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. The maximum concentration of its active metabolite, MPA, is attained 60 to 90 minutes following an oral dose. The average bioavailability of orally administered mycophenolate mofetil in a pharmacokinetic study of 12 healthy patients was 94%. In healthy volunteers, the Cmax of mycophenolate mofetil was 24.5 (±9.5)μg/mL. In renal transplant patients 5 days post-transplant, Cmax was 12.0 (±3.82) μg/mL, increasing to 24.1 (±12.1)μg/mL 3 months after transplantation. AUC values were 63.9 (±16.2) μg•h/mL in healthy volunteers after one dose, and 40.8 (±11.4) μg•h/mL, and 65.3 (±35.4)μg•h/mL 5 days and 3 months after a renal transplant, respectively. The absorption of mycophenolate mofetil is not affected by food.
A small amount of drug is excreted as MPA in the urine (less than 1%). When mycophenolate mofetil was given orally in a pharmacokinetic study, it was found to be 93% excreted in urine and 6% excreted in feces. Approximately 87% of the entire administered dose is found to be excreted in the urine as MPAG, an inactive metabolite.
The volume of distribution of mycophenolate mofetil is 3.6 (±1.5) to 4.0 (±1.2) L/kg.
Plasma clearance of mycophenolate mofetil is 193 mL/min after an oral dose and 177 (±31) mL/min after an intravenous dose.
/Absorption/ is rapid and extensive after oral administration.
In 12 healthy volunteers, the mean absolute bioavailability of oral mycophenolate mofetil relative to intravenous mycophenolate mofetil (based on MPA AUC) was 94%. The area under the plasma-concentration time curve (AUC) for MPA appears to increase in a dose-proportional fashion in renal transplant patients receiving multiple doses of mycophenolate mofetil up to a daily dose of 3 g.
Protein binding: To plasma albumin: High (97% for mycophenolic acid (MPA) at concentration ranges normally seen in stable renal transplant patients). At higher mycophenolic acid glucuronide (MPAG) concentrations (e.g., in patients with renal impairment or delayed graft function), binding of MPA may be decreased as a result of competition between MPA and MPAG for binding sites.
The mean (+/-SD) apparent volume of distribution of MPA in 12 healthy volunteers is approximately 3.6 (+/-1.5) and 4.0 (+/-1.2) L/kg following intravenous and oral administration, respectively. MPA, at clinically relevant concentrations, is 97% bound to plasma albumin. MPAG is 82% bound to plasma albumin at MPAG concentration ranges that are normally seen in stable renal transplant patients; however, at higher MPAG concentrations (observed in patients with renal impairment or delayed renal graft function), the binding of MPA may be reduced as a result of competition between MPAG and MPA for protein binding. Mean blood to plasma ratio of radioactivity concentrations was approximately 0.6 indicating that MPA and MPAG do not extensively distribute into the cellular fractions of blood.
For more Absorption, Distribution and Excretion (Complete) data for MYCOPHENOLATE MOFETIL (9 total), please visit the HSDB record page.

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Metabolism / Metabolites
After both oral and intravenous administration mycophenolate mofetil is entirely metabolized by liver carboxylesterases 1 and 2 to mycophenolic acid (MPA), the active parent drug. It is then metabolized by the enzyme glucuronyl transferase, producing the inactive phenolic glucuronide of MPA (MPAG). The glucuronide metabolite is important, as it is then converted to MPA through enterohepatic recirculation. Mycophenolate mofetil that escapes metabolism in the intestine enters the liver via the portal vein and is transformed to pharmacologically active MPA in the liver cells.N-(2-carboxymethyl)-morpholine, N-(2-hydroxyethyl)-morpholine, and the N-oxide portion of N-(2-hydroxyethyl)-morpholine are additional metabolites of MMF occurring in the intestine as a result of liver carboxylesterase 2 activity. UGT1A9 and UGT2B7 in the liver are the major enzymes contributing to the metabolism of MPA in addition to other UGT enzymes, which also play a role in MPA metabolism. The four major metabolites of MPA are 7-O-MPA-β-glucuronide (MPAG, inactive), MPA acyl-glucuronide (AcMPAG), produced by uridine 5ʹ-diphosphate glucuronosyltransferases (UGT) activities, 7-O-MPA glucoside produced via UGT, and small amounts 6-O-des-methyl-MPA (DM-MPA) via CYP3A4/5 and CYP2C8 enzymes.
Following oral and intravenous dosing, mycophenolate mofetil undergoes complete metabolism to MPA /mycophenolic acid/, the active metabolite. Metabolism to MPA occurs presystemically after oral dosing. MPA is metabolized principally by glucuronyl transferase to form the phenolic glucuronide of MPA (MPAG) which is not pharmacologically active. In vivo, MPAG is converted to MPA via enterohepatic recirculation. The following metabolites of the 2- hydroxyethyl-morpholino moiety are also recovered in the urine following oral administration of mycophenolate mofetil to healthy subjects: N-(2-carboxymethyl)-morpholine, N-(2- hydroxyethyl)-morpholine, and the N-oxide of N-(2-hydroxyethyl)-morpholine.

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Biological Half-Life
The average apparent half-life of mycophenolate mofetil is 17.9 (±6.5) hours after oral administration and 16.6 (±5.8) hours after intravenous administration.
For mycophenolic acid (MPA):Mean apparent: Approximately 17.9 hours after oral administration and 16.6 hours after intravenous administration.
Mean (+/-SD) apparent half-life and plasma clearance of MPA are 17.9 (+/-6.5) hours and 193 (+/-48) mL/min following oral administration and 16.6 (+/-5.8) hours and 177 (+/-31) mL/min following intravenous administration, respectively.

