Aims: Nucleotide depletion induced by the immunosuppressant mycophenolate mofetil (MMF) has been shown to exert neuroprotective effects. It remains unclear whether nucleotide depletion directly counteracts neuronal demise or whether it inhibits microglial or astrocytic activation, thereby resulting in indirect neuroprotection.
Methods: Effects of MMF on isolated microglial cells, astrocyte/microglial cell co-cultures and isolated hippocampal neurones were analysed by immunocytochemistry, quantitative morphometry, and elisa.
Results: We found that: (i) MMF suppressed lipopolysaccharide-induced microglial secretion of interleukin-1β, tumour necrosis factor-α and nitric oxide; (ii) MMF suppressed lipopolysaccharide-induced astrocytic production of tumour necrosis factor-α but not of nitric oxide; (iii) MMF strongly inhibited proliferation of both microglial cells and astrocytes; (iv) MMF did not protect isolated hippocampal neurones from excitotoxic injury; and (v) effects of MMF on glial cells were reversed after treatment with guanosine.
Conclusions: Nucleotide depletion induced by MMF inhibits microglial and astrocytic activation. Microglial and astrocytic proliferation is suppressed by MMF-induced inhibition of the salvage pathway enzyme inosine monophosphate dehydrogenase. The previously observed neuroprotection after MMF treatment seems to be indirectly mediated, making this compound an interesting immunosuppressant in the treatment of acute central nervous system lesions [2].

Background: T lymphocytes induce the transformation of fibroblasts into myofibroblasts, the main mediators of fibrogenesis. The inosine 5'-monophosphate dehydrogenase inhibitor mycophenolate mofetil (MMF) and the anti-CD25 monoclonal antibody daclizumab (DCZ) have been reported to suppress the proliferation of T lymphocytes.
Aim: To evaluate the preventive effects of MMF and DCZ in early stages of bleomycin (BLM)-induced scleroderma.
Methods: This study involved five groups of Balb/c mice (n = 10 per group). Mice in four of the groups were injected subcutaneously (SC) with BLM [100 μg/day in 100 μL phosphate-buffered saline (PBS)] for 4 weeks; the remaining (control) group received only 100 μL PBS. Three of the BLM-treated groups also received either intraperitoneal MMF 50 or 150 mg/kg/day, or SC DCZ 100 μg/week. At the end of the fourth week, all mice were killed, and blood and tissue samples were obtained for further analysis.
Results: In the BLM-treated group, increases were seen in inflammatory-cell infiltration, α-smooth muscle actin-positive (α-SMA+) fibroblastic cell count, tissue hydroxyproline content, and dermal thickness. Dermal fibrosis was histopathologically prominent. In BLM-treated mice also given MMF or DCZ, inflammatory-cell infiltration, tissue hydroxyproline content and dermal thickness were decreased. In the MMF groups, decreases were also noted in α-SMA+ fibroblastic cell count.
Conclusion: In this BLM-induced dermal fibrosis model, MMF and DCZ treatments prevented the development of dermal fibrosis. Further studies are needed to evaluate whether targeting T lymphocytes is effective in resolving pre-existing fibrosis in human scleroderma.[3]

Analyses of microglial and astrocytic apoptosis and proliferation [2]
Microglial cells or astrocytes treated with Mycophenolate MofetilMMF or incubated in culture medium were used to determine the possible toxic concentration range of Mycophenolate Mofetil/MMF. Apoptotic cells were visualized by immunocytochemical demonstration of activated caspase-3. Proliferation studies on microglial cells were performed in astrocyte/microglial cell co-cultures because significant microglial proliferation was only obtained in the presence of astrocytes. Stimulation of isolated microglial cells with LPS or macrophage-colony stimulating factor alone did not induce significant proliferative activity (data not shown). To analyse proliferation of microglial cells bromo-desoxy-uridine (BrdU; 0.01 mM) was added to the culture medium for 16 h prior to fixation. Foetal calf serum (1–10%) or corticotrophin releasing factor (CRF, 10 µM) for 48 h were used to stimulate proliferation in isolated astrocyte cultures, and BrdU (0.01 mM) was added 16 h prior to fixation. The proliferation index was calculated as the percentage of proliferating cells related to the total number of cells.

Animals and experimental protocols [3]
Fifty specific‐pathogen‐free female Balb/c mice, 6 weeks old and weighing 20–25 g, were used for the experimental procedures. Defined areas of the lower back skins of the mice were shaved for subcutaneous injections. Mice in the control group received 100 μL/day phosphate‐buffered saline (PBS) subcutaneously (SC) to the shaved back skin. To induce dermal fibrosis, the remaining four groups received BLM 100 μg dissolved in 100 μL PBS and sterilized by filtration (0.2 μm filter) to the shaved skin on the back. Two groups of these BLM‐treated mice were also injected either intraperitoneally with MMF 50 or 150 mg/kg/day dissolved in 100 μL and 300 μL saline containing 0.4% Tween 80 and 0.9% benzyl alcohol, respectively, and a third BLM‐treated group was given DCZ 100 μg (100 μL) SC once weekly.

