The GLP-1 Derivative NN2211 Restores ␤-Cell
`Sensitivity to Glucose in Type 2 Diabetic Patients
`After a Single Dose
`
`Annette M. Chang,1 Grethe Jakobsen,2 Jeppe Sturis,2 Marla J. Smith,1 Cathie J. Bloem,1 Bob An,3
`Andrzej Galecki,4 and Jeffrey B. Halter1
`
`Glucagon-like peptide 1 (GLP-1) stimulates insulin se-
`cretion in a glucose-dependent manner, but its short
`half-life limits its therapeutic potential. We tested
`NN2211, a long-acting GLP-1 derivative, in 10 subjects
`with type 2 diabetes (means ⴞ SD: age 63 ⴞ 8 years, BMI
`30.1 ⴞ 4.2 kg/m2, HbA1c 6.5 ⴞ 0.8%) in a randomized,
`double-blind, placebo-controlled, crossover study. A
`single injection (7.5 ␮g/kg) of NN2211 or placebo was
`administered 9 h before the study. ␤-cell sensitivity was
`assessed by a graded glucose infusion protocol, with
`glucose levels matched over the 5–12 mmol/l range.
`Insulin secretion rates (ISRs) were estimated by decon-
`volution of C-peptide levels. Findings were compared
`with those in 10 nondiabetic volunteers during the same
`glucose infusion protocol. In type 2 diabetic subjects,
`NN2211, in comparison with placebo, increased insulin
`and C-peptide levels, the ISR area under the curve
`(AUC) (1,130 ⴞ 150 vs. 668 ⴞ 106 pmol/kg; P < 0.001),
`and the slope of ISR versus plasma glucose (1.26 ⴞ 0.36
`vs. 0.54 ⴞ 0.18 pmol 䡠 l[minⴚ1 䡠 mmolⴚ1 䡠 kgⴚ1]; P <
`0.014), with values similar to those of nondiabetic
`control subjects (ISR AUC 1,206 ⴞ 99; slope of ISR
`versus plasma glucose, 1.44 ⴞ 0.18). The long-acting
`GLP-1 derivative, NN2211, restored ␤-cell responsive-
`ness to physiological hyperglycemia in type 2 diabetic
`subjects. Diabetes 52:1786 –1791, 2003
`
`Glucagon-like peptide 1 (GLP-1) is a hormone
`
`that stimulates insulin secretion and simulta-
`neously decreases glucagon secretion (1,2).
`The insulinotropic effect is glucose dependent.
`Because GLP-1 stimulates insulin secretion primarily at
`elevated glucose levels, it is possible that GLP-1 therapy of
`type 2 diabetes might present a low risk of hypoglycemia
`(3). GLP-1 might also decrease hepatic glucose production
`indirectly (4), delay gastric emptying, and suppress appe-
`
`From the 1Department of Internal Medicine, University of Michigan, Ann
`Arbor, Michigan; 2Novo Nordisk A/S, Bagsvaerd, Denmark; 3Novo Nordisk
`Pharmaceuticals, Inc., Princeton, New Jersey; and the 4Institute of Gerontol-
`ogy, University of Michigan, Ann Arbor, Michigan.
`Address correspondence and reprint requests to Jeffrey B. Halter, MD,
`University of Michigan, 1111 CCGC Bldg., 1500 East Medical Center Dr., Ann
`Arbor, MI 48109-0926. E-mail: jhalter@umich.edu.
`G.J., J.S., and B.A. hold stock in Novo Nordisk A/S.
`Received for publication 29 October 2002 and accepted in revised form 8
`April 2003.
`ADA, American Diabetes Association; AUC, area under the curve; ELISA,
`enzyme-linked immunosorbent assay; GCRC, General Clinical Research Cen-
`ter; GLP-1, glucagon-like peptide 1; ISR, insulin secretion rate.
`© 2003 by the American Diabetes Association.
`
`tite in type 2 diabetic patients (5). This array of effects
`gives GLP-1 the potential to be an efficacious and safe
`glucose-lowering agent for type 2 diabetes. In addition,
`GLP-1 has been shown to stimulate the differentiation of
`islet progenitor cells into insulin-producing cells and may
`be important for ␤-cell neogenesis (6).
