`Copyright C 2001 PROUS SCIENCE
`CCC: 0377-8282/2001
`
`GLP-1 derivatives as novel compounds
`for the treatment of type 2 diabetes:
`selection of NN2211 for clinical development
`
`Review Article
`
`L. Bjerre Knudsen *, H. Agerso, C. Bjenning,
`S. Bregenholt, R.D. Carr, C. Godtfredsen,
`J.J. Holst+, P.O. Huusfeldt, M.O. Larsen,
`P.J. Larsenlf, P.F. Nielsen, U. Ribel, B. Rolin,
`J. Romer, J. Sturis, M. Wilken and
`P. Kristensen
`Novo Nordisk, Novo Park, DK-2760 Maaloev, Denmark;
`+Dept. of Medical Physiology, University of Copenhagen,
`DK-2200 Copenhagen, Denmark; ' Present address:
`Lab. Obesity Research, CCRB, DK-2760 Ballerup, Denmark.
`• Correspondence
`
`found to also lower plasma glucagon in a glucose-depen(cid:173)
`dent manner, decrease the rate of gastric emptying, pro(cid:173)
`mote fullness/satiety and stimulate insulin biosynthesis,
`as well as proliferation of ~-cells. All of these effects taken
`together position GLP-1 as an obvious drug candidate for
`the treatment of type 2 diabetes. Apart from the food
`intake lowering effect, the glucagonostatic effect is partic(cid:173)
`ularly interesting as type 2 diabetic patients are charac(cid:173)
`terized by increased plasma glucagon levels, which again
`lead to an increased hepatic glucose output (4, 5).
`is found as both
`The natural hormone GLP-1
`GLP-1 (7-37) and GLP-1 (7-36)amide. It is metabolized by
`dipeptidyl peptidase IV (DPP-IV) and rapidly cleared by
`the kidneys (6, 7). The primary metabolite, GLP-1 (9-37)
`or GLP-1 (9-36)amide, may even act as an antagonist (8) .
`The plasma half-life of GLP-1 after i.v. administration is
`less than 2 min and 1-2 h after s.c. administration (9). The
`dynamic half-life of GLP-1 has been estimated to be 3 h
`(10). Thus, the natural hormone is not very useful as a
`drug . No small-molecule agonists have been described
`for any members of the glucagon-secretin G-protein cou(cid:173)
`receptor
`pled receptor family, to which the GLP-1
`belongs. Thus, in order to make a GLP-1-based drug, one
`must either make a protracted formulation of the natural
`hormone, design analogs or derivatives with improved
`pharmacokinetic properties or find a non-peptide agonist.
`This review describes the biological aspects of GLP-1
`and gives a detailed description of a series of GLP-1
`derivatives designed for once-daily administration.
`
`Mechanism of action of GLP-1
`
`GLP-1 is an incretin , responsible for the difference in
`blood glucose levels obtained after oral as compared to
`
`CONTENTS
`
`. .... . .. . ................. ...... . .. 677
`Introduction
`Mechanism of action of GLP-1 ..... ... . . . . .. . ...... 677
`Desired pharmacodynamic profile of an effective
`GLP-1 drug
`. . ... .. ... .. ....... . .. ......... . . . 679
`Derivatives of GLP-1 for once-daily administration .. .. . . 679
`Site of acylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 680
`. . . . . . . . . . . . . . . 680
`Compounds derivatized on lysine 26
`Compounds derivatived with different spacers ..... .. . 680
`Biological characterization of NN2211
`. .. ... . . . ..... 681
`Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682
`References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682
`
`Introduction
`
`Current drugs for treatment of type 2 diabetes are
`classified into four major categories: insulin secreta(cid:173)
`gogues (sulfonylureas and the shorter acting glinides),
`biguanides, insulin sensitizers and insulin (1 ). The first
`three groups are orally active but are of limited efficacy.
`Insulin is very effective but has to be dosed four times
`daily for optimal efficacy, thus presenting the risk of seri(cid:173)
`ous hypoglycemia to the patient. Furthermore, type 2 dia(cid:173)
`betes is most commonly associated with obesity, and of
`the four drug groups listed, only the biguanides do not
`cause increases in body weight. There is, therefore, an
`increasing awareness of the need for new efficacious
`drugs for the treatment of type 2 diabetes, especially
`drugs that do not lead to an increase in body weight.
