CLINICAL PRACTICE
`REVIE W PAP ER
`Liraglutide: the therapeutic promise from animal models
`
`(11D THE INTERNATIONAL JOURNAL OF
`
`L. B. Knudsen
`
`Novo Nor<f~k A/S, Novo
`Nor<f~k Park, DK 2760, Mal0V,
`Denmark
`
`Correspondence to:
`Lotte Bjerre Knudsen,
`Department Biology and
`Pharmacology Mg\ Novo
`Nor<f~k A/S, Novo Notdisk
`Park, ~ I0V, DK 2760 Denmark
`Tel.: t45 444 3 47BB
`Fax: +45 3075 47BB
`Email: bkn@novonor<f~k.mm
`
`Disclosures
`This article forms part of a
`supplement funded by Novo
`Nor<f~k.
`LBK ~ an empbyee of Novo
`Nor<f~k. The author was part
`of the team that discovered
`and developed riraglutide. All
`patents naming the author as
`an inventor bebng to Novo
`Nor<f~k; the author has no
`financial interest related to afrf
`patents. LBK has shares in
`Novo Nor<f~k as part of an
`empbiee offering programme.
`
`SUMMARY
`
`Aims: To review the differences between the human glucagon-like peptide-1 (GLP-
`1) molecule and the analogue liraglutide, and to summarise key data from the lira(cid:173)
`glutide preclinical study programme showing the therapeutic promise of this new
`agent. Key findings: Liraglutide is a full agonist of the GLP-1 receptor and shares
`97% of its amino acid sequence identity with human GLP-1. Unlike human GLP-1 ,
`however, liraglutide binds reversibly to serum albumin, and thus has increased
`resistance to enzymatic degradation and a longer half-life. In preclinical studies,
`liraglutide demonstrated good glycaemic control, mediated by the glucose-depen(cid:173)
`dent stimulation of insulin and suppression of glucagon secretion and by delayed
`gastric emptying. Liraglutide also had positive effects on body weight, beta-cell
`preservation and mass, and cardiac function. Conclusions: The therapeutic prom(cid:173)
`ise of liraglutide is evident from preclinical data. Liraglutide showed the potential
`to provide good glycaemic control without increasing the risk of hypoglycaemia
`and, as with exenatide, but not dipeptidyl peptidase-4 inhibitors, to mediate
`weight loss. Although these benefits have subsequently been studied clinically,
`beta-cell mass can be directly studied only in animal models. In common with
`other incretin-based therapies, liraglutide showed the potential to modulate the
`progressive loss of beta-cell function that drives the continuing deterioration in gly(cid:173)
`caemic control in patients with type 2 diabetes. Body weight was lowered by a
`mechanism involving mainly lowered energy intake, but also potentially altered
`food preference and maintained energy expenditure despite weight loss.
`
`Introduction
`
`As explained by Dr Unger in this supplement", the
`therapeutic potential of incretin hormones for the
`treatment of type 2 diabetes is the subject of consid
`erable ongoing research. Interest has focused on the
`incretin glucagon like peptide 1 (GLP 1) in particu
`Jar and has resulted in two new classes of antihyper
`glycaemic agents: the dipeptidyl peptidase 4 (DPP 4)
`inhibitors and the GLP 1 receptor agonists. Liraglu
`tide, which belongs to the latter class of agents, is
`one of only two commercially available GLP 1 recep
`tor agonists. Liraglutide is approved for use in com
`bination with selected oral agents (Europe and USA)
`and as monotherapy (USA and Japan).
`In this first review of the supplement, differences
`between the human GLP 1 and liraglutide molecules
`are discussed and key data from the liraglutide pre
`clinical programme are summarised. Preclinical work
`
`•unger J. Liraglutide: <an ~ make a difference in the treatment of type 2 diabetes?
`kit J Clin Praa 2010; 64 (Suppl. 167}: 1 3.
