A Recombinant Human Glucagon-Like Peptide
`(GLP)-1–Albumin Protein (Albugon) Mimics
`Peptidergic Activation of GLP-1 Receptor–Dependent
`Pathways Coupled With Satiety, Gastrointestinal
`Motility, and Glucose Homeostasis
`
`Laurie L. Baggio,1 Qingling Huang,1 Theodore J. Brown,2 and Daniel J. Drucker1
`
`Peptide hormones exert unique actions via specific G
`protein– coupled receptors; however, the therapeutic
`potential of regulatory peptides is frequently compro-
`mised by rapid enzymatic inactivation and clearance
`from the circulation. In contrast, recombinant or cova-
`lent coupling of smaller peptides to serum albumin
`represents an emerging strategy for extending the cir-
`culating t1/2 of the target peptide. However, whether
`larger peptide-albumin derivatives will exhibit the full
`spectrum of biological activities encompassed by the
`native peptide remains to be demonstrated. We report
`that Albugon, a human glucagon-like peptide (GLP)-1–
`albumin recombinant protein, activates GLP-1 receptor
`(GLP-1R)-dependent cAMP formation in BHK-GLP-1R
`cells, albeit with a reduced half-maximal concentration
`(EC50) (0.2 vs. 20 nmol/l) relative to the GLP-1R agonist
`exendin-4. Albugon decreased glycemic excursion and
`stimulated insulin secretion in wild-type but not GLP-
`1Rⴚ/ⴚ mice and reduced food intake after both intrace-
`rebroventricular and intraperitoneal administration.
`Moreover, intraperitoneal injection of Albugon inhib-
`ited gastric emptying and activated c-FOS expression in
`the area postrema, the nucleus of the solitary tract, the
`central nucleus of the amygdala, the parabrachial, and
`the paraventricular nuclei. These findings illustrate
`that peripheral administration of a larger peptide-albu-
`min recombinant protein mimics GLP-1R– dependent ac-
`tivation of central and peripheral pathways regulating
`energy intake and glucose homeostasis in vivo. Diabetes
`53:2492–2500, 2004
`
`From the 1Department of Medicine, Banting and Best Diabetes Centre,
`Toronto General Hospital, Toronto, Ontario, Canada; and the 2Division of
`Reproductive Science, Samuel Lunenfeld Research Institute, Mount Sinai
`Hospital, and the University of Toronto, Toronto, Ontario, Canada.
`Address correspondence and reprint requests to Dr. Daniel J. Drucker,
`Toronto General Hospital, Banting and Best Diabetes Centre, 200 Elizabeth
`St., MBRW4R-402, Toronto, Canada M5G 2C4. E-mail: d.drucker@utoronto.ca.
`Received for publication 4 December 2003 and accepted in revised form
`28 May 2004.
`CCK, cholecystokinin; CNS, central nervous system; DPP-IV, dipeptidyl
`peptidase-IV; Ex-4, exendin-4; GLP, glucagon-like peptide; GLP-1R, GLP-1
`receptor; HSA, human serum albumin; ICV, intracerebroventricular; IP, intra-
`peritoneal; NTS, nucleus of the solitary tract.
`© 2004 by the American Diabetes Association.
`
`2492
`
`Glucagon-like peptide (GLP)-1 is a 30 –amino
`
`acid peptide hormone secreted from gut endo-
`crine cells in response to nutrient ingestion that
`promotes nutrient assimilation through regula-
`tion of gastrointestinal motility and islet hormone secre-
`tion (1). Infusion of GLP-1 into normal or diabetic human
`subjects stimulates insulin and inhibits glucagon secre-
`tion, thereby indirectly modulating peripheral glucose
`uptake and control of hepatic glucose production (2).
`GLP-1 also produces anorectic effects, and short-term
`infusion of GLP-1 is associated with diminished appetite
`and reduced energy consumption in normal, obese, and
`diabetic human subjects (3). Taken together, the actions of
`GLP-1 to reduce glycemia while preventing concomitant
`weight gain have attracted considerable interest in phar-
`maceutical approaches to enhancing GLP-1 action for the
`treatment of type 2 diabetes.
