AMYR, CTR[1][2][3].

Investigations of the SAR, leading to cagrilintide (23) (AM833 or NN0174-0833; proposed international non-proprietary name: cagrilintide, was initiated by in vitro receptor binding and potency screening to identify the best attachment point for fatty acid acylation. The positioning of the fatty acid could be speculated to affect the ability of the analogues to interact with the amylin receptors and either increase or decrease access to receptors and the brain. The common backbone chosen was similar to pramlintide (see Table 1) rather than h-amylin in order to reduce issues with fibril formation.[2]
We found that certain positions in the backbone could be mutated to lysine and subsequently acylated on the lysine sidechain with weakly albumin-binding fatty acids similar to the one used in liraglutide with no or modest loss of potency (e.g., positions 1 and 3 at the N-terminal part, 11 and 18 within the helical part, and 21, 28, and 31 in the C-terminal part), whereas modifications in other positions turned out to give reduced potency (e.g., positions 2, 10, 14–17, and 29) in the functional assay (data not shown). The N-terminal part was identified as the most promising position for attachment of the fatty acids. An apparent loss of potency was observed when analogues N-terminally lipidated with C20-diacid (strong albumin binding) were compared to the references pramlintide and s-calcitonin. The loss of potency is caused by the presence of albumin in the assay and is often seen with stronger albumin binders, for example, in the case of semaglutide. The fatty acids bind to albumin which interferes with the receptor interaction and result in an apparent loss of potency. In contrast, the binding assay is run in the presence of ovalbumin which reduces unspecific binding but does not bind to the fatty acids or affect receptor interactions. It was also confirmed that the amidated C-terminus was essential for bioactivity with very little room for modification, and the position that best combined low sensitivity to modification with easy synthesis appeared to be the N-terminus. This was a surprise because the amylin and GLP-1 receptors both belong to the subfamily B1 of GPCR receptors, yet GLP-1 cannot be lipidated in the N-terminus without loss of potency.[2]
These observations can be explained in retrospect by a homology model of cagrilintide (23) bound to AMY3R (Figure 1) together with a recent cryo-EM structure of the GLP-1 receptor. These structures predict that the CTR/RAMP complex has a more open structure than the GLP-1 receptor. Furthermore, while the N-terminus of GLP-1 penetrates as an α-helix into the transmembrane (TM) domain (see Figure S-5 in Supporting Information), the N-terminus of the amylin peptide is predicted to form a loop, stabilized by a disulfide bond, that points out of the TM area, thereby tolerating N-terminal lipidation. The homology model of AMY3R, together with an apo crystal structure of cagrilintide (23) without lipidation, also predicts that a helical segment exists in the N-terminal part of cagrilintide (23) (residues 5–18) stabilized by a salt bridge between positions 14 and 17, and that the C-terminus adopts overall an extended conformation that binds the extracellular N-terminal domain of CTR, whereby residues 20–24 form an unstructured loop.[2]

Compound 23 (cagrilintide acetate) (0.1, 1, 3, 10, 30 nmol/kg; scsingle) decreases the rat's food intake[1]. Good pharmacokinetic parameters are demonstrated by cagrilintide acetate (10 nmol/kg; iv or sc; single dose) [1].
Rats' food intake is decreased by cagrilintide (compound 23) (0.1, 1, 3, 10, 30 nmol/kg; subcutaneous injection) [2]. Caglitide exhibits good pharmacokinetic parameters when administered intravenously or subcutaneously (10 nmol/kg) [2].

In vitro assays used in the SAR: [2]
The screening plan consisted of in vitro screening on human CTRa and AMY3 receptors, and similar rat screening on the potent compounds. Thus, the selection of Cagrilintide was done using 8 different in vitro assays (functional potency and binding affinities of the compounds in the two species). The analogue development has been performed over a duration of time. To secure the quality and stability of the assay over the period, we have included pramlintide as a reference compounds in each experiment, which makes comparison over time possible and meaningful. In addition, the switch (in potency or affinity) of pramlintide was use as a validation of the AMY3R and CTR expression respectively.

