CLINICAL PRACTICE
`REVIEW PAPER
`Liraglutide: the therapeutic promise from animal models
`
`@ TH[ INTERNATIONAL JOIJRNALOF
`
`L B. Knudsen
`
`Novo Noml,k A/ S, Novo
`Nordisk Park, DK 2160, Malm,
`Denmark
`
`Corresponden<e to:
`Lotte Bjerre Knuruen,
`Depanment Biology and
`Phannacology Mgt, Novo
`Nord"tsk A/ S, Novo No!1fi51t
`Park, ~ 1011, DK 2760 Denmark
`Tel.: t454443 4788
`Fa>e +45 3075 4788
`Email: lbkn®novonord~k.mm
`
`Disclo,ures
`This article fonns part of a
`Sllj)p~mei1 1 funded by NOVO
`Nord'~k.
`LBK is an employee of Novo
`Nordisk, The aurlor was part
`of the t£am that discovered
`and developed li1a,lutlde. All
`patents naming the author· as
`an inventor belong to Novo
`Nordisk; tl1e audio, has no
`financial interest re lated to any
`patents. LBK has .hares in
`Novo Nord~k as part ol an
`emploiee offer mg program me .
`
`SUMMARY
`
`Aims: To review 1he differences between !he human glucagon-like peptide-1 (GLP·
`1) molecule and the analogue liraglutide, and to summarise key data from the Hra(cid:173)
`glutide preclinical study programme showing the therapeutic promise of thrs 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,
`hoWe\/€r,
`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 g~caemic control, mediated by the glucose-depen(cid:173)
`dent stimulation of insulin and suppression of glucagon secretion and by delayed
`gastric emptying. Liraglulide also had positive effects on body weigh~ beta-cell
`preservation and mass, arid cardiac function. Conclusions: The therapeutic prom(cid:173)
`ise of liraglutide is evident from preclinical data. Liraglutide showed the potential
`to provide good glycaemlc control without increasing the risk. of hypog~caemia
`and, as with exenatide, but not dipepticfyl peptidase-4
`inhibitors, to mediate
`weight loss-. Although these benefits have subsequent~ been studied clinically,
`beta-cell mass can be directly studled only in animal models. In common with
`other lncretin-based therapies, litilglutide showed the potential to modulate the
`progressive loss of beta-cell funclion that drives the continuing deterioration in gly(cid:173)
`Giemlc control In patients with type 2 diabetes. Body Weight was lowered by a
`mechanism involving mafnly lowered energy intake, but also potentially altered
`food preference and maintained e~ergy expenditure despite weight loss,
`
`Introduction
`
`As explained by Dr Unger in th.is supplement , the
`therapeutic po1:ential of inaetin honnones for the
`treatment of type 2 diabetes is the subject of consid
`erable ongolng research. Intel'l'st has focused on the
`incretin glucagon like peptide 1 (GLP 1) in partiru
`lar and has resulted in two new classes of antihyper
`.glycaem.ic agents: the dipeptidyl peptidase 4 (DPP 4)
`inhibitors and the GLP 1 receptor agonists. Li.raglu
`tide, which belongs to the latter class of agents, is
`one of only two commercially available GLP 1 recep
`tor agonists. Llraglutide is approved for use in com
`bination with selected oral agents (Europe a-nd USA)
`and as rnonothcrapy (USA and fapan}.
`[n this firs t review of the supplement, differences
`between the human GLP l and li.raglutide molecules
`are discussed and key data from the lira1:1lutide pre
`clinical program.me are summarised. Preclinical work
`
`"linger J. Ltagl1.11ide: tan ft make a difference in the treatment of type 2 diabetes/
`Wit J an Praa 2010: 64 (S uppl. 167): 1 3.
`
`is a necessary precursor to human stud ies: assessment
`of toxicity in a preclinical setting is a legal require
`ment, bu t prec.lin.ical studies also gather important
`preliminary data on ph:mnacolci-netics and pharma
`codynarnics. A large body- of data in any preclinical
`-programme is generated from in vitro investigation.;
`however, normal animals and animal models of the
`disease state allow us to examine the effects of a new
`drug on the comple..-...: 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 subsequ ent clinical trials, not least
`because animal modl'ls nmain the only way to
`e..."\.-µlore directly the mechanistic effects of therapy on
`different organs and tissues, such as beta cells. Ani
`mal stuclies are also essential to explore novel effects
`of a diabetes drug, such as a direc;t effect on cardiac
`function. Throughout this review, key findings fm
`other incretia based therapies and GLP 1 a.re noted
`to provide context for the liraglutide data, and the
`
`4
`
`©> 2010 Blackwell Publigiing Ltd Int 1 Clin Pract, October 2010, 64 (Suppl. 167), 4 11
`doi: 10. 11 l llj .1 742 124 1.2010.02499 .x
`
`MPI EXHIBIT 1066 PAGE 1
`
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`
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`

