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Anal. Chem. 2006, 78, 6448-6456

Direct Molecular Analysis of Whole-Body Animal Tissue Sections by Imaging MALDI Mass Spectrometry Sheerin Khatib-Shahidi,† Malin Andersson,† Jennifer L. Herman,‡ Todd A. Gillespie,‡ and Richard M. Caprioli*,†

Mass Spectrometry Research Center, Departments of Chemistry and Biochemistry, Vanderbilt University, 465 21st Avenue S, Suite 9160 MRB III, Nashville, Tennessee 37221, and Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285

Imaging mass spectrometry (IMS) that utilizes matrixassisted laser desorption/ionization (MALDI) technology can provide a molecular ex vivo view of resected organs or whole-body sections from an animal, making possible the label-free tracking of both endogenous and exogenous compounds with spatial resolution and molecular specificity. Drug distribution and, for the first time, individual metabolite distributions within whole-body tissue sections can be detected simultaneously at various time points following drug administration. IMS analysis of tissues from 8 mg/kg olanzapine dosed rats revealed temporal distribution of the drug and metabolites that correlate to previous quantitative whole-body autoradiography studies. Whole-body MALDI IMS is further extended to detecting proteins from organs present in a whole-body sagittal tissue section. This technology will significantly help advance the analysis of novel therapeutics and may provide deeper insight into therapeutic and toxicological processes, revealing at the molecular level the cause of efficacy or side effects often associated with drug administration. The introduction of a new drug to market takes on average eight years or more and costs approximately $500 million to develop.1 A bulk of the time and money is spent on following lead compounds that all too often prove to be unsuccessful in clinical trials. Improvements in the preclinical steps of drug discovery and development are needed to better identify potentially successful drug candidates, while at the same time eliminating inferior drug compounds early in the process. A key component of this process is the ability to gain knowledge about the distribution and delivery of a novel therapeutic compound to a target organ or tissue, since tissue distribution plays an essential role in the pharmacokinetic behavior of a drug. Of even greater interest would be the development of a detection system that allows for the correlation * Corresponding author. E-mail: [email protected]. Fax: 615-343-8372. † Vanderbilt University. ‡ Eli Lilly and Co. (1) DiMasi, J. A.; Hansen, R. W.; Grabowski, H. G. J. Health Economics 2003, 22, 151-185.

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between drug tissue distribution and pharmacological or toxicological effects. Here we describe the development and application of a new methodology for the analysis of whole organ and whole-body animal tissue sections by MALDI imaging mass spectrometry (IMS). Data obtained by this novel methodology demonstrate the capacity to detect and determine the relative concentrations of drug in tissues and more importantly, at the same time, the distribution of various metabolites. Such information is not easily obtainable by current imaging techniques such as autoradiography. We also demonstrate the power of IMS to detect proteomic contributions of individual organs across whole-body tissue sections. Detecting protein changes with both spatial and molecular specificity allows the correlation of drug tissue distribution and therapeutic response within the same tissue. Mass spectrometry is widely used in many aspects of pharmaceutical research and development, from structural characterization of new chemical entities to pharmacokinetic studies. Imaging mass spectrometry technology brings new dimensions and capabilities to aid in the development of new drugs. IMS employs MALDI MS for the desorption and detection of compounds with the use of a matrix, a low molecular weight organic compound that absorbs the energy of the laser. The matrix is applied to a sample to cocrystallize with the analytes. Absorption of the UV radiation from a laser pulse by the crystals subsequently causes matrix and analyte molecules to desorb from the sample surface. Gas-phase ionization occurs in the desorbed MALDI plume to generate ions, predominately singly protonated intact molecular ions ([M + H]+), that are typically analyzed by a timeof-flight (TOF) mass spectrometer. The resulting mass spectrum contains hundreds of signals representing the compounds ablated from the sample surface directly from the area irradiated by the laser, typically 30-50 µm in diameter. A molecular image of the tissue section is obtained by a raster of the section to produce a data set consisting of an ordered array of mass spectra, where each spectrum represents the local molecular composition at known x,y coordinates. Each spectrum at a specific coordinate is a pixel in a two-dimensional molecular map of the tissue section, with each pixel containing hundreds of distinct molecular peaks and associated relative abundances. 10.1021/ac060788p CCC: $33.50