Absorption, Distribution and Excretion
Mycophenolate mofetil is rapidly absorbed in the small intestine. Peak plasma concentrations of its active metabolite, mycophenolate mofetil (MPA), are reached 60 to 90 minutes after oral administration. A pharmacokinetic study in 12 healthy subjects showed a mean bioavailability of 94% for orally administered mycophenolate mofetil. In healthy volunteers, the peak plasma concentration (Cmax) of mycophenolate mofetil was 24.5 (±9.5) μg/mL. In kidney transplant recipients, Cmax was 12.0 (±3.82) μg/mL 5 days post-transplantation, increasing to 24.1 (±12.1) μg/mL 3 months post-transplantation. The AUC value after a single dose in healthy volunteers was 63.9 (±16.2) μg•h/mL, and the AUC values at 5 days and 3 months after kidney transplantation were 40.8 (±11.4) μg•h/mL and 65.3 (±35.4) μg•h/mL, respectively. Food does not affect the absorption of mycophenolate mofetil. A small amount of the drug is excreted in the urine as MPA (less than 1%). A pharmacokinetic study showed that after oral administration of mycophenolate mofetil, 93% was excreted in the urine and 6% in the feces. Approximately 87% of the administered dose is excreted in the urine as the inactive metabolite MPAG. The volume of distribution of mycophenolate mofetil is 3.6 (±1.5) to 4.0 (±1.2) L/kg. The plasma clearance of oral mycophenolate mofetil is 193 mL/min, and that after intravenous administration is 177 (±31) mL/min.
Absorption is rapid and extensive after oral administration.
In 12 healthy volunteers, the mean absolute bioavailability (based on MPA AUC) of oral mycophenolate mofetil relative to intravenous mycophenolate mofetil was 94%. In kidney transplant patients receiving multiple mycophenolate mofetil treatments, the area under the plasma concentration-time curve (AUC) of MPA appeared to increase in a dose-proportional manner, up to a maximum daily dose of 3 g.
Protein binding: High binding to plasma albumin (97% binding of MPA to plasma albumin within the concentration range typically observed in stable kidney transplant patients). At higher mycophenolate glucuronide (MPAG) concentrations (e.g., in patients with impaired renal function or delayed recovery of transplanted kidney function), MPA binding may be reduced due to competition for binding sites between MPA and MPAG. In 12 healthy volunteers, the mean (± standard deviation) apparent volume of distribution of MPA after intravenous and oral administration was approximately 3.6 (±1.5) L/kg and 4.0 (±1.2) L/kg, respectively. At clinically relevant concentrations, MPA bound to plasma albumin was 97%. Within the range of MPAG concentrations typically observed in stable renal transplant patients, MPAG bound to plasma albumin was 82%; however, at higher MPAG concentrations (seen in patients with impaired renal function or delayed recovery of renal transplant function), MPA binding may be reduced due to competition for protein binding sites between MPAG and MPA. The mean plasma radioactivity ratio was approximately 0.6, indicating that MPA and MPAG are not widely distributed into the cellular components of the blood. For more complete data on the absorption, distribution, and excretion of mycophenolate mofetil (9 in total), please visit the HSDB record page.