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Human Calcitonin Receptor-Luciferase Assay [2]
A Functional Reporter Assay Measuring cAMP Responsive Element Mediated Activity A baby hamster kidney (BHK) 570 cell line stably transfected with the human calcitonin receptor, and a cAMP responsive element luciferase reporter gene was used in this functional assay. In this cell line, the human calcitonin receptor activity is reflected in the luciferase intensity occurring in response to amylin analogs when measured as described in this section. The cells were cultured in growth medium (Dulbecco′s Modified Eagle′s Medium [DMEM]) with 10% fetal bovine serum (FBS), 1% Pen/Strep, and 1 mM Na-pyruvate). Methotrexate (250 nM) and Neomycin (500 µg/ml) were used as selection markers for luciferase and calcitonin receptor, respectively. Cells at approximately 80–90% confluence were washed with PBS and lifted from the plates with Versene. After centrifugation (2 min, 1300 rpm), the cell pellet was dissolved in 10% DMSO, 30% FBS, and 60% growth medium (see above) and frozen (–80°C) until utilization. The day before the experiment, cells were thawed, washed and seeded in 100 μl growth medium (as described above) on a white 96 well culture plates (20000 cells/well). After incubation overnight at 37°C and 5% CO2, the growth medium was replaced by 50 μl/well assay medium (DMEM [without phenol red], Glutamax, 10% FBS, and 10 mM HEPES ([4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid], pH 7.4). Further, 50 μl/well of sample diluted in assay buffer was added. After 3 h incubation at 37°C and 5% CO2, the medium was removed and replaced by 100 μl/well PBS and 100 μl/well SteadyLite. The plates were sealed and incubated at room temperature for 30 m. Finally, luminescence was measured on a TopCounter in single photon counting (SPC) mode. EC50 values were calculated in GraphPad Prism using a nonlinear regression and pEC50 values are calculated as -LogEC50.

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Human Amylin 3 Receptor-Luciferase Assay [2]
A Functional Reporter Assay Measuring cAMP Responsive Element Mediated Activity To generate a stable clone expressing AMY3R, the Hollex-1 cells were further transfected with receptor activity-modifying protein 3 (RAMP3) and pcDNA3.1/hygromycin, the latter was used as a selection marker. Briefly, the transfection was carried out in a T75 Flask seeded with 1250000 the day before transfection. The cells were transfected with 9 μg RAMP3 cDNA, 1 μg pcDNA3.1/hygromycin, and 25 μl FuGENE 6. Hereafter, a stable clone expressing RAMP3 was selected. The stable clone was cultured in DMEM with 10% FBS, 1% Pen/Strep and 1 mM Na-pyruvate, Methotrexate (250 nM), Neomycin (500 µg/ml), and Hygromycin (400 µg/ml). The Methotrexate (250 nM), Neomycin (500 µg/ml), and Hygromycin (400 µg/ml) was used as selection markers for luciferase, calcitonin receptor, and RAMP3, respectively. Cells at approximately 80–90% confluence were washed with PBS and lifted from the plates with Versene. After centrifugation (2 min, 1300 rpm), S8 the cell pellet was dissolved in 10% DMSO, 30% FBS, and 60% growth medium (see above), and frozen (–80°C) until utilization. The day before the experiment, cells were thawed, washed, and seeded in 100 μl growth medium (as described above) on a white 96 well culture plates (20000 cells/well). After incubation overnight at 37°C and 5% CO2, the growth medium was replaced by 50 μl/well assay medium (DMEM [without phenol red], Glutamax, 10% FBS, and 10 mM HEPES, pH 7.4). Further, 50 μl/well of sample diluted in assay buffer was added. After 3 h incubation at 37°C and 5% CO2, the medium was removed and replaced by 100 μl/well PBS and 100 μl/well SteadyLite. The plates were sealed and incubated at room temperature for 30 m. Finally, luminescence was measured on a TopCounter (Packard) in SPC mode. EC50 values were calculated in GraphPad Prism using a nonlinear regression and pEC50 values calculated as -LogEC50.