`A major challenge for the therapeutic use of regulatory
`peptides, including native GLP-1, is a short circulating t1/2,
`due principally to rapid enzymatic inactivation and/or re-
`nal clearance. Although infusion of native GLP-1 is highly
`effective in lowering blood glucose in subjects with type 2
`diabetes, a single subcutaneous injection of the native pep-
`tide is quickly degraded and disappears from the circulation
`within minutes (4). Hence, the majority of pharmaceutical
`approaches to the development of GLP-1 mimetic agents
`have focused on the development of long-acting degrada-
`tion-resistant peptides (5). The naturally occurring lizard
`salivary gland peptide exendin-4 (Ex-4) is a potent GLP-1
`receptor (GLP-1R) agonist and exhibits therapeutic efficacy
`in studies of patients with type 2 diabetes (6). Similarly, a
`fatty acylated human GLP-1 analog, liraglutide, exhibits a
`more sustained duration of action and potently reduces
`glycemic excursion in diabetic subjects (7).
`Although degradation-resistant GLP-1R agonists appear
`to be promising agents for the treatment of diabetes, the
`need for once- or twice-daily injection of these peptides
`has fostered complementary efforts directed at identifica-
`tion of more potent longer-acting agents with sustained
`efficacy in vivo. Given the long circulating t1/2 of albumin-
`linked drugs (8), a GLP-1–albumin protein should exhibit a
`much more prolonged circulating t1/2 and hence requires a
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`reduced frequency of parenteral administration, relative to
`native GLP-1. Nevertheless, the molecular interaction with
`the GLP-1R, volume of distribution, and access to the
`central nervous system (CNS) would be predicted to be
`markedly different for a much larger albumin-based mole-
`cule (8).
`Whether all of the desirable actions of native GLP-1,
`including the activation of the CNS centers regulating food
`intake and gastrointestinal motility, would be mimicked by
`a much larger GLP-1–albumin protein is currently unclear.
`Accordingly, we have examined the biological actions of
`Albugon, a recombinant GLP-1– human serum albumin
`(HSA) fusion protein, using a combination of cell line stud-
`ies in vitro and both wild-type and GLP-1R⫺/⫺ mice in vivo.
`
`RESEARCH DESIGN AND METHODS
`Reagents. Tissue culture medium, serum, and G418 were purchased from
`Invitrogen (San Diego, CA). Forskolin and 3-isobutyl-1-methylxanthine were
`obtained from Sigma (St. Louis, MO). HSA and Albugon were provided by
`Human Genome Sciences (Rockville, MD). Ex-4 and exendin (9-39) were
`purchased from California Peptide Research (Napa, CA).
`Measurement of cAMP. Baby hamster kidney (BHK) cells stably transfected
`with rat GLP-1R were generated and propagated in medium containing 0.05
`mg/ml G418 as described previously (9). Before analysis, BHK-GLP-1R cells
`were grown to 70 – 80% confluence in 24-well plates in the absence of G418 at
`37°C. Ex-4 and exendin (9-39) were dissolved in PBS, and cells were treated
`with control or test reagents in Dulbecco’s modified Eagle’s medium contain-
`ing serum and 100 ␮mol/l 3-isobutyl-1-methylxanthine. Cells were incubated
`with 1 ␮mol/l exendin (9-39) or medium alone for 5 min at 37°C, followed by
`an additional 10-min incubation in the presence of increasing concentrations
`of Ex-4, HSA, or Albugon. All reactions were carried out in triplicate and
`terminated by the addition of ice-cold absolute ethanol. Cell extracts were
`collected and stored at ⫺80°C until assayed. For cAMP determinations,
`aliquots of ethanol extracts were lyophilized, and cAMP levels were measured
`using a cAMP radioimmunoassay kit (Biomedical Technologies, Stoughton, MA).
`Mice. All animal experiments were carried out in accordance with protocols
`and guidelines approved by the Toronto General Hospital Animal Care
`Committee. GLP-1R⫺/⫺ mice on the C57BL/6 genetic background, and age-
`matched (8- to 15-week-old male) wild-type C57BL/6 mice (Charles River
`Laboratories, Montreal, PQ) were maintained on a 12-h light/dark cycle and
`allowed free access to standard rodent food and water, except where noted.
`Wild-type mice were acclimated to the animal facility for a minimum of 1 week
`before analysis.
