TS (Ki = 1.3 nM); DHFR (IC50 = 7.2 nM); GARFT (IC50 = 65 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.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Most sources consider breastfeeding to be contraindicated during maternal high-dose antineoplastic drug therapy. The manufacturer recommends that mothers should not to breastfeed during treatment with pemetrexed and for one week after the last dose. Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk.[1] Women who receive chemotherapy during pregnancy are more likely to have difficulty nursing their infant.[2]
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.
28170295

[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.

Pemetrexed disodium heptahydrate is an organic sodium salt that is the heptahydrate form of pemetrexed disodium. Inhibits thymidylate synthase (TS), 421 dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). It has a role as an antimetabolite, an antineoplastic agent, an EC 1.5.1.3 (dihydrofolate reductase) inhibitor, an EC 2.1.1.45 (thymidylate synthase) inhibitor and an EC 2.1.2.2 (phosphoribosylglycinamide formyltransferase) inhibitor. It is an organic sodium salt and a hydrate. It contains a pemetrexed disodium.
A guanine-derived ANTINEOPLASTIC AGENT that functions as a NUCLEIC ACID SYNTHESIS INHIBITOR through its binding to, and inhibition of, THYMIDYLATE SYNTHASE.
See also: Pemetrexed (has active moiety).

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Drug Indication
Malignant pleural mesotheliomaPemetrexed Baxter in combination with cisplatin is indicated for the treatment of chemotherapy naïve patients with unresectable malignant pleural mesothelioma. Non-small cell lung cancer Pemetrexed Baxter in combination with cisplatin is indicated for the first line treatment of patients with locally advanced or metastatic non-small cell lung cancer other than predominantly squamous cell histology (see section 5. 1). Pemetrexed Baxter is indicated as monotherapy for the maintenance treatment of locally advanced or metastatic non-small cell lung cancer other than predominantly squamous cell histology in patients whose disease has not progressed immediately following platinum-based chemotherapy (see section 5. 1). Pemetrexed Baxter is indicated as monotherapy for the second line treatment of patients with locally advanced or metastatic non-small cell lung cancer other than predominantly squamous cell histology (see section 5. 1).
Malignant pleural mesotheliomaAlimta in combination with cisplatin is indicated for the treatment of chemotherapy-naïve patients with unresectable malignant pleural mesothelioma. Non-small-cell lung cancer Alimta in combination with cisplatin is indicated for the first-line treatment of patients with locally advanced or metastatic non-small-cell lung cancer other than predominantly squamous cell histology. Alimta is indicated as monotherapy for the maintenance treatment of locally advanced or metastatic non-small-cell lung cancer other than predominantly squamous cell histology in patients whose disease has not progressed immediately following platinum-based chemotherapy. Alimta is indicated as monotherapy for the second line treatment of patients with locally advanced or metastatic non-small-cell lung cancer other than predominantly squamous cell histology.

C20H33N5NA2O13

597.49

597.187

C, 40.21; H, 5.57; N, 11.72; Na, 7.70; O, 34.81

357166-29-1

Pemetrexed;137281-23-3;Pemetrexed disodium;150399-23-8

135564751

white solid powder

11

14

7

40

737

1

[Na+].[Na+].O=C1C2=C(N=C(N([H])[H])N1[H])N([H])C([H])=C2C([H])([H])C([H])([H])C1C([H])=C([H])C(C(N([H])[C@]([H])(C(=O)[O-])C([H])([H])C([H])([H])C(=O)[O-])=O)=C([H])C=1[H].O([H])[H].O([H])[H].O([H])[H].O([H])[H].O([H])[H].O([H])[H].O([H])[H]

QJVSMHJWAOSBMD-MYXYZBIASA-L

InChI=1S/C20H21N5O6.2Na.7H2O/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);;;7*1H2/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;heptahydrate

LY231514; LY-231514; HSDB 7316; LY 231514; HSDB7316; HSDB-7316; Pemetrexed; trade name: ALIMTA; MTA

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: ~100 mg/mL (~166.8 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.)