One Week’s Treatment With the Long-Acting
`Glucagon-Like Peptide 1 Derivative Liraglutide
`(NN2211) Markedly Improves 24-h Glycemia and
`␣- and ␤-Cell Function and Reduces Endogenous
`Glucose Release in Patients with Type 2 Diabetes
`
`Kristine B. Degn,1 Claus B. Juhl,1 Jeppe Sturis,2 Grethe Jakobsen,2 Birgitte Brock,1
`Visvanathan Chandramouli,3 Joergen Rungby,1 Bernard R. Landau,3 and Ole Schmitz1
`
`Glucagon-like peptide 1 (GLP-1) is potentially a very
`attractive agent for treating type 2 diabetes. We ex-
`plored the effect of short-term (1 week) treatment with
`a GLP-1 derivative, liraglutide (NN2211), on 24-h dy-
`namics in glycemia and circulating free fatty acids, islet
`cell hormone profiles, and gastric emptying during
`meals using acetaminophen. Furthermore, fasting en-
`dogenous glucose release and gluconeogenesis (3-3H-
`glucose infusion and 2H2O ingestion, respectively) were
`determined, and aspects of pancreatic islet cell function
`were elucidated on the subsequent day using homeosta-
`sis model assessment and first- and second-phase insu-
`lin response during a hyperglycemic clamp (plasma
`glucose ⬃16 mmol/l), and, finally, on top of hyperglyce-
`mia, an arginine stimulation test was performed. For
`accomplishing this, 13 patients with type 2 diabetes
`were examined in a double-blind, placebo-controlled
`crossover design. Liraglutide (6 ␮g/kg) was adminis-
`tered subcutaneously once daily. Liraglutide signifi-
`cantly reduced the 24-h area under the curve for glucose
`(P ⴝ 0.01) and glucagon (P ⴝ 0.04), whereas the area
`under the curve for circulating free fatty acids was
`unaltered. Twenty-four-hour insulin secretion rates as
`assessed by deconvolution of serum C-peptide concen-
`trations were unchanged, indicating a relative increase.
`Gastric emptying was not influenced at the dose of
`liraglutide used. Fasting endogenous glucose release
`was decreased (P ⴝ 0.04) as a result of a reduced
`glycogenolysis (P ⴝ 0.01), whereas gluconeogenesis was
`unaltered. First-phase insulin response and the insulin
`response to an arginine stimulation test with the pres-
`ence of hyperglycemia were markedly increased (P <
`
`From the 1Department of Endocrinology (M & C), University Hospital of
`Aarhus, and Department of Clinical Pharmacology, University of Aarhus,
`Aarhus, Denmark; 2Novo Nordisk A/S, Bagsværd, Denmark; and 3Case West-
`ern Reserve University School of Medicine, Cleveland, Ohio.
`Address correspondence and reprint requests to Ole Schmitz, MD, Depart-
`ment of Medicine M (Endocrinology & Diabetes), University Hospital of
`Aarhus, AKH, Nørrebrogade 42-44 DK-8000 Aarhus C, Denmark. E-mail:
`ole.schmitz@iekf.au.dk.
`Received for publication 14 October 2003 and accepted in revised form
`23 January 2004.
`AUC, area under the curve; DPP-IV, dipeptidylpeptidase IV; EGR, endoge-
`nous glucose release; ELISA, enzyme-linked immunosorbent assay; FFA, free
`fatty acid; GLP-1, glucagon-like peptide 1; GLY, glycogenolysis; GNG, gluco-
`neogenesis; HMT, hexamethylenetetramine; HOMA, homeostasis model as-
`sessment; ISR, insulin secretion rate; OHA, oral hypoglycemic agent; tmax, time
`of occurrence for maximum drug concentration.
`© 2004 by the American Diabetes Association.
`
`0.001), whereas the proinsulin/insulin ratio fell (P ⴝ
`0.001). The disposition index (peak insulin concentra-
`tion after intravenous bolus of glucose multiplied by
`insulin sensitivity as assessed by homeostasis model
`assessment) almost doubled during liraglutide treat-
`ment (P < 0.01). Both during hyperglycemia per se and
`after arginine exposure, the glucagon responses were
`reduced during liraglutide administration (P < 0.01 and
`P ⴝ 0.01). Thus, 1 week’s treatment with a single daily
`dose of the GLP-1 derivative liraglutide, operating
`through several different mechanisms including an ame-
`liorated pancreatic islet cell function in individuals with
`type 2 diabetes, improves glycemic control throughout
`24 h of daily living, i.e., prandial and nocturnal periods.