`Short-term (12-h) infusion of GLP-1 as well as 6-week
`continuous subcutaneous infusion of GLP-1 has been
`shown to significantly improve insulin secretion in type 2
`diabetic patients (7,8). However, native GLP-1 has a very
`short half-life because of its rapid degradation by dipepti-
`dyl peptidase IV and, thus, is unlikely to be used as a
`therapeutic drug in diabetes treatment. NN2211 is an
`acylated derivative of GLP-1 with full agonistic activity at
`the GLP-1 receptor in vitro (9). NN2211 is slowly degraded
`because of a combination of albumin binding, metabolic
`stability, and gradual release from the injection site. Phar-
`macokinetic profiles in healthy volunteers and type 2
`diabetic subjects have shown that NN2211 is suitable for
`once-daily injection (10 –12). NN2211, in a single dose of 10
`␮g/kg, has been found to effectively reduce fasting and
`postprandial hyperglycemia, delay gastric emptying, and
`suppress prandial glucagon secretion in type 2 diabetic
`patients (12).
`In the present study, we assessed the effect of a single
`subcutaneous injection of NN2211 on ␤-cell sensitivity to
`glucose using a graded glucose infusion protocol in a
`group of adults with type 2 diabetes. The trial was a
`randomized, double-blind, placebo-controlled crossover
`trial. The degree of improvement of ␤-cell function was
`also assessed by comparison with a control group of
`healthy volunteers of similar age who did not receive the
`drug.
`
`RESEARCH DESIGN AND METHODS
`Type 2 diabetic subjects. The protocol was approved by the University of
`Michigan Institutional Review Board and performed in accordance with the
`Declaration of Helsinki. All subjects gave their informed consent after the
`nature of the study was explained in detail to them. Type 2 diabetic subjects
`(n ⫽ 10; 6 men, 4 women) were evaluated in a randomized, double-blind,
`placebo-controlled, two-period crossover trial. Baseline clinical characteris-
`tics for diabetic and control subjects are summarized in Table 1. All diabetic
`subjects met American Diabetes Association (ADA) criteria for type 2 diabetes
`with a fasting plasma glucose ⱖ7.0 mmol/l or a 2-h plasma glucose ⱖ11.1
`mmol/l, determined by oral glucose tolerance testing. Subjects were treated
`for a minimum of 2 months with diet therapy or 3 months of oral hypoglyce-
`mic monotherapy before entering the study. Of the 10 subjects, 9 were treated
`with oral hypoglycemic agents (sulfonylurea, n ⫽ 4; metformin, n ⫽ 4;
`␣-glucosidase inhibitor, n ⫽ 1) and 1 was treated with diet alone. Treatment
`
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`TABLE 1
`Clinical characteristics of diabetic and control subjects
`
`n
`Age (years)
`Sex (male/female)
`Weight (kg)
`BMI (kg/m2)
`HbA1c (%)
`Duration of diabetes (years)
`
`Data are mean ⫾ SD.
`
`Diabetic
`subjects
`
`10
`62.9 ⫾ 7.6
`6/4
`88.1 ⫾ 18.9
`30.1 ⫾ 4.2
`6.5 ⫾ 0.8
`5.4 ⫾ 5.7
`
`Control
`subjects
`
`10
`61.3 ⫾ 7.3
`5/5
`75.2 ⫾ 13.6
`26.0 ⫾ 2.8
`5.1 ⫾ 0.4
`—
`
`with a sulfonylurea was discontinued 1 week before study days and other oral
`antidiabetic agents were discontinued 1 day before study days.
`Subjects ate their usual evening meal and then fasted after 20:00 on the day
`before study days. They arrived at the University of Michigan General Clinical
`Research Center (GCRC) at 20:00. Catheters were inserted in antecubital veins
`and contralateral hand/wrist veins for blood sampling and infusion purposes.
`At 23:00, 7.5 ␮g/kg of NN2211 or placebo were injected subcutaneously into
`the abdomen in random order using NovoPen 1.5 with NovoFine needles.
