Analysis of Xanomeline, a Potential Drug for Alzheimer's Disease, by

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Downloaded by UNIV MASSACHUSETTS AMHERST on October 11, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch016

Analysis of Xanomeline, a Potential Drug for Alzheimer's Disease, by Electrospray Ionization Tandem Mass Spectrometry Todd A. Gillespie, Thomas J. Lindsay, J . David Cornpropst, Peter L. Bonate, Theresa G. Skaggs, Allyn F. DeLong, and Lisa A. Shipley Department of Drug Disposition, Lilly Research Laboratories, Indianapolis, IN 46285 Xanomeline, (3-[4-(hexyloxy)-1, 2, 5-thiadiazol-3-yl]-1, 2, 5, 6tetrahydro-1-methylpyridine) is a selective M muscarinic agonist currently under investigation for the potential symptomatic treatment of Alzheimer's disease. The characterization of the metabolites of xanomeline has been accomplished utilizing electrospray tandem mass spectrometry (ES-MS/MS). The use of ES-MS/MS has been employed in the identification of numerous oxidative metabolites of xanomeline resulting from extensive first pass metabolism. In addition, on-line liquid chromatography (LC) coupled with ES-MS/MS has been utilized to further characterize the metabolites of xanomeline after subcutaneous dosing in rats and transdermal dosing in humans. The advantages and limitations of on-line LC/ES-MS/MS for biotransformation studies are demonstrated in this application. 1

Electrospray (ionspray) ionization coupled with tandem mass spectrometry (ESMS/MS) has proven to be an invaluable tool in the characterization of trace level mammalian metabolites of pharmaceutical candidates (i, 2). Furthermore, the combination of on-line LC with ES-MS/MS has become the method of choice for analysis of polar, thermolabile drug candidates and their respective metabolites in complex biological matrices (3, 4). The utilization of this methodology from the initial discovery phase of a potential drug candidate through human clinical trials and beyond can enhance a drug's rapid development (5, 6). Electrospray typically generates abundant [M+H] species and avoids the use of high temperatures which may cause thermal degradation of metabolites (particularly polar conjugates). In addition, tandem mass spectrometry provides both the selectivity and sensitivity required for detection of trace level (ng/mL) metabolites in the presence of large concentrations of endogenous components. Identification of the metabolites from a new drug substance typically follows three steps in our laboratories. Initially animal urine, plasma or microsomal incubates are examined to tentatively identify major metabolites. Second, in vivo metabolism and balance studies are performed in different animal species utilizing radiolabeled drug substance and third, human studies are performed with +

0097-6156/95/0619-0315$12.00/0 © 1996 American Chemical Society

In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; Snyder, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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radiolabeled material when possible. The applications discussed in this work will focus on the second and third steps of this process using specific examples to illustrate these steps. Xanomeline, (3-[4-(hexyloxy)-l, 2, 5-thiadiazol-3-yl]-l, 2, 5, 6-tetrahydro-lmethylpyridine) is a selective M i muscarinic agonist currently under investigation for the potential symptomatic treatment of Alzheimer's disease.

Xanomeline (* denotes l^C-radiolabel) Previous pharmacokinetic/balance studies conducted in rats and monkeys with radiolabeled drug suggested extensive metabolism of the parent compound (7). ES/MS/MS has been utilized to characterize the urine and plasma metabolites of xanomeline in humans (8-10). Furthermore, on-line LC/ES-MS/MS has been used to identify the urinary metabolites in rat after a subcutaneous dose of xanomeline in preparation for metabolite identification in humans after transdermal dosing of xanomeline. This work shows the advantages and limitations of using on-line LC/ES-MS/MS for characterization of numerous oxidative metabolites of xanomeline. Experimental A biotransformation study was conducted in human volunteers using ^Cxanomeline at an oral dose of 75 mg (50μΟί). Both urine and plasma samples were collected for analysis, however, only the urine was utilized for metabolite identification due to the low dose of xanomeline administered. Therefore, pooled human plasma for metabolite identification was obtained from a pharmackinetic study in human volunteers administered an oral dose of 150 mg (free base) of xanomeline. In addition, urine and plasma from rats was collected after subcutaneous administration of 10 mg/kg (free base) of ^c-xanomeline to F344 rats. Sample Preparation. Initial work with xanomeline utilized LC with fraction collection. Twenty mL of urine from each subject's 0-6 hour urine collection was pooled (80 mL),freeze-driedand the residue extracted with 1-2 mL of methanol (MeOH)/water (50/50;v/v), then centrifuged. A 200 μL aliquot of the supernatent was injected onto a Zorbax RX-C8 column (25cm χ 0.46mm id) and separated by using a linear gradient consisting of 80/20 50 mM ammonium acetate (NH40Ac)/MeOH for 30 minutes, then ramped to 100% MeOH over 75 minutes. Fractions were then collected in 1.25 minute increments and based on the radiochromatogram were then dried for analysis.



