Metabolite of mycophenolic acid

With an IC50 of 3.3 mg/L, mycophenolic acid glucuronide exhibits a modest type II inhibition of IMP dehydrogenase (IMPDH)[3].

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Effect of MPA and MPAG/Mycophenolic acid 7-O-glucuronide on hNaDC3 [1]
In hNaDC3-expressing oocytes, addition of 0.1 mM MPA induced a small inward current compared with ORI-2 alone ( Figure 1 A). When sodium was replaced by N -methyl-D-glucamine, the MPA-associated inward current was abolished, indicating sodium-dependent translocation of MPA ( Figure 1 A). Figure 1 also shows the currents induced by 0.1 mM MPA (B, open circles) and 0.1 mM MPAG (C, open circles) in hNaDC3-oocytes as a function of membrane potential, each in comparison with those elicited by 1 mM succinate in the same oocytes (closed circles). At all potentials tested, the succinate-dependent inward currents were larger in magnitude as compared with MPA- or MPAG-induced currents. No inward currents were observed in H 2 O-injected oocytes when succinate, MPA, or MPAG was added (data not shown). When applied together with 1 mM succinate, MPA attenuated the inward currents by 16 ± 10% and 28 ± 8% at 0.1 and 1 mM, respectively. MPAG was somewhat more potent, eliciting a current decrease of 32 ± 19% already at 0.1 mM ( Figure 2 ). These data demonstrate that MPA and MPAG interact with, and are transported by human NaDC3.

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Effect of MPAG and AcMPAG on hOAT1- and hOAT3-mediated transport [1]
With respect to renal elimination, the glucuronide metabolites of MPA, especially MPAG, which are normally delivered to the kidney from the liver [ 1 ], are of particular importance. In contrast to MPA, the glucuronides MPAG and AcMPAG did not significantly attenuate PAH uptake by hOAT1-expressing oocytes at clinically relevant concentrations ( Figure 5 ). However, ES uptake in hOAT3-oocytes was significantly inhibited by 100 µM AcMPAG (80.9 ± 6.5%, P < 0.01), as well as by 10 µM and 100 µM MPAG (53.7 ± 5.2%, P < 0.01, and 69.2 ± 12.7%, P < 0.05, respectively) ( Figure 5 ). In these experiments, inhibition by 10 µM AcMPAG was slightly less than that by 10 µM MPAG (46.8 ± 14.5%) and, due to its high variability, not statistically significant. However, the dose response curves indicate that AcMPAG is a more potent hOAT3 inhibitor (IC 50 2.88 ± 0.42 µM) than MPAG (IC 50 15.2 ± 18.5 µM) ( Figure 6 ). Mycophenolic acid glucuronide (MPAG) inhibition kinetics were evaluated using purified recombinant human type II inosine monophosphate dehydrogenase (IMPDH). MPAG inhibitory concentrations (IC50) were found to be 532- to 1022-fold higher than those for MPA. As expected, according to tight-binding inhibitor kinetics, mycophenolic acid (MPA) IC50 values increased as IMPDH concentrations increased, whereas IC50 values for xanthosine monophosphate (competitive IMPDH inhibitor used as control), an inhibitor known not to be tight binding, remained independent of enzyme concentration. Although MPAG exhibited only weak inhibition of IMPDH activity, in comparison with MPA, IC50 values increased with increasing enzyme concentration. The presence of trace quantities of MPA (0.2% on a molar basis) in the MPAG preparation, detected by high-performance liquid chromatography analysis, could account for this observation. These data support the proposal that MPAG is a pharmacologically inactive metabolite of MPA.[2]

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Results: MPA and AcMPAG showed an inhibition of rhIMPDH II (IC(50) 25.6 microg/L and 301.7 microg/L, respectively; the K(i) of MPA for NAD and IMP was 50.8 and 57.7 nmol/L, respectively; and that of AcMPAG for NAD and IMP was 382.0 and 511.0 nmol/L. MPAG had no significant effect on the enzyme. AcMPAG apparently acts by the same uncompetitive inhibition mechanism as MPA, with a 12-fold higher IC(50) and an 8-10 times higher K(i). When coincubated with MPA, AcMPAG activity was negligible at pharmacological concentrations. Furthermore, after 6-h incubation at their respective maximum concentration (C(max)), MPA was 10 times more concentrated in Jurkat cells than AcMPAG. Conclusions: AcMPAG is a weaker inhibitor of rhIMPDH II than MPA and is less concentrated in lymphocytes in vitro, suggesting that it would not be pharmacologically active in vivo and might not need to be monitored in MPA-treated patients [3].