`There was a 3– 6 week interval between dosing periods. Insulin was adminis-
`tered as a low-dosage (0.5–5.0 units/h) continuous infusion overnight to
`ensure baseline glucose levels were near normal and comparable before the
`graded glucose infusion protocol during each study day.
`A graded glucose infusion protocol was initiated at 08:00 the following day.
`The aim of this procedure was to assess insulin secretion in response to
`gradually raising plasma glucose levels from ⬃5 to 12 mmol/l over 3 h.
`Subjects were studied in the supine position. The antecubital intravenous
`catheter was used for insulin and glucose infusions. The second intravenous
`catheter in a dorsal hand/wrist vein of the contralateral arm was placed into
`a warming box heated to 60°C to obtain arterialized blood samples for glucose
`and insulin. After an initial baseline sample was taken, a small intravenous
`dose of insulin (0.007– 0.014 units/kg) was administered to lower the glucose
`level to ⬃5 mmol/l. The insulin was allowed to decay (20 min after bolus
`insulin), after which samples were drawn at 5-min intervals for 15 min to
`define baseline levels of glucose,
`insulin, and C-peptide. An intravenous
`infusion of 20% dextrose was then started. Every 5 min, samples were drawn
`for determination of insulin, C-peptide, and glucose. For each of the 5-min
`samples, plasma glucose was measured using a Beckman glucose analyzer
`(Beckman, Palo Alto, CA), and the glucose infusion rate was adjusted
`accordingly to gradually raise plasma glucose levels from 5 to 12 mmol/l over
`3 h. Periodic additional blood samples were drawn for glucagon and NN2211
`concentrations.
`Normal control subjects. Healthy subjects (n ⫽ 10; 5 men, 5 women) who
`did not receive the trial medication also underwent the graded glucose
`infusion protocol to provide reference data on ␤-cell function. All control
`subjects met ADA criteria for normal glucose tolerance with a fasting plasma
`glucose ⱕ6.1 mmol/l and 2-h plasma glucose ⬍7.8 mmol/l, as determined with
`an oral glucose tolerance test. Subjects were selected to approximate the
`demographic characteristics of the diabetic patients. As shown in Table 1, the
`diabetic and control subjects were well matched except for BMI, which was
`higher in diabetic subjects. The control subjects were admitted to the GCRC
`the morning after an overnight fast. The graded glucose infusion protocol was
`carried out as described for the diabetic patients, except control subjects did
`not receive the overnight insulin infusion or the trial medication.
`Assays. Plasma glucose was measured immediately at bedside with a
`Beckman glucose analyzer using the glucose oxidase technique. HbA1c was
`measured by high-performance liquid chromatography, with a normal range of
`3.8 – 6.4%. All other blood samples were centrifuged and serum was stored at
`–20°C until analysis. Serum insulin was quantified using a highly specific and
`sensitive two-site enzyme-linked immunosorbent assay (ELISA) with an
`interassay coefficient of variation of 3% (DAKO Diagnostics, Cambridgeshire,
`U.K.). C-peptide was measured by a two-site monoclonal-based ELISA with an
`interassay coefficient of variation (in triplicate) of 3% (DAKO Diagnostics).
`Glucagon was assessed by radioimmunoassay (GL-32K; Linco Research, St.
`Charles, MO.). NN2211 concentrations were analyzed by ELISA using a
`capture monoclonal antibody against GLP-1/NN2211 and a detection mono-
`clonal antibody specific for the NH2-terminal portion of GLP-1/NN2211. Before
`pharmacokinetic analysis, the samples were incubated at 37°C to remove
`endogenous GLP-1, as NN2211 is stable with incubation. The mean accuracy
`(recovery) of the NN2211 assay has been reported as 102% (range 93–113%)
`(11).
