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In vitro enzymic synthesis of mammalian liver xenobiotic metabolites catalysed by ovine liver microsomal cytochrome P450
Enzyme and Microbial Technology 29 (2001) 306 –311
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In vitro enzymic synthesis of mammalian liver xenobiotic metabolites catalysed by ovine liver microsomal cytochrome P450 Ulrike Hubl, David E. Stevenson* Industrial Research Limited, PO Box 31–310, Lower Hutt, New Zealand Received 31 August 2000; received in revised form 3 May 2001; accepted 4 June 2001
Abstract Ovine microsomal Cytochrome P450 was used preparatively in vitro to make milligram quantities of Nordiazepam, a known metabolite of the anxiolytic drug Diazepam. The product was purified by reversed phase chromatography and preparative HPLC. This synthetic approach should be of use in the preparation of purified metabolites for analytical reference purposes or research. Using demethylation of p-nitroanisole as a model reaction, the conditions required for optimum activity of ovine liver microsomal cytochrome P450 in vitro have been determined. Variation of both enzyme and substrate concentrations revealed saturation behavior in both cases. Above the optimal concentrations, no increase in yield of product was obtained. End-product inhibition was shown to occur and this accounts for the lower conversion rate at higher substrate concentrations. The enzyme showed reasonable resistance to denaturation by organic cosolvents (essential for substrate solubilisation). Up to 3% v/v of acetonitrile, methanol or DMSO had no apparent effect on the enzyme, reaction yields being unaffected. Another xenobiotic metabolizing enzyme, Glucuronyl transferase is also present in microsomes. Attempted simultaneous hydroxylation and glucuronidation, in the presence of UDPGA, the cofactor for Glucuronyl transferase was not successful, due to the effects of contaminating -glucuronidase in the microsomes. The glucuronidation/de-glucuronidation did, however, increase the yield of hydroxylation product. Conditions which favour glucuronidation allowed some glucuronide to accumulate but the total yield of hydroxylation products (glucuronidated and free) was significantly reduced. © 2001 Elsevier Science Inc. All rights reserved. Keywords: cytochrome P450, p-nitroanisole; diazepam; hydroxylation; demethylation; enzymic; ovine
1. Introduction Hydrophobic xenobiotic compounds such as drugs and toxins are metabolised in the mammalian liver into more polar derivatives. The main metabolic pathway involves hydroxylation (if required) by the cytochrome P450 hydroxylase and associated NADPH cytochrome reductase, followed by conjugation to form water soluble species [1]. Cytochrome P450 activates molecular oxygen for insertion into a carbonhydrogen bond. The conjugates, glucuronides being the most common, are then readily excreted by the renal system [1]. Hydroxylated or glucuronidated metabolites are useful in clinical trials of the parent drug, or as analytical reference standards in the testing for illegal drugs. There are many reports of the in vitro use of glucuronyl transferase in preparative glucuronide synthesis [2– 4], but cytochrome P450 has mainly been used in enzyme electrodes [5] and
when immobilised, in trials of artificial livers for extra-corporeal detoxification of blood during liver disease [6 –9]. In one such report [9] cytochrome P450 was co-immobilised with glucuronyl transferase and the combination was able to hydroxylate and subsequently glucuronidate benzo(a)pyrene. In this report we describe the use of ovine liver microsomes for the in vitro demethylation of p-nitroanisole, to form p-nitrophenol and the optimisation of the reaction conditions for preparative scale synthesis. The demethylation of p-nitroanisole is, strictly, a hydroxylation of the methyl group followed by its non-enzymic loss as formaldehyde to liberate the phenolic hydroxyl group. We also report the preparative scale demethylation of the drug Diazepam.
2. Materials and Methods 2.1. Materials
Corresponding author. Tel.: ⫹64 – 4-569 – 0000; fax: ⫹64 – 4-566 – 6004. E-mail address: [email protected] (D. Stevenson). *
NADP, Glucose-6-phosphate dehydrogenase, glucose-6phosphate and UDP-glucuronate (UDPGA) were obtained
0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 1 ) 0 0 3 8 9 - 1
U. Hubl, D.E. Stevenson / Enzyme and Microbial Technology 29 (2001) 306 –311
from Boehringer Mannheim (Mannheim, Germany). Buffers, solvents and general chemicals were obtained from Sigma/Aldrich. Diazepam was a gift from Douglas Pharmaceuticals (Auckland, New Zealand), Nordiazepam and Temazepam were gifts from B Dent Global Ltd, (Wellington, NZ). Ovine liver microsome suspension (“microsomes”; containing cytochrome P450, UDP-glucuronyl transferase and NADPH reductases) was prepared according to Gibson and Skett [1], from fresh ovine livers obtained from Taylor Preston Ltd. (Wellington, NZ). Microsomes were collected by flocculation with Calcium Chloride solution and centrifugation at 27,000 g, rather than by ultra-centrifugation. The microsomal pellet was resuspended in 50 mM Tris buffer, pH 7.4, containing 0.1 M KCl and 2 mM DTT. This resulted in a protein concentration of 11–13 mg/ml and approx 7% total solids. Microsomes were frozen in 3 ml plastic vials, using liquid nitrogen and stored at –70°C. 2.2. General method for trial hydroxylation reactions Tris buffer (0.1 M, pH 7.4, 240 l), co-factor regeneration mix (50 l from 1 ml of the same Tris buffer, containing 10 l glucose-6-phosphate dehydrogenase suspension, 6 mM glucose-6-phosphate, 2 mM NADP, 50 mM nicotinamide and 1 mM DTT) and microsome suspension (200 l, to give a protein concentration of 4.8 mg/ml in a total volume of 0.5 ml) were incubated at 37°C in an open 7 ml glass vial for 10 min. The reaction was started by adding the required volume of substrate solution (p-nitroanisole in acetonitrile or diazepam in DMSO; 10 l, unless stated otherwise). After incubation with magnetic stirring overnight, the reaction was stopped by adding acetonitrile/ethanol (1:1 v/v; 1 ml). The sample was mixed and centrifuged in a microcentrifuge (13,000 rpm/5 min./room temperature). The supernatant was sampled for HPLC analysis.
