DHFR/dihydrofolate reductase; DNA synthesis; antimetabolite; antifolate
The primary target of methotrexate is dihydrofolate reductase (DHFR), an enzyme that catalyzes the reduction of dihydrofolate to tetrahydrofolate, a critical step in the synthesis of purines and pyrimidines. Methotrexate binds to DHFR with very high affinity, acting as a competitive inhibitor. In addition to DHFR, methotrexate also inhibits other enzymes involved in nucleotide synthesis, including thymidylate synthase, aminoimidazole carboxamide ribonucleotide transformylase (AICART), and amidophosphoribosyltransferase. These multiple enzyme inhibitions collectively disrupt DNA synthesis and cell proliferation. The drug's effects on folate metabolism also contribute to its anti-inflammatory properties in autoimmune conditions.

In vitro activity: Methotrexate (0.1-10 mM) induces apoptosis of in vitro activated T cells from human peripheral blood. Methotrexate achieves clonal deletion of activated T cells in mixed lymphocyte reactions. Methotrexate can selectively delete activated peripheral blood T cells by a CD95-independent pathway. Methotrexate is taken up by cells via the reduced folate carrier and then is converted within the cells to polyglutamates. Methotrexate leads to diminished production of leukotriene B4 by neutrophils stimulated ex vivo. Methotrexate polyglutamates inhibit the enzyme aminoimidazolecarboxamidoadenosineribonucleotide (AICAR) transformylase more potently than the other enzymes involved in purine biosynthesis. Methotrexate is also known to suppress TNF activity by suppressing TNF-induced nuclear factor-κB activation in vitro, in part related to a reduction in the degradation and inactivation of an inhibitor of this factor, IκBα, and probably related to the release of adenosine. Methotrexate suppresses the production of both TNF and IFN-γ by T-cell-receptor-primed T lymphocytes from both healthy human donors and RA patients. Methotrexate treatment is associated with a significant decrease of TNF-α-positive CD4+ T cells, while the number of T cells expressing the anti-inflammatory cytokine IL-10 increased.

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For life science-related study, methotrexate (hydrate) is a biochemical reagent that can be utilized as an organic substance or biological material.
In vitro, methotrexate inhibits the growth of various cancer cell lines by inducing cell cycle arrest and apoptosis. It effectively blocks the proliferation of leukemia, lymphoma, and solid tumor cells at nanomolar to micromolar concentrations. The drug's cytotoxicity is mediated through the depletion of reduced folates, leading to impaired nucleotide biosynthesis and DNA damage. Methotrexate also exhibits immunomodulatory effects in vitro, including suppression of T-cell activation and inhibition of pro-inflammatory cytokine production. The compound shows synergistic effects when combined with other chemotherapeutic agents in cell-based assays. Resistance to methotrexate in vitro can arise through decreased drug uptake, increased DHFR expression, or altered folate metabolism.

Amethopterin, or methotrexate, lowers mice's thymus and spleen indices. At doses ≥5 mg/kg, methotrexate significantly reduces splenic, thymic, and white blood cells. The model group and the treatment plus control group, however, vary significantly (p <0.01). It is evident that the administration of grape seed proanthocyanidins along with Siberian ginseng eleutherosides reduces the negative effects of methotrexate on mouse thymus and spleen indices[2]. For five weeks, methotrexate (MTX) (2 mg/kg; i.p. ; once weekly) effectively treats Freund's complete adjuvant-induced arthritis. Curcumin (30 mg/kg and 100 mg/kg, three times a week for five weeks; ip) and methotrexate (1 mg/kg; ip; once in a week for five weeks) together have a strong anti-arthritic effect and guard against hematological toxicity[4].
In vivo, methotrexate demonstrates significant antitumor activity in various xenograft and syngeneic mouse models of cancer, including leukemia, lymphoma, and solid tumors. The drug effectively inhibits tumor growth through its anti-proliferative and pro-apoptotic mechanisms. In animal models of rheumatoid arthritis and other autoimmune diseases, methotrexate reduces inflammation, joint destruction, and disease severity. The compound is typically administered via intraperitoneal, intravenous, or oral routes in preclinical studies. Pharmacodynamic studies in animals show dose-dependent suppression of DHFR activity and nucleotide synthesis in target tissues. Methotrexate's in vivo efficacy is influenced by its pharmacokinetic properties, including tissue distribution and clearance rates.