`Glucose tolerance tests and measurement of plasma insulin. Oral or
`intraperitoneal (IP) glucose tolerance tests were carried out after an overnight
`fast (16 –18 h). Glucose (1.5 mg/g body wt) was administered orally through a
`gavage tube or by injection into the peritoneal cavity. A blood sample was
`drawn from the tail vein at 0, 10, 20, 30, 60, 90, and 120 min after glucose
`administration, and blood glucose levels were measured using a Glucometer
`Elite blood glucose meter (Bayer, Toronto, Ontario, Canada). For plasma
`insulin determination, a blood sample (100 ␮l) was removed from the tail vein
`during the 10- to 20-min time period after glucose administration and
`immediately mixed with a 10% volume of a chilled solution containing 5,000
`KIU/ml Trasylol (Bayer), 32 mmol/l EDTA, and 0.1 nmol/l Diprotin A (Sigma).
`Plasma was separated by centrifugation at 4°C and stored at ⫺80°C until
`assayed. Plasma insulin levels were measured using a rat insulin enzyme-
`linked immunosorbent assay kit (Crystal Chem, Chicago, IL) with mouse
`insulin as standard.
`Measurement of plasma glucagon. After an overnight fast (16 h), mice were
`given an IP injection of 1 mg/kg HSA or Albugon. At 20 min after HSA or
`Albugon administration, mice were killed and cardiac blood was obtained and
`mixed with a 10% volume of a chilled solution containing 5,000 KIU/ml
`Trasylol, 32 mmol/l EDTA, and 0.1 nmol/l Diprotin A. Plasma was separated by
`centrifugation at 4°C and stored at ⫺80°C until assayed. Plasma glucagon
`levels were measured using a glucagon radioimmunoassay kit (Linco Re-
`search, St. Charles, MO).
`Feeding studies. For analysis of food intake, mice were fasted overnight
`(16 –18 h), and control (PBS or HSA) or test (Ex-4 or Albugon) reagents were
`administered by intracerebroventricular (ICV) or IP injection. For ICV injec-
`tions, mice were lightly anesthetized using isoflurane inhalation (Abbott
`Laboratories, Saint-Laurent, Quebec, Canada), and reagents were adminis-
`tered in a total volume of 5 ␮l by injection into the lateral ventricles using a
`2.5 mm ⫻ 30-gauge needle attached to a Hamilton syringe as described (10).
`
`L.L. BAGGIO AND ASSOCIATES
`
`Mice were allowed to recover from the anesthetic (⬃10 min) before assess-
`ment of food intake. For IP injections, 100 ␮l control or test reagent was
`injected into the peritoneal cavity. Immediately after ICV or IP injection, mice
`were weighed and then placed into individual cages containing preweighed
`rodent food, with free access to water. At 2, 4, 7, and 24 h after reagent
`administration, the food was reweighed and total food intake (g/g of body wt)
`was calculated.
`Gastric emptying. The gastric emptying rate was determined as described
`(11). Briefly, mice were fasted overnight (18 h) and then allowed free access
`to preweighed rodent food for 1 h. After the 1-h refeeding, the remaining food
`was weighed and food intake was determined. Mice were then given IP
`injections of PBS, Ex-4 (0.17 mg/kg), HSA (2.7 mg/kg), Albugon (3 mg/kg), or
`cholecystokinin (CCK)-8 (4 ␮g) and deprived of food for an additional 4 h.
`Mice were anesthetized with Somnotol (sodium pentobarbital solution; MTC
`Pharmaceuticals, Cambridge, Ontario, Canada), their stomachs were re-
`moved, and stomach content wet weight was determined. Gastric emptying
`rate was calculated using the following: gastric emptying rate (%) ⫽ [1 ⫺
`(stomach content wet weight/food intake)] ⫻ 100.
`Measurement of c-FOS activation in the murine CNS. The number of
`c-FOS immunoreactive neurons in specific brain regions was assessed quan-
`titatively in both wild-type C57BL/6 and GLP-1R⫺/⫺ mice as described (12,13).
