debrisoquine metabolism and cyp2d expression in marmoset … · 2011. 10. 5. · dmd #41566 3...

1 DMD #41566

Title page

Debrisoquine metabolism and CYP2D expression in marmoset liver

microsomes

Brian R Cooke, S W Annie Bligh, Z Richard Cybulski, Costas Ioannides and

Michael Hall

Department of In Vitro Metabolism, Huntingdon Life Sciences Ltd., Woolley Road,

Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, United Kingdom (B.R.C.,

Z.R.C., M.H.), Institute for Health Research and Policy, London Metropolitan

University, 166-220 Holloway Road, London, N7 8DB, United Kingdom (S.W.A.B.)

and Molecular Toxicology Group, Faculty of Health and Medical Sciences,

University of Surrey, Guildford, Surrey, GU2 7XH, United Kingdom (C.I.).

DMD Fast Forward. Published on October 5, 2011 as doi:10.1124/dmd.111.041566

Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on October 5, 2011 as DOI: 10.1124/dmd.111.041566

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Running title page

Running title: Marmoset debrisoquine metabolism and CYP2D expression

Corresponding author: Dr M Hall Department of In Vitro Metabolism Huntingdon Life Sciences Ltd. Woolley Road, Alconbury Huntingdon, Cambridgeshire PE28 4HS, UK Tel. No: +44 (0) 1480 892143 Fax No: +44 (0) 1480 892281 E-mail: [email protected]

Number of text pages: 11

Number of tables: 2

Number of Figures: 6

Number of References: 24

Number of words in Abstract: 171

Number of words in Introduction: 518

Number of words in Results and Discussion: 1,021

Nonstandard abbreviations:

2D COSY Two-Dimensional Correlation Spectroscopy

CID collisionally induced dissociation

ESI electrospray ionisation

HPLC high-performance liquid chromatography

LC-MS/MS liquid chromatography-tandem mass spectrometry


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Abstract

The objective of this study was to define CYP2D enzymes in marmoset (Callithrix

jacchus) liver microsomes, both at the activity level using debrisoquine as the model

substrate, and at the protein level employing antibodies raised to human CYP2D6.

Marmoset liver microsomes were incubated with [14C]debrisoquine and the structure of

the generated metabolites determined using liquid chromatography-tandem mass

spectrometry and NMR. Marmoset liver microsomes were very effective in

hydroxylating debrisoquine at various positions. Although 4-hydroxydebrisoquine was

formed, in contrast to rat and human it was only a minor metabolite. Debrisoquine was

more extensively hydroxylated in the 7-, 5-, 6- and 8-positions. In addition to the

mono-hydroxylated metabolites, a dihydroxy metabolite, namely 6,7-

dihydroxydebrisoquine was identified. Finally, metabolites that had undergone ring-

opening were also detected but were not investigated further. Antibodies to CYP2D6

immunoreacted with protein in marmoset and human, but not rat hepatic microsomes.

In conclusion, we have demonstrated that marmoset liver microsomes are effective in

hydroxylating debrisoquine at various positions and that they contain a protein that is

immunorelated to human CYP2D6.


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Introduction

Non-human primates are employed regularly as non-rodent human surrogates during

drug development. The most commonly used non-human primates are monkeys of the

genus Macaca, including the cynomolgus monkey (Macaca fascicularis), rhesus

monkey (Macaca mulatta) and Japanese macaque (Macaca fuscata). The macaque

cytochromes P450, the most important enzyme system in drug metabolism, share high

amino acid sequence identity with the human orthologue proteins (Uno et al., 2007).

However, despite this similarity, these old world monkeys do not always generate

metabolic data predictive of human metabolism (Guengerich, 1997; Bogaards et al.,

2000), they are relatively large in size and display other characteristics that can cause

them to be challenging to use (Jacqz et al., 1988). In contrast, the common marmoset

(Callithrix jacchus), a new world monkey, is small and easier to handle, and is

characterised by good fertility so that captive breeding is generally successful.