Methotrexate enters tissues and is converted to a methotrexate polyglutamate by folylpolyglutamate. Methotrexate's mechanism of action is due to its inhibition of enzymes responsible for nucleotide synthesis including dihydrofolate reductase, thymidylate synthase, aminoimidazole caboxamide ribonucleotide transformylase (AICART), and amido phosphoribosyltransferase. Inhibtion of nucleotide synthesis prevents cell division. In rheumatoid arthritis, methotrexate polyglutamates inhibit AICART more than methotrexate. This inhibition leads to accumulation of AICART ribonucleotide, which inhibits adenosine deaminase, leading to an accumulation of adenosine triphosphate and adenosine in the extracellular space, stimulating adenosine receptors, leading to anti-inflammatory action.
The in vitro enzyme assay for methotrexate typically involves measuring the inhibition of dihydrofolate reductase (DHFR) activity. The assay is performed in a buffer system containing NADPH, dihydrofolate, and purified DHFR enzyme. Methotrexate is added at various concentrations, and the reaction is monitored spectrophotometrically by following the decrease in absorbance at 340 nm due to NADPH oxidation. The IC50 value is determined from the concentration-dependent inhibition curve. The assay can also be performed using recombinant DHFR from different species to assess selectivity. Positive controls such as aminopterin are included for comparison. The reaction mixture is incubated at 37 °C for a specified period, and the initial reaction rates are calculated.

Cell Assay: Each cell line is studied in growth inhibition experiments using 96-well microtiter plates. As antifols are schedule dependent, preliminary experiments are aimed at defining the longest duration of exposure that would allow for continuous logarithmic phase growth of cells without changing of the culture media while maintaining a linear relationship between SRB optical density and cell number. Twenty-four hours after cell plating, the cell lines are exposed to the antifol for 120 h (three replicates per experiment). To ensure that a complete sigmoidal survival-concentration curve could be observed, the following drug concentrations are studied: Methotrexate (0.002-5 μM), AMT (0.0001-1 μM), PXD (0.0003-10 μM), TLX (0.0002-0.5 μM). Experiments are repeated at least twice.
In vitro cell-based assays for methotrexate typically use cancer cell lines such as L1210 murine leukemia, HeLa, or human leukemia cell lines. Cells are seeded in 96-well plates and treated with serial dilutions of methotrexate for 48-72 hours. Cell viability is assessed using MTT, CCK-8, or resazurin-based assays to determine the half-maximal inhibitory concentration (IC50). Apoptosis is evaluated by flow cytometry using Annexin V/PI staining or by detecting caspase activation. Cell cycle analysis is performed using propidium iodide staining to assess cell cycle arrest. For combination studies, methotrexate is tested together with other agents to evaluate synergistic or antagonistic effects. All experiments include vehicle controls and are performed in triplicate for statistical reliability.

The combination of bioactive phytochemicals is administered one week prior to the Methotrexate exposure. Treatment group I: mice are given a combination of green tea polyphenols and eleutherosides from Siberian ginseng (0.2 mL/10 g, i.g. once daily) for 15 days, and a single dose of Methotrexate (2 mg/kg, i.p. once daily) is added on the 8th day. Treatment group II: mice are given a combination of grape seed proanthocyanidins and eleutherosides from Siberian ginseng for 15 days, and Methotrexate is administered on the 8th day in a similar manner. Model group: animals received distilled water instead of bioactive phytochemicals combinations for 15 days and the same Methotrexate protocol applied to this group on the 8th day. Control group: mice are given distilled water through 15 days and physiological saline instead of Methotrexate is administered on the 8th day in a similar manner. Twelve hours after the final doses, the animals are euthanized by cervical dislocation.

Absorption, Distribution and Excretion
Methotrexate has a bioavailability of 64-90%, but bioavailability decreases at oral doses exceeding 25 mg due to saturation of methotrexate carrier-mediated transport. The time to peak concentration (Tmax) of methotrexate is 1-2 hours. After an oral dose of 10-15 µg, serum concentrations can reach 0.01-0.1 µM. More than 80% of methotrexate is excreted unchanged, with approximately 3% excreted as a 7-hydroxylated metabolite. Methotrexate is primarily excreted in the urine, with 8.7-26% of intravenously administered doses appearing in the bile. The steady-state volume of distribution of methotrexate is approximately 1 L/kg. The clearance rate of methotrexate varies considerably among patients and decreases with increasing dose. Currently, predicting methotrexate clearance is difficult, and even with all preventative measures, extremely high serum methotrexate concentrations can still occur. In adults, oral absorption of methotrexate appears to be dose-related. Peak serum concentrations are reached within 1 to 2 hours. Methotrexate is generally well absorbed at doses of 30 mg/m² or lower, with an average bioavailability of approximately 60%. Absorption decreases significantly at doses exceeding 80 mg/m², likely due to a saturation effect. Following intravenous administration, the initial volume of distribution is approximately 0.18 L/kg (18% of body weight), and the steady-state volume of distribution is approximately 0.4 to 0.8 L/kg (40% to 80% of body weight). Protein binding: moderate (approximately 50%), primarily bound to albumin. When serum methotrexate concentrations exceed 0.1 μmol/mL, passive diffusion becomes the dominant intracellular transport pathway. The drug is widely distributed throughout the body, with the highest concentrations found in the kidneys, gallbladder, spleen, liver, and skin. For more complete data on the absorption, distribution, and excretion of methotrexate (out of 10), please visit the HSDB records page.