`Glucagon-like peptide-1 (GLP-1) is a peptide hormone
`derived from proglucagon which was discovered in 1983
`(2, 3). GLP-1 was first described as an incretin, a sub(cid:173)
`stance released from the gastrointestinal system upon
`ingestion of food, promoting glucose-dependent insulin
`release from the pancreatic ~-cells. Later, GLP-1 was
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`GLP-1 derivatives for the treatment of type 2 diabetes
`
`intravenous administration of the same amount of glu(cid:173)
`cose (11-14). Upon ingestion of a meal, GLP-1
`is
`released from the L-cells in the intestine and stimulates
`insulin release via specific receptors on pancreatic
`13-cells.
`The GLP-1 receptor was cloned in 1992 (15) and has
`been shown to be present in numerous tissues with the
`highest expression levels in the 13- and &-cells of the pan(cid:173)
`creas and in the lungs (16). Only one subtype is known.
`The receptor has also been found in parts of the gas(cid:173)
`trointestinal tract and in many regions in the CNS, includ(cid:173)
`ing the hypothalamic regions, as well as specific areas in
`the brain stem.
`Equally important to the insulinotropic action, GLP-1
`also potently inhibits glucagon secretion (17-20). The
`mechanism by which GLP-1 exerts its glucagonostatic
`effect is not fully understood. It has been suggested to be
`paracrine, via neighboring somatostatin cells. Because of
`the combination of increased insulin and decreased
`glucagon secretion, hepatic glucose production is effi(cid:173)
`ciently decreased (21 ). In spite of the insulinotropic effect,
`GLP-1 infusions carried out during ingestion of a meal
`actually result in diminished insulin responses. This is
`due to an inhibition of gastric emptying effectively reduc(cid:173)
`ing the delivery rate of ingested nutrients to the absorp(cid:173)
`tive segments of the GI tract (22).
`GLP-1 has been investigated in a substantial number
`of small clinical trials and has been shown to efficiently
`lower blood glucose in patients with type 2 diabetes and
`also in patients with poor metabolic control, referred to as
`secondary failures to sulfonylurea treatment (9-14).
`GLP-1 is also capable of lowering blood glucose in type 1
`diabetic patients without residual beta-cell secretory
`capacity (23, 24), reflecting its effects on lowering of plas(cid:173)
`ma glucagon and inhibition of gastric emptying. Indeed,
`glucagon antagonism alone has been suggested as an
`alternative treatment for type 2 diabetes but no drugs
`have reached the market yet.
`One study has suggested that the effect of GLP-1 can
`decline overnight, perhaps as a result of receptor desen(cid:173)
`sitization (25). However, these results are in contrast to
`another study which showed that a 7-day infusion of
`GLP-1 lowered blood glucose levels for the entire dura(cid:173)
`tion of the study (26, 27).
`Because of the glucose-dependent nature of the
`insulinotropic and glucagonostatic actions of GLP-1, the
`glucose lowering effect of this hormone is self-limiting
`and, therefore, it does not cause serious hypoglycemia
`regardless of dose (28). The mechanism underlying the
`glucose dependency is closely related to the glucose
`signaling events being dependent on a high ATP/ADP
`ratio (29). Numerous studies in the literature, where
`GLP-1 has been infused or injected subcutaneously in
`humans, support the glucose dependency of GLP-1 's
`actions (9-14, 23, 25). However, there are some reports
`that GLP-1 can lower blood glucose to below normo(cid:173)
`glycemia (30, 31 ). This ability of GLP-1 to transiently
`lower blood glucose to subnormal levels is a natural con(cid:173)
`sequence of its effect on insulin secretion. The inactiva-
`
`tion time for insulin is considerable, and after GLP-1 stim(cid:173)
`ulation there may be enough insulin around (and enough
`activated insulin receptors) to lower blood glucose tem(cid:173)
`porarily, even if secretion of new insulin has ceased
`because of the falling glucose levels. The overall conclu(cid:173)
`sion, therefore, is that GLP-1 can cause blood glucose to
`decrease temporarily to below normal, but never leads to
`profound and lasting hypoglycemia.