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2.5. Concentration of organic solvent p-nitroanisole solutions in acetonitrile containing concentrations of 20 to 250 mM were prepared in acetonitrile, DMSO and methanol. For starting the enzyme reaction 2 l (in the case of the highest concentration) to 25 l (in the case of the lowest substrate concentration) of the solutions were added to give a final substrate concentration of 1 mM. The solvent concentration was thus varied from 0.4 to 5%, for DMSO and methanol and 0.4 to 20% for acetonitrile. The other reaction conditions were as described in Section 2.2. 2.6. Influence of UDPGA on p-nitroanisole hydroxylation Substrate solutions containing concentrations of 50 to 200 mM were prepared. UDPGA solution (10 mM in Tris buffer; 50 l) was added to Tris buffer (see above; 190 l), cofactor regeneration mixture (see above, 50 l) and microsomes (see above; 200 l, to give 4.8 mg protein/ml). The reaction was started by adding 10 l of different substrate solutions following the same procedure as described above (Section 2.4). The same experiment was done using glucuronidation buffer (0.2 M Tris, pH 8.0 containing 0.1 M glucuronic acid). 2.7. Determination of product inhibition p-Nitrophenol solutions (10 to 50 mM, 10 l) were added to Tris-buffer (230 l), cofactor regeneration mixture (50 l) and microsomes (200 l). The reaction was started by the addition of p-nitroanisole solution (100 mM; 10 l) as described above. For determination of any decomposition of p-nitrophenol, acetonitrile (10 l) was added instead of the substrate solution. The activity of the enzyme preparation was also tested with 20 l substrate solution (50 mM) added to the same reaction mixture, without adding p-nitrophenol. 2.8. HPLC analysis
2.3. Variation of enzyme amount Volumes of enzyme suspension between 50 and 400 l (giving 1.2–9.6 mg protein/ml) were used for the reaction. The volume of Tris buffer was varied accordingly, so that the final volume of the reaction mixture remained at 0.5 ml. The other reaction conditions were as described in Section 2.2. 2.4. Variation of substrate concentration p-nitroanisole solutions in acetonitrile containing concentrations in a range of 5 to 250 mM were prepared. The enzyme reaction was carried out as above, with 10 l of the different substrate solutions being added.
Centrifuged solution from the quenched enzyme reaction (see section 2.2; 20 L) was injected onto a Phenomenex (Torrance, CA) Nucleosil (5 M, 10 nM (100 Å), C18 column (250 ⫻ 4.6 mm plus 30 ⫻ 4.6 mm guard) fitted to a Gilson (Middleton, WI) dual pump HPLC system fitted with an autoinjector and UV-visible diode array detector. For p-nitroanisole reaction samples, the solvent flow rate was kept constant at 0.7 ml min⫺1 and the initial solvent composition was aqueous trifluoroacetic acid (TFA; 0.1% v/v)/acetonitrile, 75: 25 v/v. The acetonitrile content was increased linearly to 90% over a period of 20 min and back to 25% over 1 min. Peaks were detected by UV absorption at 311 nm. Under these conditions, the retention times of p-nitroanisole, p-nitrophenol and p-nitrophenyl glucuronide were 20.7, 14.7 and 8.4 min respectively. Their relative
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extinction coefficients, determined by injecting an equimolar mixture of the three compounds, were 1:1.2:1.7, respectively. Diazepam reactions were analysed similarly, except that the acetonitrile content of the eluting solvent was varied linearly from 40 – 80% over a period of 14 min. UV detection was at 238 nm. Under these conditions the retention times for Nordiazepam, Diazepam and Temazepam were 7.7, 10.2 and 13 min, respectively and the relative extinction coefficients were 1:1.12:1.09, respectively. 2.9. Purification and characterisation of products from preparative diazepam reaction The hydroxylation reaction of Diazepam was a direct scale up of the analytical scale reaction. Conditions were as described in Section 2.2 except that a total volume of 330 ml was used with 2 mM (190 mg) Diazepam, 2% v/v DMSO and 60% v/v microsome suspension (final protein conc. 7.2 mg/ml). The mixture of microsomes, buffer and cofactors was stirred rapidly at 37°C and the substrate added dropwise in DMSO (6.6 ml). The mixture was then split between 9 ⫻ 100 ml unstoppered conical flasks and stirred at 37°C overnight. The crude yield of Nordiazepam was determined to be 9.5% by analytical HPLC. The mixture was then freeze-dried, redissolved in water (400 ml) and freeze-dried again to remove the bulk of the DMSO. The benzodiazepines were extracted with hot (50°C) ethyl acetate (2 ⫻ 200 ml), filtered and the solvent removed under vacuum. The residue (1.96 g) was dissolved in 50% aqueous methanol (150 ml) and applied to a 2.5 ⫻ 20 cm column of LiChroprep RP-18 (40 – 63 M; Merck, Darmstadt, Germany) equilibrated with the same solvent. The column was eluted with 150 ml each of 50%, 75% and 100% methanol. The benzodiazepines eluted in 100% methanol. Fractions were monitored by TLC on foil backed silica plates (Merck) using ethyl acetate/hexane 1:1 v/v as solvent. A slight separation was achieved between Diazepam and Nordiazepam on the column, but a lot of other material, presumably lipid from the microsomes, was removed. The fractions containing Nordiazepam were pooled and evaporated to dryness under vacuum. The mixture of benzodiazepines (0.48 g) was separated by preparative HPLC. The method was identical to the analytical method (Section 2.8) except that a 21 ⫻ 250 mm C-5 column was used with isocratic solvent (acetonitrile/ water 32.5:67.5) at 10 ml/min. The sample was dissolved in Methanol (2.5 ml) and applied to the column in five 0.5 ml injections. The retention times of Nordiazepam and Diazepam were 30 and 43 mins respectively. Fractions were collected manually. Purified Nordiazepam (8 mg/4% yield) was obtained after pooling and evaporation of solvents and crystallisation from aqueous methanol. The 1H- and 13C-NMR spectra [10] and analytical HPLC retention time of the isolated material were identical to those of the authentic standard.
