Systemic Administration of the Long-Acting GLP-1
`Derivative NN2211 Induces Lasting and Reversible
`Weight Loss in Both Normal and Obese Rats
`
`Philip J. Larsen,1,2 Christian Fledelius,3 Lotte Bjerre Knudsen,4 and Mads Tang-Christensen1
`
`Postprandial release of the incretin glucagon-like pep-
`tide-1 (GLP-1) has been suggested to act as an endoge-
`nous satiety factor in humans. In rats, however, the
`evidence for this is equivocal probably because of very
`high endogenous activity of the GLP-1 degrading en-
`zyme dipeptidyl peptidase-IV. In the present study, we
`show that intravenously administered GLP-1 (100 and
`500 ␮g/kg) decreases food intake for 60 min in hungry
`rats. This effect is pharmacologically specific as it is
`inhibited by previous administration of 100 ␮g/kg ex-
`endin(9-39), and biologically inactive GLP-1(1-37) had
`no effect on food intake when administered alone (500
`␮g/kg). Acute intravenous administration of GLP-1 also
`caused dose-dependent inhibition of water intake, and
`this effect was equally well abolished by previous ad-
`ministration of exendin(9-39). A profound increase in
`diuresis was observed after intravenous administration
`of both 100 and 500 ␮g/kg GLP-1. Using a novel long-
`acting injectable GLP-1 derivative, NN2211, the acute
`and subchronic anorectic potentials of GLP-1 and deriv-
`atives were studied in both normal rats and rats made
`obese by neonatal monosodium glutamate treatment
`(MSG). We showed previously that MSG-treated ani-
`mals are insensitive to the anorectic effects of centrally
`administered GLP-1(7-37). Both normal and MSG-
`lesioned rats were randomly assigned to groups to
`receive NN2211 or vehicle. A single bolus injection of
`NN2211 caused profound dose-dependent inhibition of
`overnight food and water intake and increased diuresis
`in both normal and MSG-treated rats. Subchronic mul-
`tiple dosing of NN2211 (200 ␮g/kg) twice daily for 10
`days to normal and MSG-treated rats caused profound
`inhibition of food intake. The marked decrease in food
`intake was accompanied by reduced body weight in both
`groups, which at its lowest stabilized at ⬃85% of initial
`body weight. Initial excursions in water intake and
`diuresis were transient as they were normalized within
`a few days of treatment. Lowered plasma levels of
`triglycerides and leptin were observed during NN2211
`treatment in both normal and MSG-treated obese rats.
`
`From the 1Laboratory of Obesity Research, Center for Clinical and Basic
`Research, Ballerup, Denmark; and 2Neuroendocrine Pharmacology, 3Pharma-
`cological Research 2, and 4Molecular Pharmacology, Novo Nordisk A/S,
`Copenhagen, Denmark.
`Address correspondence and reprint requests to Philip J. Larsen, Dr, MSci,
`Laboratory of Obesity Research, Centre for Clinical and Basic Research,
`Ballerup Byvej 222, 2750 Ballerup, Denmark. E-mail: pjl@ccbr.dk.
`Received for publication 24 January 2001 and accepted in revised form 26
`July 2001.
`L.B.K and C.F. are employees of and hold stock in Novo Nordisk.
`ANOVA, analysis of variance; DEXA, dual energy X-ray absorptiometry;
`DPP-IV, dipeptidyl peptidase-IV; EE, energy expenditure; FFA, free fatty acids;
`GLP-1, glucagon-like peptide-1; MSG, monosodium glutamate; RER, respira-
`tory exchange ratio; TG, triacylglycerol.
`
`In a subsequent study, a 7-day NN2211 treatment period
`of normal rats ended with measurement of energy ex-
`penditure (EE) and body composition determined by
`indirect calorimetry and dual energy X-ray absorptiom-
`etry, respectively. Compared with vehicle-treated rats,
`NN2211 and pair-fed rats decreased their total EE
`corresponding to the observed weight loss, such that EE
`per weight unit of lean body mass was unaffected.
`Despite its initial impact on body fluid balance, NN2211
`had no debilitating effects on body water homeostasis as
`confirmed by analysis of body composition, plasma elec-
`trolytes, and hematocrit. This is in contrast to pair-fed
`animals, which displayed hemoconcentration and ten-
`dency toward increased percentage of fat mass. The
`present series of experiments show that GLP-1 is fully
`capable of inhibiting food intake in rats via a peripher-
`ally accessible site. The loss in body weight is accompa-
`nied by decreased levels of circulating leptin indicative
`of loss of body fat. The profound weight loss caused by
`NN2211 treatment was without detrimental effects on
`body water homeostasis. Thus, long-acting GLP-1 deriv-
`atives may prove efficient as weight-reducing therapeu-
`tic agents for overweight patients with type 2 diabetes.