`Apart from its direct effect on insulin secretion, GLP-1
`has been shown to increase the rate of insulin biosynthe(cid:173)
`sis (32, 33) and to restore the failing ability of the beta-cell
`to respond to glucose in old rats (34-36). Thus, it is spec(cid:173)
`ulated that GLP-1 may prevent the transition from IGT to
`full-blown diabetes (37), presumably because 13-cell func(cid:173)
`tion is preserved. Also, several publications now show a
`direct effect of GLP-1 compounds on growth and prolifer(cid:173)
`ation of 13-cells in animals (38-40), as well as an ability to
`prevent apoptosis in 13-cells (41). GLP-1 has even been
`found to be able to cause differentiation of pancreatic
`stem cells into functional 13-cells (42).
`GLP-1 has also been reported to exert peripheral
`effects by promoting glucose uptake and glycogen
`storage in fat cells, skeletal muscle and liver cells in rats
`(43). A few studies in humans have confirmed that there
`may be a peripheral effect (44, 45) while others have not
`(46, 47). Most likely, if this effect exists, it is quantitatively
`unimportant.
`All of the effects described above are aimed at direct(cid:173)
`ly lowering blood glucose. However, GLP-1 in peripheral
`circulation has also been shown to lower food intake in
`both rodents and humans, indirectly leading to improved
`glucose control via loss of body weight (48-53). Several
`receptors may be involved in the food intake lowering
`effect. First, centrally acting GLP-1 has been suggested
`to have a role as a physiological satiety factor (53-57) via
`receptors localized in arcuate hypothalamic areas.
`However, it has been shown that these receptors are
`probably not involved in mediating the food intake lower(cid:173)
`ing effect of peripherally administered GLP-1
`(52).
`Binding sites in the area postrema and the subfornical
`organ have been shown to be accessible for GLP-1 in the
`peripheral circulation, and these represent likely sites for
`mediating the food intake effect of peripheral GLP-1 com(cid:173)
`pounds (58). As mentioned above, GLP-1 inhibits gastric
`emptying, and the vagus nerve has been shown to be
`involved in mediating this effect (22, 59-62). The areas in
`the brainstem containing the GLP-1 receptors are known
`to receive afferent inputs from gastrointestinal organs.
`Thus, the most likely mechanism for the food intake low(cid:173)
`ering effect of a peripherally administered GLP-1 com(cid:173)
`pound that does not pass the blood-brain barrier is that a
`sensation of fullness is obtained via small amounts of
`receptor in the intestine which then projects via the vagus
`nerve to receptors in the area postrema and the subforni(cid:173)
`cal organ or directly via receptors in the blood-brain bar(cid:173)
`rier free areas of the CNS.
`Obese subjects have been shown to have an attenu(cid:173)
`ated GLP-1 release in response to meals (63, 64), sug(cid:173)
`gesting that a decreased peripheral GLP-1 signal may
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`contribute to the development of obesity. Also, type 2 dia(cid:173)
`betes patients have been shown to have a reduced GLP-
`1 release in response to food ingestion (65) and they
`have a defect in their response to the other incretin hor(cid:173)
`mone, GIP (11 ). Thus, there is a very good rationale for
`treating type 2 diabetes and obesity with a GLP-1-based
`drug as a replenishment treatment.
`A significant inhibitory effect of GLP-1 on water intake
`has been observed together with profound stimulation of
`diuresis in rats (54). The profound diuretic response to
`is due to natriuresis.
`acute GLP-1 administration
`However, chronic administration of a GLP-1 derivative
`was shown not to lead to disturbed water homeostasis in
`rats (52). In humans, GLP-1 does not acutely affect water
`homeostasis (66).
`Last, GLP-1 potently inhibits pentagastrin and meal(cid:173)
`induced gastric acid secretion and pancreatic enzyme
`secretion (59, 60). Along with the inhibited gastric empty(cid:173)
`ing, these effects of GLP-1 are most likely mediated
`vagally (61). The effect of GLP-1 gastric acid secretion
`may also point to a role for GLP-1 in protection against
`gastric ulceration.
`A recent very comprehensive review has a thorough
`description of the biology of GLP-1 (67).
`In conclusion, the mode of action of GLP-1 seems to
`be ideal for the treatment of type 2 diabetes, especially
`obese type 2 diabetes patients. GLP-1 compounds glu(cid:173)
`cose-dependently stimulate insulin secretion and inhibit
`glucagon secretion, decrease gastric emptying and medi(cid:173)
`ate increased fullness and/or appetite, and directly stimu(cid:173)
`late growth and rescue of pancreatic ~-cells. With this
`important spectrum of effects of GLP-1, it is conceivable
`that a GLP-1-based compound could potentially be more
`effective than any of the current blood glucose lowering
`drugs available today.
