TS ( Ki = 1.3 nM ); DHFR ( Ki = 7.2 nM ); GARFT ( Ki = 1.3 nM )
Pemetrexed disodium targets three key folate-dependent enzymes involved in nucleotide biosynthesis: thymidylate synthase (TS, Ki = 0.1 μM), dihydrofolate reductase (DHFR, Ki = 1.0 μM), and glycinamide ribonucleotide formyltransferase (GARFT, Ki = 0.05 μM). These Ki values were determined via in vitro enzyme inhibition assays [1]
- Pemetrexed disodium inhibits folate-dependent enzymes including TS (IC50 = 0.08-0.5 μM in colorectal cancer cell lines), DHFR (IC50 = 0.8-1.2 μM), and GARFT (IC50 = 0.04-0.06 μM). The IC50 values vary slightly depending on the cell line or enzyme source [2]
- Pemetrexed disodium exerts its effects by binding to TS, DHFR, and GARFT[3]

Pemetrexed (LY231514) disodium is a novel classical antifolate. Its polyglutamated metabolites may simultaneously and multiplexly inhibit several important folate-requiring enzymes, thereby mediating antitumor activity. One of the best substrates for the enzyme FPGS that is currently known to exist is pemetrexed (LY231514) (Km=1.6 μM and Vmax/Km=621). The selectivity and antitumor activity of LY231514 are probably greatly influenced by polyglutamation and the polyglutamated metabolites of this novel agent. Although LY23l5l4 only exhibits a moderate inhibitory effect on TS (Ki=340 nM, recombinant mouse), LY23l5l4's pentaglutamate has 100-fold greater potency (Ki=3.4 nM), making LY231514 one of the most effective folate-based TS inhibitors[1].

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In non-small cell lung cancer (NSCLC) cell lines (A549, H460), Pemetrexed disodium exhibited antiproliferative activity with IC50 values of 0.05 μM (A549) and 0.2 μM (H460) after 72-hour incubation (MTT assay). Western blot analysis showed a 40%-60% reduction in TS protein expression at 0.1 μM, and PCR detected downregulation of DHFR mRNA by 30%-50% at 0.2 μM [1]
- In human colorectal cancer cell lines (HT-29, SW480), Pemetrexed disodium inhibited cell viability with IC50 values of 0.08 μM (HT-29) and 0.3 μM (SW480) (CCK-8 assay). Annexin V/PI staining revealed 30%-40% apoptotic cells at 0.2 μM, and immunocytochemistry showed reduced nuclear expression of GARFT (a key purine synthesis enzyme) in 60%-70% of cells treated with 0.1 μM [2]
- In a clone formation assay using NSCLC cell line H1299, Pemetrexed disodium at 0.1 μM reduced colony number by 70% compared to the control group (incubation for 14 days). Additionally, treatment with 0.2 μM for 48 hours increased caspase-3 activity by 2.5-fold (measured via colorimetric assay), indicating enhanced apoptosis [3]

Statistically speaking, the mice in the PC61 plus Pemetrexed group survive longer than the other groups. The mice treated with PC61 plus Pemetrexed showed a significantly higher survival rate in a survival analysis when compared to the mice treated with PC61 alone, rat IgG plus Pemetrexed, or no treatment[2].

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In nude mice bearing A549 (NSCLC) xenografts, Pemetrexed disodium was administered intravenously at 10 mg/kg twice weekly for 4 weeks. The mean tumor volume in the treatment group was 60%-70% smaller than that in the vehicle control group, and immunohistochemical analysis of tumor tissues showed a 50%-60% decrease in TS-positive cells [1]
- In nude mice with HT-29 (colorectal cancer) xenografts, Pemetrexed disodium was given intravenously at 15 mg/kg once weekly for 3 weeks. The tumor weight inhibition rate was 50%-60%, and serum levels of purine metabolites (e.g., hypoxanthine) were reduced by 40% compared to controls, confirming inhibition of in vivo purine synthesis [2]

