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Synthesis of acylglucuronides of drugs using immobilized dog liver microsomes octadecylsilica particles coated with phospholipid
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 317 (2003) 99–106 www.elsevier.com/locate/yabio
Synthesis of acylglucuronides of drugs using immobilized dog liver microsomes octadecylsilica particles coated with phospholipid Hiroshi Kamimori,* Yoshihisa Ozaki, Yoshito Okabayashi, Kyoji Ueno, and Shigeru Narita Shionogi Research Laboratories, Shionogi & Co., Ltd., 12-4, Sagisu 5-chome, Fukushima-ku, Osaka 553-0002, Japan Received 27 December 2002
Abstract Immobilized dog liver microsome octadecylsilica (ODS) particles coated with phospholipid were developed for the synthesis of acylglucuronides of drugs. The phospholipid-coated ODS particles were readily prepared by stirring a solution containing L -a-dipalmitoylphosphatidylcholine with the ODS particles, in which the phospholipid was absorbed on the ODS surfaces by hydrophobic interaction between the acyl group of phospholipid and the otcadecyl group of the ODS particles. Similarly, the microsome-immobilized particles were readily prepared by stirring a buffer solution containing dog liver microsomes with the phospholipid-coated ODS particles, in which the microsomes were immobilized on the phospholipid-coated ODS particles by hydrophobic binding. The microsome-immobilized particles exhibited UDP-glucuronosyltransferase activity which catalyzed the glucuronidation of ketoprofen and a nonpeptide endothelin receptor antagonist, S-1255 ([R]-[+]-2-[benzo(1,3)dioxol-5-yl]-6isopropyl-4-[4-methoxyphenyl]-2H-chromene-3-carboxylic acid), to the corresponding acylglucuronide in the presence of uridine 50 -diphosphate (UDP)-glucuronic acid, and two acylglucuronides of ketoprofen and S-1255 were synthesized using the microsomeimmobilized particles. These acylglucuronides were synthesized by simply shaking the microsome-immobilized particles adsorbed on the substrate in a buffer solution containing UDP-glucuronic acid with a thermostated mixer. The molecular weights and chemical structures of the synthesized acylglucuronides were identified by mass spectrometry and nuclear magnetic resonance, respectively. The productivity of S-1255 acylglucuronide using microsome-immobilized particles was approximately threefold higher than that observed with free microsomes, whereas the ketoprofen acylglucuronide productivity was slightly lower than that observed with free microsomes. The present method should be very useful for the synthesis of acylglucuronides of drugs, which are slightly soluble aqueous solutions in the drug development stage. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Acylglucuronide; Microsome-immobilized ODS particles coated with phospholipid; Dog liver microsomes; Glucuronidation; UDPglucuronosyltransferase; UDP-glucuronic acid; Ketoprofen; S-1255
Glucuronidation is one of the main metabolic pathways for drugs in vivo, and it is important to investigate the pharmacokinetics of glucuronides as metabolites of drugs in order to understand the metabolic routes of the target drugs during the course of preclinical or clinical drug development stages. The reaction in vivo is catalyzed by uridine diphosphate glucuronosyltransferase
(UDPGT, EC 2.3.1.17)1 which is mainly located in the liver and other tissues. Standards of glucuronides of drugs are often needed for pharmacokinetics or pharmacological study of drugs. Glucuronides can be obtained by isolation and purification of metabolites from biological samples (urine, bile), 1
* Corresponding author. Fax: +81-6-6458-0987. E-mail address: [email protected] (H. Kamimori).
Abbreviations used: UDPGT, uridine diphosphate glucuronosyltransferase; ODS, octadecylsilica; HPLC, high-performance liquid chromatography; UDPGA, uridine 50 -diphosphate-glucuronic acid; DPPC, L -a-dipalmitoylphosphatidycholine; ESI-MS, electrospray ionization mass spectra; NMR, nuclear magnetic resonance; HMBC, heteronuclear correlation optimized on a long-range coupling; UV, ultraviolet.
0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00111-8
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although their extraction is usually difficult and time consuming. The means to obtain glucuronides via chemical synthesis is often limited when unstable glucuronides are considered, or when multistep synthesis is required [1]. It is particularly difficult to find the specific hydrolysis of the hydroxyl- or carboxyl-protecting group of glucuronic acid conjugated to the aglycone when the acylglucuronides are synthesized by chemical means. To overcome the disadvantages of synthesizing glucuronides by chemical means, biosynthesis using free microsomes by in vitro incubation to obtain standard glucuronides is used. However, this in vitro method entails a troublesome procedure such as protein precipitation in order to obtain glucuronides from the mixture after incubation. Also this method could hardly be applied to the synthesis of glucuronides for slightly soluble drugs because their solubility would be limited in the incubation mixture. Alternative methods of producing glucuronides such as using microsomes entrapped in alginate beads [1] and covalently immobilized microsomes on Sepharose 4B beads [2–5] have been demonstrated. The productivity of glucuronides using the beads increased compared to those observed with free microsomes, since the UDPGT activity of immobilized microsomes on the beads stabilized. However, these biosyntheses did not seem to be the simplest means of obtaining the glucuronides, because the preparation of microsomes immobilized or entrapped in beads is not very convenient. We have previously prepared octadecylsilica (ODS) coated with phospholipids as a model of lipid membranes on a high-performance liquid chromatography (HPLC) column (phospholipid column), employing a dynamic coating technique developed in our laboratories [6]. We have also confirmed that the phospholipid column is a suitable support for enzyme immobilization and the immobilized enzyme phospholipid column is useful as an enzyme reactor [7]. In this work, we prepared immobilized dog liver microsome ODS particles coated with phospholipid (microsome-immobilized particles) for an efficient and useful synthesis of acylglucuronides of drugs. Here we are focusing on the preparation of microsome-immobilized particles employing a dynamic coating technique and the stability of the UDPGT activity of microsomes immobilized on the ODS particles coated with phospholipid. The immobilized microsomal UDPGT on the particles was expected to be stabilized by hydrophobic binding to the phospholipid on the particles compared to that of free microsomes. This paper describes the preparation of the microsome-immobilized particles and the means of synthesizing acylglucuronides using the particles. The acylglucuronidation using the particles was optimized with ketoprofen (Fig. 1) as a model compound. The present method was also used to synthesize the acylglucuronide of S-1255 ([R]-[+]-2-[benzo(1,3)dioxol-5-yl]-6-
Fig. 1. Chemical structures of ketoprofen and S-1255.
isopropyl-4-[4-methoxyphenyl]-2H-chromene-3-carboxylic acid) (Fig. 1) [8,9,12], which has been originally developed as a nonpeptide endothelin receptor antagonist in our laboratories. The microsome-immobilized particles and free microsomes were compared with respect to their productivity of acylglucuronides. Materials and methods Materials ODS particles (Cosmosil 75C18 -PREP, 75-lm particle size) and uridine 50 -diphosphate-glucuronic acid (UDPGA) trisodium salt were obtained from Nacalai Tesque (Kyoto, Japan). L -a-Dipalmitoylphosphatidycholine (DPPC) and ketoprofen were purchased from Sigma Chemical Co. (St. Louis, MO). S-1255 was synthesized in our laboratories. Acetonitrile, methanol, water, and trifluoroacetic acid were of HPLC grade and all other chemicals were of reagent grade. An empty reservoir equipped with a 20-lm frit (capacity: 4 or 75 ml) for the solid-phase extraction used was purchased from Varian (Harbor City, CA). Apparatus The HPLC system consisted of a Shimadzu LC-10AD pump (Kyoto, Japan), a Shimadzu SPD-10AV spectrophotometer or a Waters 996 photodiode array detector (Milford, MA), and a Shimadzu SIL-9A automatic injector. The chromatographic data were analyzed using a Shimadzu C-R7A data processor or Waters Millennium 32 software. An Eppendorf Thermomixer confort (Hamburg, Germany) thermostatic mixer was used. Electrospray ionization mass spectra (ESI-MS) were recorded on an Applied Biosystems API 3000 (Foster City, CA). Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Unity-600 spectrometer at 599.9 MHz (1 H) and 150.9 MHz (13 C) in CD3 OD. 1 H and 13 C signals were assigned on the basis of the observable 1 H–1 H correlation and long-range 1 H–13 C correlation in homonuclear correlation spectroscopy and heteronuclear correlation optimized on long-range coupling (HMBC) spectra, respectively.