Furthermore, given the fact that the marmoset size is similar to that of rat, techniques

and methodologies developed in rat can be readily adapted for use in marmosets

(McAnulty, 1996; Smith et al., 2001). Most studies on cytochrome P450 expression in

primates have been carried out in the cynomolgus and rhesus monkeys (Uno et al.,

2006, 2007; Iwasaki and Uno 2009; Uehara et al., 2010). Only a limited number of

investigations have been devoted to delineating the cytochrome P450 enzymes in

marmoset liver, and these have been largely confined to immunological studies

employing antibodies raised to rat and human cytochrome P450 enzymes (Schulz et

al., 1996, 1998, 2001; Igarashi et al., 1997; Uno et al., 2010).

One of the most important cytochrome P450 subfamilies involved in drug

metabolism is CYP2D (Zanger, 2008). The human CYP2D6 was the first drug


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metabolising enzyme for which polymorphic expression was described, the

antihypertensive debrisoquine being the drug employed in these studies, whose 4-

hydroxylation is catalysed by this enzyme (Mahgoub et al., 1977). At least two

enzymes of the CYP2D subfamily, CYP2D17 and CYP2D44 have been identified in

cynomolgus monkey (Iwasaki and Uno 2009; Uno et al., 2010). These showed

homologies in nucleotide and amino acid sequences of 94% and 93% between

monkey CYP2D17 and human CYP2D6 and of 93% and 91% between monkey

CYP2D44 and human CYP2D6, respectively. Moreover, both CYP2D17 and

CYP2D44, similar to the human orthologue, could metabolise dextromethorphan and

bufuralol (Iwasaki and Uno 2009; Uno et al., 2010). Similarly the cDNA of two

CYP2D enzymes, CYP2D19 and CYP2D30 from marmoset liver have been cloned

and the proteins expressed in yeast cells (Hichiya et al., 2004). These showed

homologies in nucleotide and amino acid sequences of 92% and 91% between

monkey CYP2D19 and human CYP2D6 and of 96% and 95% between monkey

CYP2D30 and human CYP2D6, respectively. Both expressed enzymes demonstrated

the capability to metabolise bufuralol and debrisoquine, albeit the regioselectivity of

hydroxylation of debrisoquine was quite different between the two. Further, expressed

CYP2D19 has been shown to hydroxylate propranolol (Narimatsu et al., 2011). The

objective of this study was to define CYP2D enzymes in marmoset liver microsomes,

both at the activity and protein level, using debrisoquine as the model substrate and

antibodies raised to human CYP2D6.


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Materials and Methods

Chemicals

Debrisoquine sulphate (3,4-dihydro-2(1H)-isoquinolinecarboximidamide sulphate

(2:1); ICN Biomedicals Ltd, Thame, Oxfordshire, UK), 4-hydroxydebrisoquine

(Ultrafine Chemicals Ltd, Salford, UK), [guanidine-14C]debrisoquine (53 μCi/μmol,

50 μCi/ml) (Amersham International plc, Amersham, Buckinghamshire, UK), 1,3-

dichloro-9,9-dimethylacridin-2-one-7-yl phosphate, diammonium salt (DDAO

phosphate) (Invitrogen, Carlsbad, CA, USA) and all other chemicals were purchased

at the highest commercially available grade. Only the 4-hydroxy metabolite of

debrisoquine is available commercially as an authentic reference standard.

Monoclonal anti-human CYP2D6 antibody raised in mouse ascites fluid, rat liver

microsomes and recombinant human CYP2D6 expressed in insect cells

(Supersomes™) (BD Biosciences, Bedford, MA, USA), rabbit anti-mouse IgG alkaline

phosphatase conjugated antibody (Invitrogen, Carlsbad, CA, USA), and full range

molecular weight markers (10,000-250,000 Da) (Amersham International plc,

Amersham, Buckinghamshire, UK) were purchased from the commercial suppliers

listed.

