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`
`Development and Characterization of a Glucagon-Like Peptide 1-Albumin
`Conjugate The Ability to Activate the Glucagon-Like Peptide 1 Receptor In
`Vivo
`
`Article  in  Diabetes · March 2003
`
`DOI: 10.2337/diabetes.52.3.751 · Source: PubMed
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`MPI EXHIBIT 1033 PAGE 1
`
`

`

`Development and Characterization of a Glucagon-Like
`Peptide 1-Albumin Conjugate
`The Ability to Activate the Glucagon-Like Peptide 1
`Receptor In Vivo
`
`Jung-Guk Kim,1 Laurie L. Baggio,1 Dominique P. Bridon,2 Jean-Paul Castaigne,2 Martin F. Robitaille,2
`Lucie Jette´ ,2 Corinne Benquet,2 and Daniel J. Drucker1
`
`The rapid degradation of native glucagon-like peptide 1
`(GLP-1) by dipeptidyl peptidase-IV (DPP-IV) has fos-
`tered new approaches for generation of degradation-
`resistant GLP-1 analogues. We examined the biological
`activity of CJC-1131, a DPP-IV–resistant drug affinity
`complex (DAC) GLP-1 compound that conjugates to
`albumin in vivo. The CJC-1131 albumin conjugate bound
`to the GLP-1 receptor (GLP-1R) and activated cAMP
`formation in heterologous fibroblasts expressing a GLP-
`1R. CJC-1131 lowered glucose in wild-type mice, but not
`in GLP-1Rⴚ/ⴚ mice. Basal glucose and glycemic excur-
`sion following glucose challenge remained significantly
`reduced 10 –12 h following a single injection of CJC-
`1131. Twice daily administration of CJC-1131 to db/db
`mice significantly reduced glycemic excursion following
`oral and IP glucose challenge (P < 0.01 to 0.05) but did
`not significantly lower body weight during the 4-week
`study period. Levels of random fed glucose were signif-
`icantly lower in CJC-1131–treated ⴙ/ⴙ and db/db mice
`and remained significantly lower even 1 week following
`discontinuation of CJC-1131 administration. CJC-1131
`increased levels of pancreatic proinsulin mRNA tran-
`scripts, percent islet area, and the number of bromode-
`oxyuridine-positive islet cells. These findings demon-
`strate that an albumin-conjugated DAC:GLP-1 mimics
`the action of native GLP-1 and represents a new ap-
`proach for prolonged activation of GLP-1R signaling.
`Diabetes 52:751–759, 2003
`
`Glucagon-like peptide 1 (GLP-1) is a progluca-
`
`gon-derived peptide secreted from intestinal
`L-cells in response to nutrient ingestion (1,2).
`GLP-1 acts as an incretin to lower postprandial
`glycemic excursion via stimulation of insulin secretion and
`inhibition of glucagon secretion. GLP-1 also exerts actions
`
`From the 1Banting and Best Diabetes Centre, Department of Medicine,
`University of Toronto, Toronto General Hospital, Toronto, Ontario; and
`2Conjuchem, Montreal, Quebec.
`Address correspondence and reprint requests to Dr. Daniel J Drucker, 200
`Elizabeth St., MBRW-4R-402, Toronto, Ontario, Canada M5G 2C4. E-mail:
`d.drucker@utoronto.ca.
`Received for publication 16 July 2002 and accepted in revised form 6
`November 2002.
`AUC, area under the curve; CHO, Chinese hamster ovary; DAC, drug affinity
`complex; DPP-IV, dipeptidyl peptidase-IV; GIP, glucose-dependent insulino-
`tropic peptide; GLP-1, glucagon-like peptide 1; GLP-1R, GLP-1 receptor; HSA,
`human serum albumin.
`
`independent of islet hormone secretion, including inhibi-
`tion of both gastric emptying and food intake (3,4), and
`stimulation of ␤-cell proliferation (5,6).
`Although the structurally related gut hormone glucose-
`dependent insulinotropic peptide (GIP) also potentiates
`glucose-dependent insulin secretion (7), unlike GLP-1, the
`insulinotropic actions of GIP are diminished in diabetic
`rodents or in human subjects with type 2 diabetes (8,9). In
`contrast, GLP-1 administration rapidly lowers glucose in
`both normal and diabetic subjects (9 –11), and 6 weeks of
`continuous subcutaneous infusion of native GLP-1 signif-
`icantly decreased blood glucose and HbA1c in human
`patients with type 2 diabetes (12). Hence there is consid-
`erable enthusiasm for the development of GLP-1– based
`pharmaceutical agents for the treatment of type 2 diabetes
`(2,13).
