Reviewe Anthony L Parola, PhD
`
`NDA No. 22-341
`
`mu {5‘s «grains nfierfi weeks exposure
`bmmmm "ML
`.mieeme riboprcbe Io IGLPJR mRNA: cigar minus»: i
`
`Figure $6 Gl‘P-IR mRNA expression in rat pancreatic islets
`
`[N000 4.2.3.7.3 P30]
`
`sit
`
`
`award fluorescence microscope.
`figure 35 Rat zlvyzaié co-Iocalisution of calcitonin mRNA and immutmmictivity
`
`[N000 4.2.3.7.3 P22]
`
`In mouse thyroid tissue sections, high backgroundanti-calcitonin antibody staining
`precludes the identification of c-cells (Figures 1 and 2). Since GLP-1 receptor mRNA was at or
`below the level of visual detection using the anti-sense probe and high background calcitonin
`immunoreactivity, colocalization of calcitonin immunoreactivity and GLP-1 receptor mRNA was
`equivocal. Non-specific mRNA detection using a sense probe confirms that GLP—1 receptor
`mRNA was at or below the level of detection (Figure 2).
`
`31
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22-341
`
`01: Ml. Calcitoniu demon:
`liver grains in auromdiagmpbs after in siru hybridisation
`\lcxMXS complex: tower left
`ml: ”5 labelled antisense ribuprobe r0 miGLP-i R: right: composilcofmc lFL and in-
`. itu hybridisation frames made by ‘ndding‘ihc two pictures after a small adjustment or
`- he commsr to tower the blurr (if {he gem) fluorescence. Cryostat sections.
`
`Figure 1
`iybridismicn frames Cryostat sections
`
`vii 1 S labcitcd s.
`
`~cr lcfi: silwr grains in automdibgmphs after in sin: hybridisation
`\L tibnprobc lo mLGLP-i R: right: composite oflhc IFL and iii-Sim
`
`Figure 2 Mouse thyroid GLP-iR mRNA in-situ hybridisation sense control with C-cell
`calcitonin lFL
`
`[NOOO 4.2.3.7.3 P16]
`
`As a positive control, anti-sense human GLP—1 receptor RNA probes labeled cells in rat
`pancreas and an antisense calcitonin probe labeled anti-calcitonin immunoreactive cells in human
`thyroid tissue sections. 35’S-labeled calcitonin riboprobes labeled specific cells in mouse thyroid
`tissue sections, but due to high background staining with the anti-calcitonin antibody, it was not
`possible to discern if mRNA labeled cells were positive for calcitonin immunoreactivity (Figure
`7).
`
`32
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22-341
`
`alum»: tn-sinx hybridisation with sens
`
`grains alter 1‘»
`right
`
`Figure 7 Mnuse thyroid cn-Iomlisntion of calcitmiin mRNA mid immlmoreactivity
`
`mooo 4.2.3.7.3 P21]
`
`Figure 5 shows colocalization of GLP- 1 receptor mRNA and calcitonin immunoreactivity
`was negative in monkeys As a positive control, anti--sense monkey GLP- 1 receptor RNA probes
`labeled specific cellsin monkey pancreas and an antisense calcitonin probe labeled calcitonin
`immunoreactive cellsin monkey thyroid tissue sections (not shown).
`
`Ion M211 zlsmsmsc nhfiwo‘v‘c 10 [10117431
`
`amply“; mm 31 thyroid C<
`all Column; m-sim ityhri-‘J
`
`figures Cynomoigns monkey thyroid GLPJR mRNA cwiovzalisafion with (Scot! calcitonin
`
`bounm aw. writ field \‘icw ofsilvcr
`
`[N000 4.2.3.7.3 P20]
`
`rand human cell in
`
`Functional GLP- 1 receptors in rat thyroid 0--cell lines (MTC 6-23 and CA—77), a human
`c-cell line (TT), and a rat pancreatic beta cell line (INS-1E) were characterized by saturation
`radioligand binding of [1251]GLP-1 (7—37) to estimate the number of receptors/cell and radioligand
`affinity. Non-specific binding was determined in the presence of unlabeled GLP-l.
`Rat MTC 6—23 cells had 16,000 GLP-l receptors/cell with an affinity of Kd 47.0 pM
`(Figure 1). The number and receptors / cell may be underestimated because MTC 6—23 cells were
`
`33
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22-341
`
`detached from culture plates by protease treatment 24 hours prior to performing radioligand
`binding experiments.
`
`5“?
`
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`
`Figure 1 GLP-1 receptor saturation plain for MTC 6-23 cell line.
`[N000 4.2.3.7.3 P10]
`
`Rat CA-77 cells expressed 13,000 GLP-l binding sites/cell with and affinity of Kd 31
`
`pM.