Animals

Marmoset livers were taken from five male animals, ranging from 6 years 11 months

to 8 years 8 months in age and 376 g to 571 g body weight (batch 1), and from 1 year

10 months to 11 years in age and 289 g to 397 g body weight (batch 2), housed at a

UK breeding colony. Animals were sacrificed under barbiturate anaesthesia, and

livers were excised immediately and snap frozen in liquid N2. Resected male human


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liver samples were obtained frozen from the UK Human Tissue Bank. Livers were

transferred frozen for storage at -75° until required. Microsomes were isolated by

differential centrifugation. Pooled liver microsomes (n=5) were used throughout,

unless otherwise stated.

Debrisoquine hydroxylation

The hydroxylation of debrisoquine was determined by a method based on that

described by Kronbach et al. (1987). For determination of the kinetics of debrisoquine

hydroxylation, marmoset hepatic microsomal protein (0.75 mg; batch 1) was mixed

with NADPH (6 mM) and Tris-HCl buffer (50 mM), pH 7.69 at 25°C in a total

volume of 250 µl and pre-incubated for 3 minutes at 37°C. The reaction was initiated

by the addition of [14C]debrisoquine (25 µl; final concentrations: 50 to 5000 µM) and

was terminated after 10 minutes by the addition of chilled perchloric acid (70%, w/v).

To generate material for the partial identification of hydroxylated debrisoquine

metabolites by LC-MS/MS (Finnigan TSQ7000), [14C]debrisoquine (500 µM) was

incubated with marmoset liver microsomes (5 mg/ml; batch 1) for 120 minutes under

similar conditions. Following precipitation of the protein and centrifugation, the

supernatant was transferred to an HPLC vial, which was loaded onto an autosampler.

An aliquot (60 µl) was injected directly onto the HPLC system (Waters Alliance

2690) equipped with a Nucleosil 120-5 C18 EXCEL column (100 × 4.6 mm) fitted with

a C18 pre-column. Mobile phase consisted of solvent A (10 mM trifluoroacetic acid)

and solvent B (acetonitrile), and the analyte was eluted using a linear gradient over 40

minutes; flow rate through the column was 0.5-1.0 ml/minute (HPLC method 1). The

eluate was monitored for UV absorbance at 211 nm and for radioactivity using a β-

RAM radioisotope system and data collected using Laura system software. Unreacted


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[14C]debrisoquine and metabolites were quantified by dividing the

radiochromatogram into regions of interest and converting the % of total radioactivity

in each region of interest into amount of debrisoquine equivalents using the starting

concentration of [14C]debrisoquine.

LC-MS/MS

For on-line LC-MS analysis the HPLC eluate from the column passed through a

splitter, in the ratio of 9:1, with the majority of the eluate passing to waste (via UV

and radio detectors) and the remainder to a Finnigan TSQ7000 mass spectrometer for

positive ESI (ESI+). The spray voltage and the capillary temperature were set to 4.5

kV and 250°C, respectively. Nitrogen was used as sheath gas at a head pressure of 60

psi and also as auxiliary gas at 10 units. Mass spectrometric data were acquired over

an appropriate mass range at a scan rate of 1 s/scan. The mass spectrum of metabolites

was obtained by averaging several scans across the appropriate region of the mass

chromatogram, with appropriate background subtraction. This mass spectrum was

examined to identify a candidate protonated molecular ion, MH+ for the compound,

and any other structurally significant fragment ions resulting from in-source CID. The

ESI+ mass spectrometric data were examined to identify candidate molecular ions for

the metabolite(s) of interest, from which particular ions were selected for MS/MS

analysis in the product ion scan mode. To achieve this, the Q1 mass analyser was set

to pass only ions of mass corresponding to one of the candidate molecular ion species

identified from the mass spectrum. This precursor ion was fragmented by CID in the

Q2 region at a collision energy that was empirically optimised to provide maximum

structural information (Argon, 2 mTorr). A mass spectrum of the fragments produced

was recorded by scanning Q3 across a mass range from m/z 10 to a point several mass

units above the selected precursor ion mass, employing a scan rate of 1 s/scan. Several


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scans were averaged to produce the product ion mass spectra of the putative

metabolites.