In an Ehrlich murine spontaneous adenocarcinoma model, mycophenolic acid glucuronide (intraperitoneal injection; 6 mg/mouse; once two days; 6 d) therapy inhibits tumor growth[2].

Assay of IMPDH Activity [2]
IMPDH activity was determined as described previously. Briefly, the assay mixture (final volume, 1 ml) contained 400 nmol NAD, 200 nmol IMP, 0.1 mol potassium phosphate at pH 7.8, 0.5 mol potassium chloride, 3 mmol EDTA, IMPDH at appropriate concentrations, and a range of concentrations of the enzyme inhibitors MPA, MPAG, or XMP). NADH formation was monitored at 340 nm for 20 minutes at 37°C on a Beckman DU 640 spectrophotometer. Median inhibitory concentrations (IC50) were calculated statistically (least-squares fit) using the PS Plot computer program.

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RHimpdh ii enzyme assay [3]
The rhIMPDH II assay was performed in 100 mmol/L Tris-HCl buffer (pH 8 at 37 °C), containing 100 mmol/L KCl, 2 mmol/L EDTA, and 1 mmol/L dithiothreitol. We added a volume of 100 μL rhIMPDH II (4% vol/vol) to 850 μL enzyme buffer and vortex-mixed it thoroughly with 20 μL IMP (0.01–10 mmol/L).The reaction medium was prewarmed at 37 °C using an incubator block. We added 10 μL potential inhibitors (MPA, AcMPAG, or MPAG) diluted in acetonitrile (final percentage 1% vol/vol) to the incubation mixture. Control incubations were spiked with the same amount of acetonitrile. We then started the reaction by adding 20 μL NAD+ (0.25–20 mmol/L). The mixture was immediately mixed thoroughly, transferred into a 1-mL UV microvial, and placed in a thermoregulated spectrophotometer. As the rhIMPDH II catalyzed reaction is equimolar, NADH formation is directly linked to the amount of IMP and NAD+ used. Reaction monitoring consisted of spectrophotometric measurement of NAD+ reduction (molar absorptivity 6220 at λ = 340 nm). Two measurements per second were performed for 5 min. We computed the results using an in-house program that calculates the initial velocity rate of each reaction using a linear regression model.
To test AcMPAG and MPAG stability during the assay, both molecules were separately incubated in triplicate at 2 different concentrations (10 μg/L and 10 mg/L) in 1 mL of the reaction mixture at 37 °C for 30 min. Every 5 min, 100 μL was sampled, mixed with 100 μL acetonitrile/formic acid (97%/3% vol/vol), and immediately chilled on ice to avoid further degradation. Negative controls were carried out in triplicates by incubating AcMPAG and MPAG in water at 37 °C at the same concentrations. MPA, AcMPAG, and MPAG were then determined in each sample using a validated HPLC-MS/MS method.

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inhibition experiments [3]
rhIMPDH II was incubated in triplicate as described above with a saturating concentration of both cosubstrates (IMP 200 μmol/L; NAD+ 400 μmol/L) and 10 increasing concentrations of the 3 potential inhibitors: MPA (1 μg/L to 1 mg/L), AcMPAG (10 μg/L to 10 mg/L), and MPAG (200 μg/L to 20 mg/L). We calculated the half-maximal inhibitory concentration (IC50) for each potential inhibitor using the Hill model and Winreg 3.1. To calculate the inhibition constant (Ki) with respect to both IMPDH II cosubstrates NAD+ and IMP, each inhibitor was incubated at 5 increasing concentrations (50 μg/L to 3.0 mg/L for AcMPAG and 1 μg/L to 1.0 mg/L for MPA). We first performed experiments at a fixed saturating concentration (200 μmol/L) of IMP and increasing concentrations of NAD+ (5–400 μmol/L), then performed at a saturating NAD+ concentration (400 μmol/L) and 6 increasing concentrations of IMP (2 μmol/L to 200 μmol/L). We calculated the Ki of each inhibitor for each cosubstrate by nonlinear regression using in-house software called Inhib 1.1. To compare the effect on rhIMPDH II of AcMPAG alone or in the presence of MPA, AcMPAG was incubated with rhIMPDH II at concentrations ranging from 0.05- to 10-fold the MPA IC50 value, with MPA or AcMPAG at approximately its IC50 (25 μg/L and 300 μg/L, respectively). The inhibition rates were calculated with respect to a positive control without any inhibitor.