H. Kamimori et al. / Analytical Biochemistry 317 (2003) 99–106
HPLC system The HPLC column used was an YMC pack Pro C18 (250 10 mm i.d., 5 lm, YMC, Kyoto, Japan). The mobile phases used were acetonitrile/0.1% trifluoroacetic acid solution (50/50, v/v) or acetonitrile/methanol/0.1% trifluoroacetic acid solution (50/30/20, v/v/v) at a flow rate of 2 ml/min, and the detector was set at a wavelength of 300 nm. Preparation of dog liver microsomes Dog liver samples were removed from female beagle dogs without the treatment of drugs in our laboratories. Dog liver microsomes were prepared as described previously [10] and the microsomal pellets obtained were stored in a freezer at )80 °C until use. The microsomal protein concentration was determined using the Micro Bicinchoninic Acid Protein Assay Reagent Kit (Pierce, Rockford, IL) with bovine serum albumin as standard [11]. The microsomal protein concentration used in this study was approximately 20 mg/ml. Preparation of microsome-immobilized particles The procedure was done under an ambient temperature of 25 1 °C. To prepare phospholipid-coated ODS particles, a solution of 56 mg of DPPC dissolved in 75 ml of 80% methanol was stirred with 0.1 g of ODS particles for 40 min in a beaker. After stirring, the solution containing the ODS particles was filtered through the empty reservoir, and the particles were collected. They were then sufficiently washed with 5 ml of water followed by 5 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4). Microsome-immobilized particles were prepared by adding to 200 ll of microsomes (protein content approximately 4 mg), 20 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4). The suspension was stirred with the phospholipid-coated particles prepared from 0.1 g of ODS particles for 60 min in a beaker. After stirring, the suspension containing the particles was filtered through the empty reservoir, and the microsome-immobilized particles were collected. Synthesis of acylglucuronide using microsome-immobilized particles To microsome-immobilized particles prepared from 0.1 g of ODS particles 20 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4) containing 10 lmol of substrate was added, and the mixture was stirred for 5 min in a beaker. After this, the mixture containing the particles was filtered through the empty reservoir, and the particles absorbed with substrate were collected. To the particles, 1 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4)
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containing 50 mM UDPGA and 10 mM magnesium chloride was added in a polypropylene microcentrifuge tube, which was then shaken with a thermostatic mixer at 37 °C for 6–24 h. After stirring, the mixture containing the particles was filtered through the empty reservoir and the desired fraction was eluted from the particles with 10 ml of 80% methanol for a few times. The eluates were injected several times onto the HPLC system and the peak fractions corresponding to acylglucuronide were collected. The fraction solutions were evaporated and lyophilized. Evaluation of preparation of microsome-immobilized particles The amount of DPPC coating the ODS particles was calculated from the difference between DPPC concentrations of the solution before and after the preparation procedure. DPPC concentrations in the coating solution were determined by HPLC with ultraviolet (UV) detection as described previously [6]. The amount of immobilized microsomes on the phospholipid-coated particles was estimated as the protein content. This was calculated from differences in the concentrations of the solutions containing microsomes before and after preparing the immobilized microsomes. The concentrations of the solutions were determined using the Micro Bicinchoninic Acid Protein Assay Reagent Kit as described above. Evaluation of production of acylglucuronide The production of acylglucuronides using the microsome-immobilized particles was calculated from the peak area of the formed products compared with that of the standard solution injected onto the HPLC system under the same analysis conditions. No significant UV spectral shift between each acylglucuronide and its substrate was confirmed by HPLC with photodiode array detection. Also, the productivity of acylglucuronides was expressed as the production per milligram of the immobilized microsomal protein on the particles. The productivity of acylglucuronide observed with free microsomes was estimated in the same manner.
Results and discussion Evaluation of preparation of microsome-immobilized particles The coating amount of DPPC on the ODS particles (0.1 g) was calculated to be approximately 20 mg. The amount of microsomes immobilized on the phospholipidcoated particles prepared from 0.1 g of ODS particles was calculated to be approximately 2 mg as the protein
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content. As a result, the microsome-immobilized particles could readily be prepared by stirring the particles with the solution containing the desired ligands in a beaker. Identification of ketoprofen acylglucuronide Ketoprofen acylglucuronide is produced from ketoprofen with dog liver microsomes by in vitro incubation in the presence UDPGA [12]. We tried synthesizing the ketoprofen acylglucuronide using microsome-immobilized particles. As the eluate from the microsome-immobilized particles was injected onto the HPLC system, the peak corresponding to the acylglucuronide was detected around a retention time of 9 min while the peak of ketoprofen was detected around a retention time of 22 min (data not shown). The peaks of keprofen and its acylglucuronide could be completely separated under the present HPLC conditions. The production obtained from the fraction solution corresponding to the peak of the acylglucuronide was identified by ESI-MS and NMR. Analysis by ESI-MS of the production revealed the presence of molecular ions at m=z 255 ½M þ H 176þ and m=z 431 ½M þ Hþ , corresponding to the molecular weights of ketoprofen and its acylglucuronide, respectively. The relative intensities of peaks of molecular ions were 40% for ketoprofen acylglucuronide, compared with 100% for ketoprofen. Fig. 2 shows the chemical structure of ketoprofen acylglucuronide, and the 1 H and 13 C NMR chemical
Fig. 2. Chemical structure of ketoprofen acylglucuronide. The arrow shows the long-range 1 H–13 C correlation.
shifts of ketoprofen and its acylglucuronide synthesized using microsome-immobilized particles are summarized in Table 1. The 1 H signals between 7.5 and 7.8 ppm, the doublet at 1.537 ppm, and the quartet at 3.964 ppm were assigned to protons of ketoprofen. The H-2 of ketoprofen (d3.819) was shifted downfield to d3.964 by substitution of the molecule. The 1 H signals of d5.499 (d), d3.332 (t), d3.414 (t), d3.497 (br), and d3.868 (br) were assigned to H-10 , H-20 , H-30 , H-40 , and H-50 of glucronic acid, respectively. The H-10 anomeric proton, at 5.499 ppm, has a coupling constant of 8.0 Hz, which is characteristic of diaxial coupling in the b-glucuronic acid [13,14]. As the H-10 proton of free b-glucuronic acid is known to display a chemical shift of 4.7 ppm, the chemical downfield shifts at H-10 suggested acylation of 1-OH of ketoprofen [13,14]. Also, long-range 1 H–13 C correlation was observed between d5.499 (b-glucuronic acid anomeric proton) and 174.22 (C-1 of ketoprofen) in the HMBC spectrum. The correlation proved that C-1
Table 1 1 H and 13 C NMR chemical shifts of ketoprofen and ketoprofen acylglucuronide Positiona
Ketoprofen
Ketoprofen acylglucuronide J/Hz
dH 1 2 2-CH3 3 4 5 6 7 8 10 20 30 40 50 60 5-CO 100 200 & 600 300 & 500 400 a
dC
—
—
—
—
—
—
—
—
—
—
—
—
7.776 7.573 7.653
198.45 138.83 131.07 129.57 133.89
198.19 138.60 131.01 129.46 133.76
q d
7.2 7.2
7.744
t
1.6
7.658 7.499 7.617
brd t dt
8 7.7 7.6, 1.6
d-like t-like m
8 8
177.74 46.51 19.00 143.13 130.19 139.10 129.81 129.78 133.10
J/Hz
dH
174.22 46.44 19.11 141.82 130.27 138.99 129.79 129.71 133.16 95.86 73.62b 77.68 72.99b NDc 174.22
3.819 1.479
The number of positions is indicated in Fig. 2. Exchangeable. c Not detected. b
dC
3.964 1.537
q d
7.2 7.2
7.744
t
1.8
7.672 7.503 7.642 5.499 3.332 3.414 3.497 3.868
brd t m d d brt br br
8 7.7
7.788 7.544 7.654
d-like t-like m
8.0 8.4 9
8 8
H. Kamimori et al. / Analytical Biochemistry 317 (2003) 99–106
of ketoprofen was linked to C-10 of b-glucuronic acid through the oxygen atom [13]. As a result, the chemical structure of ketoprofen acylglucuronide synthesized using the particles could be completely identified using ESI-MS and NMR. Since the ketoprofen acylglucuronide could be synthesized using the microsome-immobilized particles, we next tried to optimize the acylglucuronidation with ketoprofen as the substrate using the microsomeimmobilized particles. Optimization of glucuronidation using microsome-immobilized particles Time of microsome immobilization on the phospholipid-coated particles. Fig. 3 shows the relationship between the time of microsome immobilization on the phospholipid-coated particles and the productivity of ketoprofen acylglucuronide. As the stirring time of microsome immobilization increased, the amount of immobilized microsomal protein on the phospholipidcoated particles and the productivity of ketoprofen acylglucuronide increased. The amount of microsomal protein reached a plateau when the time of microsome immobilization was more than 60 min, although the productivity decreased somewhat at an immobilization time of 120 min. As a result, the time of microsome immobilization on the phospholipid-coated particles was set at 60 min when the microsomes were efficiently immobilized on the particles for the most suitable productivity of glucuronidation.