`
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`Figure 2
`
`GLP— I receptor saturation plots for the CA-77 cell line.
`[N000 4.2.3.7.3 P11]
`
`Rat INS—1E cells, derived from pancreatic beta cells, expressed 8,780 GLP—l binding
`sites/cell with and affinity of Kd 82 pM.
`A
`E
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`
`Figure 4
`
`GLP—1 receptor saturation plots for the INS-1E cell line.
`
`[NOOO 4.2.3.7.3 P13]
`
`The finding that rat thyroid c-cell lines and a pancreatic beta cell line express similar
`levels of GLP—1 receptors, despite a large difference in GLP-1 receptor antibody reactivity and
`mRNA levels in thyroid and pancreas tissue sections suggests the density of GLP-1 receptors in
`rat lines may not reflect the receptor density in thyroid c—cells or rat pancreas in vivo.
`Figure 3 shows specific saturable binding of [1251]GLP-1 (7-37) was not demonstrated in
`human TT cells.
`
`34
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22—341
`
`.I
`
`I
`
`25
`
`so
`
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`Figure 3 GLPw! receptor saturation plots for the TT cell line
`
`[NOOO 4.2.3.7.3 P12]
`
`
`
`
`
`Functlonal GLP-l receptors in thyroid c-cell lines were quantified in a flow cytometric
`fluorescent ligand—binding assay. Specifically bound fluorescence of rat MTC 6—23 and CA-77
`cells or human TT cells labeled with varying concentrations of fluorescently labeled GLP-l
`[GLP—l (7—36)—Lys(6-FAM)] in the absence or presence of 2 LLM exendin(9-39) was quantified
`by flow cytometry to estimate the number of GLP-1 receptor sites and ligand affinity.
`#GLP-1Rlcelt on 3 different Gael; lines
`
`5050
`
`
`
`
`
`
`
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`
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`
`Figure 4 Number of gamma-expressed GLPJR on the. rat Gee]! lines rMTC 6—23 and CAv
`77. and on the human C-cell fine TT.
`Results are means 033 independent experiments. The expression ol‘GLP-l R on the rat C-cell lines
`was significantly higher that GLP-E R expression on the human C-ccll line (see legal}.
`[NOOO 4.2.3.7.3 P17]
`
`GLP-1 receptor sties on thyroid c—cell lines were demonstrated on rat MTC 6-23 and CA-
`77 cells, but not on human TT cells (Figure 4). Estimated GLP-1 receptor density on rat MTC 6—
`23 cells and CA-77 cells was similar (3,499 and 4,369 GLP-l receptors / cell, respectively).
`Compared to radioligand binding saturation studies, the number of GLP-1 receptor per cell
`estimated by flow cytometry of fluorescent GLP-l labeled cells was 4.6 fold lower for MTC 6-23
`cells and 2 fold lower for CA-77 cells.
`
`
` W4 JJ;/
`9 I
`§§
`The sponsor claims GLP—1 receptor was identified in Western blots of rat thyroid and rat
`thyroid c-cell lines MTC 6-23 and CA77, but not human c—cell line TT using anti-human GLP-1
`receptor antibody K102B. However, these results are equivocal because the specificity of the
`antibody for GLP—l receptors was not demonstrated and the protein size and SDS-PAGE pattern
`were not consistent with published results for GLP-l receptors (Widman et al, Biochem. J. (1995)
`310, 203-214).
`
`35
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22-341
`
`GLP-1 receptor protein in rat thyroid c-cell lines MTC 6—23 and CA-77 and human c-cell
`line TT was quantified by western blotting using rabbit anti-human GLP—1 receptor antibody
`targeted to the peptide sequence TVSLWETVQKWREYRRQC corresponding to human GLP—1
`receptor amino acids 29 — 46 in the receptor’s amino terminus (rabbit antibody 102B, 2 non-
`identical amino acids in the corresponding sequence of the rat GLP-1 receptor), an HRP—
`conjugated horse anti-rabbit IgG secondary antibody, and a chemiluminescent substrate (ECL,
`GE Healthcare).
`Antibody IOZB identified a 51 kDa protein in rat thyroid and rat c—cell lines and
`immunoreactivity with the 51 kDa protein was reduced by preabsorbing it with the peptide
`antigen (Figure 1).
`
`
`
`GLP~1R (51 kDa)
`6LP-1 R
`
`(Peptide absorption of antibody)
`
`GAPDR (35 kDa}
`
`
`
`
`at)?!
`urkfiC 8'33
`
`
`
` GUMRlavala(AU‘BGImmzl
`
`AWWWWW. 1w...
`1mm.
`«v “gov “(not
`\zl"
`\fi"
`
`WWW.
`
`Figure l GLMR cannot be detected in a human C-c'ell line.