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Metabolism/Metabolites
After oral and intravenous administration, mycophenolate mofetil is completely metabolized by hepatic carboxylesterases 1 and 2 to the active parent drug mycophenolic acid (MPA). Subsequently, it is metabolized by glucuronyltransferase to produce the inactive MPA phenol glucuronide (MPAG). This glucuronide metabolite is important because it is subsequently converted to MPA via enterohepatic circulation. Mycophenolate mofetil (MMF), which is not metabolized in the intestine, enters the liver via the portal vein and is converted to pharmacologically active MPA within hepatocytes. N-(2-carboxymethyl)-morpholine, N-(2-hydroxyethyl)-morpholine, and their N-oxide moieties are other metabolites produced by the metabolism of MMF in the intestine by hepatic carboxylesterase 2. UGT1A9 and UGT2B7 in the liver are the main enzymes for MPA metabolism; in addition, other UGT enzymes are involved in MPA metabolism. The four major metabolites of mycophenolic acid (MPA) are: 7-O-MPA-β-glucuronide (MPAG, inactive), MPA acylglucuronide (AcMPAG) produced by uridine diphosphate glucuronyltransferase (UGT), 7-O-MPA glucoside produced by UGT, and a small amount of 6-O-demethyl-MPA (DM-MPA) produced by CYP3A4/5 and CYP2C8 enzymes. After oral and intravenous administration, mycophenolic esters are completely metabolized to the active metabolite MPA (mycophenolic acid). After oral administration, MPA metabolism occurs in the first-pass phase. MPA is mainly metabolized by glucuronyltransferase to produce the pharmacologically inactive MPA phenolglucuronide (MPAG). In vivo, MPAG is converted to MPA via enterohepatic circulation. Following oral administration of mycophenolate moiety to healthy subjects, the following metabolites of the 2-hydroxyethylmorpholine moiety were also detected in urine: N-(2-carboxymethyl)-morpholine, N-(2-hydroxyethyl)-morpholine, and N-oxide of N-(2-hydroxyethyl)-morpholine.
Biological half-life
The mean apparent half-life of mycophenolate moiety after oral administration was 17.9 (±6.5) hours, and after intravenous administration it was 16.6 (±5.8) hours.
Mycophenolic acid (MPA): Mean apparent half-life: approximately 17.9 hours after oral administration and approximately 16.6 hours after intravenous administration.
The mean (±SD) apparent half-life and plasma clearance of MPA after oral administration were 17.9 (±6.5) hours and 193 (±48) mL/min, respectively. After intravenous administration, its absorption time was 16.6 (±5.8) hours and the flow rate was 177 (±31) mL/min.

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Pharmacokinetic characteristics[2]
Mycophenolate mofetil is well absorbed after oral administration and is rapidly converted to the active metabolite mycophenolic acid. The area under the plasma concentration-time curve (AUC) is generally proportional to the dose; however, there are some differences in AUC values among different patients. The AUC and peak plasma concentration (Cmax) of mycophenolic acid in patients with stable renal transplantation (>3 months post-transplantation) are about 50% higher than those in patients in the early post-transplantation period.
Mycophenolic acid is mainly excreted in the urine as mycophenolic acid glucuronide (about 87%); 6% is excreted in the feces. After oral administration, the mean apparent half-life and plasma clearance of mycophenolic acid are 17.9 hours and 11.6 L/h, respectively.
After a single dose, the AUC of mycophenolic acid and its glucuronide metabolites was higher in patients with renal impairment than in patients with normal renal function. However, the pharmacokinetics of mycophenolic acid esters were not altered in patients with cirrhosis after a single dose. Data in children are limited, but the AUC and Cmax of mycophenolic acid appear to increase with age.

Protein Binding
Mycophenolic acid (a metabolite of mycophenolic acid esters) has a protein binding rate of 97%, primarily binding to albumin. Its inactive metabolite, MPAG, binds to plasma albumin at normal therapeutic concentrations at 82%. When MPAG concentrations are elevated due to various reasons such as renal impairment, the protein binding rate of MPA may decrease due to competitive binding. Mycophenolic acid esters (morpholinoethyl ester of mycophenolic acid) inhibit de novo purine synthesis by inhibiting inosine monophosphate dehydrogenase. Its selective lymphocyte antiproliferative effect involves T cells and B cells, preventing antibody formation. Mycophenolic acid esters have immunosuppressive effects when used alone, but are most commonly used in combination with other immunosuppressants. Mycophenolic acid esters in combination with cyclosporine and glucocorticoids have been studied in large randomized clinical trials involving nearly 1500 kidney transplant recipients. These trials showed that mycophenolic acid esters were significantly more effective than placebo or azathioprine in reducing treatment failure rates and the incidence of acute rejection. Furthermore, mycophenolate mofetil may help reduce the incidence of chronic rejection. Mycophenolate mofetil is relatively well tolerated. The most common adverse reaction is gastrointestinal intolerance; hematological abnormalities have also been observed. The reversible cytosolic effect of mycophenolate mofetil allows for dose adjustment or discontinuation, thus avoiding serious toxicity or excessive suppression of the immune system. The occurrence of cytomegalovirus-induced tissue invasive disease and malignancy is an issue that needs to be evaluated through long-term follow-up studies. Mycophenolate mofetil does not cause the side effects common to other commercially available immunosuppressants, such as nephrotoxicity, hepatotoxicity, hypertension, neurological disorders, electrolyte disturbances, skin diseases, hyperglycemia, hyperuricemia, hypercholesterolemia, dyslipidemia, and bone loss. Based on preliminary information, the benefit-risk ratio of mycophenolate mofetil for preventing rejection in deceased kidney transplants has been confirmed to be positive. Data from studies on other types of organ transplantation are encouraging, but limited in number and insufficient to draw definitive conclusions. Long-term follow-up studies are needed to confirm these observations. Despite its high price, mycophenolate mofetil's benefits in reducing rejection, treatment failure, and associated costs suggest it may be cost-effective. [1]