`
`is a necessary precursor to human studies: assessment
`of toxicity in a preclinical setting is a legal require
`ment, but preclinical studies also gather important
`preliminary data on pharmacokinetics and pharma
`codynamics. A large body of data in any preclinical
`programme is generated from in vitro investigations;
`however, normal animals and animal models of the
`disease state allow us to examine the effects of a new
`drug on the complex interplay of metabolic pro
`cesses. This review focuses largely on the in vivo
`studies from the liraglutide preclinical programme,
`which demonstrate the therapeutic promise of lira
`glutide. These data continue to be relevant to the
`physician despite subsequent clinical trials, not least
`because animal models remain the only way to
`explore directly the mechanistic effects of therapy on
`different organs and tissues, such as beta cells. Ani
`ma! studies are also essential to explore novel effects
`of a diabetes drug, such as a direct effect on cardiac
`function. Throughout this review, key findings for
`other incretin based therapies and GLP 1 are noted
`to provide context for the liraglutide data, and the
`
`4
`
`© 2010 Blackwell Publishing Ltd Int J Clin Pract, October 2010, 64 (Suppl. 167). 4 11
`dioi: 10.1111/j.1742 1241.2010.02499.x
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`Liraglutide: the therapeutic promise from animal models
`
`5
`
`reader is referred to enstmg reviews and/or key
`papers for more information. Subsequent articles in
`this supplement by Dr Schmidt, Drs Raskin and
`Mora, and Dr McGill consider the performance of
`liraglutide in clinical trials.
`
`The liraglutide molecule
`
`Liraglutide is a full agonist of the GLP 1 receptor
`(1) . The liraglutide and human GLP 1 molecules
`also share a 97% amino acid sequence identity.
`However, although the GLP 1 molecule is rapidly
`degraded in the body and has a half life of approx
`imately 2 min following intravenous administration
`(2), the liraglutide molecule has a half life of 13 h
`following subcutaneous administration (3). Struc
`turally, the molecules differ in only two respects
`(Figure 1) . First, a C16 fatty acid chain (palmitic
`acid) is attached via a glutamic acid linker to lysine
`at position 26. Second, lysine is replaced with argi
`nine at position 34, ensuring that the C16 side
`chain attaches only at position 26. The fatty acid
`chain allows reversible binding of liraglutide to
`albumin in the bloodstream, prolonging the action
`of liraglutide and increasing its resistance to degra
`dation by the DPP 4 enzyme, and thus avoiding
`renal elimination. The fatty acid chain also allows
`liraglutide molecules to self associate into heptamers
`at the injection site, delaying absorption from the
`subcutis ( 4) .
`
`Native
`Human GLP-1
`
`Liraglutide
`
`Figure 1 Amino acid structure of human glucagon like
`peptide 1 (GIP 1) and liraglutide. Amino acids that are
`shaded in the liraglutide molecule differ from those in the
`GIP 1 molecule. (Reprinted from Mol Cell Endocrinol.
`Russell Jones D, Molecular, pharmacological and clinical
`aspects of liraglutide, a once daily GIP 1 analogue, pages
`137 40, ©2009, with permission from Elsevier)
`
`Key findings from the liraglutide
`preclinical study programme
`
`Preclinical data, particularly from animal models of
`diabetes and obesity, have revealed the considerable
`therapeutic potential of liraglutide. Beneficial effects
`are apparent in terms of glycaemic control, weight
`loss, beta cell regulation and cardiovascular function.
`
`Glycaemic control and hypoglycaemia
`
`Background
`The cornerstone of current antidiabetic therapy is
`aggressive control of hyperglycaemia. Achieving and
`maintaining control are commonly frustrated by
`treatment related increases in the risk of hypoglyca
`emia and in body weight and by the continued
`decline in beta cell function. GLP 1 based therapies
`are attractive therapeutic options because the stim
`ulation of insulin secretion and suppression of
`glucagon release with human GLP 1 are glucose
`dependent (5), providing a degree of protection
`against hypoglycaemia. GLP 1 also impacts glycae
`mic control by slowing gastric emptying (6), thus
`reducing postprandial glucose excursions. The lira
`glutide preclinical programme examined its antihy
`perglycaemic and body weight lowering potential.
