DMF, MMF and MEF remained completely intact in a buffer of pH 1 mimicking the pH in the stomach (Figure 1A and 1F). However, at pH 8 resembling the pH in the small intestines DMF, the most abundant component of the FAE tablet, was hydrolyzed to MMF (the half-life of DMF amounted to 1.5 hr) (Figure 1B). Addition of MEF, the other component of the FAE tablet, did not affect the half-life of DMF (1.7 hr) (Figure 1G). MMF remained intact (Figure 1B) in this buffer during the period of analysis (6 hr) as did MEF (Figure 1G).

To further examine the pH-dependency of the hydrolysis of DMF to MMF, we measured concentrations of DMF and MMF in phosphate buffers (with pH ranging from 6.5–8) supplemented with DMF or the combination of DMF and MEF. The results revealed that the half-life of DMF dramatically decreased with increasing pH values and the maximal hydrolysis of DMF to MMF was seen at pH 8 (half life of DMF was 1.5 hr). For example, at pH 7.4 the half-life of DMF amounted to 12.7 ± 1.0 hr (n = 3) (Figure 1C). In agreement with these results we observed that the Fumaraat 120 tablet disintegrated completely between 1.5 and 2.5 hr in the alkaline, but not in the acidic, environment. The half-life of DMF in the tablet amounted to approximately 2.3 hr (data not shown).

Since FAE must enter the circulation to exert their anti-psoriatic effects at the affected skin site [14], we determined the hydrolysis of DMF to MMF and examined the stability of MMF, DMF and MEF in both normal human serum and whole blood (both with a pH of 7.4). The half-life of DMF in serum (Table 1 and Figure 1D) is dramatically shorter (p < 0.05) than that in a buffer of the same pH. MMF (Figure 1D and 1I) and MEF (Figure 1I) concentrations slowly decreased in serum during the period of analysis (6 hr). Furthermore, the half-life of DMF was even shorter (p < 0.05) in whole blood than in serum (Table 1 and Figure 1E), indicating that circulating cells are also involved in the hydrolysis of DMF to MMF. Furthermore, concentrations of MMF (Figure 1E and 1J) and MEF (Figure 1J) in whole blood decreased steadily during the period of analysis (6 hr), indicating that they may be metabolized by blood cells as well.

To find out which blood cell type(s) is (are) responsible for the hydrolysis of FAE in whole blood, hydrolysis of DMF in a buffer of pH 7.4 by purified blood cell fractions was analyzed. The results revealed that monocytes/lymphocytes (Figure 2A), but not granulocytes (Figure 2B) and erythrocytes (Figure 2C), at concentrations present in whole blood effectively hydrolyzed DMF to MMF.

The following FAE were used: dimethylfumarate (DMF; purity > 97%, TioFarma, Oud-Beijerland, The Netherlands), calcium-monoethylfumarate (MEF; purity > 97%, Tiofarma), monomethylfumarate (MMF; purity > 97%, AstraZeneca R&D, Charnwood, Loughborough, UK). In addition, the enteric-coated, magisterial manufactured tablet (named Fumaraat 120^®; TioFarma), containing 120 mg of DMF and 95 mg of calcium-MEF was investigated in this study.

To investigate the stability of DMF, MMF and MEF and the hydrolysis of DMF to MMF in several environments representing various aspects of different body compartments, 0.1 N HCl with pH 1 and 0.1 M sodium phosphate buffer with pH 8 were spiked with 2 mg/L of DMF, MMF, MEF or the combination of 2 mg/L of DMF and 1.4 mg/L MEF, to resemble the ratio of these two components in the Fumaraat 120^® tablet. In addition, to determine the release of the contents of a Fumaraat 120^® tablet and the hydrolysis of DMF to MMF at pH 1 (0.1 N of HCl) and pH 8 (0.1 M of sodium phosphate buffer), the tablet was placed in these buffers and at various intervals samples were taken and prepared for measurement of the concentrations of DMF, MMF and MEF by high-performance liquid chromatography (HPLC) as described below.

