On the sulfation of O-desmethyltramadol by human cytosolic sulfotransferases

Accepted Manuscript Title: On the sulfationPlease check the dochead and correct if necessary–> of O-desmethyltramadol by human cytosolic sulfotransferases Authors: Mohammed I. Rasool, Ahsan F. Bairam, Katsuhisa Kurogi, Ming-Cheh Liu PII: DOI: Reference:

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On the Sulfation of O-desmethyltramadol by Human Cytosolic Sulfotransferases Mohammed I. Rasoola,b, Ahsan F. Bairama,c, Katsuhisa Kurogia,d, Ming-Cheh Liua,* a

Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, University of Toledo Health Science Campus, Toledo, OH 43614 USA b Department of Pharmacology and Toxicology, College of Pharmacy, University of Karbala, Karbala, Iraq c Department of Pharmacology and Toxicology, College of Pharmacy, University of Kufa, Kufa, Iraq d Biochemistry and Applied Biosciences, University of Miyazaki, Miyazaki 889-2192 Japan

*Corresponding Author:

Ming-Cheh Liu, Ph.D. Professor Department of Pharmacology College of Pharmacy and Pharmaceutical Sciences University of Toledo Health Science Campus 3000 Arlington Avenue Toledo, OH 43614 USA

Tel: Fax: E-mail:[email protected]

(419) 383-1918 (419) 383-1909

Graphical abstract

Short Title: On the sulfation of O-desmethyltramadol

Abbreviations: O-DMT, O-desmethyltramadol; ATP, adenosine 5’-triphosphate; PAPS, 3’phosphoadenosine-5’- phosphosulfate; SULT, cytosolic sulfotransferase; TLC; thin-layer chromatography.

1

1 2 3

Abstract

4

Background: Previous studies have demonstrated that sulfate conjugation is involved in the

5

metabolism of the active metabolite of tramadol, O-desmethyltramadol (O-DMT). The current

6

study aimed to systematically identify the human cytosolic sulfotransferases (SULTs) that are

7

capable of mediating the sulfation of O-DMT.

8

Methods: The sulfation of O-DMT under metabolic conditions was demonstrated using HepG2

9

hepatoma cells and Caco-2 human colon carcinoma cells. O-DMT-sulfating activity of thirteen

10

known human SULTs and four human organ specimens was examined using an established

11

sulfotransferase assay.

12

respectively, buffers at different pHs and varying O-DMT concentrations in the assays.

13

Results: Of the thirteen human SULTs tested, only SULT1A3 and SULT1C4 were found to

14

display O-DMT-sulfating activity, with different pH-dependency profiles.

15

revealed that SULT1C4 was 60 times more catalytically efficient in mediating the sulfation of O-

16

DMT than SULT1A3 at respective optimal pH. Of the four human organ specimens tested, the

17

cytosol prepared from the small intestine showed much higher O-DMT-sulfating activity than

18

cytosols prepared from liver, lung, and kidney. Both cultured HepG2 and Caco-2 cells were

19

shown to be capable of sulfating O-DMT and releasing sulfated O-DMT into cultured media.

20

Conclusion: SULT1A3 and SULT1C4 were the major SULTs responsible for the sulfation of

21

O-DMT. Collectively, the results obtained provided a molecular basis underlying the sulfation

22

of O-DMT and contributed to a better understanding about the pharmacokinetics and

23

pharmacodynamics of tramadol in humans.

pH-dependency and kinetic parameters were also analyzed using,

2

Kinetic analysis

1 2

Keywords: Tramadol; O-desmethyltramadol; sulfation; cytosolic sulfotransferase; SULT.

3

Introduction

4 5

Tramadol, (2-[(dimethylamino)methyl]-1-(3-methoxyphenyl)cyclohexanol; cf. Figure 1),

6

is a centrally acting synthetic opioid analgesic which is widely used in the management of

7

moderate to moderately severe types of acute and chronic pain [1, 2]. There are two suggested

8

mechanisms through which tramadol may produce its effect. One, which is believed to be the

9

primary mode, involves the µ-receptor-mediated opioid analgesic effect. The other involves the

10

inhibition

11

accumulated/released norepinephrine and serotonin may in turn contribute to the antinociceptive

12

effects of tramadol [3-5].

of

norepinephrine

reuptake

and

serotonin

release

stimulation.