`Diabetes 50:2530 –2539, 2001
`
`Peripheral administration of glucagon-like pep-
`
`tide-1 (GLP-1) acutely affects food intake in hu-
`mans, but
`the underlying mechanisms that
`decrease food intake concomitant with earlier
`onset of subjective sensation of fullness are not fully
`understood (1– 4). The anorectic effects of continuous
`intravenous administration of GLP-1 are present in indi-
`viduals who are lean or obese or have type 2 diabetes
`(1,3,4), suggesting that the observed effects are part of a
`physiologically relevant meal-terminating system. Results
`from similar experiments in rats have been ambiguous,
`probably because of high activity of GLP-1 degrading
`enzyme dipeptidyl peptidase-IV (DPP-IV) (5,6). Thus, early
`experiments were unable to demonstrate peripheral ef-
`fects of intraperitoneal GLP-1 injections on feeding be-
`havior (7,8), whereas later experiments have shown
`significant but short-lasting anorectic effects of subcuta-
`neous administration of GLP-1 (9). Anorexia induced by
`peripheral administration of GLP-1 involves vagal control
`of gastric motility (10,11).
`Central administration of 1–3 ␮g of GLP-1 specifically
`inhibits food intake in rats via a hypothalamic site that is
`sensitive to neonatal monosodium glutamate (MSG) le-
`sioning (12). However, central administration of slightly
`
`2530
`
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`

`higher doses of GLP-1 leads to taste aversion, but because
`this latter effect is unaffected by MSG treatment, it further
`stresses the specificity of the central GLP-1–induced an-
`orexia (12). In addition, it is possible to elicit anorexia
`without concomitant taste aversion if GLP-1 is injected
`directly into the hypothalamic paraventricular nucleus
`(13). Acute injections of both GLP-1 and NN2211 exerts
`profound adipsia and diuresis. These effects on body water
`homeostasis could potentially hamper long-term treatment
`of patients with type 2 diabetes with GLP-1 agonists, and
`the potential anorectic effects of these agonists may be
`accompanied with debilitating affects on body water ho-
`meostasis.
`Given that peripheral administration of GLP-1 affects
`food intake in humans, we decided to study further the
`anorectic potential of this peptide in the laboratory rat.
`Dose-response studies investigating the effect of intrave-
`nously administered GLP-1(7-37) or a novel long-acting
`acylated GLP-1 derivative NN2211 (14) on food intake
`were performed. In continuation of acute pharmacological
`studies, we examined the effect on food intake and body
`weight of twice daily subcutaneous administration of
`NN2211 for 10 days followed by a 5-day recovery period.
`To study the impact of 7 days of NN2211 treatment on
`energy expenditure (EE) and body composition, we stud-
`ied normal rats by indirect calorimetry and subsequently
`subjected them to dual energy X-ray absorptiometry
`(DEXA) scanning. Before, during, and after treatment,
`blood biochemical markers of energy and fluid homeosta-
`sis metabolic state were monitored.
`
`RESEARCH DESIGN AND METHODS
`Animals. All experiments were carried out on male Wistar rats. Normal male
`rats arrived at the animal facilities 2 weeks before experimentation, and until
`use, animals were housed three to four per cage under standard laboratory
`conditions (12:12 light:dark cycle, lights on 6:00 A.M.). Before experimentation,
`animals had free access to a standard rat diet (Altromin 1324) and water ad
`libitum. Hypothalamically obese rats were obtained by treating neonatal rats
`with MSG. Pregnant Wistar rats (Møllegården, Ll; Skensved, Denmark) arrived
`at the animal unit 1 day before timed labor (E21). Neonatal Wistar pups
`received a number of subcutaneous injections with MSG (L-glutamic acid,
`G-1626; 4 mg/g body wt; Sigma, Vallensbaek Strand, Denmark) dissolved in
`sterile distilled H2O. Injections were given at days P1, P3, P5, P7, and P9
`according to a previously published method (12). After weaning, pups were
`separated by sex and allowed to grow to the age of 12–14 weeks before
`experiments were initiated. Neonatal MSG treatment is accompanied by adult
`obesity and a number of neuroendocrine dysfunctions (hypothalamic hypo-
`gonadism, low fertility, stunted growth, chronic HPA-axis activation), and
`currently used MSG-treated rats all displayed characteristic stunted growth,
`adiposity, short tail caused by self-mutilation, and apparent blindness. All
`experiments were carried out in accordance with guidelines provided by the
`Danish Justice Department and approved by a personal license to P.J.L.