`Although native GLP-1 effectively lowers blood glucose
`following acute peptide administration (14,15), both en-
`dogenous and exogenously administered GLP-1 exhibit a
`short t1/2 in vivo due primarily to NH2-terminal cleavage
`and inactivation by the enzyme dipeptidyl peptidase (DPP-
`IV) (16,17). The physiological importance of DPP-IV for
`GLP-1 degradation and glucose homeostasis is exemplified
`by studies of mice or rats with inactivating mutations in
`the DPP-IV gene. These rodents exhibit enhanced glucose
`clearance following glucose challenge and increased cir-
`culating levels of intact GLP-1 in vivo (18,19). Similarly,
`administration of DPP-IV enzyme inhibitors is associated
`with reduced glycemic excursion, enhanced insulin secre-
`tion, and reduced degradation of GLP-1 in normal and
`diabetic rodents (20 –22) and in human subjects (23).
`Accordingly, there is considerable interest in comple-
`mentary strategies for circumventing the rapid cleavage of
`GLP-1, including the development of GLP-1– based analogs
`with enhanced resistance to degradation and increased
`biological potency in vivo (13,24). The majority of these
`analogs exhibit one or more amino acid substitutions that
`reduce the affinity of the peptide for DPP-IV and subse-
`quent cleavage both in vitro and in vivo. Similarly, the
`naturally occurring lizard peptide exendin-4 is a potent
`GLP-1R agonist that exhibits reduced DPP-IV mediated
`cleavage and a longer duration of action in both rodents
`and human subjects (25,26). We have recently initiated
`studies of GLP-1 derivatives that are resistant to DPP-IV
`and are synthesized with a short covalent reactive chem-
`
`DIABETES, VOL. 52, MARCH 2003
`
`751
`
`MPI EXHIBIT 1033 PAGE 2
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`

`

`CJC-1131, A GLP-1 ALBUMIN GLP-1R AGONIST
`
`ical linker that interacts with a specific cysteine residue in
`the albumin molecule following parenteral administration
`of the modified GLP-1 peptide. The resultant GLP-1–
`albumin drug affinity complex (DAC) is predicted to retain
`the actions of GLP-1, yet exhibit a more prolonged dura-
`tion of action due to a combination of DPP-IV resistance
`and the longer t1/2 conferred by serum albumin in vivo. To
`test the hypothesis that albumin-bound DAC–GLP-1 deriv-
`atives retain the biological properties of native GLP-1, we
`studied the activity of a DAC–GLP-1 compound, CJC-1131,
`using cells and in normal GLP-1R⫺/⫺ and diabetic ro-
`dents.
`
`RESEARCH DESIGN AND METHOD
`Animals. All animal experiments were carried out in accordance with
`protocols approved by the Toronto General Hospital Animal Care Committee
`or the Comite´ Institutionnel de Protection des Animaux de l’UQAM. C57BL/6
`db/db mice (The Jackson Laboratory, Bar Harbor, ME) and age- and sex-
`matched C57BL/6 wild-type mice from the same genetic background were
`used for chronic administration studies following a minimum 1-week accli-
`matization period in the animal facility. Wild-type CD-1 mice and GLP-1R⫺/⫺
`mice (27) in the CD-1 background were used for acute peptide administration
`experiments. Mice were allowed ad libitum access to food and water, except
`where noted. Animals were on a 12-h light, 12-h dark cycle (lights on 0700 h).
`For dose-response experiments shown in Fig. 2B, female CD-1 mice (Charles
`River Canada, St-Constant, QC), 7- to 10-weeks old, were studied, whereas for
`glycemic measurements shown in Figs. 2C and D, experiments were carried
`out with female db/db mice aged 7–10 weeks. CJC-1131 is a synthetic
`modification of GLP-1 (Fig. 1A) consisting of a single amino acid substitution
`of L-Ala8 to D-Ala8 at position 2, enabling some additional protection from
`DPP-IV and the addition of a lysine (Lys37) to the COOH-terminal of the
`peptide. The other modification involves the selective attachment of a
`[2-[2-[2-maleimidopropionamido(ethoxy)ethoxy]acetamide to the epsilon
`amino group of Lys37.
`In vitro experiments: binding affinity and cyclic AMP generation.