`Briefly, animals were given IP injections of PBS, Ex-4, HSA, or Albugon in a
`100 ␮l volume. At 10 and 60 min after injection, mice were anesthetized with
`Somnotol. All mice were perfused intracardially with ice-cold normal saline
`followed by 4% paraformaldehyde solution. Brains were removed immediately
`at the end of perfusion, kept in ice-cold 4% paraformaldehyde solution for 3
`days, and then transferred to a solution containing paraformaldehyde and 10%
`sucrose for 12 h. Brains were cut into 25-␮m sections using a Leica SM2000R
`sliding microtome (Leica Microsystems, Richmond Hill, Ontario, Canada) and
`stored at ⫺20°C in a cold cryoprotecting solution. Sections were processed
`for immunocytochemical detection of FOS using a conventional avidin-biotin-
`immunoperoxidase method (Vectastain ABC Elite Kit; Vector Laboratories,
`Burlingame, CA) as described (12). The FOS antibody (Sigma-Aldrich,
`Oakville, Ontario, Canada) was used at a 1:50,000 dilution. Brain sections
`corresponding to the level of the area postrema, the nucleus of the solitary
`tract (NTS), the central nucleus of the amygdala, and the parabrachial and
`paraventricular nuclei were defined according to the mouse brain atlas of
`Franklin and Paxinos (14) and selected for analyses.
`Statistical analysis. All data are presented as means ⫾ SE. Statistical
`significance was determined by ANOVA and Bonferroni post-test using Prism
`version 3.03 software (GraphPad Software, San Diego, CA). A P value ⬍0.05
`was considered to be statistically significant.
`
`RESULTS
`Albugon is a recombinant human protein that contains a
`dipeptidyl peptidase-IV (DPP-IV)-resistant human GLP-1
`analog encoded in the same open reading frame as the
`HSA amino acid sequence. To evaluate whether the bioac-
`tive domain(s) of a much smaller peptide hormone could
`still recognize and functionally interact with its cognate
`receptor when constrained within a much larger heterol-
`ogous protein, we examined whether Albugon activated
`GLP-1R in vitro. Albugon produced a dose-dependent
`stimulation of cAMP accumulation in BHK-GLP-1R cells
`that was significantly diminished by coincubation with the
`GLP-1R antagonist exendin (9-39) (Fig. 1). Nevertheless,
`Albugon was not as effective in activating the GLP-1R
`when compared with the much smaller more potent
`lizard-derived GLP-1R agonist Ex-4 (Fig. 1; EC50 ⫽ 0.2 vs.
`20 nmol/l for Ex-4 vs. Albugon, respectively).
`These experiments demonstrate that the considerably
`altered peptide conformation that arises after insertion of
`GLP-1 sequences into a much larger albumin open reading
`frame does not eliminate the ability of essential GLP-1
`motif(s) to recognize and activate GLP-1Rs. To ascertain
`whether circulating Albugon is capable of reaching key
`sites and activating GLP-1R– dependent actions in vivo, we
`carried out oral glucose tolerance testing in mice. Glyce-
`mic excursion was significantly reduced after parenteral
`Albugon administration to wild-type mice (Fig. 2A). Further-
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`DIABETES, VOL. 53, SEPTEMBER 2004
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`ACTIONS OF A RECOMBINANT GLP-1 ALBUMIN PROTEIN
`
`FIG. 1. Albugon exhibits similar efficacy but lower potency than Ex-4 at
`the rat GLP-1R in vitro. A stable BHK cell line expressing the rat
`GLP-1R was pretreated for 5 min with 1 ␮mol/l exendin (9-39) [Ex(9-
`39)] or medium alone before a 10-min treatment with increasing
`concentrations of Ex-4, Albugon, or HSA. cAMP levels in lyophilized
`aliquots of cell extracts were measured by radioimmunoassay and used
`to calculate total cAMP content per well. Values are expressed as
`means ⴞ SE and are representative of data from two independent
`experiments, each performed in triplicate. *P < 0.05, **P < 0.01,
`***P < 0.001 for Ex-4 – vs. Albugon-treated cells.
`
`more, the glucose-lowering properties of Albugon depend-
`ed on a functional GLP-1 receptor because Albugon had no
`effect on blood glucose in GLP-1R⫺/⫺ mice (Fig. 2B). Con-
`sistent with the known actions of smaller GLP-1R peptide
`agonists, Albugon, at doses ranging from 0.1 to 10 mg/kg,
`markedly reduced glycemic excursion after not only oral but
`also IP glucose loading, in association with a significant
`increase in the insulin-to-glucose ratios (Fig. 3A and B).