NMR

For the analysis of hydroxylated metabolites of debrisoquine by NMR, larger scale

incubations were performed. Initially, a 3-minute pre-incubation was carried out of

the mixture comprising 50 mM Tris HCl buffer, pH 7.69 at 25°C, 6 mM NADPH, and

4 mg/ml pooled marmoset liver microsomal protein (batch 1) in a total volume of 35

ml. Reaction was initiated by the addition of 3.5 ml of 5 mM [14C]debrisoquine in Tris

buffer, giving a final concentration of 500 μM. Following a 180-minute incubation the

reaction was terminated by the addition of an equal volume of chilled acetonitrile, and

precipitated protein removed by centrifugation. The supernatant was then lyophilised.

The dried sample was re-suspended in 5 ml 0.1% (v/v) trifluoroacetic acid (HPLC

solvent A). Aliquots were analysed by HPLC running a linear solvent gradient over 60

minutes (HPLC solvent B was acetonitrile), at 4 ml/minute flow rate using an Inertsil

ODS3 C18 column (250 × 9 mm), with eluate monitored for UV at 211 nm (HPLC

method 2). Eight individual metabolite fractions plus unchanged parent debrisoquine

were collected and pooled from the eluates (Figure 1). Total volume and radioactivity

were determined in each fraction and used to estimate the mass of individual

metabolite fractions based upon the original total starting mass of [14C]debrisoquine.

Proton NMR spectra were obtained at 500 MHz by dissolving standards

(debrisoquine and 4-hydroxydebrisoquine) and the dried HPLC fractions in D2O using

water as an internal reference. Analysis was conducted in a Bruker Avance NMR 500

MHz spectrometer equipped with a 5 mm broadband BBO probe. The position of

hydroxyl substitution was deduced by proton coupling patterns and connectivity of


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protons 1 and 4 with the aromatic protons 8 and 5, respectively. The assignments of

the NMR signals were made by long-range 2D COSY experiments.

Western blotting

For the immunoblot analysis marmoset (batch 2), rat and human hepatic microsomal

proteins were transferred electrophoretically to a Hybond-P polyvinylidene difluoride

membrane. The amount of protein loaded was determined during preliminary

investigations. Duplicate aliquots (20 µl) of pooled microsomal protein, negative

control (no microsomal protein) and Supersomes™ (as positive control and for

standard curve construction) were each loaded into the appropriate wells. A 10 µl

aliquot of a protein molecular weight marker (10,000-250,000 Da) was also included.

Electrophoresis was performed at 150 V (200 mA) for 70 minutes. The immunoblot

was probed with the primary antibody (monoclonal anti-human CYP2D6 antibody

raised in mouse) for 30 minutes followed by the alkaline phosphatase-labelled

secondary antibody (rabbit anti-mouse antibody). The blot was then exposed to

DDAO phosphate, and digital images of the fluorescent hydrolysis product were

captured immediately on a Fujifilm FLA-5000 imaging system incorporating a red

laser (635 nm). Protein molecular weights were calculated by comparing Rf values of

individual protein bands to Rf values of the molecular weight markers run

concurrently on the same gel. These values were used to construct a weighted

algorithmic standard curve.


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Results and Discussion

CYP2D6 is the only human enzyme within this cytochrome P450 subfamily, and it is

a strictly constitutive enzyme which, however, makes an important contribution to

drug metabolism despite its relatively low level of expression (Zanger 2008). Its role

in the bioactivation of chemical carcinogens is very limited (Ioannides and Lewis

2004). The enzyme has received extensive attention, being the first cytochrome P450

that was demonstrated to be polymorphically expressed, which is the basis for the

presence of poor and extensive metabolisers of the antihypertensive drug debrisoquine

within the human population (Eiermann et al., 1998). The purpose of the present study

was to define the CYP2D subfamily in the liver of the marmoset, a potential surrogate

primate animal species for human.

