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Imaging of Distribution of Topically Applied Drug Molecules in Mouse Skin by Combination of Time-of-Flight Secondary Ion Mass Spectrometry and Scanning Electron Microscopy Peter Sjövall,*,† Tanja M. Greve,‡ Susanne K. Clausen,‡ Kristian Moller,‡ Stefan Eirefelt,‡ Björn Johansson,§ and Kim T. Nielsen*,‡ †

SP Technical Research Institute of Sweden, Post Office Box 857, SE-50115 Borås, Sweden LEO Pharma A/S, Industriparken 55, 2750 Ballerup, Denmark § Karolinska Institutet, SE-171 76 Stockholm, Sweden ‡

S Supporting Information *

ABSTRACT: In the development of topical drugs intended for local effects in the skin, one of the major challenges is to achieve drug penetration through the external barrier of the skin, stratum corneum, and secure exposure to the viable skin layers. Mass spectrometric imaging offers an opportunity to study drug penetration in a variety of skin models by mapping the spatial distribution in different skin layers after topical application of the drug. In this study, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) and scanning electron microscopy (SEM) to image the distribution of three drug molecules in skin tissue cross sections of inflamed mouse ear. The three compounds, roflumilast, tofacitinib, and ruxolitinib, were topically administered to the mouse ears, which were subsequently cryosectioned and thawed for the analyses. The results reveal that the combination of TOF-SIMS and SEM was beneficial for interpretation of drug distribution. SEM identified the different skin layers, while spatial distributions of all three compounds could be visualized by TOF-SIMS, showing that the drug was primarily distributed into, or on the top of, the stratum corneum. Imaging of endogenous skin components like cholesterol, phospholipids, ceramides, and free fatty acids showed distributions in good agreement with the literature. One limitation of the TOF-SIMS method is sensitivity, typically allowing for analysis in the millimolar range rather than the pharmacologically relevant micromolar range. However, the data presented demonstrate the potential of the technique for studying the penetration of drugs with different physicochemical properties in skin.

T

of transdermal penetration of medicinal products. It is of particular importance to determine the penetration characteristics of the active pharmaceutical ingredient (API) of topically applied drugs in order to judge whether the API reaches the desired molecular target at pharmacologically relevant concentrations. Penetration studies using, for example, pig or human excised skin in Franz-type diffusion cells6 and bioanalysis of heat-separated skin layers7 are standard methods to address these types of questions. However, these results have relatively low spatial resolution, as they are limited to the thickness of the different skin layers. Furthermore, the heat separation method is limited to skin of certain thickness, such as human or pig skin, leaving out skin samples from rodents. One way to achieve higher spatial resolution is to use imaging techniques. Previous studies of skin tissue and percutaneous penetration by imaging techniques include confocal laser scanning microscopy (CLSM),8,9 infrared (IR) imaging,10−13 Raman imaging,14−16 and various imaging techniques based on mass

he development of drugs for topical administration is a challenging task, with penetration of the skin being one of the major issues.1 Acting as a barrier to water loss and chemical or microbial attack, mammalian skin is a complex organ that essentially consists of three layers: epidermis, dermis, and subcutaneous layer. The physical barrier of the skin is mainly located in the stratum corneum (SC), which is the outermost layer of the epidermis.2 The barrier function of the skin to prevent loss of water and electrolytes is provided by the hydrophobic extracellular lipid matrix, which surrounds the corneocytes in stratum corneum.3 The lipid composition of stratum corneum is very different in comparison to other tissues and most biological membranes. Thus, human stratum corneum contains very little phospholipid and predominantly consists of ceramides (50% w/w), cholesterol (25% w/w), and free fatty acids (15% w/w).4 Furthermore, the molecular structures of the ceramides are specific to the skin5 and the carbon chains of the free fatty acids are relatively long, with 22 and 24 carbon atoms being the most abundant chain lengths. In order to optimize treatment of dermatological diseases, it is important to have a thorough understanding of the composition of different skin compartments and mechanisms © 2014 American Chemical Society

Received: December 3, 2013 Accepted: February 25, 2014 Published: February 25, 2014 3443