`Other available GLP 1 based therapies
`DPP 4
`inhibitors (sitagliptin, saxagliptin and vildagliptin)
`that enhance the actions of the incretin hormones
`and the GLP 1 receptor agonist exenatide that
`mimics endogenous GLP 1
`have shown glucose
`dependent antihyperglycaemic properties in preclin
`ical trials (7 9) and are included as comparators
`in some of the studies discussed below.
`
`Glycaemic control and hypoglycaemia with
`liraglutide
`Liraglutide showed potent, long lasting, and both
`dose
`and glucose dependent antihyperglycaemic
`effects in numerous animal models of diabetes and
`obesity.
`Mouse models. Ob/ob and db/db mice have
`increased body fat and insulin resistance compared
`with normal mice, with the severity of diabetes
`dependent on the age of the mouse. The increased
`body fat results from natural mutations in either the
`gene for leptin (ob/ob mice) or the leptin receptor
`(db/db mice) .
`Liraglutide showed a dose dependent and long
`lasting antihyperglycaemic effect in ob/ob mice (10) .
`The mean area under the curve (AUC) for blood
`glucose, a measure of glucose excursion, was signifi
`cantly lower after a single subcutaneous (s.c.) injec
`tion of liraglutide (30, 100, 300 or 1000 µg/kg) than
`
`© 2010 Blackwell Publishing Lid Int J Clln Pract, October 2010, 64 (Suppl. 167), 4 11
`
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`8
`
`Liraglutide: the therapeutic promise from animal models
`
`Weight loss
`
`Background
`A large proportion of patients with type 2 diabetes
`are overweight or obese, and it is estimated that even
`a modest weight loss of 1 kg will result in decreases
`in fasting plasma glucose of 0.2 mmol ⁄ l (15). In
`humans, GLP 1 enhances satiety, resulting in weight
`loss (16,17). The mechanism for GLP 1 induced low
`ering of body weight may involve both gastric emp
`tying and an effect in the brain.
`Importantly, preclinical data also reveal key differ
`ences between GLP 1 receptor agonists and DPP 4
`inhibitors in terms of their effects on body weight.
`Only GLP 1 receptor agonists mediate reduced food
`intake and weight loss (7,18,19). This corresponds
`with clinical data showing weight
`loss with the
`GLP 1 receptor agonists, but weight neutrality with
`the DPP 4 inhibitors*. Preclinical data further show
`that although the effects of GLP 1,
`liraglutide and
`exenatide on insulin release are glucose dependent
`(see earlier), their effects on appetite regulation are
`not (18).
`
`Liraglutide in normal rats and rat models
`The effects of liraglutide on food intake and body
`weight were investigated in normal rats. Investiga
`tions were also undertaken in a number of rat mod
`els:
`rats made obese by neonatal
`exposure
`to
`monosodium glutamate;
`female
`rats
`showing
`increased food intake, weight and fat gain, and
`impaired mean glucose tolerance after
`receiving
`olanzapine; candy fed rats showing increased calorie
`consumption; ZDF rats with insulin resistance
`(11,12,18 21). These studies showed that liraglutide
`reduced food intake, body weight, fat mass and glu
`cose tolerance. Body weight was lowered by a mecha
`nism involving mainly lowered energy intake, but
`also potentially altered food preference and main
`tained energy expenditure despite weight loss. The
`findings are exemplified below by the studies in
`candy fed and ZDF rats.
`The effects of liraglutide and vildagliptin on body
`weight and food intake were compared in candy fed
`rats (18). First, the rats were fed chow and supple
`mentary candy, which resulted in increased weight
`(mostly attributable to an increase in fat mass) and a
`slight increase in feeding associated energy expendi
`ture. Over
`the
`following 12 weeks, mean body
`weights returned to normal in rats who received lira
`glutide (0.2 mg ⁄ kg bid s.c.) alongside supplementary
`candy and also in rats reverting to a chow only diet.
