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Introduction

ZJM-289, [2-(1-diethylaminoacetoxy)pentyl] benzoic acid-{2-methoxy-4-[2-(4-nitrooxybutoxy carbonyl)-vinyl]}phenyl ester hydrochloride (Figure 1a), is a novel nitric oxide–donating derivative of 3-n-butylphthalide (Figure 1b) and is reported to alleviate the cerebral ischemic-reperfusion injury in rats (Zhuang et al. 2010). ZJM-289 consists of three ester bonds (Figure 1a), and the-oretically may be hydrolysed into ferulic acid (Figure 1c), 2-(1-hydroxypentyl)-benzoate and nitrate by esterase (Min et al. 2008). In vivo and in vitro data indicate that the anti-platelet aggregation activity of ZJM-289 is improved above that of 3-n-butylphthalide (Zhang and Peng 2006; Min et al. 2008). Furthermore, 2-(1-hydroxypentyl)-benzoate is also a novel derivative of 3-n-butylphthalide

for the treatment of cerebral ischemia (Zhang et al. 2004). Nitric oxide is a potent inhibitor of the aggrega-tion of platelets (Al-Sa’doni and Ferro 2005; Sara et al. 2006), and ferulic acid may scavenge free radicals and prevent increased oxidation of lipids (Laranjinha et al. 1994; Srinivasan et al. 2007; Maurya and Devasagayam 2010), while 3-n-butylphthalide exhibits anti-thrombotic and anti-platelet activities (Chang and Wang 2003; Peng et al. 2004; Peng et al. 2009) as well as improves cognitive impairment (Peng et al. 2010). These three components may have synergetic effects on the platelets. Zhuang et al. (2010) have reported ZJM-289 as a nitric oxide donor. Therefore, it is necessary to identify the possible metabolites, both in circulatory and excretory system, to evaluate whether the expected pharmacologically active

RESEARCH ARTICLE

Identification of circulatory and excretory metabolites of a novel nitric oxide donor ZJM-289 in rat plasma, bile, urine and faeces by liquid chromatography–tandem mass spectrometry

Ning Li1, Xuliang Wang2, Tingting Li3, Hui Ji3, Yihua Zhang2, Zhixia Qiu1, Di Zhao1, and Xijing Chen1

1Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, China, 2Center of drug discovery, China Pharmaceutical University, Nanjing, China, and 3Department of Pharmacology, China Pharmaceutical University, Nanjing, China

Abstract1. ZJM-289, [2-(1-diethylaminoacetoxy)pentyl] benzoic acid-{2-methoxy-4-[2-(4-nitrooxybutoxy carbonyl)-vinyl]}

phenyl ester hydrochloride, is a novel nitric oxide–donating derivative of 3-n-butylphthalide synthesised on the hypothesis that it may be hydrolysed in vivo into 3-n-butylphthalide, ferulic acid and nitric oxide in hope that the three components may exert effects on the platelets as well as on central nervous system synergistically.

2. In this study, ZJM-289 was extensively metabolised in rats. Eight major metabolites were identified by liquid chromatography (LC)–mass spectrometry (MS)/MS in rat plasma, bile, urine and faeces after intravenous administration. Metabolites M1, M2, M3, M4 and M5 were hydrolytic products of ZJM-289, M6 and M7 was a hydroxylation product of M5, and M8 was a glucuronide of M1.

3. The pharmacologically active metabolite ferulic acid (M3) was a major metabolite in all the biological matrixes examined. 3-n-Butylphthalide was also present at a moderate level in the circulation. And along with the previous research, the anti-platelet activity of ZJM-289 was more potent than that of 3-n-butylphthalide both in vivo and in vitro. All these findings validated the theory of drug design.

Keywords: ZJM-289, 3-n-butylphthalide, ferulic acid, metabolites identification, fragmentation pathway, rat

Address for Correspondence: Prof. Xijing Chen, PhD, China Pharmaceutical University, Center of Drug Metabolism and Pharmacokinetics, Mailbox 210, #24 Tongjiaxiang, Nanjing 210009, Jiangsu, China. Tel.: 86 25 83271286; Fax: 86 25 83271335. E-mail: [email protected]

(Received 18 March 2011; revised 05 April 2011; accepted 08 April 2011)

Xenobiotica, 2011; 41(9): 805–817© 2011 Informa UK, Ltd.ISSN 0049-8254 print/ISSN 1366-5928 onlineDOI: 10.3109/00498254.2011.580385

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metabolites such as 3-n-butylphthalide and ferulic acid are generated in vivo.