`Binding affinity was determined by incubating either native GLP-1 or CJC-1131
`and 0.03 nmol/l 125I-labeled GLP-1(7–36) amide with Chinese hamster ovary
`(CHO) cells stably transfected with the human GLP-1R cDNA using an
`incubation buffer of 20 mmol/l Tris-HCl, pH 7.4, 5 mmol/l MgCl2, 20 mmol/l
`NaCl, 2% BSA for 90 min at 37°C. For analysis of cAMP generation, CHO–
`GLP-1R cells were incubated for 20 min with various peptide ligands in the
`presence of 100 mmol/l IBMX followed by analysis of cAMP as described (28).
`Experimental protocol for db/db mouse studies. Baseline plasma glucose,
`oral and intraperitoneal glucose tolerance testing, and food intake were
`assessed before beginning CJC-1131 treatment. During the 4-week treatment
`period, wild-type and db/db mice were given an intraperitoneal injection of
`either 25 ␮g of CJC-1131 or an equal volume of saline twice daily at 800 and
`1600 h. Following the 4-week treatment period, a subset of mice from each
`group was sacrificed for analyses, whereas an additional set of mice continued
`to be monitored for another 3 weeks.
`Glucose tolerance tests and measurement of plasma insulin. Oral and
`intraperitoneal glucose tolerance tests were carried out following an over-
`night fast (16 –18 h) as described (27,29). Glucose (1.5 mg/g body wt) was
`administered orally through a gavage tube or via injection into the peritoneal
`cavity, and blood was drawn from a tail vein at 0, 10, 20, 30, 60, 90, and 120 min
`after glucose administration. Blood glucose levels were measured by the
`glucose oxidase method using a glucose meter (Glucometer Elite; Bayer,
`Toronto, Canada). For plasma insulin determinations, a blood sample was
`removed from the tail vein during the 10- to 20-min period following glucose
`administration, and plasma insulin was determined using a rat insulin enzyme-
`linked immunosorbent assay kit (Crystal Chem, Chicago, IL) with mouse
`insulin as a standard.
`RNA isolation and Northern blot analysis. Mice were anesthetized with
`CO2, and pancreata were removed immediately for RNA extraction by the
`acid-guanidinium isothiocyanate method. Total RNA (10 ␮g) was electropho-
`resed in a 1% (wt/vol) formaldehyde-agarose gel and transferred to a nylon
`membrane (Nytran Plus; Schleicher & Schuell, Keene, NH). For Northern blot
`analysis, the blot was hybridized to 32P-labeled random-primed complemen-
`tary DNA probes corresponding to rat proglucagon, rat insulin, or 18S RNA.
`Analysis of islet size and ␤-cell proliferation. Mice were injected with 100
`mg/kg 5-bromo-2⬘-deoxyuridine (BrdU, Sigma-Aldrich, St. Louis, MO) intra-
`peritoneally 6 h before being sacrificed. The pancreas was removed, fixed
`overnight in either 10% buffered formalin or 4% paraformaldehyde, and
`
`FIG. 1. A: Structural representation of CJC-1131 consisting of a single
`amino acid substitution of L-Ala8 to D-Ala8 at position 2 and a Lys37
`addition to the COOH-terminus with selective attachment of a [2-
`[2-[2-maleimidopropionamido(ethoxy)ethoxy]acetamide to the epsilon
`amino group of Lys37. B: Binding affinity of native GLP-1 (f) or
`CJC-1131:HSA (E) in CHO cells transfected with the human recombi-
`nant GLP-1R. C: cAMP response of native GLP-1 (f) or CJC-1131:HSA
`(E) in CHO cells transfected with the human recombinant GLP-1R.
`
`embedded in paraffin. Sections were obtained and stained with hematoxylin
`and eosin using standard protocols. Histological sections were immuno-
`stained for insulin using guinea-pig anti-insulin (Dako Diagnostics Canada,
`Mississauga, ON, Canada) as primary antibody (1:100 dilution) and rabbit
`anti– guinea-pig immunoglobulin (Dako Diagnostics Canada) as secondary
`antibody (1:50 dilution). Antibody binding was visualized by 3,3-diaminoben-
`zidine, and sections were counterstained by Meyers hematoxylin. Islet histo-
`morphometry was carried out according to the principles of Delesse (30). The
`sections were examined using a Leica (Leitz Labor Lux S; Leica Microsystem,
`Heerbrugg, Switzerland) microscope equipped with a video camera and
`connected to a computer with imaging software (Q500MC; Leica Microsys-
`tem). Estimates of islet area, islet number, number of BrdU immunopositive
`cells, and total pancreatic area were determined in a blinded manner as
`described (29,31).
`Statistics. Results are expressed as mean ⫾ SD or mean ⫾ SE. Statistical
`significance was calculated by Student’s t test using SPSS windows version 10
`(SPSS, Chicago, IL). P ⬍ 0.05 was considered to be statistically significant.