To further investigate the effect of the pH on the hydrolysis of DMF to MMF, 0.1 M sodium phosphate buffers with pH values ranging from 6.5–8 were spiked with DMF and the combination of DMF and MEF. At several intervals thereafter, samples were taken, and then prepared for measurement of the various FAE by HPLC.

As the current buffers lack proteins, no extraction procedure was necessary and the concentrations of the various FAE in the samples could be directly quantified by HPLC (see below).

As described above, serum and whole blood from 3 volunteers was spiked with 2 mg/L of DMF, MMF, MEF or the combination of 2 mg/L of DMF and 1.4 mg/L MEF. All volunteers were healthy as assessed by a full medical screening.

At several intervals, samples were taken, and then prepared for measurement of the various FAE by HPLC. In short, serum and whole blood contained proteins known to interfere with the measurement of FAE. To overcome this problem, the various FAE were extracted from serum and whole blood samples and subsequently the concentrations were measured by HPLC (see below).

The various blood cell fractions were obtained from blood of healthy volunteers using centrifugational techniques as described earlier [17,19]. In short, blood was subjected to Ficoll Amidotrizoate (ρ = 1.077 gm/L; Dept. of Pharmacy, Leiden University Medical Center, Leiden, The Netherlands) density gradient centrifugation (440 g 20 min at 18°C). After resuspension of the cells in the pellet in phosphate buffered saline (PBS; pH 7,4) the granulocytes were purified by plasmasteril (Fresenius AG, Bad Homburg, Germany) sedimentation (1 g) for 10 min at 37°C, washed with PBS and the contaminating erythrocytes were lysed with distilled water. Erythrocytes were obtained after washing the cells in the Ficoll-Amidotrizoate pellet three times with PBS supplemented with 0.1 IU heparin. Cells in the Ficoll-Amidotrizoate interphase (monocytes/lymphocytes) were washed three times with PBS containing 0.5 IU heparin and then resuspended in PBS pH 7.4. Next, suspensions of 1 × 10⁶ monocytes and lymphocytes/mL PBS, 4 × 10⁶ granulocytes/mL PBS, and 5 × 10⁹ erythrocytes/mL PBS were spiked with 2 mg/L DMF. Again, at several intervals samples were taken and concentrations of the various FAE were measured as described below.

The concentrations of the various fumarates in serum samples were determined as described (Litjens et al., submitted for publication). Briefly, after precipitation of serum proteins with acetonitrile, DMF in the samples was quantitated by HPLC. The sample preparation for MMF and MEF required a protein precipitation step with metaphosphoric acid followed by extraction with diethylether and additional pH-lowering to pH 0.5. Next, sodium chloride was added before centrifugation at 12,000 g. Thereafter, the ether layer was transferred to a glass vial and after evaporation the residue reconstituted in methanol: 0.1 M potassium phosphate buffer (KH₂PO₄/K₂HPO₄; pH 7.5) supplemented with 5 mM tetrabutylammonium dihydrogen phosphate 1:1 (v/v).

Concentrations of DMF, MMF, and MEF were determined on a HPLC apparatus (Spectra SERIES P100, Thermo Separation Products, Breda, The Netherlands) equipped with an Alltima C18 (5 μ 250*4.6; Alltech, Lokeren, Belgium) column and an Alltima Guard C18 precolumn (5 μ 7.5*4.6; Alltech, Lokeren, Belgium) using methanol:water 30:70 (v/v) as an eluent for DMF and methanol: potassium phosphate buffer supplemented with 5 mM tetrabutylammonium dihydrogen phosphate 20:80 (v/v) as eluent for MMF and MEF. The limit of detection for all three compounds amounted to 0.01 mg/L, the coefficient of variation for MMF, DMF and MEF was 7%, 8% and 9% at 0.5 mg/L, respectively (n = 4), and the recovery of MMF, DMF and MEF amounted to 75 ± 7%, 98 ± 3%, 67 ± 7% (n = 6). Standard curves constructed with purified FAE in buffers or human serum were used to quantify the concentrations of FAE in the various samples of these buffers and human serum or whole blood, respectively.