capacity of aCmpag and mpa to cross cellular membranes [3]
We cultured Jurkat cells from a human cell line of lymphoma in a 75-cm2 culture flask at a concentration of 2 × 106 cells/mL in RPMI 1640 medium supplemented with 10% FBS, 2 mmol/L glutamine, 1% amino acids, 1% vitamins, 1% pyruvate, 100 U/mL penicillin, and 100 mg/L streptomycin. Incubations were performed at 37 °C and 5% CO2 for 6 h with MPA (final concentration 20 mg/L), AcMPAG, and MPAG (taken here as a chemically stable control for AcMPAG; final concentration 2 mg/L). The medium was then centrifuged; the supernatant was sampled and analyzed to measure the extracellular concentration, and the cell pellet was used to measure the intracellular concentration after lysis by addition of 200 μL of a 50/50 (vol/vol) hypotonic water/formic acid solution and incubation at −20 °C for 48 h. We quantified MPA, MPAG, and AcMPAG using a published LC-MS/MS technique. The limit of quantification of the technique was 10 μg/L. The linearity was verified up to 10 mg/L (r = 0.999). The intraassay CVs were <10% and the interassay CVs <15% over this linearity range. The recovery was good, with mean relative errors <15% over the linearity range.
We could not calculate the actual intracellular concentration because the volume of intracellular medium, or even the cells’ volume or mass, cannot be precisely measured. Therefore, we calculated the ratio of the drug amount found in the cells after 6 h of incubation (in μg) to the total amount of compound present in the incubation medium at time 0 (e.g., the spiked amount), to compare the cell passage of AcMPAG relative to those of MPA and MPAG.

Animal/Disease Models: Swiss albino strain mouse implanted with Ehrlich ascites tumor cells[2]
Doses: 6 mg/mouse
Route of Administration: intraperitoneal (ip) injection; 6 mg/mouse; once two days; 6 d
Experimental Results: demonstrated 76.8% tumor inhibition compared to the untreated control.

Metabolism / Metabolites
Mycophenolic acid glucuronide is a known human metabolite of mycophenolic acid.

[1]. Mycophenolic acid (MPA) and its glucuronide metabolites interact with transport systems responsible for excretion of organic anions in the basolateral membrane of the human kidney. Nephrol Dial Transplant. 2007 Sep;22(9):2497-503.

[2]. Synthesis of mycophenolic acid beta-D-glucuronide and its antitumor activity. J Antibiot (Tokyo). 1970 Aug;23(8):408-13.

[3]. Effect of mycophenolate acyl-glucuronide on human recombinant type 2 inosine monophosphate dehydrogenase. Clin Chem. 2009 May;55(5):986-93.

In summary, mycophenolate interacts with high affinity with both human OAT1 and OAT3, and inhibits substrate transport. The metabolites MPAG and AcMPAG were found to interact with hOAT3, but not with hOAT1. The potent inhibition with IC 50 values well within the range of plasma concentrations encountered during mycophenolate therapy make hOAT1 and hOAT3 sensitive targets for interference with the renal elimination of co-administered hOAT1/hOAT3 drug substrates, such as virustatics, corticosteroids or diuretics.[1]

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Background: Mycophenolic acid (MPA), the active moiety of the prodrug mycophenolate mofetil, is widely used in immunosuppressive regimens after kidney, liver or heart transplantation. MPA is metabolized predominantly to the inactive 7-O-glucuronide (MPAG). A minor fraction is converted to the pharmacologically active acyl glucuronide (AcMPAG). All compounds ultimately are eliminated via the kidneys. Due to their structures, MPA and its metabolites are candidate substrates for the human organic anion transporters 1 (OAT1) and 3 (OAT3) as well as for the Na+-dicarboxylate cotransporter 3 (NaDC3). Methods: Human (h)OAT1, hOAT3 and hNaDC3 were expressed from in vitro synthesized cRNA in collagenase-defolliculated Xenopus laevis oocytes. On day 3 post-injection, measurements were made of (i) substrate-associated currents using MPA and MPAG (only in hNaDC3-expressing oocytes) and (ii) uptake of [3H]p-aminohippurate (hOAT1) or [3H]estrone sulfate (hOAT3) in the absence or presence of either MPA, MPAG or AcMPAG. Results: In hNaDC3-expressing oocytes at -60 mV, MPA (0.1 mM) as well as MPAG (0.1 mM) induced inward currents that were 17 and 25% of the currents evoked by succinate (1 mM). Vice versa, currents induced by succinate (1 mM) were partially inhibited by MPA and MPAG. hOAT1 and hOAT3 were potently inhibited by MPA (IC50 1.24 and 0.52 microM, respectively). Human OAT3, but not hOAT1, was additionally inhibited by both glucuronide metabolites of MPA in a concentration-dependent manner (IC50 15.2 microM for MPAG and 2.88 microM for AcMPAG), consistent with a preference of hOAT3 for more bulky substrates compared with hOAT1. Conclusions: MPA and its metabolites potently interact with renal organic anion transporters hOAT1 and hOAT3, and thereby may interfere with the renal secretion of antiviral drugs, cortisol and other organic anions.[1]