Fig. 1. Effect of microsomal protein concentration on p-nitrophenol yield (%) from p-nitroanisole (as determined by HPLC).
3. Results and Discussion 3.1. Effect of microsomal enzyme concentration Microsomal protein concentrations between 1.2 and 9.6 mg/ml were added to reaction mixtures containing 5 mM p-nitroanisole. Product formation increased with increasing amounts of added microsomes, following a linear relation for volumes under 200 l (Fig. 1). At concentrations above 7 mg of added microsomal protein, a maximum conversion of 25.8% was reached. The yield was still 86% of the maximum at 4.8 mg/ml protein. This latter amount was used for all subsequent experiments, unless stated otherwise. 3.2. Effect of p-nitroanisole substrate concentration The enzymic reaction was carried out with substrate concentrations between 0.1 and 5 mM. An almost complete conversion to p-nitrophenol was found for concentrations below 0.2 mM (Fig. 2). With increasing substrate concentrations the yield of product decreased. The product concentration reached a maximum of 0.95 mM at a substrate concentration of 5 mM, however, the yield of product at this substrate concentration was only 20%. 3.3. Effect of different co-solvents Acetonitrile, methanol and dimethyl sulphoxide (DMSO) were compared as co-solvents. The solvent is necessary for dissolving the water insoluble substrate p-nitroanisole. The solvent concentration was varied between 0.4 and 3% (v/v), keeping the substrate constant at 1 mM. In this range the reaction yield did not vary significantly (from 50 – 60%) for any of the three solvents. The relative resistance of the enzyme to solvent denaturation is of great practical value as substrates are by definition poorly water soluble. The influence of acetonitrile was investigated over a wider range, up
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309
Fig. 3. Inhibition effect of reaction product (p-nitrophenol) on ovine microsomal Cytochrome P450. The product yield is corrected for the amount added at the start of the reaction.
Fig. 2. Effect of substrate (p-nitroanisole) concentration on % yield (upper) and product (p-nitrophenol) concentration (lower).
idase inhibitor [11] and should also promote the accumulation of the product glucuronide in the presence of UDPGA. It appears that, unless steps are taken to inhibit glucuronidase, the glucuronide does not accumulate, but its transient formation promotes hydroxylation, probably (see below) by relieving product inhibition. Unfortunately, it appears that conditions that favour glucuronide accumulation inhibit the hydroxylation reaction and lower overall yields. It does not, therefore, appear to be efficient to combine hydroxylation and glucuronidation in vitro as a “one pot” reaction. Addition of UDPGA (which is relatively expensive) likewise, would be an uneconomical way to promote hydroxylation. 3.5. Product inhibition
to 20% (v/v). At concentrations above 3% (v/v), the yield decreased steadily, being below 5% at 10% (v/v) acetonitrile and zero at concentrations above 15%. 3.4. Effect of addition of UDPGA on hydroxylation UDPGA is the glucuronyl donor for the glucuronyl transferase which is also present in the microsome preparation [1]. The hydroxylation was carried out at substrate concentrations between 0.4 and 4 mM in the presence of 1 mM UDPGA. The presence of UDPGA increased the yield of nitrophenol by between 5 and 8%, but the variation with concentration was otherwise very similar to that shown in Fig 2. No p-nitrophenyl glucuronide was detected. Under conditions previously found to favour glucuronidation (pH 8.0, 0.2 M buffer, 100 mM glucuronic acid [11]), however, a decrease of p-nitrophenol and formation of 2–9% glucuronide was observed. These conditions inhibit -glucuronidase, naturally present in the microsomes [11]. The overall yield of products, however, was still 10 –15% lower than under standard hydroxylation conditions. D-saccharic acid 1,4-lactone, although not tested here, is a known glucuron-
The possibility of product inhibition of the hydroxylation reaction was tested by adding different concentrations of p-nitrophenol (up to 2 mM) in acetonitrile to enzyme reactions containing 2 mM p-nitroanisole and 200 l microsomes. The solvent concentration was kept constant at 4%. As controls, the reactions were carried out under the same conditions but without substrate. The yield of the hydroxylation reactions was determined by subtracting the level of pnitrophenol in each control (determined by HPLC) from that in the corresponding reaction with substrate. p-nitrophenol inhibited the enzyme reaction in a concentration-dependent manner (Fig. 3). When adding equimolar amounts of p-nitrophenol a 50% reduction in yield was found. These results also help explain the general observation (see above) that higher substrate concentrations lead to much lower percentage yields of hydroxylation products. 3.6. Effect of amount of co-factor mix For regeneration of NADPH, which is essential as electron donor in the hydroxylation reaction, a co-factor mixture
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Fig. 4. Structures of Benzodiazepines. R1 ⫽ Me, R2 ⫽ H; Diazepam (I); R1 ⫽ Me, R2 ⫽ OH; Temazepam (II); R1 ⫽ H, R2 ⫽ H; Nordiazepam (III).
