E m e r g i n g T r e a t m e n t s a n d T e c h n o l o g i e s
`O R I G I N A L
`A R T I C L E
`
`Pharmacokinetics, Pharmacodynamics,
`Safety, and Tolerability of a Single-Dose
`of NN2211, a Long-Acting Glucagon-
`Like Peptide 1 Derivative, in Healthy
`Male Subjects
`
`1
`BODIL ELBRØND, MD
`GRETHE JAKOBSEN, MSC
`1
`SØREN LARSEN, MSC
`1
`HENRIK AGERSØ, PHD
`LISBETH BJERRING JENSEN, PHD
`
`1
`
`1
`
`2
`PAUL ROLAN, MD
`1
`JEPPE STURIS, PHD
`1
`VIBEKE HATORP, MSC
`MILAN ZDRAVKOVIC, PHD
`
`1
`
`OBJECTIVE — The primary objective of the present study was to investigate the safety, tolerabil-
`ity, and pharmacokinetics of a single dose of NN2211, a long-acting glucagon-like peptide 1
`(GLP-1) derivative, in healthy male subjects. The secondary objective was to investigate the
`pharmacodynamics of NN2211.
`
`RESEARCH DESIGN AND METHODS — In a double-blind, randomized dose, escala-
`tion, placebo-controlled study, healthy male subjects were enrolled at eight consecutive dose levels
`(1.25, 2.5, 5.0, 10.0, 12.5, 15.0, 17.5, and 20.0 ␮g/kg) with eight subjects per dose level at a 3:1
`active:placebo randomization. After subcutaneous dosing with NN2211, 48-h pharmacokinetic, and
`24-h glucose, insulin and glucagon profiles were assessed. In addition, three subjects at each dose
`level were randomly assigned (one placebo/two active) to an intravenous glucose tolerance test
`(IVGTT) 9 h after the dose (corresponding to the time to maximal plasma concentration of NN2211).
`RESULTS — After subcutaneous administration, the half-life of NN2211 was found to be
`11–15 h. Overall, although there were no statistically significant differences compared with
`placebo in the area under the curve (0 –9 h for insulin or glucagon), there was a borderline-
`significant lowering of glucose levels (P ⫽ 0.066). During the IVGTT, there was a statistically
`significant increase in insulin secretion (P ⫽ 0.0002), but there was no significant effect on
`glucagon levels. Although no significant effect was observed on glucose levels during the IVGTT,
`there was a dose-dependent increase in the glucose disappearance constant. Whereas no serious
`adverse events were observed, there was a higher incidence of adverse events after active treat-
`ment compared with placebo treatment (notably headache, dizziness, nausea, and vomiting).
`
`CONCLUSIONS — This study provides evidence that NN2211 has a pharmacokinetic pro-
`file consistent with once-daily dosing in humans.
`
`Diabetes Care 25:1398 –1404, 2002
`
`G lucagon-like peptide 1 (GLP-1) is a
`
`sess antidiabetogenic effects in both
`animal models and patients with type 2
`polypeptide hormone secreted
`diabetes (2–7). The mechanism of action
`from the L-cells in the gastrointes-
`rests on a suite of effects: notably, a glu-
`tinal tract (1) that has been shown to pos-
`● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
`
`From 1Novo Nordisk A/S, Health Care Development, Bagsvaerd, Denmark; and 2Medeval Ltd., Manchester
`Science Park, Manchester, U.K.
`Address correspondence and reprint requests to Milan Zdravkovic, PhD, Novo Nordisk A/S, Health Care
`Development, Novo Alle´, 2880 Bagsvaerd, Denmark. E-mail: mzd@novonordisk.com.
`Received for publication 30 October 2001 and accepted in revised form 2 May 2002.
`B.E., G.J., S.L., H.A., L.B.J., J.S., V.H., and M.Z. hold stock in Novo Nordisk A/S.
`Abbreviations: AUC, area under the curve; Cmax, maximal plasma concentration; DPP-IV, dipeptidyl
`peptidase IV; ECG, electrocardiogram; GLP-1, glucagon-like peptide 1; IVGTT, intravenous glucose toler-
`ance test; Kg, glucose disappearance constant.
`A table elsewhere in this issue shows conventional and Syste`me International (SI) units and conversion
`factors for many substances.
`
`cose-dependent stimulation of insulin se-
`cretion (8),
`inhibition of glucagon
`secretion (9), inhibition of gastric empty-
`ing (10), and a decrease in appetite
`(11,12). In addition, a direct stimulation
`of growth and proliferation of the ␤-cells
`has recently been demonstrated in mice
`(13). Taken together, these effects suggest
`that GLP-1 is a promising candidate for
`treating type 2 diabetes. However, the
`rapid clearance and degradation of GLP-1
`by dipeptidyl peptidase IV (DPP-IV) re-
`sults in a half-life of ⬃1 h in humans after
`subcutaneous administration, compro-
`mising its therapeutic potential as a new
`treatment modality (2).