The

13 14

When taken orally, tramadol is well absorbed and extensively metabolized, with

15

metabolites excreted mostly in the urine [6, 7]. The metabolism of tramadol has been studied in

16

humans and other mammalian animal models including mice, hamsters, rats, guinea pigs, rabbits,

17

and dogs [6-8]. Studies using human subjects have revealed 11 Phase I metabolites (designated

18

M1-11) and 12 Phase II metabolites (including seven glucouronides and five sulfate conjugated

19

products) [7]. Five major tramadol Phase I metabolic products identified were O-DMT (M1), N-

20

desmethyl tramadol (M2) which may undergo further metabolism to generate di-N-desmethyl

21

tramadol (M3), tri-N, O-desmethyltramadol (M4), and di-N, O-desmethyltramadol (M5) [5-7].

22

In vitro studies revealed CYP2D6 and CYP3A4 as the primary cytochrome P450 enzymes

23

responsible for catalyzing O-demethylation and N-demethylation, respectively, of tramadol in

3

1

human liver microsomes [5,7,9]. Additional studies using mice and rats showed that among the

2

five major Phase I tramadol metabolites, O-DMT was the only one that retained the analgesic

3

activity, being in fact two to four times more effective than tramadol in reducing pain [10, 11].

4

Furthermore, the affinity of O-DMT for the µ-opioid receptors has been shown to be 200 times

5

higher than that of the parent compound [10-12]. These studies thus indicated that O-DMT may

6

play a major role in the analgesic effect of tramadol [6, 7, 10, 11]. It is therefore an important

7

issue regarding the metabolism of O-DMT. Sulfated O-DMT has been reported to be present in

8

human urine [1, 7].

9

sulfation of O-DMT, however, remain(s) unknown.

The cytosolic sulfotransferase (SULT) enzyme(s) that mediate(s) the

10 11

Sulfation is considered as a critical step in the biotransformation and homeostasis of

12

some key endogenous compounds such as catecholamine neurotransmitters and thyroid/steroid

13

hormones [13-20]. Additionally, it plays an important role in the detoxification of xenobiotic

14

compounds such as drugs and environmental toxicants [16, 19, 21-23]. The sulfation reactions

15

are catalyzed by the cytosolic sulfotransferase (SULT) enzymes [21, 22]. Sulfate or sulfamate

16

conjugates are produced following the transfer of the sulfonate group from the physiological

17

donor 3-phosphoadenosine-5-phosphosulphate (PAPS) to the hydroxyl or amino group of the

18

endogenous and xenobiotic substrates through the action of the SULTs [16, 21, 22]. It has been

19

reported that SULTs and UDP glucuronosyltransferases catalyze approximately 40% of drug

20

conjugation reactions, accounting for a large portion of the Phase II conjugation reactions in the

21

body [23, 24].

22

4

1

In this communication, we report a systematic investigation of the sulfating activity of all

2

known human SULTs towards O-DMT. HepG2 human hepatoma cells and Caco-2 human colon

3

carcinoma cells were tested for their capacity to metabolize O-DMT through sulfation.

4

Moreover, the O-DMT-sulfating activity of cytosols of human brain, small intestine, kidney,

5

liver, and lung was examined.

6 7

Materials and Methods

8 9

Materials.

Ecolume scintillation cocktail was a product of MP Biomedicals (Solon, OH).

10

HepG2 human hepatoma cells (ATCC HB-8065) and Caco-2 human colon carcinoma cells

11

(ATCC-HTB-37) were purchased from American Type Culture Collection (Manassas, VA). O-

12

DMT (≥ 98% in purity) was from Cayman Chemical Company (Ann Arbor, MI).