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Effect of support material for microsome immobilization on the productivity of ketoprofen acylglucuronide. To estimate the effect of support material for microsome immobilization on the productivity of ketoprofen acylglucuronide, the immobilized microsome ODS particles were prepared by the same procedure and the ketoprofen acylglucuronide was synthesized using the particles. As shown in Table 2, the productivity of ketoprofen acylglucuronide using the phospholipid-coated particles as the support was approximately threefold higher than that obtained using the ODS particles while the amount of immobilized microsomal protein was almost the same between the supports. Thus, phospholipid as a coating support stabilized the UDPGT in the immobilized microsomes and its immobilization produced intrinsic UDPGT activity, compared to the ODS particles not coated with phospholipid. These findings suggested that the phospholipid-coated particles could be an efficient support for microsome immobilization. Concentration of UDPGA and enzyme reaction time. The enzyme reaction time was investigated to optimize the glucuronidation using the microsome-immobilized particles in the presence of 10–100 mM UDPGA of the enzyme reaction mixture. Fig. 4 shows the relationship among the concentration of UDPGA, the enzymatic reaction time, and the productivity of ketoprofen acylglucuronide. The results indicated that the maximum productivity of ketoprofen acylglucuronide was approximately 370 nmol/mg protein when the reaction condition was at 37 °C for 6 h in the presence of 50 mM of UDPGA. Reproducibility of synthesis of ketoprofen acylglucuronide To test the reproducibility of synthesis of ketoprofen acylglucuronide using the microsome-immobilized particles, the ketoprofen acylglucuronide was synthesized in triplicate over two runs. The amount of immobilized
Table 2 Effect of support material for microsome immobilization on productivity of ketoprofen acylglucuronide
Fig. 3. Relationship between time of microsome immobilization on the phospholipid-coated particles and the productivity of ketoprofen acylglucuronide. The microsome-immobilized particles were prepared from the phospholipid-coated ODS particles (0.1 g). Ketoprofen acylglucuronide was synthesized using the microsome-immobilized particles prepared from 0.1 g of ODS particles in 1 ml of 50 mM Trishydrochloric acid buffer (pH 7.4) containing 50 mM UDPGA and 10 mM magnesium chloride at 37 °C for 2 h.
Support material for microsome
Amount of immobilized microsomal protein on support material (mg)a
Productivity of ketoprofen acylglucuronide (nmol/mg protein)b
ODS particles Phospholipid-coated particles
2.98 3.02
59 165
a
The microsome-immobilized particles were prepared from the phospholipid-coated ODS particles (0.1 g) or ODS particles (0.1 g). b Ketoprofen acylglucuronide was synthesized using the microsome-immobilized particles prepared from 0.1 g of ODS particles or the phospholipid-coated ODS particles (0.1 g) in 1 ml of 50 mM Trishydrochloric acid buffer (pH 7.4) containing 50 mM UDPGA and 10 mM magnesium chloride at 37 °C for 2 h.
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Synthesis of S-1255 acylglucuronide using microsomeimmobilized particles
Fig. 4. Relationship among the concentration of UDPGA, enzymatic reaction time, and the productivity of ketoprofen acylglucuronide. Key: 10 mM UDPGA (j), 50 mM UDPGA (d), 100 mM UDPGA (s). The microsome-immobilized particles were prepared from the phospholipid-coated ODS particles (0.1 g). Ketoprofen acylglucuronide was synthesized using the microsome-immobilized particles prepared from 0.1 g of ODS particles in 1 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4) containing 10, 50, or 100 mM UDPGA and 10 mM magnesium chloride at 37 °C for 1–24 h.
Table 3 Reproducibility of synthesis of ketoprofen acylglucuronide using the microsome-immobilized particles Run no.
Amount of immobilized microsomal protein (mg)a
Productivity of ketoprofen acylglucuronide (nmol/mg protein)b
1
1.49 1.19 1.94 1.54 0.38
528 414 542 495 70
Mean (n ¼ 3) SD
2.48 2.33 2.31 2.37 0.09
248 231 238 239 9
Total Mean (n ¼ 6) SD
1.96 0.52
367 147
Mean (n ¼ 3) SDc 2
S-1255 (Fig. 1) was originally developed in our laboratories as a nonpeptide endothelin receptor antagonist, and glucuronidation is its major metabolite pathway in dogs [15]. We tried to synthesize S-1255 acylglucuronide using the microsome-immobilized particles by the same procedure. The eluate obtained from the particles was injected onto the HPLC system, the peak fractions corresponding to acylglucuronide were collected, and the fraction solutions were evaporated and lyophilized. The production was identified by MS and NMR. The analysis by ESI-MS of the production revealed the presence of the molecular ions at m=z 459 ½M H 176 and m=z 653 ½M H , corresponding to the molecular weights of S-1255 and its acylglucuronide, respectively. The relative intensities of peaks of molecular ions were 15% for S-1255, compared with 100% for S-1255 acylglucuronide. Fig. 5 shows the chemical structure of S-1255 acylglucuronide, and the 1 H and 13 C NMR chemical shifts of S-1255 and its acylglucuronide synthesized using microsome-immobilized particles are summarized in Table 4. The 1 H signals of d5.387 (d), d2.882 (dd), d3.319 (t), d3.383 (t), and d3.721 (d) were assigned to H10 , H-20 , H-30 , H-40 , and H-50 of glucronic acid, respectively. The H-10 anomeric proton, at 5.387 ppm, has a coupling constant of 8.1 Hz, which is characteristic of a diaxial coupling in the b-glucuronic acid [13,14]. Also, the chemical downfield shifts at H-10 of b-glucuronic acid proposed acylation of 1-OH of S-1255 [13,14] as the result of ketoprofen acylglucuronide. Long-range 1 H–13 C correlation was observed between d5.387 (bglucuronic acid anomeric proton) and 165.68 (3-COO of S-1255) in the HMBC spectrum. The correlation proved that 3-COO of S-1255 was linked to C-10 of b-glucuronic acid through the oxygen atom [13]. As a result, the chemical structure of S-1255 acylglucuronide synthe-
a The microsome-immobilized particles were prepared from the phospholipid-coated ODS particles (0.1 g). b Ketoprofen acylglucuronide was synthesized using the microsomeimmobilized particles prepared from 0.1 g of ODS particles in 1 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4) containing 50 mM UDPGA and 10 mM magnesium chloride at 37 °C for 6 h. c Standard deviation.
microsomal protein, productivity of ketoprofen acylglucuronide, and their standard deviations were calculated. As shown in Table 3, the results indicated that the reproducibility of this procedure was acceptable enough in view of the enzyme reaction.