`This study further emphasizes GLP-1 and its derivatives
`as a promising novel concept for treatment of type 2
`diabetes. Diabetes 53:1187–1194, 2004
`
`Type 2 diabetes is characterized by insulin resis-
`
`tance and progressive islet cell dysfunction lead-
`ing to insulin deficiency (1). Increased hepatic
`glucose release is considered to play a key role
`in fasting, as well as postprandial hyperglycemia, and
`increased gluconeogenesis seems to be essential in this
`scenario (2,3). In addition, elevated glucagon levels (4,5)
`and a reduced response of glucagon-like peptide 1 (GLP-1)
`to meals (6) are common features.
`The importance of aggressive glucose-lowering therapy
`to prevent late diabetes complications in type 2 diabetes
`has been convincingly established (7,8). However, the U.K.
`Prospective Diabetes Study also demonstrated that the
`antidiabetic treatment used failed to maintain acceptable
`glycemic control in the vast majority of the patients, em-
`phasizing the need for more effective antidiabetic agents.
`GLP-1 is an incretin hormone secreted from the intesti-
`nal mucosa in response to meal
`ingestion (9). Insulin
`secretion is stimulated, and glucagon secretion is inhib-
`ited; both actions are glucose dependent. Also, GLP-1 has
`trophic effects on pancreatic ␤-cells and inhibits their
`apoptosis (10). Furthermore, it delays gastric emptying
`(9,11) and may even be a satiety factor. These observa-
`tions support GLP-1 as a novel candidate for treatment of
`type 2 diabetes, and the beneficial effects of GLP-1 on
`
`DIABETES, VOL. 53, MAY 2004
`
`1187
`
`MPI EXHIBIT 1081 PAGE 1
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`

`

`LIRAGLUTIDE TREATMENT AND TYPE 2 DIABETES
`
`FIG. 1. Flow sheet illustrating the procedures on study day 9. See text for details.
`
`glycemic control in individuals with type 2 diabetes have
`been demonstrated (12,13). However, native GLP-1 has a
`very short half-life because of its rapid degradation by the
`ubiquitous enzyme dipeptidylpeptidase IV (DPP-IV) (14),
`making GLP-1 per se unsuitable as a therapeutic drug.
`Derivatives that are resistant to DPP-IV are one way to
`overcome this failing. Liraglutide is an acylated GLP-1
`derivative that binds to albumin. This limits its sensitivity
`to DPP-IV and delays absorption from its injection site. A
`prolonged pharmacokinetic profile in humans is attained,
`the half-life after subcutaneous administration being
`⬃12 h (15,16). A single-dose study demonstrated signif-
`icant glucose lowering in individuals with type 2 diabetes
`in both the fasting and postprandial states (16), and in a
`12-week repeated-dose study, liraglutide lowered blood
`glucose to the same extent as a sulfonylurea compound
`(glimepiride) and the prevalence of hypoglycemia was
`very low (17).
`The present study was undertaken to gain further in-
`sight into GLP-1 derivatives as novel antidiabetic drugs.
`We sought to do this by administering liraglutide to
`individuals with type 2 diabetes for 7 days and assessing
`the impact on 24-h glucose and hormone levels during
`daily life conditions, gastric emptying rate, endogenous
`glucose release (EGR), and various aspects of the pancre-
`atic islet cell function.
`
`RESEARCH DESIGN AND METHODS
`The protocol was approved by the local ethics committee and performed in
`accordance with the Helsinki Declaration.
`Patients. Thirteen patients (5 women and 8 men) with type 2 diabetes
`according to World Health Organization criteria were examined. Their age
`(mean ⫾ SD) was 56.4 ⫾ 9.2 years, BMI was 31.2 ⫾ 3.6 kg/m2, last measured
`HbA1c before inclusion was 7.3 ⫾ 0.4% (normal range ⬍6.4%), and the duration
`of diabetes was 3.0 ⫾ 2.6 years (range 5 months to 8 years). At study entry, six
`patients were treated with diet alone and seven were additionally treated with
`oral hypoglycemic agents (OHAs; sulfonylurea n ⫽ 3, metformin n ⫽ 3,
`sulfonylurea and metformin n ⫽ 1). Concomitant medications were angioten-
`sin-converting enzyme inhibitors (n ⫽ 4), thiazide diuretics (n ⫽ 1), ␤-block-
`ers (n ⫽ 1), calcium antagonists (n ⫽ 1), HMG-CoA reductase inhibitors (n ⫽
`2), and acetyl salicylic acid (n ⫽ 3). Three patients had retinopathia simplex,
`and three had nephropathy (microalbuminuria).
`Experimental design. This crossover trial was double-blinded, placebo-
`controlled, and randomized. After inclusion, the patients discontinued their
`OHA for 2 weeks before beginning study medication. Liraglutide (6 ␮g/kg body
`wt) or placebo was injected subcutaneously into the abdomen once daily (at
`
`⬃0745) for 9 days using a NovoPen (1.5 with Novofine 30-G, 0.3- to 8-mm
`needle) as the dispensing device. After 7 days of treatment, the patients
`arrived at the clinical research unit at 2200. In the next 2 days (days 8 and 9),
`the following experiments were carried out during continuous treatment.