13

morpholinoethanesulfonic acid (MES), penicillin G, dimethyl sulfoxide (DMSO), 2-(cyclohexyl-

14

amino)

15

piperazineethanesulfonic acid (HEPES), fetal bovine serum (FBS), silica gel thin-layer

16

chromatography (TLC) plates, adenosine-5’-triphosphate (ATP), streptomycin sulfate, sodium

17

fluoride

18

dithiothreitol (DTT), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and minimum

19

essential medium (MEM) were products from Sigma Chemical Company (St. Louis, MO).

20

Carrier-free sodium [35S]sulfate was purchased from American Radiolabeled Chemicals (St.

21

Louis, MO). Recombinant human bifunctional PAPS synthase was cloned and expressed as

22

described previously [25]. Recombinant human SULTs (≥ 95% in purity as judged by sodium

23

dodecyl sulfate-polyacrylamide gel electrophoresis), including SULT1A1 (GenBank Accession #

ethanesulfonic

(NaF),

acid

(CHES),

Trizma

base,

4-(2-hydroxyethyl)-1-

N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic

5

2-

acid

(TAPS),

1

AAI10888.1), SULT1A2 (GenBank Accession # NP_001045.1), SULT1A3 (GenBank

2

Accession # AAH78144.1), SULT1B1 (GenBank Accession # NP_055280.2), SULT1C2

3

(GenBank Accession # AAH05353.1), SULT1C3 (GenBank Accession # NP_001307807.1),

4

SULT1C4 (GenBank Accession # AAI25044.1), SULT1E1 (GenBank Accession #

5

EAX05597.1), SULT2A1 (GenBank Accession # AAH20755.1), SULT2B1a (GenBank

6

Accession # AAC78498.1), SULT2B1b (GenBank Accession # AAC78499.1), SULT4A1

7

(GenBank Accession # CAG30474.1), and SULT6B1 (GenBank Accession # AAI40798.1),

8

were cloned, expressed, and purified as described previously [26-28]. Pooled human lung S9

9

fraction (Lot No. 0710281), small intestine (duodenum and jejunum) S9 fraction (Lot No.

10

0710351), liver cytosol (Lot No. 09103970), and kidney S9 fraction (Lot No. 0510093) were

11

purchased from XenoTech, LLC (Lenexa- KS). Other chemicals used were of the highest grade

12

commercially available.

13 14

Metabolic labeling of Caco-2 human colon carcinoma cells and HepG2 human hepatoma

15

cells. The generation and release of [35S]sulfated O-DMT by HepG2 cells and Caco-2 cells,

16

labeled with 0.3 mCi/ml [35S]sulfate and 0, 1, 5, 25, 50, or 100 µM of O-DMT, was carried out

17

based on a previously established procedure [29].

18 19

Enzymatic assay. The O-DMT-sulfating activity of purified recombinant human SULTs was

20

assayed based on a previously established protocol, with radioactive PAP[ 35S] as the sulfate

21

donor [18]. The same protocol was employed for the analysis of pH-dependency of the O-DMT-

22

sulfating activity of SULT1A3 and SULT1C4 using different buffers (sodium acetate at 4.5, 5,

23

and 5.5; MES at 6, and 6.5; HEPES at 7, 7.5, and 8; TAPS at 8.5; CHES at 9, 9.5, and 10; and

6

1

CAPS at 10.5, 11, and 11.5). In the study on the kinetics of the sulfation of O-DMT by

2

SULT1A3 or SULT1C4, the assays were performed using varying concentrations (10, 12.5,

3

16.67, 25, 33.33, 50, 66.67, 100 or 200 µM) of O-DMT as substrates. Michaelis-Menten kinetics

4

using nonlinear-regression (GraphPad Prism) was employed for the analysis of the activity data

5

obtained. To assay for O-DMT-sulfating activity of human organ cytosols or S9 fractions, the

6

assay mixture was supplemented with NaF (a phosphatase inhibitor).

