overweight or obese tested (5, 10, 30, 100, 150 and 300 μg) and reported a dose-dependent subjects in 2016, no further development reported [127]. reduction in blood glucose and food intake. This study also suggested Hanmi Pharma and Janssen Pharmaceuticals (Beerse, Belgium) the need for slower dose up-titration due to few vomiting events re- were jointly developing once-weekly, long-acting GLP-1R/GCGR dual ported at a higher dose (300 μg) [119]. Moreover, Phase 2a MAD study agonist peptide, Efinopegdutide (HM12525A/JNJ64565111) in which in obese or overweight T2D patients showed 32.78 % reduction in OXM-analogue (HMOXM25) is conjugated to non-glycosylated human blood glucose (placebo 10.16 %), and 3.84 kg body weight loss (pla- Fc fragment by a non-peptidyl linker. During mid-2019, Janssen cebo 1.70 kg) after 41 days of treatment. The study also reported a decided to step back from its on-going Phase 2 development and re- trend towards a reduction in the appetite (20 %) and placebo corrected turned rights to Hanmi. The later now intends to determine future de- HbA1c (0.3 %) levels [117]. Appetite suppression driven weight loss by velopment plans further, after reviewing clinical outputs [128]. The MEDI0382 is likely to be mediated via GLP-1R/GCGR co-agonism, preliminary animal studies showed the potential of this candidate in where the contribution of delayed gastric emptying is expected [120]. weight-loss and improved glycaemia [129]. In DIO mice, its treatment The company is optimistically pursuing the future development of Co- showed higher bodyweight reduction (32 %) as compared to liraglutide tadutide in NAFLD/NASH population as a monotherapy (15 %) by increasing FGF-21 levels and UCP-1/2 expression. Moreover, (NCT04019561) [121] owing to its ability to reduce hepatic collagen it also demonstrated positive impact in NASH animal models by redu- [122], enhance mitochondrial function and glucagon-mediated inhibi- cing hepatic triglyceride (TG) accumulation and inflammation [130]. tion of lipogenesis [123]. Additionally, HM12525A treatment reported beneficial effects on cho- Identifying the desired optimal ratio of GLP-1R and GCGR agonism lesterol profile in hyperlipidemic hamsters via increased β-oxidation,

5 M. Patil, et al. Peptides 127 (2020) 170296 decreased hepatic low-density lipoprotein (LDL) receptor clearance response to nutrients [149]. It exerts various metabolic actions. The through reduction of Proprotein convertase subtilisin/Kexin type 9 utilisation of distinct additional metabolic functions of GIP from GLP-1 (PCSK-9) and hepatic cholesterol biosynthesis inhibition [131]. is thus far encouraging in combination strategy for T2D [150]. Re- Besides a joint venture with Hanmi, Janssen Pharma was in- cently, Eli Lilly reported positive outcomes for Tirzepatide, a novel dual dependently developing JNJ-54728518, a dual agonist which is a pe- GIP and GLP-1 receptor agonist in Phase 2 study conducted on T2D gylated form of OXM. Its chronic dosing in DIO mice exhibited super- patients [151]. However, enhanced endogenous secretion of incretins iority over liraglutide in terms of body weight reduction (26.91 vs 7.44 (GLP-1, GIP, and glucagon) observed post- Roux-en-Y gastric bypass %), glucose-lowering (59 vs 118 mg/dL), anorectic effect (0.04 vs 0.07 (RYGBP) surgery raised the possibility of weight management by mi- g/g body weight) and fat loss (12.3 vs 6.0 g) [132]. However, no further micking bariatric surgery conditions using a unimolecular tri-agonist to updates are available on its development [133]. Merck was also in the stimulate physiological responses of these three incretins. [152]. Har- race with a lead candidate, MK-8521. In early-2017, the company dis- monised metabolic activity due to tri-agonism is likely to enhance continued the Phase 2 trial in T2D patients aiming to assess its safety bodyweight reduction to a more physiological condition [114]. and efficacy in comparison with liraglutide and metformin YAG-glucagon, a DPP-IV resistant peptide acting as tri-agonist, was (NCT02492763) [134]. Eventually, during mid-2019, Merck decided to shown to ameliorate glucose homeostasis in DIO mice after 14 days of stop its further