As the 4-hydroxylation of debrisoquine, the major metabolite in both rat and

humans (Allen et al., 1975), is used routinely to monitor CYP2D6 activity, the

metabolism of debrisoquine was investigated in NADPH-fortified pooled male

marmoset liver microsomes. Analysis of microsomal incubates using HPLC method 1

revealed the presence of three metabolite fractions (MF), one of which eluted with the

same retention time as authentic 4-hydroxydebrisoquine (Figure 2). The other two

were designated as MF-1 and MF-3, with the latter apparently being present at

markedly higher levels compared with 4-hydroxydebrsoquine. MF-3 is likely to have

been a complex of more than one metabolite and was formed with an estimated Km of

94 µM and Vmax of 696 pmoles/min/mg protein. A similar profile of hydroxylated

metabolites was seen when [14C]debrisoquine was incubated separately with each of

the five individual marmoset liver microsomes that were used to form the pool (data

not shown).


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LC-MS analysis of the incubate showed a peak with a mass of m/z 176 eluting

with the same retention time as authentic debrisoquine, and two peaks with a mass of

m/z 192, one of which eluted with the same retention time as authentic 4-

hydroxydebrisoquine; the second corresponded to MF-3 (Figure 3). It was postulated,

therefore that the metabolite fractions of mass m/z 192 were likely to be different

isomers of hydroxydebrisoquine, due to the increase of 16 mass units compared to

debrisoquine. Similarly, there was evidence for the presence of a possible dihydroxylated

debrisoquine with a mass of m/z 208, 32 mass units larger than debrisoquine. This

metabolite fraction corresponded to MF-1 on the original radiochromatogram. Product

ion mass spectra produced by LC-MS/MS analysis (Figure 4) provided some

structural information. The prominent loss of m/z 42 indicated the cleavage of CN2H2,

while other fragmentations resulted in formation of m/z 72, m/z 105, m/z 121 and m/z

133. Prominent product ions of m/z 132 and 150 were seen in the metabolite fraction

eluting at 6 minutes, indicating a mixture of aliphatic and aromatic

hydroxydebrisoquines, respectively. Similarly, the metabolite fraction eluting at 7

minutes comprised only aromatic hydroxydebrisoquine(s). However, none of these

fragmentations provided information as to the precise location of the hydroxyl

group(s).

In order to define more precisely the hydroxylation positions, NMR analysis

of metabolite fractions isolated using HPLC method 2 (Figure 1) from the large scale

incubation was performed. The assignment of the NMR signals was made by long-

range 2D COSY experiments (Table 1). The results of this analysis show that,

although debrisoquine was hydroxylated at the 4-position, this was not the principal

metabolite but in fact a relatively minor metabolite (Table 2). Debrisoquine was

hydroxylated by marmoset liver microsomes more extensively at the 7-, 5-, 6- and 8-


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positions, whereas these are only trace metabolites in rat and human urine (Allen et

al., 1975, 1976; Idle et al., 1979). In humans and rats 1- and 3-hydroxydebrisoquine

that are subjected to ring-opening have also been reported (Eiermann et al., 1998).

HPLC fractions 5, 7 and 8 were identified as possible open ring degradation products

of debrisoquine in the present study, but these were not further identified by NMR

analysis due to the lack of associated groups within their structure. Finally, a

dihydroxy metabolite produced by marmoset liver microsomes, namely 6,7-

dihydroxydebrisoquine, was also identified (Table 2). A dihydroxy metabolite has

been reported in a single human individual, characterised by the presence of 13 active

CYP2D6 genes, but the position of the hydroxyl groups was not delineated (Eiermann

et al., 1998). The hydroxylated metabolites generated by marmoset liver microsomes

are shown in Figure 5.

Antibodies raised to CYP2D6 failed to immunoreact with rat liver

microsomes, but single bands were evident when human (53.4 kDa) and marmoset

(49.8 kDa) liver microsomes were probed (Figure 6), in agreement with a previous

report (Uno et al., 2010). A liver enzyme in marmoset monkey sharing high amino

acid sequence similarity with human CYP2D6 has been reported by Igarashi et al.