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Pharmacokinetic Properties [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 dosage; however, there is some interpatient variation in values. The AUC and peak plasma concentration (Cmax) of mycophenolic acid are approximately 50% higher in stable renal transplant patients (>3 months post-transplantation) than in patients during the immediate post-transplant period.

Mycophenolic acid is primarily eliminated (≈87%) in the urine as mycophenolic acid glucuronide; 6% is eliminated in the faeces. The mean ‘apparent’ half-life and plasma clearance of mycophenolic acid are 17.9 hours and 11.6 L/h, respectively, after oral administration.

The AUC of mycophenolic acid and its glucuronide metabolite were higher in patients with renal impairment than in patients with normal renal function following single dose administration. However, the pharmacokinetic s of mycophenolic mofetil after a single dose are not altered in patients with cirrhosis. There are limited data in children but AUC and Cmax of mycophenolic acid appear to rise with increasing age.

Protein Binding
The protein binding of mycophenolic acid, the metabolite of mycophenolate mofetil, is 97% and it is mainly bound to albumin. MPAG, the inactive metabolite, is 82% bound to plasma albumin at normal therapeutic concentrations. At elevated MPAG concentrations due to various reasons, including renal impairment, the binding of MPA may be decreased due to competition for binding.

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Mycophenolate mofetil (the morpholinoethyl ester of mycophenolic acid) inhibits de novo purine synthesis via the inhibition of inosine monophosphate dehydrogenase. Its selective lymphocyte antiproliferative effects involve both T and B cells, preventing antibody formation. Mycophenolate mofetil has immunosuppressive effects alone, but is used most commonly in combination with other immunosuppressants. Mycophenolate mofetil, in combination with cyclosporin and corticosteroids, has been studied in large, randomised clinical trials involving nearly 1500 renal allograft transplant recipients. These trials demonstrated that mycophenolate mofetil is significantly more effective in reducing treatment failure and acute rejection episodes than placebo or azathioprine. Additionally, mycophenolate mofetil may be able to reduce the occurrence of chronic rejection.
Mycophenolate mofetil is relatively well tolerated. The most common adverse effect reported is gastrointestinal intolerance; haematological aberrations have also been noted. The reversible cytostatic action of mycophenolate mofetil allows for dose adjustment or discontinuation, preventing serious toxicity or an overly suppressed immune system. Cytomegalovirus tissue invasive disease and the development of malignancies are concerns that merit evaluation in long term follow-up studies.
Mycophenolate mofetil does not cause the adverse effects typically associated with other commercially available immunosuppressant medications such as nephrotoxicity, hepatotoxicity, hypertension, nervous system disturbances, electrolyte abnormalities, skin disorders, hyperglycaemia, hyperuricaemia, hypercholesterolaemia, lipid disorders and structural bone loss.
Based on preliminary information, a positive benefit-risk ratio has been demonstrated with the use of mycophenolate mofetil in the prophylaxis of rejection in cadaveric renal allograft transplantation. Data from studies in other types of organ transplants are promising, but are too limited to draw clear conclusions. Long term follow-up studies are required to confirm these observations. Although mycophenolate mofetil is expensive, the beneficial effects on the reduction of rejection, treatment failure and related expenses suggest that it is most likely to be cost effective.[1]

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Tolerability [2]
Rates of adverse events associated with mycophenolate mofetil appear to be dose related: 2 g/day is generally better tolerated than 3 g/day. Gastrointestinal (diarrhoea, vomiting), haematological and lymphatic (leucopenia, anaemia), and infectious (sepsis, opportunistic infections) events are most common. Diarrhoea and sepsis (most commonly cytomegalovirus viraemia) were slightly more common in patients receiving mycophenolate mofetil than in those receiving azathi-oprine. There was also an increased proportion of patients with leucopenia after treatment with mycophenolate mofetil 3 g/day compared with azathioprine treatment. The overall risk of malignancies associated with mycophenolate mofetil was 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.

Compound derived from Penicillium stoloniferum and related species. It blocks de novo biosynthesis of purine nucleotides by inhibition of the enzyme inosine monophosphate dehydrogenase (IMP DEHYDROGENASE). Mycophenolic acid exerts selective effects on the immune system in which it prevents the proliferation of T-CELLS, LYMPHOCYTES, and the formation of antibodies from B-CELLS. It may also inhibit recruitment of LEUKOCYTES to sites of INFLAMMATION.
See also: Mycophenolic Acid (has active moiety); Mycophenolate Mofetil (is salt form of).

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In summary, our results indicate that MMF: (i) inhibits the secretion of TNF-α, IL-1β and NO of microglial cells; (ii) inhibits TNF-α secretion of astrocytes; (iii) suppresses the proliferation of microglial cells and astrocytes; (iv) has no direct neuroprotective effects on cultured, excitotoxically injured hippocampal neurones; and (v) acts by inhibiting glial IMPDH. Against a background of promising animal experiments and open label clinical trials on the use of MMF in various CNS disorders, MMF seems to be a promising candidate for further investigations on the treatment of acute brain and spinal cord pathologies.[2]

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MMF and DCZ, which target T lymphocytes, inhibit inflammatory activity, and thus prevent the development of skin fibrosis in early stages of a mouse model of BLM‐induced scleroderma. However, MMF is more effective at suppressing fibroblastic activation than in DCZ, and has a more prominent antifibrotic effect. These results support the assumption that T lymphocytes play an important role in the pathogenesis of scleroderma, and that suppression of T lymphocytes may be an effective strategy for treatment of human scleroderma, when started during the early stages of the disease. However, targeting T lymphocytes alone may not be an adequate treatment approach for 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.)