`It has previously been demonstrated
`that derivatization of insulin with fatty ac-
`ids facilitates binding to serum albumin
`and provides a protracted action (14,15).
`This approach has also been reported to
`result in prolongation of the half-life of
`GLP-1 after subcutaneous administration
`in dogs (16). NN2211 is an acylated long-
`acting GLP-1 derivative, which, in pre-
`clinical experiments, has shown
`prolonged pharmacokinetic properties
`compared with the native GLP-1 (17)
`while maintaining its biological action
`both in vitro (17) and in vivo (18 –20).
`The primary objective of the present
`study was to investigate the safety, toler-
`ability, and pharmacokinetics of a single
`dose of NN2211 in healthy male subjects,
`and the secondary objective was to inves-
`t i g a t e t h e p h a r m a c o d y n a m i c s o f
`NN2211.
`
`RESEARCH DESIGN AND
`METHODS
`
`Study protocol
`The protocol was approved by the Inde-
`pendent Ethics Committee in Manches-
`ter, U.K., and conducted in accordance
`with the Helsinki Declaration and Good
`Clinical Practice. Subjects consented to
`
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`DIABETES CARE, VOLUME 25, NUMBER 8, AUGUST 2002
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`MPI EXHIBIT 1083 PAGE 1
`
`

`

`participating in the study in writing after a
`full explanation of the study had been
`given.
`
`Subjects
`Subjects were recruited from a volunteer
`database kept at Medeval Ltd. A total of 72
`healthy male subjects in good general
`health, as verified by medical history,
`physical examination, clinical chemistry,
`hematology, and electrocardiogram
`(ECG), aged 18 – 45 years with BMI
`20 –27 kg/m2, were included in this
`study. Mean age was 27 years (range 19 –
`41) and weight 74.5 kg (range 58.0 –
`98.0).
`
`Study design
`This was a single-center, randomized,
`double-blind, placebo-controlled, single-
`dose, sequential-dose level escalation
`study. Eight different dose levels of
`NN2211 were studied: 1.25 (performed
`twice), 2.5, 5.0, 10.0, 12.5, 15.0, 17.5,
`and 20.0 ␮g/kg with eight subjects at each
`dose level, and a 3:1 active:placebo ran-
`domization within each dose level. Sub-
`jects were only enrolled at one dose level.
`In addition, at each dose level, three sub-
`jects were randomly assigned (one pla-
`cebo/two active) to receive a 2-h intra-
`venous glucose tolerance test (IVGTT).
`Finally, to determine the absolute bio-
`availability of NN2211, all eight subjects
`allocated at the 5 ␮g/kg subcutaneous
`dose level returned after a 7-day washout
`to receive an intravenous dose of 5 ␮g/kg
`NN2211 or placebo.
`
`Study medication and blinding
`NN2211 at a concentration of 5 mg/ml or
`matching placebo, manufactured in ac-
`cordance with good manufacturing prac-
`tice, was provided in cartridges. These
`cartridges fit into the NovoPen 1.5, which
`was used for subcutaneous dose adminis-
`tration. The medication was labeled in ac-
`cordance with the blinded status of the
`study. For the intravenous administration
`of NN2211/placebo performed at the 5
`␮g/kg dose level to determine the abso-
`lute bioavailability, a dilution of the trial
`medication was performed correspond-
`ing to 0.1 mg/ml. The intravenous infu-
`sion was performed over 1 h (to avoid
`high peak plasma concentrations), with
`an infusion rate calculated based on the
`weight of the individual subject. Sealed
`codes were kept at the trial site and at
`Novo Nordisk in the U.K. and Denmark.
`
`A copy of the code was provided only to
`the responsible person performing the
`analysis of the concentration of NN2211
`in blood and was not further disclosed
`(because this analysis would unblind the
`study anyway, it was done to reduce the
`number of placebo samples analyzed).
`The code was only broken after database
`lock but could have been broken for indi-
`vidual subjects in case of an emergency.
`
`Experimental procedures
`The subjects were admitted to the clinic
`on the day before dose administration and
`given a standardized meal at ⬃7:00 P.M.
`on the day of admittance and at 6:00 A.M.