`
`RESULTS
`To circumvent the short biological t1/2 of native GLP-1 due
`principally to DPP-IV–mediated degradation, we designed
`a new DPP-IV–resistant GLP-1 derivative that would form
`
`752
`
`DIABETES, VOL. 52, MARCH 2003
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`MPI EXHIBIT 1033 PAGE 3
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`

`

`KIM AND ASSOCIATES
`
`FIG. 2. A: Blood glucose levels in 9-week-old wild-type C57ⴙ/ⴙ male
`mice during an oral glucose tolerance test performed 30 min after
`either intraperitoneal or subcutaneous administration of saline or
`CJC-1131 (25 ␮g); n ⴝ 5 mice/group. Values are expressed as means ⴞ
`SE. B: Blood glucose levels in 7- to 10-week-old female CD-1 mice
`during an oral glucose tolerance test 5 min after an intravenous
`administration of saline (f) or 1 (䡺), 10 (F), or 100 (E) nmol/kg of
`CJC-1131; n ⴝ 10 mice/group. Values are expressed as mean ⴞ SE; *P <
`0.05, **P < 0.02, ***P < 0.001 for differences in area under the curve
`(AUC) glucose for mice treated with saline vs. 1, 10, and 100 nmol/kg
`CJC-1131, respectively. C: Basal glycemia in 6- to 7-week-old female
`diabetic db/db mice after a single subcutaneous injection of either
`vehicle (f) or 100 nmol/kg CJC-1131 (E); n ⴝ 5 mice/group. Values are
`expressed as means ⴞ SE; *P < 0.02 for AUC glucose in saline versus
`CJC-1131–treated mice. D: Blood glucose levels in 7- to 10-week-old
`female db/db mice during an oral glucose tolerance test performed 12 h
`after intravenous administration of saline (f), 100 nmol/kg of native
`GLP-1 (䡺), or 100 nmol/kg of CJC-1131 (E); n ⴝ 5 mice/group. Values
`are expressed as mean ⴞ SE; *P < 0.05 for AUC glucose in saline versus
`CJC-1131–treated mice. E: Blood glucose levels in wild-type CD-1ⴙ/ⴙ
`(E) and GLP-1R ⴚ/ⴚ (F) mice during an oral glucose tolerance test
`performed 60 min after intraperitoneal administration of CJC-1131
`(25 ␮g); n ⴝ 9 –10 mice/group. Values are expressed as mean ⴞ SE; *P <
`0.05; **P < 0.01. #P < 0.001 for AUC glucose in GLP-1Rⴙ/ⴙ vs.
`GLP-1Rⴚ/ⴚ mice.
`
`a covalent bond with albumin in vivo, thereby conferring
`to covalently linked GLP-1 the longer circulating t1/2 of
`albumin (32). A D-alanine residue at position 2 was substi-
`tuted for the native alanine, and the COOH-terminal end of
`the GLP-1 molecule was coupled to a reactive chemical
`linker capable of forming a 1:1 covalent bond to the Cys
`residue in serum albumin (Fig. 1A). The resultant GLP-1
`derivative, designated CJC-1131, should retain the biolog-
`ical properties of native GLP-1 yet exhibit DPP-IV resis-
`tance and a prolonged t1/2 consistent with the clearance of
`native serum albumin in vivo (33).
`To assess the properties of a CJC-1131– human serum
`albumin (HSA) conjugate, we incubated HSA with CJC-
`1131 in vitro and utilized the purified CJC-1131:HSA con-
`
`jugate for studies of GLP-1R binding using CHO cells
`transfected with human recombinant GLP-1R (Fig. 1B).
`The displacement of 125I-labeled GLP-1 by native GLP-1
`versus the CJC1131:HSA complex was highly similar over
`a range of CJC-1131 concentrations (Ki ⫽ 5.16 nmol/l for
`native GLP-1 vs. 12 nmol/l for CJC-1131:HSA). To assess
`the bioactivity of CJC-1131, we measured cAMP accumu-
`lation using CHO cells transfected with human recombi-
`nant GLP-1R. These experiments demonstrated virtually
`identical dose-response relations for cAMP accumulation,
`with native GLP-1 exhibiting an EC50 of 13 nmol/l, whereas
`the EC50 for CJC-1131–HSA was 11–13 nmol/l (Fig. 1C).