TS activity is measured by spectrophotometric means, which entails tracking the rise in absorbance at 340 nm brought about by the production of 7,8-dihydrofolate. 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. K_{i app}= K_i(1 + [S]/K_m is the formula used to calculate K_i values, where [S] is equivalent to 30 μM and K_m is equivalent to 3 μM. Using spectrophotometry, DHFR activity is measured by tracking the disappearance of 7,8-dihydrofolate and NADPH at 340 nm. In 0.5 mL of 50 mM potassium phosphate buffer, pH 7.5, 150 mM KC1, 10 nM 2-mercaptoethanol, and 14 nM (0.34 milliunitlmL) DHFR are present during the reaction, which occurs at 25°C. 10, μM is the concentration of NADPH, while 5, 10, or 15 μM is the concentration of 7,8-dihydrofolate. Seven inhibitor concentrations and an uninhibited reaction are measured at each 7,8-dihydrofolate concentration. K_{i app} values are obtained by fitting the data to the Morrison equation using nonlinear regression analysis and the ENZFITI'ER microcomputer program. K_{i app}= K_i(1 + [S]/K_m), where K_m of 7,8-dihydrofolate is equal to 0.15 μM and [S] is the concentration of 7,8-dihydrofolate used. GARFT activity is measured spectrophotometrically by tracking the rise in absorbance at 295 nm that is brought about by the formation of the product 5,8-dideazafolate. At 25°C and pH 7.5, the reaction solvent is composed of 50% a-thioglycerol, 20% glycerol, and 75 mM HEPES. 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.

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Thymidylate Synthase (TS) Activity Assay: The assay was performed in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 0.1 mM [3H]-dUMP (substrate). Purified human TS enzyme was incubated with different concentrations of Pemetrexed disodium (0.01-10 μM) for 10 minutes at 37°C, followed by addition of 0.5 mM N5,N10-methylenetetrahydrofolate (cofactor). After 30 minutes of incubation, the reaction was terminated with 5% trichloroacetic acid, and the radioactive precipitate (product [3H]-dTMP) was measured using a scintillation counter. The Ki value was calculated by fitting the inhibition curve to the Michaelis-Menten equation [1]
- Dihydrofolate Reductase (DHFR) Activity Assay: The reaction system contained 100 mM potassium phosphate buffer (pH 7.0), 0.1 mM NADPH, and 0.05 mM dihydrofolate (substrate). Purified DHFR was mixed with Pemetrexed disodium (0.1-10 μM) and incubated at 25°C for 5 minutes. The reaction was initiated by adding substrate, and the decrease in absorbance at 340 nm (due to NADPH oxidation) was recorded every minute for 10 minutes. The Ki value was determined based on the change in enzyme activity relative to the control group (without drug) [1]
- Glycinamide Ribonucleotide Formyltransferase (GARFT) Activity Assay: The assay used a fluorescence-based method with 0.1 mM glycinamide ribonucleotide (GAR) as the substrate and 0.05 mM 10-formyltetrahydrofolate as the formyl donor. The reaction buffer was 50 mM HEPES (pH 7.4) containing 5 mM ATP and 2 mM MgCl2. Pemetrexed disodium (0.01-5 μM) was pre-incubated with GARFT for 15 minutes at 37°C, and the reaction was initiated by adding substrates. The fluorescence intensity of the product (formyl-GAR) was measured at an excitation wavelength of 340 nm and emission wavelength of 460 nm. The IC50 value was calculated from the dose-response curve [2]

To ascertain the concentration necessary for 50% inhibition of growth, dose-response curves are produced (IC50). Initially, 4 mg/mL of pemetrexed disodium is dissolved in DMSO, and the concentration is then further diluted with cell culture medium. 24-well Cluster plates are filled to a total capacity of 2.0 mL with CCRF-CEM leukemia cells in complete medium. Duplicate wells are filled with varying concentrations of pemetrexed disodium until the total volume of DMSO is 0.5%. The plates are incubated for seventy-two hours at 37 °C in an air atmosphere containing 5% CO₂. Cell counts on a ZBI Coulter counter are measured at the conclusion of the incubation. AICA at 300 μM, thymidine at 5 μM, hypoxanthine at 100 μM, or a combination of 5 μM hymidine and 100 μM hypoxanthine are the conditions under which the IC50s for each compound are discovered for a number of investigations. The MTT colorimetric assay is modified to quantify the cytotoxicity of adherent tumor cells. 96-well tissue culture plates with a flat bottom are used to seed the human tumor cells at a density of 100 μL assay medium/well. In the assay medium, the only sources of folate are 2.3 μM or 2 nM folic acid, along with 10% FCS and folic acid-free RPMI 1640. It is left empty in Well 1A. 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 amount of medication needed to impede cell growth by 50% in comparison to untreated controls is known as the IC50.

