Pemetrexed disodium (LY-231514)

• Catalog No.: S1135
• CAS Number: 150399-23-8
• M. F.: C20H19N5Na2O6
• M. W.: 471.3743
• Storage: -20°C

SYNONYMS

• IUPAC name: 1,5-disodium (2S)-2-{[4-(2-{2-amino-4-oxo-1H,4H,7H-pyrrolo[2,3-d]pyrimidin-5-yl}ethyl)phenyl]formamido}pentanedioate
• IUPAC Traditional name: 1,5-disodium (2S)-2-{[4-(2-{2-amino-4-oxo-1H,7H-pyrrolo[2,3-d]pyrimidin-5-yl}ethyl)phenyl]formamido}pentanedioate
• Synonyms: LY-231514; Alimta

DATABASE IDS

• CAS Number: 150399-23-8

PROPERTIES

• Target: Dihydrofolate Reductas
• Target: Antimetabolites
• Salt Data: Disodium
• Solubility: DMSO
• Storage Condition: -20°C

DETAILS

Description (English)

Research Area

• Description: Cancer

Biological Activity

• Description: Pemetrexed disodium (Alimta) is a novel antifolate and antimetabolite for TS, DHFR and GARFT with K_i of 1.3 nM, 7.2 nM and 65 nM, respectively.
• Targets: TS; DHFR; GARFT
• IC50: 1.3 nM (K_i); 7.2 nM (K_i); 65 nM (K_i) ^[1]
• In Vitro: Pemetrexed disodium shows the antiproliferative activity in CCRF-CEM leukemia, GC3/C1 colon carcinoma, and HCT-8 ileocecal carcinoma cells with IC50 of 25 nM, 34 nM and 220 nM, respectively. ^[1] A recent study shows that cisplatin plus Pemetrexed combined with SOCS-1 gene delivery shows the antitumor effect by inhibition of cell proliferation, invasiveness, and induction of apoptosis in MPM cells infected with adenovirus-expressing SOCS-1 vector. ^[2]
• In Vivo: In the human H460 non-small cell lung carcinoma xenograft, Pemetrexed disodium produces a duration-dependent tumor growth delay (TGD). ^[3]
• Clinical Trials: Pemetrexed disodium is currently in Phase I clinical trials in patients with Head and Neck Cancer.

Combination Therapy

• Description: The combination of thymidine plus hypoxanthine totally reverses the cytotoxicity caused by LY231514 in all three cell lines above, and polyglutamation is a major determinant of cytotoxicity for Pemetrexed disodium. ^[1] Furthermore, Pemetrexed disodium combined with paclitaxel produces greater-than-additive H460 TGD and additive responses with vinorelbine and carboplatin. ^[3]