`Albugon increased the levels of plasma insulin after an IP
`glucose challenge (Fig. 3C) but did not significantly reduce
`the levels of plasma glucagon in mice after an overnight
`fast (Fig. 3D). Taken together, these results demonstrate
`that Albugon lowers blood glucose and enhances insulin
`secretion through GLP-1R–dependent mechanisms in vivo.
`Smaller 30 – to 40 –amino acid GLP-1R agonists, includ-
`ing native GLP-1, Ex-4, and liraglutide, have been shown to
`inhibit food intake, presumably via activation of central
`hypothalamic centers regulating satiety (15). To assess
`whether a much larger recombinant GLP-1–albumin pro-
`tein would also exert anorectic effects, we studied food
`intake in normal mice after ICV or IP peptide administra-
`tion. ICV Albugon significantly lowered food intake in
`mice (relative to the HSA control) in a dose-dependent
`manner, and this effect was detectable for up to 7 h (Fig.
`4A) but was not sustained over the entire 24-h observation
`period. In contrast, Ex-4 exerted a more potent and
`sustained anorectic effect after ICV administration, with a
`highly significant reduction of food intake observed even
`at the end of the 24-h study period (Fig. 3).
`The blood-brain barrier is relatively impermeable to
`larger proteins such as albumin under both normal phys-
`iological conditions and in the setting of diabetes (16,17).
`Although GLP-1Rs are expressed on hypothalamic neurons
`regulating satiety (18,19), whether GLP-1R agonists re-
`quire direct access to the brain for activation of the central
`anorexic pathway remains uncertain (20). Accordingly, we
`examined whether Albugon would also exhibit anorectic
`effects after peripheral administration. Although lower
`doses of IP Albugon, 0.16 –11 nmol/kg, did not affect food
`
`intake, the highest dose tested (110 nmol/kg) reduced food
`intake at all time points studied, from 2 to 24 h after
`Albugon administration (Fig. 4C). In contrast, much small-
`er doses of Ex-4 significantly reduced food ingestion af-
`ter IP administration in the same experiments (Fig. 4D).
`Hence, although Albugon is capable of exerting anorectic
`actions after both central and peripheral administration, it
`is less potent compared with the smaller GLP-1R agonist,
`Ex-4. To ascertain whether the anorectic actions of periph-
`erally administered Albugon were due to the nonspecific
`effects of the larger amount of injected protein, we re-
`peated the experiments using age- and sex-matched wild-
`type control and GLP-1R⫺/⫺ mice. Albugon reduced food
`intake at all time points in wild-type mice but had no effect
`on food intake in GLP-1R⫺/⫺ mice. These findings demon-
`strate that IP Albugon inhibits food intake in a GLP-1R–
`dependent manner.
`GLP-1R agonists markedly inhibit gastric emptying (21),
`and gastric distension induces c-FOS in GLP-1– expressing
`neurons in the rat medulla (22). However, whether direct
`CNS access is required for either GLP-1R– dependent
`inhibition of food intake or gastric emptying has not been
`determined. The basal rate of gastric emptying was com-
`parable in wild-type and GLP-1R⫺/⫺ mice (Fig. 5). Both
`Ex-4 and CCK-8 significantly inhibited gastric emptying in
`wild-type mice; however, CCK-8, but not Ex-4, also inhib-
`ited gastric emptying in GLP-1R⫺/⫺ mice. Similarly, Al-
`bugon potently inhibited gastric emptying in wild-type but
`not in GLP-1R⫺/⫺ mice (Fig. 5).
`The anorectic actions of small peptide GLP-1R agonists
`are associated with c-FOS activation in CNS centers
`
`FIG. 2. Albugon lowers blood glucose in wild-type but not in GLP-1Rⴚ/ⴚ
`mice. Oral glucose tolerance in wild-type GLP-1Rⴙ/ⴙ (A) and GLP-1Rⴚ/ⴚ
`(B) mice given IP injections of 5 mg/kg of HSA (E) or Albugon (ALB; f)
`60 min before an oral glucose load. Values are expressed as means ⴞ
`SE; n ⴝ 5– 6 mice/group. **P < 0.01, ***P < 0.001 for Albugon- vs.
`HSA-treated mice.