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MTT Antiproliferation Assay (A549 and H460 Cells): Cells were seeded in 96-well plates at a density of 5×103 cells/well and cultured overnight in RPMI 1640 medium containing 10% fetal bovine serum. Pemetrexed disodium was added at concentrations ranging from 0.01 to 1 μM, and the cells were incubated for 72 hours at 37°C with 5% CO2. After incubation, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, and the plates were incubated for another 4 hours. The supernatant was removed, and 150 μL of dimethyl sulfoxide was added to dissolve the formazan crystals. The absorbance at 570 nm was measured using a microplate reader, and the IC50 was calculated as the drug concentration that inhibited cell viability by 50% [1]
- Annexin V/PI Apoptosis Assay (HT-29 Cells): HT-29 cells were seeded in 6-well plates at 2×105 cells/well and treated with Pemetrexed disodium (0.05, 0.1, 0.2 μM) for 48 hours. Cells were harvested by trypsinization, washed twice with cold PBS, and resuspended in binding buffer. Annexin V-FITC and propidium iodide (PI) were added to the cell suspension, which was then incubated in the dark for 15 minutes at room temperature. The percentage of apoptotic cells (Annexin V-positive/PI-negative and Annexin V-positive/PI-positive) was analyzed using flow cytometry [2]
- Clone Formation Assay (H1299 Cells): H1299 cells were seeded in 6-well plates at 200 cells/well and allowed to attach for 24 hours. Pemetrexed disodium was added at concentrations of 0.05, 0.1, 0.2 μM, and the cells were cultured for 14 days (medium was changed every 3 days). After incubation, colonies were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 30 minutes. Colonies containing more than 50 cells were counted, and the clone formation rate was calculated as (number of colonies in treatment group/number of colonies in control group) × 100% [3]

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.

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A549 Xenograft Model in Nude Mice: Female nude mice (6-8 weeks old, 18-22 g) were subcutaneously injected with 5×106 A549 cells (suspended in 0.2 mL PBS mixed with Matrigel at 1:1 ratio) into the right flank. When tumors reached a volume of 100-150 mm³, mice were randomly divided into two groups (n=6 per group): treatment group and vehicle control group. Pemetrexed disodium was dissolved in 0.9% physiological saline containing 0.1% dimethyl sulfoxide and administered via intravenous injection at a dose of 10 mg/kg twice weekly for 4 weeks. The control group received the same volume of vehicle. Tumor volume was measured every 3 days using a caliper (volume = length × width² / 2), and body weight was recorded weekly to monitor toxicity. At the end of the experiment, mice were euthanized, and tumors were excised for weight measurement and histological analysis [1]
- HT-29 Xenograft Model in Nude Mice: Male nude mice (6-8 weeks old, 20-24 g) were subcutaneously implanted with 1×107 HT-29 cells (in 0.2 mL PBS/Matrigel 1:1) into the left flank. When tumors grew to 200-250 mm³, mice were assigned to two groups (n=5 per group). Pemetrexed disodium was dissolved in 5% glucose solution and administered intravenously at 15 mg/kg once weekly for 3 weeks. The control group received 5% glucose solution. Tumor volume and body weight were measured twice weekly. After treatment, mice were sacrificed, and tumors were collected for immunohistochemistry and metabolite analysis [2]

In nude mice treated with pemetrexed disodium (10 mg/kg, intravenously), plasma concentration-time curves conformed to a two-compartment model. The terminal half-life (t1/2β) was 3.5 ± 0.4 h, and the area under the concentration-time curve (AUC0-∞) was 25.6 ± 3.2 μg·h/mL. Approximately 80% of the administered dose was excreted unchanged in the urine within 24 hours, indicating that renal excretion is the primary route of clearance [1]. In a rat pharmacokinetic study (data cited in this paper), oral administration of pemetrexed disodium (20 mg/kg) resulted in oral bioavailability of less than 10%, due to poor absorption in the gastrointestinal tract. The volume of distribution (Vd) of intravenous administration (10 mg/kg) was 0.3 ± 0.05 L/kg, indicating limited tissue distribution [2].

Effects During Pregnancy and Lactation
◉ Overview of medication use during lactation Most sources suggest that mothers should not breastfeed while receiving high-dose antitumor drugs. Manufacturers recommend that mothers should not breastfeed during pemetrexed treatment and within one week after the last dose. Chemotherapy may adversely affect the normal microbiota and chemical composition of breast milk. [1] Women receiving chemotherapy during pregnancy are more likely to experience breastfeeding difficulties. [2] ◉ Effects on breastfed infants No relevant published information was found as of the revision date. ◉ Effects on lactation and breast milk No relevant published information was found as of the revision date. 28170295 In the A549 xenograft model (nude mice), pemetrexed disodium (10 mg/kg, twice weekly) caused a transient weight loss of 10%–15% during the first two weeks of treatment, but the weight returned to normal at the end of the experiment. Compared with the control group, no significant changes were observed in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), or creatinine (indicators of liver and kidney function) levels [1].
- The plasma protein binding rate of pemetrexed disodium was determined in human plasma in vitro: approximately 80%-85% of the drug was bound to plasma proteins, and the binding was not concentration-dependent (test concentration range: 0.1-10 μM). [2]

[1]. Cancer Res . 1997 Mar 15;57(6):1116-23.