© 2006 American Chemical Society Published on Web 08/03/2006

IMS has been successfully used to map different anatomic substructures of a mouse brain consisting of normal and tumor bearing regions2 and has been used to obtain information on the local proteomic composition across a sexually mature mouse epididymis.3 Detection of pharmaceutical compounds in tissue by MALDI MS was reported by Troendle et al.4 and has been further extended by Reyzer et al. to the imaging of anticancer drug OSI774 and associated protein changes in tumor tissue from dosed mouse.5 The authors were able to detect tumor response to therapeutics based on early changes in the tumor proteome. Some preliminary reports from the authors’ laboratory, as well as others, have demonstrated the feasibility of the analysis of whole-body tissue sections.6-8 In the study presented here, efforts were focused on developing a method to detect endogenous (proteins) and exogenous (drug) compounds directly from multiple tissue morphologies present in whole-body animal tissue sections. Rats dosed with pharmacologically equivalent amounts of olanzapine (OLZ) were chosen since previous data9 using quantitative wholebody autoradiography (WBA) were available as reference for the IMS method development. WBA is a widely accepted technique to determine spatial and quantitative information about a compound of interest and is a mandatory procedure for drug approval by the FDA.10 WBA monitors the distribution of a radiolabeled drug compound on a time scale equivalent to the compound’s known pharmacokinetics, which is often obtained by tissue homogenization and analysis by high-performance liquid chromatography tandem mass spectrometry (HPLC MS/MS).11 Much information about the absorption and distribution of the radiolabeled drug can be acquired by WBA with an average turnover time of 5 days. However, the detected signal does not distinguish between the original radiolabeled compound from metabolites that have retained the radiolabel and represents a significant limitation of WBA. Conversely, IMS has the inherent capability of differentiating molecular species at similar spatial resolutions as quantitative WBA (typically 50 µm), in some cases at fractions of a single mass unit when using instruments of high resolving power, such as a Fourier transform MS. On modern state-of-the-art MALDI mass spectrometers equipped with lasers operating at 1 kHz, a whole-body mouse or rat section would take less than 4 h to image. The individual molecular distribution information acquired from the images will serve as a powerful molecular compliment to WBA for the design and development of new drugs. IMS analysis of whole animal tissue sections also allows the detection of protein changes within (2) Chaurand, P.; Schwartz, S. A.; Caprioli, R. M. J. Proteome Res. 2004, 3, 245-252. (3) Chaurand, P.; Fouchecourt, S.; DaGue, B. B.; Xu, B. J.; Reyzer, M. L.; Orgebin-Crist, M.-C.; Caprioli, R. M. Proteomics 2003, 3, 2221-2239. (4) Troendle, F. J.; Reddick, C. D.; Yost, R. A. J. Am. Soc. Mass Spectrom. 1999, 10, 1315-1321. (5) Reyzer, M. L.; Caldwell, R. L.; Dugger, T. C.; Forbes, J. T.; Ritter, C. A.; Guix, M.; Arteaga, C. L.; Caprioli, R. M. Cancer Res. 2004, 62, 9093-9100. (6) Khatib-Shahidi, S.; Reyzer, M. L.; Herman, J. L.; Gillespie, T. A.; Caprioli, R. M., Nashville, TN, 2004. (7) Khatib-Shahidi, S.; Herman, J. L.; Wickremsinhe, E.; Gillespie, T. A.; Caprioli, R. M., San Antonio, TX, 2005. (8) Rohner, T. C.; Staab, D.; Stoeckli, M. Mech. Ageing Dev. 2005, 126, 177185. (9) Chay, S. H.; Herman, J. L. Arzneimittel-Forschung/Drug Res, 1998, 48, 446454. (10) Food and Drug Administration; www.fda.gov, 2005; 21CFR312.23. (11) Solon, E. G.; Balani, S. K.; Lee, F. W. Curr. Drug Metab. 2002, 3, 451-462.