`the following day. NN2211 or placebo
`was administered between 8:00 and
`10:00 A.M. as a single subcutaneous dose
`in the abdomen. At the first dose level
`(1.25 ␮g/kg), the IVGTT was started 5 h
`postdose; however, because blinded
`pharmacokinetic data showed that the
`time to maximal plasma concentration of
`NN2211 was 9 h in humans, the protocol
`was amended to start the 2-h IVGTT at 9 h
`postdose in all subsequent dose levels, in-
`cluding a repeat of the 1.25 ␮g/kg dose
`level. A standardized meal was served
`11 h after dosing, i.e., after completion of
`the IVGTT. Blood samples for determina-
`tion of the concentration of NN2211 were
`collected at regular intervals from before
`dosing to 48 h after dosing. Furthermore,
`a predose to 24-h postdose glucose, insu-
`lin, and glucagon profile was obtained in
`all subjects. In subjects randomized to
`undergo the IVGTT, additional blood
`samples for a 2-h glucose, insulin, and
`glucagon profile were drawn, i.e., be-
`tween 9 and 11 h after dosing. All subjects
`were monitored for safety during the
`study. Adverse events either observed by
`the investigator or reported spontane-
`ously by the subjects were recorded. In
`addition, subjects were asked at predose
`and at 1, 2, 4, 6, 8, 10, 12, 14, 16, 24, and
`48 h postdose if they had had any adverse
`events (including any changes in concom-
`itant illness or new illnesses) since the last
`evaluation. Clinical laboratory tests (he-
`matology, biochemistry, and urinalysis)
`were carried out before, during, and after
`dosing. Blood glucose was measured at
`regular intervals after dose administration
`and before discharge using a HemoCue
`device. Physical examination, ECG (be-
`fore dosing and 4, 8, 12, 16, 24, and 36 h
`after dosing), and vital signs, including
`blood pressure, pulse, and temperature,
`
`Elbrønd and Associates
`
`were recorded. Urine volumes were as-
`sessed for the period 0 –24 h after dose
`administration. At the 5-␮g/kg dose level,
`the subjects returned after 1 week for the
`intravenous administration of NN2211/
`placebo. At this visit, all procedures were
`performed as described above, with the
`exception of the IVGTT, which was not
`performed.
`
`Laboratory procedures
`The concentration of NN2211 was deter-
`mined by a validated two-site immuno-
`assay using a capturing antibody
`(GLPB1F1) and a detection antibody (bi-
`otin-labeled Mab26.1) as described previ-
`ously (21). The selectivity of the assay
`toward NN2211 compared with endoge-
`nous GLP-1 was ensured by removing en-
`dogenous GLP-1 activity by incubating
`the samples for 4 h at 37°C before per-
`forming the assay. In addition, the inter-
`ference from a number of peptides was
`investigated. NN2211 (7-37) isomer in-
`terfered positively but ⬍4%, NN2211 (9-
`37) isomer had negative interference
`⬍12%, GLP-1 (15-37) had negative inter-
`ference ⬍3%, and major pro-glucagon
`fragment had negative interference ⬍4%.
`Because all of these interferences were
`low, and the concentrations of the pep-
`tides in the majority of the samples were
`low compared with the NN2211 concen-
`trations, interference was considered ir-
`relevant. Serum glucose was measured on
`a Beckman CX5 Delta analyzer using the
`glucose oxidase technique. Serum insulin
`was analyzed using the Abbott IMx assay,
`which shows ⬍0.005% cross-reactivity
`with human pro-insulin, and glucagon
`was analyzed with Linco’s glucagon ra-
`dioimmunoassay in accordance with the
`manufacturer’s instructions.
`
`Statistical analysis
`Because of the exploratory status of this
`study (first administration to humans),
`the sample size of eight subjects per dose
`level was not based on formal statistical
`considerations but was considered suffi-
`cient to meet the primary objectives of the
`study. A significance level of 5% was
`used. Results from the statistical analysis
`are presented with the mean and 95% CI.
`Safety. Urine volume (0 –24 h) was an-
`alyzed by ANOVA with the following
`model: the response for each subject was
`the sum of an overall mean, a fixed dose
`effect (placebo ⫽ zero dose), a random
`block effect of the dose level groups (in-
`
`DIABETES CARE, VOLUME 25, NUMBER 8, AUGUST 2002
`
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`MPI EXHIBIT 1083 PAGE 2
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`

`

`Single-dose study of NN2211 in healthy male subjects
`
`NN2211 plasma concentration time pro-
`file for the single-dose subcutaneous and
`intravenous administration. Dose propor-
`tionality for subcutaneous dosing was as-
`sessed for area under the curve (AUC) to
`infinity and maximal plasma concentra-
`tion (Cmax) by performing linear regres-
`sion analysis on the log-transformed
`parameters versus log-transformed dose.
`An estimate of the slope of the regression
`line and corresponding 95% CI was cal-
`culated. Dose proportionality was as-
`sumed if the slope was not statistically
`significantly different from unity. Of the
`54 subjects exposed to active treatment, 3
`and 2 subjects were excluded at the 1.25-
`␮g/kg (first cohort only) and 5-␮g/kg
`dose levels, respectively. This exclusion
`was due to dosing problems (low dosing
`volume) that caused low exposure when
`compared with other subjects at the same
`dose level.
`Pharmacodynamics. Only pharmaco-
`dynamic data after subcutaneous admin-
`istration were included in the statistical
`analysis. The AUC0 –9 h (all subjects) and
`AUC9 –10 h and AUC9 –11 h (IVGTT-
`treated subjects only) for glucose, gluca-
`gon, and insulin were derived from the
`respective profiles, applying the trapezoi-
`dal rule, and log-transformed. The glu-
`cose disappearance constant (Kg) was
`calculated by linear regression analysis of
`natural log-transformed glucose data
`from the IVGTT between 10 and 60 min
`after glucose administration. For each in-
`dividual subject, the Kg was defined as the
`negative slope of the regression line. In
`one subject receiving placebo, the glucose
`concentration did not peak until 15 min,
`and the 10-min data point was therefore
`omitted for that subject. In another sub-
`
`Figure 1—Mean pharmacokinetic profiles after subcutaneous administration of eight different
`dose levels of NN2211 to healthy male volunteers (A) and a scatter plot showing dose versus AUC
`of NN2211 after subcutaneous administration (B).