`Liraglutide: the therapeutic promise from animal models
`
`5
`
`reader is referred to eXIstrng 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 I). First, a Cl6 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 Cl 6 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 l (GIP I) and liraglutide. Amino acids that are
`shaded in the liraglutide molecule differ from those in the
`GIP l molecule. (Reprinted from Mo/ Cell Endocrinol.
`Russell Jones D, Molecular, pharmacological and clinical
`aspects of liraglutide, a once daily GIP I 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 stun
`ulation of insulin secretion and suppression of
`glucagon release with human GIP l are glucose
`dependent (5), providing a degree of protection
`against hypoglycaemia. GLP 1 also impacts glycae
`rnic 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 Ltd Int J Clln Pract, October 2010, 64 (Suppl. 167), 4 11
`
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`
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`

`6
`
`liraglutide: the therapeutic promise from ariimal models
`
`tbat after a single injection of vehicle. Moreover,
`24 h after injection, blood glucose levels were still
`significantly lower than they were for controls. To
`investigate the effects of 24 b dosing in the same ani
`mal model, liraglutide was administered twice daily
`(bid) for 2 weeks. This contrasts with once daily dos
`ing in humans because the half life of liraglutide is
`much longer in humans than in rodents. In this 24 h
`dosing study, blood glucose AUCs were significantly
`reduced with 100 ~1g/kg of liraglutide compared with
`vehicle at all assessment points (days 1, 8 and 15).
`Mean plasma insulin levels were also 60% higher in
`the liraglutide group than those in the vehicle group
`after 2 weeks.
`in
`Antihyperglycaemic effects were confirmed
`db/db mice
`treated for 15 days with
`liraglutide
`(200 µg/kg bid s.c. ) or vehicle (bid s.c.) (10). How
`ever, a shorter duration of effect was observed with
`the active comparator, exenatide (100 µg/kg bid
`s.c.). Furthermore, blood glucose levels in the exena
`tide group were similar to those in the vehicle group
`IO 12 h after dosing, whereas they were maintained
`throughout the 24 h monitoring period in the lira
`glutide group (Figure 2). The selected dose for exe
`natide was high in this study to facilitate a fair
`comparison. Normally, liraglutide is dosed 50 times
`higher than exenatide because most of the drug is
`bound to albumin.
`Rat models. Liraglutide showed antihyperglycaemic
`effects in two rat models of diabetes: younger Zucker
`diabetic fatty (ZDF) rats showing insulin resistance
`without hyperglycaemia, and older, more overtly dia
`betic ZDF rats generally considered to be models for
`treatment resistant type 2 diabetes.
`Younger ZDF rats were treated for 6 weeks with
`liraglutide (30 and 150 µg/kg bid s.c.) or vehicle
`
`QJ
`
`§
`is20 +
`"8~
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`15
`5
`25
`0
`Tim e (h)
`-•- Li raglutide
`
`- - Vehic le
`
`··• -- Exenatide
`
`Figure 2 Blood glucose levels in dbl db mice receiving
`twice daily subrutaneous administration of either
`liraglutide 200 µg/kg, exenatide 100 µg/kg or vehicle ( IO).
`Data are for day l, time 0 is at 9.00 AM and arrows
`indicate injection times; n = 10 per group. (Adapted from
`Rolin B et al. Am J P/iysiol Et1docri11ol Metab 2002; 283:
`E745 52. Reproduced with permission; conveyed through
`Copyright Oearance Center, Inc. )
`
`(bid s.c.) (11). After glucose challenges on days 21
`and 36, blood glucose levels were markedly lower for
`the liraglutide groups than for the vehicle group
`(Figure 3). Mean AUCs for blood glucose were sig
`.nificantly different among groups: lowest with the
`higher dose of liraglutide, intermediate with the
`lower dose of liraglutide and highest with vehicle.
`Dose dependent effects were also apparent for mean
`AUCs for plasma insulin after glucose challenge. In
`addition, although
`the average
`insulin AUCs
`increased by 66% between the first and second glu
`cose challenges with the higher dose of liraglutide,
`AUCs decreased by 40% with vehicle. After 41 days
`of treatment, glucose and insulin AUCs for the
`higher dose liraglutide group were significantly lower
`than corresponding AUCs for the lower dose liraglu
`tide and vehicle groups. Pair feeding, in which the
`daily food consumption for each ZDF rat in the lira
`glutide group was measured and then made available
`to a rat in the pair fed group (matched on the basis
`of initial body weight), showed that approximately
`53% of the antibyperglycaemic effect on 24 h glucose
`profiles was mediated by a reduction in food intake.
`Liraglutide retained some efficacy even in the older
`ZDF rats (12,13). In the 6 week study by Larsen
`et al, for example, a number of measures of plasma
`glucose were significantly reduced with liraglutide
`(200 µg/kg bid s.c.) , but there were few differences
`between liraglutide and vehicle groups for measures
`of plasma insulin (12). Combination therapy with
`liraglutide and pioglitawne, however, synergistically
`
`""A
`125
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`40
`80
`120
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`40
`80
`120
`~
`Time (m in)
`Time (min)
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`-+- Vehicle
`-•- Liraglutide
`30 µglkg bid
`150 µg/kg bid
`
`C
`
`i
`~
`
`Figure 3 Blood glucose levels after glucose challenge on
`(A) day 21 and (B) day 36 in fasted Zucker diabetic fatty
`rats ( 11 ). Rats were receiving subrutaneous injection of
`liraglutide (at one of two dose levels) or vehicle. Glucose
`was administered by gavage at time O. Areas under the
`curve for blood glucose were significantly different for the
`three treatment groups (p < 0.0005 and 0.0002 for days 21
`and 36, respectively). n = 6 per group. Bid, twice daily.
`(Adapted from Sturis J et al. Br I P/iarmacol 2003; 140:
`123 32. Reproduced with permission from Wiley
`lnterscience)
`
`© 20 10 Blackwell Publishing Ltd Int 1 Clin Praet, October 2010, 64 (Suppl. 167), 4 11
`
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`
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`
`