(1997) and identified as CYP2D19. Interestingly, this CYP2D19 enzyme, unlike

human CYP2D6, was found to have no debrisoquine 4-hydroxylase activity but high

debrisoquine 5-, 6-, 7-, and 8-hydroxylase activities. Subsequently, Hichiya et al.

(2004) reported another marmoset liver enzyme belonging to the CYP2D family in

the liver of a single female marmoset bred in the Primate Research Institute, Kyoto

University (Aichi, Japan), identified as CYP2D30, which like human CYP2D6

exhibited high debrisoquine 4-hydroxylase activity and relatively low debrisoquine 5-,

6-, 7- and 8-hydroxylase activities. The metabolic profile observed in the current


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study would suggest that CYP2D19 and CYP2D30 enzymes may be co-expressed in

the marmoset livers used here. Liver microsomes from female marmosets bred at both

the Primate Research Institute, Kyoto University and the Faculty of Medicine,

Kagoshima University (Kagoshima, Japan) presented a single immunoreactive band

when probed with polyclonal antibodies to rat CYP2D1 (Hichiya et al., 2004).

In conclusion, we have demonstrated that marmoset liver microsomes are

effective in hydroxylating debrisoquine at various positions and that they contain a

protein that is immunorelated to human CYP2D6. The monohydroxylation of

debrisoquine by marmoset liver is qualitatively similar but quantitatively different to

human, with the major human metabolite (4-hydroxydebrisoquine) representing only

about 2% of the total metabolites generated by marmoset liver microsomes.

Nonetheless the presence of CYP2D6-like metabolic activity in marmoset makes it a

relevant surrogate species to use in drug development. Whether CYP2D is responsible

for the hydroxylation of debrisoquine in the marmoset remains to be established.


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Authorship contributions

Participated in research design: Cooke, Ioannides, Hall

Conducted experiments: Cooke, Bligh, Cybulski

Performed data analysis: Cooke, Bligh, Cybulski

Wrote or contributed to the writing: Cooke, Ioannides, Hall


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Zanger UM (2008) The CYP2D subfamily, in Cytochromes P450: Role in the

Metabolism and Toxicity of Drugs and other Xenobiotics (Ioannides C ed) pp 241-

275, RSC Publishing, Cambridge.


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Footnotes

Address reprint requests to: Dr Brian R Cooke, PathWest Core Clinical Pathology and

Biochemistry, Level 2 North Block, Royal Perth Hospital, Wellington St Campus,

Western Australia; [email protected]

The authors acknowledge respectfully the significant contribution of the late Professor

Gordon G Gibson to the planning of this study and discussion of the results.


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Legends for Figures

Figure 1: Radio-HPLC analysis (HPLC method 2) of marmoset liver microsomal

incubation sample with debrisoquine

Marmoset liver microsomes (4 mg/ml) were incubated with [14C]debrisoquine (500

µM) in the presence of NADPH for 180 minutes. HPLC analysis was carried out

using HPLC method 2 following volume reduction, lyophilisation and reconstitution

of sample in trifluoroacetic acid (0.1%, v/v).

Figure 2: Radio-HPLC chromatogram (HPLC method 1) of debrisoquine

metabolite formation and UV chromatogram of authentic reference standards

Radio-HPLC profile was obtained using HPLC method 1 following the incubation of

marmoset liver microsomes (5 mg/ml) with [14C]debrisoquine (500 μM) for 120

minutes. UV detection was conducted at 211 nm.

Figure 3: Mass chromatograms of m/z 208, m/z 192 and m/z 176

Electrospray ionisation mass spectra chromatograms (ESI+) of [14C]debrisoquine

(500 μM) incubation sample with marmoset liver microsomal protein (5 mg/ml) for

120 minutes. Trace A: ESI+ total ion current (TIC) chromatogram, Trace B: ESI+

scan of mass m/z 207.5 – 208.5, Trace C: ESI+ scan of mass m/z 191.5 – 192.5, Trace

D: ESI+ scan of mass m/z 175.5 – 176.5, Trace E: UV absorbance at 211 nm and

Trace F: radiochromatogram of incubate.