`We next assessed whether the route of CJC-1131 admin-
`istration was an important determinant of bioactivity in
`
`DIABETES, VOL. 52, MARCH 2003
`
`753
`
`MPI EXHIBIT 1033 PAGE 4
`
`

`

`CJC-1131, A GLP-1 ALBUMIN GLP-1R AGONIST
`
`C57BL/6 mice following glucose loading. CJC-1131 mark-
`edly reduced glycemic excursion following intraperitoneal
`or subcutaneous administration (Fig. 2A). Similarly, intra-
`venous CJC-1131 administration produced a dose-depen-
`dent reduction in glycemic excursion following glucose
`loading (Fig. 2B). To ascertain whether a single injection
`of CJC-1131 would lower blood glucose in diabetic mice,
`we administered CJC-1131 by subcutaneous injection to
`db/db mice. Remarkably, basal random glycemia de-
`creased rapidly and remained lower in db/db mice for up to
`10 h following a single subcutaneous injection of CJC-1131
`(Fig. 2C). Furthermore, the glucose-lowering effect of
`CJC-1131 was still evident during an oral glucose tolerance
`test performed 12 h after a single intravenous CJC-1131
`injection (Fig. 2D). Although CJC-1131 consistently low-
`ered glucose in wild-type mice, no effect was observed in
`GLP-1R⫺/⫺ mice (Fig. 2E), demonstrating the critical
`importance of an intact GLP-1R for the biological activity
`of CJC-1131 in vivo.
`These findings demonstrated that single injections of
`CJC-1131 exert glucose-lowering effects in normal and
`diabetic mice. We next assessed whether more prolonged
`repeated administration of CJC-1131 would lower glucose
`in mice with severe diabetes. Wild-type control C57BL/6
`(C57⫹/⫹) ordb/db mice were treated with saline or
`CJC-1131 twice daily for 4 weeks. Before initiation of
`CJC-1131, mean fasting glucose was 4.3 ⫾ 0.9 mmol/l in
`C57⫹/⫹ vs. 17.9 ⫾ 6.7 mmol/l in db/db mice (Fig. 3A). After
`2 weeks of twice daily saline or CJC-1131 administration,
`fasting blood glucose was significantly lower in both
`control C57⫹/⫹ and db/db mice treated with CJC-1131
`(4.5 ⫾ 0.7 vs. 3.3 ⫾ 0.5 mmol/l and 19.2 ⫾ 5.8 vs. 12.5 ⫾ 3.7
`mmol/l, saline vs. CJC-1131 in C57⫹/⫹ vs. db/db mice,
`respectively; P ⬍ 0.01, Fig. 3A). Fasting glucose remained
`significantly lower in C57⫹/⫹ mice but not in db/db mice
`treated with CJC-1131 at the end of the 4-week treatment
`period (Fig. 3A). In contrast, fed blood glucose was
`significantly lower in CJC-1131–treated C57⫹/⫹ and db/db
`mice throughout the 4-week experiment (Fig. 3B; P ⬍ 0.01,
`saline versus CJC-1131). Furthermore, fed blood glucose
`remained significantly lower in db/db mice 1 week follow-
`ing the last injection of CJC-1131 (Fig. 3B, P ⬍ 0.05).
`Although CJC-1131 produced a small but significant de-
`crease in body weight in C57⫹/⫹ mice, no differences in
`body weight were detected in db/db mice treated with
`saline versus CJC-1131 over the 4-week experiment (Fig.
`3C).
`To determine whether repeated administration of CJC-
`1131 was associated with improvement in glucose toler-
`ance, we performed oral and intraperitoneal glucose
`tolerance testing in C57⫹/⫹ and db/db mice treated with
`saline or CJC-1131 for 2 weeks. Blood glucose excursion
`was significantly lower
`in CJC-1131–treated control
`C57⫹/⫹ mice following either oral or intraperitoneal
`glucose loading (Fig. 4A and B). Similarly, both fasting
`glucose and the glycemic excursion following glucose
`loading were modestly but significantly reduced in CJC-
`1131–treated db/db mice at multiple time points (Fig. 4A
`and B). Although plasma insulin levels were significantly
`greater in db/db mice compared with C57⫹/⫹ mice both in
`the fasting state and after glucose loading, plasma insulin
`
`FIG. 3. A: Weekly fasting blood glucose in C57ⴙ/ⴙ (E, F) and db/db (‚,
`Œ) mice before and during i.p treatment with saline (E, ‚) or 25␮g
`CJC-1131 (F, Œ) twice daily for 4 weeks and for several weeks
`following discontinuation of active treatment. The arrow denotes the
`end of the 4-week treatment period. B: Weekly fed blood glucose in
`C57ⴙ/ⴙ (E, F) and db/db (‚, Œ) mice treated with saline (E, ‚) or 25
`␮g CJC-1131 (F, Œ) twice daily observation for 4 weeks and observation
`for 3 weeks. C: Weekly body weight in C57ⴙ/ⴙ (E, F) and db/db (‚, Œ)
`mice treated with saline or 25 ␮g CJC-1131 twice daily by intraperitoneal
`injection for 4 weeks. Values are expressed as means ⴞ SE; *P < 0.05,
`**P < 0.01; n ⴝ 10–20 mice per group for experiments depicted in A–C.