Fig. 5. Chemical structure of S-1255 acylglucuronide. The arrow shows the long-range 1 H–13 C correlation.
H. Kamimori et al. / Analytical Biochemistry 317 (2003) 99–106
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Table 4 1 H and 13 C NMR chemical shifts of S-1255 and S-1255 acylglucuronide Positiona
S-1255
S-1255 acylglucuronide J/Hz
dH 2 3 4 4a 4b 5 6 7 8 3-COO 10 20 30 40 50 60 100 200 300 400 500 600 OCH2 O 1000 2000 and 6000 3000 and 5000 4000 4000 -OCH3 OCH2 ðCH3 Þ2
a b
6.125
6.224 6.733 6.704
s
d dd d
2.8 8.7, 2.8 8.7
dC
dH
76.51 123.84 145.38 126.38 148.53 116.65 153.36 120.53 118.68 168.92
6.165
—
—
—
—
—
—
—
—
—
—
—
—
6.948
brd
0.8
6.722 6.955 5.881 5.888
dd d-like d d
7.8, 0.8 8 1.3 1.3
7.17 6.992
br m
3.836 4.188 1.117 1.140
s m d d
6.0 6.0
J/Hz s
— — — —
6.265
d
2.8
dd d
8.7, 2.8 8.7
5.387 2.882 3.319 3.383 3.721
d dd t t d
8.1 9.1, 8.1 9.1 9.4 9.5
6.969
brs
6.725 6.978 5.891 5.896
d d-like d d
7.21 7.018
br brd
8
3.869 4.204 1.133 1.153
s m d d
6.0 6.0
—
6.786 6.749 —
134.26 109.13 149.20 149.20 108.81 122.57 102.51 130.34 131.3 (br) 114.60 161.01 55.75 71.79 22.17 22.33
7.8 8 1.3 1.3
dC 76.11 121.87 148.10 126.29 149.02 116.93 153.48 121.50 118.95 165.68 95.62 73.63 77.66 72.90 77.03 172.95 134.15 109.16 149.20b 149.22b 108.83 122.64 102.50 129.99 131.7 (br) 114.80 161.38 55.93 72.02 22.18 22.36
The number of positions is indicated in Fig. 5. Exchangeable.
sized using the particles has been completely identified using ESI-MS and NMR. The maximum productivity of S-1255 acylglucuronide was approximately 1400 nmol/mg protein using the microsome-immobilized particles (in the presence of 50 mM UDPGA, at 37 °C for 24 h). Comparison of productivity of glucuronidation with free microsomes The productivities of acylglucuronides of ketoprofen and S-1255 as the substrates using the microsome-immobilized particles were compared to those observed with free microsomes. The productivities of two acylglucuronides obtained using the free microsomes are indicated under the optimum conditions using the same content of microsomes immobilized or substrate adsorbed on the particles. As shown in Fig. 6, the productivity of S-1255 acylglucuronide using the microsome-immobilized particles was approximately threefold higher than that observed with the free
microsomes, although ketoprofen acylglucuronide productivity was slightly lower than that observed with the free microsomes. We assume that the significant difference of S-1255 acylglucuronide productivity between the microsome-immobilized particles and the free microsomes would be caused by the difference of the concentration of substrate in the enzymatic reaction mixture. In the case of the synthesis of glucuronides with free microsomes, the concentration of substrate in the mixture was limited in order to effectively promote the glucuronidation as a higher concentration would inhibit the enzyme activity. The maximum productivity of S1255 acylglucuronide with the free microsomes was obtained when the concentration of S-1255 in the mixture was approximately 1.0 lmol/ml. On the other hand, in the case of the microsome-immobilized particles, the glucuronidation could be sufficiently promoted using the particles adsorbed on the excessive S-1255, whose concentration in the mixture was approximately 9.8 lmol/ ml. The excessive concentration of substrate in the mixture would not inhibit the enzyme activity because
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References
Fig. 6. Comparison of the productivity of acylglucuronides of ketoprofen and S-1255 by microsome-immobilized particles and free microsomes. Microsome-immobilized particles: ketoprofen; adsorbed substrate on the particles, 3.5 lmol; immobilized microsomal protein, 2.00 mg; S-1255; adsorbed substrate on the particles, 9.8 l mol; immobilized microsomal protein, 2.83 mg. Free microsomes: ketoprofen; added substrate to the enzymatic reaction mixture, 3.5 lmol; microsomal protein, 2.00 mg; S-1255; added substrate to the enzymatic reaction mixture, 1.0 lmol; microsomal protein, 2.80 mg. Enzymatic reaction mixture: 1 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4) containing 50 mM UDPGA and 10 mM magnesium chloride; reaction conditions, 37 °C, 6–24 h.
its substrate was adsorbed on the particles. As a result, the production of S-1255 acylglucuronide with the particles increased compared to that observed with the same protein content of free microsomes. These findings indicated that the immobilized microsomal UDPGT on the particles was stabilized and the microsomes immobilized on the particles could be efficiently utilized for the synthesis of acylglucuronides, even when there was an excessive concentration of substrate in the mixture. Therefore, this method should be especially useful for the synthesis of acylglucuronides of drugs that are slightly soluble in aqueous solution. Conclusion Acylglucuronides of ketoprofen and S-1255 were synthesized using microsome-immobilized particles. The microsome-immobilized particles can readily be prepared by stirring the particles with a solution containing the desired ligands, and acylglucuronide can easily be synthesized by stirring the microsome-immobilized particles absorbed on substrate in a buffer solution containing UDPGA. The productivity of S-1255 acylglucuronide using microsome-immobilized particles was approximately threefold higher than that observed with free microsomes. The present method should be helpful for obtaining the standards of acylglucuronides, ether glucuronides or other metabolites of drugs, which are slightly soluble in aqueous solution or cannot be synthesized by chemical means for drug development stages.