the various organs and throughout the entire animal. This will significantly aid in the advancement of novel therapeutics by providing insight into therapeutic and toxicological responses associated with drug administration. Identification of protein alterations may lead to verification of drug efficacy and early signs of drug toxicity and, generally, will allow refinement of the initial strategy during the drug discovery and development process. EXPERIMENTAL SECTION Materials. The MALDI matrixes, sinapinic acid (SA) and 2,5dihydroxybenzoic acid (DHB), were purchased from Sigma Chemical Co. (St. Louis, MO). OLZ drug standard was produced by Lilly Research Laboratories (Eli Lilly and Co., Indianapolis, IN). Whole-Body Tissue Preparation. Collaborators at Eli Lilly administered all physiologically equivalent OLZ dosing (po 8 mg/ kg) to 10-week-old male Fischer 344 rats, which had fasted overnight prior to start of study. Animals were euthanized at 2 and 6 h postdose by isoflurane anesthesia followed by exsanguination via cardiac puncture. Control and dosed rats were frozen in hexane/dry ice and stored at -20 °C. Individual rats were frozen in a block of ice, and 20-µm-thick whole-body sagittal tissue sections were collected on acetate film tape (3M, St. Paul, MN) using a cryomacrocut (Leica CM3600, Leica Microsystems Inc., Bannockburn, IL) at -20 °C. The tissue sections were then either thaw-mounted (for protein imaging) or mounted using conductive double-sided tape (drug imaging) onto MALDI target plates. A single sagittal section of a whole rat spanned four MALDI plates (measuring 4 × 4 cm). All plates were placed into a vacuum desiccator for ∼1 h before matrix application. Protein Imaging. For the first set of imaging experiments, 12-µm coronal brain sections from control male Sprague-Dawley rats (∼300 g) were cut on a Leica CM 3050 S cryotome at an anterior-posterior level that encompasses the frontal cortex, nucleus accumbens, and septum. The tissue sections were thawmounted onto a MALDI target plate and washed briefly in 70% ethanol (30s) and twice in 95% ethanol (30 s). The sections were allowed to dry in a desiccator for 1 h prior to applying a seeding layer of ground SA. Arrays of discreet matrix spots (200 µm in diameter; 20 mg/mL SA in 50:50 acetonitrile/0.2% trifluoroacetic acid (TFA) in dH2O) were deposited across the section using an acoustic microdispenser.12 For the whole-body imaging, each plate from a single sagittal section was individually spray coated using a glass spray nebulizer with 20 mg/mL SA and 5 mg/mL DHB (same solvent conditions) matrix combination prior to analysis.13-15 At 30 cm from the target plate, a cycle (3 spray passes) of matrix coatings was applied for 25-30 cycles, with 1- min ambient drying time between cycles. All imaging experiments were performed on either an Ultraflex II MALDI-TOF/TOF-MS (Bruker Daltonics, Billerica, MA) equipped with a solid-state Smartbeam laser operating at 200 Hz or a Voyager DE STR MALDI-TOF-MS (Applied Biosystems) equipped with a 337-nm nitrogen laser operating at 20 Hz. (12) Aerni, H.-R.; Cornett, D. S.; Caprioli, R. M. Anal. Chem. 2006, 78, 827834. (13) Cohen, S. L.; Chait, B. T. Anal. Chem. 1996, 68, 31-37. (14) Gonnet, F.; Lemaitre, G.; Waksman, G.; Tortajada, J. Proteome Sci. 2003, 1, 1-7. (15) Laugesen, S.; Roepstorff, P. J. Am. Soc. Mass Spectrom. 2003, 14, 9921002.

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Figure 1. Coronal forebrain section from control rat analyzed by IMS. Optical image of brain tissue section; anatomical structures are visible such as the nucleus accumbens (1), septum (2), and frontal cortex (3) (A). MALDI MS protein ion image of nucleus accumbens (red, m/z 6928) (B). MALDI MS protein ion image of septum (green, m/z 6981) (C). MALDI MS protein ion image of prefrontal and frontal cortex (blue, m/z 6916) (D). Composite ion density image for relative distributions of the protein ion intensities (E). Spectrum highlights peaks selected for ion images. Bar, 0.5 cm.

