TS (Ki = 1.3 nM); DHFR (IC50 = 7.2 nM)

Pemetrexed (LY231514) disodium is a novel classical antifolate, and its antitumor activity may come from its polyglutamated metabolites, which may simultaneously and multiplely inhibit several important folate-requiring enzymes. One of the best substrates for the enzyme FPGS that is currently known to exist is pemetrexed (LY231514) (K_m=1.6 μM and V_max/K_m=621). The selectivity and antitumor activity of LY231514 are probably greatly influenced by polyglutamation and the polyglutamated metabolites of this novel agent. The pentaglutamate of LY23l5l4 is 100-fold more potent (K_i=3.4 nM) than LY23l5l4, which only moderately inhibits TS (K_i=340 nM, recombinant mouse)[1]. This makes LY231514 one of the most potent folate-based TS inhibitors.

Pemetrexed disodium causes a duration-dependent tumor growth delay (TGD) in the human H460 non-small cell lung carcinoma xenograft.

The formation of the product, 7,8-dihydrofolate, was observed to result in an increase in absorbance at 340 nm, which was then used to measure TS activity. The assay buffer has the following contents: 25 mM MgC1₂, 6.5 mM formaldehyde, 1 mM EDTA, 75 mM 2-mercaptoethanol, 50 mM N-tris[hydroxymethyljmethyl-2-aminoethanesulfonic acid]. The concentrations of hIS, 6R-MTHF, and deoxyuridylate monophosphate are 30 μM, 100 μM, and 30 nM (1.7 milliunits/mL), respectively. An uninhibited reaction and six inhibitor concentrations are tested at the 6R-MTHF concentration. K_{i app} values are obtained by applying nonlinear regression analysis, with the assistance of the ENZFITTER program, to fit the data to the Morrison equation. The following equation is used to calculate Ki values: With [S] equal to 30 μM and Km equal to 3 μM, K_{i app}= K_i(1 + [S]/K_m). NADPH and 7,8-dihydrofolate, the substrates, disappear at 340 nm, which is how spectrophotometric analysis of DHFR activity is done. The reaction occurs in 0.5 mL of 50 mM potassium phosphate buffer at 25°C. The buffer has a pH of 7.5, 150 mM KC1, 10 nM 2-mercaptoethanol, and 14 nM (0.34 milliunitlmL) DHFR. 7,8-dihydrofolate is varied at 5, 10, or 15 μM, while NADPH is present at a concentration of 10 μM. Seven inhibitor concentrations are tested along with an uninhibited reaction at each 7,8-dihydrofolate saturation. Fitting the data to the Morrison equation through nonlinear regression analysis is how the ENZFITI'ER microcomputer program determines the K_{i app} values. For every 7,8-dihydrofolate used, [S] represents its concentration, and K_m is equivalent to 0.15 μM. K_{i app}= K_i(1 + [S]/K_m). When 5,8-dideazafolate is formed at 295 nm, an increase in absorbance is observed, which is how spectrophotometric analysis of GARFT activity is quantified. 50% glycerol, 25% HEPES, and 50% a-thioglycerol at a pH of 7.5 at 25°C make up the reaction solvent. Substrates and enzyme were used at the following concentrations: 10 μM α,β-glycinamide ribonucleotide, 0–10 μM 10-formyl–5,8–dideazafolic acid, and 10 nM (1.9 milliunits/mL) GARFT. Ki values are determined using the Beckman DU640 spectrophotometer's Enzyme Mechanism program, which fits data to the Michaelis-Menten equation for competitive inhibition using nonlinear regression analysis.

The concentration necessary for 50% inhibition of growth (IC50) is found by creating dose-response curves. At a starting concentration of 4 mg/mL in DMSO, pemetrexed disodium is dissolved. The concentration is then adjusted with cell culture medium. One hundred twenty-four well Cluster plates are filled with 2.0 mL of CCRF-CEM leukemia cells in complete medium. To make DMSO final volume of 0.5%, replicate wells are filled with pemetrexed disodium at different concentrations. In a 5% CO₂ in air atmosphere, the plates are incubated for 72 hours at 37°C. Cell counts on a ZBI Coulter counter are measured at the conclusion of the incubation. In multiple investigations, the half-life of each compound is ascertained when subjected to 300 μM AICA, 5 μM thymidine, 100 μM hypoxanthine, or a mix of 5 μM hymidine and 100 μM hypoxanthine. Cell cytotoxicity for adherent tumor cells is determined by modifying the original MTT colorimetric assay. In 96-well tissue culture plates with a flat bottom, 100 μL of assay medium is used to seed human tumor cells per well. The only sources of folate in the assay medium are 2.3 μM or 2 nM folic acid, along with 10% FCS and folic acid-free RPMI 1640. Well 1A is not filled in. Antifolate stock solutions (one milligram per milliliter) are prepared in Dulbecco's PBS, and then successive 2-fold dilutions are made in PBS. Triplicate wells are filled with ten-μL aliquots of each concentration. Plates are incubated at 37 °C for 72 hours in an atmosphere that is humidified with 5% CO2 in the air. 10 μL of stock MTF solution is added to each well of an assay after MTT has been dissolved in PBS at a concentration of 5 mg/mL. The plates are then incubated for an additional two hours at 37 °C. 100 μL of DMSO is added to each well after incubation. The plates are read on a Dynatech MR600 reader using a test wavelength of 570 nm and a reference wavelength of 630 nm following complete formazan solubilization. The drug concentration needed to impede cell growth by 50% in comparison to untreated controls is known as the inhibitory concentration (IC50).

Mice: The mice used are female CBA mice and female NOD/SCID mice (NOD.CB17-Prkdc^scid) that are 6–8 weeks old. In order to investigate the synergistic effect of premetrexed (100 mg/kg) in combination with anti-CD25 Ab or IgG control, tumor-bearing mice receive it intraperitoneally (i.p.) from days 4–8 (5 consecutive days). Based on earlier research conducted on mice, the current study's Pemetrexed dosage and schedule were chosen.

[1]. LY231514, a pyrrolo[2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes. Cancer Res. 1997 Mar 15;57(6):1116-23.

[2]. Synergistic antitumor effects of regulatory T cell blockade combined with pemetrexed in murine malignantmesothelioma. J Immunol. 2010 Jul 15;185(2):956-66.

C20H21N5O6

427.41

427.1492

C, 56.20; H, 4.95; N, 16.39; O, 22.46

137281-23-3

Pemetrexed disodium;150399-23-8;Pemetrexed disodium heptahydrate;357166-29-1;Pemetrexed disodium hemipenta hydrate;357166-30-4;Pemetrexed-d5;1129408-57-6

Solid powder

C1=CC(=CC=C1CCC2=CNC3=C2C(=O)NC(=N3)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O

WBXPDJSOTKVWSJ-ZDUSSCGKSA-N

InChI=1S/C20H21N5O6/c21-20-24-16-15(18(29)25-20)12(9-22-16)6-3-10-1-4-11(5-2-10)17(28)23-13(19(30)31)7-8-14(26)27/h1-2,4-5,9,13H,3,6-8H2,(H,23,28)(H,26,27)(H,30,31)(H4,21,22,24,25,29)/t13-/m0/s1

(2S)-2-[[4-[2-(2-amino-4-oxo-3,7-dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]amino]pentanedioic acid

Pemetrexed; HSDB 7316; HSDB7316; HSD-7316; Alimta

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: ~250 mg/mL (~584.9 mM)
H2O: < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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