BMC Pharmacology
`
`BioMed Central
`
`Open Access
`Research article
`In vitro pharmacokinetics of anti-psoriatic fumaric acid esters
`Nicolle HR Litjens1, Elisabeth van Strijen1, Co van Gulpen1, Herman Mattie1,
`Jaap T van Dissel1, H Bing Thio2 and Peter H Nibbering*1
`
`Address: 1Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands and 2Department of Dermatology and
`Venereology, Erasmus Medical Center, Rotterdam, The Netherlands
`
`Email: Nicolle HR Litjens - nicollelitjens@hotmail.com; Elisabeth van Strijen - e.van_strijen@lumc.nl; Co van Gulpen - cvgulpen@xs4all.nl;
`Herman Mattie - h.mattie@wxs.nl; Jaap T van Dissel - j.t.van_dissel@lumc.nl; H Bing Thio - H.thio@erasmusmc.nl;
`Peter H Nibbering* - p.h.nibbering@lumc.nl
`* Corresponding author
`
`Published: 12 October 2004
`
`BMC Pharmacology 2004, 4:22
`
`doi:10.1186/1471-2210-4-22
`
`This article is available from: http://www.biomedcentral.com/1471-2210/4/22
`
`Received: 06 February 2004
`Accepted: 12 October 2004
`
`© 2004 Litjens et al; licensee BioMed Central Ltd.
`This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
`which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
`
`Abstract
`Background: Psoriasis is a chronic inflammatory skin disease that can be successfully treated with
`a mixture of fumaric acid esters (FAE) formulated as enteric-coated tablets for oral use. These
`tablets consist of dimethylfumarate (DMF) and salts of monoethylfumarate (MEF) and its main
`bioactive metabolite is monomethylfumarate (MMF). Little is known about the pharmacokinetics of
`these FAE. The aim of the present study was to investigate the hydrolysis of DMF to MMF and the
`stability of MMF, DMF and MEF at in vitro conditions representing different body compartments.
`Results: DMF is hydrolyzed to MMF in an alkaline environment (pH 8), but not in an acidic
`environment (pH 1). In these conditions MMF and MEF remained intact during the period of analysis
`(6 h). Interestingly, DMF was hardly hydrolyzed to MMF in a buffer of pH 7.4, but was rapidly
`hydrolyzed in human serum having the same pH. Moreover, in whole blood the half-life of DMF
`was dramatically reduced as compared to serum. The concentrations of MMF and MEF in serum
`and whole blood decreased with increasing time. These data indicate that the majority of the FAE
`in the circulation are metabolized by one or more types of blood cells. Additional experiments with
`purified blood cell fractions resuspended in phosphate buffered saline (pH 7.4) revealed that at
`concentrations present in whole blood monocytes/lymphocytes, but not granulocytes and
`erythrocytes, effectively hydrolyzed DMF to MMF. Furthermore, in agreement with the data
`obtained with the pure components of the tablet, the enteric-coated tablet remained intact at pH
`1, but rapidly dissolved at pH 8.
`Conclusion: Together, these in vitro data indicate that hydrolysis of DMF to MMF rapidly occurs
`at pH 8, resembling that within the small intestines, but not at pH 1 resembling the pH in the
`stomach. At both pHs MMF and MEF remained intact. These data explain the observation that after
`oral FAE intake MMF and MEF, but not DMF, can be readily detected in the circulation of human
`healthy volunteers and psoriasis patients.