`
`A.M. CHANG AND ASSOCIATES
`
`FIG. 1. Profile of plasma glucose concentrations during graded glucose
`infusion studies after a single injection of NN2211 (f) or placebo (䡺)
`given 9 h before the study in type 2 diabetic subjects. Results are
`compared with those of healthy control subjects (‚) who did not
`receive the drug. Baseline glucose values were normalized by overnight
`insulin infusion in diabetic subjects. Glucose levels during variable
`rate glucose infusion begun at time 0 were well matched during the
`NN2211 and placebo studies in diabetic subjects and were also matched
`with values for control subjects from 80 to 220 min. Data are means ⴞ
`SE, n ⴝ 10 for each group.
`
`Statistical analysis. The primary objective of the efficacy analysis was to
`compare the effects of NN2211 versus placebo on ␤-cell responsiveness to
`graded glucose infusion as assessed by the insulin secretion rate (ISR) area
`under the curve (AUC) over the 40- to 220-min time interval of graded glucose
`infusion studies. The ISR during the graded glucose infusion protocol was
`derived by deconvolution of peripheral C-peptide concentrations and previ-
`ously determined C-peptide kinetics (13,14). The total ISR over the 40- to
`220-min time interval was estimated for each subject by calculating the AUC
`using the trapezoidal rule.
`The secondary objectives of the efficacy analysis were to compare NN2211
`with placebo with respect to the following: 1) slope of the ISR versus plasma
`glucose level for each subject (estimated by a regression model of ISR on
`plasma glucose level), 2) glucagon AUC over the 40- to 220-min time interval
`(calculated in a similar way to that of ISR AUC), and 3) insulin clearance
`(mean ISR divided by mean insulin concentration).
`Adverse events were recorded on the treatment days. All efficacy end
`points for NN2211 versus placebo were analyzed using an ANOVA model for
`the crossover design. Two-sided tests were performed with P ⫽ 0.05 as the
`level of significance. Results for diabetic subjects are given as NN2211 versus
`placebo.
`
`RESULTS
`Plasma glucose, insulin, and C-peptide levels. Profiles
`of plasma glucose, insulin, and C-peptide during graded
`glucose infusion studies after NN2211 or placebo admin-
`istration in diabetic subjects are displayed in Figs. 1 and 2.
`Results are compared with those of healthy control sub-
`jects who did not receive the drug. Fasting plasma glucose
`levels in diabetic subjects at initiation of the graded
`glucose infusion protocol (9 h after dosing and overnight
`insulin infusion) were slightly lower with NN2211 than
`with placebo (6.28 ⫾ 0.29 vs. 7.03 ⫾ 0.39 mmol/l). How-
`ever, glucose levels were well matched in diabetic subjects
`over the 40- to 220-min time interval of the graded glucose
`infusion protocols, which was included in ISR AUC deter-
`mination, and were also matched with control subjects
`over the 80- to 220-min time interval. Variable glucose
`infusion rates were used to achieve the matched glucose
`levels during graded glucose infusion studies, with mean
`total glucose infusion rates of 233 ⫾ 13 ml with NN2211
`and 167 ⫾ 11 ml with placebo in the diabetic subjects (P ⬍
`0.001) and 338 ⫾ 16 ml in the control subjects.
`Fasting levels of insulin (90 ⫾ 41 vs. 70 ⫾ 26 pmol/l) and
`C-peptide (0.8 ⫾ 0.1 vs. 0.7 ⫾ 0.1 nmol/l) were similar for
`NN2211 and placebo. Insulin and C-peptide levels signifi-
`
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`GLP-1, TYPE 2 DIABETES, AND INSULIN
`
`FIG. 2. Profiles of plasma insulin (A) and C-peptide (B) concentrations
`during graded glucose infusion studies after a single injection of
`NN2211 (f) or placebo (䡺) in type 2 diabetic subjects. Results are
`compared with those of healthy control subjects (‚) who did not
`receive the drug. Insulin and C-peptide levels significantly increased in
`response to the graded hyperglycemic stimulus after NN2211 compared
`with after placebo (P < 0.001 for insulin AUC and C-peptide AUC) to
`values similar to those of healthy control subjects. Data are means ⴞ
`SE, n ⴝ 10 for each group.
`
`cantly increased in response to the graded hyperglycemic
`stimulus after NN2211 compared with placebo in diabetic
`subjects (P ⬍ 0.0001 for both insulin and C-peptide AUC).