`On day 8 at 0730, a catheter was placed in an antecubital vein for blood
`sampling. Three standard meals were served at 0800, 1200, and 1800, to be
`finished within 20 min. Breakfast contained 2,660 kJ (protein 14 E%, carbo-
`hydrate 55 E%, and fat 31 E%), lunch contained 2,865 kJ (protein 16 E%,
`carbohydrate 50 E%, and fat 34 E%), and dinner contained 3,397 kJ (protein 28
`E%, carbohydrate 53 E%, and fat 19 E%). With breakfast and dinner, 1 g of
`acetaminophen dissolved in 150 ml of water was given to assess gastric
`emptying rate (18). Serum acetaminophen was determined every 15–30 min
`during the following 4 h, and area under the curve (AUC) for acetamino-
`phen (AUCacetaminophen) and the time of occurrence for maximum drug
`concentration (tmax) were calculated. During day 8, blood was collected with
`changing intervals for determination of glucose, insulin, C-peptide, proinsulin
`(only fasting and after breakfast), glucagon, free fatty acids (FFAs), and
`liraglutide.
`On day 9, the following procedures were performed (Fig. 1).
`Gluconeogenesis. At 0300 and 0500, patients drank 2 ml of 2H2O/kg body
`water (99.9% H; Sigma Aldrich; body water was estimated to be 50% of total
`body weight in women and 60% in men). Water ingested ad libitum thereafter
`was enriched with 0.4% 2H2O to maintain isotopic steady state. At 0800, 0830,
`and 0900, blood was drawn for determination of gluconeogenesis (GNG).
`EGR. EGR was estimated by use of an isotope dilution technique. At 0600, a
`bolus of 3-3H-glucose (adjusted priming: 6 ␮Ci ⫻ plasma glucose level in
`mmol/l) was given followed by a continuous infusion (0.3 ␮Ci/min) until 0900.
`Blood samples were drawn before the bolus and every 15 min from 0800 to
`0900 for determination of glucose 3H specific activity. Steady state was
`achieved during this period in both situations, the average specific activity of
`glucose being 2,078, 2,180, and 2,160 cpm/mg at time points 0800, 0830, and
`0900 during active treatment and 1,927, 1,822, and 1,906 cpm/mg at the same
`time points during placebo.
`First-phase insulin secretion. First-phase insulin secretion was deter-
`mined using an intravenous glucose bolus. At 0915, 25 g of glucose was
`administered intravenously over 2 min. Blood was then collected every 2–5
`min until 0932 for determining glucose and insulin.
`Hyperglycemic clamp. From 0932 to 1145, 20% glucose was infused to
`maintain plasma glucose at 16 mmol/l. After 75 min, when approximate steady
`state was attained, blood was drawn every 10 min (from 1045 to 1115) for
`determining plasma glucose and serum insulin, C-peptide, and proinsulin.
`Arginine stimulation test. At 1115, a bolus of 5 g ofarginine was
`given intravenously over 30 s followed by collection of blood every 5 min until
`1145 for determining circulating glucose, insulin, C-peptide, glucagon, and
`proinsulin.
`Safety. Adverse events, vital signs, hematology, and biochemistry were
`monitored throughout the study.
`Assays. All biochemical analyses were performed in duplicate. Plasma
`glucose was measured immediately on a glucose analyzer (Beckman Instru-
`ments, Palo Alto, CA) using the glucose oxidase technique. All other serum
`and plasma samples were stored at ⫺20°C (C-peptide at ⫺80°C) until analyzed.
`Serum insulin was determined using a highly specific and sensitive two-site
`
`1188
`
`DIABETES, VOL. 53, MAY 2004
`
`MPI EXHIBIT 1081 PAGE 2
`
`

`

`K.B. DEGN AND ASSOCIATES
`
`enzyme-linked immunosorbent assay (ELISA; DAKO Diagnostics, Cam-
`bridgeshire, U.K.), and serum C-peptide was measured by a two-site mono-
`clonal-based ELISA with an intra-assay coefficient of variation of 5.1% (DAKO
`Diagnostics). Serum proinsulin was analyzed by specific immunoassay with
`no cross-reaction with insulin and C-peptide (DAKO Diagnostics). Serum
`acetaminophen was determined by high-performance liquid chromatography
`after extraction. Plasma glucagon was measured using a radioimmunoassay
`kit (Linco Research). Liraglutide concentrations were analyzed by ELISA
`using a monoclonal antibody against GLP-1/liraglutide as capture antibody
`and another monoclonal antibody specific for the NH2-terminal part of
`GLP-1/liraglutide for detection. Before the latter analysis, samples were
`incubated at 37°C to remove endogenous GLP-1, liraglutide being stable
`toward this incubation. Determination of 3-3H-glucose activity was as previ-
`ously described (19). Measurement of deuterium enrichment at carbons 2 and
`5 glucose was as described by Landau et al. (20). In brief, 15 ml of blood was
`diluted with 30 ml of demineralized water and deproteinized using 15 ml of 0.3
`N ZnSO4 and 15 ml of 0.3 N Ba(OH)2. The samples were then centrifuged at
`2,000 rpm for 15 min, and the pellet was diluted in 15 ml of demineralized
`water to wash out the remaining glucose. Glucose was isolated by high-
`performance liquid chromatography. For determination of deuterium enrich-
`ment on C5, glucose was first converted to xylose, and the carbon 5 with its
`hydrogens was cleaved by periodate oxidation to formaldehyde, which was
`condensed with ammonium hydroxide to form hexamethylenetetramine
`(HMT). The 2H bound to C2 of glucose was isolated after conversion of
`glucose to ribitol-5-phosphate and arabitol-5-phosphate and treated to form
`HMT. Enrichments in the HMTs were measured using a Hewlett-Packard mass
`spectrometry system with standards of HMTs analyzed along with the
`unknown samples.