`Formulation of GLP-1(7-37), GLP-1(1-37), exendin(9-39), and NN2211.
`GLP-1(7-37) and GLP-1(1-37) were obtained from L. Thim (Novo Nordisk);
`exendin(9-39) was purchased from Bachem (Bissendorf Biochemicals, Ham-
`burg, Germany). High-performance liquid chromatography analyses of all
`peptides claim ⬎90% purity. Peptides were dissolved in sterile isotonic saline
`to which 1% bovine serum albumin (BSA) was added (Fraction V, #735 086;
`Boehringer Mannheim, Mannheim, Germany). The GLP-1 derivative NN2211
`(Arg34, Lys26-[N-⑀(␥-Glu[N-␣-hexadecanoyl])]-GLP-1[7-37]) was synthesized
`according to a previously described procedure (14). The compound NN2211 is
`a member of a group of pharmacologically active GLP-1 derivatives that have
`long plasma half-lives (14). Thus, NN2211 is an acylated GLP-1 derivative with
`a plasma half-life of ⬃14 h in pigs. Pharmacokinetic data from rats show that
`⫽ 4 h probably as a result of considerably higher endogenous DPP-IV
`t1/2
`activity in this species (L.B.K., unpublished observations). NN2211 was
`dissolved in sterile phosphate-buffered saline (50 mmol/l, pH 7.4) to a final
`concentration of either 0.1 or 1.0 mg/ml. Solutions were always made fresh ⬃1
`h before use and stored at 4°C in sterile tubes. Material from several batches
`
`P.J. LARSON AND ASSOCIATES
`
`of NN2211 was used, and corrections for impurity were always performed.
`Subcutaneous injections were administered using standard 1-ml syringes
`equipped with 25-G needles.
`Experiment 1: single dose of GLP-1. Sixteen adult male Wistar rats were
`equipped with jugular intravenous catheters (Department of Pharmacology,
`The Panum Institute, University of Copenhagen). Catheters were implanted
`under Avertin (tribromoethanol, 200 mg/kg) anesthesia in the right jugular
`vein with the tip aiming at the right atrium. After placement, the catheter was
`externalized via subcutaneous tunneling to the interscapular area. Catheter
`patency was secured by instillation of heparinized (1,000 IU/l) isotonic sterile
`saline into the catheter before closure with a metal rod. After 7 days of
`postoperative recovery, animals were housed individually in standard meta-
`bolic cages (Techniplast, Gazzoda, Italy) and acclimatized over a period of 7
`days to a 5-h restricted feeding scheme with access to food from 8:00 A.M. to
`1 P.M. and water ad libitum.
`Seven days after initiation of the restricted feeding scheme, animals were
`assigned to a random crossover dosing paradigm. Five minutes before
`presentation of food, animals received an intravenous injection of GLP-1 (5,
`100, or 500 ␮g/animal). Statistical analysis of intergroup treatment variation
`was carried out using factorial analysis of variance (ANOVA) followed by
`Scheffe post hoc analysis.
`Experiment 2: single dose of NN2211. Normal adult male Wistar rats (n ⫽
`16) and MSG-treated rats (n ⫽ 16) were housed individually in metabolic
`cages with free access to a rat diet and water for at least 1 week before
`experimentation. Animals were kept in a 12:12 light:dark cycle, and the effect
`of NN2211 on nighttime food intake was assessed by injecting animals
`subcutaneously 2–3 h before lights out. Animals were left without food and
`water in the period from dosing to the onset of darkness. Three doses of
`NN2211 and vehicle were tested in a random crossover experiment (10, 50,
`and 200 ␮g/kg). Food and water intake and diuresis were monitored every 30
`min for the first 2 h after onset of nighttime (lights out) and finally 12 h later
`at lights on (t720). All measurements were done in complete darkness with the
`assistance of night vision goggles (Bausch and Lomb, Rochester, NY). The
`minimum interval between experiments was 48 h. Statistical analysis of
`intergroup treatment variation was carried out using factorial ANOVA fol-
`lowed by Scheffe post hoc analysis.
`Experiment 3: continuous administration of NN2211. Beginning 1 week
`before the first dose was administered, normal adult male Wistar rats (n ⫽ 16)
`and MSG-treated rats (n ⫽ 16) were housed individually in metabolic cages
`with free access to a rat diet and water. Animals were kept on a 12:12
`light:dark cycle, and the subchronic effect of two daily subcutaneous injec-
`tions of NN2211 or vehicle on body weight, food and water intake, diuresis,
`and feces excretion was monitored. All parameters were measured between 9
`and 11 A.M. while animals received their morning dose. On the basis of daily
`food intake, animals were stratified to different treatment groups ensuring
`comparable baseline values for food and water intake. Animals received two
`daily injections of NN2211 (100 or 200 ␮g/kg b.i.d.) at 8:00 A.M. and 7:00 P.M.