7 8

Results

9 10

Survey of the O-DMT-sulfating activity of the human SULTs. A systematic study of the O-

11

DMT-sulfating activity of all thirteen known human SULTs was performed in order to identify

12

the one(s) that is(are) capable of catalyzing the sulfation of O-DMT. Enzymatic assays were

13

performed at both neutral pH (7.0) and physiological pH (7.4), and three different substrate

14

concentrations (5, 25, and 50 M) were tested. Results showed that of the thirteen human

15

SULTs (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1C3, SULT1C4,

16

SULT1E1, SULT2A1, SULT2B1a, SULT2B1b, SULT4A1 and SULT6B1), only SULT1A3 and

17

SULT1C4 exhibited sulfating activities toward O-DMT. Activity data compiled in Table 1

18

indicated that the O-DMT-sulfating activity of SULT1C4 was considerably higher than that of

19

SULT1A3 at both neutral and physiological pH and with all three O-DMT concentrations tested.

20 21

Characterization of the O-DMT-sulfating activity of human SULT1C4 and SULT1A3. As

22

shown in Figure 2A, SULT1C4 displayed O-DMT-sulfating activity over a wide pH range

23

spanning pH 6.5-10.5, with the optimal activity observed at pH 9-9.5. SULT1A3 also displayed

7

1

O-DMT-sulfating activity, albeit at considerably lower levels than SULT1C4, over a wide pH

2

range spanning pH 6.5-10, with the optimal activity observed at pH 8-9 (Figure 2B). The

3

kinetics of the sulfation of O-DMT by SULT1C4 or SULT1A3 was analyzed using different

4

concentrations (ranging from 10 to 200 µM) of O-DMT as substrates at neutral pH (7.0) and

5

physiological pH (7.4), as well as their respective optimal pH (8.5 for SULT1A3 and 9.5 for

6

SULT1C4; cf. Figure 2). Table 2 summarizes the kinetic constants calculated based on the data

7

obtained. It is noted that compared with SULT1A3, SULT1C4 showed much lower Km and

8

considerably higher Vmax. Based on the calculated Vmax/Km, SULT1C4 is 4.92, 4.96, and 59.83

9

times more efficient in mediating the sulfation of O-DMT than SULT1A3 at, respectively,

10

neutral, physiological, and optimal pH.

11 12

Sulfation of O-DMT in cultured human cells and by human organ specimens. Cultured

13

HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells were tested in a

14

metabolic labeling experiment. The results shown in Figure 3 indicated that both HepG2 and

15

Caco-2 cells were capable of producing [35S] sulfated O-DMT in a concentration-dependent

16

manner. It was noted that [35S] sulfated O-DMT was detected in the labeling media containing

17

as low as 5 M of O-DMT for both cell types. Cytosol or S9 fractions prepared from human

18

small intestine, kidney, liver, or lung were analyzed for O-DMT-sulfating activity. Results

19

obtained are shown in Table 3. Of the four organ samples tested, the small intestine displayed

20

the strongest O-DMT-sulfating activity (at 11.05 pmol/min/mg), followed by liver (at 0.76

21

pmol/min/mg), lung (at 0.15 pmol/min/mg), and kidney (at 0.06 pmol/min/mg).

22 23

Discussion

8

1 2

The Phase II sulfate conjugation reaction as mediated by the SULTs is known to be an

3

important pathway in the metabolism and inactivation of a variety of endogenous and xenobiotic

4

compounds [13, 14, 19, 20]. The centrally acting analgesic drug, tramadol, is widely used in

5

pain management [1, 2]. Previous studies indicated that O-DMT, the active Phase I metabolite

6

of tramadol, may in fact play a critical role in the analgesic effect of tramadol [6, 7, 10, 11].

7

Sulfated O-DMT has been identified in human urine, indicating that sulfation is critically

8

involved in its disposal from the body [1, 7]. However, the SULT enzyme(s) that is (are)

9

responsible for the sulfation of O-DMT remain(s) obscure. The current study was performed to

10

systematically evaluate the O-DMT-sulfating activity of known human SULTs and to

11

characterize the enzymatic features of those SULTs that are capable of catalyzing the sulfation of

12

O-DMT.

13

metabolic conditions. Moreover, the presence of O-DMT-sulfating activity in human organ

14

specimens was examined.