containing glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP⫹ and DTT was added to each enzyme reaction. The reaction of the glucose-6-phosphate dehydrogenase produces ribulose-5-phosphate, CO2 and NADPH. The NADP⫹ is thus recycled to NADPH. Insufficient glucose-6-phosphate could limit the reaction yield at higher substrate concentrations. When the concentration of glucose-6-phosphate was doubled, however, only a 1–2% increase in product yield was found. 3.7. Scale up of p-nitroanisole reaction The incubation of 5 mM p-nitroanisole with microsomes under the described conditions (see Materials and Methods) resulted a preparatively useful yield of 20% p-nitrophenol. A potential limiting factor to scale up of this reaction would be oxygen diffusion into the reaction mixture. This is likely to become less efficient as the reaction volume increases. To evaluate the scalability of the reaction, it was carried at a 100-fold larger scale (20 ml reaction volume, in an open 100 ml conical flask with stirring). As a control, an incubation on the original small scale was carried out simultaneously using the same microsome preparation. The yields of the reactions were 21% (0.89 mM product concentration) and 15% (0.59 mM product concentration) for the control. It appears that oxygen diffusion is not a limiting factor in the larger scale reaction. For p-nitrophenol, at least, the reaction can be carried out at a synthetically relevant scale. 3.8. Hydroxylation of diazepam Diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H1,4-benzodiazepin-2-one; I, Fig. 4), a benzodiazepine anxi-
olytic drug, is metabolised in humans by two pathways involving cytochrome P450 action [12]. These are a hydroxylation at C-3 to form a pharmacologically active metabolite, Temazepam (II) and removal of the N-methyl group to form the predominant, also pharmacologically active metabolite, Nordiazepam (Nordazepam, Desmethyldiazepam; III). Diazepam is therefore a good model compound to determine the practicality of preparative enzymic hydroxylation for in vitro synthesis of drug metabolites. The optimised reaction conditions found for p-nitroanisole were adapted for the hydroxylation of Diazepam. Diazepam is poorly soluble in acetonitrile, but DMSO proved to be a suitable cosolvent. Trial reactions were carried out as described in Section 2.2 but using Diazepam in DMSO as substrate, at final concentrations of 2 and 4 mM, with DMSO at concentrations of 1, 2 or 4% (v/v). The yields were generally lower than with p-nitroanisole, but still preparatively useful; 9.5, 12.5 and 4% Nordiazepam from 2 mM Diazepam with 1, 2 or 4% (v/v) DMSO, respectively and 3, 4.5 and 2% from 4 mM Diazepam, respectively. Only traces of Temazepam (⬍1%) were observed. The optimal conditions (2 mM Diazepam, 2% DMSO/ 12.5% yield) were scaled up to give a reaction volume of 330 ml. HPLC analysis indicated a crude yield of 9.5%. After isolation and purification (Section 2.9) an isolated yield of Nordiazepam of 4% was obtained. Since the materials required for this reaction are all relatively inexpensive or regenerable, it could be carried out at any manageable scale to make larger amounts of product.
4. Conclusion The conditions required for optimum activity of ovine liver cytochrome P450 in vitro have been determined using demethylation of p-nitroanisole to p-nitrophenol as a model reaction. Variation of both enzyme and substrate concentrations revealed saturation behavior in both cases. Maximal product yields were obtained with microsome suspension comprising at least 60% of the total reaction volume and with least 5 mM p-nitroanisole. One reason for the saturation effect of substrate appears to be end-product inhibition. The presence of 2 mM p-nitrophenol greatly reduced the reaction rate and consequently, the yield of product. The enzyme showed reasonable resistance to denaturation by organic cosolvents (essential for substrate solubilisation). Up to 3% (v/v) of acetonitrile, methanol or DMSO had no apparent effect on the enzyme, with reaction yields being unaffected. Ovine liver microsomes contain glucuronyl transferase as well as cytochrome P450 and it is possible in principle to hydroxylate/dealkylate a compound and glucuronidate it in a “one pot” reaction. Under the optimal conditions for hydroxylation, however, no glucuronidation was observed in the presence of UDPGA, the cofactor for glucuronyl transferase. This is thought to be due to the effects of the
U. Hubl, D.E. Stevenson / Enzyme and Microbial Technology 29 (2001) 306 –311
-glucuronidase which is also present in microsomes. The glucuronidation/de-glucuronidation did, however, increase the yield of hydroxylation product. Conditions which favour glucuronidation [11] allowed some glucuronide to accumulate but the total yield of hydroxylation products was significantly reduced. The in vitro reaction can be used preparatively to make milligram quantities of metabolites. Nordiazepam, a known metabolite [12] of the anxiolytic drug Diazepam, was thus prepared and purified by reversed phase chromatography and preparative HPLC. Compared with glucuronide synthesis using microsomal enzymes [2– 4], yields of hydroxylation are generally much lower, but hydroxylation does not require large amounts of expensive cofactors, NADPH being readily regenerable. The synthetic approach reported here should be of use in the preparation of purified metabolites for analytical reference purposes or research.
[3]
[4]
[5] [6]
[7]
[8]
[9]
Acknowledgments This work was supported by the NZ Foundation for Research, Science and Technology under contract C08620.