`
`cluding the two placebo subjects), and a
`random error. All 72 subjects enrolled
`were included.
`
`Pharmacokinetics. The pharmacoki-
`netic parameters were assessed by non-
`compartmental methods from the 48-h
`
`Table 1—Pharmacokinetic parameters after NN2211 administration to healthy male subjects
`
`Clearance
`(ml 䡠 min⫺1 䡠 kg⫺1)
`
`Volume of
`distribution (l/kg)
`
`t1/2* (h)
`
`Dose (␮g/kg)
`
`Absolute
`bioavailability (%)
`
`AUC
`(n 䡠 nmol⫺1 䡠 l⫺1)
`tmax (h)
`Cmax (nmol/l)
`9.6 ⫾ 1.9
`0.49 ⫾ 0.19
`15 ⫾ 6
`14
`—
`—
`—
`1.25 s.c.†
`10 ⫾ 3.4
`2.3 ⫾ 0.70
`60 ⫾ 29
`11
`—
`—
`—
`2.5 s.c.
`0.07 ⫾ 0.01
`0.10 ⫾ 0.02
`216 ⫾ 43
`8.1
`—
`—
`—
`5.0 i.v.
`9.3 ⫾ 3.2
`4.5 ⫾ 3.4
`55 ⫾ 37
`134 ⫾ 108
`15
`—
`—
`5.0 s.c.‡
`12 ⫾ 6.0
`7.9 ⫾ 2.7
`235 ⫾ 33
`14
`—
`—
`—
`10.0 s.c.
`11 ⫾ 2.4
`10 ⫾ 2.7
`283 ⫾ 31
`12
`—
`—
`—
`12.5 s.c.
`11 ⫾ 1.1
`13 ⫾ 2.0
`384 ⫾ 57
`13
`—
`—
`—
`15.0 s.c.
`11 ⫾ 1.0
`17 ⫾ 3.0
`451 ⫾ 65
`11
`—
`—
`—
`17.5 s.c.
`10 ⫾ 1.3
`20 ⫾ 3.0
`523 ⫾ 105
`11
`—
`—
`—
`20.0 s.c.
`Data are mean pharmacokinetic parameters ⫾ SD after intravenous and subcutaneous administration of NN2211 to healthy male subjects (n ⫽ 6 unless otherwise
`indicated). tmax, time to maximal plasma concentration; t1/2, half-life. *Harmonic mean; † n ⫽ 9; ‡ n ⫽ 4.
`
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`

`

`Elbrønd and Associates
`
`curred from 10 and 15 ␮g/kg, respec-
`tively. At the highest dose level, 6 of 6
`subjects experienced nausea and/or vom-
`iting compared with 2 subjects experienc-
`ing nausea and 1 experiencing vomiting
`out of 18 subjects in the placebo-treated
`group. There were no clinically signifi-
`cant changes in vital signs, ECG parame-
`ters, physical examination, or safety
`laboratory parameters (hematology, bio-
`chemistry, and urinalysis). There was a
`tendency toward lower urine volumes at
`doses ⱖ12.5 ␮g/kg, but there was no
`overall significant difference in urine vol-
`umes 0 –24 h after dose administration
`between active and placebo treatment
`(mean ⫺167; 95% CI [⫺650 to 316]).
`Figure 1A shows the mean plasma
`concentration time profiles of NN2211
`after subcutaneous administration, and
`the derived pharmacokinetic parameters
`are given in Table 1. NN2211 was slowly
`absorbed after subcutaneous administra-
`tion, with maximum plasma concentra-
`tions obtained ⬃9 –12 h after dosing and
`a subsequent mean elimination half-life in
`the range of 11–15 h. After intravenous
`administration, NN2211 was shown to
`have a low volume of distribution (0.07
`l/kg) and a mean elimination half-life of
`8.1 h. The absolute bioavailability based
`on the intravenous and subcutaneous
`crossover administration of 5.0 ␮g/kg was
`found to be 55%. Figure 1B shows a plot
`of AUC of NN2211 versus dose, indicat-
`ing a linear relationship. However, statis-
`tical analysis showed linearity only in
`the dose range of 2.5–20 ␮g/kg for both
`Cmax (mean slope 1.07 [95% CI 0.91–
`1.24]) and AUC (1.09 [0.94 –1.24]) but
`not in the range of 1.25–20 ␮g/kg (1.28
`[1.17–1.40] and 1.26 [1.15–1.37] for
`Cmax and AUC, respectively).
`The mean 24-h insulin, glucose, and
`glucagon time profiles are shown in Figs.