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Rat calcitonin receptor [2]
cAMP assay BHK 13 cells were transiently transfected with rat calcitonin receptor using FuGENE 6, in a ratio of 1 µg cDNA:1.5 µl FuGENE 6, according to the manufacturer’s recommendations (BHK tk-ts 13 is a temperature-sensitive clone of the BHK cell line, described in Talavera and Basilico, 1977).1 Cells were grown in DMEM with 10% FBS and 1% Pen/Strep. Approximately 24 h after transfection, the cells were harvested and frozen (–80°C) until utilization. On the day of experimentation, cells are thawed, washed twice and then washed in PBS buffer (2% human serum albumin [HSA], 0.5% Tween 20). Cells were seeded (100000 cells/well) into 96 well FlashPlates with samples or standard. Here, 50 µl cell of the suspension was added to the FlashPlates containing 50 µl of test-compound or reference compound (2% HSA, 0.5% Tween 20). The mixture was shaken for 5 min and allowed to stand for 25 min at room temperature. The reaction was stopped with 100 µl detection mix-pro-well (detection mix; 11 ml detection buffer and 100 µl [~2 µCi] cAMP [125I] Tracer). The plates were then sealed with plastic, shaken for 30 min, and allowed to stand overnight (or alternatively, for at least 2 h), and scintillation was measured in the Topcounter (2 min/well). In general, the assay procedure described in the FlashPlate kit-protocol was followed (FlashPlate cAMP assay [NEN Life Science Products cat no SMP004]). The cAMP amount was determined using a standard curve and curves were presented in GraphPad PRISM. EC50 values were calculated in GraphPad Prism using a nonlinear regression and pEC50 values calculated as ‑LogEC50.

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Rat amylin receptor 3 [2]
cAMP assay BHK tk-ts 13 cells (Talavera and Basilico, 1977) were transiently transfected with rat calcitonin receptor (150 µg rat CTR cDNA pr 10000000 cells) and rat RAMP 3 (the cDNA ratio was 1 µg CTR cDNA pr 1.5 µg rat RAMP3) using 2.5 µl FuGENE 6 pr µg cNDA. Cells were grown in DMEM with 10% FBS and 1% Pen/Strep. Approximately 24 h after transfection, the cells were harvested and frozen (–80°C) until utilization. On the day of experimentation, cells are thawed, washed twice, and then washed in PBS buffer (2% HSA, 0.5% Tween 20). Cells were seeded (100000 cells/well) into 96 well FlashPlates (Perkin Elmer) with samples or standard. Here, 50 µl cell of the suspension was added to the FlashPlates containing 50 µl of test-compound or reference compound (2% HSA, 0.5% Tween 20). The mixture was shaken for 5 min and allowed to stand for 25 min at room temperature. The reaction was stopped with 100 µl detection mix-pro-well (detection mix; 11 mL detection buffer and 100 µl [~2 µCi] cAMP [125I] Tracer). The plates were then sealed with plastic, shaken for 30 min, and allowed to stand overnight (or alternatively, for at least 2 h), and scintillation was measured in the Topcounter (2 min/well). In general, the assay procedure described in the FlashPlate kit-protocol was followed (Plate cAMP assay. The cAMP amount was determined S9 using a standard curve and curves were presented in GraphPad PRISM. EC50 values were calculated in GraphPad Prism using a nonlinear regression and pEC50 values calculated as -LogEC50.

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Calcitonin receptor binding assay (human and rat) [2]
The binding assay was performed using scintillation proximity assay (SPA) beads (RPNQ0001) from PerkinElmer and cell membranes containing either the human or rat calcitonin receptor. BHK tk-ts 13 cells were transiently transfected with the human or rat calcitonin receptor and cultured as described above. Membranes were prepared in the following way: the cells were rinsed with PBS and incubated with Versene for approximately 5 min before harvesting. The cells were flushed with PBS and the cell-suspension was centrifuged for 5 min at 1000 rpm. Cells were homogenized in a buffer containing 20 mM Na-HEPES and 10 mM EDTA (pH 7.4), and centrifuged at 20000 rpm for 15 min. The resulting pellet was resuspended, homogenized, and centrifuged (20000 rpm, 15 min) in a buffer containing 20 mM Na-HEPES and 0.1 mM EDTA (pH 7.4, buffer 2). The resulting pellet was resuspended in buffer 2 and protein concentration was measured. The homogenate was kept cold during the whole procedure. The membranes were kept at –80°C until use. Assay was performed in a 384 well Optiplate in a total volume of 40 µl. Membranes were mixed with SPA beads in a 1:1 ratio. Final concentration of SPA beads was 0.05 mg/well. Testcompounds were dissolved in DMSO and further diluted in assay buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 0.1% ovalbumin, and 0.02% Tween20). Radioligand 125I-Calcitonin was dissolved in assay buffer and added to the Optiplate at a final concentration of 75 pM/well (30000 cpm/10 µl). The final mixture was incubated for 120 min at 25°C prior to centrifugation (1500 rpm, 10 min). Samples were analyzed on TopCounter. The IC50 was calculated using (one site binding competition analysis) GraphPad Prism5 and pIC50 values calculated as -LogIC50.