[10]
References
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[1] Gibson GG, Skett P. Introduction to drug metabolism, Chapter 1 “Pathways of drug metabolism,” 2nd Ed. Blackie, Glasgow, 1994. [2] Jackson C-JC, Hubbard JW, Midha KK. Biosynthesis and characterisation of glucuronide metabolites of fluphenazine: 7-hydroxyflu-
[12]
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phenazine glucuronide and fluphenazine glucuronide. Xenobiotica 1991;21:383–93. Gygax D, Spies P, Winkler T, Pfaar U. Enzymatic synthesis of  -D-glucuronides with in situ regeneration of uridine 5⬘-diphosphoglucuronic acid. Tetrahedron 1991;47:5119 –22. Stevenson DE, Hubl U. Optimisation of -D-Glucuronide Synthesis Using UDP-Glucuronyl Transferase. Enz Microb Technol 1999;24: 388 –96. Schubert F, Scheller F, Mohr P. Application of cytochrome P-450 in enzyme reactors and enzyme electrodes. Pharmazie 1985;40:235–39. Denti E, Freston JW, Marchisio M, Kolff WJ. Toward a bioartificial drug metabolizing system: gel immobilised liver cell microsomes. Trans Amer Soc Artif Int Organs 1976;22:693–700. Yawetz A, Perry AS, Freeman A, Katchalski-Katzir E. Monooxygenase activity of rat liver microsomes immobilised by entrapment in a crosslinked prepolymerised polyacrylamide hydrazide. Biochim Biophys Acta 1984;798:204 –9. Brugmann E, Stange J, Falkenhagen D, Dummler W, Ernst B, Dautzenberg H. encapsulaiton of liver microsomes–a further procedure for detoxication. Biomat Art Cells Art Org 1990;18:513–5. Mackenzie P, Lang M, Hanninen O. Metabolism of benzo(a)pyrene by liposomes containing purified microsomal drug metabolising enzymes. In: Coon MJ, Conney AH, Estabrook RW, editors. Microsomes Drug Oxid, Chem Carcinog. New York: Academic Press, 1980:659 – 62. Okamura T, Sugiura W, Miyazawa M. Reduction of oxazepam to desmethyldiazepam by human intestinal bacteria. Biol Pharm Bull 1996;19:647– 8. Stevenson DE. Optimisation of UDP-Glucuronyl Transferase-Catalysed Synthesis of Testosterone--D-glucuronide by Inhibition of Contaminating -Glucuronidase. Biotechnol Techniques 1999;13: 17–21. The Merck Index. 11th Ed. S Budavari, Ed. Merck, Co, Rahway, NJ,. pp 472, 1057, 1439, 1989.
www.elsevier.com/locate/enzmictec
In vitro enzymic synthesis of mammalian liver xenobiotic metabolites catalysed by ovine liver microsomal cytochrome P450 Ulrike Hubl, David E. Stevenson* Industrial Research Limited, PO Box 31–310, Lower Hutt, New Zealand Received 31 August 2000; received in revised form 3 May 2001; accepted 4 June 2001
Abstract Ovine microsomal Cytochrome P450 was used preparatively in vitro to make milligram quantities of Nordiazepam, a known metabolite of the anxiolytic drug Diazepam. The product was purified by reversed phase chromatography and preparative HPLC. This synthetic approach should be of use in the preparation of purified metabolites for analytical reference purposes or research. Using demethylation of p-nitroanisole as a model reaction, the conditions required for optimum activity of ovine liver microsomal cytochrome P450 in vitro have been determined. Variation of both enzyme and substrate concentrations revealed saturation behavior in both cases. Above the optimal concentrations, no increase in yield of product was obtained. End-product inhibition was shown to occur and this accounts for the lower conversion rate at higher substrate concentrations. The enzyme showed reasonable resistance to denaturation by organic cosolvents (essential for substrate solubilisation). Up to 3% v/v of acetonitrile, methanol or DMSO had no apparent effect on the enzyme, reaction yields being unaffected. Another xenobiotic metabolizing enzyme, Glucuronyl transferase is also present in microsomes. Attempted simultaneous hydroxylation and glucuronidation, in the presence of UDPGA, the cofactor for Glucuronyl transferase was not successful, due to the effects of contaminating -glucuronidase in the microsomes. The glucuronidation/de-glucuronidation did, however, increase the yield of hydroxylation product. Conditions which favour glucuronidation allowed some glucuronide to accumulate but the total yield of hydroxylation products (glucuronidated and free) was significantly reduced. © 2001 Elsevier Science Inc. All rights reserved. Keywords: cytochrome P450, p-nitroanisole; diazepam; hydroxylation; demethylation; enzymic; ovine
1. Introduction Hydrophobic xenobiotic compounds such as drugs and toxins are metabolised in the mammalian liver into more polar derivatives. The main metabolic pathway involves hydroxylation (if required) by the cytochrome P450 hydroxylase and associated NADPH cytochrome reductase, followed by conjugation to form water soluble species [1]. Cytochrome P450 activates molecular oxygen for insertion into a carbonhydrogen bond. The conjugates, glucuronides being the most common, are then readily excreted by the renal system [1]. Hydroxylated or glucuronidated metabolites are useful in clinical trials of the parent drug, or as analytical reference standards in the testing for illegal drugs. There are many reports of the in vitro use of glucuronyl transferase in preparative glucuronide synthesis [2– 4], but cytochrome P450 has mainly been used in enzyme electrodes [5] and
when immobilised, in trials of artificial livers for extra-corporeal detoxification of blood during liver disease [6 –9]. In one such report [9] cytochrome P450 was co-immobilised with glucuronyl transferase and the combination was able to hydroxylate and subsequently glucuronidate benzo(a)pyrene. In this report we describe the use of ovine liver microsomes for the in vitro demethylation of p-nitroanisole, to form p-nitrophenol and the optimisation of the reaction conditions for preparative scale synthesis. The demethylation of p-nitroanisole is, strictly, a hydroxylation of the methyl group followed by its non-enzymic loss as formaldehyde to liberate the phenolic hydroxyl group. We also report the preparative scale demethylation of the drug Diazepam.