`Calculations and statistics. Fasting data are presented as the mean values
`at 0730 and 0800 on days 8 and 9. Furthermore, fasting plasma glucose on day
`9 is presented. AUCs of substrates and hormones were calculated by applying
`the trapezoidal rule and represent the total AUC. EGR was determined using
`Steele’s equations for steady state, as modified by Finegood et al. (21). The
`fractional contribution of GNG to glucose production equals the ratio between
`deuterium bound to carbon 5 in glucose and that bound to carbon 2. The rate
`of GNG in the fasting state was obtained by multiplying the mean EGR from
`0800 to 0900 by the mean fractional contribution of GNG during that same
`hour. The rate of glycogenolysis (GLY) was calculated by subtracting the rate
`of GNG from total EGR. Insulin secretion rates (ISRs) were estimated by
`mathematical analysis (deconvolution) of peripheral C-peptide concentrations
`using a two-compartment model, as described by Polonsky et al. (22) and the
`standard C-peptide kinetic parameters published by Van Cauter et al. (23).
`This model allows accurate estimation of ISR also under non–steady-state
`conditions. Another estimate of ␤-cell function and an estimate of insulin
`resistance were calculated by homeostasis model assessment (HOMA-B and
`R) (24), using glucose and insulin values after a 14-h long fast and 24 h after
`the last liraglutide injection. As a supplement to the latter, insulin sensitivity
`was calculated by dividing glucose infusion rate during steady state of the
`hyperglycemic clamp with mean serum insulin level. Finally, an analog to the
`disposition index was calculated by multiplying 1/HOMA-R by the peak insulin
`concentrations during the first-phase insulin secretion test (25).
`Statistical analysis was performed using a mixed model with sequence of
`treatment, visit, and treatment as fixed factors and patient as a random factor.
`Differences were considered significant at P ⬍ 0.05. All results are given as
`liraglutide versus placebo.
`
`RESULTS
`Twenty-four-h profiles of substrates and hormones.
`During the 24-h profile, the average concentration of plas-
`ma glucose (P ⫽ 0.01) and postprandial plasma glucose
`concentrations after all three meals were decreased by
`⬃20% (P ⫽ 0.01, P ⫽ 0.02, and P ⫽ 0.02, respectively)
`during liraglutide treatment. Fasting plasma glucose also
`declined (Fig. 2, Table 1).
`Circulating concentrations of fasting insulin, 24-h AUC
`insulin, postprandial insulin concentrations, and ISR were
`comparable during treatment with liraglutide and placebo,
`despite the lower glycemia during active treatment sug-
`gesting a relative increase in insulin secretion. The ratio of
`
`FIG. 2. Twenty-four-hour profiles of plasma glucose (A), serum FFAs
`(B), serum insulin (C), and plasma glucagon (D) (means ⴞ SEM), day
`8. E, placebo; F, liraglutide;
`, meals. (Statistical details are listed in
`Table 1.)