`
`*McGill JB. Liraglutide: effects beyond glycaemic control in diabetes treatment. Int
`J Clin Pract 2010; 64 (Suppl. 167): 28 34.
`
`Most of the weight loss in the liraglutide group was
`attributable to a relative decrease in fat mass,
`assessed by dual energy X ray absorptiometry. As
`expected, there was no increase in plasma insulin
`levels in the liraglutide group to mediate the weight
`loss, as the effect of liraglutide on insulin release is
`glucose dependent (see earlier) and these rats were
`normoglycaemic. Instead, the weight loss seems likely
`to have resulted from the decreased calorie intake,
`with a shift in favour of chow over candy, and raised
`energy expenditure.
`In contrast,
`the vildagliptin
`(10 mg ⁄ kg bid orally) + supplementary candy group
`gained weight over the 12 week period, as did the
`group continuing to receive no treatment + supple
`mentary candy. Furthermore, whole body fat masses
`at end point were significantly higher in these two
`groups compared with the liraglutide + candy group.
`In a 6 week study of ZDF rats, animals receiving
`liraglutide (150 lg ⁄ kg bid s.c.) had a significantly
`reduced mean daily food intake compared with rats
`receiving vehicle (11). The increase in mean body
`weight was also significantly less for the liraglutide
`group compared with the
`vehicle
`group after
`10 days. Although the between group difference had
`disappeared by day 42, this reflected the reduced loss
`of calories attributable to glycosuria in the liraglutide
`group.
`In older and more overtly diabetic ZDF rats, treat
`(200 lg ⁄ kg bid s.c.)
`ment with liraglutide
`for
`6 weeks was associated with significantly decreased
`daily food intake and body weight and some reduc
`tion in fat depots compared with vehicle (12).
`
`Liraglutide in a minipig model
`Weight loss studies were also conducted in minipigs.
`Obesity and feeding behaviour in these animals more
`closely resemble those of humans than those of
`rodents. Pigs mainly eat in meals during the light
`period and do not eat in the dark period; they also
`show increases in body fat that resemble humans,
`which rodents do not unless they have a severe
`monogenic form of obesity. Liraglutide reduced food
`intake and body weight in severely obese, hyper
`phagic minipigs (22). Mean food intake was greatly
`reduced during 7 weeks of treatment with liraglutide
`(7 lg ⁄ kg qd s.c.) compared with pre and post treat
`ment periods (Figure 6A) and mean body weights
`were generally stable before treatment, but decreased
`during treatment (Figure 6B).
`
`Beta-cell regulation
`
`Background
`The progressive loss of beta cell function ultimately
`drives
`the continuing deterioration in glycaemic
`
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`10
`
`Liraglutide: the therapeutic promise from animal models
`
`bearing kidneys of animals treated with liraglutide
`was significantly lower than that in control mice after
`48 h (24). Liraglutide was also associated with a
`reduced time to achieve normoglycaemia and better
`long term glycaemic control than vehicle (24), sug
`gesting therapeutic potential for liraglutide in islet
`transplantation.
`Normal rats and rat models. In two 6 week studies
`of ZDF rats with insulin resistance and beta cell
`defects, there were no significant differences between
`liraglutide (30 or 150 lg ⁄ kg bid s.c.) and vehicle
`groups
`for beta cell proliferation (11). However,
`beta cell mass was significantly greater for two of the
`three liraglutide groups compared with respective
`vehicle controls. Importantly,
`in non diabetic rats,
`the mean beta cell mass was significantly greater in
`the group treated with liraglutide 200 lg ⁄ kg bid s.c.
`for 1 week than in the group receiving vehicle, but
`beta cell mass was normalised after a total of 6 weeks
`of treatment (20), suggesting that effects in normo
`glycaemic animals are temporary.