`2A, 3A, and 4A, respectively. Further-
`more, the derived AUC0 –9 h/9 h is shown
`in Table 2. Overall, in the period before
`the first meal and the IVGTT correspond-
`ing to AUC0 –9 h, there was a borderline-
`significant lowering of glucose levels (P ⫽
`0.066; mean 0.97 [95% CI 0.94 –1.00])
`but no statistically significant difference
`between active and placebo treatment for
`insulin (0.97 [0.81–1.16]) or glucagon
`(0.98 [0.91–1.05]). Furthermore, in con-
`nection with the meal (corresponding to
`11-h postdose) and at doses ⬎10.0 ␮g/
`kg, there was a tendency toward lower
`insulin and glucose levels after active
`
`Figure 2—A: Mean concentration time profiles of insulin after NN2211/placebo administration
`to healthy male subjects (subjects randomized to the IVGTT have not been included in the figure)
`(n ⫽ 4 in all dose groups except the placebo, where n ⫽ 8, and at the 5.0 ␮g/kg dose level, where
`n ⫽ 3). B: IVGTT profiles of insulin after NN2211/placebo administration to healthy male subjects
`(n ⫽ 2 in all dose groups except the placebo, where n ⫽ 7, and at the 5.0 ␮g/kg dose level, where
`n ⫽ 1).
`
`ject receiving placebo, the glucose con-
`centration had rebounded markedly at
`the 60-min time point, which was there-
`fore omitted for that subject. Exclusion of
`these data points represents a conserva-
`tive approach for the assessment of a pos-
`sible positive effect of NN2211 on Kg. In
`the statistical model for end points, the
`response for each subject was the sum of
`an overall mean, a fixed-dose effect (pla-
`cebo ⫽ zero dose), a random block effect
`of the dose level groups (including the
`one or two placebo subjects), and a ran-
`dom error, and the data were analyzed
`using ANOVA. All subjects from the first
`cohort of 1.25 ␮g/kg were excluded be-
`cause of the timing of the IVGTT. For the
`AUC0 –9 h glucose, insulin, and glucagon
`analysis, two subjects at 5.0 ␮g/kg were
`excluded because of low exposure, and
`
`one additional subject was excluded be-
`cause he did not fast (randomized to pla-
`cebo at the 17.5-␮g/kg dose level), giving
`a total population of 61 subjects. Because
`no comparison to placebo could be made,
`all subjects receiving an IVGTT at the
`17.5-␮g/kg dose level were additionally
`excluded in the AUC9 –11 h analysis, giv-
`ing a total population of 20 subjects.
`
`RESULTS — N o s e r i o u s a d v e r s e
`events were reported, and all subjects
`completed the study. There was a higher
`number of adverse events reported in
`subjects on active treatment versus pla-
`cebo treatment. The higher frequency was
`noted for headache, dizziness, nausea,
`and vomiting. Whereas headache and
`dizziness occurred on the majority of dose
`levels, nausea and vomiting mainly oc-
`
`DIABETES CARE, VOLUME 25, NUMBER 8, AUGUST 2002
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`

`Single-dose study of NN2211 in healthy male subjects
`
`estimate 0.0713 ⫾ 0.0238 [mean ⫾ SE]),
`such that increasing doses of NN2211 in-
`creased the Kg value. However, the statis-
`tical analysis did not show a significant
`overall difference between NN2211 and
`placebo treatment (P ⫽ 0.16; 0.58
`[⫺0.26 to 1.42]),
`indicating that
`NN2211 had an effect on Kg at higher, but
`not at lower, doses.
`
`CONCLUSIONS — The observations
`in this study of an increase in gastrointes-
`tinal side effects and dizziness after
`NN2211 administration are in accor-
`dance with previously published observa-
`tions after both GLP-1(7-36) amide
`infusion and NN2211 administration to
`healthy male subjects and patients with
`type 2 diabetes (3,9,21,22). The mecha-
`nism behind the occurrence of nausea
`and vomiting is most likely mediated via
`inhibition of gastric emptying (11,12,23,
`24).
`Native GLP-1 is a polypeptide that
`undergoes rapid metabolism by DPP-IV
`and, in addition, is rapidly cleared by
`renal elimination (1). The half-life after
`intravenous or subcutaneous administra-
`tion in humans has been reported to be
`⬃5 min (1) and 1 h (2), respectively.