Cultured human cells were used to evaluate the sulfation of O-DMT under the

15 16

To

screen

human

SULTs

for

O-DMT-sulfating

activity,

different

substrate

17

concentrations, 5, 25, and 50 M, were tested. Moreover, the enzymatic assays were performed

18

at both neutral (7.0) and physiological (7.4) pH. It is noted that plasma levels of tramadol in

19

patients administered with the drug have been reported to be in the range of 0.38-2.24 M) [30-

20

33].

21

metabolism of tramadol, may be in the similar range. Of the thirteen human SULTs tested, only

22

SULT1A3 and SULT1C4 exhibited sulfating activities toward O-DMT. Results compiled in

23

Table 1 indicated that the O-DMT-sulfating activity of SULT1C4 was much higher than that of

It is conceivable that the level of O-DMT generated inside the cells, upon Phase I

9

1

SULT1A3 for all substrate concentrations tested at both neutral and physiological pH. It is noted

2

that, while SULT1C4 was originally described as being able to catalyze the sulfation of N-

3

hydroxy-2-acetylaminofluorene [26], it has since been shown to use a number of phenolic

4

hydroxyl group-containing drugs, such as acetaminophen, oxymorphone, naloxone, and

5

hydromorphone, as substrates [19, 29]. In regard to its tissue specificity of expression, high

6

levels of SULT1C4 mRNA were previously reported to be present in human fetal lung, liver,

7

small intestine, and kidney, whereas lower, yet significant, levels of SULT1C4 mRNA were

8

detected in adult kidney, ovary, and spinal cord [20, 23, 26, 34]. SULT1A3, generally known as

9

the dopamine sulfotransferase [35], and had been shown to be expressed at high levels in the

10

gastrointestinal tract, platelets, brain, and at low levels in the fetal liver [29, 36]. In regard to its

11

sulfating activity, SULT1A3 has been reported to be capable of mediating the sulfation of

12

compounds containing phenolic hydroxyl groups in their chemical structures [37, 38]. It is

13

therefore not surprising that O-DMT serves as a substrate for SULT1A3.

14 15

pH-dependency experiments revealed that both SULT1C4 and SULT1A3 were active

16

toward O-DMT over a wide range of pH (Figure 2). These results implied that both SULT1C4

17

and SULT1A3 may function to mediate the sulfation of O-DMT inside the cells where local pH

18

may fluctuate under changing metabolic conditions.

19

SULT1C4 is considerably more efficient in mediating the O-DMT sulfation than SULT1A3.

20

The kinetic constants shown in Table 2 fall within the range of Km (2.2 to 4091 µM) and Vmax (3

21

to 501 nmol/min/mg) values previously reported for different SULT enzymes toward different

22

substrate compounds [35].

23

10

According to the calculated Vmax/Km,

1

While purified SULT1A3 and SULT1C4 were shown to be capable of sulfating O-DMT,

2

it is important to find out whether they may function inside the cells. To obtain this information,

3

cultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells were tested in a

4

metabolic labeling experiment. It is noted that HepG2 cells and Caco-2 cells are cells derived

5

from the liver and intestine, two major organs involved in the first-pass metabolism of many

6

drugs. Previous studies had demonstrated the expression of several SULT isoforms, including

7

SULT1A1, SULT1A2, SULT1A3, SULT1E1, and SULT2A1 in HepG2 cells [39-41]. Caco-2

8

cells had also been shown to express SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2,

9

SULT1C4, and SULT2A1 [29, 42]. Therefore, both of these two cell lines are equipped with

10

enzymes that are capable of sulfating O-DMT. Since the SULTs are differentially expressed in

11

different organs of the human body [41, 43], it is interesting to compare the O-DMT-sulfating

12

activity of different human organ specimens.

13

intestine sample (Table 3) was compatible with the high levels of expression of SULT1A3 in

14

gastrointestinal track, particularly the small intestine [41]. It is possible that the liver, lung, and

15

kidney, while likely operating at smaller scales, may also play a role in the metabolism of O-

16

DMT through sulfation.