`throughout a 10-day period followed by a 5-day drug-free recovery period.
`Animals were weighed every morning (between 9 and 11 A.M.) together
`with measurement of daily food and water intake as well as diuresis and feces
`excretion. At day 0, orbital blood samples were taken from all animals before
`the first dose was administered. Further orbital blood samples were taken at
`day 7 and 14 (i.e., during and after treatment with NN2211). At the final day of
`experimentation, animals were decapitated and trunk blood was collected as
`described. Different dose administrations were conducted in two separate
`experiments, each with its own vehicle-treated control groups. Data from
`these control groups were pooled.
`Statistical analysis of effect of treatment on body weight, food and water
`intake, diuresis, and feces excretion was carried out using factorial ANOVA
`followed by Bonferroni correction for multiple comparison. Biochemical data
`from plasma samples were analyzed using factorial ANOVA followed by
`Fisher’s or Scheffe’s post hoc analysis.
`Experiment 4: effect of subchronic NN2211 treatment on energy ex-
`penditure and body composition. Twenty-four 12-week-old male rats were
`used in this study. The rats were housed individually in a temperature- (20°C)
`and light-controlled environment (12:12 light:dark cycle; lights on from 7:00
`A.M.) with free access to food and water for at least 7 days before experimen-
`tation. The rats were stratified into three groups (G1, G2, and G3) according
`to weight 3 days before study start (n ⫽ 7 per group). The rats in G1 and G3
`were treated with vehicle, and the rats in G2 were treated with NN2211 (200
`␮g/kg b.i.d.). The rats in G1 and G2 had free access to food and water during
`the 7-day treatment period, whereas the rats in G3 were “single” pair fed after
`the rats in G2. Body weight and food and water intake were recorded daily. By
`the end of the study (day 7), oxygen consumption and body composition were
`determined by indirect calorimetry and DEXA, respectively. Likewise, blood
`samples were collected for determination of hematocrit as well as for plasma
`
`DIABETES, VOL. 50, NOVEMBER 2001
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`

`WEIGHT LOSS THERAPY WITH GLP-1 DERIVATIVE
`
`FIG. 1. Single intravenous infusions of GLP-1(7-37) cause dose-depen-
`dent anorexia in rats that are kept on a restricted feeding schedule.
`Food intake over the initial 30 min of the feeding session was recorded
`for all doses tested (5, 100, and 500 ␮g/rat). *, statistically significant
`differences from vehicle-treated animals (P < 0.05 as determined by
`ANOVA followed by Scheffe’s post hoc analysis).
`
`levels of triacylglycerol (TG), glycerol, free fatty acids (FFAs), and total
`cholesterol.
`DEXA. Body composition was determined by DEXA (pDEXA Sabre, Stratec
`Medizintechnic; Norland Medical Systems, Pho¨ rzheim, Germany). The instru-
`ment settings used were as follows: a scan speed of 40 mm/s, a resolution of
`1.0 ⫻ 1.0 mm, and automatic/manual histogram width estimation. The
`coefficient of variation as assessed by 10 repeated measurements (with
`repositioning of the rat between each measurement) was 3.48, 3.17, and 3.73%
`for bone mineral content, lean tissue mass, and fat tissue mass, respectively.
`By the end of the study (day 7), the rats were killed. The carcasses were stored
`in plastic bags at ⫺20°C before determination of body composition; which
`was determined on defrosted carcasses.
`Indirect calorimetry. Oxygen consumption, CO2 production, EE, and the
`respiratory exchange ratio (RER) were determined by indirect calorimetry
`(Oxymax System; Columbus Instruments, Columbus, OH). The rats (n ⫽ 1 per
`chamber) were placed in airtight acrylic chambers (10.5 l). Oxygen and CO2
`concentrations in the chamber in- and outlet gas were determined simulta-
`neously every 20.25 min over a period of 4.4 h. Instrument settings used were
`as follows: a gas flow rate of 1.86 l/min, settle time of 90 s; measure time of
`40 s, and system recalibration for each eight-chamber measuring cycle. By the
`end of the study (day 7), the nonfasted rats were subjected to indirect
`calorimetry (from 8:00 A.M. 1:00 P.M.). In contrast to the previous 6 days, the
`nonfasted rats did not receive NN2211 between 7:30 and 8:30 before indirect
`calorimetry. On the day of indirect calorimetry, the rats received treatment at
`9:30 —after four pretreatment measurements. The rats had no access to food
`or water during their stay in the acrylic chamber. The indirect calorimetric
`measurements were performed over 3 days. As a “positive” instrument
`control, every day included a reference animal “treated” with the EE increas-
`ing compound 2,4-dinitrophenol (DNP, Sigma). On the basis of the measure-
`ments of O2 and CO2 in the chamber in- and outlet gas, estimates of O2
`consumption, CO2 production, EE, and RER were calculated.