`Findings in this study show an elimina-
`tion half-life of 8.1 h after intravenous ad-
`ministration of NN2211. This increase in
`the half-life of NN2211 compared with
`native GLP-1 is most likely mediated via a
`combination of factors. Notably, NN2211
`has been shown to have a lower suscepti-
`bility to metabolism by DPP-IV (25) and a
`high degree of albumin binding of
`NN2211 (as has been shown for other
`fatty acid derivatives [14,15]), and after
`subcutaneous administration, an addi-
`tional prolongation of the half-life is me-
`diated by a slow absorption of NN2211
`from the injection site, as evidenced by
`the further increase in half-life observed
`from subcutaneous to intravenous ad-
`ministration. Thus, the pharmacokinetic
`terminal half-life of NN2211 after the
`subcutaneous administration observed in
`this study is in accordance with previous
`findings in pigs, where the half-life was
`found to be 14 h (17), and studies with
`NN2211 in humans (21,22) and strongly
`supports once-daily administration of
`NN2211. In addition to improvements in
`compliance and convenience to the pa-
`tients by the once-daily formulation, an
`increase in exposure from 16 to 24 h of
`native GLP-1 showed further improve-
`
`Figure 3—A: Mean concentration time profiles of glucose after NN2211/placebo administration
`to healthy male subjects (subjects randomized to the IVGTT have not been included in the figure)
`(n ⫽ 4 in all dose groups except the placebo, where n ⫽ 8, and at the 5.0 ␮g/kg dose level, where
`n ⫽ 3). B: IVGTT profiles of glucose after NN2211/placebo administration to healthy male
`subjects (n ⫽ 2 in all dose groups except the placebo, where n ⫽ 7, and at the 5.0 ␮g/kg dose level,
`where n ⫽ 1).
`
`treatment. Concentration time profiles for
`insulin, glucose, and glucagon during the
`IVGTT (9 –11 h after dose) are shown in
`Figs. 2B, 3B, and 4B, respectively. Fur-
`thermore, the derived AUC9 –10 h/1 h and
`AUC9 –11 h/2 h are shown in Table 2, i.e.,
`corresponding to the first hour of the
`IVGTT and the complete 2-h IVGTT. Al-
`though there was a tendency toward
`higher glucose levels within the first 5
`min after the initiation of the IVGTT in
`the actively treated subjects, a lowering of
`glucose occurred from 30 to 120 min in
`the higher-dose groups. There was a clear
`dose-dependent increase in insulin se-
`cretion during the IVGTT, but there was
`no effect on glucagon levels. Statistical
`analysis showed a significant increase in
`insulin levels after NN2211 administra-
`
`tion during the IVGTT (AUC9 –11 h) (P ⫽
`0.0002; mean 2.29 [95% CI 1.80 –2.92])
`but no significant effect on glucose (0.97
`[0.82–1.14]) or glucagon (1.13 [0.89 –
`1.42]). Statistical analysis of the AUC9 –10 h
`during the IVGTT showed a similar pat-
`tern to that for the complete IVGTT.
`Thus, there was a statistically significant
`increase in insulin levels in the NN2211-
`treated group compared with the placebo
`group (P ⫽ 0.0002; 2.74 [1.75– 4.28]),
`whereas no significant effect on glucose
`(1.05 [0.89 –1.23]) or glucagon (1.06
`[0.87–1.29]) was observed. The effect of
`NN2211 on intravenous glucose toler-
`ance, as assessed by Kg up to 1 h after
`glucose administration, is shown in Table
`2. There was a clear and significant dose-
`response relationship (P ⫽ 0.0078; slope
`
`1402
`
`DIABETES CARE, VOLUME 25, NUMBER 8, AUGUST 2002
`
`MPI EXHIBIT 1083 PAGE 5
`
`

`

`Elbrønd and Associates
`
`1.98⫾1.11
`1.05⫾0.085
`1.00⫾0.92
`Kg(%/min)
`
`2.75⫾0.71
`
`NA
`
`2.66⫾1.03
`2.39⫾0.14
`1.58⫾0.23
`
`1.24
`
`67.3⫾29.8
`
`NA
`
`49.7⫾6.58
`77.9⫾7.42
`62.3⫾4.17
`
`81.4
`
`55.0⫾1.13
`63.4⫾0.57
`57.7⫾11.9
`
`(pg/ml)
`Glucagon
`
`h,andn⫽7fortheremainingparameters(AUC9–11h/2h,AUC9–10h/1h,andKg).†n⫽4fortheAUC0–9h/9h,andn⫽1fortheremainingparameters(AUC9–11h/2h,AUC9–10h/1h,andKg).
`Dataaremeans⫾SD(n⫽6unlessotherwiseindicated).ForsubjectsreceivingtheIVGTT,theAUC9–10h/1h,AUC9–11h/2h,andKgareshown(n⫽2unlessotherwiseindicated).*n⫽15fortheAUC0–9h/9
`20.0␮g/kg
`17.5␮g/kg
`15.0␮g/kg
`12.5␮g/kg
`10.0␮g/kg
`5.0␮g/kg†
`2.5␮g/kg
`1.25␮g/kg
`Placebo*
`
`AUC9–11h/2h(IVGTTsubjects)
`
`AUC9–10h/1h(IVGTTsubjects)
`
`AUC0–9h/9h
`
`Table2—Glucose,insulin,andglucagonAUC0–9h/9inallsubjects
`
`Figure 4—A: Mean concentration time profiles of glucagon after NN2211/placebo administration
`to healthy male subjects (subjects randomized to the IVGTT have not been included in the figure)
`(n ⫽ 4 in all dose groups except the placebo, where n ⫽ 8, and at the 5.0 ␮g/kg dose level, where
`n ⫽ 3). B: IVGTT profiles of glucagon after NN2211/placebo administration to healthy male
`subjects (n ⫽ 2 in all dose groups except the placebo, where n ⫽ 7, and at the 5.0 ␮g/kg dose level,
`where n ⫽ 1).