The highest activities detected for the small

17 18

Conclusions. The present study demonstrated clearly that of the thirteen known human SULTs,

19

only SULT1C4 and SULT1A3 were capable of mediating the sulfation of O-DMT. Results

20

obtained using cultured HepG2 cells and Caco-2 cells indicated a concentration-dependent

21

production and release of sulfated O-DMT into culture media. O-DMT-sulfating activities were

22

detected, in decreasing order, in the cytosol or S9 fractions of human small intestine, liver, lung,

11

1

and kidney. These results provided a molecular basis for further investigation on the metabolism

2

of O-DMT by sulfation in vivo.

3 4

Conflict of interest: None declared.

5 6

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16 17 18 19 20 21 22 23 24 25

17

1 2 3 4 5 6 7

Tables

8 9

Specific Activity (nmol/min/mg) Substrate (O-DMT) Concentration (μM)

pH

SULT1A3

SULT1C4

7

0.07 ± 0.01

0.20 ± 0.02

7.4

0.10 ± 0.02

0.32 ± 0.03

7

0.16 ± 0.01

0.45 ± 0.02

7.4

0.29 ± 0.02

0.81 ± 0.13

7

0.18 ± 0.01

1.24 ± 0.03

7.4

0.28 ± 0.01

2.12 ± 0.15

5

25

50

10 11 12

Table 1. Specific Activities of Human SULT1A3 and SULT1C4 with O-DMT as a substrate. Data shown represent mean ± SD derived from three determinations.

13 14 15

18

1 2 3

Enzyme

SULT1A3

SULT1C4

4 5 6 7 8 9 10 11 12

pH

Vmax (nmol/min/mg)

Km (µM)

Vmax/Km (ml/min/mg)

7

1.29 ± 0.16

331.07 ± 54.35

0.0039

7.4

3.40 ± 0.23

435.00 ± 35.14

0.0078

Optimal

7.66 ± 0.04

475.21 ± 7.18

0.0161

7

7.51 ± 0.04

390.07 ± 0.9

0.0192

7.4

11.90 ± 0.20

307.58 ± 2.56

0.0387

Optimal

17.89 ± 1.45

18.57 ± 2.41

0.9633

Table 2. Kinetic constants of the sulfation of O-DMT by human SULT1C4 and SULT1A3 at neutral, physiological, and optimal pH. Kinetic parameters were determined based on MichaelisMenten kinetics. Results represent means ± SD derived from three determinations. Optimal pH tested for SULT1A3 and SULT1C4 were 8.5 and 9.5, respectively.

Specific Activity (pmol/min/mg) Substrate

Small intestine

Kidney

19

Liver

Lung

O-DMT 1 2 3 4

11.05 ± 0.64

0.06 ± 0.01

0.76 ± 0.06

0.15 ± 0.04

Table 3. Sulfating activities of human small intestine, kidney, liver, and lung cytosols with ODMT as a substrate. Data shown represent mean ± SD derived from three determinations. The final concentration of the substrate used in the assay mixture was 50 µM.

5 6 7

20

1 2

21

1 2 3 4

Figure 1. Chemical structures of tramadol (A) and O-DMT (B).

5 6

Figure 2. pH-dependency of O-DMT sulfating activity of the human SULT1C4 (A) and

7

SULT1A3 (B). Enzymatic assays were carried out under standard assay conditions as described

8

in the materials and methods section using different buffer systems as indicated. Data shown

9

represent calculated mean ± SD derived from three independent experiments.

10 11 12 13 14 15 16 17

Figure 3. Analysis of [35S]sulfated O-DMT generated and released by HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells labeled with [35S]sulfate in the presence of ODMT. The figure shows the autoradiograph taken from the plate at the end of the TLC analysis. Confluent HepG2 cells and Caco-2 cells were labeled with [35S]sulfate for 18 hours in the presence of different concentrations (1, 5, 25, 50, and 100 µM; corresponding to lanes 1-5) of ODMT. C indicates the control labeling medium sample without O-DMT. The arrows indicate the position of sulfated O-DMT.

22