Approximately 70-100 laser shots were averaged per spectrum to obtain optimal signal-to-noise ratios, and sensitivity and data were acquired in the linear mode, under delayed extraction conditions. Prior to image analysis of each plate, external calibration files were created using a protein standard mix consisting of insulin β chain (bovine, m/z 3497), cytochrome c (bovine, m/z 12 232), and apomyoglobin (equine, m/z 16 953). Custom software was developed for image acquisition.16-18 The acquisition software controls the instrumental parameters, as well as the x,y movement of the sample stage at a specified lateral resolution within the borders of a defined area. At 250-µm lateral resolution, the average number of pixels per plate was ∼30 000 or a total of ∼115 000 pixels for the entire sagittal rat section. The processing of image data was performed using custom software and ProTS-Data from Biodesix Inc. (Steamboat Springs, CO). The custom software allows for the batch processing of each spectrum associated with a pixel. All spectra were processed for baseline correction (32 peak-width detection) with a nine-point Gaussian smooth. Twodimensional ion density maps were created using the image reconstruction software (BioMap, Novartis, Basel, Switzerland). For the whole-body images, spectra from each plate were examined for interplate normalization by choosing the two most abundant signals represented in both spectra for intensity value comparison. Negligible intensity variations were observed between all four plates, and therefore, an arbitrary intensity threshold value was selected for all organ images spanning multiple plates. All 2D protein images were saved as TIFF files. Low-level image (16) Stoeckli, M.; Farmer, T. B.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1999, 10, 67-71. (17) Stoeckli, M.; Chaurand, P.; Hallahan, D. E.; Caprioli, R. M. Nat. Med. 2001, 7, 493-496. (18) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751-4760.

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processing was performed for removal of background noise and registration of individual images was done using Adobe Photoshop 7.0 software. Interanimal variability was assessed by comparing spectra from the striatum of individual animals (n ) 6), where each spectrum is an average of data obtained from nine matrix spots in the striatum (200 laser shots per spot). Drug Imaging. OLZ standard solutions of 0.02, 0.2, 2, and 20 pmol/µL were prepared in 100% methanol. A 1-µL aliquot from each dilution was combined with an equal volume of matrix (40 mg/mL DHB in 75% methanol), vortexed, and centrifuged for 30 s. Each matrix/drug dilution was spotted (250 nL) on a gold MALDI target plate in 10 individual wells using an automatic pipettor. Upon formation of cocrystals, the spots were analyzed on a QStar XL (MDS Sciex, Concord, ON, Canada) equipped with an oMALDI source (20 Hz nitrogen laser 337 nm) and a hybrid QqTOF mass analyzer to obtain MS and MS/MS data. OLZ was analyzed by selected reaction monitoring to augment the sensitivity for all analyses. Fragmentation of OLZ was achieved using a collision energy of 25 eV with an argon collision gas at a CID gas pressure of (3-4) × 10-5 Torr. A MS/MS spectrum was recorded for each spot by ablating the sample until all matrix crystals had been depleted (60 s). For each OLZ concentration, OLZ signal intensity (ion counts) of all 10 spectra was averaged and this average intensity value was plotted against the final concentration of OLZ on plate to produce a standard curve. For the drug imaging experiments, each plate from a single 20-µm-thick dosed whole-body tissue section were spray-coated with DHB (40 mg/mL) in 75% methanol using the abovementioned procedure. Image analyses were performed using multiple reaction monitoring mode (OLZ m/z 313 f 256; Ndesmethyl OLZ m/z 299 f 256; 2-hydroxymethyl OLZ m/z 329

Figure 2. IMS protein analysis of whole-body sagittal tissue sections from rat. Rat head whole-body tissue subsection optical and ion images (A). Ion images from left to right: brain, m/z 20 736; gray matter, m/z 10 271; white matter, m/z 5978; eye socket, m/z 6858. Rat abdomen whole-body tissue subsection optical and ion images (B). Ion images from left to right: liver, m/z 14 370; kidney cortex, m/z 4638; cecum wall, m/z 5155; muscle, m/z 11 836. Bars, 1 cm (A, B).