`

`70
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`••◊•• Piog litazone + veh icle (PV)
`-□- Li rag lutide + vehicle (LV)
`-• ·· Li rag lutide + piog litazone (LP)
`
`Liraglutide: the therapeutic promise from animal models
`
`7
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`
`Figure 4 Blood glucose levels and areas under the crnve
`(AUG) in Zucker diabetic fatty rats after glucose challenge
`on day 42 (12). Rats received twice daily vehicle, liraglutide
`(200 µg/kg), pioglitazone (5 mg/kg) or a combination of
`liraglutide and pioglitazone. Gluco e was administered at
`time 0. ~ttp < 0.001 vs. vehicle; n = IO per group.
`(Adapted from Larsen PJ et al. Combination of the insulin
`sensitizer, pioglitazone, and the long acting GLP 1 human
`analogue, liraglutide, exerts potent synergistic glucose
`lowering efficacy in severely diabetic ZDF rats. Diabetes
`Obes Metab © 2008, with pennission from Wiley
`Blackwell)
`
`at week 6
`
`improved glycaemic control even
`(Figure 4).
`Minipig model. Studies of the efficacy of liraglutide
`were conducted in minipigs made diabetic with
`streptozotocin (14). Minipigs and humans have simi
`lar skin, and injectable componnds therefore display
`similar phannacokinetics. Short term investigations
`showed that liraglutide has a glucose dependent anti
`hyperglycaemic effect, whereas longer
`term studies
`showed that liraglutide reduces gastric emptying and
`improves glucose tolerance and insulin sensitivity
`(14).
`A group of minipigs receiving intravenous (i.v.)
`liraglutide
`(2 µg/kg)
`required almost a 200%
`increase in the glucose infusion rate compared with
`the vehicle group during a hyperglycaemic clamp,
`and even then bad slightly lower blood glucose levels
`(Figure 5). Mean AUCs for plasma
`insulin were
`markedly higher and mean AUCs for plasma gluca
`gon markedly lower during the clamp for the liraglu
`tide group than for the vehicle group. Moreover,
`data from animals under a hyperglycaemic clamp
`and animals receiving a low dose glucose infusion
`showed that the correlations between insulin and
`glucagon values were linear for minipigs receiving
`liraglutide or vehicle. However, the steeper slope with
`liraglutide indicated
`that responses were glucose
`dependent. Importantly, at low glucose levels, plasma
`
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`
`80
`
`100 120 140
`
`20
`
`40
`60
`Time (min)
`- •- Lirag lut ide
`
`Figure 5 (A) Glucose infusion rates during and (BJ plasma
`glucose levels before, during and after a hyperglycaemic
`clamp in fasted minipigs (1 4). Minipigs received either
`liraglutide or glucose intravenously (i.v.) before the damp.
`Animals treated with liraglutide required a significantly
`greater glucose in.fusion rate (p < 0.005), and still had
`slightly lower plasma glucose levels, than animals
`receiving vehicle. n = 6. (Adapted from Ribel U et al.
`Eur J Phannacol 2002; 45 I: 217 25. Reprinted from Eur J
`P/111rmacol., Ribel U et al., NN221 l: a long acting glucagon
`like pep tide I de riv ati ve with an ti diabetic effects in
`glucose intolerant pigs, pages 217 25, © 2002, with
`permission from Elsevier)
`
`gl.ucagon concentrations rose, contributing to the
`maintenance of norrnoglycaemia.
`In a chronic dosing study, minipigs received either
`liraglutide [3.3 µg/kg s.c. once daily (qd)] or vehicle
`for 4 weeks (14). The liraglutide group had signifi
`cantly reduced gastric emptying (assessed as the AUC
`for paracetamol) and iniproved glucose tolerance at
`2 and 4 weeks compared with the vehicle group. Fur
`thermore, insulin sensitivity (assessed as glucose to
`insulin ratio) improved for the liraglutide group dur
`ing the study, but was unchanged in the vehicle
`group.
`
`© 2010 Blackwell Publishing Lid Int 1 Gin 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
`
`ª 2010 Blackwell Publishing Ltd Int J Clin Pract, October 2010, 64 (Suppl. 167), 4 11
`
`
`
`e
`
`MPI EXHIBIT 1066 PAGE 5
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1066-0005
`
`