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Amylin 3 receptor binding assay (human and rat) [2]
The binding assay was performed using SPA beads (RPNQ0001) from PerkinElmer and cell membranes containing either the human or rat amylin 3 receptor. BHK13 cells were transiently transfected with the human or rat calcitonin receptor and human or rat RAMP 3 at an equimolar ratio (1:3 µg) and cultured as described above. Membranes were prepared in the following way: the cells were rinsed with PBS and incubated with Versene for approximately 5 min before harvesting. The cells were flushed with PBS and the cell-suspension was centrifuged for 5 min at 1000 rpm. Cells were homogenized (ultrathurrax) in a buffer containing 20 mM Na-HEPES and 10 mM EDTA (pH 7.4) and centrifuged at 20000 rpm for 15 min. The resulting pellet was resuspended, homogenized, and centrifuged (20000 rpm, 15 min) in a buffer containing 20 mM Na-HEPES and 0.1 mM EDTA (pH 7.4, buffer 2). The resulting pellet was resuspended in buffer 2 and protein concentration was measured (BCA protein Assay, Pierce). The homogenate was kept cold during the whole procedure. The membranes were kept at –80°C until use. Assay was performed in a 384 well Optiplate in a total volume of 40 µl. Membranes were mixed with SPA beads in a 1:1 ratio. Final concentration of SPA beads was 0.05 mg/well. Testcompounds were dissolved in DMSO and further diluted in assay buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 0.1% Ovalbumin, and 0.02% Tween20). Radioligand 125I-rat amylin was dissolved in assay buffer and added to the Optiplate at a final S10 concentration of 50 pM/well (20000 cpm/10 µl). The final mixture was incubated for 120 min at 25°C prior to centrifugation (1500 rpm, 10 min). Samples were analyzed on TopCounter. The IC50 was calculated using (one site binding competition analysis) GraphPad Prism5 and pIC50 values calculated as -LogIC50.

Tests for Fibril Formation Propensity (ThT Assay) [2]
The stability properties of the individual amylin analogues were assessed with respect to their propensity toward fibril formation, expressed as time until fibril formation (lag-time) and loss of dissolved peptide (peptide recovery). Propensity toward formation of fibrils upon exposure to mechanical stress was assessed, as previously described for insulin formulations using the thiazole dye ThT, which exhibits specific fluorescence characteristics in the presence of amyloid fibrils. Samples were prepared by dissolving each compound in 10 mM glycylglycine buffer (pH 4.0) or 10 mM HEPES buffer (pH 7.5) to a nominal concentration of 250 μM, followed by the addition of a ThT stock solution (0.1 mM) to a final ThT concentration of 1 μM. Each solution is aliquoted (200 μL/well; n = 4) to a microtiter plate sealed with a transparent foil. Mechanical stress was applied using a Fluoroskan Ascent FL fluorescence plate reader at 37 °C incubation, 960 rpm, 1 mm amplitude, with 20 min fluorescence reading intervals for 45 h (filters: excitation 444 nm; emission 485 nm). The lag-time until increasing fluorescence was assessed by visual inspection of the fluorescence–versus–time plots for each sample. If no fibril formation was detected, the lag-time was set to equal the length of the test (45 h). After analysis, wells representing individual replica were pooled and residual soluble peptide was isolated using centrifugation (20,000g for 30 min at room temperature) followed by 0.22 μm filtration. The amount of residual dissolved peptide was determined using a Waters Alliance 2695 system equipped with an XBridge column (bridged ethylsiloxane/silica hybrid 130 C18 3.5 μm to 4.6 × 50 mm) with a flow rate of 2 mL/min at 30 °C and detection at 215/276 nm. A gradient combining eluent A (7.7% w/w acetonitrile, 200 mM Na2SO4, 20 mM NaH2PO4, 20 mM Na2HPO4, pH 7.2) and eluent B (65.5% w/w acetonitrile) was applied (%A/%B: 0–11/2 min: 80/20; 11/2–2 min: linear to 50/50; 2–51/2 min: 50/50; 51/2–6 min: linear to 80/20; and 6–9 min: 80/20). The amount of residual dissolved peptide was reported on a relative scale (peptide recovery) to the amount of dissolved peptide prior to exposure to mechanical stress.