2. Materials and Methods 2.1. Materials
Corresponding author. Tel.: ⫹64 – 4-569 – 0000; fax: ⫹64 – 4-566 – 6004. E-mail address: [email protected] (D. Stevenson). *
NADP, Glucose-6-phosphate dehydrogenase, glucose-6phosphate and UDP-glucuronate (UDPGA) were obtained
0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 1 ) 0 0 3 8 9 - 1
U. Hubl, D.E. Stevenson / Enzyme and Microbial Technology 29 (2001) 306 –311
from Boehringer Mannheim (Mannheim, Germany). Buffers, solvents and general chemicals were obtained from Sigma/Aldrich. Diazepam was a gift from Douglas Pharmaceuticals (Auckland, New Zealand), Nordiazepam and Temazepam were gifts from B Dent Global Ltd, (Wellington, NZ). Ovine liver microsome suspension (“microsomes”; containing cytochrome P450, UDP-glucuronyl transferase and NADPH reductases) was prepared according to Gibson and Skett [1], from fresh ovine livers obtained from Taylor Preston Ltd. (Wellington, NZ). Microsomes were collected by flocculation with Calcium Chloride solution and centrifugation at 27,000 g, rather than by ultra-centrifugation. The microsomal pellet was resuspended in 50 mM Tris buffer, pH 7.4, containing 0.1 M KCl and 2 mM DTT. This resulted in a protein concentration of 11–13 mg/ml and approx 7% total solids. Microsomes were frozen in 3 ml plastic vials, using liquid nitrogen and stored at –70°C. 2.2. General method for trial hydroxylation reactions Tris buffer (0.1 M, pH 7.4, 240 l), co-factor regeneration mix (50 l from 1 ml of the same Tris buffer, containing 10 l glucose-6-phosphate dehydrogenase suspension, 6 mM glucose-6-phosphate, 2 mM NADP, 50 mM nicotinamide and 1 mM DTT) and microsome suspension (200 l, to give a protein concentration of 4.8 mg/ml in a total volume of 0.5 ml) were incubated at 37°C in an open 7 ml glass vial for 10 min. The reaction was started by adding the required volume of substrate solution (p-nitroanisole in acetonitrile or diazepam in DMSO; 10 l, unless stated otherwise). After incubation with magnetic stirring overnight, the reaction was stopped by adding acetonitrile/ethanol (1:1 v/v; 1 ml). The sample was mixed and centrifuged in a microcentrifuge (13,000 rpm/5 min./room temperature). The supernatant was sampled for HPLC analysis.
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2.5. Concentration of organic solvent p-nitroanisole solutions in acetonitrile containing concentrations of 20 to 250 mM were prepared in acetonitrile, DMSO and methanol. For starting the enzyme reaction 2 l (in the case of the highest concentration) to 25 l (in the case of the lowest substrate concentration) of the solutions were added to give a final substrate concentration of 1 mM. The solvent concentration was thus varied from 0.4 to 5%, for DMSO and methanol and 0.4 to 20% for acetonitrile. The other reaction conditions were as described in Section 2.2. 2.6. Influence of UDPGA on p-nitroanisole hydroxylation Substrate solutions containing concentrations of 50 to 200 mM were prepared. UDPGA solution (10 mM in Tris buffer; 50 l) was added to Tris buffer (see above; 190 l), cofactor regeneration mixture (see above, 50 l) and microsomes (see above; 200 l, to give 4.8 mg protein/ml). The reaction was started by adding 10 l of different substrate solutions following the same procedure as described above (Section 2.4). The same experiment was done using glucuronidation buffer (0.2 M Tris, pH 8.0 containing 0.1 M glucuronic acid). 2.7. Determination of product inhibition p-Nitrophenol solutions (10 to 50 mM, 10 l) were added to Tris-buffer (230 l), cofactor regeneration mixture (50 l) and microsomes (200 l). The reaction was started by the addition of p-nitroanisole solution (100 mM; 10 l) as described above. For determination of any decomposition of p-nitrophenol, acetonitrile (10 l) was added instead of the substrate solution. The activity of the enzyme preparation was also tested with 20 l substrate solution (50 mM) added to the same reaction mixture, without adding p-nitrophenol. 2.8. HPLC analysis
2.3. Variation of enzyme amount Volumes of enzyme suspension between 50 and 400 l (giving 1.2–9.6 mg protein/ml) were used for the reaction. The volume of Tris buffer was varied accordingly, so that the final volume of the reaction mixture remained at 0.5 ml. The other reaction conditions were as described in Section 2.2. 2.4. Variation of substrate concentration p-nitroanisole solutions in acetonitrile containing concentrations in a range of 5 to 250 mM were prepared. The enzyme reaction was carried out as above, with 10 l of the different substrate solutions being added.
Centrifuged solution from the quenched enzyme reaction (see section 2.2; 20 L) was injected onto a Phenomenex (Torrance, CA) Nucleosil (5 M, 10 nM (100 Å), C18 column (250 ⫻ 4.6 mm plus 30 ⫻ 4.6 mm guard) fitted to a Gilson (Middleton, WI) dual pump HPLC system fitted with an autoinjector and UV-visible diode array detector. For p-nitroanisole reaction samples, the solvent flow rate was kept constant at 0.7 ml min⫺1 and the initial solvent composition was aqueous trifluoroacetic acid (TFA; 0.1% v/v)/acetonitrile, 75: 25 v/v. The acetonitrile content was increased linearly to 90% over a period of 20 min and back to 25% over 1 min. Peaks were detected by UV absorption at 311 nm. Under these conditions, the retention times of p-nitroanisole, p-nitrophenol and p-nitrophenyl glucuronide were 20.7, 14.7 and 8.4 min respectively. Their relative
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extinction coefficients, determined by injecting an equimolar mixture of the three compounds, were 1:1.2:1.7, respectively. Diazepam reactions were analysed similarly, except that the acetonitrile content of the eluting solvent was varied linearly from 40 – 80% over a period of 14 min. UV detection was at 238 nm. Under these conditions the retention times for Nordiazepam, Diazepam and Temazepam were 7.7, 10.2 and 13 min, respectively and the relative extinction coefficients were 1:1.12:1.09, respectively. 