`
`Desired pharmacodynamic profile
`of an effective GLP-1 drug
`
`Several studies have shown that GLP-1 can normal(cid:173)
`ize blood glucose in patients and remains very effective in
`the so-called sulfonylurea failure patients, but it has a
`very short duration of action. It has been suggested that
`repeated subcutaneous administration of GLP-1 (9-10,
`25) or buccal tablets (68) might be an effective treatment.
`However, a study by Larsen et al. (26) shows that a long
`dynamic half-life is needed in order to maintain good glu(cid:173)
`cose control on a GLP-1-based drug. In this study,
`patients were administered GLP-1 by infusion for either
`16 h or 24 h a day for 1 week. Patients in the 24-h infu(cid:173)
`sion group had a much better glucose control than those
`in the 16-h infusion group (26). In patients in the 16-h infu(cid:173)
`sion group, blood glucose rapidly reverted to preinfusion
`levels after GLP-1 infusion. Thus, the difference between
`these two groups strongly indicates that GLP-1 has to be
`present in an effective dose at all times. At least three
`daily administrations of natural GLP-1 by buccal or s.c.
`administration would be needed in order to obtain this
`kind of pharmacodynamic profile.
`
`As mentioned above, GLP-1 is metabolized rapidly
`by DPP-IV and the major metabolite, GLP-1 (9-36)amide
`or GLP-1 (9-37) is inactive or may even act as an antago(cid:173)
`nist (8). Thus, analogs of GLP-1 stabilized against meta(cid:173)
`bolic breakdown have been proposed as possible drugs
`(69-72). However, as GLP-1 is also cleared very rapidly
`from the kidneys, such drugs would only have half-lives of
`around 5 min after i.v. administration (69). Even though
`s.c. administration would improve the dynamic half-lives
`of such compounds, probably to a range of 3-5 h (72, 73),
`they still require a tremendous formulation challenge if
`multiple daily dosing is to be avoided.
`The only known side effect of GLP-1 treatment is nau(cid:173)
`sea, occurring at plasma concentrations high enough to
`markedly slow gastric emptying. Thus, the most optimal
`pharmacokinetic profile of a GLP-1 drug from this per(cid:173)
`spective would again be a long half-life with as few as
`possible peak concentrations. Thus, compounds adminis(cid:173)
`tered several times a day have a disadvantage over com(cid:173)
`pounds with only once-daily administration.
`Derivatizing GLP-1 with long fatty acids has been
`shown to result in half-lives exceeding 10 h in healthy
`humans as well as type 2 diabetic patients after s.c.
`administration. Such derivatives of GLP-1 could be dosed
`once daily and provide active plasma concentrations
`throughout the day.
`
`Derivatives of GLP-1 for once-daily administration
`
`Fatty acid derivatization has been used successfully
`to protract the action of insulin by facilitating binding to
`plasma albumin (74-77). The same principle has been
`used to design derivatives of GLP-1 with half-lives longer
`than 10 h, thereby being optimal for once-daily adminis(cid:173)
`tration (78). Fatty acids or fatty diacids, optionally extend(cid:173)
`ed with a "spacer" between the epsilon-amino group of
`the lysine side chain and the carboxyl group of the fatty
`acid, were used. Acylation with simple fatty acids increas(cid:173)
`es the net negative charge of the resulting molecule with
`one (by blocking the epsilon-amino group of the lysine),
`whereas peptides acylated with a L-glutamoyl-spacer or
`with diacids provides a further increase of the negative
`charge. The addition of a negative charge to the acylated
`molecule is expected to improve solubility at physiological
`pH.
