>	UnAcylated Ghrelin (UAG)

The ghrelin program at AlizéPharma aims at developing an analog of unacylated ghrelin (UAG), a first product of a new therapeutic class for the treatment of metabolic disorders including Type 2 diabetes and the PraderWilli Syndrome. It has been conducted for the last 5 years in collaboration with Erasmus Medical Center (Rotterdam) and the University of Turin.

Preclinical and clinical data suggest that UAG and its analogs have the potential to fulfill unsatisfied medical needs in type 2diabetes and the PraderWilli syndrome, through a novel mechanism of action that includes:  marked decrease in circulating levels of acylated ghrelin (AG), a known orexigenic and diabetogenic hormone; improved glucose control; insulin-sensitizing actions; trophic effect on beta cells; reduction in fat mass deposition; and positive impact on vascular remodeling and on recovery following ischemia. AZP-531, a stabilized UAG analog with improved pharmacokinetic properties, was designed and is currently undergoing preclinical development. UAG, its analogs including AZP-531 and their therapeutic uses are protected by 5 families of patents totaling 34 patents and patents applications worldwide.

 

Background: the Ghrelin system

Ghrelin is a 28-amino acid circulating peptide that was initially discovered in 1999 as the endogenous ligand of an orphan receptor, the Growth Hormone (GH) Secretagogue type 1a Receptor (GHS-R1a) (1). Ghrelin is naturally secreted by the stomach, pancreas and a wide variety of cells and tissues, and circulates into two different forms:

• Acylated Ghrelin (AG), commonly referred to as ghrelin, is acylated (octanoyl moiety) on the Serine in position 3,
• UnAcylated Ghrelin (UAG) is the ghrelin peptide devoid of the acyl moiety.

 

 

 

The acylation of UAG into AG is catalyzed by Ghrelin O-AcylTransferase (GOAT) and is essential for binding to GHS-R1a (2;3). Thus AG, but not UAG, induces secretion of Growth Hormone (GH) and is responsible for other peripheral and central activities mediated by GHS-R1a (4). AG is an orexigenicand diabetogenichormone that stimulates appetite and induces insulin resistance (1;5). Despite initial interest, the development of AG as a therapeutic drug has been limited by its diabetogenic properties (6-9). Therefore inhibiting AG’s diabetogenic and adipogenic effects is now recognized as a therapeutic target for the treatment of metabolic disorders, such as type 2diabetes and obesity (10).

 

Pharmacological profile of UAG

Unacylated ghrelin (UAG) is the predominant form of circulating ghrelin (80-90%). UAG, although devoid of GHS-R1a-binding affinity, is an active peptide exerting specific biological activities through a yet undetermined receptor, sometimes supporting, but most often counteracting and opposing the effects of AG (10;11). Hence, while AG and UAG promote pancreatic ß-cell survival (12;13), UAG has been reported to counteract AG-induced insulin resistance and hyperglycemia (14;15) and to suppress AG-induced glucose output by primary hepatocytes (16). These initial observations are further supported by subsequent pharmacological studies performed in animals and humans which emphasize the antidiabetogenic potential of UAG.

 

Preclinical data

In vitro, UAG, in contrast to AG, promotes glucose and FFA uptake by cardiomyocytes, myotubes or adipocytes (17;18). In STZ-induced diabetic rats, UAG administration counteracted STZ-induced hyperglycemia and protected the animals from the STZ-induced decrease in insulin levels, while exerting clear trophic effects on pancreatic islet cell mass(19). In mice on a high-fat diet, UAG administration improved insulin sensitivity and prevented increases in fat mass and body weight without affecting food intake (20). Supporting animal data from literature indicate that over-expression of UAG in transgenic mice promotes glucose tolerance and insulin sensitivity, while reducing fat deposition and triglycerides levels (21;22).

 

Clinical data

In healthy volunteers, UAG administration as an IV bolus or as continuous infusion has been shown to inhibit the hyperglycemic effect of AG and also to lower glucose levels (14;23). In T2DM patients, infusion of UAG for up to 16 hours resulted in a reduction of post-prandial hyperglycemia, in line with earlier observations(24).Moreover, a strong inhibitory effect on blood AG concentrations was observed in these patients (24). To date, UAG as bolus injection or short-term infusion has been administered to more than 100 individuals and has been reported to have a safety profile comparable to placebo.

Cardiovascular benefits

 

In addition to its metabolic effects, UAG also exerts beneficial cardiovascular effects(25-27). Recently, it was reported that acute administration of UAG, but not AG, restores Endothelial Progenitor Cells (EPC) mobilization in diabetic animals and in T2DM patients, resulting in improved vascular remodeling(28). UAG has also been shown to protect EPCs from oxidative stress and from senescence, two important unwanted processes that are present in diabetic patients. In hindlimb ischemia mouse model, UAG improves functional recovery and muscle regeneration post-ischemia in wild type and ob/ob mice (29). Considering the fact that circulating EPCs are biomarkers for vascular functions and cardiovascular outcomes(30;31), these results support the rationale for investigating the effects of UAG analogs on cardiovascular outcome, a highly unmet need in the treatment of the complications of diabetes. In addition, it may pave the way to the development of UAG analogs in ischemic diseases such as peripheral arterial diseases or myocardial infarction.