`
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`L.L. BAGGIO AND ASSOCIATES
`
`FIG. 3. Albugon dose-dependently lowers the
`glucose excursion and increases plasma insulin
`and insulin-to-glucose ratios after an oral or
`peripheral glucose challenge. Oral glucose toler-
`ance test (A) and IP glucose tolerance test (B) in
`wild-type mice treated with different doses of IP
`HSA or Albugon (ALB) 60 min before a glucose
`load. The bottom panel in A and B indicates the
`plasma insulin-to-glucose ratios (ng/ml:mmol/l)
`at the 20-min time point after glucose adminis-
`tration in HSA- or ALB-treated mice. C: Plasma
`insulin levels at the 20-min time point after IP
`glucose administration in mice treated with 1
`mg/kg HSA (䡺) or ALB (f). D: Plasma glucagon
`levels in fasted mice at 20 min after IP adminis-
`tration of 1 mg/kg HSA (䡺) or ALB (f). Values
`are expressed as means ⴞ SE; n ⴝ 5–12 mice/
`group. *P < 0.05, **P < 0.01, ***P < 0.001 for
`Albugon- vs. HSA-treated mice.
`
`coupled with the control of energy intake (18,20,23). To
`determine whether peripherally administered Albugon
`was capable of activating neuronal FOS expression, we
`examined the pattern and extent of CNS FOS expression
`after IP Albugon administration. Ex-4 markedly increased
`c-FOS expression in the area postrema, the NTS, the
`central nucleus of the amygdala, the parabrachial nucleus,
`and the hypothalamic paraventricular nuclei (Fig. 6A–E).
`Similarly, Albugon significantly activated c-FOS expres-
`sion in the identical brain regions, although much less
`robustly in the NTS and paraventricular nucleus than in
`
`Ex-4 (Fig. 6A–E). Analysis of the murine CNS after periph-
`eral (IP) administration of HSA did not detect immuno-
`reactive HSA in brain parenchyma (data not shown),
`consistent with the inability of HSA to rapidly cross the
`normal blood-brain barrier (8).
`
`DISCUSSION
`GLP-1– based therapies for type 2 diabetes are attracting
`increasing attention in part because of the preliminary
`efficacy demonstrated in clinical studies (6) and because
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`ACTIONS OF A RECOMBINANT GLP-1 ALBUMIN PROTEIN
`
`FIG. 4. Albugon (ALB) reduces food intake in
`fasted mice but is less anorectic than Ex-4. After
`an overnight fast, wild-type mice were given ICV
`(A and B) or IP (C–E) injections of PBS or
`increasing doses (nmol/kg) of Ex-4, HSA, or ALB.
`E: Wild-type and GLP-1Rⴚ/ⴚ mice were fasted
`overnight and then given IP injections of 110
`nmol/kg of HSA or ALB. Food intake was mea-
`sured at 2, 4, 7, and 24 h after recovery from
`injection. Values are expressed as means ⴞ SE;
`n ⴝ 4 –5 mice/group. *P < 0.05, **P < 0.01, ***P <
`0.001 vs. control-treated (PBS or HSA) mice.
`
`of unique yet complementary mechanisms of action (1).
`Unlike some antidiabetic agents that reduce blood glucose
`while promoting weight retention, GLP-1R activation is
`coupled with short-term inhibition of food intake in both
`rodent and human studies (15). Moreover, prolonged
`GLP-1 administration for 6 weeks to diabetic human sub-
`jects was associated with a significant reduction in body
`
`weight over the study period (24). The anorectic proper-
`ties of GLP-1 and its peptide analogs are thought to be due
`in part to both inhibition of gastric emptying and activa-
`tion of central satiety centers coupled with reduction of
`energy intake (20,25). Although development of GLP-1–
`based small peptide analogs resistant to enzymatic inacti-
`vation is a major focus of current pharmaceutical activity
`
`2496
`
`FIG. 5. Albugon (ALB) reduces the
`gastric emptying rate in wild-type but
`not GLP-1Rⴚ/ⴚ mice. Gastric emptying
`rate in wild-type (WT) (A) and GLP-
`1Rⴚ/ⴚ (B) mice at 4 h after IP admin-
`istration of PBS, Ex-4 (0.17 mg/kg),
`HSA (2.7 mg/kg), ALB (3 mg/kg), or
`CCK octapeptide (CCK-8; 4 ␮g/
`mouse) is shown. Values are ex-
`pressed as means ⴞ SE; n ⴝ 3– 4 mice/
`group. *P < 0.05, ***P < 0.001 vs.