`Blood sampling and biochemical assays
`Blood sampling. Orbital blood samples were obtained by puncture of the
`orbital venous plexus with glass capillary tubes. Samples were taken in
`standard heparinized EDTA (0.18 mol/l) glass tubes (Vacutainer) to which
`aprotinin (1,500 KIE/ml) and bacitracin (3%) were added. After sampling,
`tubes were kept on ice before being centrifuged (4°C at 5,000g for 10 min), and
`the resulting plasma was stored at ⫺80°C before being analyzed. Trunk blood
`was obtained by decapitating animals and sampling into heparinized (⬃500
`IU/tube) glass tubes to which aprotinin (1,500 KIE/ml) and bacitracin (3%)
`were added. Glycerol and FFA concentrations were determined in EDTA (0.18
`mol/l) plasma containing 1% NaF (wt/vol).
`Plasma glucose. Plasma glucose was measured on a standard COBAS
`analyzer (Toxicology Projects & Planning, Novo Nordisk, Copenhagen, Den-
`mark).
`Plasma leptin. Plasma leptin was measured using a commercially available
`
`mouse leptin enzyme-linked immunosorbent assay kit (Crystal Chemical,
`Chicago, IL), showing ⬎95% cross-reactivity to rat leptin.
`Plasma biochemistry. Orbital blood samples (days 0, 7, and 14) were taken
`from rats that were receiving 100 ␮g/kg b.i.d. NN2211 and a set of correspond-
`ing vehicle-treated animals. Plasma values of sodium, TG, cholesterol, creat-
`inine, carbamide, and total protein were measured on a standard COBAS
`analyzer. FFA and glycerol were measured on a standard Hitachi Automatic
`analyzer.
`Plasma potassium. Orbital blood samples (days 0, 7, and 14) were taken
`from rats that were receiving 200 ␮g/kg b.i.d. NN2211 and a set of correspond-
`ing vehicle-treated animals. Blood was collected in heparinized glass tubes
`(Vacutainer), and the potassium content in resulting plasma was measured
`potentiometrically with an ion selective probe on a standard COBAS analyzer.
`
`RESULTS
`Experiment 1
`Effects of a single dose of GLP-1 on feeding behav-
`ior. Acute intravenous administration of high doses of
`GLP-1 (100 and 500 ␮g/animal) decreased food intake at 30
`and 60 min after onset of feeding period in rats that were
`kept on a restricted feeding scheme (Fig. 1). The anorectic
`effect of the highest dose of GLP-1 (500 ␮g/animal) was
`completely abolished by previous administration of 100 ␮g
`of exendin(9-39) (6.1 ⫾ 0.4 vs. 5.9 ⫾ 0.3 g). Also, the
`biologically inactive peptide GLP-1(1-37) (500 ␮g/animal)
`had no acute effect on food intake (6.1 ⫾ 0.4 vs. 6.2 ⫾
`0.3 g). Water intake was similarly decreased in rats that
`were receiving an intravenous injection of GLP-1, and the
`pharmacological characteristics of water consumption
`mirrored that of feeding.
`Intravenous administration of GLP-1 also affected diure-
`sis. Water excretion was markedly and dose-dependently
`increased in rats that were receiving intravenous bolus
`injections of GLP-1 (100 and 500 ␮g/animal; Fig. 2). The
`effect displayed pharmacological specificity in as much it
`was not elicited by 500 ␮g/animal GLP-1(1-37). However,
`an attempt to antagonize the diuretic effect of 500 ␮g of
`GLP-1 with exendin(9-39) (100 ␮g/animal) was only partial-
`ly successful. Thus, exendin(9-39) completely abolished
`anorectic GLP-1 actions, whereas the diuretic actions of
`500 ␮g GLP-1 were only partially abolished (Fig. 2).