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These data support the hypothesis that MPAG has little or no direct pharmacologic activity. Because MPAG exhibited apparently weak inhibition of IMPDH, the observed increase in IC50 with higher concentrations of IMPDH cannot be caused by MPAG itself, because only tight-binding inhibitors can cause this effect. An alternative explanation is that the apparently weak inhibition was caused by the presence of small amounts of the tight-binding inhibitor MPA. The finding of a small amount of MPA in the MPAG using HPLC analysis supports this hypothesis. Further support is derived from the observation that when the concentration of MPA, as a trace contaminant in the MPAG preparation, is calculated and substituted for MPAG in the IC50 determination, the IC50 (MPAcontam) values were very similar to the IC50 MPA results (Table 1). These data support the hypothesis that MPAG, in comparison with MPA, exerts little direct effect on the pharmacologic target. Nevertheless, in patients who achieve high plasma concentrations of the MPA metabolite (e.g., renal transplant patients with delayed graft function), the possible contribution to the active drug concentration via the action of glucuronidase needs to be explored.[2]

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Background: The immunosuppressive effect of mycophenolic acid (MPA) is essentially attributed to IMPDH II inhibition, which leads to a reduction of lymphocyte proliferation. We investigated the action of the MPA metabolites MPA-phenyl-glucuronide (MPAG) and MPA-acyl-glucuronide (AcMPAG) on recombinant human IMPDH II (rhIMPDH II), as well as their passage into lymphocytes in vitro. Methods: We measured rhIMPDH II activity spectrophotometrically through the initial velocity of NADH formation, leading to the computation of the kinetic parameters K(m), IC(50), and K(i) (Michaelis constant, half-maximal inhibition concentration, and inhibition constant). We measured intracellular and extracellular concentrations of MPA, MPAG, and AcMPAG after incubation of Jurkat lymphoma cells with each compound separately, using liquid chromatography-tandem mass spectrometry.[3]

C23H28O12

496.46

496.158

C, 55.64; H, 5.68; O, 38.67

31528-44-6

Mycophenolic acid glucuronide-d3

6442661

White to off-white solid powder

1.502g/cm3

875.7ºC at 760mmHg

98-100ºC

299.6ºC

1.624

0.298

5

12

9

35

827

5

CC1=C2COC(=O)C2=C(C(=C1OC)C/C=C(\C)/CCC(=O)O)O[C@H]3[C@@H]([C@H]([C@@H]([C@H](O3)C(=O)O)O)O)O

BYFGTSAYQQIUCN-HGIHDBQLSA-N

InChI=1S/C23H28O12/c1-9(5-7-13(24)25)4-6-11-18(32-3)10(2)12-8-33-22(31)14(12)19(11)34-23-17(28)15(26)16(27)20(35-23)21(29)30/h4,15-17,20,23,26-28H,5-8H2,1-3H3,(H,24,25)(H,29,30)/b9-4+/t15-,16-,17+,20-,23+/m0/s1

(2S,3S,4S,5R,6S)-6-[[5-[(E)-5-carboxy-3-methylpent-2-enyl]-6-methoxy-7-methyl-3-oxo-1H-2-benzofuran-4-yl]oxy]-3,4,5-trihydroxyoxane-2-carboxylic acid

mycophenolic acid glucuronide; 31528-44-6; Mycophenolic acid glucosiduronate; Mycophenolic acid 7-O-glucuronide; 54TS5J9T0K; UNII-54TS5J9T0K; MPAG cpd; .BETA.-D-GLUCOPYRANOSIDURONIC ACID, 5-((2E)-5-CARBOXY-3-METHYL-2-PENTEN-1-YL)-1,3-DIHYDRO-6-METHOXY-7-METHYL-3-OXO-4-ISOBENZOFURANYL;

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)

DMSO: 33.33 mg/mL (67.14 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.04 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.04 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.04 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)