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In Vitro Characterization for SAR Analysis [2]
The potency on the human AMY3R and CTR’s was obtained in cAMP-responsive element luciferase reporter assay performed on baby hamster kidney (BHK) cells stably expressing the reporter and the designated receptors. The potency on the rat AMY3R and CTR was determined in a cAMP assay on BHK cells transiently expressing the receptors. The luciferase assay cells were thawed on the day before the experiment and incubated overnight. On the day of the experiment, cells were washed and incubated for 3 h with an agonist. The medium was removed and replaced by phosphate-buffered saline and steadylite in a ratio of 1:1 and incubated at room temperature for 30 min before luminescence was measured. In the cAMP assay, the transfected cells were thawed and seeded into FlashPlates on the day of the experiment and incubated with agonist for 30 min. The reaction was stopped with detection mix. The plates were then sealed with plastic, shaken, and then allowed to stand at least 2 h before scintillation was measured. EC50 values were calculated in GraphPad Prism. The apparent binding affinities were determined using the scintillation proximity assay with beads from PerkinElmer and cell membranes containing the AMY3R or CTR’s. BHK cells were transiently transfected with the human or rat receptor and cultured for 24 h. Membranes were harvested, prepared, and kept at −80 °C until use. The assay was performed in a 384 well Optiplate. Membranes were mixed with scintillation proximity assay beads in a 1:1 ratio. Test compounds were dissolved in DMSO, further diluted in assay buffer, and added to the Optiplate together with the dissolved radioligand. In the CTR-binding assays, 125I-Calcitonin was used as the radioligand, and 125I-rat amylin was used as the radioligand for the AMY3R assays. The final mixture was incubated for 120 min at 25 °C prior to centrifugation. Samples were analyzed on TopCounter (Packard). The IC50 values were calculated using GraphPad Prism5. To secure the quality and stability of the assay, we included pramlintide as a reference compounds in each experiment in all assays used in the SAR. The switch in potency or binding affinity observed for pramlintide was used as a validation of the AMY3R and CTR(a), respectively. No response was observed in untransfected cells when stimulated with amylin or calcitonin peptide analogues, and no signal was observed in untransfected cells when exposed to radioligands of the amylin or calcitonin peptide families.

Animal/Disease Models: Sprague Dawley male rats (12weeks old; ~400 g)[1]
Doses: 0.1, 1, 3, 10, 30 nmol/kg
Route of Administration: subcutaneous (sc) injection; single
Experimental Results: decreased food intake in the rat for several days at doses in the range of 1-10 nmol/kg.

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Animal/Disease Models: Sprague Dawley male rats (12weeks old; ~400 g)[1]
Doses: 10 nmol/kg
Route of Administration: intravenous (iv) injection or subcutaneous (sc) injection; single
Experimental Results: demonstrated good pharmacokinetic/PK parameters with T1/2 of 20, 27 h for iv and sc, respectively.

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In Vivo Experiments [2]
In vivo experiments were approved under a license from the Danish Animal Experiments Inspectorate. All animals had access to shelter, nesting material, and chewing sticks and were acclimatized and getting used to handling at least 1 week prior to any experiments. Rats were housed in groups, except for 1 week prior to and during the monitoring of the food intake.

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Acute Food Intake in Rats [2]
Effect on appetite in lean rats measured up to 60 h after single administration of selected amylin analogues was analyzed using an automated food intake monitoring system, in which up to 32 rats (male Sprague Dawley, Taconic Europe, body weight: 200–250 g), with ad libitum access to standard chow and tap water from water bottles, were single housed for individual registration of food consumption. The rats were acclimatized to reverse light cycle (12 h light and 12 h dark) and single housing at least 5 days prior to testing at controlled temperature conditions (20 °C ± 1 °C). Amylin analogues were administered subcutaneously immediately before lights were turned off. Food intake was recorded for 48 or 60 h post-dosing. Dose levels were 3 and 30 nmol/kg for screening purposes, n = 5–7. Control rats were dosed with vehicle. Test substances were formulated in 2 mM acetate, 250 mM glycerol, 0.025% Tween-20, and pH 4. Accumulated food intake was calculated for the periods 0–24 and 24–48, 48–60 h, respectively. Mean accumulated food intake in each dose group was compared to vehicle and reported as a percentage of mean food intake in the vehicle group, which was defined as 100%.