Nowadays, tandem mass spectrometry (MS) tech-niques are widely applied for the qualitative and quanti-tative analyses in many fields and play a vital role in drug metabolism research, such as the structural elucidation of drug metabolites (Zelinski and Borlak 2005; Han et al. 2007; Huang et al. 2010). Identification and structure elucidation of metabolites by liquid chromatography (LC)–MS/MS is preceded on the premise that parent compounds retain their core structures after in vivo or in vitro biotransformation. Hence, their metabolites may be characterised by comparing product ion spectra of the metabolites to those of the parent compounds (Ning et al. 2010), even when an authentic standard is not avail-able for each metabolite.

Metabolite identification for 3-n-butylphthalide and ferulic acid using high-performance liquid chroma-tography (HPLC)–UV, HPLC–fluorescence detection, HPLC–MS/MS and isotope labelling has been well documented (Wang et al. 1997; Zhao et al. 2003; Jong et al. 2006; Niu et al. 2008). However, metabolism of 3-n-butylphthalide novel derivative ZJM-289 has not been reported. In the present study, an LC–MS/MS method was developed to identify and characterise the major metabolites in rat plasma, bile, urine and faeces following

intravenous administration of ZJM-289. The structures of eight metabolites, including the pharmacologically active metabolites 3-n-butylphthalide and ferulic acid were elucidated for the first time by tandem MS.

Materials and methods

Reagents and chemicalsZJM-289 and 3-n-butylphthalide with a purity of 98% was kindly offered by the Center of Drug Discovery, China Pharmaceutical University. Authentic stan-dards of metabolite M1, and 3-n-butylphthalide (M5) were also synthesised by Center of Drug Discovery, China Pharmaceutical University. β-Glucuronidase (041K70321, type B-1, from bovine liver) was purchased from Sigma-Aldrich (St. Louis, MO). Acetonitrile and methanol of HPLC grade were purchased from Tedia (Fairfield, OH). Formic acid and other chemicals were of analytical grade. Ferulic acid (>98%) was obtained from National Institutes for Food and Drug Control of China.

Animals and drug administrationMale Sprague-Dawley rats (200 ± 20 g) were sup-plied by Shanghai SIPPR/BK Experimental Animal Co (Shanghai, China). All experimental protocols and procedures were approved by the Animal Ethics

Figure 1. Structures of ZJM-289. (a) 1, 2 and 3 represent three different ester bonds, respectively; (b) 3-n-butylphthalide and (c) ferulic acid.

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Identification of ZJM-289 metabolites in rat 807

© 2011 Informa UK, Ltd.

Committee of China Pharmaceutical University. Rats were kept humanely with free access to water and food for 7 successive days before the experiment. The rats were fasted 12 h before dose administration. ZJM-289 was dissolved in physiological saline water at a target concentration of 16 mg/ml, and the rats received ZJM-289 solution by intravenous bolus administration with a single dose of 80 mg/kg.

The blood samples were collected from three rats into heparinised tubes via jugular vein pre-dose and at 2, 5, 10, 15, 20, 30, 45, 60, 90 and 120 min post-dose for identifica-tion of circulatory metabolites. The blood samples were centrifuged at 12,000 r.p.m. for 2 min to obtain plasma, and 50 μL plasma of each subject was mixed immediately and added into triple volumes of acetonitrile to precipi-tate proteins. The mixture was vortexed and centrifuged at 16,000 r.p.m. for 10 min. An aliquot of 10 μL of the supernatants was transferred to inject into LC–MS/MS system.

Another group of three rats were housed in meta-bolic cage individually, and then administrated ZJM-289 for identification of excreted metabolites in urine and faeces. The urine and faeces samples from three different subjects were pooled at the interval of 4 h from the intravenous dosing to 24 h in clean tubes. For the

urine samples, they were centrifuged at 16,000 r.p.m. for 10 min immediately, then an aliquot of 10 μL was injected into the LC–MS/MS system. The faeces samples were added to triple volume water and acetonitrile (1:1, v/v) to generate faecal homogenates by tissue grinder, then centrifuged at 16,000 r.p.m. for 10 min. The super-natants were vaporised to dryness, and then the resi-dues were reconstituted in acetonitrile and water (1:1, v/v) for LC–MS/MS analysis.

After overnight fasting, three rats were anaesthetised with by intraperitoneal administration of urethane at 1 g/kg and kept anaesthetised throughout the whole experiment period. The bile duct was cannulated by a polyethylene tube (inner diameter, 0.28 mm; outer diam-eter, 0.61 mm; Becton Dickinson), and the bile samples were collected before and 0.25, 0.5, 1.0, 2, 4 and 6 h after a single dose intravenous administration of ZJM-289 via the caudal vein. Bile samples were processed by adding triple volumes of acetonitrile, and the mixture vortexed for 1 min and centrifuged at 16,000 r.p.m. for 10 min. The supernatant (10 μL) was injected for LC–MS/MS for analysis.