Figure 3. Whole-body protein analysis of rat sagittal tissue section by IMS. Optical image of rat sagittal tissue section across four gold MALDI target plates (A). Ion image overlay of unique organ signals: brain, green m/z 21 952; thymus, light blue m/z 6893; thoracic cavity, purple m/z 7921; liver, blue m/z 14 321; kidney cortex, yellow m/z 4643; cecum wall, cyan m/z 5155; testis, orange m/z 14 400; and muscle, red m/z 11 836 (B). Bar, 1 cm.

f 272) using the oMALDI Server 4.0 software. The software allows the user to define the boundaries of the area to be imaged and the lateral resolution of the laser (400 µm × 400 µm). The oMALDI Server works to direct the Analyst QS acquisition software to acquire data at each pixel (signal summed at 4 s/transition; total 12 s/pixel). Similar to the image reconstruction software previously described, ion density maps can be created after data acquisition has completed using the oMALDI Server 4.0 imaging software. Low-level processing of the images (removal of background noise and image alignment) was done using Adobe Photoshop 7.0 software. RESULTS AND DISCUSSION Protein Imaging. All protein images were acquired on a MALDI-TOF MS in the linear mode under delayed extraction

conditions at an image resolution of 250-280 µm. The formation of a homogeneous matrix crystal layer across a tissue section is essential for reproducible results, and for single-organ analyses, an application of SA matrix solution has been shown to provide the necessary crystal coverage to achieve high-quality ion images.19 We demonstrate this on a tissue section from control rat brain, where ion signals are detected and found to be localized to regions or substructures such as the frontal cortex (m/z 6916), septum (m/z 6981), and nucleus accumbens (m/z 6928), as shown in Figure 1. The same ion sensitivity and structural resolution is needed for whole-body sections comprised of many organs, each with different surface chemistries. (19) Chaurand, P.; Caprioli, R. M. Electrophoresis 2002, 23, 3125-3135.

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Figure 4. Interanimal variability and IMS reproducibility. Overlay of striatal spectra from six animals (A). Expanded mass region of striatal spectra in panel A (B). Average striatal spectra from all animals reveals a low interanimal variability (C).

In the first set of experiments aimed at protocol development, liver, kidney, and brain tissue sections were thaw-mounted to a single MALDI target plate to act as models for the anticipated variations in chemical composition within the whole animal tissue section. It was determined that SA in combination with 20% DHB provided a relatively homogeneous matrix layer across all organ tissues and IMS revealed hundreds of protein signals from each tissue type. Some of these ion signals were found to be unique to a region and could be used to produce organ- and region-specific images. We evaluated the successful protocol using subsections of whole-body sagittal tissue from rat, such as the head and abdomen, since organs of varying surface properties were represented. Homogeneous matrix coverage was achieved across 6452

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the entire section for IMS analysis to produce two-dimensional ion maps (Figure 2). Signals were found to be specific to substructures of organs, such as the gray matter (m/z 10 271) and white matter (m/z 5978) of the brain, as well as the eye socket (m/z 6858), kidney cortex (m/z 4638), and cecum wall (m/z 5155). Other signals were found to be specific to an entire organ, such as the brain (m/z 20 736), liver (m/z 14 370), and muscle (m/z 11 836). For whole-body sagittal tissue sections, using the same sample preparation and analysis conditions, signals unique to individual organs were detected and used to produce a partial twodimensional protein map of a rat (Figure 3). Organ-specific ion signals were reliably detected when spanning multiple MALDI

Figure 5. Whole-body protein analysis of glioma tumor mouse sagittal tissue section by IMS. Optical image of mouse head subsection on gold MALDI target plate. Tumor-bearing region outlined in red (A). Ion image overlay of unique protein signals: bone, white m/z 5796; glioma tumor, green m/z 11 375; muscle, red m/z 11 902; and brain, blue m/z 22 257 (B). Protein ion spectrum of glioma tumor region (C). Bar, 5 mm.