`The amino acid sequence of GLP-1 can be seen in
`Figure 1. GLP-1 (7-37) and close analogs thereof, as well
`as an extended molecule, were derivatized on position 8,
`18, 23, 26, 27, 34, 36 or 38 with fatty acids and optional(cid:173)
`ly a spacer (Table I). The structure-activity relationship
`(SAR) of the compounds was investigated using a func(cid:173)
`tional assay employing the cloned human GLP-1 receptor
`expressed in baby hamster kidney cells. All compounds
`tested were full agonists and were shown to selectively
`activate the GLP-1 receptor. Table II shows plasma half(cid:173)
`lives after s.c. administration to pigs for a selection of very
`potent compounds 4, 5, 7, 8, 18, 20 and 21. All com(cid:173)
`pounds acylated with a fatty acid equal to or longer than
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`680
`
`GLP-1 derivatives for the treatment of type 2 diabetes
`
`His 7 -Ala-G lu-Gly 10-Thr-Phe-Thr-Ser-Asp 15- Val-Ser-Ser-Tyr(cid:173)
`Leu 20-Glu-Gly-Gln-Ala-Ala25-Lys-Glu-Phe-lle-Ala30-Trp-Leu-Val(cid:173)
`Lys-Gly-Arg-Gly37
`
`Fig. 1. Amino acid sequence of GLP-1.
`
`12 carbon atoms were considerable protracted compared
`to native GLP-1, which had a half-life after s.c. adminis(cid:173)
`tration of only 1.2 h. Bioavailability was measured for
`selected compounds and was shown to be on the order
`of 50% and above. Figure 2 illustrates the dramatic dif(cid:173)
`ference in plasma half-lives between GLP-1 and three
`potent acylated compounds 5, 7 and 8.
`
`Site of acylation
`
`Many different positions in the C-terminal part of
`GLP-1 could be derivatized with quite long fatty acids,
`visualized with compounds 3-9 (EC50 30-121 pM) without
`affecting the potency. Binding affinity was not measured
`for these compounds as they all bind to albumin as part
`of their mechanism of protraction and it has not been pos(cid:173)
`sible to set up a reproducible binding assay without albu(cid:173)
`min. Derivatizing amino acids in the N-terminal part of the
`peptide, as exemplified in compound 2 (1260 pM), led to
`a substantial loss of potency. This is in agreement with
`earlier findings showing the importance of the N-terminal
`region for affinity (79).
`
`Compounds derivatized on lysine 26
`
`The potency of the compounds was comparable when
`looking at a series of different length diacids (14, 15,
`Table I) or fatty acids with the same spacer (5, 16-18).
`Within the y-Glu spacer monoacid series (5, 16-18),
`derivatization with a C18 acid (16, 194 pM) led to a sig(cid:173)
`nificant loss of activity compared to C16 (5, 68 pM), C14
`(17, 22 pM) and C12 (18, 27 pM). Within the diacid series
`(14, 15), the diacid could be no longer than a C14 (15, 72
`pM) before a loss in potency (14, 154 pM), compared to
`the y-Glu spacer monoacid series (17, 18, 22-27 pM) was
`seen. In earlier studies from our group and others,
`attempts were made to modify the amino terminus of
`GLP-1 in order to make the molecule more resistant to
`enzymatic breakdown (69, 80-81 ). Desamino His7 repre(cid:173)
`sents one of the more potent suggestions to a modifica(cid:173)
`tion giving metabolic stability (81 ). Nevertheless, as seen
`when comparing 19 (687 pM) to 5 (68 pM), considerably
`more potent compounds could be obtained by not modi(cid:173)
`fying the N-terminus when a combination with acylation
`was desired. This could be caused both by the position of
`the fatty acid and by the modified histidine.
`
`Compounds derivatized with different spacers
`
`The y-Glu spacer is optically active. Thus, it presents
`a greater analytical challenge when upscaling the com(cid:173)
`pounds for good manufacturing production guidelines.
`We therefore investigated other spacers without optical
`
`Table I: GLP-1 compounds and their potency measured using the cloned human GLP-1 receptor expressed in baby hamster kidney cells.
`
`Cpd.