dx.doi.org/10.1021/ac403924w | Anal. Chem. 2014, 86, 3443−3452

Analytical Chemistry

Article

spectrometry, such as matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI)17,18 and timeof-flight secondary ion mass spectrometry (TOF-SIMS).19 These studies have mainly focused on the characterization of skin constituents10,11,15,18 or the penetration of fluorescent molecules,8,9 nonactive ingredients (e.g., solvents12), surfactants,14 or penetration enhancers.13,16 Compared to other imaging techniques, mass spectrometry has the advantage of high chemical specificity without requiring the use of labels, such as isotopes or fluorescent tags, thereby ensuring the analysis of the parent molecule and not a combination of the parent molecule and metabolites containing the isotope or tag.20 Mass spectrometric imaging techniques like MALDI-MSI and TOF-SIMS have recently proven their value for imaging of drugs and metabolites in biological tissues.20−23 However, only a limited number of these studies have reported on imaging of drug distributions in skin.21,23−25 The primary advantages of TOF-SIMS compared to MALDI are that TOF-SIMS has a submicrometer spatial resolution and that no chemical preparation of the sample is required, whereas MALDI has a spatial resolution on the order of 25−50 μm and requires matrix deposition for extraction of the analytes, which further lower the resolution. The disadvantages of TOF-SIMS compared to MALDI-MSI include lower sensitivity and mass range and, for the majority of instruments, lack of tandem mass spectrometry (MS/MS) capability for providing absolute identification of the API or metabolites in the tissue.21,26,27 Regarding spatial resolution, it should be mentioned that dynamic SIMS has been reported to have the ability to image isotopic labeled compounds at subcellular levels in biological tissue.28−30 However, this technique has not yet been used for skin sample analysis. In the comparison of spatial resolution for different techniques, it should be noted that the technically determined resolution of the instrument may not be achieved due to alterations caused by sample preparation like drying/ freezing, thawing, vacuum, etc.29 In this study, we present imaging TOF-SIMS data on skin cross sections of mouse ears sensitized to the hapten oxazolone and, subsequently, treated topically with APIs. Mouse ear is routinely used for analyses of inflammation, such as in the chronic oxazolone model,31 which is a well-established hapteninduced skin inflammation model. Furthermore, the TOFSIMS data are combined with high-resolution SEM micrographs to determine the position of the different skin layers and the penetration and relative distribution of APIs within the skin. Distributions of various skin lipids like cholesterol, fatty acids, ceramides, and phospholipids are also visualized.

Chart 1. Structures of (A) Roflumilast, (B) Tofacitinib, and (C) Ruxolitinib

ical properties that make them suitable for skin penetration (e.g., MW < 500 g/mol and log D7.4 between 1 and 4; see Table 1)35−39 and are known to be able to penetrate the skin to some extent. Table 1. Selected Physical Chemistry Properties of the three Active Pharmaceutical Ingredients API

mol weight (g/mol)

ion (M + H)+ m/z

ion (M − H)− m/z

log D, pH 7.4

protein binding, rat (%)

roflumilast tofacitinib ruxolitinib

403.207 312.370 306.365

403.04 313.18 307.16

401.02 311.16 305.15

4 1.2 2.9

99.7 57.6 91.9

Mouse Treatment. Female BALB/cABomTac mice, 7 weeks old at arrival, were purchased from Taconic Europe. The experiment was conducted under a license approved by The Danish Animal Experiments Inspectorate. All mice were sensitized with oxazolone [4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (≥90% by HPLC), Sigma Aldrich] dissolved in acetone (15 mg/mL) by applying 100 μL of the solution to the shaved abdominal skin. One week later, the mice were challenged topically with 10 μL of oxazolone in acetone (25 mg/mL) on each side of the right ear. Thirty minutes later, each mouse was dosed with 40 μL of a saturated acetone solution on the inside of the right ear, with either roflumilast (0.13 mg), tofacitinib (0.4 mg), ruxolitinib (0.56 mg), or pure acetone (control), two mice in each group. Two hours after dosing with the API, the mice were terminated by cervical dislocation and the ear tissues were collected and snap-frozen in liquid nitrogen. The tissue was not tape-stripped and all skin layers were intact. The API concentration in the ear tissue was measured in one mouse ear from each API treatment group (see protocol under Bioanalysis of Ear Tissues) and the remaining mouse ear from each group was analyzed by TOFSIMS and SEM techniques. For ethical reasons, since this study is mainly focusing on the feasibility of using TOF-SIMS for imaging of drug distribution in mouse skin, we have decided to use as few mice as possible. Hence the number of mice used does not fulfill the requirement of minimum three mice to ensure sufficient repeatability of the results.40 Tissue Sections. Frozen cross sections of ear tissue (thickness 20 μm) were cut through the central part, perpendicular to the long axis, by use of a cryostat (Leica Jung CM 3000, Leica Microsystems, Wetzlar, Germany) at −20 °C. The sections were stably apposed onto the glass slides (Superfrost Plus; Gerhard Menzel GmbH, Braunschweig, Germany) by gentle warming of the tissue by pressure of a finger on the back side of the slide, allowing the tissue just to

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EXPERIMENTAL SECTION Selected Active Pharmaceutical Ingredients. The APIs studied in the present work are roflumilast, 3-(cyclopropylmethoxy)-N-(3,5-dichloropyridin-4-yl)-4-(difluoromethoxy)benzamide, 32 approved by the U.S. Food and Drug Administration (FDA) for treatment of chronic obstructive pulmonary diseases (COPD); tofacitinib, 3-[(3R,4R)-4-methyl3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1yl]-3-oxopropanenitrile,33 approved by FDA for treatment of rheumatoid arthritis and under development for treatment of psoriasis; and ruxolitinib, (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propanenitrile,34 approved by FDA for treatment of intermediate or high-risk myelofibrosis and also investigated for treatment of plaque psoriasis (see Chart 1). All of the studied compounds display physicochem3444