`
`ments in glycemic control, indicating the
`therapeutic importance of full 24-h
`GLP-1 exposure (26).
`In the present study, a borderline sig-
`nificant lowering in 0 –9 h glucose levels
`after NN2211 administration was ob-
`served, whereas no significant effect on
`0 –9 h insulin or glucagon levels was
`found. This assessment period was cho-
`sen because the subjects were fasted ex-
`cept for water (before IVGTT and meal).
`Therefore, the previously reported effects
`in healthy subjects on inhibition of gastric
`emptying by GLP-1 causing a lower meal-
`stimulated insulin secretion outweighing
`the stimulation on insulin secretion
`caused by GLP-1 would not be present
`(27). This blunting effect was observed in
`
`this study in connection with the meals at
`⬃11 h after dosing. Previous studies with
`GLP-1 infusion in healthy subjects have
`shown an acute effect on insulin and glu-
`cose during fasting conditions (4,5),
`whereas the present study only demon-
`strated a borderline significant effect on
`glucose. The basis for this difference re-
`mains speculative but could relate to dif-
`ferences in administration form, notably
`intravenous versus subcutaneous, or sam-
`pling frequency differences. However, in
`accordance with previous findings in
`both animal models and humans (28),
`significant increases in insulin levels were
`observed during the IVGTT in this study.
`Even though the initial higher glucose
`values observed during the IVGTT, for
`
`43.3⫾6.16
`
`NA
`
`20.5⫾2.00
`30.5⫾8.16
`35.7⫾13.0
`
`38.1
`
`31.7⫾4.39
`16.2⫾0.51
`13.4⫾4.7
`(mU/l)
`Insulin
`
`6.30⫾1.60
`6.05⫾0.40
`7.81⫾0.071
`
`6.03⫾1.00
`
`NA
`
`7.94
`
`7.18⫾0.88
`8.76⫾0.67
`7.34⫾1.05
`(mmol/l)
`Glucose
`
`60.9⫾24.4
`
`NA
`
`46.4⫾5.4
`68.7⫾14.0
`59.6⫾1.18
`
`74.8
`
`50.1⫾1.75
`62.3⫾0.38
`55.9⫾11.8
`
`78.1⫾14.2
`
`NA
`
`35.4⫾8.22
`54.8⫾15.4
`54.0⫾19.4
`
`53.8
`
`43.0⫾0.77
`19.8⫾0.32
`17.5⫾8.23
`
`8.14⫾1.21
`
`NA
`
`8.83⫾2.79
`7.89⫾0.88
`10.5⫾0.42
`
`10.0
`
`9.23⫾0.94
`9.53⫾0.82
`8.90⫾1.40
`
`75.7⫾18.1
`82.2⫾13.9
`70.8⫾16.2
`87.8⫾16.0
`81.0⫾9.18
`78.5⫾17.0
`69.9⫾8.82
`75.4⫾10.4
`72.1⫾12.4
`
`6.32⫾1.85
`12.1⫾2.01
`7.47⫾3.68
`7.71⫾2.52
`7.74⫾3.79
`7.27⫾2.11
`5.00⫾1.78
`7.25⫾3.61
`8.67⫾4.53
`
`4.25⫾0.21
`4.22⫾0.32
`4.16⫾0.37
`4.27⫾0.33
`4.42⫾0.29
`4.54⫾0.25
`4.42⫾0.23
`4.78⫾0.27
`4.50⫾0.32
`
`(pg/ml)
`Glucagon
`
`(mU/l)
`Insulin
`
`(mmol/l)
`Glucose
`
`(pg/ml)
`Glucagon
`
`(mU/l)
`Insulin
`
`(mmol/l)
`Glucose
`
`DIABETES CARE, VOLUME 25, NUMBER 8, AUGUST 2002
`
`1403
`
`MPI EXHIBIT 1083 PAGE 6
`
`

`

`Single-dose study of NN2211 in healthy male subjects
`
`which the explanation is not known,
`could have confounded the observed
`stimulation of insulin secretion, the effect
`on insulin was clearly dose related (in
`contrast to the glucose increase). Al-
`though the increased insulin secretion
`mediated by NN2211 did not signifi-
`cantly decrease glucose levels, there was a
`dose-dependent increase in the glucose
`disappearance constant after NN2211 ad-
`ministration, indicating that the lack of a
`measurable effect on glucose was not
`caused by NN2211 decreasing insulin
`sensitivity. In a recent study with
`NN2211 in patients with type 2 diabetes,
`NN2211 was found to reduce fasting and
`meal-related glycemia. This effect was
`found to be mediated via an increase in
`insulin secretion rate, suppression of glu-
`cagon secretion, and a delay in gastric
`emptying (22). Thus, in contrast to the
`present study, suppression of glucagon
`secretion was found in type 2 diabetic pa-
`tients after NN2211 administration.