Protocol

• Protocol: Kinase Assay ^[1]
• Enzyme Assays and Methods.: TS activity is assayed using a spectrophotometric method, which involved monitoring the increase in absorbance at 340 nm resulting from formation of the product, 7,8-dihydrofolate. The assay buffer contains 50 mM N-tris[hydroxymethyljmethyl-2-aminoethanesulfonic acid, 25 mM MgC1₂, 6.5 mM formaldehyde, 1 mM EDTA, and 75 mM 2-mercaptoethanol, pH 7.4. The concentrations of deoxyuridylate monophosphate, 6R-MTHF, and hIS are 100 μM, 30μM and 30 nM (1.7 milliunits/mL), respectively. At the 6R-MTHF concentration, an uninhibited reaction and six concentrations of inhibitor are assayed. K_{i app} values are determined by fitting the data to the Morrison equation using nonlinear regression analysis with the aid of the program ENZFITTER. K_i values are calculated using the equation: K_{i app}= K_i(1 + [S]/K_m), where [S] is equal to 30 μM and K_m is equal to 3 μM. DHFR activity is assayed spectrophotometrically by monitoring the dis appearance of the substrates NADPH and 7,8-dihydrofolate at 340 nm. The reaction takes place at 25°C in 0.5 mL of 50 mM potassium phosphate buffer, which contains 150 mM KC1 and 10 nM 2-mercaptoethanol, pH 7.5, and 14 nM (0.34 milliunitlmL) DHFR. The NADPH concentration is 10 μM and 7,8-dihydrofolate is varied at 5, 10, or 15 μM. At each 7,8-dihydrofolate concentration, an uninhibited reaction and seven concentrations of inhibitor are assayed. The ENZFITI'ER microcomputer program is used to obtain K_{i app} values by fitting the data to the Morrison equation by nonlinear regression analysis. K_{i app}= K_i(1 + [S]/K_m), where [S] is equal to the concentration of 7,8-dihydrofolate used and K_m of 7,8-dihydrofolate is equal to 0.15 μM. GARFT activity is assayed spectrophotometrically by monitoring the increase of absorbance resulting from formation of the product 5,8-dideazafolate at 295 nm. The reaction solvent contains 75 mM HEPES, 20% glycerol, and 50 mM a-thioglygerol, pH 7.5, at 25°C. The concentrations of substrates and enzyme used are 10 μM α,β-glycinamide ribonucleotide, 0-10 μM 10-formyl-5,8-dideazafolic acid, and 10 nM (1.9 milliunits/mL) GARFT. K_i values are calculated using the Enzyme Mechanism program of the Beckman DU640 spectrophotometer, which uses nonlinear regression analysis to fit data to the Michaelis-Menten equation for competitive inhibition.
• Protocol: Cell Assay ^[1]
• Cell Lines: CCRF-CEM leukemia, GC3/C1 colon carcinoma, and HCT-8 ileocecal carcinoma cells.
• Concentrations: 0-30 μM
• Incubation Time: 72 hours
• Methods: Dose-response curves are generated to determine the concentration required for 50% inhibition of growth (IC50). Pemetrexed disodium is dissolved initially in DMSO at a concentration of 4 mg/mL and further diluted with cell culture medium to the desired concentration. CCRF-CEM leukemia cells in complete medium are added to 24-well Cluster plates in a total volume of 2.0 mL. Pemetrexed disodium at various concentrations are added to duplicate wells so that the final volume of DMSO is 0.5%. The plates are incubated for 72 hour at 37 °C in an atmosphere of 5% CO₂ in air. At the end of the incubation, cell numbers are determined on a ZBI Coulter counter. For several studies, IC50s are determined for each compound in the presence of either 300 μM AICA, 5 μM thymidine, 100 μM hypoxanthine, or combination of 5 μM hymidine plus 100 μM hypoxanthine. For adherent tumor cells, a modification of the original MTT colorimetric assay is used to measure cell cytotoxicity. The human tumor cells are seeded in 100 μL assay medium/well in 96-well flat-bottomed tissue culture plates. The assay medium contains folic acid-free RPMI 1640 supplemented with 10% FCS and either 2 nM folinic acid or 2.3 μM folic acid as the sole folate source. Well 1A is left blank. Stock solutions of antifolates are prepared in Dulbecco's PBS at 1 mg/mL, and a series of 2-fold dilutions are subsequently made in PBS. Ten-μL aliquots of each concentration are added to triplicate wells. Plates are incubated for 72 hours at 37 °C in a humidified atmosphere of 5% CO₂-in-air. MTT is dissolved in PBS at 5 mg/mL, 10 μL of stock MTF solution are added to each well of an assay, and the plates are incubated at 37 °C for 2 additional hours. Following incubation, 100 μL of DMSO are added to each well. After thorough formazan solubilization, the plates are read on a Dynatech MR600 reader, using a test wavelength of 570 nm and a reference wavelength of 630 nm. The IC50 is determined as the concentration of drug required to inhibit cell growth by 50% compared to an untreated controls.
• Protocol: Animal Study ^[3]
• Animal Models: EMT-6 mammary carcinoma, the human HCT 116 colon carcinoma, and the human H460 non-small cell lung carcinoma are injected s.c. into the nude mice.
• Formulation: Pemetrexed disodium is dissolved in DMSO and then diluted in water.
• Doses: 100 mg/kg or 150 mg/kg
• Administration: Administered via i.p.

References

• References: [1] Shih C, et al. Cancer Res. 1997, 57(6), 1116-1123.
• References: [2] Iwahori K, et al. Int J Cancer. 2012, doi: 10.1002/ijc.27611.
• References: [3] Teicher BA, et al. Clin Cancer Res. 2000, 6(3), 1016-1023.

REFERENCES

• Shih C, et al. Cancer Res. 1997, 57(6), 1116-1123.
• Iwahori K, et al. Int J Cancer. 2012, doi: 10.1002/ijc.27611.
• Teicher BA, et al. Clin Cancer Res. 2000, 6(3), 1016-1023.