Introduction

Biotransformations have implicitly served as fundamental and successful tool in the fields of new drug discovery, where the objective is to obtain pharmacologically improved metabolites [1–3] or a suitable alternative metabolic system ideally mimicking mammalian/human metabolism [1, 4–12]. In these two aspects, steroids are the first compounds which have been established as ideal substrates for biocatalysts, thus paving the way for innumerous possibilities garnered by the techniques of biotransformation [1, 13]. These facts prompted us to conduct metabolic studies on dexamethasone as biotransformation of synthetic glucocorticoids is an area less explored (Fig. 1).

Fig. 1
figure 1

Structural formula of compounds II, III, IV.

Full size image

Dexamethasone is a potent synthetic member of a glucocorticoid class of corticosteroid compounds. It acts as an anti-inflammatory and immunosuppressant. Dexamethasone is a synthetic fluoro-glucocorticoid. Dexamethasone (9-fluoro-11_,17,21-trihydroxy-16a-methylpregna-1,4-diene-3,20-dione) has a basic structure with 21 carbon atoms, and it is 20 to 25 times more potent than the natural corticosteroid cortisol. Biotransformation of dexamethasone has been studied in cross-bred castrated male horses, which resulted in determining several metabolites including 17-oxodexamethasone, 11-dehydrodexamethasone, 20-dihydrodexamethasone, 6-hydroxydexamethasone and 6-hydroxy-17-oxodexamethasone [14].

Metabolism of dexamethasone was also studied in human liver microsomal incubations in vitro, which resulted in finding new metabolites, including 6 beta-hydroxydexamethasone, 6 alpha-hydroxydexamethasone, 6-hydroxy-9-alpha-fluoro-androsta-1,4-diene-11-beta-hydroxy-16-alpha-methyl-3,17-dione (6-hydroxy-9-alpha-F-A) and 9-alpha-fluoro-androsta-1,4-diene-11 beta-hydroxy-16-alpha-methyl-3,17-dione (9-alpha-F-A). Dexamethasone underwent sidechain cleavage to form 9-alpha-F-A. This metabolite was then a substrate for 6-hydroxylation [15].

Biotransformation of dexamethasone in human liver in vitro resulted in extensive production of 6-hydroxylated and side-chain-cleaved metabolites. Dexamethasone goes through extensive side-chain breakage to form 9-alpha-F-A in human kidney samples. CYP3A4 is responsible for 6α-and 6β-hydroxylation of dexamethasone, and CYP17 is supposed to initiate side-chain segmentation to produce 9α-fluoro-androsta-1,4-diene-11β-hydroxy-16α-methyl-3,17-dione (9-alpha-F-A). Even though 9-alpha-F-A has not been separated before as a metabolite in its non-hydroxylated form in samples of human liver, it is formed as an intermediate metabolite that is further rapidly hydroxylated to OH-9-alpha-F-A. Furthermore, molecular models of CYP3A4 and CYP17 (17,20-lyase) have been used to model the enzyme fits of dexamethasone. From these modelling studies it has been found that dexamethasone complements both putative enzyme active sites in orientations likely to lead to the formation of metabolites identified in vitro [16].

Dexamethasone is mostly transformed to 6-hydroxydexamethasone and side-chain segmented products in human liver both in vivo and in vitro with CYP3A4 responsible for making 6-hydroxylated compounds. In conclusion, male rats produce a metabolic pattern which is closely similar to that in humans. Even so, 6-hydroxylation was most abundant in hamster which may therefore be an appropriate model to use for further studies on dexamethasone metabolism by CYP3A [17].

Materials and Methods

General

Instrumentation. The 1H-NMR and 13C-NMR spectra were recorded in deuterated CDCI3 on Bruker Avance 600-MHz instrument using TMS as an internal standard. Chemical shifts were determined in parts per million (δ), and coupling constants (J) were expressed in hertz. Evaporation was conducted by an Heidolph Laborata 4000 rotary evaporator system under reflux in vacuo. Thin layer chromatographic separation was conducted on precoated silica gel plates (0.25 mm thick). Thin layer chromatographic separation was conducted on precoated silica gel plates (0.25 mm thick, E. Merck). Column chromatography was performed by using silica gel (70 – 230 mesh, E. Merck) as an adsorbent. Purity of compounds was evaluated on pre-coated TLC silica gel plates. Recycling preparative HPLC was performed on LC-9101 instrument using JAIGEL – GS320 column and UV-3702 UV/VIS detector. Methanol was used as an eluent.

Materials and microorganisms. Dexamethasone was purchased from Sigma-Aldrich. Bacillus subtilis (ATCC 6051) was obtained from American Type Culture Collection Centre. Other chemicals were of analytical grade.

Microbial culture. A culture medium for B. subtilis was prepared by dissolving glucose (5 g), yeast extract (5 g), peptone (1 g), sucrose (2 g) in distilled water (4.0 L). Stock cultures of B. subtilis were stored on slants of nutrient agar at 4°C. 0.1 M phosphate buffer (pH 7.2) used for resting-cell suspensions of B. subtilis consisted of 10.6 g K2HPO4, 4.08 g KH2PO4, and 1000 mL distilled H2O.