Figure 4: Product ion mass spectra of peaks eluting at 4, 6, 7 and 16 minutes

LC-MS/MS of [14C]debrisoquine (500 μM) incubation sample with marmoset liver

microsomal protein (5 mg/ml) for 120 minutes; Spectrum A: dihydroxydebrisoquine

product ion of m/z 166 eluting at 4 minutes, Spectrum B: monohydroxydebrisoquine

product ions of m/z 150 and m/z 132 eluting at 6 minutes, Spectrum C:


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monohydroxydebrisoquine product ion of m/z 150 eluting at 7 minutes, and Spectrum

D: debrisoquine product ion of m/z 134 eluting at 16 minutes.

Figure 5: Postulated pathway for the metabolism of debrisoquine by marmoset

liver microsomes

Figure 6: Immunoblot analysis of CYP2D proteins

Immunoblot analysis was carried out using antibodies to human CYP2D6 protein

followed by rabbit anti-mouse antibody labelled with alkaline phosphatase. Lane 1

(M) contained molecular weight markers, lanes 2 and 3 were loaded with 50 µg

marmoset liver microsomal protein, lanes 4 and 5 with 20 µg human liver microsomal

protein, lane 6 contained 0.2 pmoles CYP2D6 Supersomes™ as a positive (+ve)

control, lanes 7 and 8 were loaded with 50 µg rat liver microsomal protein and lane 9

acted as a negative (–ve) control (microsomal protein replaced by water).


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Table 1: Long-Range 2D COSY 1H NMR Assignments

Two-Dimensional Correlation Spectroscopy (2D COSY) 1H NMR assignment of debrisoquine hydroxyl groups of metabolites generated

following incubation of debrisoquine (500 µM) with marmoset hepatic microsomal protein (4 mg/ml) for 180 minutes. Multiplicity of resonance

is given by the abbreviations: s, single; d, doublet; dd, doublet of doublets; t, triple; dt, doublet of triplets and m, multiplet.

Proton Debrisoquine 4-Hydroxy 5-Hydroxy 6-Hydroxy 7-Hydroxy 8-Hydroxy 6,7-Dihydroxy

1

4.45 s 4.66 d 15.9

4.47 d 15.9

4.45 s 4.35 s 4.39 s 4.38 s 4.29 s

3

3.47 t 5.92

3.47 t 5.92

3.81. dd 13.8, 3.81

3.47 dd 13.8, 2.75

- 3.51 t 5.92 3.42 t 5.92 3.46 t 5.92 3.47 t 5.92

4

2.85 t 5.92 4.85 t 3.2 4.45 s 2.78 t 5.92 2.77 t 5.92 2.80 t 5.92 2.69 t 5.92

5

7.11-7.19 m 7.28 dd 7.32, 1.83 - 6.66 s 7.04 d 8.39 6.71 d 7.78 6.61 s

6 7.11-7.19 m 7.32 dt 7.32, 1.83 6.70 d 7.78 - 6.69 dd 8.39, 2.59 7.03 t 7.78 -

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7

7.11-7.19 m 7.25 dt 7.63, 1.37 7.05 t 7.78 6.65 dd 7.78, 2.59 - 6.67 d 7.78 -

8

7.11-7.19 m 7.15 dd 7.63, 1.22 6.72 d 7.78 6.98 d 7.78 6.62 d 2.44 - 6.65 s




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Table 2: Metabolites identified from each of the HPLC fractions by NMR analysis

Fraction1 Total estimated mass of fraction

(µg)

Metabolite % Present in fraction2

1 133 6,7-Dihydroxydebrisoquine 100

2 230 6-Hydroxydebrisoquine 88

4-Hydroxydebrisoquine 12



5 39 Not detected -


7 & 8 35 & 39 Not detected -

1 Isolated metabolite fraction, as presented in Figure 1

2 Based upon the percent of the fraction containing metabolite




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