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Metabolic/Metabolic Substances
Methotrexate is metabolized in the liver and tissues by folate polyglutamate synthase to methotrexate polyglutamate. Gamma-glutamyl hydrolase hydrolyzes the glutamate chain of methotrexate polyglutamate, converting it back to methotrexate. Small amounts of methotrexate are also converted to 7-hydroxymethotrexate.
After absorption, methotrexate is metabolized in the liver and intracellularly to produce methotrexate polyglutamate, which can be hydrolyzed back to methotrexate. Methotrexate polyglutamate inhibits dihydrofolate reductase and thymidylate synthase. Small amounts of these polyglutamate metabolites may remain in tissues for extended periods; the retention and persistent effects of these active metabolites vary depending on the cell, tissue, and tumor. In addition, small amounts of methotrexate polyglutamate can be converted into 7-hydroxymethotrexate; because 7-hydroxymethotrexate is three to five times less water-soluble than the parent compound, the accumulation of this metabolite can be quite significant after high doses of methotrexate. After oral administration of methotrexate, the drug is also partially metabolized by the gut microbiota. After absorption, methotrexate is metabolized in the liver and intracellularly to produce methotrexate polyglutamate, which can be hydrolyzed back into methotrexate. Methotrexate polyglutamate inhibits dihydrofolate reductase and thymidylate synthase. Small amounts of these polyglutamate metabolites may remain in tissues for extended periods; the retention and sustained effects of these active metabolites vary depending on the cell, tissue, and tumor. Furthermore, small amounts of methotrexate polyglutamate can be converted into 7-hydroxymethotrexate; because 7-hydroxymethotrexate is three to five times less water-soluble than the parent compound, the accumulation of this metabolite can be quite significant after high doses of methotrexate. Following oral administration of methotrexate, the drug is also partially metabolized by the intestinal flora. Renal excretion is the primary route of elimination, and the amount excreted depends on the dose and route of administration (A620).
Elimination route: Renal excretion is the primary route of elimination, and the amount excreted depends on the dose and route of administration. After intravenous administration, 80% to 90% of the administered dose is excreted unchanged in the urine within 24 hours. Bile excretion is limited, not exceeding 10% of the administered dose.
Half-life: Low dose (below 30 mg/m²): 3 to 10 hours; High dose: 8 to 15 hours.
Biological half-life
The half-life of low-dose methotrexate in adults is 3 to 10 hours. The half-life of high-dose methotrexate is 8 to 15 hours. The terminal half-life in pediatric patients treated with methotrexate for acute lymphoblastic anemia is 0.7 to 5.8 hours. The terminal half-life of methotrexate in pediatric patients treated with juvenile idiopathic arthritis is 0.9 to 2.3 hours. Terminal half-life: Low dose: 3 to 10 hours. High dose: 8 to 15 hours. Note: Clearance varies considerably between individuals. Small amounts of methotrexate and its metabolites bind to proteins and may remain in tissues (kidneys, liver) for weeks to months; fluid overload (such as ascites or pleural effusion) and renal impairment can also delay clearance.
Methotrexate exhibits complex pharmacokinetics with dose-dependent absorption and elimination. Following oral administration, absorption is variable and often incomplete, with bioavailability ranging from 60-80% at low doses but decreasing at higher doses. The drug is extensively distributed into tissues, with high concentrations in the liver, kidneys, and spleen. Methotrexate is primarily excreted unchanged in the urine via glomerular filtration and active tubular secretion, with a terminal half-life of approximately 3-12 hours depending on the dose. The compound undergoes intracellular polyglutamation, which enhances its retention and activity in target cells. Methotrexate shows low protein binding (approximately 50%) and can accumulate in third-space fluids such as pleural effusions.