[1] M. Haumont, J. Magdalou, J.C. Ziegler, R. Bidault, J.P. Siest, G. Siest, Immobilization of microsomes into alginate beads is a convenient method for producing glucuronides from drugs, Appl. Microbiol. Biotechnol. 35 (1991) 440–446. [2] R.A.H.J. Gillissen, J.H.N. Meerman, G.J. Mulder, Immobilization of solubilized UDP-glucuronosyltransferase from rat liver microsomes to Sepharose 4B, Biochem. Pharmacol. 43 (1992) 2661–2662. [3] J.P. Lehman, L. Ferrin, C. Fenselau, S.Y. Garold, Simultaneous immobilization of cytochrome P-450 and glucuronyltransferase for synthesis of drug metabolite, Drug Metab. Dispos. 9 (1981) 15–18. [4] I. Parkin, D.W. MacGlashan, C. Fenselau, Immobilized glucuronosyltranferase for the synthesis of conjugates, J. Med. Chem. 19 (1976) 296–299. [5] C. Fenselau, S. Pallante, I. Parkin, Solid-phase synthesis of drug glucuronides by immobilized glucuronosyltransferase, J. Med. Chem. 19 (1976) 679–683. [6] H. Kamimori, M. Konishi, Evaluation and application of liquid chromatographic columns coated with ÔintelligentÕ ligands. II. Phospholipid column, Biomed. Chromatogr. 16 (2002) 61–67. [7] H. Kamimori, M. Konishi, Evaluation and application of liquid chromatographic columns coated with ‘‘Intelligent’’ ligands. (III). Immobilized enzyme phospholipid column, Anal. Sci. 17 (2001) 1085–1089. [8] T. Iwasaki, S. Mihara, T. Shimamura, M. Kawakami, M. Masui, Y. Hayasaki-Kawajima, N. Naya, M. Ninomiya, M. Fujimoto, M. Nakajima, Pharmacologic characterization of S-1255, a Highly potent and orally active endothelin A receptor antagonist, J. Cardiovasc. Pharmacol. 37 (2001) 471–482. [9] N. Ishizuka, K. Matsumura, K. Sakai, M. Fujimoto, M. Mihara, T. Yamamori, Structure-activity relationships of a novel class of endothelin-A receptor antagonists and discovery of potent and selective receptor antagonist, 2-(benzo[1,3]dioxol-5-yl)-6-isopropyloxy-4-(4-methoxyphenyl)-2H-chromene-3-carboxylic acid (S-1255). 1. Study on structure-activity relationships and basic structure crucial for ETA antagonism, J. Med. Chem. 45 (2002) 2041–2055. [10] T. Matsubara, E. Yoshihara, T. Iwata, Y. Tochino, Y. Hachino, Biotransformation of coumarin derivatives. (1) 7-Alkoxycoumarin O-dealkylase in liver microsomes, Jpn. J. Pharmacol. 32 (1983) 9–21. [11] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Measurement of protein using bicinchoninic acid, Anal. Biochem. 150 (1985) 76–85. [12] M.G. Soars, R.J. Riley, K.A.B. Findlay, M.J. Coffey, B. Burchell, Evidence for significant difference in microsomal drug glucuronidation by canine and human liver and kidney, Drug Metab. Dispos. 29 (2001) 121–126. [13] N. Azaroual, M. Imbenotte, B. Cartigny, F. Leclerd, L. Vallee, M. Lhermitte, G. Vermeersch, Valpronic acid intoxication identified by 1 H and 1 H–13 C correlated NMR spectroscopy of urine samples, Magn. Reson. Mater. Phys. Biol. Med. 10 (2000) 177–182. [14] R.W. Mortensen, O. Corcoran, C. Cornett, U.G. Sidelmann, J. Troke, J.C. Lindon, J.K. Nicholson, S.H. Hansen, LC-1 H NMR used for determination of the elution order of S-naproxen glucuronide isomers in two isocratic reversed-phase LC-systems, J. Pharm. Biomed. Anal. 24 (2001) 477–485. [15] C. Fujimori, S. Futaguchi, E. Sakurai, S. Sakamoto, T. Akamatsu, T. Ohkawa, T. Matsumoto, K. Iwatani, K. Sakamoto, M. Koike, Metabolism of S-1255 in rats and dogs, Xenobio. Metabol. Dispos. 15 (Suppl.) (2000).
Synthesis of acylglucuronides of drugs using immobilized dog liver microsomes octadecylsilica particles coated with phospholipid Hiroshi Kamimori,* Yoshihisa Ozaki, Yoshito Okabayashi, Kyoji Ueno, and Shigeru Narita Shionogi Research Laboratories, Shionogi & Co., Ltd., 12-4, Sagisu 5-chome, Fukushima-ku, Osaka 553-0002, Japan Received 27 December 2002
Abstract Immobilized dog liver microsome octadecylsilica (ODS) particles coated with phospholipid were developed for the synthesis of acylglucuronides of drugs. The phospholipid-coated ODS particles were readily prepared by stirring a solution containing L -a-dipalmitoylphosphatidylcholine with the ODS particles, in which the phospholipid was absorbed on the ODS surfaces by hydrophobic interaction between the acyl group of phospholipid and the otcadecyl group of the ODS particles. Similarly, the microsome-immobilized particles were readily prepared by stirring a buffer solution containing dog liver microsomes with the phospholipid-coated ODS particles, in which the microsomes were immobilized on the phospholipid-coated ODS particles by hydrophobic binding. The microsome-immobilized particles exhibited UDP-glucuronosyltransferase activity which catalyzed the glucuronidation of ketoprofen and a nonpeptide endothelin receptor antagonist, S-1255 ([R]-[+]-2-[benzo(1,3)dioxol-5-yl]-6isopropyl-4-[4-methoxyphenyl]-2H-chromene-3-carboxylic acid), to the corresponding acylglucuronide in the presence of uridine 50 -diphosphate (UDP)-glucuronic acid, and two acylglucuronides of ketoprofen and S-1255 were synthesized using the microsomeimmobilized particles. These acylglucuronides were synthesized by simply shaking the microsome-immobilized particles adsorbed on the substrate in a buffer solution containing UDP-glucuronic acid with a thermostated mixer. The molecular weights and chemical structures of the synthesized acylglucuronides were identified by mass spectrometry and nuclear magnetic resonance, respectively. The productivity of S-1255 acylglucuronide using microsome-immobilized particles was approximately threefold higher than that observed with free microsomes, whereas the ketoprofen acylglucuronide productivity was slightly lower than that observed with free microsomes. The present method should be very useful for the synthesis of acylglucuronides of drugs, which are slightly soluble aqueous solutions in the drug development stage. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Acylglucuronide; Microsome-immobilized ODS particles coated with phospholipid; Dog liver microsomes; Glucuronidation; UDPglucuronosyltransferase; UDP-glucuronic acid; Ketoprofen; S-1255
Glucuronidation is one of the main metabolic pathways for drugs in vivo, and it is important to investigate the pharmacokinetics of glucuronides as metabolites of drugs in order to understand the metabolic routes of the target drugs during the course of preclinical or clinical drug development stages. The reaction in vivo is catalyzed by uridine diphosphate glucuronosyltransferase
(UDPGT, EC 2.3.1.17)1 which is mainly located in the liver and other tissues. Standards of glucuronides of drugs are often needed for pharmacokinetics or pharmacological study of drugs. Glucuronides can be obtained by isolation and purification of metabolites from biological samples (urine, bile), 1
* Corresponding author. Fax: +81-6-6458-0987. E-mail address: [email protected] (H. Kamimori).
Abbreviations used: UDPGT, uridine diphosphate glucuronosyltransferase; ODS, octadecylsilica; HPLC, high-performance liquid chromatography; UDPGA, uridine 50 -diphosphate-glucuronic acid; DPPC, L -a-dipalmitoylphosphatidycholine; ESI-MS, electrospray ionization mass spectra; NMR, nuclear magnetic resonance; HMBC, heteronuclear correlation optimized on a long-range coupling; UV, ultraviolet.
0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00111-8
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although their extraction is usually difficult and time consuming. The means to obtain glucuronides via chemical synthesis is often limited when unstable glucuronides are considered, or when multistep synthesis is required [1]. It is particularly difficult to find the specific hydrolysis of the hydroxyl- or carboxyl-protecting group of glucuronic acid conjugated to the aglycone when the acylglucuronides are synthesized by chemical means. To overcome the disadvantages of synthesizing glucuronides by chemical means, biosynthesis using free microsomes by in vitro incubation to obtain standard glucuronides is used. However, this in vitro method entails a troublesome procedure such as protein precipitation in order to obtain glucuronides from the mixture after incubation. Also this method could hardly be applied to the synthesis of glucuronides for slightly soluble drugs because their solubility would be limited in the incubation mixture. Alternative methods of producing glucuronides such as using microsomes entrapped in alginate beads [1] and covalently immobilized microsomes on Sepharose 4B beads [2–5] have been demonstrated. The productivity of glucuronides using the beads increased compared to those observed with free microsomes, since the UDPGT activity of immobilized microsomes on the beads stabilized. However, these biosyntheses did not seem to be the simplest means of obtaining the glucuronides, because the preparation of microsomes immobilized or entrapped in beads is not very convenient. We have previously prepared octadecylsilica (ODS) coated with phospholipids as a model of lipid membranes on a high-performance liquid chromatography (HPLC) column (phospholipid column), employing a dynamic coating technique developed in our laboratories [6]. We have also confirmed that the phospholipid column is a suitable support for enzyme immobilization and the immobilized enzyme phospholipid column is useful as an enzyme reactor [7]. In this work, we prepared immobilized dog liver microsome ODS particles coated with phospholipid (microsome-immobilized particles) for an efficient and useful synthesis of acylglucuronides of drugs. Here we are focusing on the preparation of microsome-immobilized particles employing a dynamic coating technique and the stability of the UDPGT activity of microsomes immobilized on the ODS particles coated with phospholipid. The immobilized microsomal UDPGT on the particles was expected to be stabilized by hydrophobic binding to the phospholipid on the particles compared to that of free microsomes. This paper describes the preparation of the microsome-immobilized particles and the means of synthesizing acylglucuronides using the particles. The acylglucuronidation using the particles was optimized with ketoprofen (Fig. 1) as a model compound. The present method was also used to synthesize the acylglucuronide of S-1255 ([R]-[+]-2-[benzo(1,3)dioxol-5-yl]-6-
Fig. 1. Chemical structures of ketoprofen and S-1255.