`Two sets ofC-ccll line lysutcs; Iysis group l and lysis group 2, with high and low passage numbers, respectively {see
`table 2 for passage numbers), were analysed by Western immtmoblotting for GLP- tR cxprcssiortl Lysate of rat thyroid
`was included as a positive control. A, representative result from three independent Western blot experiments (see
`appendix 8 {or whole filter image). Top panel. the GLP— l R baud location is indicated by an arrow. Middle panel,
`absorption with $4 0M human GLP-IR, peptide lead to reduction at? the 5! kDa GLP-ER banal demonstrating the
`specificity of the reaction. Bottom panel. a parallel, membrane was probed with ttnti-GAPDH antibody, to verify equal
`protein loading. Bi Quantitative analysis of 613le expression levels. Data from but!) C~cell lint: lysis groups for three
`independent Westcm blotting experiments are shown in the bar diagram
`
`[NOOO 4.2.3.7.3 P14]
`
`Reviewer note: Because ofthe heterogeneity ofglycosylated receptors, immunoreactive GLP-I
`receptors would be expected to be diffuse bands at a molecular weight > 55 with nonglycosylated
`receptor appearing as more discrete bands at a molecular weight < 55 (Widmann et al, Biochem.
`J. (1995) 310, 203-214). The staining pattern in Figure 1 not consistent with a glycosylated G—
`protein coupled receptor.
`Images of the entire western blot from a representative experiment showed intense,
`specific immunoreactivity in TT cells at > 250 KDa, probably consisting of protein complexes
`that didn’t enter or barely entered the gel, and between 105 and 160 kDa. This data doesn’t
`support the sponsor’s conclusions about the absence of specific staining in TT cells or the
`specificity of antibody 102B.
`'
`
`36
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22-34]
`
`km:
`250 —
`Experiment 2Pn’mary antibody: 150 ____
`rabbit 1028 anti-GLP-lR
`105 __ ‘
`
`'f'
`
`75 -
`
`
`
`Primary antibody
`(rabbit 10213 anti~GLP~1R)
`preadsorbed with 84 nM im-
`munogen- peptide
`.
`[N000 4.2.3.7.3 P19]
`
`
`
`Consrstent with low to undetectable functional GLP-l receptors in TT cells, a human
`thyroid c-cell line, GLP-1 receptor transcript levels in TT cells were much lower compared to
`transcript levels in rat thyroid c—cell lines MTC 6-23 or CA77. Relative GLP-1 receptor transcript
`levels in rat and human c-cell lines were measured by real-time quantitative RT—PCR
`incorporating fluorescent primers into amplified cDNA and normalizing GLP—1 receptor
`transcript levels to transcript levels encoding for beta-actin. Summary results in Figure 3 show
`relative levels of GLP-1 receptor mRNA were significantly higher in rat c-cell lines MTC 6-23
`and CA77 (l 8 — 27 GLP-1 receptor transcripts / 1000 beta actin transcripts) compared to human
`TT cells (1 GLP-1 receptor transcript / 1000 beta actin transcripts).
`
`005 ~
`
`,,
`
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`
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`
`Comparison of GLP-IR mRNA expression levels between rat amt human
`
`Figure .3
`chell lines
`Relative GLP— lR mRNA levels were determined by xmmnlimtion to beta actin, and
`expressed as 2““ (arbitrary units‘ Table 3) The columns represent mean 2"“ values
`from table 3; Bars, one standard deviation: *Both mt C~cell lines had significantly
`higher relative GLP—IR mRNA content that the hmmm T’l‘ C-cell line (941.01. student‘s
`I-tcstli
`
`[Nooo 4.2.3.7.3 P17]
`
`37
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22-341
`
`
`
` tiona LP-l receptor coupled to adenylyl cyclase activation and calcitonin
`secretion was demonstrated in rat MTC 6-23 and CA77 thyroid c—cell lines, but not in human TT
`cells. Pentagastrin, a calcitonin secretagogue used as a positive control, did not stimulated
`calcitonin release from any of the 3 c-cell lines suggesting these cell lines are not representative
`of receptor coupled calcitonin secretion in vivo.
`'
`The rank order potency of the GLP-1 receptor agonists human GLP-l (7 — 37), exenatide,
`or liraglutide to stimulate CAMP accumulation and calcitonin secretion in rat and human c—cell
`lines was determined. In these in vitro experiments, the lower potency of liraglutide compared to
`GLP-l (7 — 37) and exenatide was attributed to liraglutide binding to protein in culture media
`supplemented with 15% horse serum.
`In rat MTC 6-23 cells, GLP-l (7 — 37), exenatide, and liraglutide dose-dependently
`.