`
`levels were not further increased in db/db mice treated
`with CJC-1131 (data not shown).
`GLP-1R agonists have been shown to increase proinsu-
`lin gene expression and promote islet neogenesis and
`␤-cell proliferation (5,6,34). To determine whether a larger
`CJC-1131:albumin conjugate exhibits comparable actions
`on the diabetic pancreas in vivo, we carried out Northern
`blot analysis using pancreatic RNA from C57⫹/⫹ and
`db/db mice. Levels of pancreatic insulin mRNA transcripts
`were comparable in normoglycemic wild-type C57⫹/⫹
`
`754
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`DIABETES, VOL. 52, MARCH 2003
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`MPI EXHIBIT 1033 PAGE 5
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`KIM AND ASSOCIATES
`
`compared with control C57⫹/⫹ mice, but was not signif-
`icantly different in saline– vs. CJC-1131–treated C57⫹/⫹ or
`db/db mice (Fig. 5B).
`To determine whether CJC-1131 administration was
`associated with changes in the number or size of pancre-
`atic islets, we carried out morphometric analysis of pan-
`creata from C57⫹/⫹ and db/db mice following 4 weeks of
`treatment with either saline or CJC-1131 (Fig. 6). Total
`percent islet area was increased in C57⫹/⫹ mice and
`significantly increased in db/db mice following CJC-1131
`administration (P ⬍ 0.01; Fig. 6B). We next assessed the
`proportion of small, medium, and large islets, as previ-
`ously described (35). CJC-1131 treatment significantly
`increased the percent large islet area in db/db mice (Fig.
`6C; P ⬍ 0.01). The percentage of large islets was also
`increased in control mice treated with CJC-1131, but this
`difference did not achieve statistical significance (Fig. 6C).
`In contrast, the number of small islets was significantly
`increased in both C57⫹/⫹ (212.2%, P ⬍ 0.05) and db/db
`mice (94.6%, P ⬍ 0.05) treated with CJC-1131 (Fig. 6D).
`Furthermore, CJC-1131 significantly increased the total
`number of islets in both C57⫹/⫹ and db/db mice (Fig. 6E).
`To determine whether the observed differences in per-
`cent islet area and number of islets were attributable in
`part to increased rates of islet cell replication, we assessed
`the numbers of BrdU⫹ islet cells in saline– or CJC-1131–
`treated mice (Fig. 6F and G). CJC-1131 administration
`significantly increased the number of BrdU⫹ islet cells in
`db/db mice (413.1%, P ⬍ 0.01; Fig. 6G).
`
`FIG. 4. Blood glucose levels following oral (A) or intraperitoneal (B)
`glucose challenge in C57ⴙ/ⴙ (E, F) and db/db mice (‚, Œ), n ⴝ 20 mice
`per group, after 2 weeks of twice daily administration of either saline
`(E, ‚) or 25␮g CJC-1131 (F, Œ). AUC for oral glucose tolerance
`testing in C57ⴙ/ⴙ mice (saline treated 825.4 ⴞ 36.3 and CJC-1131–
`treated 550.2 ⴞ 29.0; P < 0.001) and db/db mice (saline treated
`3,620.6 ⴞ 55.0 and CJC-1131–treated 3,081.4 ⴞ 161.0; P < 0.01). AUC
`for intraperitoneal glucose tolerance testing in C57ⴙ/ⴙ mice (saline-
`treated 985.1 ⴞ 65.6 and CJC-1131–treated 434.2 ⴞ 28.0; P < 0.001)
`and db/db mice (saline-treated 3,298.1 ⴞ 204.8 and CJC-1131–treated
`2,824.6 ⴞ 218.6; P < 0.05). Values are expressed as mean ⴞ SE; *P <
`0.05, **P < 0.01.
`
`mice treated with either saline or CJC-1131. In contrast, 4
`weeks of CJC-1131 administration significantly increased
`the levels of proinsulin mRNA transcripts in db/db mice
`(Fig. 5A). Pancreatic insulin content was reduced in db/db
`
`DISCUSSION
`The observation that GLP-1 administration effectively re-
`duces blood glucose in human subjects with type 2 diabe-
`tes has
`stimulated considerable effort
`toward the
`development of GLP-1– based agonists for pharmaceutical
`administration. Although infusion of native GLP-1 is highly
`effective in lowering blood glucose in diabetic patients
`(12,36), the native peptide is an excellent substrate for
`DPP-IV (16,17), and exhibits a very short t1/2 in vivo.