In order to avoid the possible degradation of the metabolites in plasma, bile, urine and faeces, the col-lected samples were processed immediately.

Figure 2. LC chromatogram of metabolites in rat (a) plasma and (b) urine. LC, liquid chromatography.

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Enzyme hydrolysis of the bile samplesAn aliquot (150 μL) bile sample was spiked with 50 μL β-glucuronidase (2000 units) prepared in the sodium acetate buffer (pH 5.0), and incubated in a shaking water bath at 37°Cfor 12 h. After incubation, the hydro-lysis was terminated with triple volumes of acetonitrile, and samples were processed for analysis as described above.

HPLC–UV analysisThe LC system consisted of an LC10AD binary pump system, a SIL10AD autosampler and a CTO 10A oven (Shimadzu, Kyoto, Japan). The analytical column was Shimadzu Shim-pack VP-ODS (column size 5 μm, 150 × 2.0 mm; serial no. 9042026), coupled with a Security Guard C18 guard column (4 × 3.0 mm; Phenomenex, Torrance, CA). The mobile phase consisted of water

Figure 3. LC chromatogram of metabolites in rat (a) bile and (b) faeces. LC, liquid chromatography.

Table 1. Comparison of retention time and MS/MS data in positive mode for parent compound and the proposed metabolites in LC–MS/MS.Compound name Matrix Retention time (min) Parent ion (m/z) Major fragment ions (m/z)ZJM-289 Plasma 27.1 615 484,408,337,322,173,145,117M1 Plasma, bile 23.3 498 367,173,145,117,91M2 Plasma, bile 23.3 367 173,145,117,91,105M3 Plasma, bile, urine, faeces 13.7 195(+)193(−) 177,145 117,89,178,149,134M4 Plasma, bile, urine, faeces 17.9 322 173, 145,117, 86,132M5 Plasma, bile, urine, faeces 24.1 191 173, 145,117, 91,77,105M6 Plasma, bile, urine, faeces 15.4 207 189,171,143,128,105,91,77M7 Plasma, bile, urine, faeces 21.9 207 189,171,143,128,105,91,77M8 Bile 21.1 674 543,498,367,173, 145, 117,132, 86LC, liquid chromatography; MS, mass spectrometry.

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(contained 1.0% formic acid, mobile phase A) and metha-nol (mobile phase B) with flow rate set at 0.3 mL/min. The wavelength of the UV detector (SPD 10Avp; Shimadzu) was set at 304 nm. The autosampler temperature was maintained at 5°C and injection volume was 10 μL. The composition of gradient elution increased from 10% to 90% of mobile phase B over 32 min, and maintained at 90% B for 2 min, followed by change to the initial condi-tion and re-equilibrated. The temperature of the column was maintained at 40°C.

Mass spectrometric conditionsSamples were analysed by a Thermo Scientific TSQ Quantum MS/MS system equipped with electro-spray ionisation interface. The spray voltage was set at 4000 V for positive mode and 3400 V for negative mode. The

temperature of capillary was maintained at 350°C. The fluid was nebulised by high-purity nitrogen, and sheath gas and auxiliary gas were set at 30 and 10 arbitrary units, respectively. The product ions were generated by colli-sion-induced dissociation of the selected precursor ions using ultra purity argon (Ar) as collision gas (pressure set at 1.5 mTorr).

Results

Metabolic profiles of ZJM-289 in ratMetabolic profiles of ZJM-289 in plasma and urine are presented in Figure 2. Seven metabolites (M1–M7) were identified in plasma, with M1, M2 and M3 being rela-tively more abundant metabolites than others. Except M1 and M2, the metabolites observed in plasma were also

Figure 4. (a) Extracted ion chromatogram and (b) chemical structure and MS/MS spectrum of parent compound in rat plasma. MS, mass spectrometry.

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detected in urine, with M3 being the most abundant uri-nary metabolite. In addition to the metabolites observed in plasma, M1 glucuronide (M8) was also detected in bile (Figure 3). Faecal metabolites included M3, M4, M5, M6, and M7 (Figure 3). Metabolite M3 was also the most abundant metabolite in bile and faeces. ZJM-289 was extensively metabolised in rats, with only a minor amount of the parent drug detected in plasma, and no parent drug detected in urine, bile or faeces.