2.9. Purification and characterisation of products from preparative diazepam reaction The hydroxylation reaction of Diazepam was a direct scale up of the analytical scale reaction. Conditions were as described in Section 2.2 except that a total volume of 330 ml was used with 2 mM (190 mg) Diazepam, 2% v/v DMSO and 60% v/v microsome suspension (final protein conc. 7.2 mg/ml). The mixture of microsomes, buffer and cofactors was stirred rapidly at 37°C and the substrate added dropwise in DMSO (6.6 ml). The mixture was then split between 9 ⫻ 100 ml unstoppered conical flasks and stirred at 37°C overnight. The crude yield of Nordiazepam was determined to be 9.5% by analytical HPLC. The mixture was then freeze-dried, redissolved in water (400 ml) and freeze-dried again to remove the bulk of the DMSO. The benzodiazepines were extracted with hot (50°C) ethyl acetate (2 ⫻ 200 ml), filtered and the solvent removed under vacuum. The residue (1.96 g) was dissolved in 50% aqueous methanol (150 ml) and applied to a 2.5 ⫻ 20 cm column of LiChroprep RP-18 (40 – 63 M; Merck, Darmstadt, Germany) equilibrated with the same solvent. The column was eluted with 150 ml each of 50%, 75% and 100% methanol. The benzodiazepines eluted in 100% methanol. Fractions were monitored by TLC on foil backed silica plates (Merck) using ethyl acetate/hexane 1:1 v/v as solvent. A slight separation was achieved between Diazepam and Nordiazepam on the column, but a lot of other material, presumably lipid from the microsomes, was removed. The fractions containing Nordiazepam were pooled and evaporated to dryness under vacuum. The mixture of benzodiazepines (0.48 g) was separated by preparative HPLC. The method was identical to the analytical method (Section 2.8) except that a 21 ⫻ 250 mm C-5 column was used with isocratic solvent (acetonitrile/ water 32.5:67.5) at 10 ml/min. The sample was dissolved in Methanol (2.5 ml) and applied to the column in five 0.5 ml injections. The retention times of Nordiazepam and Diazepam were 30 and 43 mins respectively. Fractions were collected manually. Purified Nordiazepam (8 mg/4% yield) was obtained after pooling and evaporation of solvents and crystallisation from aqueous methanol. The 1H- and 13C-NMR spectra [10] and analytical HPLC retention time of the isolated material were identical to those of the authentic standard.
Fig. 1. Effect of microsomal protein concentration on p-nitrophenol yield (%) from p-nitroanisole (as determined by HPLC).
3. Results and Discussion 3.1. Effect of microsomal enzyme concentration Microsomal protein concentrations between 1.2 and 9.6 mg/ml were added to reaction mixtures containing 5 mM p-nitroanisole. Product formation increased with increasing amounts of added microsomes, following a linear relation for volumes under 200 l (Fig. 1). At concentrations above 7 mg of added microsomal protein, a maximum conversion of 25.8% was reached. The yield was still 86% of the maximum at 4.8 mg/ml protein. This latter amount was used for all subsequent experiments, unless stated otherwise. 3.2. Effect of p-nitroanisole substrate concentration The enzymic reaction was carried out with substrate concentrations between 0.1 and 5 mM. An almost complete conversion to p-nitrophenol was found for concentrations below 0.2 mM (Fig. 2). With increasing substrate concentrations the yield of product decreased. The product concentration reached a maximum of 0.95 mM at a substrate concentration of 5 mM, however, the yield of product at this substrate concentration was only 20%. 3.3. Effect of different co-solvents Acetonitrile, methanol and dimethyl sulphoxide (DMSO) were compared as co-solvents. The solvent is necessary for dissolving the water insoluble substrate p-nitroanisole. The solvent concentration was varied between 0.4 and 3% (v/v), keeping the substrate constant at 1 mM. In this range the reaction yield did not vary significantly (from 50 – 60%) for any of the three solvents. The relative resistance of the enzyme to solvent denaturation is of great practical value as substrates are by definition poorly water soluble. The influence of acetonitrile was investigated over a wider range, up
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309
Fig. 3. Inhibition effect of reaction product (p-nitrophenol) on ovine microsomal Cytochrome P450. The product yield is corrected for the amount added at the start of the reaction.
Fig. 2. Effect of substrate (p-nitroanisole) concentration on % yield (upper) and product (p-nitrophenol) concentration (lower).
idase inhibitor [11] and should also promote the accumulation of the product glucuronide in the presence of UDPGA. It appears that, unless steps are taken to inhibit glucuronidase, the glucuronide does not accumulate, but its transient formation promotes hydroxylation, probably (see below) by relieving product inhibition. Unfortunately, it appears that conditions that favour glucuronide accumulation inhibit the hydroxylation reaction and lower overall yields. It does not, therefore, appear to be efficient to combine hydroxylation and glucuronidation in vitro as a “one pot” reaction. Addition of UDPGA (which is relatively expensive) likewise, would be an uneconomical way to promote hydroxylation. 3.5. Product inhibition
to 20% (v/v). At concentrations above 3% (v/v), the yield decreased steadily, being below 5% at 10% (v/v) acetonitrile and zero at concentrations above 15%. 3.4. Effect of addition of UDPGA on hydroxylation UDPGA is the glucuronyl donor for the glucuronyl transferase which is also present in the microsome preparation [1]. The hydroxylation was carried out at substrate concentrations between 0.4 and 4 mM in the presence of 1 mM UDPGA. The presence of UDPGA increased the yield of nitrophenol by between 5 and 8%, but the variation with concentration was otherwise very similar to that shown in Fig 2. No p-nitrophenyl glucuronide was detected. Under conditions previously found to favour glucuronidation (pH 8.0, 0.2 M buffer, 100 mM glucuronic acid [11]), however, a decrease of p-nitrophenol and formation of 2–9% glucuronide was observed. These conditions inhibit -glucuronidase, naturally present in the microsomes [11]. The overall yield of products, however, was still 10 –15% lower than under standard hydroxylation conditions. D-saccharic acid 1,4-lactone, although not tested here, is a known glucuron-
The possibility of product inhibition of the hydroxylation reaction was tested by adding different concentrations of p-nitrophenol (up to 2 mM) in acetonitrile to enzyme reactions containing 2 mM p-nitroanisole and 200 l microsomes. The solvent concentration was kept constant at 4%. As controls, the reactions were carried out under the same conditions but without substrate. The yield of the hydroxylation reactions was determined by subtracting the level of pnitrophenol in each control (determined by HPLC) from that in the corresponding reaction with substrate. p-nitrophenol inhibited the enzyme reaction in a concentration-dependent manner (Fig. 3). When adding equimolar amounts of p-nitrophenol a 50% reduction in yield was found. These results also help explain the general observation (see above) that higher substrate concentrations lead to much lower percentage yields of hydroxylation products. 3.6. Effect of amount of co-factor mix For regeneration of NADPH, which is essential as electron donor in the hydroxylation reaction, a co-factor mixture
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Fig. 4. Structures of Benzodiazepines. R1 ⫽ Me, R2 ⫽ H; Diazepam (I); R1 ⫽ Me, R2 ⫽ OH; Temazepam (II); R1 ⫽ H, R2 ⫽ H; Nordiazepam (III).