General Fermentation Procedure

Microbial biotransformation studies were carried out by incubating cultures on shaker operating at 150 rpm and 25°C. Preliminary screening experiments were carried out in 250-mL cotton plugged culture flasks containing 100 mL nutrient agar medium. The medium was sterilized at 121°C for 15 min. Fermentations were accomplished according to a standard two-stage protocol [15, 25, 28]. B. subtilis stock inoculum was first prepared by suspending the cells from one agar slant in 1 mL of sterile distilled water. Submerged stage I cultures were then initiated by adding 0.1 mL of the B. subtilis stock inoculum to a 250-mL flask containing 100 mL of nutrient agar medium. Following incubation of stage I cultures for 3 days on a shaker, stage II cultures were initiated by inoculating 100 mL of fresh, sterile complex medium with 1 mL of stage I culture broth. Dexamethasone at a concentration of 25 mg in 0.5 mL of methanol was added to 26 mL of incubation medium 2 days after inoculation of stage II cultures. Cultures were sampled at 24-h intervals by extracting 5 mL of the broth with 5 mL n-butanol. The extracts were concentrated and chromatographed on TLC plates. Positive/substrate controls consisted of autoclaved medium which was incubated with dexamethasone in total absence of B. subtilis. Negative/culture controls consisted of fermentation blanks in which B. subtilis was grown under identical conditions without addition of dexamethasone.

Metabolism of Dexamethasone

Preliminarily prepared 1-mL stock inoculum was distributed equally among ten stage-I 250-mL flasks, each containing 100 mL of nutrient agar medium. Stage I cultures were then incubated on the shaker for 3 days at room temperature. After completion of 3-day incubation period of stage I culture flasks, stage II cultures were initiated by inoculating 40

1-L flasks, each containing 200 mL of fresh, sterile, beef extract-enriched complex medium, with stage I culture broth. Each stage II flask was inoculated with 5 mL of stage I culture broth. The stage II cultures were incubated on a shaker, after which the optical density of the medium was monitored. Subsequently, 8 g of dexamethasone was dissolved in 20 mL of methanol and dispensed equally among 40 1-L stage II flasks. After 7-day incubation on a shaker, the suspensions were pooled and extracted three times with 15 L of n-butanol. N-butanol extract was dried over anhydrous Na2SO4, filtered, and concentrated at reduced pressure to afford a yellowish brown gum (2.5 g).

Isolation and Purification of Metabolites II, III and IV

n-Butanol extract was subjected to a silica gel column chromatography (6 × 80 cm) with n-hexane – EtOAc gradient (100% n-hexane up to 100% EtOAc) to yield two fractions: (a) 52.5 mg and (b) 36.2 mg. Fraction (a) was chromatographed on silica gel column (2 × 30 cm; 15 g of silica gel) with n-hexane–EtOAc gradient (0 : 100 to 10 : 90 v/v; total volume, 2 L), which yielded fractions 1a (18 mg) and 2a (28.5 mg).

Fraction 1a was further purified by recycling HPLC isocratic elution with EtOAc/CH3OH (75: 25) at a flow rate of 10 mL/min. The recycling process was repeated three times, after which HPLC peaks were separated into four (A – D) parts. Part C was recrystallized to afford compound II (3 mg). Fraction 2a was subjected to silica gel column chromatography followed by crystallization from methanol to obtain white amorphous compound III (5 mg).

Fraction b (36.2 mg) was chromatographed on silica gel column (1.8 × 31 cm; 10 g of silica gel) with n-hexane – EtOAc gradient (100% n-hexane up to 100% EtOAc) which afforded homogeneous sub-fractions comprising metabolite 5. Further purification on a Sephadex LH-20 column (2 × 26 cm) with 500 mL of n-butanol yielded compound IV (7 mg).

Dexamethasone (I). White solid, mol. wt., 408.46; m.p., 268 – 271°C; 1H NMR (600 MHz, CDCl3; δ, ppm): 7.38 (d, 1 H, J = 10.1, H-1), 6.20 (dd, 1H, J = 2.0, 10.1, H-2), 6.01 (d, 1H, J = 2.0, H-4), 5.61 (m, OH, H-11), 5.1 (m, OH, H-21), 4.78 (m, 1H, H-21), 4.22 (m, 1H, H-11), 2.83 (m, 1H, H-14), 2.70 (m, 1H, H-6), 2.57 (m, 1H, H-8), 2.45 (dd, J = 3.0, 14.0, 1H, H-6), 2.1(m, 1H, H-15), 2.0 (m, 1H, H-7), 1.81(d, J = 1.5,14.0,1H, H-12), 1.59 (m, 1H, H-16), 1.50 (m, 1H, H-12), 1.49 (s, 3H, H-18), 1.44 (m, 1H, H-15), 1.42 (m, 1H, H-7), 1.02 (s, 3H, H-19), 0.98 (s, 3H, H-19); for 13C NMR data see Table 1.