`increased CAMP accumulation (ECsos 120, 90, and 5,800 pM, respectively, Figure 3) with a
`maximum response < 50% of the 100 uM forskolin control, a phorbol ester that directly activates
`adenylyl cyclase (Figure 4A).
`'
`
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`20
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`
`
`
`Figure 3 GLP-l receptor signalling and pharmacology is; the rat thyroid C-celi line MTC 6—
`23.
`
`[NOOO 4.2.3.7.3 P14]
`
`To demonstrate GLP-1 receptor specificity of liraglutide’s effect in MTC 6-23 cells, the
`liraglutide dose—response curve shifted right—ward in the presence of 10 nM exendin (9 — 39), a
`GLP-1 receptor antagonist (Figure 4B).
`50
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`
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`
`g
`
`10
`
`cAMP(pmal)
`
`A
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`I Gly.1(7.37)
`
`55
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`o LlragMide ¢ 10nM 649-39)
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`L'l Forskolln+10rM5t(S-39)
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`.3
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`Concentration (log(M))
`
`.5
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`.10
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`Concentraflnn (log(M))
`
`-s
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`
`4
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`55
`59
`.o
`m
`
`a33.‘5“
`
`
`
`cAMP(pmol)
`
`Figure 4 Camparison of GLP—1 and forskolin induced cAMP activation in the rat thyroid
`cell line, MTC 6-23.
`
`[N000 42.3.7.3 P15]
`
`38
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22-341
`
`GLP-l (7-37), exenatide, and liraglutide had a similar rank order potency and ECsos for calcitonin
`secretion (Figure 5A, 80, 55, and 5,300 pM, respectively) and the maximal effect of liraglutide
`was up to 67% of the maximum response to 10 HM forskolin.
`s
`
`A
`
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`
`9
`
`
` g
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`-5 4
`Concentration (log (Mn
`Concentration (log (ND)
`
`Figure 5 Calcitonin release from the rat C-ccll line MTC 6—23.
`
`Data are from cxpcn'mcnl 14725.0()! and 13737-880. In A, the 5mm: concentration was 1% and in B
`15%.
`
`[N000 4.2.3.7.3 P16]
`
`Exendin(9—39), a GLP-1 receptor specific agonist, inhibits liraglutide stimulated
`calcitonin secretion from MTC 6-23 cells (Figure 6).
`7
`
`- magmas
`4-manganese)
`‘ 3W Umgufiéo
`z +Emndin(9-39)
`+ 0.3IIM Emaiida
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`* 1nM (ELF-1
`
`
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`
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`palm!Calclmnlnrelease
`
`WW
`.1:
`.12
`.11
`.10
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`6
`-5
`Concentration (109 (M))
`
`Figure 6 Exendin(9-39) antagonised GLP-l, exenatidc and liraglutlde induced calcitonin
`release.
`
`[N000 4.2.3.7.3 P17]
`
`Pentagastrin stimulates calcitonin release by activating thyroid CCKZ receptors in humans
`and rats. Pentagastrin didn’t stimulate calcitonin release from rat MTC 6-23 cells (Figure 7), rat
`CA-77 cells, or human TT c-cell lines (figures not shown).
`
`39
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22-341
`
`release
`ngmlCalcitonlrI
`
`I ”JG-37)
`v Liraglulide
`V Exenah'de
`+Pemagasuln
`
`
`
`
`
`
`WWW17 .15 .15 .14 .13 42 -11 40 .9 -s -7 -s .5
`
`
`
`
`Concentration (log (W)
`
`Figure 7 Pentagastrin did not stimulate calcitonin release from MTC 6—23 cells.
`
`Data are from experiment 14725-001.
`
`[N000 4.2.3.7.3 P18]
`Calcium dose-dependently stimulates calcitonin release from MTC 6-23 cells (Figure 8A).
`Liraglutide enhances calcium-stimulated calcitonin release, and the effect is greater at higher
`calcium concentrations (Figure 8B)
`
`A
`
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`
`fro
`33
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`,9
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`a basal (0,8mMCaCI2)
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`
`Figure 8 Calcitonin release from MTC 6-23 cells with varying calcium concentrations.
`[NOOO 4.2.3.7.3 P19]
`
`GLP-1 receptor agonists had the same rank order potency of agonist—induced CAMP
`accumulation (GLP-1(7-37) > exenatide >> liraglutide) in CA-77 cells with similar potencies
`compared to MTC 6-23 cells (Figure 10). GLP-1 receptor agonists didn’t stimulate adenylyl
`cyclase in human TT cells, but the positive control, forskolin, did. GLP-l(7-37), but not
`pentagastrin, stimulated calcitonin secretion from CA-77 cells.
