Anticancer; amylin analog; diabetes
Amylin receptors (AMY receptors), calcitonin receptor (CTR). Pramlintide is an amylin analog and agonist. Amylin receptors are heterodimers of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs), giving rise to the AMY1, AMY2, and AMY3 subtypes.

In a dose-dependent manner, pramlintide suppresses the growth of HCT-116 and HT-29, with a greater efficacy against the latter (IC50 values of 48.67 and 9.10 μg/mL, respectively) [1]. The antiproliferative effects of 5-fluorouracil, oxaliplatin, or irinotecan were synergistically induced when 5, 10, and 20 μg/mL of Pramlintide was added to HCT-116 and HT-29 [1].

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Pramlintide is a potent amylin receptor agonist. It exerts its effects by binding to and activating these receptors, which are found in several tissues including the pancreas, brain, and gastrointestinal tract. This leads to a reduction in postprandial glucagon secretion, slowing of gastric emptying, and promotion of satiety.

In clinical studies involving patients with type 1 diabetes, pramlintide reduces postprandial blood glucose excursions. It also decreases postprandial glucagon secretion (which is abnormally elevated in type 1 diabetes) and slows the rate of gastric emptying, resulting in better glycemic control and modest weight loss.

A cell-free binding assay for amylin receptors is complex due to their requirement for multiple proteins (CTR + RAMP). Typically, membranes from cells co-expressing human CTR and RAMP1/2/3 are incubated with [125I]-amylin and varying concentrations of pramlintide. After incubation, bound and free ligands are separated by filtration, and the Ki is determined.

MTT assay [1]
The HCT-116 (wild-type p53) and HT-29 (mutant p53) cells were plated into the 96 well plates at a density of 5×103 in 200 μL of medium per well and the cells were incubated and allowed to attach overnight. The attached cells in the plates were treated with a series of drug concentrations: pramlintide (0–102.4 μg/mL), 5-FU (0–200 μM), OXA (0–300 μM), or IRN (0–160 μM) alone or in combination with three different concentrations of pramlintide (5, 10, and 20 μg/mL) that correspond to 0.5×IC50, IC50, and 2×IC50 in HT-29. Cells grown in medium alone (for treatment with pramlintide only) or containing an equivalent amount of DMSO served as control (for other treatment conditions). [1]
Cells were incubated with the drugs at the indicated concentrations for 72 hours. All measurements were done in triplicate. After that, cell proliferation assay was performed per the manufacturer’s protocol. Briefly, MTT dye was added to the treated cells at a final concentration of 0.5 mg/mL in PBS. Then, the plates were incubated at 37°C for 3 hours and the MTT was discarded and the formazan product was dissolved by adding 100 μL of DMSO to each well, followed by shaking for 5 minutes. Then, the plates were read using an enzyme-linked immunosorbent assay plate reader at 570 nm with a reference wavelength of 690 nm. Cell viability was calculated as follows: absorbance of the experimental group/absorbance of the control group. The IC50 value was defined as the concentration needed for a 50% reduction in cell viability. Dose–effect analyses and IC50 calculations were performed using Compusyn software 1.0

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For a cell-based functional assay, cells (e.g., CHO-K1) co-expressing the calcitonin receptor (CTR) and a receptor activity-modifying protein (RAMP1, 2, or 3) are seeded in 96-well plates and loaded with a calcium indicator dye (e.g., Fluo-4). Cells are treated with varying concentrations of pramlintide, and the increase in intracellular calcium is measured to determine the agonist's EC50.

Pramlintide is administered by subcutaneous (SC) injection in both research and clinical settings. In diabetic rodent models, its effects are assessed by measuring postprandial blood glucose levels, glucagon concentrations, gastric emptying rate (using a labeled meal), and body weight over time. Efficacy is also assessed via intravenous glucose tolerance tests (IVGTT).

Pramlintide is administered subcutaneously and is unsuitable for oral administration due to its peptidic nature. In humans, its absorption is relatively slow, with a peak concentration (Cmax) occurring approximately 1 hour after SC injection. It is rapidly cleared (half-life ~48 minutes) and is degraded primarily in the kidneys.

In clinical use, pramlintide is associated primarily with gastrointestinal side effects, including nausea, vomiting, and anorexia, which are dose-dependent and tend to subside over time. The most serious risk is severe hypoglycemia, particularly when used in conjunction with insulin, necessitating careful dose adjustments.

[1]. Pramlintide, an antidiabetic, is antineoplastic in colorectal cancer and synergizes with conventional chemotherapy. Clin Pharmacol. 2018 Mar 5;10:23-29.