`Values of insulin and C-peptide with NN2211 in diabetic
`subjects were similar to those of healthy control subjects
`who did not receive the drug.
`Insulin secretion. As shown in Fig. 3, the ISR increased
`substantially with NN2211 administration compared with
`placebo during the graded glucose infusion studies over
`
`FIG. 3. Relation between ISR and plasma glucose levels during the
`graded glucose infusion protocol in type 2 diabetic subjects who
`received NN2211 (f) or placebo (䡺). ISR was derived by deconvolu-
`tion of peripheral C-peptide concentrations. ISR was substantially
`increased with NN2211 compared with placebo over the glucose range
`6 –12 mmol/l and was similar to values in healthy control subjects (‚)
`who did not receive the drug. Data are means ⴞ SE, n ⴝ 10 for each
`group.
`
`the glucose range 6 –12 mmol/l, and was similar to values
`in healthy control subjects who did not receive the drug.
`The effects of NN2211 in enhancing insulin secretory
`response became evident after 40 – 60 min of glucose
`infusion, when the plasma glucose levels reached 6 –7
`mmol/l. The drug effect was increasingly apparent as
`plasma glucose levels increased further to 12 mmol/l, thus
`displaying the glucose-dependent action of NN2211. As
`summarized in Fig. 4, ␤-cell sensitivity to glucose as
`assessed by the ISR AUC was increased by ⬃70% with
`NN2211 treatment (1,130 ⫾ 150 vs. 668 ⫾ 106 pmol/kg; P ⬍
`0.001). The ISR AUC with NN2211 in diabetic subjects was
`similar to values in healthy control subjects who did not
`receive the drug (1,206 ⫾ 99). The ISR AUC was an
`estimate of the total amount of insulin secreted per
`kilogram of body weight over the 40- to 220-min time
`interval of the graded glucose infusion studies. The effect
`of NN2211 on the slope of ISR versus plasma glucose level
`is summarized in Fig. 5. The slope of ISR versus plasma
`glucose level increased by ⬃133% with NN2211 (1.26 ⫾
`0.36 vs. 0.54 ⫾ 0.18 pmol 䡠 l[min⫺1] 䡠 mmol⫺1 䡠 kg⫺1]; P ⬍
`0.014). The slope of ISR versus plasma glucose level with
`NN2211 in diabetic subjects was comparable to the value
`in healthy control subjects (1.44 ⫾ 0.18).
`The higher glucose infusion rate with NN2211 versus
`placebo in diabetic subjects was compatible with the
`greater ISR after NN2211. However, the glucose infusion
`rate in diabetic subjects remained considerably less than
`that of healthy control subjects, whereas serum insulin
`curves were virtually superimposable. Thus, the diabetic
`subjects were likely to have a greater degree of insulin
`resistance compared with the healthy control subjects,
`despite similar insulin secretory responses in the two
`groups. This observation was compatible with reduced
`sensitivity to endogenously secreted insulin in the diabetic
`subjects compared with healthy control subjects.
`Other measures. As shown in Fig. 6, glucagon levels were
`similar with NN2211 and placebo administration in the
`fasting state and did not change over time in the diabetic
`subjects. The glucagon AUC remained unchanged with
`NN2211 compared with placebo (14,665 ⫾ 1,350 vs.
`15,761 ⫾ 1,788 pg 䡠 min⫺1 䡠 ml⫺1; P ⫽ 0.24). Plasma
`glucagon levels were higher in the fasting state and during
`graded glucose infusion studies in diabetic subjects com-
`pared with control subjects, although the glucagon AUC
`was not significantly different (11,827 ⫾ 768 pg 䡠 ml⫺1 in
`healthy control subjects; P ⫽ 0.11). Insulin clearance
`(mean ISR divided by mean insulin concentration) was
`similar with NN2211 and placebo (0.06 ⫾ 0.01 vs. 0.05 ⫾
`0.01 l 䡠 min⫺1 䡠 kg⫺1; P ⫽ 0.35). The pharmacokinetic
`profile of NN2211 was evaluated by 10-point profiles for up
`to 17 h after dosing. The AUC of NN2211 plasma concen-
`tration from 0 to 17 h was 70,742 ⫾ 19,256 pmol/l, the Cmax
`of NN2211 was 5,884 ⫾ 1,778 pmol/l, and the Tmax of
`NN2211 was 13.1 ⫾ 2.8 h. The t1/2 was not determined
`because of the limited duration of postdosing profiling and
`because this parameter has been reported in previous
`studies with NN2211 in healthy (t1/2 ⫽ 11–15 h) and
`diabetic subjects (t1/2 ⫽ 10.0 ⫾ 3.5 h) (10 –12).