`
`Background
`Psoriasis is a chronic inflammatory skin disease character-
`
`ized by epidermal hyperplasia and infiltration of inflam-
`matory cells into skin lesions. Anti-psoriatic therapies are
`
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`mainly anti-inflammatory. Long-term use of many of
`these anti-psoriatic therapies is often hampered by serious
`adverse effects [1-5]. In this connection it is of interest that
`already in 1959, Schweckendiek introduced fumaric acid,
`an intermediate of the citric acid cycle, for the treatment
`of his psoriasis [6]. The main adverse effect of fumaric acid
`therapy, i.e. induction of gastric ulcers, was overcome by
`application of a mixture of fumaric acid esters (FAE) with
`great bioavailability [7]. This mixture, consisting of
`dimethylfumarate (DMF) and salts of monoethylfuma-
`rate (MEF), was formulated as enteric-coated tablets. This
`systemic therapy, successfully applied by several German
`[8,9] and Dutch [10,11] dermatologists, can be taken by
`patients for a long period due to the excellent safety pro-
`file [12]. Adverse effects that do occur are mostly mild and
`transient and include facial flushing and gastro-intestinal
`complaints. Pharmacokinetic data of FAE therapy are very
`limited and mainly based on personal communications
`[8,13]. For such a pharmacokinetic study, we first devel-
`oped a highly sensitive method to determine concentra-
`tions of FAE in human blood (Litjens et al., manuscript
`submitted). In the present study, we investigated the
`hydrolysis of DMF to its most bioactive metabolite mon-
`omethylfumarate (MMF) and the stability of MMF, DMF
`and MEF in different environments representing various
`body compartments using this methodology.
`
`Results
`Stability of FAE and hydrolysis of DMF to MMF in buffers
`of various pH
`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 anal-
`ysis (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 dra-
`matically 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, environ-
`ment. The half-life of DMF in the tablet amounted to
`approximately 2.3 hr (data not shown).
`
`Changes in the concentrations of DMF, MMF and MEF in
`serum and whole blood
`Since FAE must enter the circulation to exert their anti-
`psoriatic effects at the affected skin site [14], we deter-
`mined 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 dramati-
`cally shorter (p < 0.05) than that in a buffer of the same
`pH. MMF (Figure 1D and 1I) and MEF (Figure 1I) concen-
`trations 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. Fur-
`thermore, 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/lym-
`phocytes (Figure 2A), but not granulocytes (Figure 2B)
`and erythrocytes (Figure 2C), at concentrations present in
`whole blood effectively hydrolyzed DMF to MMF.
`
`Discussion
`A major finding of the present study is that hardly any
`DMF was hydrolyzed in a buffer of pH ≤ 7.4, whereas at
`pH 8, resembling the pH of the small intestines, this FAE
`was effectively hydrolyzed to its active metabolite MMF. It
`should be noted that MMF (and MEF) remained stable in
`these buffers. We realize that using acidic or alkaline buff-
`ers to mimick the conditions in body compartments, like
`the stomach and the small intestines, is only a first
`attempt to investigate the in vitro pharmacokinetics of
`FAE. For example, no enzymes, e.g. esterases, are present
`in these buffers whereas they are in these body compart-
`ments. In this connection, Werdenberg and collegues [15]
`recently showed that in the small intestines, the concen-
`trations of MEF and MMF remained unaffected, whereas
`concentrations of DMF decreased by the action of este-
`rases, such as carboxyl- and choline-esterases in this com-
`partment. Esterase activity is also present in the liver
`which can cause a rapid disappearance of the various FAE
`from the circulation. Absorption of FAE from the small
`intestines into the circulation is not only dependent on
`the permeability of the intestinal membrane for the vari-
`ous FAE (permeability increases with increased acyl-chain
`length and increased lipophilicity), but also on the stabil-
`ities of the various FAE in the small intestines and liver.
`Clearly, hydrolysis of DMF to MMF is not only dependent
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`
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`time (hours)
`
`Changes in the concentrations of the various FAE in different environmentsFigure 1
`
`Changes in the concentrations of the various FAE in different environments. DMF at a concentration of 2 mg/L or
`the combination of 2 mg/L DMF and 1.4 mg/L MEF were placed at 37°C in 0.1 N HCl; pH 1 (A, F), 0.1 M sodium phosphate
`buffer; pH 8 (B, G), 0.1 M sodium phosphate buffer; pH 7.4 (C, H), normal human serum (D, I) or whole blood (E, J). At various
`intervals thereafter samples were collected and the MMF (squares), DMF (circles) and MEF (triangles) concentrations were
`measured using HPLC. Results are a representative experiment of at least 3 independent experiments.