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Tolerability [2]
The incidence of adverse events associated with mycophenolate mofetil appears to be dose-related: 2 g/day is generally better tolerated than 3 g/day. The most common adverse events include gastrointestinal adverse events (diarrhea, vomiting), hematologic and lymphatic adverse events (leukopenia, anemia), and infectious adverse events (sepsis, opportunistic infections). Patients receiving mycophenolate mofetil had a slightly higher incidence of diarrhea and sepsis (most commonly cytomegalovirus viremia) compared to those receiving azathioprine. The proportion of patients receiving 3 g/day of mycophenolate mofetil also increased compared to those receiving azathioprine. The overall risk of malignant tumors associated with mycophenolate mofetil is similar to that of azathioprine.

[1]. Effect of the inosine 5'-monophosphate dehydrogenase inhibitor BMS-566419 on rat cardiac allograft rejection. Int Immunopharmacol, 2010. 10(1): p. 91-7.

[2]. Inhibition of microglial and astrocytic inflammatory responses by the immunosuppressant mycophenolate mofetil. Neuropathol Appl Neurobiol, 2010. 36(7): p. 598-611.

[3]. Mycophenolate mofetil and daclizumab targeting T lymphocytes in bleomycin-induced experimental scleroderma. Clin Exp Dermatol, 2012. 37(1): p. 48-54.

Mycophenolic acid is a compound derived from Penicillium stoloniferum and its close relatives. It blocks the de novo synthesis of purine nucleotides by inhibiting inosine monophosphate dehydrogenase (IMP dehydrogenase). Mycophenolic acid has selective effects on the immune system, inhibiting the proliferation of T cells and lymphocytes and antibody production by B cells. It may also inhibit the recruitment of leukocytes to sites of inflammation.
See also: Mycophenolic acid (with the active moiety); Mycophenolic esters (its salt form).

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In summary, our results indicate that mycophenolic esters: (i) inhibit the secretion of TNF-α, IL-1β, and NO by microglia; (ii) inhibit the secretion of TNF-α by astrocytes; (iii) inhibit the proliferation of microglia and astrocytes; (iv) have no direct neuroprotective effect on cultured, excitotoxically damaged hippocampal neurons; and (v) exert their effects by inhibiting glial IMPDH. Given the encouraging results from animal studies and open-label clinical trials of MMF in various central nervous system diseases, MMF appears to be a promising candidate for further research into the treatment of acute brain and spinal cord diseases. [2] Both MMF and DCZ target T lymphocytes and inhibit inflammatory activity, thereby preventing the development of early skin fibrosis in a bleomycin (BLM)-induced mouse scleroderma model. However, MMF is more effective than DCZ in inhibiting fibroblast activation and has a more significant anti-fibrotic effect. These results support the hypothesis that T lymphocytes play an important role in the pathogenesis of scleroderma and that inhibiting T lymphocytes in the early stages of the disease may be an effective strategy for treating human scleroderma. However, targeting T lymphocytes alone may not be a sufficient approach to treating scleroderma. [3]

C23H32CLNO7

469.96

469.187

C, 58.78; H, 6.86; Cl, 7.54; N, 2.98; O, 23.83

116680-01-4

Mycophenolate Mofetil;128794-94-5

6441022

White to off-white solid powder

1.222g/cm3

637.6ºC at 760mmHg

339.4ºC

7.51E-17mmHg at 25°C

1.557

3.263

2

8

10

32

646

0

O=C(OCCN1CCOCC1)CC/C(C)=C/CC2=C(O)C3=C(COC3=O)C(C)=C2OC.[H]Cl

OWLCGJBUTJXNOF-HDNKIUSMSA-N

InChI=1S/C23H31NO7.ClH/c1-15(5-7-19(25)30-13-10-24-8-11-29-12-9-24)4-6-17-21(26)20-18(14-31-23(20)27)16(2)22(17)28-3;/h4,26H,5-14H2,1-3H3;1H/b15-4+;

2-morpholin-4-ylethyl (E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1H-2-benzofuran-5-yl)-4-methylhex-4-enoate;hydrochloride

Mycophenolate mofetil hydrochloride; 116680-01-4; Mycophenolate mofetil HCl; UNII-UXH81S8ZVB; UXH81S8ZVB; RS 61443-190; 2-(4-Morpholinyl)ethyl ester (E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoic acid, hydrochloride; 2-Morpholinoethyl (E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-5-phthalanyl)-4-methyl-4-hexenoate hydrochloride;

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.)