development [135]. treatment. In this study, YAG-glucagon treatment reduced plasma glu- Zealand Pharma has developed ZP2929, a once-daily, subcutaneous cose (40 %), increased circulating insulin levels (1.8 fold) and enhanced therapy designed by using the glucagon sequence as a scaffold. Its insulin secretion (1.5 fold) in response to a glucose load [153]. Con- chronic treatment in DIO mice mitigated body weights, improved glu- sistent with these findings, Finan and colleagues also reported im- cose tolerance, restored HDL/LDL ratio, and corrected TG levels [136]. provement in glucose tolerance and insulin sensitivity along with a Moreover, ZP2929 showed encouraging results in NASH animal models reduction in body weight (26.6 %), and hepatic lipid content in DIO by improving liver function and lipid profile [137]. Till August 2019, mice after 21 days treatment with another tri-agonist peptide. This the company listed this peptide in the pipeline under Phase 1 devel- chimeric peptide was engineered by incorporating GIP residue into the opment [138]. The company also invented another subcutaneously existing GLP-1R/GCGR co-agonist core sequence along with Aib at administered GLP-1R/GCGR dual agonist, BI456906, which is currently position two and palmitic acid (C16:0) at Lys10 position [154]. A me- being developed by Boehringer Ingelheim (Ingelheim, Germany). chanism for enhanced insulinotropic action with tri-agonism appears Boehringer has announced its Phase 1 trial in obese patients via Phospholipase C (PLC) activation [155]. These pre-clinical proof-of- (NCT03591718) [139]. concept studies encouraged researchers to pursue further investigations Sanofi was developing SAR425899, once-daily, subcutaneously ad- on the tri-agonist concept as a strategy for diabesity. As a result, few ministered GLP-1R/GCGR dual agonist peptide engineered based on the drug candidates are currently under clinical development (Table 4). exendin-4 sequence. Its 33 days treatment in DIO mice elicited sig- Hanmi Pharma is developing HM15211, a subcutaneously ad- nificantly better body weight loss (29.1 % from basal) as compared to ministered, once-weekly GLP-1R/GCGR/GIPR tri-agonist peptide as a liraglutide (13.6 %) [140]. Phase 1 study was conducted in healthy therapy for T2D, obesity, and NAFLD/NASH. HM15211 is a modified volunteers as well as in overweight or obese patients with T2D. In both human glucagon, designed by conjugating to the human IgG Fc frag- the population, SAR425899 was found tolerable. Its treatment ex- ment via a flexible PEG linker. Four weeks treatment of HM15211 (1.44 hibited 5.46 kg body weight loss accompanied by favourable glycaemic nmol/kg, Q2D equivalent human dose 2 mg/week) in DIO mice re- effects in T2D patients, whereas, in healthy volunteers, it led to 5.32 kg sulted in a three folds higher weight reduction compared to liraglutide weight loss [141]. However, the company terminated its Phase 2 trial in (50 nmol/kg, BID; equivalent human dose 3 mg/day). The drug also patients with NAFLD even before patient enrolment, stating the deci- neutralized high GCGR-induced hyperglycaemic risk [156]. Moreover, sion was not related to safety concern (NCT03437720) [142]. Even- it also demonstrated therapeutic benefits in NASH and fibrosis animal tually, Sanofi discontinued its development [143]. Conversely, Novo models by reducing hepatic TG (82.6 %), thiobarbituric acid reactive Nordisk is optimistically pursuing development on NN9277 (NNC9204- substances levels (TBARS), and inflammation. It appears that drug 1177), a GLP-1R/GCGR dual agonist as a subcutaneous mono-therapy treatment-induced reduction in expression of hepatic collagen-1α1, for obesity [144]. A Phase 1 study is currently recruiting overweight or TIMP-1, and hydroxyproline levels collectively contribute to improve- obese patients to assess its safety, tolerability, and pharmacokinetics for ment in fibrosis. Intriguingly, results translated from mice to obese/ multiple doses (NCT03308721) [145]. NASH non-human-primates for correction in lipid profi le, body weight, Xenetic Biosciences (Massachusetts, USA) is developing PSA-oxy- and fat mass loss [157]. Lipid-lowering mechanism of