`
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`Table 1: Hydrolysis rate of DMF to MMF and half-lives of DMF in different environments. To analyze under which circumstances DMF
`can be hydrolyzed to MMF and whether MEF affects the hydrolysis of DMF into MMF, we determined the hydrolysis rates for DMF in
`different environments. In short, a 0.1 M sodium phosphate buffer, human serum and whole blood (all pH 7.4) were spiked with either
`2 mg/L of DMF or with the combination of 2 mg/L of DMF and 1.4 mg/L of MEF and at several intervals thereafter, samples were taken
`and prepared in order to measure the concentration of DMF, MMF and MEF by HPLC. Subsequently, after calculating the area under
`the curves for DMF (AUC_DMF) and MMF (AUC_MMF), the following model [16] was used to fit the concentrations of MMF and to
`estimate the kDMF (rate of hydrolysis of DMF into MMF) in these solutions: [MMF]t = i = (kDMF*AUC_DMF)-(kMMF*AUC_MMF) + [MMF]t
`= 0. In addition, the half-life was calculated using the following formula: t1/2 = ln(2)/k. Data are means and SD (n = 3). # and * significant
`(p < 0.05) different value between buffer and and serum and serum and whole blood, respectively.
`
`Buffer
`Spiked with:
`DMF
`DMF+MEF
`Serum
`Spiked with:
`DMF
`DMF+MEF
`Whole blood
`Spiked with:
`DMF
`DMF+MEF
`
`kdmf (h-1)
`
`0.06 (0.004)
`0.05 (0.01)
`
`1.96 (0.47)
`2.20 (0.25)
`
`8.01 (3.78)
`10.08 (2.74)
`
`t1/2 (h)
`
`12.72 (1.04)
`15.17 (1.88)
`
`0.37 (0.08) #
`0.32 (0.05) #
`
`0.10 (0.04)*
`0.07 (0.02)*
`
`on the pH of the environment but also on the activities of
`esterases.
`
`Another important finding of this study is that the half-
`life of DMF in whole blood is considerably shorter than
`that in serum, although the pH of both blood and serum
`is 7.4. To explain this difference in hydrolysis of DMF in
`whole blood and serum we considered the possibility that
`blood cells also hydrolyze DMF to MMF. Using purified
`blood cell fractions resuspended in PBS (pH 7.4) we
`found that monocytes/lymphocytes, but not granulocytes
`and erythrocytes, at concentrations present in whole
`blood effectively hydrolyzed DMF to MMF. The rapid
`removal of DMF from PBS after addition of granulocytes
`and erythrocytes suggests that these blood cells bind DMF.
`
`It should be realized that MMF (and MEF) most likely
`enter the circulation of psoriasis patients in order to exert
`their antipsoriatic effects in the skin lesions. In agreement
`we detected MMF and MEF, but not DMF, in the circula-
`tion of healthy volunteers and psoriasis patients after oral
`intake of Fumaraat 120® tablets [[16], Litjens et al., manu-
`script submitted; Litjens et al., unpublished data]. Our
`observation that the MMF is more rapidly removed from
`whole blood than from serum could indicate that MMF
`(and MEF) is taken up by blood cells and perhaps further
`metabolized into FA, which subsequently fuels the citric
`acid cycle, as suggested earlier by Joshi (personal commu-
`nication). The different interactions between FAE and
`
`blood cells may affect their functional activities, as has
`been reported earlier [14,17-19], thus contributing to the
`beneficial effects of FAE therapy.
`
`Conclusions
`Together, these in vitro data indicate that DMF is almost
`completely hydrolyzed to MMF at an alkaline pH, but not
`at an acidic pH, suggesting that this hydrolysis occurs
`mainly within the small intestines and not in the stom-
`ach. Most likely, MMF and MEF are then absorbed in the
`circulation where they interact with blood cells and per-
`haps cells in the psoriatic lesions. The different interac-
`tions between these FAE and the various cell types may
`explain the beneficial effects of FAE in psoriasis. Finally,
`these in vitro experimental data will be key to the pharma-
`cokinetic analysis of oral FAE in human healthy volun-
`teers and psoriasis patients.