Metabolites M1–M8 and parent compound were char-acterised by LC–MS/MS analysis in positive mode, while M3 was also characterised in negative mode. The metab-olites along with their retention times and the represen-tative fragment ions are shown in Table 1. Metabolite identification and characterisation are discussed below in details.

Parent compound (ZJM-289)The parent compound ZJM-289 was observed in plasma and eluted at around 27.1 min, with a protonated

molecular ion at m/z 615. The product ion spectrum of m/z 615 is shown in Figure 4. The most abundant frag-ment ion at m/z 484 was a result of losing the diethylam-inoacetyl moiety (molecular weight 131) and further loss of a water molecule. Loss of the methoxy group and NO

2

in the nitrate group from m/z 484 yielded the product ion m/z 408. The fragment ion m/z 336 was produced from m/z 484 by losing the methoxy group and the nitrooxybu-tanyl moiety. The product ion m/z 322 was produced from m/z 615 by losing the nitrooxybutanyl moiety and the ferulic acid moiety. The m/z 173 ion was generated from m/z 336 by cleavage of the ester bond 2, and the m/z 145 formed by further loss of the carbonyl group. The major fragment ions are listed in Table 1 and the fragmentation patterns are depicted in Figure 5.

M1M1 was detected in plasma and bile, and eluted at 23.3 min with a protonated molecular ion at m/z 498, suggesting loss of the nitrooxybutanyl moiety through

Figure 5. Proposed fragmentation pathway of parent compound.

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hydrolysis of the ester bond 3. The extracted ion chro-matogram, MS/MS spectrum and proposed structure of M3 are depicted in Figure 6. The most abundant fragment ion m/z 367 was formed due to loss of the diethylaminoacetyl moiety and further loss of a water molecule. Other product ions at m/z 173, m/z 145 and m/z 117 were identical with those for ZJM-289 (Figure 4 and Table 1). Moreover, m/z 117 was disassociated into m/z 92, the tropylium ion, by breaking the alkyl chain. Therefore, M1 was characterised as a hydrolytic metab-olite of ZJM-289, formed through hydrolysis of the ester bond 3. The retention time and the fragmentation pattern of M1 were identical to those for the authentic standard (data not shown).

M2Metabolite M2 was detected in plasma and bile, and eluted at 23.3 min had a molecular ion of m/z 367, which was the same as the most abundant product ion of M1. The major fragment ions at m/z 173, 145, 117, 92 were identical with those for M1. The product ion at m/z

105 was formed from m/z 173 a result of the loss of the alkyl chain. Therefore, M2 was characterised as ZJM-289 metabolite formed through hydrolysis of the ester bonds 1 and 3, and further loss of a water molecule. The extracted ion chromatogram, MS/MS spectrum and the proposed structure of M2 are shown in Figure 7.

M3Metabolite M3 was detected in plasma, bile, urine and faeces, and eluted at 13.7 min with a protonated molecular ion at m/z 195. The total ion chromatogram and extracted ion chromatogram in positive mode are depicted in Figure 8. The most abundant fragment ion at m/z 177 was formed by losing one water molecule in the positive mode, as depicted in Figure 8b. Fragment ions at m/z 145 and m/z 117 were identical with those for ZJM-289. In the negative mode (Figure 8), M3 had a molecular ion at m/z 193, and yielded characteristic fragment ions at m/z 178 (losing CH

3 of the methoxy

group), m/z 149 (losing carbon dioxide), m/z 134 (los-ing both methyl and carbon dioxide). The retention

Figure 6. (a) Extracted ion chromatogram and (b) chemical structure and MS/MS spectrum of M1 in rat plasma. MS, mass spectrometry.

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time of M3 and the product ion spectrum in both posi-tive and negative modes were identical to the reference standard ferulic acid (data not shown). Therefore, M3 was identified as ferulic acid.

M4Metabolite M4 was observed in plasma, bile, urine and faeces, and eluted at 17.9 min with a protonated molecu-lar ion at m/z 322, which was the same as one of the product ion for ZJM-289. The extracted ion chromato-gram, MS/MS spectrum and proposed structure of M4 are shown in Figure 9. The fragment ions at m/z 173, m/z 145 and m/z 117 are the same as those for ZJM-289. The fragment ion at m/z 132 was the diethylaminoacetic acid moiety, and its most abundant fragment ion m/z 86 was diethyl methylamine moiety.

M5Metabolite M5 was observed in plasma, bile, urine and faeces, and eluted at 24.1 min with a protonated molecular ion at m/z 191. The retention time and product ion spectrum were identical with those for the

authentic standard of 3-n-butylphthalide, as shown in Figure 10. Therefore, M5 was characterised as 3-n-butylphthalide.