`Taken together, these data indicate that
`the pharmacodynamic response of
`NN2211 in patients with type 2 diabetes
`is more pronounced that that in healthy
`subjects and also corroborates the glu-
`cose-dependent mechanism of action of
`GLP-1 and NN2211.
`
`Acknowledgments — Parts of this study were
`presented in abstract form at the American Di-
`abetes Association Annual Meeting and Scien-
`tific Sessions, Philadelphia, Pennsylvania,
`22–26 June 2001.
`
`References
`1. Kiefer TJ, Habener JF: The glucagon-like
`peptides. Endocr Rev 20:876 –913, 1999
`2. Gutniak MK, Linde B, Holst JJ, Efendic S:
`Subcutaneous injection of the incretin
`hormone GLP-1 abolishes postprandial
`glycaemia in NIDDM. Diabetes Care 17:
`1039 –1044, 1994
`3. Nauck MA, Wollschla¨ger D, Werner J,
`Holst JJ, Orskov C, Creutzfeldt W, Willms
`B: Effects of subcutaneous GLP-1 [7-36
`amide] in patients with NIDDM. Diabeto-
`logia 39:1546 –1553, 1996
`4. Nathan DM, Schreiber E, Fogel H, Mojsov
`S, Habener JF: Insulinotropic action of
`glucagonlike peptide-1-(7-37) in diabetic
`and nondiabetic subjects. Diabetes Care
`15:270 –276, 1992
`5. Qualman C, Nauck M, Holst JJ, Ørskov C,
`Creutzfeldt W: Insulinotropic actions of
`intravenous glucagon-like-peptide [7-36
`amide] in the fasting state in healthy sub-
`jects. Acta Diabetol 32:13–16, 1995
`6. Toft-Nielsen MB, Madsbad S, Holst JJ:
`
`Continuos subcutaneous infusion of glu-
`cagon-like-peptide 1 lowers plasma glu-
`cose and reduces appetite in type 2
`diabetic patients. Diabetes Care 22:1137–
`1143, 1999
`7. Nauck MA, Kleine N, Ørskov C, Holst JJ,
`Willms B, Creutzfeldt W: Normalisation
`of fasting hyperglycaemia by exogenous
`GLP-1 [7-36 amide] in type 2 diabetic pa-
`tients. Diabetologia 36:741–744, 1993
`8. Kreymann B, Williams G, Ghatei MA,
`Bloom SR: Glucagon-like-peptide 1 7-36:
`a physiological incretin in man. Lancet
`2:1300 –1304, 1987
`9. Ritzel R, Ørskov C, Holst JJ, Nauck MA:
`Pharmacokinetic, insulinotropic, and glu-
`cagonostatic properties of GLP-1 [7-36
`amide] after subcutaneous injection in
`healthy volunteers: dose relationships.
`Diabetologia 38:720 –725, 1995
`10. Wettergren A, Schjoldager B, Mortensen
`PE, Myhre J, Christiansen J, Holst JJ:
`Truncated GLP-1 (proglucagon 72–107
`amide) inhibits gastric and pancreatic func-
`tion in man. Dig Dis Sci 38:665–673, 1993
`11. Na¨slund E, Gutniak MK, Skogar S, Ro¨ ss-
`ner S, Hellstro¨ m PM: GLP-1 increases the
`period of postprandial satiety and slows
`gastric emptying in obese humans. Am J
`Clin Nutr 68:525–530, 1998
`12. Na¨slund E, Barkeling B, King N, Gutniak
`M, Blundell JE, Holst JJ, Rossner S, Hell-
`strom PM: Energy intake and appetite are
`suppressed by glucagon-like-peptide 1
`(GLP-1) in obese men. Int J Obes Relat
`Metab Disord 23:304 –311, 1999
`13. Edvell A, Lindstro¨ m P: Initiation of in-
`creased pancreatic islet growth in young
`normoglycaemic mice (Umeå ⫹/?). Endo-
`crinology 140:778 –783, 1999
`14. Kurtzhals P, Havelund S, Jonassen I,
`Kiehr B, Larsen UD, Ribel U, Markussen J:
`Albumin-binding of
`insulins acylated
`with fatty acids: characterization of the li-
`gand protein-interaction and correlation
`between binding-affinity and timing of
`the insulin effect in-vivo. Biochem J 312:
`725–731, 1995
`15. Markussen J, Havelund S, Kurtzhals P,
`Andersen AS, Halstrom J, Hasselager E,
`Larsen UD, Ribel U, Schaffer L, Vad K,
`Jonassen I: Soluble fatty-acid acylated in-
`sulins bind to albumin and show pro-
`tracted action in pigs. Diabetologia
`39:281–288, 1996
`16. Chou JZ, Place GD, Waters DG, Kirk-
`wood JA, Bowsher RR: A radioimmunoas-
`say for LY315902, an analogue of glu-
`cagon-like insulinotropic peptide, and its
`application in the trial of canine pharma-
`cokinetics. J Pharm Sci 86:768 –773, 1997
`17. Knu