To investigate the synergistic effect of pramlinide with chemotherapy drugs in colorectal cancer cell lines, we attempted to test three different concentrations of pramlinide, corresponding to 0.5×IC50, IC50, and 2×IC50 for each cell line. However, due to the high concentration requirement of pramlinide in the HCT-116 cell line and the limited drug supply, to maintain consistency with the comparisons studied, we ultimately used pramlinide concentrations of 5, 10, and 20 μg/mL, corresponding to 0.5×IC50, IC50, and 2×IC50 for the HT-29 cell line, respectively. We demonstrated for the first time that, at clinically achievable low concentrations, pramlinide, when used in combination with 5-fluorouracil (5-FU), oxaliplatin (OXA), and irinotecan (IRN), synergistically inhibits the proliferation of colorectal cancer cells in HCT-116 and HT-29 cell lines in a concentration-dependent manner. These results suggest that pramlintide is a novel potential adjuvant anticancer drug with beneficial effects in overcoming resistance to 5-fluorouracil (5-FU), oxaliplatin (OXA), and irinotecan (IRN). Further in vivo and clinical studies are needed to establish pramlintide as an effective chemoprevention and chemotherapy drug for colorectal cancer. [1]
Despite the encouraging results obtained in this study, there are still some limitations. First, we only used the short-term MTT assay to detect the antitumor potential of pramlintide. Second, we only used two representative cell lines to study the differential effects of pramlintide based on p53 status; therefore, the difference in the response of HT-29 and HCT-116 cells to pramlintide may not be caused by p53. [1]
Conclusion: This study is the first to show that pramlintide has anticancer activity against colorectal cancer and has a synergistic effect with 5-FU, oxaliplatin, and irinotecan. Future studies will explore the antiproliferative mechanism of pramlintide and the potential molecular mechanism of its synergistic effect more comprehensively. In addition, other long-term trials (such as colony formation trials) and in vivo colorectal cancer models will be used to analyze the antitumor potential of pramlinin. [1]

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Pramlintide (Symlin) was approved by the FDA in 2005 for both type 1 and type 2 diabetes. It is the first and only approved synthetic analog of the pancreatic hormone amylin. By mimicking the natural effects of amylin, it complements insulin therapy to better control blood sugar levels, especially after meals. Its ability to induce weight loss is another key clinical benefit, making it valuable for overweight patients with diabetes.

C173H271N51O55S2

4009.44157624245

4007.9451

187887-46-3

Pramlintide;151126-32-8;Pramlintide TFA

118984456

Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH2 (Disulfide bridge:Cys2-Cys7); H-Lys-Cys(1)-Asn-Thr-Ala-Thr-Cys(1)-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH2.CH3CO2H; L-lysyl-L-cysteinyl-L-asparagyl-L-threonyl-L-alanyl-L-threonyl-L-cysteinyl-L-alanyl-L-threonyl-L-glutaminyl-L-arginyl-L-leucyl-L-alanyl-L-asparagyl-L-phenylalanyl-L-leucyl-L-valyl-L-histidyl-L-seryl-L-seryl-L-asparagyl-L-asparagyl-L-phenylalanyl-glycyl-L-prolyl-L-isoleucyl-L-leucyl-L-prolyl-L-prolyl-L-threonyl-L-asparagyl-L-valyl-glycyl-L-seryl-L-asparagyl-L-threonyl-L-tyrosinamide (2->7)-disulfide acetic acid

KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY-NH2 (Disulfide bridge:Cys2-Cys7); KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY

White to off-white solid powder

57

61

110

281

9690

41

S1C[C@@H](C(N[C@@H](C)C(N[C@@H]([C@@H](C)O)C(N[C@@H](CCC(N)=O)C(N[C@H](C(N[C@H](C(N[C@@H](C)C(N[C@@H](CC(N)=O)C(N[C@@H](CC2C=CC=CC=2)C(N[C@@H](CC(C)C)C(N[C@@H](C(C)C)C(N[C@@H](CC2=CNC=N2)C(N[C@@H](CO)C(N[C@@H](CO)C(N[C@@H](CC(N)=O)C(N[C@@H](CC(N)=O)C(N[C@@H](CC2C=CC=CC=2)C(NCC(N2CCC[C@H]2C(N[C@@H]([C@@H](C)CC)C(N[C@@H](CC(C)C)C(N2CCC[C@H]2C(N2CCC[C@H]2C(N[C@H](C(N[C@H](C(N[C@H](C(NCC(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N)=O)CC2C=CC(=CC=2)O)=O)[C@@H](C)O)=O)CC(N)=O)=O)CO)=O)=O)C(C)C)=O)CC(N)=O)=O)[C@@H](C)O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)CC(C)C)=O)CCCNC(=N)N)=O)=O)=O)=O)NC([C@H]([C@@H](C)O)NC([C@H](C)NC([C@H]([C@@H](C)O)NC([C@H](CC(N)=O)NC([C@H](CS1)NC([C@H](CCCCN)N)=O)=O)=O)=O)=O)=O.OC(C)=O