 

The UAG development program

Original and promising pharmacological profile

• [AG] inhibitory effect, insulin sensitizing effect & ß-cell protection
• Expected positive effects on several cardiovascular risk factors

Lead analog ready to enter preclinical development

• A cyclic 8-AA lead compound has been selected from a Structure-Activity-Relationship and design program(32). This analog, derived from a UAG fragment, exhibits the same pharmacological profileas UAGand improved pharmacokinetic properties (33)and is currently undergoing preclinical development. First Phase I clinical trial in healthy volunteers is expected to be performed in 2013.

 

Targeted Indications

Because of their mechanism of action, UAG and analogs are proposed as drug candidate in metabolic indications where inhibiting or antagonizing acylated ghrelin might be beneficial, such as the PraderWilli Syndrome and type 2 diabetes.

• Type 2 diabetes is a disease characterized by hyperglycemia, which is to say an excessively high level of glucose (sugar) in the blood. This disease generally occurs in adults of an advanced age, and tends to affect obese or overweight people. The number of patients suffering from type 2 diabetes is constantly rising as a result of the spread of obesity and the aging of the population. The International Diabetes Federation expects the number of diabetic patients to rise worldwide from 285 million in 2010 to 438 million in 2030. Treating diabetes and its cardiovascular complications is a major public health challenge. At present, there is no treatment that can properly cure diabetes and its complications. In this context, there is a major medical need for developing innovative drugs that are based on new mechanisms of action and target several cardiovascular risk factors.
• The PraderWilli syndrome is a rare genetic condition with an estimated incidence of approximately 8/100 000. At birth, the child exhibits a diminution of muscle tone (hypotonia) causing in particular sucking problems. After the age of two, the hypotonia diminishes and a severe hyperphagia is observed, correlated with abnormally high levels of circulating acylated ghrelin. The needs in calories intake declines which, together with the hyperphagia, results in an obesity condition with its associated life-threatening complications (diabetes, cardiovascular complications). The child also exhibits endocrine problems (short stature and hypogonadism), learning difficulties and behavioral problems.There is currently no specific treatment for this syndrome, and no effective treatment for eating disorders. Patients’ quality of life can only be improved by early multidisciplinary care (growth hormone, adequate diet, orthophony, etc.).

 

Strong IP position

AlizéPharma holds a strong IP position on the UAG program consisting of 5 families with a total of 34 patents and patent applications internationally and that are related to UAG, its analogs and their potential clinical indications.

 

References

(1) Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999 Dec 9;402(6762):656-60.

 

(2)    Gutierrez JA, Solenberg PJ, Perkins DR, Willency JA, Knierman MD, Jin Z, et al. Ghrelin octanoylation mediated by an orphan lipid transferase. Proc Natl Acad Sci U S A 2008 Apr 29;105(17):6320-5.

 

(3)    Yang J, Brown MS, Liang G, Grishin NV, Goldstein JL. Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 2008 Feb 8;132(3):387-96.

 

(4)    Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, et al. A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 1996 Aug 16;273(5277):974-7.

 

(5)    Date Y, Kojima M, Hosoda H, Sawaguchi A, Mondal MS, Suganuma T, et al. Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 2000 Nov;141(11):4255-61.

 

(6)    Tong J, Prigeon RL, Davis HW, Bidlingmaier M, Kahn SE, Cummings DE, et al. Ghrelin suppresses glucose-stimulated insulin secretion and deteriorates glucose tolerance in healthy humans. Diabetes 2010 Sep;59(9):2145-51.

 

(7)    Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000 Oct 19;407(6806):908-13.

 

(8)    Broglio F, Arvat E, Benso A, Gottero C, Muccioli G, Papotti M, et al. Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab 2001 Oct;86(10):5083-6.

 

(9)    Broglio F, Prodam F, Riganti F, Gottero C, Destefanis S, Granata R, et al. The continuous infusion of acylated ghrelin enhances growth hormone secretion and worsens glucose metabolism in humans. J Endocrinol Invest 2008 Sep;31(9):788-94.

 

(10)    Delhanty PJ, van der Lely AJ. Ghrelin and glucose homeostasis. Peptides 2011 Mar 21.

 

(11)    Kumar R, Salehi A, Rehfeld JF, Hoglund P, Lindstrom E, Hakanson R. Proghrelin peptides: Desacyl ghrelin is a powerful inhibitor of acylated ghrelin, likely to impair physiological effects of acyl ghrelin but not of obestatin A study of pancreatic polypeptide secretion from mouse islets. Regul Pept 2010 Sep 24;164(2-3):65-70.

 

(12)    Granata R, Settanni F, Biancone L, Trovato L, Nano R, Bertuzzi F, et al. Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 3',5'-cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inositol 3-Kinase/Akt signaling. Endocrinology 2007 Feb;148(2):512-29.

 

(13)    Granata R, Settanni F, Trovato L, Destefanis S, Gallo D, Martinetti M, et al. Unacylated as well as acylated ghrelin promotes cell survival and inhibit apoptosis in HIT-T15 pancreatic beta-cells. J Endocrinol Invest 2006 Oct;29(9):RC19-RC22.