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Pharmacokinetic Evaluation of Cagrilintide [2]
Sprague Dawley male rats (approx. 400 g, 12 week old) were familiarized with handling procedures during a 2-week period prior to the PK experiment. An intravenous bolus injection was performed via a catheter Venflon in the tail vein of the rat, while the rat was temporarily restrained, and subcutaneous injection was performed in the neck. Dosing volume was 2 mL/kg of 10 nmol/kg 23. Blood samples (200 μL) were taken from the tongue vein at 5, 15, and 30 min and 1, 11/2, 2, 4, 6, 12, 24, 48, 72, and 96 h. All blood samples were collected into test tubes containing EDTA for stabilization and kept on ice until centrifugation. Plasma was separated from whole blood by centrifugation, and the plasma was stored at −20 °C or lower until analysis. Plasma samples were analyzed by LC–MS on either a triple quadrupole instrument from Applied Biosystems or an Orbitrap. For analysis on the triple quadrupole, a multiple reaction monitoring (MRM) transition from m/z 1103 → 1075 was used, whereas a scan range from m/z 1102–1107 (selected ion monitoring) was used on the Orbitrap. Plasma samples were diluted with three volumes of ethanol containing 1% formic acid, and the resultant supernatant was subjected to online sample clean-up by turbulent flow chromatography on a Cyclone (50 × 0.5 mm) column from Thermo Fisher prior to LC–MS analysis. For LC–MS analysis, an Onyx Monolithic C18 column from Phenomenex (50 × 2.0 mm ID) was used. The mobile phases for gradient HPLC comprised 5 and 95% organic solvent (50:50 mixture of acetonitrile and methanol), and formic acid was added to both eluents at a concentration of 0.1%. The lower limit of quantification was 2 nM. Plasma concentration–time profiles were analyzed by a non-compartmental analysis using Phoenix WinNonlin Professional 6.2 (Pharsight, Mountain View, CA, US). Calculations were performed using individual concentration–time values from the animals. The AUC was calculated and given as AUCinf_pred unless otherwise stated. The percentage of extrapolated AUC was less than 25% in all studies. The s.c. bioavailability was calculated as (AUC/dose) s.c./(AUC/dose) i.v. The given mean values are all arithmetric except for T1/2 and Tmax, which are given as harmonic mean and geometric mean, respectively.

The duration of action of 23 was indicated from the long-lasting reduction of appetite in the food intake screening model (Figure 3) and was further confirmed in a pharmacokinetic study in rats (Figure 4 and Table 7). In addition, the lag-time in ThT assay at pH 7.5 was shorter for 22 than that for 23. Consequently, 23 was chosen for clinical development and completed clinical phase 2 in 2020. A particular focus was on the combination of semaglutide and cagrilintide as the combination of amylin and GLP-1 therapy has been suggested to work using partly complimentary mechanisms. The clinical data published for the combination of cagrilintide and semaglutide indeed indicate that cagrilintide in a 20 week phase 1B study was able to induce further 7.4% weight loss on top of semaglutide to a total weight loss of 17.1%, thereby warranting further studies in obesity. The half-life was found to be 159–195 h. This validates lipidation with fatty di-acids as a mean to prolong half-life of peptide hormones, though albumin binding of cagrilintide was not directly measured. There is rich evidence that long-acting peptides and proteins conjugated to negatively charged fatty acid derivatives exhibit strong and reversible albumin binding. In addition, we have studied similar in vitro receptor assays as reported here but in the presence of varying amounts of albumin, and the data (not shown here) support that cagrilintide is a reversible albumin binder like the GLP-1 analogue semaglutide.[2]

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species; n; RoA; dose (nmol/kg); AUC/D (h·kg/L); Vz (L/kg); Cl (L/h/kg); T1/2 (h) Sprague Dawley rat; 5; i.v.; 10; 266; 0.109; 0.00377; 20 ± 2
Sprague Dawley rat; 5; s.c.; 10; 87; N/A; N/A; 27 ± 3

[1]. AM833 Is a Novel Agonist of Calcitonin Family G Protein-Coupled Receptors: Pharmacological Comparison with Six Selective and Nonselective Agonists. J Pharmacol Exp Ther. 2021 Jun;377(3):417-440.