`
`40
`
`

`

`950
`33°
`750
`§
`
`
`
`cAMP(mo!):2§
`
`m
`
`150
`
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`-6
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`Concentration (Iog(M))
`
`
`
`
`
`we
`
`no
`
`Reviewer: Anthony L Parola, PhD
`
`
`NDA No. 22-341
`
`1
`
`1300
`
`8
`
`'1 madam).
`+Pen£agaslrm
`
`§
`
`pglmlCatatoniarelease
`
`”WWW
`~17 .13 .15 .14 .1: 42 d1 40 -a
`-c
`:1 4 5
`Concentration (109 (Ml)
`
`Figure 10 cAMP and caicitonin data from the rat thyroid C~oeli line, CA-77.
`
`The data are from experiments 13737-056 and 14725-002.
`
`W000 4.2.3.7.3 P21]
`
`Forskolin increased cAMP accumulation (not shown) and calcitonin secretion from
`human TT cells (Figure 12), but GLP—1 receptor agonists GLP-1(7-37), exenatide, or liraglutide
`did not.
`
`A
`
`Tag?”
`.
`WW3”)
`*uraglufida
`" 8359'
`
`A
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`2
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`g
`2
`8
`
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`i Forskolin
`+Exenatide
`w—Llragtutide
`II Basal
`
`a
`
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`
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`
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`~11 .16 .15 .14 .13 .12 .11 .10 .o .0 -7
`-s
`.5
`~15
`.14 ~13
`.12 .11
`.10
`.9
`.a q .s 6
`Concentration (log (Ml)
`Concentration (tog (Nu)
`
`Figure 12 Calcitouin release from human Gee}! line, TT.
`
`[N000 4.2.3.7.3 P23]
`
`The rank order potency and absolute potencies of GLP—1 receptor agonists GLP-l,
`exenatide, and liraglutide to stimulate intracellular CAMP accumulation were similar in a rat
`pancreatic beta cell line, RIN2A18, and rat thyroid c-cell MTC 6—23 cells (Table 1, below)
`
`41
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22—341
`
`Liraglutide
`— GLPJ
`
`
`
`
`1400.600
`cAMP EC” (pM)
`591227
`
`RINZAIS
`n=8
`
`
`
`cAMI’ EC” (pM)
`580%2800
`
`rM‘K‘C 6-23
`new
`
`
`Calcitonin 2C5" (pM)
`530053400
`
`
`rM‘E‘C 6-23
`n=6
`
`7600i600
`(10%HS, n=2)
`(10%HS, n=2)
`
`
`
`4200x3600
`68:62
`
`
`(5%HS, n==5)
`(5%HS, n=5)
`
`
`
`88:33
`8303:270
`
`(l%I—IS, n=5)
`(1%HS. n=5)
`
`
`
`Potency for GLP-l, exenatide and liraglmide in rat pancreas and thyroid cell fines.
`Table 1
`cAMP experimems were conducted using 0.1% serum albumin and calcitonin release experiments 15%
`serum (horse serum, HS) when nothing else is indicated.
`
`[N000 4.2.3.7.3 P24]
`
`
`
`
`.
`,
`N. 319
`, W ..
`.
`.
`ts,
`
`
`
`he mitogenic potential 0
`irag ut1 e, uman GLP—l (7—37), a d exenatide were
`determined by [3H]thymidine incorporation into DNA of thyroid c-cell lines rat CA77, rat MTC
`6-23, and human TT cells and as a positive control, rat insulinoma INS-1E cells. Although fetal
`calf serum caused a proliferative response in rat and human c-cell lines, none of the GLP-1
`receptor agonists were mitogenic (Figure 5). To demonstrate activity of GLP—1 receptor agonists
`in this assay, GLP—l (7-37) was a mitogen in rat INS-1E cells, but only at low glucose
`concentrations. Gastrin and EGF were previously characterized mitogens in human TT cells, but
`TT cells used in this assay did not proliferate in response to either and these cells were devoid cf
`gastrin receptors in a functional binding assay.
`
`RAT CA-‘l'l
`RAT MTG 643
`e
`
`.0»ch (74:7;
`W“ Draglume
`-0- Emuétnnd.
`*ch
`“owner;
`
`1
`n
`.1
`'09 Icons: raw:
`
`2
`
`3
`
`I
`4,
`96‘
`‘
`
`«2
`
`x
`g E
`53,
`’5
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`2
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`,3:a
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`a
`
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`mam (7.37;
`no»magma}.
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`a
`2
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`2
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`log [com (nun
`
`e3
`*
`
`HUMAN TT
`
`-.- GLP—‘l (3537)
`—b-Klagntide
`-a—Emm‘m-é
`«—FCS
`-—-vehicle
`
`2
`
`.
`
`r—-—r—:-——r—r-—1
`
`10!! {WM (“W1
`
`
`
`Figure 5 CHM and GLP-1 analnguc mediated pmlifemtion mam three C-cell zines.