`PBS; ##P < 0.01 vs. HSA.
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`L.L. BAGGIO AND ASSOCIATES
`
`FIG. 6. IP Albugon and Ex-4 increase c-FOS levels in the mouse CNS. Representative photomicrographs are shown of c-FOS–stained coronal brain
`sections of area postrema (AP) (A), NTS (B), hypothalamic parabrachial nucleus (PB) (C), central nucleus of the amygdala (CeA) (D), and
`paraventricular nucleus of the hypothalamus (PVH) (E) from wild-type mice at 60 min after IP injection of PBS, Ex-4 (11 nmol/kg), HSA (110
`nmol/kg), or Albugon (110 nmol/kg). No hypoglycemia was detected after administration of either Albugon or Ex-4 in these experiments. Original
`magnification, ⴛ200. CC, central canal; 3V, third ventricle. The number of c-FOS immunopositive (Fosⴙ) cells are depicted below the
`corresponding CNS section. Data are presented as means ⴞ SE; n ⴝ 3 mice/treatment. ***P < 0.001 for Ex-4 – vs. PBS-treated mice at 60 min; #P <
`0.05, ###P < 0.001 for Albugon- vs. HSA-treated mice at 60 min; ^^P < 0.01, ^^^P < 0.001 for Ex-4 – vs. Albugon-treated mice at 60 min.
`
`(1), the need for once- or twice-daily injection of these
`peptides has stimulated efforts toward development of
`even longer-acting molecules that retain the ability to
`activate GLP-1Rs.
`The relatively prolonged circulating t1/2 of endogenous
`and exogenously administered albumin has fostered at-
`tempts directed at coupling peptide moieties to albumin,
`thereby increasing the circulating t1/2 of the albumin-
`peptide complex (26). Albuferon is an interferon-␣–albu-
`min fusion protein that retains the biological activity of
`interferon yet exhibits a markedly extended pharmacoki-
`netic profile relative to native interferon in Cynomolgus
`monkeys (27). Similarly, fusion of the amino acid sequence
`of human growth hormone to albumin prolonged the
`circulating t1/2 of Albutropin relative to the native growth
`hormone, yet Albutropin retained the ability to stimulate
`IGF-I and body weight gain in vivo (28). Moreover, peptide
`binding to albumin extended the pharmacokinetic proper-
`ties of several smaller proteins including insulin (29), Fab
`antibody fragments (26), and coagulation factor VIIa
`inhibitor 1a (30). In contrast to Albugon, however, these
`albumin-peptide derivatives exert their predominant ac-
`tions outside the CNS, and activation of the CNS is not
`critical for their therapeutic actions.
`
`More recent efforts have been directed at extending the
`t1/2 of the much smaller GLP-1 molecule using albumin-
`based approaches. Liraglutide is a fatty acylated human
`DPP-IV–resistant GLP-1 analog that binds to albumin and
`exhibits a t1/2 of ⬃11–15 h after parenteral administration
`in humans (31). Liraglutide inhibits food ingestion in rats
`(32) and decreases gastric emptying in human subjects
`(33); hence, the transient GLP-1–albumin interaction does
`not preclude communication with CNS centers important
`for control of satiety and gastrointestinal motility. How-
`ever, liraglutide binds to albumin in a noncovalent disso-
`ciable manner, with the actions of liraglutide attributed to
`a free peptide unconstrained by its intermittent associa-
`tion with albumin in the circulation.
`We recently studied the properties of CJC-1131, a DPP-
`IV–resistant human GLP-1 analog that covalently couples
`with HSA after parenteral administration (34). CJC-1131
`binds to the GLP-1R and activates a broad spectrum of
`GLP-1R– dependent actions associated with glucose reduc-
`tion in db/db mice, including stimulation of insulin secre-
`tion and insulin gene expression and expansion of islet
`mass (34). Intriguingly, CJC-1131 also activates FOS ex-
`pression in hypothalamic neurons and reduces food intake
`and weight gain in normal and diabetic mice (23,34).