containing glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP⫹ and DTT was added to each enzyme reaction. The reaction of the glucose-6-phosphate dehydrogenase produces ribulose-5-phosphate, CO2 and NADPH. The NADP⫹ is thus recycled to NADPH. Insufficient glucose-6-phosphate could limit the reaction yield at higher substrate concentrations. When the concentration of glucose-6-phosphate was doubled, however, only a 1–2% increase in product yield was found. 3.7. Scale up of p-nitroanisole reaction The incubation of 5 mM p-nitroanisole with microsomes under the described conditions (see Materials and Methods) resulted a preparatively useful yield of 20% p-nitrophenol. A potential limiting factor to scale up of this reaction would be oxygen diffusion into the reaction mixture. This is likely to become less efficient as the reaction volume increases. To evaluate the scalability of the reaction, it was carried at a 100-fold larger scale (20 ml reaction volume, in an open 100 ml conical flask with stirring). As a control, an incubation on the original small scale was carried out simultaneously using the same microsome preparation. The yields of the reactions were 21% (0.89 mM product concentration) and 15% (0.59 mM product concentration) for the control. It appears that oxygen diffusion is not a limiting factor in the larger scale reaction. For p-nitrophenol, at least, the reaction can be carried out at a synthetically relevant scale. 3.8. Hydroxylation of diazepam Diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H1,4-benzodiazepin-2-one; I, Fig. 4), a benzodiazepine anxi-
olytic drug, is metabolised in humans by two pathways involving cytochrome P450 action [12]. These are a hydroxylation at C-3 to form a pharmacologically active metabolite, Temazepam (II) and removal of the N-methyl group to form the predominant, also pharmacologically active metabolite, Nordiazepam (Nordazepam, Desmethyldiazepam; III). Diazepam is therefore a good model compound to determine the practicality of preparative enzymic hydroxylation for in vitro synthesis of drug metabolites. The optimised reaction conditions found for p-nitroanisole were adapted for the hydroxylation of Diazepam. Diazepam is poorly soluble in acetonitrile, but DMSO proved to be a suitable cosolvent. Trial reactions were carried out as described in Section 2.2 but using Diazepam in DMSO as substrate, at final concentrations of 2 and 4 mM, with DMSO at concentrations of 1, 2 or 4% (v/v). The yields were generally lower than with p-nitroanisole, but still preparatively useful; 9.5, 12.5 and 4% Nordiazepam from 2 mM Diazepam with 1, 2 or 4% (v/v) DMSO, respectively and 3, 4.5 and 2% from 4 mM Diazepam, respectively. Only traces of Temazepam (⬍1%) were observed. The optimal conditions (2 mM Diazepam, 2% DMSO/ 12.5% yield) were scaled up to give a reaction volume of 330 ml. HPLC analysis indicated a crude yield of 9.5%. After isolation and purification (Section 2.9) an isolated yield of Nordiazepam of 4% was obtained. Since the materials required for this reaction are all relatively inexpensive or regenerable, it could be carried out at any manageable scale to make larger amounts of product.
4. Conclusion The conditions required for optimum activity of ovine liver cytochrome P450 in vitro have been determined using demethylation of p-nitroanisole to p-nitrophenol as a model reaction. Variation of both enzyme and substrate concentrations revealed saturation behavior in both cases. Maximal product yields were obtained with microsome suspension comprising at least 60% of the total reaction volume and with least 5 mM p-nitroanisole. One reason for the saturation effect of substrate appears to be end-product inhibition. The presence of 2 mM p-nitrophenol greatly reduced the reaction rate and consequently, the yield of product. The enzyme showed reasonable resistance to denaturation by organic cosolvents (essential for substrate solubilisation). Up to 3% (v/v) of acetonitrile, methanol or DMSO had no apparent effect on the enzyme, with reaction yields being unaffected. Ovine liver microsomes contain glucuronyl transferase as well as cytochrome P450 and it is possible in principle to hydroxylate/dealkylate a compound and glucuronidate it in a “one pot” reaction. Under the optimal conditions for hydroxylation, however, no glucuronidation was observed in the presence of UDPGA, the cofactor for glucuronyl transferase. This is thought to be due to the effects of the
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-glucuronidase which is also present in microsomes. The glucuronidation/de-glucuronidation did, however, increase the yield of hydroxylation product. Conditions which favour glucuronidation [11] allowed some glucuronide to accumulate but the total yield of hydroxylation products was significantly reduced. The in vitro reaction can be used preparatively to make milligram quantities of metabolites. Nordiazepam, a known metabolite [12] of the anxiolytic drug Diazepam, was thus prepared and purified by reversed phase chromatography and preparative HPLC. Compared with glucuronide synthesis using microsomal enzymes [2– 4], yields of hydroxylation are generally much lower, but hydroxylation does not require large amounts of expensive cofactors, NADPH being readily regenerable. The synthetic approach reported here should be of use in the preparation of purified metabolites for analytical reference purposes or research.
[3]
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[5] [6]
[7]
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[9]
Acknowledgments This work was supported by the NZ Foundation for Research, Science and Technology under contract C08620.
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