`
`[N000 4.2.3.7.3 P18]
`
`42
`
`

`

`
` Table i ' Result sunning _
`”Fatima
`Gastrin/CCKZ receptor
`anbesinlBBZreceptor
`my
`“:50"
`[95% CE]
`195% C1]
`
`(nMJ
`(RM)
`> 1.000
`‘2 1,000
`> 1.000
`0.54
`10» 14 — 2.0L
`
`1:133; Amide
`15253119194
`
`Pemagaslrm
`Gastriu-i‘f
`
`i
`i
`1‘"
`
`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22—341
`
`
`
`Liraglutide doesn’t elicit calcitonin secretion from c—cells by activating CCKZ (gastrin
`receptor) or bombesin (gastrin releasing peptide receptor) receptors. Varying concentrations up to
`1 uM GLP-l (7-37), exenatide, or liraglutide did not displace specific 125I—CCK-8 or [1251]Tyr4-
`bombesin binding to cell membranes from rat AR42J cells, a pancreatic acinar cell line
`endogenously expressing CCKZ and bombesin receptors, respectively. Both CCKZ and bombesin
`receptors mediate cell proliferation through a phospholipase C dependent, Gq/Gl l-coupled
`pathway.
`
`
`
`a
`i
`
`
`
`
`D-(Plic“,l.cu-NH.E!’{dos-Mel“)Bombcsinto-l 4)
`
`*lCm values are calculated from the mean of Logic”) from 3. experiments. except. for pcmagaszrin. which is
`{1171114 experiments,
`_..
`
`_,-,-.
`
`. -..,.-..c.-...*.
`
`mooo 4.2.3.7.3 P9]
`
`1
`
`—.
`2??
`75 E
`5 3
`u 0
`.3 E
`§§
`m 5
`
`‘ Gill}
`0 Exandind
`A Liraqfanlde
`
`>
`I Panagasmn
`. Gasu‘xn-v
`
`2:
`g;
`E 5
`a 3.
`n o
`"E. E
`a}
`'0 g
`
`.13
`
`4
`.12 41 no .9
`L99 (Cone. {Ml}
`
`I
`.7 4 .5
`
`w
`
`5
`
`
`
`n GLPA
`n Emadznd
`A Liragm‘de
`
`- 1344.393
`‘ ass Aniag
`
`.3
`.9
`43 «12 m .19
`Log (Canal (33])
`
`.1
`
`4;
`
`.5
`
`Displacement by GLP~L exendin-st and limglmide of A) [Isl—CCKas and B) ‘zsl-Tyr’.
`Figure 1
`bombesin binding to gastrtn and bombesin receptors. respcclively. from the mt pancreatic neinar cell line
`A'RSlZl
`
`[N000 4.2.3.7.3 P9]
`
`
`
`43
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22-341
`
`In male Sprague Dawley rats, a single subcutaneous dose of 0.75 mg/kg liraglutide
`transiently increased plasma calcitonin < 2 fold peaking 1 hour after dosing. Increased plasma
`PTH occurring 6 hours after dosing was considered a counter-regulatory response, but it occurred
`in the absence of substantial changes in plasma calcium concentrations. Consistent with
`liraglutide-induced increased calcitonin secretion, liraglutide decreased calcitonin peptide levels
`in thyroid, but it also decreased thyroid calcitonin mRNA levels within 6 hours of dosing. In male
`rats calcium loaded by calcium gluconate ip injection, a single 50 dose of 0.75 mg/kg liraglutide
`transiently increased calcitonin secretion < 2 fold above that induced by calcium gluconate alone.
`Within 6 hours of dosing, calcium loading alone decreased thyroid calcitonin peptide levels and
`increased calcitonin mRNA levels. In calcium loaded rats, liraglutide increased calcitonin peptide
`levels in thyroid and increased calcitonin mRNA levels above those elicited by calcium loading
`alone. These results suggest in calcium loaded rats, liraglutide increased calcitonin transcription
`and translation more than it increased calcitonin secretion, and its effects on calcitonin secretion,
`transcription, and translation were greater than for calcium loading alone.
`Plasma calcitonin, PTH, and calcium levels were measured up to 6 hours after
`administration of a single subcutaneous dose of 0 (vehicle) or 0.75 mg/kg liraglutide (2 mL/kg,
`neck injection site) in fasted male Sprague Dawley rats (30/dose, fasted 12 — 14 hours prior to
`dosing, study 203281). A second study used the same study design, except rats were also given a
`single intraperitoneal injection of 1 mM /kg calcium after vehicle or liraglutide treatment to
`determine if increased calcium levels affect the response to liraglutide (study 203282). At the end
`of the study, thyroid calcitonin protein and transcript levels were quantified. Study observations
`were body weight, ionized calcium, plasma pH, adjusted ionized calcium (pH 7.4), plasma intact
`PTH, and plasma calcitonin with orbital plexus blood samples taken fromisoflurane/OZ/NZO
`anesthetized rats prior to dosing and 0.25, 0.5, 1, 3, or 6 hours after dosing.