isopropyl-4-[4-methoxyphenyl]-2H-chromene-3-carboxylic acid) (Fig. 1) [8,9,12], which has been originally developed as a nonpeptide endothelin receptor antagonist in our laboratories. The microsome-immobilized particles and free microsomes were compared with respect to their productivity of acylglucuronides. Materials and methods Materials ODS particles (Cosmosil 75C18 -PREP, 75-lm particle size) and uridine 50 -diphosphate-glucuronic acid (UDPGA) trisodium salt were obtained from Nacalai Tesque (Kyoto, Japan). L -a-Dipalmitoylphosphatidycholine (DPPC) and ketoprofen were purchased from Sigma Chemical Co. (St. Louis, MO). S-1255 was synthesized in our laboratories. Acetonitrile, methanol, water, and trifluoroacetic acid were of HPLC grade and all other chemicals were of reagent grade. An empty reservoir equipped with a 20-lm frit (capacity: 4 or 75 ml) for the solid-phase extraction used was purchased from Varian (Harbor City, CA). Apparatus The HPLC system consisted of a Shimadzu LC-10AD pump (Kyoto, Japan), a Shimadzu SPD-10AV spectrophotometer or a Waters 996 photodiode array detector (Milford, MA), and a Shimadzu SIL-9A automatic injector. The chromatographic data were analyzed using a Shimadzu C-R7A data processor or Waters Millennium 32 software. An Eppendorf Thermomixer confort (Hamburg, Germany) thermostatic mixer was used. Electrospray ionization mass spectra (ESI-MS) were recorded on an Applied Biosystems API 3000 (Foster City, CA). Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Unity-600 spectrometer at 599.9 MHz (1 H) and 150.9 MHz (13 C) in CD3 OD. 1 H and 13 C signals were assigned on the basis of the observable 1 H–1 H correlation and long-range 1 H–13 C correlation in homonuclear correlation spectroscopy and heteronuclear correlation optimized on long-range coupling (HMBC) spectra, respectively.
H. Kamimori et al. / Analytical Biochemistry 317 (2003) 99–106
HPLC system The HPLC column used was an YMC pack Pro C18 (250 10 mm i.d., 5 lm, YMC, Kyoto, Japan). The mobile phases used were acetonitrile/0.1% trifluoroacetic acid solution (50/50, v/v) or acetonitrile/methanol/0.1% trifluoroacetic acid solution (50/30/20, v/v/v) at a flow rate of 2 ml/min, and the detector was set at a wavelength of 300 nm. Preparation of dog liver microsomes Dog liver samples were removed from female beagle dogs without the treatment of drugs in our laboratories. Dog liver microsomes were prepared as described previously [10] and the microsomal pellets obtained were stored in a freezer at )80 °C until use. The microsomal protein concentration was determined using the Micro Bicinchoninic Acid Protein Assay Reagent Kit (Pierce, Rockford, IL) with bovine serum albumin as standard [11]. The microsomal protein concentration used in this study was approximately 20 mg/ml. Preparation of microsome-immobilized particles The procedure was done under an ambient temperature of 25 1 °C. To prepare phospholipid-coated ODS particles, a solution of 56 mg of DPPC dissolved in 75 ml of 80% methanol was stirred with 0.1 g of ODS particles for 40 min in a beaker. After stirring, the solution containing the ODS particles was filtered through the empty reservoir, and the particles were collected. They were then sufficiently washed with 5 ml of water followed by 5 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4). Microsome-immobilized particles were prepared by adding to 200 ll of microsomes (protein content approximately 4 mg), 20 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4). The suspension was stirred with the phospholipid-coated particles prepared from 0.1 g of ODS particles for 60 min in a beaker. After stirring, the suspension containing the particles was filtered through the empty reservoir, and the microsome-immobilized particles were collected. Synthesis of acylglucuronide using microsome-immobilized particles To microsome-immobilized particles prepared from 0.1 g of ODS particles 20 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4) containing 10 lmol of substrate was added, and the mixture was stirred for 5 min in a beaker. After this, the mixture containing the particles was filtered through the empty reservoir, and the particles absorbed with substrate were collected. To the particles, 1 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4)
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containing 50 mM UDPGA and 10 mM magnesium chloride was added in a polypropylene microcentrifuge tube, which was then shaken with a thermostatic mixer at 37 °C for 6–24 h. After stirring, the mixture containing the particles was filtered through the empty reservoir and the desired fraction was eluted from the particles with 10 ml of 80% methanol for a few times. The eluates were injected several times onto the HPLC system and the peak fractions corresponding to acylglucuronide were collected. The fraction solutions were evaporated and lyophilized. Evaluation of preparation of microsome-immobilized particles The amount of DPPC coating the ODS particles was calculated from the difference between DPPC concentrations of the solution before and after the preparation procedure. DPPC concentrations in the coating solution were determined by HPLC with ultraviolet (UV) detection as described previously [6]. The amount of immobilized microsomes on the phospholipid-coated particles was estimated as the protein content. This was calculated from differences in the concentrations of the solutions containing microsomes before and after preparing the immobilized microsomes. The concentrations of the solutions were determined using the Micro Bicinchoninic Acid Protein Assay Reagent Kit as described above. Evaluation of production of acylglucuronide The production of acylglucuronides using the microsome-immobilized particles was calculated from the peak area of the formed products compared with that of the standard solution injected onto the HPLC system under the same analysis conditions. No significant UV spectral shift between each acylglucuronide and its substrate was confirmed by HPLC with photodiode array detection. Also, the productivity of acylglucuronides was expressed as the production per milligram of the immobilized microsomal protein on the particles. The productivity of acylglucuronide observed with free microsomes was estimated in the same manner.
Results and discussion Evaluation of preparation of microsome-immobilized particles The coating amount of DPPC on the ODS particles (0.1 g) was calculated to be approximately 20 mg. The amount of microsomes immobilized on the phospholipidcoated particles prepared from 0.1 g of ODS particles was calculated to be approximately 2 mg as the protein
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content. As a result, the microsome-immobilized particles could readily be prepared by stirring the particles with the solution containing the desired ligands in a beaker. Identification of ketoprofen acylglucuronide Ketoprofen acylglucuronide is produced from ketoprofen with dog liver microsomes by in vitro incubation in the presence UDPGA [12]. We tried synthesizing the ketoprofen acylglucuronide using microsome-immobilized particles. As the eluate from the microsome-immobilized particles was injected onto the HPLC system, the peak corresponding to the acylglucuronide was detected around a retention time of 9 min while the peak of ketoprofen was detected around a retention time of 22 min (data not shown). The peaks of keprofen and its acylglucuronide could be completely separated under the present HPLC conditions. The production obtained from the fraction solution corresponding to the peak of the acylglucuronide was identified by ESI-MS and NMR. Analysis by ESI-MS of the production revealed the presence of molecular ions at m=z 255 ½M þ H 176þ and m=z 431 ½M þ Hþ , corresponding to the molecular weights of ketoprofen and its acylglucuronide, respectively. The relative intensities of peaks of molecular ions were 40% for ketoprofen acylglucuronide, compared with 100% for ketoprofen. Fig. 2 shows the chemical structure of ketoprofen acylglucuronide, and the 1 H and 13 C NMR chemical
Fig. 2. Chemical structure of ketoprofen acylglucuronide. The arrow shows the long-range 1 H–13 C correlation.