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Human plasma collected at 1, 1.5, 2, and 4 hour time points was pooled (30 mL) for analysis. The pooled plasma was diluted with an equal volume of 50 mM NH4OAC (pH 5) split into approximately 5 mL aliquots and applied to CN/C18 (lcc, Bond Elut) combination SPE ("piggy-backed") cartridges. The "piggybacked" cartridges were washed with 50 mM NH4OAC (pH 5) then separated and the CI8 cartridges eluted with 2 mL of MeOH. The CN cartridges were eluted with 2 mL of 200 mM NH40Ac/MeOH (13.8 mL of 56.6% concentrated NH4OH brought to 1 L with MeOH). The eluents in both cases were collected in silanized glass tubes and evaporated to dryness using nitrogen (N2). The dryed eluents were reconstituted with MeOH, combined and centrifuged. The supernatent was then transferred to a silanized glass tube and taken to dryness with N2. (Recovery data obtained for this procedure indicated a 66% recoveryfromthe elution step and a 31% loss in the load and wash steps.) This residue was then reconstituted with 2.5 mL of 50 mM NH4OAC (pH 5), then injected (200 μL injections until complete) onto a Zorbax RX-C8 column (25cm χ 0.46mm id). Separation was obtained using a linear gradient consisting of 100% 50 mM NH4OAC ramped to 100% MeOH over 60 minutes and held for an additional 5 minutes at a flow rate of 1 mL/min. Fractions were then collected and dried for analysis. All other sample analyses were performed utilizing on-line LC/MS and LC/MS/MS. The rat urine and human urine collectedfromvoluteers administered xanomeline via a patch were centrifuged, transferred to glass test tubes and then diluted 1:1 with 100 mM NH4OAC. LC/MS and MS/MS. All experiments were performed on a Finnigan MAT TSQ700 triple stage quadrupole (MS/MS). Initial work with xanomeline in human urine and plasma utilized an Analytica of Branford electrospray (ES) interface. In most cases, samples (collected fractions) were directly infused with a Harvard Apparatus syringe pump at a rate of 2 pJL/min after reconstitution with 100 - 300 μΐ, of MeOH/1% acetic acid (50/50;v/v). Positive ion detection was achieved with a voltage between -3500 to -3800 V applied to the Analytica ES interface and with 20 mL/min of nitrogen (N2) drying gas @ 250°C. All direct infusion analyses were performed in the profile mode from m/z 100 - m/z 500 at 1 sec/scan and averaged for 1 min. Select human urine samples (fractions) were analyzed by LC-ES/MS and LC/ES-MS/MS. Liquid chromatographic analyses were performed using a Waters 600-MS liquid chromatographic pump. A Basic column (5cm χ 0.40mm id) from YMC, Inc. was coupled to the Analytica ES interface. An isocratic system of acetonitrile/0.2M acetic acid (50/50;v/v) was used at a flow rate of 1 mL/min. A flow of 2.5 μΙ7πήη was introduced into the Analytica ES interface by splitting the LC flow of 1 mL/min with a low dead volume tee. A voltage of -4200 V (applied to the canister) was used with 30 mL/min of nitrogen (N2) drying gas @ 250°C to obtain positive ion detection. Analyses were performed in centroid mode at 1 sec/scan. The collisionally activated dissociation (CAD) analyses were obtained using a collision offset energy of -20 eV and a collision pressure of 2.0 mTorr of argon (Ar). On-line LC/MS and LC/MS/MS were performed using a Finnigan atmospheric pressure ionization (API) source with a "high flow" ES interface. The diluted xanomeline urine samples were analyzed on a Zorbax RX-C8 column (25cm χ 4.6mm id) using 100 pL injections. A linear gradient was employed consisting of 100% 50 mM NH4OAC ramped to 100% MeOH over 60 minutes and held for an additional 5 minutes at a flow rate of 1 mlVmin. The LC eluant was split 1:4 to allow 250 pIVmin into the Finnigan ES interface and 750 pL/min to


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Peak 10 100

1 Peak 7

Peaks Peak 2 3 & 4

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5 0

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A

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100-

J

Full Scan Positive Ion LC/ES/VIS Radioclironiatog

50-

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58:20

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Figure 1. Full scan positive ion LC/ES-MS trace and the radiochromatogram for 24 hour rat urine after subcutaneous dosing.

mlz 284

^Peak 2

1 m/z 298 ^ m/z

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Figure 2. LC/ES-MS mass chromatograms of selected ions for 24 hour rat urine after subcutaneous dosing.


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either a Raytest Ramona 5-LS radio chemical detector with a solid flow cell or a Waters 490-MS UV multiwavelength detector. Positive ion detection was achieved with the Finnigan ES using a spray voltage of +4500 V (applied to the needle), capillary heater temperature of 275°C, sheath gas of 70 psi (N2)> and an auxiliary gas flow of 15-20 mL/min (N2). Analyses were performed in centroid mode at 1 sec/scan. The CAD analyses utilized a collision offset energy of -30 eV and a collision gas pressure of 1.6 mTorr (argon).


Results and Discussion Rat Urine. At present, all ADME (absorption, distribution, metabolism, and elimination) work performed within our laboratories involves metabolism and balance studies which utilize radiolabeled compound. Figure 1 shows the full scan electrospray positive ion RIC (reconstructed ion chromatogram) trace and radiochromatogram from a 24 hour urine sample (~1 mL) collectedfroma rat after subcutaneous dosing of radiolabeled xanomeline (l^C-xanomeline). A gradient consisting of 50 mM NH4OAC and MeOH was used over a period of 60 minutes to separate potential metabolites. In our laboratory we typically use shorter (

Analysis of Xanomeline, a Potential Drug for Alzheimer's Disease, by

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