`
`Methods
`Fumaric acid esters (FAE)
`The following FAE were used: dimethylfumarate (DMF;
`purity > 97%, TioFarma, Oud-Beijerland, The Nether-
`lands), 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.
`
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`0
`
`1
`
`2
`
`3
`
`0
`
`1
`
`2
`
`3
`
`A
`
`4
`
`B
`
`4
`
`C
`
`3
`
`2
`
`1
`
`0
`
`3
`
`2
`
`1
`
`0
`
`3
`
`2
`
`1
`
`0
`
`Concentration (mg/L)
`
`0
`
`1
`
`2
`
`3
`
`4
`
`time (hours)
`
`Hydrolysis of DMF to MMF by various types of blood cellsFigure 2
`
`Hydrolysis of DMF to MMF by various types of blood cells. Monocytes/lymphocytes, granulocytes, and erythrocytes
`were purified from blood of healthy volunteers using centrifugational techniques. Next, the various cell types were resus-
`pended in PBS pH 7.4 to concentrations present in whole blood, e.g. 1 × 106/mL monocytes/lymphocytes (A), 5 × 106/mL gran-
`ulocytes (B) and 5 × 109/mL erythrocytes (C), and then DMF was added to a final concentration of 2 mg/L. At various intervals
`thereafter samples were collected and the MMF (squares) and DMF (circles) concentrations were measured using HPLC.
`Results are a representative experiment of 3–4 independent experiments.
`
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`DMF, MMF and MEF in acidic and alkaline environments
`To investigate the stability of DMF, MMF and MEF and the
`hydrolysis of DMF to MMF in several environments repre-
`senting 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 concen-
`trations of DMF, MMF and MEF by high-performance liq-
`uid chromatography (HPLC) as described below.
`
`To further investigate the effect of the pH on the hydroly-
`sis 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 proce-
`dure was necessary and the concentrations of the various
`FAE in the samples could be directly quantified by HPLC
`(see below).
`
`Concentrations of DMF, MMF and MEF in serum and
`whole blood
`As described above, serum and whole blood from 3 vol-
`unteers 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 pre-
`pared 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 concen-
`trations were measured by HPLC (see below).
`
`Effects of purified blood cell fractions on the hydrolysis of
`DMF in PBS (pH 7.4)
`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 Nether-
`lands) 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 Hom-
`burg, 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-Amidotri-
`zoate 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 concen-
`trations of the various FAE were measured as described
`below.
`
`Sample preparation and HPLC analysis
`The concentrations of the various fumarates in serum
`samples were determined as described (Litjens et al., sub-
`mitted 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 meta-
`phosphoric 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 metha-
`nol: 0.1 M potassium phosphate buffer (KH2PO4/
`K2HPO4; pH 7.5) supplemented with 5 mM tetrabutylam-
`monium 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, Bel-
`gium) 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: potas-
`sium 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 con-
`centrations of FAE in the various samples of these buffers
`and human serum or whole blood, respectively.
`
`Authors' contributions
`NL participated in the design of the study, analysis of the
`data and drafted the manuscript. ES carried out the opti-
`misation of the HPLC method and performed all HPLC
`
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`analyses. CvG was responsible for the optimisation of the
`HPLC method and participated in the design of the study.
`HM participated in the design of the study and analysis of
`the data. JvD, HT and PN conceived of the study and par-
`ticipated in its design and coordination. All authors read
`and approved the final manuscript.
`
`Acknowledgements
`We would like to thank Dr. J. Tio (TioFarma) for providing the enteric-
`coated tablets and purified components of the tablet. This study was finan-
`cially supported by a grant from AstraZeneca R&D Charnwood, Loughbor-
`ough, UK).
`
`19.
`
`ylfumarate on human granulocytes. Eur J immunol 1996,
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`Litjens NHR, Rademaker M, Ravensbergen B, Rea D, van der Plas
`MJA, Thio B, Walding A, van Dissel JT, Nibbering PH: Monomethyl-
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