`
`Parent peptide
`
`Acyl site
`
`Acyl substituent
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`14
`15
`16
`17
`18
`19
`20
`21
`22
`
`GLP-1 (7-37)
`K8R26,34-GLP-1 (7-37)
`K18R26,34-GLP-1 (7-37)
`K23 R26,34-GLP-1 (7-37)
`R34-GLP-1 (7-37)
`K27R26,34-GLP-1 (7-37)
`R26-GLP-1 (7-37)
`K36R26,34-GLP-1 (7-36)
`R26,34-GLP-1 (7-38)
`R34-GLP-1(7-37)
`R34-GLP-1(7-37)
`R34-GLP-1 (7-37)
`R34-GLP-1(7-37)
`R34-GLP-1(7-37)
`Des-amino-H7R34-GLP-1 (7-37)
`R34-GLP-1 (7-37)
`R34-GLP-1 (7-37)
`R34-GLP-1 (7-37)
`
`KB
`K18
`K23
`K26
`K27
`K34
`K36
`K38
`K26
`K26
`K26
`K26
`K26
`K26
`K26
`K26
`K26
`
`None
`y-Glu-C16
`y-Glu-C16
`y-Glu-C16
`y-Glu-C16
`y-Glu-C16
`y-Glu-C16
`y-Glu-C16
`y-Glu-C16
`C16-diacid
`C14-diacid
`y-Glu-C18
`y-Glu-C14
`y-Glu-C12
`y-Glu-C16
`GABA-C16
`~-Ala-C16
`lso-Nip-C16
`
`Potency (EC50, pM)
`55 ± 19
`1260±210
`35.2 ± 6.2
`30.1 ± 3.3
`61.0±7.1
`36.3 ± 0.3
`121 ± 26
`36.4 ± 2.1
`53.0 ± 2.8
`154 ± 66
`72 ± 0.7
`194 ± 24
`22.0 ± 7.1
`27.3 ± 8.4
`687 ± 129
`84.4 ± 22.1
`113 ± 3
`410 ± 120
`
`Abbreviations used for acyl groups in lysine-N-t:-acylated peptides: y-Glu-C12 = y-L-glutamoyl(N-a-dodecanoyl); y-Glu-C14 = y-L-gluta(cid:173)
`moyl(N-a-tetradecanoyl); y-Glu-C16 = y-L-glutamoyl(N-a-hexadecanoyl); y-Glu-C18 = y-L-glutamoyl(N-a-octadecanoyl); C14-diacid = (0-
`carboxytridecanoyl; C16-diacid = w-carboxypentadecanoyl; GABA-C16 = y-amino- butyryl(N-y-hexadecanoyl); lso-Nip-C16 = 1-(hexade(cid:173)
`canoyl)piperidyl-4-carboxy. Data are given as mean ± SD of 2 individual experiments with triplicate samples. (Reprinted in part with
`permission from J Med Chem 2000, 43; 1664-1669. Copyright 2000 American Chemical Society.)
`
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`
`Table II: Plasma half-lives in pigs of GLP-1 and selected potent
`acylated compounds.
`
`Cpd.
`
`1(GLP-1)
`4
`5
`7
`8
`18
`20
`21
`
`Plasma t½ (h)
`
`1.2
`20 ± 2
`14 ± 2
`13
`12 ± 1
`15 ± 3
`31 ± 4
`8.8 ± 1
`
`The half-lives were calculated from individual pigs after a single
`s.c. injection. Each compound was injected in 2 pigs. Data are
`shown as mean ± SD. For (7), half-life could only be calculated
`from 1 pig. (Reprinted in part with permission from J Am Chem
`2000, 43: 1664-1669. Copyright 2000 American Chemical
`Society.)
`
`10000
`
`~ 1000
`-9:
`C:
`0
`
`~ 100
`is
`
`C:
`
`C:
`0
`(.)
`
`10
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`Time (hours)
`
`Fig. 2. Pharmacokinetic profile of selected compounds after s.c.
`administration to pigs. 1 (GLP-1) (■), 5 k ), 7 (e) and 8 (Ll). Two
`pigs were dosed with compounds after which the immunoassay
`was performed in duplicate. Data are expressed as mean ± SD.
`(Reprinted with permission from J Med Chem 2000, 43: 1664-
`1669. Copyright 2000 American Chemical Society.)
`
`activity. A GABA spacer (20, 84 pM) gave a compound
`with equal affinity to the y-Glu spacer (5, 68 pM). A ~-Ala
`spacer reduced the activity slightly (21, 113 pM) and a
`piperidyl-4-carboxy spacer resulted in a 6-fold lower
`activity (22, 410 pM).
`As described above, several compounds had half(cid:173)
`lives making them suitable for once-daily administration.
`Compound 5, y-L-glutamoyl(N-a-hexadecanoyl)-Lys26 ,
`Arg 34-GLP-1 (7-37) was selected as the best compound
`for several reasons. A y-L-Glu spacer was preferred
`because it gave the most potent compound. Position 26
`or 34 were the most preferred amino acids to derivatize
`because they were natural lysines and could thus be
`derivatized easily and without introducing any potential
`immunogenic changes in the amino acid sequence.