Toxicity Summary
Methotrexate's antitumor activity is due to the inhibition of folate reductase, thereby inhibiting DNA synthesis and cell replication. Its mechanism of action against rheumatoid arthritis is unclear. Toxicity Data
Humans (intravenous): TD: 740 mg/kg Mouse (intraperitoneal): LD50 mg/kg Rat (oral): LD50 135 mg/kg Rat (intraperitoneal): LD50 6 mg/kg LD50: 43 mg/kg (oral, rat) (A308) Interactions Oral neomycin may decrease the absorption of oral methotrexate. Severe, sometimes fatal, toxicities (including hematologic and gastrointestinal toxicities) have been observed in patients with various malignancies, psoriasis, or inflammatory diseases after concomitant use of nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., indomethacin, ketoprofen) and methotrexate (especially at high doses). Rheumatoid arthritis.
Concomitant use of penicillin-type drugs (e.g., amoxicillin, carbenicillin, meropenem) may reduce the renal clearance of methotrexate, presumably through inhibition of renal tubular secretion of the drug. There have been reports of elevated serum methotrexate concentrations in patients receiving low- or high-dose methotrexate concurrently with penicillin-type drugs, leading to gastrointestinal or hematologic toxicity; therefore, patients receiving both drugs concurrently should be closely monitored.
Concomitant intrathecal administration of methotrexate and acyclovir may cause neurological abnormalities; use with caution.
For more complete data on interactions of methotrexate (16 in total), please visit the HSDB record page.

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Non-human toxicity values
Oral LD50 in rats: 180 ± 45 mg/kg body weight
Intraperitoneal LD50 in rats: 6-25 mg/kg body weight
Intraperitoneal LD50 in mice: 94 ± 9 mg/kg body weight
Methotrexate has a well-characterized toxicity profile. Acute toxicity includes gastrointestinal effects such as nausea, vomiting, and mucositis, as well as bone marrow suppression leading to leukopenia, thrombocytopenia, and anemia. Hepatic toxicity can manifest as elevated liver enzymes and, with chronic use, fibrosis or cirrhosis. Nephrotoxicity occurs due to precipitation of methotrexate and its metabolites in the renal tubules, particularly at high doses. Pulmonary toxicity includes pneumonitis and interstitial lung disease. Neurotoxicity may present as leukoencephalopathy with long-term use. Folic acid or folinic acid rescue is often administered to mitigate toxicity. The drug is contraindicated in pregnancy due to teratogenic effects. All toxicities are dose-dependent and manageable with appropriate monitoring.

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

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

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

[4]. Evaluation of the concomitant use of methotrexate and curcumin on Freund's complete adjuvant-induced arthritis and hematological indices in rats. Indian J Pharmacol. 2011;43(5):546-550.

