MTX/methotrexate analog; DHFR/dihydrofolate reductase; anticancer
The compound is primarily an intermediate or a prodrug version of methotrexate; its ultimate target, after deprotection, is dihydrofolate reductase (DHFR). Methotrexate is a potent inhibitor of DHFR, an enzyme essential for the synthesis of tetrahydrofolate and, consequently, for DNA biosynthesis. The OtBu protection of the alpha-carboxyl group masks this critical functionality and reduces the compound's direct affinity for the DHFR enzyme in vitro.

Compared to MTX-PVG, OtBu-MTX-PVG has a smaller HT1080 effect on HT1080 cells. According to the findings, there is less activity in vitro when the MTX α-carboxyl group is protected [1].

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Compared to the native MTX-PVG dendrimer conjugate, the OtBu-MTX-PVG conjugate shows lower in vitro cytotoxicity against HT1080 cells. This demonstrates that the protection of the alpha-carboxyl group of MTX results in reduced direct in vitro activity. However, when conjugated via a cleavable linker, the OtBu-protected MTX retains its activity after deprotection, confirming its utility as a cleavable, pro-moiety in drug delivery systems.

Lys-MTXcleavable- (OtBu) effectively inhibits tumor growth in HT1080 tumor-bearing mice [1]. Protection of the α-carboxyl group of methotrexate can be employed to improve circulation half-life and prevent liver accumulation of comparable MTX-conjugated dendrimers while still keeping anticancer efficacy in vivo [1].

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Unlike the native drug, the OtBu-protected methotrexate analog has been designed to improve systemic exposure and reduce off-target accumulation. A dendrimer conjugate of this protected MTX significantly reduces tumor growth in HT1080 tumor-bearing mice. The protection of the alpha-carboxyl group enhances the circulatory half-life and reduces liver accumulation of the conjugated construct while still releasing the active drug to exert antitumor activity in vivo.

A typical biochemical DHFR inhibition assay is performed with the fully deprotected methotrexate, not its tert-butyl ester. The binding affinity is assessed by incubating purified DHFR enzyme with the reduced cofactor NADPH and varying concentrations of methotrexate to monitor the decrease in absorbance at 340 nm (deltaA340) as the NADPH is consumed. The Ki is determined from the competition with the substrate dihydrofolate.

A cellular assay can be used to compare the activity of MTX and its OtBu-protected version. HT1080 fibrosarcoma cells are seeded in a 96-well plate and treated with varying concentrations of methotrexate, OtBu-MTX, or their dendrimer conjugates for 72 hours. Cell viability is then determined using a standard MTT or CCK-8 assay to calculate IC50 values and assess the impact of the OtBu protection on cellular potency.

Animal/Disease Models: Female, Balb/c nu/nu, 6 weeks (carrying HT1080 cells) [1]
Doses: 30 mg/kg
Route of Administration: Once a week for 2 weeks
Experimental Results: Demonstrated significant tumor size (63%) Decrease day 12.

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A generation 5 PEGylated (PEG 1100) polylysine dendrimer, conjugated via a stable amide linker to OtBu protected methotrexate (MTX), was previously shown to have a circulatory half-life of 2 days and to target solid tumors in both rats and mice. Here, we show that deprotection of MTX and substitution of the stable linker with a matrix metalloproteinase (MMP) 2 and 9 cleavable linker (PVGLIG) dramatically increased plasma clearance and promoted deposition in the liver and spleen (50-80% of the dose recovered in the liver 3 days post dose). Similar rapid clearance was also seen using a scrambled peptide suggesting that clearance was not dependent on the cleavable nature of the linker. Surprisingly, dendrimers where OtBu capped MTX was linked to the dendrimer surface via the hexapeptide linker showed equivalent in vitro cytotoxicity against HT1080 cells when compared to the uncapped dendrimer and also retained the long circulating characteristics of the stable constructs. The OtBu capped MTX conjugated dendrimer was subsequently shown to significantly reduce tumor growth in HT1080 tumor bearing mice compared to control. In contrast the equivalent dendrimer comprising uncapped MTX conjugated to the dendrimer via the same hexapeptide linker did not reduce tumor growth, presumably reflecting very rapid clearance of the construct. The results are consistent with the suggestion that protection of the α-carboxyl group of methotrexate may be used to improve the circulatory half-life and reduce the liver accumulation of similar MTX-conjugated dendrimers, while still retaining antitumor activity in vivo.[1]

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The in vivo efficacy of a dendrimer conjugate bearing the OtBu-protected MTX is evaluated in an HT1080 tumor-bearing mouse model. Female Balb/c nu/nu mice (6 weeks old) bearing established HT1080 xenografts (approximately 100 mm3) are administered the MTX-OtBu conjugate intravenously at a dose of 30 mg/kg once per week for two weeks. Tumor volume is measured with calipers three times per week. At the end of the study (e.g., day 12), a significant (63%) reduction in tumor size is observed.