HM15211 is ntomodulin, a long-acting subcutaneous injection. It is a chemically plausibly a sum of its ability to reduce lipid absorption, cholesterol polysialylated, proteases-resistant form of native OXM peptide with synthesis, increase LDL clearance, and HDL synthesis [158]. The drug extended half-life. Its single injection (15 mmol/kg) induced anorectic was found safe and well-tolerable in Phase 1 SAD study conducted in effect lasting for eight hours in normal C57BL/6 N mice [146]. In mid- obese healthy subjects. The study reported no significant changes in 2014, the company reported its safety and tolerability in a Phase 1 heart rate, blood pressure, and electrocardiogram (ECG). However, it study in healthy volunteers [147]. suppressed endogenous GLP-1, glucagon, and GIP concentrations in a Velocity Pharma (California, USA) is developing long-acting, OXM- dose-dependent manner, confirming its hypothesised effects on en- based GLP-1R/GCGR dual agonist, VPD-107 (SP-1373) by utilising dogenous incretin levels [159]. Hanmi intends to further evaluate this EuPort technology. In rodent models of obesity and NASH, it demon- strated superiority over semaglutide. In DIO mice, 28 days equimolar Table 4 (12 nmole/kg; QD SC) dosing of SP-1373 and semaglutide resulted in GLP-1R/GCGR/ GIPR Tri-agonists under-development. 25 % and 10 % body weight loss, respectively. However, both treat- ments exhibited similar effects on glycaemia and food intake. A similar Drug Name Inventor Indications Development Status fi superiority pro le observed in an obese rat model with respect to HM15211 Hanmi Obesity, NAFLD/ Phase 1 bodyweight reduction (40 % vs 13 %) and improvement in liver stea- Pharma NASH tosis (59 % vs 52 %) [148]. NN9423/NNC9204- Novo Obesity Phase 1 1706 Nordisk 2.4.2. GLP-1R/GCGR/GIPR tri-agonists GGG Tri-agonist Eli Lilly Diabetes Phase 1 SAR441255 Sanofi- T2D, Obesity, Discontinued The glucose-dependent insulinotropic polypeptide (GIP) is an in- Aventis cretin hormone secreted in the K-cells of the proximal small intestine in

6 M. Patil, et al. Peptides 127 (2020) 170296 candidate in NAFLD/NASH patients (NCT03744182) [160]. Besides its stimulating endogenous glucagon release [179]. It will be interesting to role in metabolic disorders, HM15211 also reported to possessing know if glucagon and SGLT2i/ or DPP-IV inhibitors augment mutual neuroprotective and bone protective properties [161,162]. potential to provide broader coverage in diabesity and related com- Novo Nordisk is pursuing development on an injectable tri-agonist plications. candidate, NNC9204-1706 (NN9423) as a treatment option for obesity. Structural similarity of GLP-1, glucagon and their receptors allows Recently, Novo completed two separate Phase 1 studies, in overweight glucagon to bind GLP-1R at ten times lesser extent of GLP-1, whereas, or obese patients, assessing safety, tolerability, and PK upon its single GLP-1 also engages GCGR albeit at a lesser extent. The contribution of (NCT03095807), and multiple (NCT03661879) dosing. However, study this cross-reactive agonism in mono/dual/poly agonist needs a thor- outputs are yet to be published [163,164]. The research pipeline of Eli ough evaluation. According to recently proposed paradoxical theories, Lilly indicates Phase 1 development of a biologic entity, "GGG Tri- GCGR agonism enhances glucose tolerance, insulin secretion, and in- Agonist" for the treatment of diabetes. However, no information is sulin tolerance, in part by amplifying insulin action [180,181]. In- available in the public domain [165]. During late-2019, Sanofi dis- tensified future understanding of this concept may extend the current continued the development on Phase 1 tri-agonist drug candidate, role of glucagon by changing its position in health and diabetes. SAR441255 [166]. Altogether, hitherto understanding of glucagon biology has changed canonical views about the hormone. Glucagon biology seems to be 2.4.3. Future perspectives on dual and tri-agonists complicated, and still, many things are yet to be discovered and un- Pharmacological marriages of glucagon with GLP-1 (dual agonist) derstood. and GLP-1/GIP (tri-agonist) are in their early phases, with