`There were no treatment-related deaths, clinical signs or body weight changes with or
`without injected calcium. One rat in the liraglutide + calcium group died during anesthesia, but its
`death was not attributed to treatment.
`
`In the absence of injected calcium, there were no changes in plasma ionized calcium
`compared to calcium concentrations prior to dosing (uncorrected or pH corrected calcium, see the
`summary table below where group 1 is control and group 2 is liraglutide treated). Liraglutide
`appeared to increase blood pH between 0.5 and 3 hours after dosing (Figure 3).
`
`Group mean Ca‘WpH 7.4)
`Group mean Ca“
`as “Jo
`as % of
`of levels before dosing
`levels before dosing
`
`Group 1
`
`WW.......me
`
`(’35
`m..!?.'i‘.":.....-.si. W ’
`.221,
`
`0%
`3%
`
`Blood samptiug
`time 1min:
`
`
`
`
`
`
`
`
`
`
`g
`g
`
`2%
`
`g
`
`[N000 4.2.3.7.3 P28]
`
`44
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22—341
`
`
`
`Figure3
`
`pH ofblood
`
`[N000 4.2.3.7.3 P32]
`
`Compared to the control group, plasma calcium levels in the liraglutide treated group
`trended lower from 0.5 hours onward in rats concurrently administered calcium (below). There
`were no liraglutide-related changes in pH in rats concurrently injected with calcium (Figure 3,
`below).
`
`Group mean Ca"
`Blood sampling
`
`(ntmolz‘h) as % of
`timcpnim
`
`levels before rinsing
`{hows after dosing)
`
`Group I
`Group 2
`
`
`3§a
`0'5
`
`ia
`«WMMWMNWMMWMmummi
`
`"
`3
`
`13
`
`Group mean Ca“(pH 7.4)
`
`(inmolfL) as "A
`
`nf icvek before rinsing
`
`Group I
`3
`Group 2
`32 %
`12%
`7%
`4 (:4,
`
`
`
`
`
`ms 7 '
`
`L35 WW
`
`'Figure3
`
`p1] of blood
`
`
`
`[N000 4.2.3.7.3 P30]
`
`Plasma calcitonin was significantly increased 1 hour after dosing in the 0.75 mg/kg
`liraglutide treated group, but the increase wasn’t sustained, probably because pH adjusted plasma
`ionized calcium levels were decreased. The sponsor hypothesized decreased plasma calcium was
`secondary to increased excretion in urine, but this was not demonstrated in this study. Decreased
`
`45
`
`

`

`
`Reviewer: Anthony L Parola, PhD
`
`NDA No. 22-341
`
`plasma calcium may have countered any sustained effect of liraglutide to increase calcitonin.
`Increased plasma intact PTH occurring 6 hours after dosing in the liraglutide treated group was
`consistent with lower plasma calcium.
`
`Reviewer note: The sponsor determined statistical significance oftreatment related changes in
`plasma calcitonin, calcium, and intact PTH using natural log transformed values.
`
`In the liraglutide treated group, plasma calcitonin increased 0.5 and 1 hour after dosing
`(Figure 2, Table 2). Increased calcitonin levels did not occur at liraglutide’s Tmax, which
`typically occurs 3 — 6 hours after subcutaneous dosing. PTH levels in the liraglutide group were
`increased 6 hours after dosing. Increased PTH in the liraglutide treated group is consistent with a
`counter—regulatory response to minimally decreased blood calcium concentration.
`
`
`
`’3 52M)
`:23:ng 2' Caicimnils "‘9‘!me ‘0 “mm" ““d Liragimidc “NC 90'] '70) (mean +}. Figure I. )uiact PTH Response in \‘chidc and liraglutide (NNC 994 £70) (mean +1-
`
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`Table 2. Calcilunin and VI}! Lu'cls in Vehicicv and Lintglmidc {NSC 90-1170)» Table 2. (Calcitonin nml yTH Levels in Vehidc- in“! liraglutide (N’fi‘C 90-4 170)»
`Treated A Iimais
`Treated Animals (cont)
`
`
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`Treuimcm Tim: E
`g
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`11.54
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`165.02
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`M85
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`
`In calcium loaded rats, calcitonin levels were markedly increased within 1 hour after
`calcium loading and returned to baseline levels within 3 hours (Figure 1a, Table 3a, b below).