[2]. Int J Cancer . 2013 Jan 15;132(2):459-71.

[3]. Clin Cancer Res . 2000 Mar;6(3):1016-23.

Pemetrexed disodium is an organosodium salt, the disodium salt of N-{4-[2-(2-amino-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic acid. It inhibits thymidine synthase (TS), dihydrofolate reductase (DHFR), and glycine ribonucleotide formyltransferase (GARFT). It has antitumor, antimetabolite, EC 1.5.1.3 (dihydrofolate reductase) inhibitor, EC 2.1.2.2 (phospribosylglycine formyltransferase) inhibitor, and EC 2.1.1.45 (thymidine synthase) inhibitor activities. It contains pemetrexed (2-). Pemetrexed disodium is the disodium salt for synthesizing pyrimidine antifolate. Pemetrexed binds to and inhibits thymidine synthase (TS), an enzyme that catalyzes the methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) to 2'-deoxythymidine-5'-monophosphate (dTMP), a crucial precursor in DNA synthesis. Pemetrexed is a guanine-derived antitumor drug that acts as a nucleic acid synthesis inhibitor by binding to and inhibiting the activity of thymidine synthase. See also: Pemetrexed (broadly defined). Drug Indications Malignant pleural mesothelioma: Pemetrexed Krka in combination with cisplatin is used to treat patients with unresectable malignant pleural mesothelioma who have not undergone chemotherapy. Non-small cell lung cancer: Pemetrexed Krka in combination with cisplatin is indicated as first-line treatment for locally advanced or metastatic non-small cell lung cancer (non-squamous predominant histological type). Pemetrexed Krka is also indicated for maintenance therapy in patients with locally advanced or metastatic non-small cell lung cancer (non-squamous predominant histological type) whose disease has not progressed immediately after platinum-based chemotherapy. Additionally, pemetrexed Krka is indicated as second-line treatment for locally advanced or metastatic non-small cell lung cancer (non-squamous predominant histological type). Malignant pleural mesothelioma: Pemetrexed (Alimta) in combination with cisplatin is indicated for the treatment of patients with unresectable malignant pleural mesothelioma who have not previously received chemotherapy. Non-small cell lung cancer: Pemetrexed in combination with cisplatin is indicated as first-line treatment for patients with locally advanced or metastatic non-small cell lung cancer, excluding squamous predominant histological types. Pemetrexed monotherapy is also indicated for maintenance therapy in patients with locally advanced or metastatic non-small cell lung cancer, excluding squamous predominant histological types, and is indicated for patients whose disease has not progressed immediately after platinum-based chemotherapy. Alimta is indicated as second-line monotherapy for patients with locally advanced or metastatic non-small cell lung cancer (excluding squamous cell carcinoma).
Head and neck cancer (class exemptions cover: oropharyngeal epithelial carcinoma, excluding nasopharyngeal carcinoma), malignant pleural mesothelioma

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Pemetrexed disodium is a multi-target antifolate drug designed to overcome resistance to single-target antifolate drugs (such as methotrexate) by inhibiting multiple enzymes in nucleotide synthesis. The 1997 study was one of the early preclinical studies supporting its development for the treatment of non-small cell lung cancer (NSCLC)[1]
- A 2013 study showed that pemetrexed disodium exhibited higher sensitivity in colorectal cancer cell lines with low TS expression (IC50 0.08 μM), while cell lines with high TS expression did not have this sensitivity (IC50 0.5 μM), suggesting that TS expression may be a potential predictive biomarker for drug response[2]
- A 2000 study indicated that pemetrexed disodium enhanced the apoptotic effect of cisplatin in H1299 cells (combination index <1), suggesting a possible synergistic effect between the two drugs, which supported clinical trials of pemetrexed in combination with cisplatin for the treatment of NSCLC[3]

C20H19N5NA2O6

471.37

471.113

150399-23-8

135413520

Solid powder

160°C 20mm

36-38°C

160°C/20mm

4

7

7

33

737

1

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

NYDXNILOWQXUOF-GXKRWWSZSA-L

InChI=1S/C20H21N5O6.2Na/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);;/q;2*+1/p-2/t13-;;/m0../s1

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

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)

Solubility in Formulation 1: 50 mg/mL (106.07 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: 100 mg/mL (212.15 mM) in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
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 3: Saline: 30 mg/mL

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