limited clinical data. In the coming years, adequate information will be avail- 3. Conclusions able from long-term clinical studies. As of now, available initial clinical outputs are encouraging to retain enthusiasm in the field. However, the The importance of glucagon therapy has expanded considerably long-term safety, especially CV and GI-related concerns, need to be over the past five years. As a result, we expect to see inhaled and stable thoroughly evaluated. All emerging dual/tri-agonist are injectables, and glucagon-based products entering the market soon. The availability of therefore, concerns related to allergic reactions remain to be assessed in these new glucagon products will not only reduce the fear of hy- chronic settings and leaves room for the future explorations on other poglycaemia in diabetic patients but also expand its horizon by ex- delivery routes. tending its usage in an artificial pancreas system and other delivery OXM-induced glucagon agonism is responsible for increased in- forms. trinsic heart rate in animals but not in humans [167,168]. Furthermore, After a decade of translational research in the field of dual/poly Axelsen and colleagues speculated the possible benefit of dual agonism- incretin agonist, now we are on the verge to find new generation induced inotropic and energy-preserving actions in insulin-resistant therapy for diabesity and related complications. So far, the dual/poly hearts in the treatment of severe heart failure in T2D patients [169]. agonism approach seems to be promising, and performance of emerging Such theories strengthen the impression that GLP-1R/GCGR dual ago- drug candidates in advanced-stage appears to be competent. nists may have a favourable long-term CV effect in diabetic patients. It Nevertheless, we have to wait until the availability of long-term clinical is also possible that GLP-1 dominance and its glucagonostatic nature safety data. Indeed, the development of such innovative dual/multi- may mask the unwanted activities of glucagon and solely utilise GLP-1- agonist drugs has not only changed the “diabetogenic” image of glu- independent benefits. Therefore, dual-agonist may retain CV and GI- cagon but also raised its importance. related effects of native GLP-1, as observed in initial clinical studies of Cotadutide. Funding Multi-receptor agonists may exert diverse pharmacological effects, possibly due to the receptor’s expression in multiple tissues, leaving This work was supported by internal funding from Wockhardt room for dual/tri-agonists to cover a broad range of diseases. There is Limited (Mumbai, India). an evident role of incretins in the pathophysiology of neurological [170,171] and bone-related disorders [172]. Hanmi’s tri-agonist can- Contributions didate (HM15211) is effective in Parkinson’s, and osteoporosis animal models. Such extra-metabolic indications should be taken into con- Mohan Patil and Nitin Deshmukh prepared the first version of the sideration while designing future studies on multi-receptor agonists. article after gathering information and data mining. Mahesh Patel Mostly, native peptides (glucagon/exendin-4/GLP-1/OXM) tem- provided scientific inputs and recourses. Ganesh Sangle involved in plates were used to design dual/triple incretin agonists. An alternative conceptualization, review, editorial process, and final approval of the approach could be to hunt for animal-sourced peptide templates which manuscript. have higher structural similarities with human incretins. For instance, dogfish glucagon is structurally similar to human GLP-1, GIP, and Declaration of Competing Interest glucagon, thus possessing various benefi cial metabolic effects [173]. In the LEAN trial, GLP-1R agonism improved NASH histology and No competing financial interests exist. arrested fibrosis progression [174]. Glucagon is also an effective he- patic lipid metabolism regulator as it stimulates lipid utilisation and References free fatty acid oxidation in the liver [175]. So far, observed promising synergism of both peptides in NAFLD/NASH animal models has uplifted [1] T.D. Müller, B. Finan, C. Clemmensen, R.D. DiMarchi, M.H. 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