plates used in the analyses, including markers for the liver (m/z 14 321) and muscle (m/z 11 836). Of course, many of the same proteins were also detected in multiple organs of the same tissue section. Interanimal variability and IMS reproducibility were assessed by comparing spectra from the striatum region of rat brain from six individual animals (Figure 4). A high degree of consistency was observed, even when selecting a mass range containing relatively low abundant peaks (Figure 4B). In fact, an average plot of all the spectra illustrates the relative standard errors for the peaks and range from 3.7 to 9.6% (Figure 4C). The capability of IMS combined with optimal sample preparation protocols to produce consistent signals between samples and across multiple MALDI plates demonstrated the reproducibility of the imaging methodology. Since proteomic information of multiple organs is collected in a single imaging experiment, a specific organ or anatomical region can be selected for more detailed analysis. For example, IMS was applied to sagittal sections of a mouse that was injected in the right hemisphere with GL261 human glioma cells to establish a mouse model for glioma (Figure 5). Protein signals unique to the tumor (m/z 11 375) were detected, as well as some organ specific ion signals, such as bone (m/z 5796), muscle (m/z 11 902), and gray matter (m/z 22 257) of brain (Figure 5B). The tumor-bearing region of the brain was easily identified by the specific signals found in the tumor and its correlation to histological staining. Once the region was identified, proteomic information was extracted from the tumor region (Figure 5C). To better understand differences in disease pathology, investigators have relied on several analytical techniques to detect differential expression patterns of genes, mRNA, and proteins. Mass spectrometry has been useful for identifying protein biomarkers that can be correlated with stage/progression, prognosis, and therapeutic targets in diseases.20-22 IMS technology as demonstrated here adds a significant capability for analysis of whole organ and whole-body tissue sections. Production of highquality spectra and high-resolution images were feasible with

careful sample preparation protocols and the use of a SA and DHB matrix combination. Through iterative measurements of whole-animal tissues by IMS, distinct protein patterns can be identified and used to monitor whole-body systems dynamics, opening the door to applications in systems biology. IMS measurements have the advantage of producing protein expression patterns for the entire animal and the potential to identify the particular disease, progression or stage of the disease, and response to therapeutic agents, if early markers of response are identified. IMS protein analyses of whole-animal sections could also aid in the design of novel therapeutic agents by providing insight into disease pathology, identifying potential drug targets, and performance evaluation of potential drug candidates and their side effects. Drug Imaging. In the case study of olanzapine, all MS/MS analyses were performed on an orthogonal-MALDI hybrid quadrupole-TOF mass spectrometer (equipped with imaging software) to monitor several fragments from collision induced dissociation (CID) of the parent compound (Figure 6). The lower limit of detection using tandem mass spectrometry (described below) was 60.0 ng of olanzapine on tissue (signal-to-noise ratio >3). The olanzapine standard curves were linear over a range of 0.0066.25 µg/mL of OLZ with a correlation coefficient of >0.992. Olanzapine (brand name Zyprexa) is a drug of the thienobenzodiazepine class and is generally used to treat mood disorders such as schizophrenia and acute mania in bipolar patients. As with many atypical antipsychotic drugs, the precise mechanism of action for olanzapine is unknown. However, it has been proposed that OLZ works by blocking certain serotonin type 2 (5HT2) and (20) Ranganathan, S.; Williams, E.; Ganchev, P.; Gopalakrishnan, V.; Lacomis, D.; Urbinelli, L.; Newhall, K.; Cudkowicz, M. E.; Jr., R. H. B.; Bowser, R. J. Neurochem. 2005, 95, 1461-1471. (21) Villanueva, J.; Shaffer, D. R.; Philip, J.; Chaparro, C. A.; Erdjument-Bromage, H.; Olshen, A. B.; Fleisher, M.; Lilja, H.; Brogi, E.; Boyd, J.; Sanchez-Carbayo, M.; Holland, E. C.; Cordon-Cardo, C.; Scher, H. I.; Tempst, P. J. Clin. Invest. 2006, 116, 271-284. (22) Yanagisawa, K.; Shyr, Y.; Xu, B. J.; Massion, P. P.; Larsen, P. H.; White, B. C.; Roberts, J. R.; Edgerton, M.; Gonzalez, A.; Nadaf, S.; Moore, J. H.; Caprioli, R. M.; Carbone, D. P. Lancet 2003, 362, 433-439.

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Figure 6. Fragmentation Spectra for OLZ and its metabolites. Olanzapine m/z 313 f 256 (A). N-Desmethyl metabolite m/z 299 f 256 (B). 2-Hydroxymethyl metabolite m/z 329 f 272 (C).

dopamine receptors.23 Pharmacokinetic studies of OLZ indicate that it is well absorbed after single-dose oral administration, reaching peak plasma concentrations within 5-8 h in humans23 and within 45 min for rats.24 At 2 h postdose, WBA data revealed that radioactivity associated with [14C]OLZ was readily distributed throughout the tissue of an entire rat;9 however, no information as to the molecular identity of the drug was obtainable by this (23) Eli Lilly and Co., Zyprexa Prescribing Information, PV 3391 AMP, 12/12/ 2003. (24) Aravagiri, M.; Teper, Y.; Marder, S. R. Biopharm. Drug Dispos. 1999, 20, 369-377.