`
`DIABETES, VOL. 53, MAY 2004
`
`1189
`
`MPI EXHIBIT 1081 PAGE 3
`
`

`

`LIRAGLUTIDE TREATMENT AND TYPE 2 DIABETES
`
`TABLE 1
`Twenty-four-hour profiles and fasting values
`
`䡠
`
`l⫺1 䡠 h)
`
`Fasting values
`Plasma glucose (mmol/l)
`Plasma glucose day 9 (mmol/l)
`FFA (mmol/l)
`Insulin (pmol/l)
`ISR (nmol/h)
`Proinsulin/insulin ratio
`Glucagon (pg/ml)
`24-h total AUCs
`Plasma glucose (mmol
`l⫺1 䡠 h)
`䡠
`FFA (mmol
`l⫺1 䡠 h)
`䡠
`Insulin (pmol
`ISR (nmol)
`Glucagon (pg 䡠 ml⫺1 䡠 h)
`Postprandial total AUCs
`Plasma glucose 8–12 h (mmol
`䡠
`Plasma glucose 12–16 h (mmol
`䡠
`Plasma glucose 18–22 h (mmol
`l⫺1 䡠 h)
`䡠
`Insulin 8–12 h (pmol
`l⫺1 䡠 h)
`䡠
`Insulin 12–16 h (pmol
`l⫺1 䡠 h)
`䡠
`Insulin 18–22 h (pmol
`Proinsulin/insulin ratio 8–12 h
`Glucagon 8–12 h (pg 䡠 ml⫺1 䡠 h)
`Glucagon 12–16 h (pg 䡠 ml⫺1 䡠 h)
`Glucagon 18–22 h (pg 䡠 ml⫺1 䡠 h)
`
`䡠
`
`l⫺1 䡠 h)
`l⫺1 䡠 h)
`l⫺1 䡠 h)
`
`Liraglutide
`
`8.06 ⫾ 0.52
`7.56 ⫾ 0.42
`0.52 ⫾ 0.03
`95.6 ⫾ 15.1
`17.7 ⫾ 2.0
`0.17 ⫾ 0.04
`92.8 ⫾ 7.2
`
`187.5 ⫾ 14.0
`8.54 ⫾ 0.51
`3,854 ⫾ 581
`566.1 ⫾ 55.1
`2,179 ⫾ 118
`
`38.66 ⫾ 3.52
`32.52 ⫾ 2.92
`33.76 ⫾ 2.50
`999 ⫾ 173
`723 ⫾ 107
`1,017 ⫾ 160
`0.12 ⫾ 0.03
`383.0 ⫾ 22.8
`362.6 ⫾ 18.6
`397.3 ⫾ 23.9
`
`Placebo
`
`9.39 ⫾ 0.76
`9.20 ⫾ 0.78
`0.51 ⫾ 0.03
`87.2 ⫾ 17.9
`16.1 ⫾ 2.1
`0.27 ⫾ 0.05
`94.5 ⫾ 5.6
`
`232.3 ⫾ 21.9
`8.65 ⫾ 0.68
`4,154 ⫾ 881
`561.6 ⫾ 72.6
`2,371 ⫾ 135
`
`47.51 ⫾ 3.95
`41.89 ⫾ 4.54
`41.10 ⫾ 3.94
`1,056 ⫾ 253
`808 ⫾ 162
`1,117 ⫾ 221
`0.19 ⫾ 0.04
`413.4 ⫾ 25.6
`374.2 ⫾ 19.5
`470.1 ⫾ 35.2
`
`P
`
`0.078
`0.025
`0.536
`0.513
`0.206
`0.009
`0.586
`
`0.014
`0.876
`0.375
`0.982
`0.037
`
`0.010
`0.017
`0.016
`0.512
`0.312
`0.392
`0.008
`0.080
`0.384
`0.009
`
`Data are means ⫾ SEM. Twenty-four-hour AUCs were calculated from samples obtained from 0800 to 0800. Fasting values were calculated
`from samples taken at 0730 and 0800 on days 8 and 9.
`
`24-h AUCinsulin/AUCglucose was significantly higher during
`liraglutide administration (P ⬍ 0.05), whereas only a trend
`was observed in the fasting insulin/glucose ratio (P ⬍
`0.15). The fasting proinsulin/insulin ratio and the proinsu-
`lin/insulin ratio after the breakfast meal were markedly
`reduced during liraglutide administration (P ⬍ 0.01).
`Liraglutide exposure significantly reduced the 24-h AUC
`of glucagon (P ⫽ 0.04), primarily as a result of a marked
`reduction in glucagon concentrations after the protein-rich
`evening meal (P ⬍ 0.01). Fasting plasma glucagon concen-
`trations were unaltered. The 24-h AUC insulin/glucagon
`ratio did not differ in the two situations (1.78 ⫾ 0.28
`pmol/␮g vs. 1.72 ⫾ 0.32 pmol/␮g; P ⫽ 0.20). Fasting value
`and 24-h AUC of FFAs were similar in the two regimens.
`Gastric emptying rate. AUCacetaminophen during the
`breakfast meal (210 ⫾ 16 vs. 210 ⫾ 12 ␮mol 䡠 l⫺1 䡠 h) and
`AUCacetaminophen during dinner (178 ⫾ 10 vs. 179 ⫾ 9 ␮mol
`䡠 l⫺1 䡠 h) were almost identical. Similarly, tmax did not show
`a significant difference after breakfast (40 ⫾ 8 min during
`liraglutide treatment vs. 32 ⫾ 4 min during placebo) or
`dinner (62 ⫾ 10 vs. 50 ⫾ 7 min).