`Adverse events. All diabetic and control subjects com-
`pleted the study. As expected, no hypoglycemic events
`occurred. Only one diabetic subject experienced a gastro-
`
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`A.M. CHANG AND ASSOCIATES
`
`FIG. 4. ISR AUC during the 40- to 220-min
`time interval of graded glucose infusion stud-
`ies in diabetic subjects treated with placebo
`(䡺) or NN2211 (f) and in control subjects
`(o) who did not receive the drug. After
`NN2211 injection, the ISR AUC was signifi-
`cantly increased in diabetic subjects com-
`pared with after placebo (*P < 0.001) and
`was similar to that of control subjects. Data
`are means ⴞ SE, n ⴝ 10 for each group.
`
`intestinal side effect of mild diarrhea on the day of active
`treatment; that subject was able to complete the study
`protocol and had resolution of gastrointestinal symptoms
`before discharge. A second diabetic subject experienced
`mild headache on the day of active treatment; that subject
`also was able to complete the study and had resolution of
`the headache before initiation of the graded glucose
`infusion protocol.
`
`DISCUSSION
`In this study, we investigated the effect of a single dose of
`NN2211, a long-acting GLP-1 derivative, on ␤-cell respon-
`siveness to physiological hyperglycemia in a group of
`adults with type 2 diabetes. GLP-1 is a potent glucose-
`dependent,
`insulinotropic hormone that is of potential
`interest for the treatment of type 2 diabetes. GLP-1 admin-
`istered by continuous infusion and repeated subcutaneous
`injection has been shown to significantly reduce fasting
`and postprandial hyperglycemia in type 2 diabetic patients
`(2,3,5).
`Continuous infusion of GLP-1 for 12 h has been shown
`to improve basal and stimulated ␤-cell function, as as-
`sessed by hyperglycemic clamp and arginine stimulation in
`type 2 diabetic patients (7). A 6-week continuous sub-
`cutaneous infusion of GLP-1 (compared with saline infu-
`sion) has been shown to significantly improve insulin
`secretion, as assessed by the hyperglycemic clamp method
`in 10 type 2 diabetic subjects, and also to decrease fasting
`glucose, HbA1c, and fructosamine levels (8). In a recent
`study, GLP-1 infusion also increased insulin secretory
`response to graded glucose infusion in a dosage-depen-
`dent manner in type 2 diabetic and healthy control sub-
`jects (15). However, the rapid degradation of GLP-1 and
`
`the need for continuous infusion prevent the hormone’s
`broader clinical use. In contrast, NN2211 is a slowly
`degraded GLP-1 analog that has been found to be suitable
`for once-daily dosing.
`In the present study, a single dose of NN2211 (compared
`with placebo) significantly increased insulin and C-peptide
`levels and substantially improved the overall insulin secre-
`tory response to a controlled, gradual increase of glucose
`levels over a physiological range during the glucose infu-
`sion protocol in type 2 diabetic subjects. NN2211 was
`administered the night before study days to achieve max-
`imum concentration of the drug during the graded glucose
`infusion protocol the following morning. One advantage of
`this study was the careful matching of glucose levels over
`time, which allowed quantitative comparisons of insulin
`secretion with placebo versus with NN2211, and also
`comparison with a nondiabetic control group over the
`same glucose range. In addition, insulin secretion was
`assessed in response to physiological postprandial glucose
`levels, which contrasts with the high nonphysiological
`glucose levels achieved with the hyperglycemic clamp
`method.