Therapeutic Uses
Nonsteroidal abortifacients; antimetabolites; antitumor drugs; antirheumatic drugs; dermatological drugs; enzyme inhibitors; folic acid antagonists; immunosuppressants; nucleic acid synthesis inhibitors.
Methotrexate is indicated for the treatment of breast cancer, head and neck cancers (epidermoid carcinoma), non-small cell lung cancer (especially squamous cell carcinoma), small cell lung cancer, and gestational trophoblastic tumors (choriocarcinoma of pregnancy, choriocarcinoma of destruction, hydatidiform mole). /Included on US product label/
Methotrexate is indicated for the treatment of cervical cancer, ovarian cancer, bladder cancer, colorectal cancer, esophageal cancer, gastric cancer, pancreatic cancer, and penile cancer. /Not included on US product label/
Methotrexate is indicated for the treatment of acute lymphoblastic leukemia and for the prevention and treatment of meningeal leukemia. /Included on US product label/
For more complete data on the therapeutic uses of methotrexate (17 types), please visit the HSDB record page.
Drug Warnings
Methotrexate is a highly toxic drug with an extremely low therapeutic index; a therapeutic response is unlikely to occur without an observed toxic reaction. …When methotrexate is used in combination with other anti-tumor drugs and/or radiotherapy, the toxic reactions may be more severe than when methotrexate is used alone. Although the dose of methotrexate used to treat psoriasis and rheumatoid arthritis is usually lower than the dose used for anti-tumor chemotherapy, serious toxic reactions can occur in any patient receiving this drug, and there have been reports of death due to the use of methotrexate in the treatment of psoriasis and rheumatoid arthritis.
Methotrexate should be used with extreme caution in patients with infections, peptic ulcers, ulcerative colitis, or those who are frail, as well as in very young or very old patients. Methotrexate should be used with extreme caution, or even contraindicated, in patients with malignant tumors and liver damage or insufficiency, bone marrow suppression, aplastic anemia, leukopenia, thrombocytopenia, or anemia; this drug is generally contraindicated in patients with renal insufficiency. In the treatment of psoriasis, methotrexate is contraindicated in patients with malnutrition or severe kidney or liver disease, patients with clear or laboratory evidence of immunodeficiency syndrome, and patients with a history of blood disorders (such as myeloproliferation, leukopenia, thrombocytopenia, or clinically significant anemia). Relative contraindications also include cirrhosis, active or recent hepatitis, and excessive alcohol consumption. In the treatment of rheumatoid arthritis, methotrexate is contraindicated in patients with a history of blood disorders (such as myeloproliferation, leukopenia, thrombocytopenia, or clinically significant anemia); patients with clear or laboratory evidence of immunodeficiency syndrome; and those who drink excessively, have alcoholic liver disease, or chronic liver disease. Patients receiving methotrexate treatment may experience elevated serum uric acid levels due to cell destruction and liver and kidney damage. In some patients, uric acid nephropathy and acute renal failure may occur. Tumor lysis syndrome associated with other cytotoxic drugs (such as fludarabine and cladribine) has also been reported in patients with rapidly growing tumors receiving methotrexate treatment. Drug therapy and appropriate supportive care can prevent or mitigate this complication. Methotrexate has also been reported to induce acute gouty arthritis in two patients treated for psoriasis. Large-volume fluid resuscitation, urine alkalization, and/or allopurinol administration may help prevent acute exacerbations of hyperuricemia and uric acid nephropathy. Patients receiving methotrexate may develop severe kidney disease presenting with azotemia, hematuria, and renal failure; deaths have been reported. Autopsy results from one study showed extensive necrosis of renal tubular epithelial cells. Patients with impaired renal function may experience methotrexate accumulation, leading to increased toxicity or further kidney damage. For more complete data on methotrexate (22 in total), please visit the HSDB records page. Pharmacodynamics: Methotrexate inhibits nucleotide synthases, thereby preventing cell division and exerting an anti-inflammatory effect. It has a long duration of action and is usually administered once weekly. Methotrexate has a narrow therapeutic index and should not be taken daily.
Methotrexate was first introduced clinically in the 1950s and remains a cornerstone in the treatment of multiple malignancies and autoimmune disorders. The drug is approved by the FDA and EMA for acute lymphoblastic leukemia, non-Hodgkin lymphoma, osteosarcoma, breast cancer, and head and neck cancer, as well as for rheumatoid arthritis, psoriasis, and Crohn's disease. The mechanism of action involves not only DHFR inhibition but also the accumulation of intracellular adenosine, which contributes to its anti-inflammatory effects. Methotrexate is available in oral, intravenous, intramuscular, and intrathecal formulations. Resistance mechanisms include impaired drug transport, increased DHFR expression, and decreased polyglutamation. The drug is often used in combination with other chemotherapeutics in multi-agent regimens.

C20H22N8O5.XH2O

472.45456

454.171

C, 50.84; H, 5.12; N, 23.72; O, 20.32

133073-73-1

Methotrexate;59-05-2;Methotrexate disodium;7413-34-5;Methotrexate monohydrate;6745-93-3; Methotrexate disodium;7413-34-5;Methotrexate hydrate;133073-73-1; Methotrexate-d3; 432545-63-6; 7532-09-4 (monosodium); 15475-56-6 (sodium)

126941

Yellow to orange solid powder

1.536g/cm3

195ºC

0mmHg at 25°C

1.821

5

12

9

33

704

1

CN(CC1=CN=C2C(=N1)C(=NC(=N2)N)N)C3=CC=C(C=C3)C(=O)N[C@@H](CCC(=O)O)C(=O)O

FBOZXECLQNJBKD-ZDUSSCGKSA-N

InChI=1S/C20H22N8O5/c1-28(9-11-8-23-17-15(24-11)16(21)26-20(22)27-17)12-4-2-10(3-5-12)18(31)25-13(19(32)33)6-7-14(29)30/h2-5,8,13H,6-7,9H2,1H3,(H,25,31)(H,29,30)(H,32,33)(H4,21,22,23,26,27)/t13-/m0/s1

(2S)-2-[[4-[(2,4-diaminopteridin-6-yl)methyl-methylamino]benzoyl]amino]pentanedioic acid

Methotrexate hydrate; Methotrexate monohydrate; Methotrexate hydrate(1:x); 133073-73-1; 6745-93-3; Methotrexate (monohydrate); 84DMZ3IHO0; (2S)-2-[[4-[(2,4-diaminopteridin-6-yl)methyl-methylamino]benzoyl]amino]pentanedioic acid;hydrate;

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~25 mg/mL

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.)