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technique. The IMS images obtained in this study are the first examples of simultaneous drug and metabolite imaging in whole animals, thus allowing for the systemwide evaluation of each compound in a single individual animal section. Careful sample preparation is necessary to obtain high-quality small-molecule MS/MS images. Various matrix/solvent combinations can influence the sensitivity of a small molecule detected from tissue and should be optimized prior to an imaging experiment. Deviations in small-molecule ionization yields can be contributed to several factors including the chemical properties of the small molecule, the ability of a solvent to extract a compound out of the tissue, and the extent of incorporation of the compound into the matrix crystals. For the whole-body images presented here, DHB in combination with 75% methanol was found to provide the highest analyte sensitivity. After a single oral dose of olanzapine (8 mg/kg), drug and metabolites were present in measurable amounts in almost all tissues at both 2 and 6 h postdose by IMS. At 2 h postdose, OLZ is observed to be ubiquitously distributed throughout the whole rat, with significant localization in specific organs (Figure 7). The highest OLZ signal was detected in the lung followed by the spleen, bladder, kidney, liver, thymus, brain and spinal cord, and testis. This observation is in full agreement with previously published OLZ quantitation and distribution data.9 The drug is clearly localized in the target organs brain and spinal cord. The prominent signal for OLZ in the liver is consistent with significant drug elimination by first-pass metabolism. However, OLZ was also seen localized to the bladder, indicating elimination in an unchanged form consistent with previous drug data, which found that 7% of the OLZ dose was recovered in urine in its original form.23 Biochemically, three families of cytochrome P450, CYP1, CYP2, and CYP3, are known to participate in the metabolism of drugs.25 Previous studies suggest that olanzapine is eliminated extensively by first-pass metabolism, acting as a substrate for the cytochrome P4501A2 and 2D1-5 enzymes in rat (analogous to human CYP1A2 and CYP2D6), with ∼40% of the OLZ dose metabolized before reaching the systemic circulation.23 Olanzapine’s tmax for peak concentration in rat tissues was determined to be 2 h postdose.9,26 For the first time, metabolite distribution was obtained using the whole-body IMS technology and revealed that the metabolites N-desmethylolanzapine and 2-hydroxymethyl olanzapine contributed 21% of the total MS/MS signal. The N-desmethyl metabolite was detected primarily in the liver, kidney, and bladder and contributed 8% of the total IMS signal. The 2-hydroxymethyl metabolite was detected with the highest signal in the bladder, followed by the liver and kidney. This second metabolite contributed 13% of the total IMS signal. It should be noted that little to none of these metabolites was detected in the brain and spinal cord. These data are also consistent with previous evidence that the metabolites have no central nervous system pharmacological activity.23 At 6 h following drug administration, static drug distribution was again detected throughout the whole-body section, with drug signal greatly decreased in the brain and spinal cord regions at 66% less ion signal than the 2-h time point (Figure 8). This observation correlated to previous studies that found a 78% (25) Lewis, D. F. V.; Ioannides, C.; Parke, D. V. Environ. Health Perspect. 1998, 106, 633-641. (26) Bao, J.; Potts, B. D. J. Chromatogr., B 2001, 752, 61-67.

Figure 7. Detection of drug and metabolite distribution at 2 h postdose in a whole rat sagittal tissue section by a single IMS analysis. Optical image of a 2 h post OLZ dosed rat tissue section across four gold MALDI target plates (A). Organs outlined in red. Pink dot used as time point label. MS/MS ion image of OLZ (m/z 256) (B). MS/MS ion image of N-desmethyl metabolite (m/z 256) (C). MS/MS ion image of 2-hydroxymethyl metabolite (m/z 272) (D). Bar, 1 cm.