[2]. Development of Cagrilintide, a Long-Acting Amylin Analogue. J Med Chem. 2021 Aug 12;64(15):11183-11194.

[3]. Amylin as a Future Obesity Treatment. J Obes Metab Syndr. 2021 Dec 30;30(4):320-325.

Obesity and associated comorbidities are a major health burden, and novel therapeutics to help treat obesity are urgently needed. There is increasing evidence that targeting the amylin receptors (AMYRs), heterodimers of the calcitonin G protein-coupled receptor (CTR) and receptor activity-modifying proteins, improves weight control and has the potential to act additively with other treatments such as glucagon-like peptide-1 receptor agonists. Recent data indicate that AMYR agonists, which can also independently activate the CTR, may have improved efficacy for treating obesity, even though selective activation of CTRs is not efficacious. AM833 (cagrilintide) is a novel lipidated amylin analog that is undergoing clinical trials as a nonselective AMYR and CTR agonist. In the current study, we have investigated the pharmacology of AM833 across 25 endpoints and compared this peptide with AMYR selective and nonselective lipidated analogs (AM1213 and AM1784), and the clinically used peptide agonists pramlintide (AMYR selective) and salmon CT (nonselective). We also profiled human CT and rat amylin as prototypical selective agonists of CTR and AMYRs, respectively. Our results demonstrate that AM833 has a unique pharmacological profile across diverse measures of receptor binding, activation, and regulation. SIGNIFICANCE STATEMENT: AM833 is a novel nonselective agonist of calcitonin family receptors that has demonstrated efficacy for the treatment of obesity in phase 2 clinical trials. This study demonstrates that AM833 has a unique pharmacological profile across diverse measures of receptor binding, activation, and regulation when compared with other selective and nonselective calcitonin receptor and amylin receptor agonists. The present data provide mechanistic insight into the actions of AM833. [1]

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A hallmark of the pancreatic hormone amylin is its high propensity toward the formation of amyloid fibrils, which makes it a challenging drug design effort. The amylin analogue pramlintide is commercially available for diabetes treatment as an adjunct to insulin therapy but requires three daily injections due to its short half-life. We report here the development of the stable, lipidated long-acting amylin analogue cagrilintide (23) and some of the structure-activity efforts that led to the selection of this analogue for clinical development with obesity as an indication. Cagrilintide is currently in clinical trial and has induced significant weight loss when dosed alone or in combination with the GLP-1 analogue semaglutide. [2]

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Obesity is defined as abnormal or excessive fat accumulation that contributes to detrimental health impacts. One-third of the population suffers from obesity, and it is important to consider obesity as a chronic disease requiring chronic treatment. Amylin is co-secreted with insulin from β pancreatic cells upon nutrient delivery to the small intestine as a satiety signal, acts upon sub-cortical homeostatic and hedonic brain regions, slows gastric emptying, and suppresses post-prandial glucagon responses to meals. Therefore, new pharmacological amylin analogues can be used as potential anti-obesity medications in individuals who are overweight or obese. In this narrative review, we analyse the efficacy, potency, and safety of amylin analogues. The synthetic amylin analogue pramlintide is an approved treatment for diabetes mellitus which promotes better glycaemic control and small but significant weight loss. AM833 (cagrilintide), an investigational novel long-acting acylated amylin analogue, acts as a non-selective amylin receptor. This calcitonin G protein-coupled receptor agonist can serve as an attractive novel treatment for obesity, resulting in reduction of food intake and significant weight loss in a dose-dependent manner.[3]

C196H316N54O61S2

4469.06

Cagrilintide;1415456-99-3

White to off-white solid powder

AM-833 acetate; AM833 acetate; Cagrilintide acetate; 1415456-99-3; NN-0174-0833; NN-01740833; NN0174-0833; LDERDVMBIYGIOI-IZVMHKDJSA-N;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~50 mg/mL (~11.19 mM)

Note: Please refer to the "Guidelines for Dissolving Peptides" section in the 4th page of the "Instructions for use" file (upper-right section of this webpage) for how to dissolve peptides.

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Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)