`Experiment 2
`Effects of a single dose of NN2211 on feeding behav-
`ior in rats. The acute effects of three doses of NN2211 on
`nighttime feeding were tested in a random crossover
`experiment (10, 50, and 200 ␮g/kg). The dose dependence
`of NN2211 on overnight food intake in normal rats is
`illustrated in Fig. 3. Two hours after onset of the feeding
`
`FIG. 2. Acute intravenous administration of GLP-1 causes profound
`diuresis. *, statistically significant difference from all other groups; a,
`statistically significant differences from vehicle-treated animals (P <
`0.05 as determined by ANOVA followed by Scheffe’s post hoc analysis).
`
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`P.J. LARSON AND ASSOCIATES
`
`FIG. 4. Subchronic administration of NN2211 dose-dependently de-
`creases body weight in both normal adult male Wistar rats and MSG-
`treated rats. Animals received two daily subcutaneous injections of
`NN2211 (100 or 200 ␮g/kg) or vehicle for 10 days followed by a 5-day
`recovery period. Body weight was monitored each morning. Values are
`mean ⴞ SE (n ⴝ 5– 8). *, significant difference from relevant vehicle-
`treated control as determined by ANOVA followed by Bonferroni
`multiple comparison post hoc analysis.
`
`ited food intake in normal rats (control: 22.4 ⫾ 0.7; 50
`␮g/kg: 16.3 ⫾ 0.7; 200 ␮g/kg: 8.7 ⫾ 1.3 g of food) as well as
`in MSG-treated rats (control: 12.5 ⫾ 1.9; 50 ␮g/kg: 10.0 ⫾
`1.0; 200 ␮g/kg: 5.8 ⫾ 0.7 g of food).
`Effects of a single dose of NN2211 on water intake
`and diuresis. In the same experiment, water intake and
`diuresis was monitored (Fig. 3). Both 50 and 200 ␮g/kg
`NN2211 significantly inhibited overnight (t720) water in-
`take in normal and MSG-treated rats (vehicle: 32.8 ⫾ 2.0;
`50 ␮g/kg: 21.1 ⫾ 1.1; 200 mg/kg: 11.5 ⫾ 2.5 g; vehicle-MSG:
`21.2 ⫾ 2.5; MSG-50 ␮g/kg: 17.1 ⫾ 1.9; MSG-200 ␮g/kg:
`11.5 ⫾ 1.3 ml; P ⬍ 0.05 as determined by ANOVA followed
`by post hoc Scheffe’s analysis). In contrast, the highest
`dose of NN2211 (200 ␮g/kg) significantly increased diure-
`sis in both normal and MSG-treated rats (vehicle: 7.6 ⫾ 0.3;
`200 ␮g/kg: 13.7 ⫾ 1.7; vehicle-MSG: 9.0 ⫾ 1.2; MSG-200
`␮g/kg: 12.3 ⫾ 0.9 ml; P ⬍ 0.05 as determined by ANOVA
`followed by post hoc Scheffe’s analysis).
`Experiment 3
`Effect of subchronic administration of NN2211 on
`food intake and body weight. Two daily injections of
`NN2211 to adult male Wistar rats dose-dependently de-
`creased body weight over the entire 10-day treatment
`period with significant differences obtained with the 200
`␮g/kg b.i.d. dosing regimen between treatment days 7 and
`14 (Fig. 4). Loss in body weight was preceded by de-
`creased food and water intake and increased diuresis (Fig.
`5). Food intake was significantly lower during the initial 3
`days of treatment for normal animals that were treated
`with 100 ␮g/kg b.i.d. (data not shown). Thereafter, the
`anorectic effect of this low dose was no longer statistically
`significant. In animals that were treated with 200 ␮g/kg
`b.i.d., food intake was significantly lower throughout the
`10-day treatment period. After cessation of the treatment,
`food intake normalized within a few days and body weight
`
`2533
`
`FIG. 3. Single subcutaneous injections of NN2211 dose-dependently
`inhibit food and water in both normal and MSG-treated rats. Dose-
`response curves for 720 min of food and water intake as well as diuresis
`in normal and MSG-treated rats receiving a single subcutaneous dose
`of NN2211. *P < 0.05 versus vehicle (ANOVA followed by Scheffe’s post
`hoc analysis); aP < 0.05 versus 10 ␮g/kg NN2211 (ANOVA followed by
`Scheffe’s post hoc analysis); bP < 0.05 versus 50 mg/kg NN2211
`followed by Scheffe’s post hoc analysis).