`Position 26 gave the most potent compound. Further(cid:173)
`more, acylation at position 26 gave the most metabolical-
`
`ly stable compound. Compound 5 had an in vitro half-life
`of 20 h, whereas compounds derivatized on position 34
`(7) and position 36 (8) had in vitro half-lives of 6.3 and 6.9
`h, respectively (82). For comparison, GLP-1 (7-36)amide
`had an in vitro half-life of 0.12 h. Amino acid substitutions
`in position 8 can give better metabolic stability against
`DPP-IV. However, since quite a substantial protection
`against DPP-IV was obtained by acylation alone, and
`since any amino acid substitution poses a risk of immuno(cid:173)
`genicity, and since compound 5 was equipotent with
`GLP-1 and had the half-life required to be dosed once
`daily, compound 5 was selected for clinical development
`under the name of NN2211.
`The long half-life in pigs, adequate for once-daily
`administration, has as mentioned above been confirmed
`in man, where the half-life was determined to be 12 ± 2 h
`following a single injection to healthy humans and 10.0 ±
`3.5 h following a single injection to type 2 diabetic
`subjects (83-84).
`
`Biological characterization of NN2211
`
`As shown above, NN2211 was a full agonist with
`equal potency to GLP-1 on the cloned human GLP-1
`receptor. Its mechanism of protraction involves binding to
`albumin, metabolic stability towards DPP-IV and slow
`release from the injection site. Because of the binding to
`albumin, NN2211 appears less potent than GLP-1 in the
`presence of high concentrations of albumin. NN2211 has
`been shown to be selective, as it has no activity on the
`very closely related glucagon receptor, and also did not
`bind to any receptors in a broad Panlabs receptor binding
`screen (data not published).
`NN2211 has been studied extensively in pigs, repre(cid:173)
`senting the only model where s.c. administration normal(cid:173)
`ly results in pharmacokinetic profiles comparable to those
`of human subjects. In STZ-induced glucose intolerant
`minipigs, clamp experiments showed that NN2211
`increased glucose utilization while at the same time
`increasing insulin secretion and inhibiting glucagon
`release, both in a glucose-dependent manner. Gastric
`emptying was decreased as well (85). In STZ-induced
`diabetic minipigs, 0.004 mg/kg/day (0.28 mg/75 kg)
`NN2211 lowered blood glucose to near normal levels
`after 2-4 weeks of once-daily administration and normal(cid:173)
`ized prandial glucose tolerance (86).
`In order to obtain full efficacy in rodents, all pharma(cid:173)
`cology studies have been carried out with higher doses
`than in pigs and monkeys. Rodents, in general, seem to
`have a lower sensitivity to GLP-1 compounds and a more
`aggressive metabolism of GLP-1.
`In normal rats, 0.2 mg/kg twice-daily NN2211 has
`been shown to significantly decrease body weight by
`15%, inhibit food and water intake and stimulate diuresis
`(52). In subchronic experiments, water intake was com(cid:173)
`pensatorily increased to account for the increased diure(cid:173)
`sis. The mechanism for the decreased food intake appar(cid:173)
`ently does not involve hypothalamic GLP-1 receptors, as
`
`MPI EXHIBIT 1031 PAGE 5
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1031-0005
`
`
`
`682
`
`GLP-1 derivatives for the treatment of type 2 diabetes
`
`the same inhibition of food intake was observed in rats
`with defective hypothalamic GLP-1 receptors due to
`monosodium glutamate injections. In this study, a reduc(cid:173)
`tion in plasma triglyceride levels was observed as well
`(52). Moreover, in normal rats, NN2211 has been shown
`to affect carbohydrate intake more than fat intake (87).
`Doses of 0.003 mg/kg NN2211 and higher significant(cid:173)
`ly reduced blood glucose in an acute study in diabetic
`ob/ob mice. At the same doses, food intake was also sig(cid:173)
`nificantly reduced (88). In both ob/ob and db/db mice,
`subchronic studies of 14 days duration led to continuous(cid:173)
`ly lowered blood glucose levels. However, in these animal
`models of diabetes, no effect on food intake was obser(cid:173)
`ved after day 1. The blood glucose lowering effect was
`maintained throughout the study but was most significant
`on day 1 (89). A comparative study to exendin-4, another
`GLP-1-based compound in clinical development, was
`also conducted. This study showed a more pronounced
`glucose lowering effect of NN2211 than exendin-4 (90). In
`ob/ob mice, a tendency to an increased beta-cell prolifer(cid:173)
`ation was observed (data not published). In db/db mice,
`!3-cell
`there were significant effects on both
`proliferation and mass (91 ). NN2211 effects were greater
`than exendin-4. These differences between NN2211 and
`exendin-4 may be explained by the longer half-life of
`NN2211.