`
`DIABETES, VOL. 53, SEPTEMBER 2004
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`MPI EXHIBIT 1007 PAGE 6
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`

`ACTIONS OF A RECOMBINANT GLP-1 ALBUMIN PROTEIN
`
`FIG. 6. Continued.
`
`Nevertheless, because CJC-1131 is administered parenter-
`ally as the free GLP-1 analog, which subsequently forms a
`covalent linkage with albumin in vivo (34), it remains
`likely that some or all of the acute effects of CJC-1131 on
`the brain reflect the rapid initial actions of free CJC-1131
`before covalent coupling with albumin.
`In contrast, Albugon contains the sequences of human
`GLP-1 linked in the same open reading frame with recom-
`binant HSA; hence, no “free” GLP-1 is associated with
`Albugon administration in vivo. Consistent with studies of
`CJC-1131 covalently conjugated to albumin (34), Albugon
`activates the cloned GLP-1R, but with a reduced affinity
`relative to the potent GLP-1R agonist Ex-4. Because the
`blood-brain barrier is relatively impermeable to albumin,
`peripheral administration of the much larger Albugon
`protein provides an opportunity to determine the relative
`importance of peripheral versus central GLP-1R networks
`for control of satiety and gut motility. Remarkably, Alb-
`ugon inhibited food intake after not only ICV but also after
`IP administration in mice. Similarly, Albugon significantly
`inhibited gastric emptying after IP administration. Further-
`more, the distribution of neuronal c-FOS activation in
`different CNS nuclei after peripheral Albugon administra-
`tion was highly similar, albeit less robust, compared with
`the pattern of FOS activation observed after Ex-4. These
`findings support a model whereby peripheral activation of
`GLP-1R– dependent vagal afferents is capable of activating
`
`CNS centers, transducing the effects of GLP-1 in the brain
`(35,36)
`Given the clinical importance of the anorectic actions of
`GLP-1R agonists for prevention of weight gain in the
`treatment of diabetes, the mechanisms and pathways
`activated by GLP-1R agonists that converge on inhibition
`of feeding centers are of considerable interest. Both native
`GLP-1 and Ex-4 cross the blood-brain barrier through a
`GLP-1R–independent pathway (37–39); hence, these pep-
`tides are capable of directly penetrating and activating
`CNS centers after exogenous administration. In contrast,
`peripheral administration of much larger albumin-based
`GLP-1R agonists, such as CJC-1131 and Albugon, at doses
`that do not induce hypoglycemia, rapidly activates neuro-
`nal c-FOS in distinct brain regions. Furthermore, intrave-
`nous but not ICV CJC-1131 induced tyrosine hydroxylase
`gene transcription in the area postrema in vivo (23).
`Hence, our data suggest that peripheral activation of
`central GLP-1R systems coupled with regulation of FOS
`expression, gastric emptying, and food intake does not
`require direct exposure to GLP-1R agonists in the CNS, but
`may be achieved through activation of ascending path-
`ways coupled with central GLP-1R– dependent networks.
`The current data demonstrate that as little as 0.1 mg/kg
`(1.4 nmol/kg) of Albugon was sufficient for lowering of
`blood glucose; however, much larger doses (110 nmol/kg),
`were required for inhibition of food intake after IP admin-
`
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`istration in mice. These findings imply that acute periph-
`eral Albugon administration is significantly more effective
`at reduction of glycemic excursion relative to inhibition of
`appetite. Nevertheless, the experimental design used here
`did not examine the anorectic effects of more prolonged
`sustained administration, a paradigm more representative
`of the potential therapeutic use of Albugon in humans. Fur-
`thermore, the t1/2 of circulating HSA, and by inference
`Albugon, is much longer in humans than in mice (8). Given
`the intense interest in the development of long-acting GLP-1R
`agonists for the treatment of type 2 diabetes, the biological
`properties and mechanisms of action of GLP-1–albumin
`derivatives merit further investigation.
`
`ACKNOWLEDGMENTS
`D.J.D. was supported in part by operating grants from the
`Juvenile Diabetes Research Foundation and the Canadian
`Diabetes Association and is a Senior Scientist of the
`Canadian Institutes for Health Research. Q.H. was sup-
`ported by a research fellowship from the Canadian Diabe-
`tes Association.
`We thank Human Genome Science scientists for the
`generous provision of Albugon and HSA used in these
`studies.
`
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