`EGR. The rate of EGR in the fasting state was significantly
`reduced by liraglutide (1.92 ⫾ 0.06 vs. 2.13 ⫾ 0.09 mg 䡠
`kg⫺1 䡠 min⫺1; P ⫽ 0.04). This was due to diminished GLY
`(0.83 ⫾ 0.04 vs. 1.02 ⫾ 0.04 mg 䡠 kg⫺1 䡠 min⫺1; P ⫽ 0.01). In
`contrast, GNG did not change (Fig. 3).
`Islet cell function. ␤-Cell function in the fasting state,
`as assessed by HOMA-B analysis, was increased by 30%
`during liraglutide administration (P ⫽ 0.01). First-phase
`insulin response after the intravenous glucose bolus was
`increased by ⬃60% (P ⬍ 0.01). During steady state of the
`hyperglycemic clamp, there was a 2- to 3.5-fold increase in
`mean insulin concentration, whereas mean circulating
`
`1190
`
`glucagon concentration was reduced by ⬃20% (P ⬍ 0.01).
`Analogously, the insulin response after arginine infusion
`was substantially increased during liraglutide treatment
`(P ⬍ 0.01), whereas the glucagon response was reduced
`(P ⫽ 0.01). The proinsulin/insulin ratio after liraglutide
`administration was reduced by 40 –50% during the hyper-
`glycemic clamp (Fig. 4, Table 2).
`Insulin sensitivity and disposition index. Insulin sen-
`sitivity (as calculated by HOMA-R and glucose infusion
`rate/mean insulin level) was unaltered during the two
`treatment regimens. The disposition index increased
`substantially, almost doubling after liraglutide (P ⬍
`0.01; Table 2).
`Pharmacokinetic properties. The half-life of liraglutide
`in steady state was 17.9 h, and tmax was 10.1 ⫾ 3 h (Fig. 5).
`
`FIG. 3. Fasting EGR, GNG, and GLY, day 9. Data are means ⴞ SEM. 䡺,
`placebo; f, liraglutide; *P < 0.05, liraglutide vs. placebo.
`
`DIABETES, VOL. 53, MAY 2004
`
`MPI EXHIBIT 1081 PAGE 4
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`

`

`K.B. DEGN AND ASSOCIATES
`
`ment week. These three were among the patients in whom
`the highest serum concentrations of
`liraglutide were
`found. No other safety concerns were identified.
`
`DISCUSSION
`In the present study, we investigated the effects of 1 week
`of treatment with the GLP-1 derivative liraglutide (NN2211)
`on 24-h substrate and hormone profiles under conditions
`that simulate daily living, on EGR, and on pancreatic islet
`cell function. A major finding is a markedly reduced
`circadian plasma glucose level during liraglutide treatment
`exhibited by fasting, prandial, and nocturnal concentra-
`tions. When evaluating the average reduction in plasma
`glucose (⬃2 mmol/l), it is important to emphasize that 6 of
`the 13 patients, even after OHA withdrawal, had a fasting
`plasma glucose ⬍8.3 mmol/l, i.e., the level above which
`action should be taken (26). Moreover, this 1-week study
`was probably too brief to harvest the additional advanta-
`geous effects of decreased glucose toxicity, i.e., no im-
`provement in insulin sensitivity. Finally, the same dose (6
`␮g/kg body wt) of liraglutide was used in all patients. It is
`likely that some patients would have a further improve-
`ment in blood glucose lowering using a higher dose. The
`current study thus confirms that liraglutide possesses the
`widely known beneficial effects of native GLP-1 on glyce-
`mia in patients with type 2 diabetes (12,13,27) and dem-
`onstrates that once-daily dosing of liraglutide is sufficient
`to ensure 24-h effectiveness (vide infra, pharmacokinet-
`ics). It is also important to highlight that no episodes of
`hypoglycemia occurred despite some of the patients’ ex-
`hibiting a remarkable glycemic control during liraglutide
`treatment.
`Improved glycemic control during liraglutide treatment
`is probably orchestrated by changes in insulin and gluca-
`gon secretion. Basal and prandial ISRs were unchanged
`despite the substantial reduction in glycemia, clearly
`indicating improved ␤-cell function during daily life
`conditions. This is in accordance with previous studies
`exploring native GLP-1 actions (12,13,27) and demon-
`strates that GLP-1 and/or GLP-1 derivatives augment the
`ability of the ␤-cell to respond to prevailing prandial
`stimuli, e.g., glycemia and glucose dynamics.
`A second important observation is that liraglutide sig-
`nificantly reduces 24-h circulating glucagon levels. In
`particular, the large postprandial glucagon excursion after
`the protein-rich evening meal was amply decreased. An
`impaired postprandial inhibition of glucagon is a common
`feature in type 2 diabetes (5,28), and this contributes
`notably to the postprandial hyperglycemia (4). Of particu-
`lar note is that hyperglycemia per se inhibits glucagon
`secretion, and the decrement in plasma glucagon during
`liraglutide administration was seen despite a lower glyce-
`mic level.