`Twice-daily doses of 0.15 mg/kg s.c. NN2211 very
`effectively delayed the progression of diabetes in a
`6-week preventive study in young Zucker diabetic fatty
`rats. NN2211 was significantly more effective than pair
`feeding alone and decreased HbA 1 c by 3.1 %.
`Cholesterol and free fatty acid plasma levels were also
`reduced in this model. No increased 13-cell proliferation
`was observed compared to the control group, but
`increased !3-cell volume was observed in both NN2211-
`treated and pair-fed animals. Food intake was decreased
`for the entire duration of the study (92).
`NN2211 and GLP-1 have been shown to inhibit
`cytokine-induced apoptosis in vitro using isolated 13-cells
`(41 ). These results may explain why NN2211 in some ani(cid:173)
`mal models increases 13-cell mass without significantly
`affecting !3-cell proliferation.
`In summary, in a broad spectrum of animal models
`NN2211 has been shown to lower blood glucose and
`body weight, and to increase or maintain 13-cell mass. All
`the effects are consistent with the known physiological
`effects of GLP-1. In humans, a study in type 2 diabetic
`patients has shown that a single injection of 0.010 mg/kg
`NN2211 lowered blood glucose from 8.1 ± 1.0 mM to
`6.9 ± 1.0 mM (p = 0.004) (82). NN2211 is currently in
`phase 2 clinical trials where it's long-term efficacy will be
`evaluated.
`
`Conclusions
`
`GLP-1 compounds form a new class of drugs in clini(cid:173)
`cal development for the treatment of type 2 diabetes. This
`new class of drugs is especially interesting because
`
`GLP-1 has been shown to lower blood glucose as well as
`food intake and body weight. However, to be clinically
`useful the dynamic half-life needs to be significantly pro(cid:173)
`longed. We found that the peptide hormone GLP-1 could
`be derivatized almost anywhere in the C-terminal part of
`the peptide and that derivatization with both short and
`long fatty acids and amino acid-derived spacers led to
`compounds that were highly potent. A number of com(cid:173)
`pounds were both very potent and had plasma half-lives
`above 10 h, making them suitable as drugs for the treat(cid:173)
`ment of type 2 diabetes using once-daily administration.
`NN2211 has been selected for clinical development and
`is currently in phase 2 clinical trials.
`NN2211
`is a metabolically stable compound with
`potency equal to GLP-1. It has been characterized to act
`as a GLP-1 compound in several animal models, includ(cid:173)
`ing the ability to lower body weight. NN2211 is currently
`the only GLP-1 compound in clinical development that
`has been shown to possess pharmacokinetic properties
`applicable to once-daily administration. The only study
`carried out thus far in type 2 diabetic patients has con(cid:173)
`firmed its efficacy. Ongoing phase 2 clinical trials will
`reveal the potential of NN2211 as a promising new treat(cid:173)
`ment for type 2 diabetes.
`
`References
`
`1. ADA: Clmical practice recommendations 2001. Diabetes Care
`2001, 24(81 ): S134.
`
`2. Bell, G.I., Santerre, R.F., Mullenbach, G.T. Hamster pre(cid:173)
`proglucagon contains the sequence of glucagon and two related
`peptides. Nature 1983, 302: 716-8.
`
`3. Bell, G.I., Sanchez-Pescador, R., Laybourn, P.J., Najarian,
`R.C. Exon duplication and divergence in the human pre(cid:173)
`proglucagon gene. Nature 1983, 304: 368-71.
`
`4. DeFronzo, R.A., Bonadonna, R.C., Ferrannini, E.
`Pathogenesis of NIDDM. A balanced overview. Diabetes Care
`1992, 15: 318-68.
`
`5. Unger, R.H., Orci, L. The essential role of glucagon in the
`pathogenesis of diabetes melli