`The effects of NN2211 in enhancing the insulin secretory
`response became evident after 40 – 60 min of glucose
`infusion when the plasma glucose levels reached 6 –7
`mmol/l. The drug effect was increasingly apparent as
`plasma glucose levels increased further to 12 mmol/l. The
`glucose level dependency of the effect of NN2211 on
`insulin secretion agrees with findings of GLP-1 studies in
`normal (6,17) and diabetic subjects (3). The lack of effect
`of NN2211 on insulin secretion at euglycemia indicates
`that this drug might not lead to inappropriate insulin
`secretion, which could limit the risk of hypoglycemia in
`
`DIABETES, VOL. 52, JULY 2003
`
`FIG. 5. Slope of ISR versus glucose during
`graded glucose infusion studies in diabetic
`subjects treated with placebo (䡺) or NN2211
`(f) and in control subjects (o) who did not
`receive the drug. After NN2211 injection,
`slope of ISR was significantly increased in
`diabetic subjects compared with after pla-
`cebo (*P < 0.014) and was similar to that of
`control subjects. Data are means ⴞ SE, n ⴝ
`10 for each group.
`
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`GLP-1, TYPE 2 DIABETES, AND INSULIN
`
`FIG. 6. Profile of plasma glucagon (A) and glucagon AUC (B) during
`graded glucose infusion studies. A: Plasma glucagon in diabetic sub-
`jects treated with placebo (䡺) or NN2211 (f) and in control subjects
`(‚) who did not receive the drug. Plasma glucagon levels in diabetic
`subjects were similar with NN2211 and placebo and did not change over
`time. B: Glucagon AUC remained unchanged with NN2211 (f) com-
`pared with placebo (䡺) in diabetic subjects (P ⴝ 0.24). Plasma
`glucagon levels were higher in diabetic subjects compared with control
`subjects (o), although glucagon AUC was not significantly different
`(P ⴝ 0.11). Data are means ⴞ SE, n ⴝ 10 for each group.
`
`type 2 diabetic patients. Administration of subcutaneous
`GLP-1 and intravenous glucose has been shown to induce
`reactive hypoglycemia in healthy volunteers, but not in
`type 2 diabetic subjects (18). However, long-term clinical
`intervention studies are needed to test this hypothesis.
`The insulin and C-peptide levels and insulin secretory
`response achieved with NN2211 in diabetic subjects were
`remarkably similar to those of healthy control subjects
`with normal glucose tolerance who did not receive the
`drug. The restoration of insulin secretion dynamics has
`not been observed with oral hypoglycemic agents in type 2
`diabetic patients (19,20). Thus, the dramatic improvement
`of insulin responses during graded hyperglycemia after
`NN2211 is encouraging. However, this comparison does
`not take into account the likely greater insulin resistance
`in diabetic subjects, given their higher BMI, increased
`fasting insulin levels, and decreased glucose infusion rates
`needed to achieve similar glucose and insulin levels during
`study. At a comparable level of insulin resistance, individ-
`uals with normal ␤-cells would likely secrete much more
`insulin in response to a challenge. Thus, despite the
`dramatic improvement of insulin secretion in the diabetic
`subjects after NN2211, it is likely that their ␤-cell function
`remained impaired.
`Impairment of islet sensitivity to glucose is an early
`abnormality of ␤-cell function, as demonstrated in studies
`of families with maturity-onset diabetes of the young.
`Subjects who have glucokinase mutations with elevated
`fasting and postprandial glucose levels were found to have
`similar first-phase insulin response to intravenous glucose
`
`tolerance testing compared with nondiabetic control sub-
`jects (21). However,
`insulin secretion rates were 61%
`lower with graded glucose infusion studies in which
`glucose infusion rates were increased in a stepwise fash-
`ion. Our subjects with relatively well-controlled type 2
`diabetes also had a very poor insulin secretion response to
`graded glucose infusion (see placebo values in Figs. 3–5
`compared with those of nondiabetic control subjects).