`Calcitonin levels in the liraglutide treated group were < 2 fold higher than in the vehicle control
`
`46
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`
`NDA No. 22-341
`
`group 0.25 hours after dosing, but liraglutide’s Tmax after subcutaneous dosing in rats typically
`occurs 3 — 6 hours after dosing. Six hours after dosing, iPTH was higher in the liraglutide group
`compared to controls, and increased iPTH was consistent with minimally lower plasma calcium
`levels in the liraglutide treated group compared to controls starting 3 hours after calcium loading.
`”11“": 1“ PT" "NW3 " A“ 95"“ (mum ‘9" 51’3“)
`Figure 11) Cnlcilonin Value-s - All Dam (mean +f~ SEM)
`600
`
` Calcitonin
`(pgl‘m
`
`IniactPm(ng00
`
` 0
`
`a
`
`6
`4
`2
`Time after compound administration (by)
`
`
`
`
`
`"fable 3!: Summarized Results
`
`
`E
`
` E
`
`
`
`
`
`
`
`
`
`
`
`
`
`Table 3a Summarized Results
`
`Time
`imc
`Treatment
`Dara Calciionin
`Treatment
`Point
`E Pain:
`
`*www _W-WW
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`236.58
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`aw i
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`10
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`Vehicle”
`0.5
`mean
`023.13
`180.14' Liraglmidc
`0.5
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`114.03
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`46.56
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`n
`I
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`n
`30
`9
`
`Vehicle
`3
`mean
`14.92
`300,02
`Liraglmidc
`3
`mean
`i
`16.39
`
`
`
`
`fitm-wtgs§..,.iF:§C9a-www sad 135......
`2.4x.
`2.90
`68.4!
`a
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`5m
`
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`
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`(3
`mean
`I
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`D
`
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`5
`4.95
`
`10
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`n
`mm
`} SEM
`3.5?
`
`[N000 4.2.3.7.3 P86-87]
`
`
`
`
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`Lam».
`
`Eg2
`
`Calcium loading reduced thyroid calcitonin peptide levels in vehicle treated rats, but in
`liraglutide treated rats, calcium loading increased thyroid calcitonin levels (Table 7). The effect of
`calcium to decrease thyroid calcitonin levels is consistent with increased calcitonin release, but
`the effect of liraglutide to increase thyroid calcitonin levels in calcium loaded rats is paradoxical,
`but to explain this effect, the sponsor suggests liraglutide increases calcitonin translation more
`than it increases secretion.
`
`47
`
`

`

`Reviewer: Anthony L Parola= PhD
`
`NDA No. 22-341
`
`Table 7
`
`The effect of calciamnluad itself on calcitonin protein level in thyroid tissue from
`I'llt.
`
`E
`Time palm t'g/icr l
`treatment
`
`
`
`
`Which: rats“
`Lil‘aglmide~lr¢3rtlvd mam
`
`(”alcium—IOm/et
`William
`
`
`Calcium-load
`(NW203 I82)
`
`
`(NN203 131)
`
`
`(f‘nlcinln—Imvded
`(MVQOSISZ)
`
`ll’itlmul
`calcium-10ml
`(NN303181)
`
`
` mum-load melt an \‘clixela nits was expressed as fold up: or dmvmcg
`
`
`
`
`
`'2 "(he effect at
`
`
`value}: rats tt‘mm study ,\’.,\’203.’:¥3) when computed m “axial: mks wixlwut calcklmvlum‘l {from study .\v’;\’.’l}33:YI
`1'”: likewise the affect ”realism”.
`load ihcli‘in limgluiidu-twuwd rat,» was expresmd as fold up or ilsiivnrvgulmian on um M‘el vi‘cztldwnin peptide in calciumvluaded and liraglutide-
`
`zrumcd rat» (‘ \3V303 ’82} when cemgxlmil m liraglutideutraaml ms. Milton: calcium-lazuli WA?“ 11:?!t. 5 Positive value 3‘: “fold upmgnlatien",
`Negative value
`“rum duivlstségnlmiun’fl
`
`[N000 4.2.3.7.3 P148]
`
`Compared to vehicle treated fasted rats, liraglutide decreased thyroid calcitonin levels up
`to 2.7 fold, consistent with it proposed effect to increase calcitonin secretion. In calcium loaded
`rats, liraglutide treatment increased thyroid calcitonin levels up to 3.8 fold. The difference in the
`effect of liraglutide on thyroid calcitonin levels in the presence and absence of calcium loading
`suggests liraglutide increases calcitonin synthesis more than it enhances calcium evoked
`calcitonin secretion.
`
`The effect of liraglutide on the levels of calcitonin peptide in thyroid tissue from
`Table 6
`rat,
`
`rum], calcium-loaded mix”
`(’i\"z\i203182)
`
`Fasten? 1'0le
`(NN203 [81)
`Tune 1min! qfier
`{l‘calm