`In vitro work. The stimulatory effect of liraglutide
`was demonstrated in a study of primary monolayer
`cultures of newborn rat islet cells incubated with
`GLP 1, glucose dependent insulinotropic polypeptide
`or liraglutide for 24 h (25). The incorporation of
`bromodeoxyuridine, a measure of DNA synthesis,
`was 50 80% above the basal rate in all cases. A GLP
`1 receptor mechanism was implicated as the effect
`was blocked with the antagonist exendin(9 39).
`An inhibitory effect on beta cell apoptosis in vitro
`was apparent in two separate studies (26,27). Liraglu
`tide had a dose dependent protective effect on cyto
`kine and free fatty acid mediated beta cell apoptosis
`in primary neonatal rat islets, and the effect was sig
`nificantly better for liraglutide than native GLP 1
`(26). Again, a GLP 1 receptor mechanism was impli
`cated as the effect was blocked with exendin(9 39).
`Liraglutide also reduced beta cell apoptosis in por
`cine islets in culture prior to transplantation (27).
`The rate of beta cell apoptosis at 24 h in cultures
`incubated with liraglutide was halved compared with
`cultures incubated with vehicle.
`
`Cardiovascular function
`
`Background
`As GLP 1 receptors are expressed in the heart, vas
`cular smooth muscle and regions of the central ner
`vous system that regulate the cardiovascular system,
`there is clinical interest in GLP 1 based therapies for
`patients with diabetes who are in poor cardiovascu
`lar health. However, there are currently few preclini
`cal
`studies
`into the effects of GLP 1, and the
`mechanisms underlying the effects are poorly under
`
`stood. To date, preclinical studies have shown that
`GLP 1 receptor agonists have cardioprotective effects
`and reduce blood pressure in hypertensive rats
`(23,28 31). Studies also show that both GLP 1 and a
`metabolite, GLP 1(9 36), mediate effects via GLP 1
`receptors and also independently of them (32).
`
`Cardioprotective effects with liraglutide
`The most extensive preclinical investigation with lira
`glutide has been conducted in a mouse model of
`myocardial infarction (33). Pretreatment with liraglu
`tide [200 lg ⁄ kg intraperitoneally (i.p.)] for 7 days
`increased cardiomyocyte survival and improved car
`diac function compared with controls. These effects
`were not an indirect effect of weight
`loss, as
`increased survival was also apparent at a weight
`neutral lower dose (75 lg ⁄ kg i.p.).
`
`Conclusions
`
`studies have
`series of preclinical
`An extensive
`revealed considerable potential benefits for liraglutide
`in the treatment of type 2 diabetes. Good glycaemic
`control was mediated by the glucose dependent stim
`ulation of insulin and suppression of glucagon secre
`tions,
`and also by delayed gastric
`emptying.
`Moreover,
`liraglutide showed positive effects on
`beta cell preservation and mass and on cardiac func
`tion. Although these benefits were also apparent with
`other incretin based therapies, exenatide and liraglu
`tide stand apart in offering the potential to reduce
`body weight, a key concern in the management of
`type 2 diabetes. Of these two agents,
`liraglutide is
`longer acting than exenatide and is dosed only once
`daily compared with twice daily for exenatide. Clini
`cal data indicate that liraglutide has a better glycae
`mic efficacy than exenatide despite the once daily
`dosing (see Raskin and Mora in this supplement*).
`
`Acknowledgements
`
`The author takes full responsibility for this article,
`but is grateful to Christine Deakin, DPhil, of Water
`meadow Medical (supported by Novo Nordisk Inc.)
`for writing assistance.
`
`Author contributions
`
`The outline and drafts were developed in conjunc
`tion with the author, who approved the final version
`before submission.
`
`*Raskin P, Mora PF. Glycaemic control with liraglutide: the phase 3 trial
`programme. Int J Clin Pract 2010; 64 (Suppl. 167): 21 7.
`
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`Liraglutide: the therapeutic promise from animal models
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`
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`Paper received June 2010, accepted June 2010
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`MPI EXHIBIT 1066 PAGE 8
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