Table 1 13C NMR Data for Compounds I – IVin CDCl3
Full size table

6-Hydroxydexamethasone (II). White solid; mol. wt., 408.46; m.p., 259 – 262°C; 1H NMR (600 MHz, CDCl3; δ, ppm): 7.32 (d, 1 H, J = 10.1, H-1), 6.20 (dd, 1H, J = 2.0, 10.1, H-2), 6.01 (d, 1H, J = 2.0, H-4), 5.61 (m, OH, H-11), 5.1 (m, OH, H-21), 4.89 (m, OH, H-6), 4.78 (m, 1H, H-21), 4.22 (m, 1H, H-11), 2.83 (m,1H, H-14), 2.57 (m, 1H, H-8), 2.1 (m, 1H, H-15), 2.0 (m, 1H, H-7), 1.81 (d, 1H, J = 1.5, 14.0, H-12), 1.59 (m, 1H, H-16), 1.50 (m, 1H, H-12), 1.49 (s, 3H, H-18), 1.44 (m, 1H, H-15), 1.42 (m, 1H, H-7), 1.02 (s, 3H, H-19), 0.98 (s,3H, H-19); for 13C NMR data see Table 1.

17-oxodexamethasone (III). White solid; mol. wt., 316; m.p., 239 – 240°C; 1H NMR (600 MHz, CDCl3; δ, ppm): 7.32 (d, 1 H, J = 10.1, H-1), 6.20 (dd, 1H, J = 2.0, 10.1, H-2), 6.01 (d, 1H, J = 2.0, H-4), 5.61 (m, OH, H-11), 4.22 (m, 1H, H-11), 2.83 (m, 1H, H-14), 2.67 (m, 1H, H-6), 2.57 (m, 1H, H-8), 2.42 (dd, 1H, J = 3.0,14.0, H-6), 2.1 (m, 1H, H-15), 2.0 (m, 1H, H-7), 1.81 (d, 1H, J = 1.5, 14.0, H-12), 1.59 (m, 1H, H-16), 1.50 (m, 1H, H-12), 1.49 (3H, s, H-18), 1.44 (m, 1H, H-15), 1.42 (m, 1H, H-7), 1.02(s,3H, H-19), 0.89 (d, 3H, J = 7.7, H-20); for 13C NMR data see Table 1.

6-Hydroxy-17-oxo-dexamethasone (IV). White solid; mol. wt., 348.41; m.p., 410 – 415°C; 1H NMR (600 MHz, CDCl3; δ, ppm): 7.32 (d, 1 H, J = 10.1, H-1) 6.20 (dd, 1H, J = 2.0, 10.1, H-2), 6.01 (d, 1H, J = 2.0, H-4), 5.61 (m, OH, H-11), 4.89 (OH, m, H-6), 4.22 (m, 1H, H-11), 2.83 (m, 1H,H-14), 2.57 (m, 1H, H-8), 2.1 (m, 1H, H-15), 2.0 (m, 1H, H-7), 1.81 (d, 1H, J = 1.5, 14.0, H-12), 1.59 (m, 1H, H-16), 1.50 (m, 1H, H-12), 1.49 (s, 3H, H-18), 1.44 (m, 1H, H-15), 1.42 (m, 1H, H-7), 1.02 (s, 3H, H-19), 0.89 (d, 3H, J = 7.7, H-20); for 13C NMR data see Table 1.

Results and Discussion

The main objective of our research efforts was to analyze the mammalian metabolism of dexamethasone by employing microbes as in vitro model systems to predict and prepare potential mammalian metabolites of this compound. Animal model have already provided significant insights into metabolism of dexamethasone [14–19]. The use of various Bacillus species in biotransformation of a broad range of compounds has established their significant potential as a model medium for investigating biotransformations [20–29]. In the present study, bacterial strain B. subtilis ATCC 6051 has successfully produced metabolites previously reported for the metabolism of dexamethasone on animal models. The structure of resulting metabolites was elucidated by 1H-NMR and 13C-NMR techniques.

The 1H-NMR spectrum of metabolite II revealed the absence of signal splitting as evident in position of C-6 in dexamethasone. It was replaced by a single signal identified as due to –OH proton. This was further verified by 13C-NMR signal of C-6, which showed an increase in the chemical shift.

Compound III showed complete loss of hydroxy group and –CH2OH-CO signals, which means no evidence of C-21 or C-22 in 13C-NMR spectra. Hence, compound III was found to be 17- oxo-dexamethasone.

Compound IV demonstrated changes similar to those found in the spectra of compounds II and III, so it was identified as 6-OH-17-oxodexamethasone.

The observed transformation of dexamethasone by B. subtilis into three metabolites gives hope that further optimization of the reaction conditions including temperature, broth media, pH, oxidation-reduction, and morphology modifications may thus ameliorate the amount and diversity of obtained metabolites.