`
`session, 200 ␮g/kg NN2211 significantly inhibited food
`intake (3.8 ⫾ 0.3 vs. 5.2 ⫾ 0.3 g of food). The following
`morning (t720), both 50 and 200 ␮g/kg significantly inhib-
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`

`WEIGHT LOSS THERAPY WITH GLP-1 DERIVATIVE
`
`FIG. 5. The effect of subchronic administration of 200 ␮g/kg NN2211 to normal male Wistar rats (circles) or MSG-treated rats (boxes) on food
`and water intake as well as diuresis and feces excretion. Open symbols represent vehicle-treated animals; closed symbols represent
`NN2211-treated animals. Shaded areas reflect baseline values obtained from all animals in the group throughout the week before onset of
`treatment.
`
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`

`TABLE 1
`NN2211 treatment (200 ␮g/kg BID) exerts no adverse effects on plasma glucose, potassium, sodium, and plasma variables that reflect
`renal function (creatinine, carbamide, and protein)
`
`Normal vehicle
`(n ⫽ 8)
`
`Normal NN2211
`(n ⫽ 8)
`
`MSG vehicle
`(n ⫽ 4)
`
`MSG NN2211
`(n ⫽ 8)
`
`P.J. LARSON AND ASSOCIATES
`
`Treatment values (day 7)
`Glucose (mmol/l)
`Potassium (K⫹) (mmol/l)
`Sodium (Na⫹) (mmol/l)
`Creatinine (mmol/l)
`Carbamide (mmol/l)
`Protein (g/l)
`Recovery-phase values (day 14)
`Glucose (mmol/l)
`Potassium (K⫹) (mmol/l)
`Sodium (Na⫹) (mmol/l)
`Creatinine (mmol/l)
`Carbamide (mmol/l)
`Protein (g/l)
`
`Data are means ⫾ SD.
`
`6.7 ⫾ 0.5
`4.6 ⫾ 0.3
`136.5 ⫾ 0.4
`87.6 ⫾ 1.7
`8.4 ⫾ 0.2
`64.0 ⫾ 1.2
`
`6.8 ⫾ 0.1
`5.2 ⫾ 0.5
`139.5 ⫾ 0.6
`85.3 ⫾ 1.2
`7.9 ⫾ 0.2
`67.2 ⫾ 1.2
`
`6.9 ⫾ 0.2
`4.2 ⫾ 0.1
`136.9 ⫾ 0.6
`75.6 ⫾ 2.4
`8.9 ⫾ 0.5
`63.4 ⫾ 1.2
`
`6.4 ⫾ 0.2
`4.6 ⫾ 0.3
`138.7 ⫾ 0.4
`81.9 ⫾ 1.4
`8.0 ⫾ 0.1
`65.0 ⫾ 0.6
`
`6.1 ⫾ 1.0
`4.3 ⫾ 0.2
`138.2 ⫾ 0.5
`76.3 ⫾ 3
`7.3 ⫾ 0.3
`65.4 ⫾ 1.0
`
`5.7 ⫾ 1.0
`4.3 ⫾ 0.4
`141.4 ⫾ 0.7
`85.0 ⫾ 1.1
`7.9 ⫾ 0.3
`66.3 ⫾ 0.7
`
`6.7 ⫾ 0.3
`4.1 ⫾ 0.1
`139.0 ⫾ 0.3
`77.5 ⫾ 1.9
`8.5 ⫾ 0.3
`64.1 ⫾ 0.6
`
`6.2 ⫾ 0.2
`5.0 ⫾ 0.1
`140.2 ⫾ 1.2
`90.4 ⫾ 3.9
`7.3 ⫾ 0.2
`64.3 ⫾ 0.7
`
`gradually increased toward that of vehicle-treated animals
`(Fig. 5).
`Similar effects of NN2211 treatment on food intake were
`seen in obese MSG-treated rats. Thus, two daily injections
`of 200 ␮g/kg NN2211 significantly decreased body weight
`throughout the 10-day treatment period, whereas the 100
`␮g/kg b.i.d. dosing regimen displayed a trend toward
`weight reduction without reaching statistical significance.
`Also, food intake was significantly lower in MSG-treated
`animals that were treated with 200 ␮g/kg b.i.d. NN2211.
`Effect of subchronic administration of NN2211 on
`water intake, diuresis, and feces excretion. In con-
`trast to food intake, water intake was lower in NN2211-
`treated animals only at the initial days of treatment,
`because from day 4 onward, NN2211-treated groups dis-
`played considerably higher water intake than correspond-
`ing vehicle-treated groups (Fig. 5). However, these effects
`were not statistically significant and had no impact on
`long-term body fluid homeostasis (see below).
`In normal rats that were receiving 100 ␮g/kg b.i.d.