M6 and M7M6 was observed in plasma, bile, urine and faeces, and eluted at15.4 min, and had a protonated molecular ion at m/z 207, which is 16 Da higher than that of M5. In the product ion spectrum, the most abundant fragment ion was m/z 189 formed by losing a water molecule from m/z 207. The other characteristic fragment ions were m/z 171 (losing H

2O from m/z 189), m/z 143 (losing

CO from m/z 171) and m/z 105 (benzonyl moiety) as depicted in Figure 11. The product ions m/z 105, m/z 91 and m/z 77 were identical to that of M5. In the LC chro-matogram and extracted ion chromatogram, there were two separate peaks at 15.4 min and 21.9 min, for M6 and M7, respectively. Therefore, M6 and M7 were tentatively proposed as the hydroxylation metabolites of M5, and there were two possible hydroxylation sites according to the previous research (Peng and Zhou 1996; Wang et al. 1997).


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M8M8 was detected in bile and eluted at 21.1 min with a protonated molecular ion at m/z 674, which was 176 Da higher than that of M1. The most abundant product ion at m/z 543 was also 176 Da higher than m/z 367, the product ion of M1. The fragment ions at m/z173 and m/z 145 were also identical to those for M1, and fragment

ions at m/z 132 and m/z 86 were identical to those of M4. After spiking bile with β-glucuronidase at 37 °C for 12 h, the peak area of M8 markedly decreased, while the peak area of M1 dramatically increased (data not shown). Based on the MS/MS spectral data and neutral loss of 176 of M8 (data not shown) as depicted in Figure 12, M8 was identified as the glucuronide conjugate of M1.

Figure 8. (a) Extracted ion chromatogram . Chemical structure and MS/MS spectrum of M3 in (b) positive mode and (c) negative mode. MS, mass spectrometry.

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Figure 11. (a) Extracted ion chromatogram and (b) chemical structure and MS/MS spectrum of M6 and M7 in rat plasma. MS, mass spectrometry.

Figure 12. (a) Extracted ion chromatogram and (b) chemical structure and MS/MS spectrum of M8 in rat bile. MS, mass spectrometry.

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Discussion

The main objective of this study was to characterise the circulatory and excretory metabolites, and to reveal the biotransformation fate of ZJM-289 in rats, in support of the hypothesis of drug design. The observed major metabolic routes of ZJM-289 were hydrolysis, with glucuronidation and hydroxylation of its hydrolytic products being relatively minor pathways. A scheme of the identified metabolites and metabolic pathways is shown in Figure 13.

After the intravenous bolus administration, the par-ent compound was hydrolysed immediately, and it was detectable only in blood, probably due to the high affinity of esterase both in rat liver and blood (Satoh and Hosokawa 2006; Berry et al. 2009). Therefore, in circula-tory system, the hydrolysis metabolites M1, M2, M3, M4 and M5 were detected and meanwhile, the hydroxylation products probably formed by the enzymes in liver were

transferred into the blood stream, but the glucuronide conjugate M8 detected in bile, was not found in plasma, urine and faeces. In in vitro plasma and liver microsomes incubation, M7 was found in liver microsomes incu-bated with NADPH, but not detected without NADPH (data not shown). Moreover, the pharmacologically potent metabolites ferulic acid and 3-n-butylphthalide were found in the blood where ferulic acid exerted antioxidant activity and 3-n-butylphthalide exhibited anti-thrombotic and anti-platelet activities, in addition, these two metabolites were also found in the bile, urine and faeces. In previous research, Min et al. have already reported that the nitric donor ZJM-289 possessed more potent anti-platelet activity than 3-n-butylphthalide both in vivo and in vitro. All these findings support the theory of drug design, and provide useful information to the subsequent in vitro and in vivo biotransformation research, including the identification of the enzymes that participated in the biotransformation process of

Figure 13. Proposed metabolic pathway of parent compound after intravenous administration of ZJM-289.

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ZJM-289 and its two major pharmacological metabolites ferulic acid and 3-n-butylphthalide.

Conclusion

In conclusion, 3-n-butylphthalide and ferulic acid, the expected pharmacologically active metabolites of the nitric oxide donor ZJM-289 were observed in circulation and excretory system.

Declaration of interest

This work was supported by China National Science and Technology Programs of Significant New Drugs to Create (2009ZX09103-095).

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identification of circulatory and excretory metabolites of ...}phenyl ester hydrochloride ... sara...

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