Protection of the alpha-carboxyl group of methotrexate as a tert-butyl ester significantly improves its pharmacokinetic properties when conjugated to a drug carrier. A dendrimer conjugate of OtBu-MTX was shown to have a long circulatory half-life of approximately 2 days in rats and mice, a dramatic improvement over the native, unprotected drug. This protection prevents rapid renal clearance and liver accumulation, allowing the drug carrier to effectively target solid tumors via the enhanced permeability and retention (EPR) effect.

The methotrexate alpha-tert-butyl ester is a chemical intermediate and is not itself a toxic therapeutic; its toxicity is inherent to the active drug methotrexate. Unprotected MTX is cytotoxic with known toxicities including myelosuppression, hepatotoxicity, and nephrotoxicity. The purpose of this protected analog is to reduce these systemic toxicities by masking the active drug until it reaches the target site, thereby providing a wider therapeutic window.

[1]. Capping methotrexate α-carboxyl groups enhances systemic exposure and retains the cytotoxicity of drug conjugated PEGylated polylysine dendrimers. Mol Pharm. 2011;8(2):338-349.

[2]. Understanding the mechanisms of action of methotrexate: implications for the treatment of rheumatoid arthritis. Bull NYU Hosp Jt Dis. 2007;65(3):168-73.

[3]. Methotrexate in rheumatoid arthritis. Pharmacol Rep. 2006 Jul-Aug;58(4):473-92.

[4]. The Effect of L-carnitine on Amethopterin-induced Toxicity in Rat Large Intestine.

Multiple disease-modifying antirheumatic drugs (DMARDs) can be used to control clinical activity in rheumatoid arthritis (RA). Methotrexate (MTX) is an analog of folic acid and aminopterin and is the most commonly used DMARD, currently being used to treat at least 500,000 RA patients worldwide. The mechanism by which low-dose MTX modulates inflammation in RA is unclear. Monitoring MTX concentrations in RA patients does not appear to have a significant effect on treatment efficacy. Two meta-analyses have shown that MTX has one of the best efficacy/toxicity ratios. It should currently be the first-line DMARD for most RA patients. However, a significant proportion of patients treated with MTX alone fail to achieve optimal disease control, so there are currently multiple DMARD combination therapy options. It is hoped that more aggressive use of traditional DMARDs and biologics can reduce disability and improve remission rates. The treatment of rheumatoid arthritis (RA) is a dynamic process that requires a delicate balance between benefits and risks. Even with the availability of new biologics, methotrexate (MTX) remains the standard of reference and continues to play a role in the treatment of RA patients. [3] Methotrexate has been widely used to treat rheumatoid arthritis (RA). Methotrexate has a complex mechanism of action. As a folic acid analogue, methotrexate inhibits the synthesis of purines and pyrimidines, which explains its efficacy in cancer treatment as well as some of its toxicities. In recent years, many studies have focused on the adenosine-mediated anti-inflammatory effects of methotrexate. Some aspects of methotrexate toxicity are also attributed to the release of adenosine. A better understanding of the mechanism of action and toxicity of methotrexate will guide clinicians in developing treatment plans and conducting toxicity monitoring. To this end, this article discusses the latest advances in the pharmacokinetics, mechanism of action, pharmacogenetics and toxicity of methotrexate. [2]

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Methotrexate alpha-tert-butyl ester is a research-grade chemical used in the study of drug delivery systems, specifically in the design of polymeric prodrugs. By capping the alpha-carboxyl group, researchers have been able to improve the systemic circulation and tumor targeting of MTX-conjugated nanocarriers. As an intermediate or protected analog of an approved drug, it is not a clinical agent itself. It is used to develop novel, cleavable prodrugs for targeted cancer chemotherapy.

C24H30N8O5

510.55

510.234

79640-70-3

12188936

Light yellow to yellow solid powder

3.078

4

12

11

37

801

1

CC(C)(C)OC(=O)[C@H](CCC(=O)O)NC(=O)C1=CC=C(C=C1)N(C)CC2=CN=C3C(=N2)C(=NC(=N3)N)N

HTMMDBDGHQUHPW-INIZCTEOSA-N

InChI=1S/C24H30N8O5/c1-24(2,3)37-22(36)16(9-10-17(33)34)29-21(35)13-5-7-15(8-6-13)32(4)12-14-11-27-20-18(28-14)19(25)30-23(26)31-20/h5-8,11,16H,9-10,12H2,1-4H3,(H,29,35)(H,33,34)(H4,25,26,27,30,31)/t16-/m0/s1

(4S)-4-[[4-[(2,4-diaminopteridin-6-yl)methyl-methylamino]benzoyl]amino]-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid

Methotrexate alpha-tert-butyl ester; 79640-70-3; Methotrexate a-tert-Butyl Ester; Methotrexatealpha-tert-ButylEster; Methotrexate ; A-tert-butyl ester; (S)-5-(tert-butoxy)-4-(4-(((2,4-diaminopteridin-6-yl)methyl)(methyl)amino)benzamido)-5-oxopentanoic acid; N-[4-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic Acid 1-(1,1-Dimethylethyl) Ester;

2934.99.9001

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)