`In a previous study, a single injection of NN2211 was
`found to reduce fasting and postprandial hyperglycemia,
`suppress prandial glucagon secretion, and delay gastric
`emptying in type 2 diabetic patients (12). This study also
`evaluated insulin secretion in response to a standard meal.
`Postprandial insulin secretion was only slightly and insig-
`nificantly increased with NN2211; however, the interpre-
`tation of these findings is difficult as postprandial glucose
`levels were also substantially reduced with NN2211 com-
`pared with placebo. In contrast, in the current study, the
`insulin secretory response increased dramatically with
`NN2211 compared with placebo when patients were stud-
`ied while glucose levels were carefully matched.
`A limitation of this study was its short duration, in that
`the effect of only a single injection of NN2211 was tested.
`Clearly, a longer-term study is needed to define the effec-
`tiveness of NN2211 in enhancing insulin secretion in type
`2 diabetic patients. Another limitation was the overall
`good glycemic control of the patients studied. The pres-
`ence of residual ␤-cell function in these patients may have
`contributed to the observed dramatic effect of NN2211
`(although ␤-cell function was grossly impaired in the
`absence of NN2211). The effectiveness of NN2211 on
`insulin secretion in patients with poorly controlled type 2
`diabetes needs to be assessed. All of the diabetic subjects
`in the current study were treated with oral monotherapy,
`except one subject who was treated through diet. A
`possible carryover effect of sulfonylurea treatment on
`insulin secretion should have been limited by discontinu-
`ation of these agents 1 week before the studies. In
`addition, the metformin used by four subjects and the
`␣-glucosidase inhibitor used by one subject were withheld
`1 day before the studies; however, these agents are not
`known to have direct effects on ␤-cell function. In the
`current study, effects on fasting hyperglycemia could not
`be assessed, as diabetic subjects received an overnight
`insulin infusion to achieve comparable glucose levels
`before initiation of graded glucose infusion studies. We
`observed similar glucagon levels with intravenous glucose
`stimulation with NN2211 and placebo, in contrast to the
`meal-related suppression of glucagon levels seen with
`NN2211 (12) and GLP-1 (2). Because hyperglycemia tends
`to suppress glucagon secretion, the current protocol was
`not well suited to testing for a possible suppressive effect
`of NN2211 on glucagon secretion. In addition, because this
`study did not compare the effects of GLP-1 and NN2211, it
`cannot be concluded that NN2211 will be as efficacious as
`GLP-1 in all circumstances.
`In summary, during controlled, matched hyperglycemia
`in patients with well-controlled type 2 diabetes, a single
`dose of NN2211 (compared with placebo) increased insu-
`lin and C-peptide levels and dramatically improved insulin
`secretory response to glucose, as assessed by the in-
`creased ISR AUC and the increased slope of ISR versus
`
`1790
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`DIABETES, VOL. 52, JULY 2003
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`MPI EXHIBIT 1080 PAGE 5
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`

`A.M. CHANG AND ASSOCIATES
`
`plasma glucose level. NN2211 was well tolerated, without
`evidence of hypoglycemia or significant side effects, and
`the pharmacokinetic properties should allow convenient
`once-daily dosing.
`We conclude that acute administration of the long-
`acting GLP-1 derivative, NN2211, restores ␤-cell respon-
`siveness to physiological hyperglycemia in patients with
`type 2 diabetes. NN2211 has been shown to have the
`potential beneficial effects of improving insulin secretion,
`decreasing fasting and postprandial hyperglycemia, de-
`creasing prandial glucagon secretion, and delaying gastric
`emptying. Long-term studies are needed to elucidate the
`full therapeutic potential of NN2211.
`
`ACKNOWLEDGMENTS
`This work was supported by University of Michigan GCRC
`Grant M01-RR0042; Novo Nordisk A/S (to J.B.H.); the
`Michigan Diabetes Research and Training Center; the
`Veterans Affairs Geriatric Research, Education and Clini-
`cal Center, Ann Arbor, Michigan; and the John A. Hartford
`Foundation.
`We would like to thank the study participants for their
`cooperation and commitment and the University of Mich-
`igan GCRC nurses and staff for their assistance.
`
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