`The lowering effect of native GLP-1 on upper gastroin-
`testinal motility is well established (29). In a single-dose
`study using liraglutide in patients with type 2 diabetes,
`reduced gastric emptying rate was also reported (16). In
`contrast, we did not in the current study observe delayed
`gastric emptying during liraglutide administration. The
`data achieved from the acetaminophen assessment tech-
`nique has been proved to agree with data from using much
`more sophisticated methods for assessment of gastric
`
`FIG. 4. Plasma glucose (A) and serum insulin (B) concentrations
`during an intravenous glucose bolus, hyperglycemic clamp, and argi-
`nine stimultion test, day 9. E, placebo; F, liraglutide,
`, glucose bolus
`(cid:0)(cid:0)(cid:0), arginine bolus (5 g). Plasma glucagon (C) is depicted from
`(25 g); (cid:0)(cid:0)(cid:0)
`1105. Steady state of hyperglycemic clamp was in the interval 1045–
`1115. The insert in the middle panel is a blow-up of the first-phase
`insulin response. Data are means ⴞ SEM. (Statistical details are listed
`in Table 2.)
`
`(cid:0)(cid:0)(cid:0)
`(cid:0)(cid:0)(cid:0)
`
`Safety. No hypoglycemic episodes occurred. Three pa-
`tients experienced gastrointestinal adverse events (nausea
`and abdominal pain) during the treatment period. In two
`cases, the symptoms disappeared after the first days of
`treatment, and in the remaining case, the severity of the
`gastrointestinal discomfort declined throughout the treat-
`
`DIABETES, VOL. 53, MAY 2004
`
`1191
`
`MPI EXHIBIT 1081 PAGE 5
`
`

`

`LIRAGLUTIDE TREATMENT AND TYPE 2 DIABETES
`
`TABLE 2
`Islet cell function tests, insulin sensitivity, and disposition index
`
`Intravenous glucose tolerance test
`l⫺1 䡠 h)
`䡠
`Insulin total AUC 9.15–9.32 h (pmol
`Maximal insulin concentration 9.15–9.32 h (pmol/l)
`Hyperglycemic clamp, steady state
`Insulin concentration (pmol/l)
`Proinsulin/insulin ratio
`Glucagon concentration (pg/ml)
`GIR/average serum insulin (arbitrary units)
`Arginine stimulation test
`Insulin total AUC 11.15–11.45 h (pmol
`Maximal insulin concentration (pmol/l)
`Glucagon total AUC 11.15–11.45 h (pg 䡠
`Maximal glucagon concentration (pg/ml)
`HOMA analysis
`␤-Cell function (% of normal)
`Insulin resistance (fold normal)
`Disposition index (pmol/lⴱHOMA-R)
`
`l⫺1 䡠 h)
`
`䡠
`
`l⫺1 䡠 h)
`
`Liraglutide
`
`Placebo
`
`55.45 ⫾ 9.93
`262.6 ⫾ 47.5
`
`929.6 ⫾ 262.9
`0.09 ⫾ 0.02
`55.5 ⫾ 3.7
`0.13 ⫾ 0.03
`
`799.6 ⫾ 190
`2,539 ⫾ 523
`40.7 ⫾ 2.5
`162.0 ⫾ 13.7
`
`59.95 ⫾ 8.84
`4.09 ⫾ 0.84
`80.4 ⫾ 14
`
`34.26 ⫾ 6.4
`166.5 ⫾ 32.4
`
`271.9 ⫾ 53.3
`0.18 ⫾ 0.03
`66.7 ⫾ 3.5
`0.15 ⫾ 0.04
`
`307.2 ⫾ 65.5
`1,518 ⫾ 296
`48.7 ⫾ 3.0
`193.4 ⫾ 14
`
`46.07 ⫾ 9.86
`4.74 ⫾ 1.15
`41.9 ⫾ 5
`
`P
`
`0.008
`0.007
`
`0.015
`0.001
`0.005
`0.392
`
`0.004
`0.005
`0.012
`0.034
`
`0.010
`0.373
`0.008
`
`Data are means ⫾ SEM. GIR, glucose infusion rate.
`
`emptying (30). A possible explanation behind the apparent
`discrepancy between the present and earlier data might be
`that the effect on upper gastrointestinal motility undergoes
`tachyphylaxis. However, two studies of liraglutide/native
`GLP-1 in animals (31) and humans (12) report an effect on
`gastric emptying after several weeks of treatment. Thus, it
`is conceivable that the lack of effect of liraglutide on
`gastric emptying observed in our study may be due to the
`dose used. Probably the dose-response relationships be-
`tween blood glucose–lowering effects and delaying gastric
`emptying differ, the latter being rightward shifted.
`Another novel finding is the decline in fasting EGR
`after liraglutide administration. This could be due to an
`increased insulin/glucagon ratio. However, a significant
`increase was not observed in peripheral blood. It is essen-
`tial to emphasize that circulating FFA levels did not differ
`in the two situations. In our diabetic individuals, fasting
`EGR was within what may be defined as the normal range.