DTPWZYSUQQHRKD-VIUAGAKSSA-N

InChI=1S/C171H267N51O53S2.C2H4O2/c1-21-81(12)130(163(268)207-110(56-78(6)7)169(274)222-53-33-42-118(222)170(275)221-52-32-41-117(221)160(265)219-135(89(20)230)167(272)206-109(66-125(180)238)151(256)212-128(79(8)9)161(266)186-68-126(239)192-111(70-223)154(259)203-107(64-123(178)236)152(257)218-134(88(19)229)166(271)195-98(136(181)241)57-92-43-45-94(231)46-44-92)214-159(264)116-40-31-51-220(116)127(240)69-187-141(246)101(58-90-34-24-22-25-35-90)199-148(253)105(62-121(176)234)201-149(254)106(63-122(177)235)202-155(260)112(71-224)209-156(261)113(72-225)208-146(251)103(60-93-67-184-75-188-93)205-162(267)129(80(10)11)213-150(255)100(55-77(4)5)198-145(250)102(59-91-36-26-23-27-37-91)200-147(252)104(61-120(175)233)196-137(242)82(13)189-144(249)99(54-76(2)3)197-142(247)96(39-30-50-185-171(182)183)193-143(248)97(47-48-119(174)232)194-165(270)132(86(17)227)215-138(243)83(14)190-157(262)114-73-276-277-74-115(210-140(245)95(173)38-28-29-49-172)158(263)204-108(65-124(179)237)153(258)217-131(85(16)226)164(269)191-84(15)139(244)216-133(87(18)228)168(273)211-114;1-2(3)4/h22-27,34-37,43-46,67,75-89,95-118,128-135,223-231H,21,28-33,38-42,47-66,68-74,172-173H2,1-20H3,(H2,174,232)(H2,175,233)(H2,176,234)(H2,177,235)(H2,178,236)(H2,179,237)(H2,180,238)(H2,181,241)(H,184,188)(H,186,266)(H,187,246)(H,189,249)(H,190,262)(H,191,269)(H,192,239)(H,193,248)(H,194,270)(H,195,271)(H,196,242)(H,197,247)(H,198,250)(H,199,253)(H,200,252)(H,201,254)(H,202,260)(H,203,259)(H,204,263)(H,205,267)(H,206,272)(H,207,268)(H,208,251)(H,209,261)(H,210,245)(H,211,273)(H,212,256)(H,213,255)(H,214,264)(H,215,243)(H,216,244)(H,217,258)(H,218,257)(H,219,265)(H4,182,183,185);1H3,(H,3,4)/t81-,82-,83-,84-,85+,86+,87+,88+,89+,95-,96-,97-,98-,99-,100-,101-,102-,103-,104-,105-,106-,107-,108-,109-,110-,111-,112-,113-,114-,115-,116-,117-,118-,128-,129-,130-,131-,132-,133-,134-,135-;/m0./s1

acetic acid;(2S)-N-[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-4-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-4-amino-1-[[(2S)-4-amino-1-[[(2S)-1-[[2-[(2S)-2-[[(2S,3S)-1-[[(2S)-1-[(2S)-2-[(2S)-2-[[(2S,3R)-1-[[(2S)-4-amino-1-[[(2S)-1-[[2-[[(2S)-1-[[(2S)-4-amino-1-[[(2S,3R)-1-[[(2S)-1-amino-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]carbamoyl]pyrrolidine-1-carbonyl]pyrrolidin-1-yl]-4-methyl-1-oxopentan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]carbamoyl]pyrrolidin-1-yl]-2-oxoethyl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]-2-[[(2S,3R)-2-[[(2S)-2-[[(4R,7S,10S,13S,16S,19R)-16-(2-amino-2-oxoethyl)-19-[[(2S)-2,6-diaminohexanoyl]amino]-7,13-bis[(1R)-1-hydroxyethyl]-10-methyl-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carbonyl]amino]propanoyl]amino]-3-hydroxybutanoyl]amino]pentanediamide

187887-46-3; Triproamylin acetate; UNII-JS41L76X7I; Tripro-amylin acetate; Pramlintide acetate anhydrous;

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, avoid exposure to moisture.

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 (12.47 mM)

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.)