`Hence, the requirement for constant GLP-1 infusion places
`potential constraints on the widespread use of native
`GLP-1 in the diabetes clinic. Accordingly, there remains
`ongoing interest in the development and characterization
`of peptidase-resistant GLP-1 analogs with longer durations
`
`FIG. 5. A: Pancreatic insulin mRNA; the lower panel depicts relative ratios of insulin/18s RNA from multiple analyses. **P < 0.01. B: Insulin
`content in C57ⴙ/ⴙ (n ⴝ 6) and db/db (n ⴝ 6) mice after 4 weeks of saline or CJC-1131 (25 ␮g twice daily) administration.
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`DIABETES, VOL. 52, MARCH 2003
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`CJC-1131, A GLP-1 ALBUMIN GLP-1R AGONIST
`
`FIG. 6. Histological and morphometric analysis of pancreatic islets
`from C57ⴙ/ⴙ and db/db mice after 4 weeks of saline (䡺) or twice daily
`CJC-1131 (25 ␮g) treatment (f). A: Immunohistochemical staining for
`insulin. B: Percent total islet area/total pancreatic area. C: Percent
`large islet area/total pancreatic area. D: Number of small islets/
`pancreatic area (1 ⴛ 106 ␮m2). E: Total islet number/pancreatic area.
`F: Histological representation of BrdU immunopositive islet cells. G:
`quantitation of BrdU positive cells per pancreatic area (1 ⴛ 107 ␮m2)
`in C57ⴙ/ⴙ (n ⴝ 6) and db/db (n ⴝ 6) mice. Values are expressed as the
`means ⴞ SE; *P < 0.05, **P < 0.01.
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`756
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`MPI EXHIBIT 1033 PAGE 7
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`

`

`of action suitable for subcutaneous administration in vivo
`(13).
`Several approaches have been undertaken to develop
`stable long-acting GLP-1 analogs, with DPP-IV–resistant
`molecules such as NN2211 currently being tested in clini-
`cal trials for the treatment of type 2 diabetes (37). Ex-
`endin-4, a naturally occurring lizard salivary gland peptide
`(38) that exhibits potent GLP-1–like activity and exerts its
`actions through the GLP-1R, is also undergoing clinical
`evaluation for the treatment of type 2 diabetes (26,39). The
`data presented here describe the results of introducing
`two specific modifications of the native GLP-1 molecule,
`one to engineer DPP-IV resistance, the second to achieve
`chemical coupling of the GLP-1 analog (CJC-1131) to
`serum albumin. Albumin is known to exhibit a longer t1/2
`in vivo, ⬃19 days in humans (40), which is much greater
`than the t1/2 of short-lived regulatory peptides such as
`GLP-1. Accordingly, peptide binding to albumin has been
`used to improve the pharmacokinetic properties of several
`smaller proteins, including Fab antibody fragments (33),
`coagulation factor VIIa inhibitor 1a (41), and insulin (42).
`The present results indicate that a much larger albumin–
`GLP-1 covalently linked conjugate retains the biological
`properties of native GLP-1, including binding to and acti-
`vation of the GLP-1R in vitro. Furthermore, the glucose-
`lowering effects of CJC-1131 were abolished in GLP-
`1R⫺/⫺ mice,
`providing
`important
`complementary
`evidence that the actions of CJC-1131 in vivo are mediated
`by the known GLP-1R.
`Several experimental results provide indirect evidence
`for a sustained duration of CJC-1131 action on glucose
`lowering in vivo. First, oral glucose tolerance remained
`significantly improved in mice even 12 h after a single
`CJC-1131 injection. Furthermore, fed glucose remained
`significantly lower in db/db mice treated with CJC-1131,
`even 1 week after the last CJC-1131 injection. These
`findings are consistent with data demonstrating sustained
`improvements in insulin secretion in Zucker diabetic fatty
`rats detectable even 1 week following a transient GLP-1
`infusion (43). Similarly, blood glucose remained lower in
`exendin-4 –treated partially pancreatectomized rats sev-
`eral weeks following discontinuation of exendin-4 admin-
`istration (5). Although fed glucose and glucose tolerance
`were clearly improved in db/db mice treated with CJC-
`1131, significant hyperglycemia persisted even during re-
`peated CJC-1131
`administration.