`NN2211,
`increased diuresis was accompanied by in-
`creased water intake from treatment day 2 onward to
`cessation of dosing (data not shown). In animals that were
`receiving 200 ␮g/kg b.i.d., however, fluid homeostasis was
`severely affected during the first 2 days of dosing, because
`a state of very low water intake coexisted with markedly
`increased diuresis (Fig. 5). For normal rats that were
`treated with 200 ␮g/kg b.i.d., apparent fluid balance with
`water consumption and diuresis at levels similar to vehi-
`cle-treated animals occurred from day 4 onward. Despite
`marked increasing effects on urinary water excretion,
`plasma sodium and potassium remained unaffected in
`animals that were treated with 200 ␮g/kg b.i.d. NN2211
`(Table 1). Also, plasma variables reflecting renal function
`(creatinine, carbamide, and total protein) were unaffected
`by NN2211 treatment (Table 1). Feces excretion was
`followed in normal animals that were receiving 200 ␮g/kg
`b.i.d. and was observed to decrease during administration
`of NN2211 coincident with the decrease in food intake
`(Fig. 5).
`In MSG-treated rats, NN2211 treatment (both 100 and
`200 ␮g/kg b.i.d.) induced a statistically significant reduc-
`
`tion of water intake and increased diuresis, leading to an
`initial negative water balance (Fig. 5). The compensatory
`water homeostatic mechanisms were clearly more undu-
`lating in MSG-treated animals that were treated with 200
`␮g/kg b.i.d. because they went through a phase character-
`ized by polyuria and hyperdipsia from day 3 to day 6
`before full compensation was obtained (Fig. 5). In the
`recovery phase after cessation of
`the treatment,
`the
`slightly lower water intake that accompanied lower food
`intake at the end of the NN2211 treatment period rapidly
`returned to control levels. As with normal animals, MSG-
`treated animals’ plasma electrolyte levels were unaffected
`by NN2211 treatment both during and after cessation of
`drug administration (Table 1).
`Experiment 4
`Effect of subchronic administration of NN2211 on
`EE, body composition,
`food intake, and body
`weight. As seen in experiment 3, 7 days of NN2211
`treatment significantly lowered body weight (373.3 ⫾ 14.7
`vs. 417.3 ⫾ 15.3 g), and the body weights in the pair-fed
`group were reduced similarly (378.5 ⫾ 10.5). After 7 days
`of treatment, EE was considerably lower in both NN2211
`and pair-fed animals (control 1,775 ⫾ 39; pair fed 1,634 ⫾
`49; NN2211 1,641 ⫾ 27 kcal/h; n ⫽ 6 –7, average of 3-h
`measurements). However, when EE was expressed as
`oxygen consumption per kilogram of body mass, no such
`differences were seen throughout the observation period
`(Fig. 6). Also, the RER was unaffected by 7 days of NN2211
`treatment (Fig. 7). In contrast, pair-fed animals displayed a
`switch toward lipid metabolism, probably reflecting that
`these animals had been starved for a longer period of time
`before the onset of experiment.
`Body composition analysis was carried out using a
`DEXA scanner specialized for small animals. Treatment
`with NN2211 reduced body weight by affecting both lean
`and adipose tissue mass, although the loss of fat mass did
`not quite reach statistical significance (Table 2). However,
`the percentages of lean and adipose tissue mass of total
`body weight changed in neither NN2211-treated nor pair-
`fed animals. The hydration status after 7 days of treatment
`was further assessed by measuring the hematocrit, and
`pair-fed animals displayed significant hemoconcentration
`
`DIABETES, VOL. 50, NOVEMBER 2001
`
`2535
`
`MPI EXHIBIT 1086 PAGE 6
`
`

`

`WEIGHT LOSS THERAPY WITH GLP-1 DERIVATIVE
`
`FIG. 6. Oxygen consumption was determined by indirect calorimetry by
`the end of the study (day 7). The rats received treatment at time point
`0 (10:00 A.M.). The rats had no access to food or water while being
`subjected to indirect calorimetry. On each day of experimentation, an
`animal treated with the chemical uncoupler 2,4-dinitrophenol (DNP)
`was included as a “positive” instrument. The results are expressed as
`mean ⴞ SE
`
`in comparison to both ad libitum–fed controls and
`NN2211-treated animals (ad libitum 45.6 ⫾ 0.8; NN2211
`47.0 ⫾ 0.9; pair-fed 49.1 ⫾ 0.9%; n ⫽ 7).
`Effect of subchronic administration of NN2211 on
`plasma glucose and lipids. A number of biochemical
`plasma variables were assessed in animals that were
`subjected to either 7 or 10 days of NN2211 treatment
`(experiments 3 and 4). Data obtained from experiment 3
`showed no effect of NN2211 on