`Presumably, a 10% reduction in fasting EGR (⬃10 g
`glucose overnight) shall have an effect on fasting glycemic
`
`levels. It is also reasonable to assume that a much greater
`decrease in EGR will be present during daytime, taking
`into consideration the importance of dynamic changes in
`circulating glucagon on hepatic glucose handling in the
`prandial state (4,32,33).
`Our interest was also in exploring the influence of
`liraglutide treatment on the contribution of GNG and GLY
`to EGR. The method allowed us to examine only fasting
`glucose kinetics. Several authors reported that the ele-
`vated EGR of individuals with type 2 diabetes is mainly
`attributable to an augmented GNG (2,3,34,35). The current
`study showed that the restraining effect of the GLP-1
`derivative liraglutide on fasting EGR was solely due to
`inhibition of GLY, fasting GNG being unchanged. The
`opposite result might have been suspected, namely a
`reduction of GNG. However, studies, e.g., by Cherrington
`et al. (36), have demonstrated that the inhibitory effect of
`physiological insulin concentrations (in contrast to supra-
`physiological
`levels) on hepatic glucose production is
`primarily mediated through decreased GLY, whereas GNG
`
`1192
`
`FIG. 5. Concentration profiles of liraglutide. Dosing
`time was 0730. Gray lines indicate individual concen-
`tration profiles; black line indicates mean ⴞ SEM.
`
`DIABETES, VOL. 53, MAY 2004
`
`MPI EXHIBIT 1081 PAGE 6
`
`

`

`is less sensitive to the direct action of insulin. Similarly,
`the stimulatory effect of glucagon on hepatic glucose
`release is primarily due to glycogen breakdown, whereas
`the hormone has less effect on GNG (36,37). Very high
`portal insulin levels are capable of suppressing hepatic
`GNG, but this may primarily be due to an indirect effect via
`decreased lipolysis and hence plasma FFA levels (36).
`Again, although there was no significant change in the
`concentrations of fasting levels of insulin and glucagon,
`these were relatively augmented or decreased, respec-
`tively, considering the lowered level of glycemia. Further-
`more, peripheral blood does not necessarily precisely
`reflect portal concentration. The effect of GLP-1/GLP-1
`derivatives on GLY and GNG in patients with type 2
`diabetes and more deranged glycemic control, i.e., with a
`substantial elevation of EGR, remains unknown.
`Progressive impaired ␤-cell function leading to insulin
`deficiency is a key feature of type 2 diabetes (1,38). Both a
`reduction in ␤-cell mass and a functional abnormality of
`the islet cells seem to be responsible for the islet cell’s
`deterioration (39). It seems to be an ongoing process
`hardly influenced by concomitant glucose lowering treat-
`ment (38). Loss of first-phase insulin response to glucose is
`an early ␤-cell defect, followed by a weakened basal and
`second-phase insulin output as the disease progresses
`(39,40). This, combined with an abnormal circadian gluca-
`gon secretory pattern (5,33) and diminished ability of
`hyperglycemia to suppress glucagon secretion, demon-
`strates the necessity for opposing both the ␣- and ␤-cell
`malfunction in the treatment of type 2 diabetes.
`Improved ␤-cell function in patients with type 2 diabetes
`has been reported after 6 –12 h of GLP-1 infusion as
`assessed by first- and second-phase insulin response (41).
`A 6-week study demonstrated similar effects on ␤-cell
`function, strongly indicating maintenance of this beneficial
`GLP-1 action (12). We found a dramatic improvement in
`␤-cell function after 8 days of liraglutide treatment. All
`␤-cell function tests were substantially ameliorated, indi-
`cating an effect on first- and second-phase insulin response
`to glucose as well as (near) maximal insulin secretory
`capacity. In addition, we found an almost 50% reduction of
`the proinsulin/insulin ratio in the circulation during both
`fasting and stimulation. The latter observation may indi-
`cate an improved processing of insulin in the ␤-cell. In
`fact, in the current study, liraglutide treatment almost led
`to a normalized fasting proinsulin/insulin ratio. An ele-
`vated proinsulin/insulin ratio is a cardinal feature in type 2
`diabetes and the pre-diabetic state (42,43) and is a well-
`established indicator of ␤-cell dysfunction (44). Further-
`more, liraglutide treatment led to an almost doubling of
`the disposition index, which reflects the ability of the
`␤-cells to adapt to the contemporary insulin resistance
`(glucose allostasis) (45), again emphasizing the powerful
`effects of GLP-1/GLP-1 derivatives on the ␤-cell.
`Another important impact of liraglutide on pancreatic
`glucoregulatory function was the significant inhibition of
`glucagon secretion during hyperglycemia and in particular
`during arginine infusion, demonstrating that liraglutide
`efficiently improves the secretory pattern