`

`Liraglutide: the therapeutic promise from animal models
`
`9
`
`Liraglutide treatment
`
`- - - - - - - - - - - - - - - - -
`
`-49
`
`-39
`
`-29
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`-9
`
`41
`
`51
`
`61
`
`71
`
`81
`
`91
`
`11
`
`31
`2 1
`Time (day)
`
`Liraglutide treatment
`
`-49
`
`- 39
`
`- 29
`
`- 19
`
`- 9
`
`11
`
`21
`31
`Time (day)
`
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`
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`61
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`71
`
`81
`
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`
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`
`Figure 6 (A) Mean fuod intake and (B) weight gain in severdy obese, hyperphagic minipigs befo.e, during and after
`7 weeks of treatment with liraglutide (7 ~•g/kg once daily administered subrutaneously) (22). The horizontal dotted lines
`represent the mean food intake (A) for the maintenance of normal body weight and the body weight (B) at the start of
`treatment; 11 = 6. (Figure previously published in Raun Ket al Obesity 2007; 15: 1710 6)
`
`control and thus repeated therapy failure. rn the
`absence of rel.iable non invasive measures of beta cell
`regulation in humans, our understanding of the
`effects of GLP l and related therapies are necessarily
`shaped by in vitro and preclinical in vivo investiga
`tions and by surrogate clinical measures. Research
`with GLP l indicates that it has a positive influence
`on beta cell mass via a combination of increased
`beta cell neogenesis and proliferation, and reduced
`beta cell apoptosis (23). Preclinical studies with exe
`natide suggest it has a similar profile of positive
`effects (7).
`
`Beta-cell regulation with liraglutide
`Liraglutide increases beta cell mass in hyperglycae,mic
`animals, but bas temporary or no effects in normo
`glycaemic animals (10,l 1,20). Liraglutide has also
`been shown to have an inhibitory effect on beta cell
`apoptosis after syngeneic mouse transplants (24).
`Both the stimulatory and inhibitory effects were
`apparent in in vitro studies (25 27).
`Mouse models. In ob/ ob mice, liraglutide treatment
`(100 µg/kg bid s.c. for 15 days) had no effect on
`beta cell mass, but there was a tendency for beta cell
`proliferation to be increased in the liraglutide group
`compared with the vehicle group (10). In db/ db
`mice, both the mean beta cell proliferation rate and
`the mean beta cell mass were significantly increased
`in the liraglutide group (200 µg/kg bid s.c.) com
`
`pared with the vehicle group after 15 days of treat
`ment (10) (Figure 7). In contrast, only the beta cell
`proliferation rate was significantly greater in the exe
`natide group (100 µg/kg bid s.c.), most likely illus
`trating the shorter duration of action.
`Liraglutide (200 µg/kg s.c. bid) has also been
`administered after a marginal mass syngeneic islet
`transplant in streptozotocin induced diabetic BALB/ c
`mice. The rate of beta cell apoptosis in the graft
`
`2.0
`
`T
`
`0
`
`Beta -ce ll
`proliferation rate
`
`■ Vehicle
`
`Liraglutide
`
`Beta -ce ll
`mass
`D Exenatide
`
`Figure 7 Beta cell proliferation rate and beta cell mass
`after 15 days of twice daily subcutaneous injection with
`liraglutide (200 µg/kg), exenatide ( 100 µg/kg) or vehicle in
`remale db/ db mice (10). (Adapted from Rolin Bet al. Am J
`Pliysiol Erulocrinol Metab 2002; 283: E745 52. Reproduced
`with permission; conveyed through Copyright Clearance
`Center, Inc. •p < 0.05; •••p < 0.001. BrdU,
`bromodeoxyuridine. 11 = 10 per group)
`
`© 2010 Blackwell Publishing Lid Int 1 Gin Pract, October 2010, 64 (Suppl. 167), 4 11
`
`MPI EXHIBIT 1066 PAGE 6
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1066-0006
`
`

`

`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 ph