decrease in [14C]OLZ concentration from 2 to 6 h in brain tissue.9 The decrease in detected OLZ signal indicated clearance of the drug from the target organ. OLZ was detected with the highest amount in lung, followed by localization in liver, bladder, testis, and thymus. Metabolite distribution remained unchanged, with localization primarily in lung, liver, and bladder. The combined metabolite signal was 28% of the total IMS signal, with individual contributions of 13 and 15% for N-desmethyl and 2-hydroxymethyl metabolites, respectively. Again, little to no metabolite signal was observed in the brain and spinal cord regions. Interestingly, the 2-hydroxymethyl metabolite was detected in the testis at 6 h postdose, although it was not detectable in the 2h postdose sections. Our studies show that liver, kidney, and bladder had prominent signals for OLZ and its metabolites, N-desmethyl and 2-hydroxymethyl olanzapine. On the basis of our findings, the 2-hydroxymethyl metabolite, produced by the CYP2D1-5 enzymes, was detected as the more abundant metabolite over the N-desmethyl

olanzapine. This may be due to several factors: (1) five isoforms of the CYP2D enzymes exist in rat25 (as apposed to the single form present in human, CYP2D6) allowing for increased substrate (OLZ) uptake and metabolite (2-hydroxymethyl olanzapine) turnover; (2) differences in the relative expression and catalytic activity of CYPs in rat liver versus human liver can result in large variations in the in vivo metabolic clearance of OLZ; and (3) external sources of variability such as diet, exercise, and environmental exposure can modulate activity of the CYP2D enzymes. It has been determined that at steady-state conditions (multiple dosing) in human the 10-N-glucuronide and N-desmethyl metabolites are the major circulating metabolites produced by direct glucuronidation and CYP1A2 pathways, respectively.23 In the case of rat metabolism, little is know about the involvement and preference of CYPs after a single oral dose of OLZ; however, the formation of glucuronide metabolites is an insignificant pathway for metabolic clearance of drugs,27 and these metabolites were not detected in our study. Additional studies will be needed to Analytical Chemistry, Vol. 78, No. 18, September 15, 2006

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Figure 8. Detection of drug and metabolite distribution at 6 h postdose in a whole rat sagittal tissue section by a single IMS analysis. Optical image of a 6 h post OLZ dosed rat tissue section across four gold MALDI target plates (A). Organs outlined in red. MS/MS ion image of OLZ (m/z 256) (B). MS/MS ion image of N-desmethyl metabolite (m/z 256) (C). MS/MS ion image of 2-hydroxymethyl metabolite (m/z 272) (D). Bar, 1 cm.

identify all major metabolic pathways and enzymes responsible for the clearance of OLZ in rat. Conclusions. We demonstrate here the successful application of imaging MALDI mass spectrometry for the detection of proteins and small molecules from whole-body rat tissue sections. Advances in instrumentation and methodology, such as faster lasers and matrix/solvent combinations that can maximize sensitivity based on drug class or compound structure promise to streamline the whole-body IMS technology. Successful applications of the wholebody IMS technology promise to accelerate drug development by providing a technique that can be used to simultaneously detect individual drug and metabolite distributions, as well as detect protein changes as a result of drug administration. This broad molecular functionality provides an ex vivo view into the complex interactions between exogenous and endogenous components of an animal and will provide a better understanding of drug efficacy, mechanisms of action (toxicological and pharmacological), target (27) Kassahun, K.; Mattiuz, E.; Franklin, R.; Gillespie, T. Drug Metab. Dispos. 1998, 26, 848-855.

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validation, and off-target accumulation and metabolism together with the corresponding proteomic changes that can be linked to unwanted and deleterious side effects or toxicity in animal models. ACKNOWLEDGMENT The authors thank Dr. Michelle L. Reyzer for assistance with the small-molecule imaging technique. We also thank Dr. Ariel Deutch and Dr. Dennis Hallahan for providing the rat brain and glioma mouse samples, respectively. Custom image data preprocessing software was provided by Dr. Dale S. Cornett. Funding for this project was provided by the NIH (NIH/NIGMS GM58008), Department of Defense (W81XWH-05-1-0179), and Eli Lilly and Co. (VUMC 31425). S.K.-S. acknowledges support from the NCI Biochemical and Chemical Training for Cancer Research Grant (CA09582). Received for review April 27, 2006. Accepted June 29, 2006. AC060788P