shifts of ketoprofen and its acylglucuronide synthesized using microsome-immobilized particles are summarized in Table 1. The 1 H signals between 7.5 and 7.8 ppm, the doublet at 1.537 ppm, and the quartet at 3.964 ppm were assigned to protons of ketoprofen. The H-2 of ketoprofen (d3.819) was shifted downfield to d3.964 by substitution of the molecule. The 1 H signals of d5.499 (d), d3.332 (t), d3.414 (t), d3.497 (br), and d3.868 (br) were assigned to H-10 , H-20 , H-30 , H-40 , and H-50 of glucronic acid, respectively. The H-10 anomeric proton, at 5.499 ppm, has a coupling constant of 8.0 Hz, which is characteristic of diaxial coupling in the b-glucuronic acid [13,14]. As the H-10 proton of free b-glucuronic acid is known to display a chemical shift of 4.7 ppm, the chemical downfield shifts at H-10 suggested acylation of 1-OH of ketoprofen [13,14]. Also, long-range 1 H–13 C correlation was observed between d5.499 (b-glucuronic acid anomeric proton) and 174.22 (C-1 of ketoprofen) in the HMBC spectrum. The correlation proved that C-1
Table 1 1 H and 13 C NMR chemical shifts of ketoprofen and ketoprofen acylglucuronide Positiona
Ketoprofen
Ketoprofen acylglucuronide J/Hz
dH 1 2 2-CH3 3 4 5 6 7 8 10 20 30 40 50 60 5-CO 100 200 & 600 300 & 500 400 a
dC
—
—
—
—
—
—
—
—
—
—
—
—
7.776 7.573 7.653
198.45 138.83 131.07 129.57 133.89
198.19 138.60 131.01 129.46 133.76
q d
7.2 7.2
7.744
t
1.6
7.658 7.499 7.617
brd t dt
8 7.7 7.6, 1.6
d-like t-like m
8 8
177.74 46.51 19.00 143.13 130.19 139.10 129.81 129.78 133.10
J/Hz
dH
174.22 46.44 19.11 141.82 130.27 138.99 129.79 129.71 133.16 95.86 73.62b 77.68 72.99b NDc 174.22
3.819 1.479
The number of positions is indicated in Fig. 2. Exchangeable. c Not detected. b
dC
3.964 1.537
q d
7.2 7.2
7.744
t
1.8
7.672 7.503 7.642 5.499 3.332 3.414 3.497 3.868
brd t m d d brt br br
8 7.7
7.788 7.544 7.654
d-like t-like m
8.0 8.4 9
8 8
H. Kamimori et al. / Analytical Biochemistry 317 (2003) 99–106
of ketoprofen was linked to C-10 of b-glucuronic acid through the oxygen atom [13]. As a result, the chemical structure of ketoprofen acylglucuronide synthesized using the particles could be completely identified using ESI-MS and NMR. Since the ketoprofen acylglucuronide could be synthesized using the microsome-immobilized particles, we next tried to optimize the acylglucuronidation with ketoprofen as the substrate using the microsomeimmobilized particles. Optimization of glucuronidation using microsome-immobilized particles Time of microsome immobilization on the phospholipid-coated particles. Fig. 3 shows the relationship between the time of microsome immobilization on the phospholipid-coated particles and the productivity of ketoprofen acylglucuronide. As the stirring time of microsome immobilization increased, the amount of immobilized microsomal protein on the phospholipidcoated particles and the productivity of ketoprofen acylglucuronide increased. The amount of microsomal protein reached a plateau when the time of microsome immobilization was more than 60 min, although the productivity decreased somewhat at an immobilization time of 120 min. As a result, the time of microsome immobilization on the phospholipid-coated particles was set at 60 min when the microsomes were efficiently immobilized on the particles for the most suitable productivity of glucuronidation.
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Effect of support material for microsome immobilization on the productivity of ketoprofen acylglucuronide. To estimate the effect of support material for microsome immobilization on the productivity of ketoprofen acylglucuronide, the immobilized microsome ODS particles were prepared by the same procedure and the ketoprofen acylglucuronide was synthesized using the particles. As shown in Table 2, the productivity of ketoprofen acylglucuronide using the phospholipid-coated particles as the support was approximately threefold higher than that obtained using the ODS particles while the amount of immobilized microsomal protein was almost the same between the supports. Thus, phospholipid as a coating support stabilized the UDPGT in the immobilized microsomes and its immobilization produced intrinsic UDPGT activity, compared to the ODS particles not coated with phospholipid. These findings suggested that the phospholipid-coated particles could be an efficient support for microsome immobilization. Concentration of UDPGA and enzyme reaction time. The enzyme reaction time was investigated to optimize the glucuronidation using the microsome-immobilized particles in the presence of 10–100 mM UDPGA of the enzyme reaction mixture. Fig. 4 shows the relationship among the concentration of UDPGA, the enzymatic reaction time, and the productivity of ketoprofen acylglucuronide. The results indicated that the maximum productivity of ketoprofen acylglucuronide was approximately 370 nmol/mg protein when the reaction condition was at 37 °C for 6 h in the presence of 50 mM of UDPGA. Reproducibility of synthesis of ketoprofen acylglucuronide To test the reproducibility of synthesis of ketoprofen acylglucuronide using the microsome-immobilized particles, the ketoprofen acylglucuronide was synthesized in triplicate over two runs. The amount of immobilized
Table 2 Effect of support material for microsome immobilization on productivity of ketoprofen acylglucuronide
Fig. 3. Relationship between time of microsome immobilization on the phospholipid-coated particles and the productivity of ketoprofen acylglucuronide. The microsome-immobilized particles were prepared from the phospholipid-coated ODS particles (0.1 g). Ketoprofen acylglucuronide was synthesized using the microsome-immobilized particles prepared from 0.1 g of ODS particles in 1 ml of 50 mM Trishydrochloric acid buffer (pH 7.4) containing 50 mM UDPGA and 10 mM magnesium chloride at 37 °C for 2 h.
Support material for microsome
Amount of immobilized microsomal protein on support material (mg)a
Productivity of ketoprofen acylglucuronide (nmol/mg protein)b
ODS particles Phospholipid-coated particles
2.98 3.02
59 165
a
The microsome-immobilized particles were prepared from the phospholipid-coated ODS particles (0.1 g) or ODS particles (0.1 g). b Ketoprofen acylglucuronide was synthesized using the microsome-immobilized particles prepared from 0.1 g of ODS particles or the phospholipid-coated ODS particles (0.1 g) in 1 ml of 50 mM Trishydrochloric acid buffer (pH 7.4) containing 50 mM UDPGA and 10 mM magnesium chloride at 37 °C for 2 h.
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Synthesis of S-1255 acylglucuronide using microsomeimmobilized particles
Fig. 4. Relationship among the concentration of UDPGA, enzymatic reaction time, and the productivity of ketoprofen acylglucuronide. Key: 10 mM UDPGA (j), 50 mM UDPGA (d), 100 mM UDPGA (s). The microsome-immobilized particles were prepared from the phospholipid-coated ODS particles (0.1 g). Ketoprofen acylglucuronide was synthesized using the microsome-immobilized particles prepared from 0.1 g of ODS particles in 1 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4) containing 10, 50, or 100 mM UDPGA and 10 mM magnesium chloride at 37 °C for 1–24 h.
Table 3 Reproducibility of synthesis of ketoprofen acylglucuronide using the microsome-immobilized particles Run no.