`In contrast, daily
`administration of exendin-4 to younger, less severely dia-
`betic db/db mice (starting fasting hyperglycemia of 5.1
`mmol/l) prevented the progression to more severe hyper-
`glycemia over the 2-week treatment period (44). However,
`a 2-week treatment of older, more severely diabetic db/db
`mice with twice daily administration of 200 ␮g/kg NN221
`or exendin-4 (100 ␮g/kg) did not prevent ongoing deterio-
`ration in glucose tolerance, with mice remaining very
`hyperglycemic even after the 2-week treatment period
`(45). Hence the effects of chronic GLP-1 agonist adminis-
`tration in the db/db mouse appear dependent, in part, on
`the clinical severity of the diabetes and age of the mice at
`the onset of treatment.
`Although treatment of rodents and human subjects with
`GLP-1R agonists has been associated with prevention of
`weight gain or modest weight loss (12,25,46), we did not
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`DIABETES, VOL. 52, MARCH 2003
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`KIM AND ASSOCIATES
`
`observe a significant reduction in body weight in db/db
`mice treated with CJC-1131 over the 4-week treatment
`period. Similarly, body weight was not significantly re-
`duced in db/db mice treated with NN2211 or exendin-4
`over comparable 2- to 4-week treatment periods (45,47). At
`present, it remains unclear whether restricted transport of
`an albumin–GLP-1 conjugate across the blood-brain bar-
`rier might compromise the ability of molecules such as
`albumin–CJC-1131 to reduce food intake and weight gain
`in vivo. Nevertheless, significant weight loss was observed
`in control C57⫹/⫹ mice treated with CJC-1131 over the
`same time period. Furthermore, intravenous infusion of
`CJC-1131 rapidly induced c-fos immunoreactivity in sev-
`eral regions of
`the rat central nervous system (H.
`Yamamoto, J. Elmquist, and D.J.D., unpublished observa-
`tions), providing complementary evidence suggesting that
`an albumin–GLP-1 conjugate is capable of activating cen-
`tral nervous system functions in vivo.
`Despite the failure to observe normalization of glycemia
`in the older db/db mouse, CJC-1131 administration in-
`creased islet area, with a significant increase in both the
`number and the relative percentage of small and large
`islets detected following 4 weeks of CJC-1131 treatment.
`Furthermore, the number of BrdU⫹ islet cells was signif-
`icantly increased in CJC-1131–treated mice, consistent
`with previous studies demonstrating that activation of the
`GLP-1R is coupled to expansion of islet mass in the db/db
`mouse (6,44,45). Hence, although fenestration of islet
`capillaries does restrict access and reduce permeability
`toward larger molecules in the islet microcirculation (48),
`the comparatively larger CJC-1131–albumin conjugate ap-
`parently retains the ability to promote islet growth and
`enhance insulin gene expression in the mouse in vivo.
`Despite the increase in islet area and proinsulin mRNA
`transcripts in CJC-1131–treated mice, we did not observe a
`significant increase in insulin content in the db/db mice
`treated with CJC-1131. Indeed, pancreatic insulin content
`was modestly decreased in control and CJC-1131–treated
`db/db mice relative to C57⫹/⫹ mice consistent with the
`functional exhaustion of ␤-cell insulin stores in db/db mice
`with blood glucose levels approaching 20 mmol/l.
`Given the increasing interest in strategies based on
`enhancement of incretin action for the treatment of dia-
`betes, there remains an unmet need for developing ap-
`proaches that circumvent the requirement for daily or
`continuous administration of GLP-1 analogs. Accordingly,
`long-acting or slow-release forms of GLP-1 analogs that
`exhibit desirable therapeutic properties are awaited with
`interest. The administration of albumin-based GLP-1 deriv-
`atives should confer a longer circulating t1/2 to the newly
`derived albumin-peptide conjugate, compared with the
`native GLP-1 peptide alone (32,33). The data presented
`here demonstrate that a prototype DPP-IV–resistant GLP-1
`analog that covalently couples to albumin, CJC-1131, re-
`tains the biological properties generally ascribed to
`GLP-1R agonists. The findings that CJC-1131 binds to and
`activates the GLP-1R,
`lowers blood glucose,
`increases
`proinsulin gene expression, and stimulates islet cell pro-
`liferation suggest that further assessment of the efficacy
`and mechanisms of action of albumin-based GLP-1 deriv-
`atives appears warranted.
`
`757
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`CJC-1131, A GLP-1 ALBUMIN GLP-1R AGONIST
`
`ACKNOWLEDGMENTS
`The w