 

(14)    Broglio F, Gottero C, Prodam F, Gauna C, Muccioli G, Papotti M, et al. Non-acylated ghrelin counteracts the metabolic but not the neuroendocrine response to acylated ghrelin in humans. J Clin Endocrinol Metab 2004 Jun;89(6):3062-5.

 

(15)    Gauna C, Meyler FM, Janssen JA, Delhanty PJ, Abribat T, van KP, et al. Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity. J Clin Endocrinol Metab 2004 Oct;89(10):5035-42.

 

(16)    Gauna C, Delhanty PJ, Hofland LJ, Janssen JA, Broglio F, Ross RJ, et al. Ghrelin stimulates, whereas des-octanoyl ghrelin inhibits, glucose output by primary hepatocytes. J Clin Endocrinol Metab 2005 Feb;90(2):1055-60.

 

(17)    Lear PV, Iglesias MJ, Feijoo-Bandin S, Rodriguez-Penas D, Mosquera-Leal A, Garcia-Rua V, et al. Des-acyl ghrelin has specific binding sites and different metabolic effects from ghrelin in cardiomyocytes. Endocrinology 2010 Jul;151(7):3286-98.

 

(18)    Granata R, Settanni F, Gallo D, Grande C, Nano R, Baragli A, et al. Unacylated ghrelin fragments promote survival of pancreatic ß-cells and human pancreatic islets and influence glucose and lipid metabolism in C2C12 myotubes and 3T3-L1 adipocytes. ENDO 2011 Jun;P2-499.

 

(19)    Granata R, Volante M, Settanni F, Gauna C, Ghe C, Annunziata M, et al. Unacylated ghrelin and obestatin increase islet cell mass and prevent diabetes in streptozotocin-treated newborn rats. J Mol Endocrinol 2010 Jul;45(1):9-17.

 

(20)    Delhanty PJ, Huisman M, van den Berge I, Abribat T, Themmen AP, van der Lely AJ. Unacylated ghrelin improves insulin sensitivity in the early phase of obesity. ENDO 2011 Jun;P1-489.

 

(21)    Zhang W, Chai B, Li JY, Wang H, Mulholland MW. Effect of des-acyl ghrelin on adiposity and glucose metabolism. Endocrinology 2008 Sep;149(9):4710-6.

 

(22)    Asakawa A, Inui A, Fujimiya M, Sakamaki R, Shinfuku N, Ueta Y, et al. Stomach regulates energy balance via acylated ghrelin and desacyl ghrelin. Gut 2005 Jan;54(1):18-24.

 

(23)    Benso A, St-Pierre D, Prodam F, Gramaglia E, Granata R, van der Lely AJ, et al. Metabolic effects of overnight continuous infusion of unacylated ghrelin in humans. Eur J Endocrinol 2012 Feb 29.

 

(24)    Özcan B, Neggers SJCMM, Miller AR, Yang H, Lucaites V, Abribat T, et al. Unacylated ghrelin is a strong suppressor of acylated ghrelin levels in obese diabetic subjects . ENDO 2012 Jun.

 

(25)    Bedendi I, Alloatti G, Marcantoni A, Malan D, Catapano F, Ghe C, et al. Cardiac effects of ghrelin and its endogenous derivatives des-octanoyl ghrelin and des-Gln14-ghrelin. Eur J Pharmacol 2003 Aug 22;476(1-2):87-95.

 

(26)    Kleinz MJ, Maguire JJ, Skepper JN, Davenport AP. Functional and immunocytochemical evidence for a role of ghrelin and des-octanoyl ghrelin in the regulation of vascular tone in man. Cardiovasc Res 2006 Jan;69(1):227-35.

 

(27)    Baldanzi G, Filigheddu N, Cutrupi S, Catapano F, Bonissoni S, Fubini A, et al. Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT. J Cell Biol 2002 Dec 23;159(6):1029-37.

 

(28)    Togliatto G, Trombetta A, Dentelli P, Baragli A, Rosso A, Granata R, et al. Unacylated ghrelin rescues endothelial progenitor cell function in individuals with type 2 diabetes. Diabetes 2010 Apr;59(4):1016-25.

 

(29)    Brizzi MF. manuscript in preparation 2012.

 

(30)    Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003 Feb 13;348(7):593-600.

 

(31)    Werner N, Kosiol S, Schiegl T, Ahlers P, Walenta K, Link A, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med 2005 Sep 8;353(10):999-1007.

 

(32)    Granata R, Settanni F, Julien M, Nano R, Togliatto G, Trombetta A, et al. Unacylated ghrelin fragments and analogues promote survival of pancreatic beta-cells and human pancreatic islets and prevent diabetes in streptozotocin-treated rats. J Med Chem 2012 Feb 21.

 

(33)    Julien M, Kay R, Delhanty P, Allas S, Granata R, Barton C, et al. In vitro and in vivo stability and pharmacokinetic profile of unacylated ghrelin (UAG) analogues. submitted 2012 Feb.