Amount of immobilized microsomal protein (mg)a
Productivity of ketoprofen acylglucuronide (nmol/mg protein)b
1
1.49 1.19 1.94 1.54 0.38
528 414 542 495 70
Mean (n ¼ 3) SD
2.48 2.33 2.31 2.37 0.09
248 231 238 239 9
Total Mean (n ¼ 6) SD
1.96 0.52
367 147
Mean (n ¼ 3) SDc 2
S-1255 (Fig. 1) was originally developed in our laboratories as a nonpeptide endothelin receptor antagonist, and glucuronidation is its major metabolite pathway in dogs [15]. We tried to synthesize S-1255 acylglucuronide using the microsome-immobilized particles by the same procedure. The eluate obtained from the particles was injected onto the HPLC system, the peak fractions corresponding to acylglucuronide were collected, and the fraction solutions were evaporated and lyophilized. The production was identified by MS and NMR. The analysis by ESI-MS of the production revealed the presence of the molecular ions at m=z 459 ½M H 176 and m=z 653 ½M H , corresponding to the molecular weights of S-1255 and its acylglucuronide, respectively. The relative intensities of peaks of molecular ions were 15% for S-1255, compared with 100% for S-1255 acylglucuronide. Fig. 5 shows the chemical structure of S-1255 acylglucuronide, and the 1 H and 13 C NMR chemical shifts of S-1255 and its acylglucuronide synthesized using microsome-immobilized particles are summarized in Table 4. The 1 H signals of d5.387 (d), d2.882 (dd), d3.319 (t), d3.383 (t), and d3.721 (d) were assigned to H10 , H-20 , H-30 , H-40 , and H-50 of glucronic acid, respectively. The H-10 anomeric proton, at 5.387 ppm, has a coupling constant of 8.1 Hz, which is characteristic of a diaxial coupling in the b-glucuronic acid [13,14]. Also, the chemical downfield shifts at H-10 of b-glucuronic acid proposed acylation of 1-OH of S-1255 [13,14] as the result of ketoprofen acylglucuronide. Long-range 1 H–13 C correlation was observed between d5.387 (bglucuronic acid anomeric proton) and 165.68 (3-COO of S-1255) in the HMBC spectrum. The correlation proved that 3-COO of S-1255 was linked to C-10 of b-glucuronic acid through the oxygen atom [13]. As a result, the chemical structure of S-1255 acylglucuronide synthe-
a The microsome-immobilized particles were prepared from the phospholipid-coated ODS particles (0.1 g). b Ketoprofen acylglucuronide was synthesized using the microsomeimmobilized particles prepared from 0.1 g of ODS particles in 1 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4) containing 50 mM UDPGA and 10 mM magnesium chloride at 37 °C for 6 h. c Standard deviation.
microsomal protein, productivity of ketoprofen acylglucuronide, and their standard deviations were calculated. As shown in Table 3, the results indicated that the reproducibility of this procedure was acceptable enough in view of the enzyme reaction.
Fig. 5. Chemical structure of S-1255 acylglucuronide. The arrow shows the long-range 1 H–13 C correlation.
H. Kamimori et al. / Analytical Biochemistry 317 (2003) 99–106
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Table 4 1 H and 13 C NMR chemical shifts of S-1255 and S-1255 acylglucuronide Positiona
S-1255
S-1255 acylglucuronide J/Hz
dH 2 3 4 4a 4b 5 6 7 8 3-COO 10 20 30 40 50 60 100 200 300 400 500 600 OCH2 O 1000 2000 and 6000 3000 and 5000 4000 4000 -OCH3 OCH2 ðCH3 Þ2
a b
6.125
6.224 6.733 6.704
s
d dd d
2.8 8.7, 2.8 8.7
dC
dH
76.51 123.84 145.38 126.38 148.53 116.65 153.36 120.53 118.68 168.92
6.165
—
—
—
—
—
—
—
—
—
—
—
—
6.948
brd
0.8
6.722 6.955 5.881 5.888
dd d-like d d
7.8, 0.8 8 1.3 1.3
7.17 6.992
br m
3.836 4.188 1.117 1.140
s m d d
6.0 6.0
J/Hz s
— — — —
6.265
d
2.8
dd d
8.7, 2.8 8.7
5.387 2.882 3.319 3.383 3.721
d dd t t d
8.1 9.1, 8.1 9.1 9.4 9.5
6.969
brs
6.725 6.978 5.891 5.896
d d-like d d
7.21 7.018
br brd
8
3.869 4.204 1.133 1.153
s m d d
6.0 6.0
—
6.786 6.749 —
134.26 109.13 149.20 149.20 108.81 122.57 102.51 130.34 131.3 (br) 114.60 161.01 55.75 71.79 22.17 22.33
7.8 8 1.3 1.3
dC 76.11 121.87 148.10 126.29 149.02 116.93 153.48 121.50 118.95 165.68 95.62 73.63 77.66 72.90 77.03 172.95 134.15 109.16 149.20b 149.22b 108.83 122.64 102.50 129.99 131.7 (br) 114.80 161.38 55.93 72.02 22.18 22.36
The number of positions is indicated in Fig. 5. Exchangeable.
sized using the particles has been completely identified using ESI-MS and NMR. The maximum productivity of S-1255 acylglucuronide was approximately 1400 nmol/mg protein using the microsome-immobilized particles (in the presence of 50 mM UDPGA, at 37 °C for 24 h). Comparison of productivity of glucuronidation with free microsomes The productivities of acylglucuronides of ketoprofen and S-1255 as the substrates using the microsome-immobilized particles were compared to those observed with free microsomes. The productivities of two acylglucuronides obtained using the free microsomes are indicated under the optimum conditions using the same content of microsomes immobilized or substrate adsorbed on the particles. As shown in Fig. 6, the productivity of S-1255 acylglucuronide using the microsome-immobilized particles was approximately threefold higher than that observed with the free
microsomes, although ketoprofen acylglucuronide productivity was slightly lower than that observed with the free microsomes. We assume that the significant difference of S-1255 acylglucuronide productivity between the microsome-immobilized particles and the free microsomes would be caused by the difference of the concentration of substrate in the enzymatic reaction mixture. In the case of the synthesis of glucuronides with free microsomes, the concentration of substrate in the mixture was limited in order to effectively promote the glucuronidation as a higher concentration would inhibit the enzyme activity. The maximum productivity of S1255 acylglucuronide with the free microsomes was obtained when the concentration of S-1255 in the mixture was approximately 1.0 lmol/ml. On the other hand, in the case of the microsome-immobilized particles, the glucuronidation could be sufficiently promoted using the particles adsorbed on the excessive S-1255, whose concentration in the mixture was approximately 9.8 lmol/ ml. The excessive concentration of substrate in the mixture would not inhibit the enzyme activity because
106
H. Kamimori et al. / Analytical Biochemistry 317 (2003) 99–106
References
Fig. 6. Comparison of the productivity of acylglucuronides of ketoprofen and S-1255 by microsome-immobilized particles and free microsomes. Microsome-immobilized particles: ketoprofen; adsorbed substrate on the particles, 3.5 lmol; immobilized microsomal protein, 2.00 mg; S-1255; adsorbed substrate on the particles, 9.8 l mol; immobilized microsomal protein, 2.83 mg. Free microsomes: ketoprofen; added substrate to the enzymatic reaction mixture, 3.5 lmol; microsomal protein, 2.00 mg; S-1255; added substrate to the enzymatic reaction mixture, 1.0 lmol; microsomal protein, 2.80 mg. Enzymatic reaction mixture: 1 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.4) containing 50 mM UDPGA and 10 mM magnesium chloride; reaction conditions, 37 °C, 6–24 h.
its substrate was adsorbed on the particles. As a result, the production of S-1255 acylglucuronide with the particles increased compared to that observed with the same protein content of free microsomes. These findings indicated that the immobilized microsomal UDPGT on the particles was stabilized and the microsomes immobilized on the particles could be efficiently utilized for the synthesis of acylglucuronides, even when there was an excessive concentration of substrate in the mixture. Therefore, this method should be especially useful for the synthesis of acylglucuronides of drugs that are slightly soluble in aqueous solution. Conclusion Acylglucuronides of ketoprofen and S-1255 were synthesized using microsome-immobilized particles. The microsome-immobilized particles can readily be prepared by stirring the particles with a solution containing the desired ligands, and acylglucuronide can easily be synthesized by stirring the microsome-immobilized particles absorbed on substrate in a buffer solution containing UDPGA. The productivity of S-1255 acylglucuronide using microsome-immobilized particles was approximately threefold higher than that observed with free microsomes. The present method should be helpful for obtaining the standards of acylglucuronides, ether glucuronides or other metabolites of drugs, which are slightly soluble in aqueous solution or cannot be synthesized by chemical means for drug development stages.
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