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Figure 1. Scanning electron micrographs of a mouse ear cross section as analyzed by TOF-SIMS. The area imaged in panel b is indicated by the yellow box in panel a. Histological features of the cross section are outlined in panel c, including stratum corneum (blue), epidermis (yellow), dermis (green), and cartilage (white).

each API was prepared with the pure crystal particles fixed on a metal block by use of double-sided tape. No major differences were observed in the spectra for these two types of samples with regard to the presence and secondary ion yield of major peaks, and the data presented below are from the acetonedissolved samples (due to a slight topography-induced reduction in mass resolution from the crystal sample). Scanning Electron Microscopy. After TOF-SIMS analysis, selected samples were sputter-coated with gold and analyzed by SEM on a Zeiss Supra 40VP FEG scanning electron microscope (2 kV, working distance ca. 7 mm, Everhardt-Thornley secondary electron detector). Combined TOF-SIMS and SEM images were prepared by manual alignment of images of the same analysis area by use of Adobe Photoshop Elements 9 (Adobe Systems Inc.), and overlay images were produced by use of the open-source software ImageJ. A perfect match between the SEM and TOFSIMS images was not possible, due to the topography of the tissue surface in combination with the fact that the incident angle of the primary ion beam in TOF-SIMS was 45° whereas the SEM images were recorded at normal incidence. Bioanalysis of Ear Tissues. The skin biopsies for compound concentration measurement were weighed and further processed for quantification of API in tissue. In short, 2 mL of digestion buffer was added to each sample. The samples were sonicated for 20 min at 55 °C. The homogenates were diluted with acetonitrile (1/9) and then centrifuged for 30 min at 4000 rpm at 10 °C, and 3 μL of supernatant was injected into the liquid chromatography (LC)/MS system. API concentrations (micromolar) in ear tissue were calculated from the total amount API in biopsy divided by biopsy volume, where 1 g of tissue was set to be equal to 1 mL.

thaw and then again freeze on top of the glass slide. The samples were stored at −80 °C until TOF-SIMS analysis. Time-of-Flight Secondary Ion Mass Spectrometry. Identification and localization of the APIs and endogenous lipids in skin samples were accomplished by analyzing the mouse ear tissue sections with TOF-SIMS. Each measurement was made over a suitable analysis area that could be selected by use of the integrated video camera in the instrument. The acquired data, comprising separate mass spectra from each pixel included in the measurement, were used to produce (i) accumulated mass spectra over the entire analysis area, (ii) ion images showing the spatial signal distribution from specific characteristic ions over the analysis area, and (iii) mass spectra from specific regions of interest selected from the ion images. Most data were acquired with the instrument optimized for high mass resolution in order to facilitate peak assignments and lipid identification in the mass spectra. TOF-SIMS analyses were carried out in a TOF-SIMS IV instrument (ION-TOF GmbH, Germany) using 25 keV Bi3+ primary ions and low-energy electron flooding for charge compensation. Data were acquired for positive and negative secondary ions, with the instrument optimized for high mass resolution (bunched mode) or for high image resolution (burst alignment mode). In bunched mode, the mass resolution was m/Δm ≈ 5000−7000 (where Δm is the full width at halfmaximum of a peak at mass m) at a beam diameter of 3−4 μm (pulsed Bi3+ current 0.1 pA, maximum primary ion dose density 1.3 × 1011 ions/cm2), while the mass resolution in the burst alignment mode was m/Δm ≈ 300 at a beam diameter of 300− 400 nm (pulsed Bi3+ current 0.05 pA, maximum primary ion dose density 4.9 × 1011 ions/cm2). Before TOF-SIMS analysis, the tissue samples were quickly thawed and dried by placing the sample glass slides on a warm (room-temperature) aluminum block, before immediate insertion into the TOF-SIMS instrument. All analyses were carried out with tissue samples at room temperature. At least three tissue sections were analyzed for each of the four treatment groups, and at least three images/spectra were acquired from different areas of each tissue section, adding up to at least nine separate measurements for each of the four treatment groups. Reference spectra of the pure API substances were acquired at high mass resolution (positive and negative ions, maximum primary ion dose density 3.2 × 1011 ions/cm2). Reference samples were prepared by depositing a 4 μL droplet of each API dissolved in acetone (ca. 3 mg/mL) on separate Si wafers and allowing them to dry. For comparison, a second sample of

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RESULTS AND DISCUSSION Active Pharmaceutical Ingredient Concentrations in Ear Tissues. Concentrations of the three APIs in the mouse ear samples were determined as 1, 8, and 5 mM for roflumilast, tofacitinib, and ruxolitinib, respectively. These numbers represent the average concentrations in the entire ears; that is, local concentrations are expected to be different in parts of the skin cross section due to inhomogeneous distribution of API, partly due to precipitation of API on the surface of the skin. Determination of Skin Morphology in Scanning Electron Microscopic Images. The morphological structure of the skin cross section samples was observed in SEM images obtained after TOF-SIMS analysis (see Figure 1). Key 3445



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Figure 2. Negative-ion TOF-SIMS images of control mouse ear cross section. Images display the distribution of the indicated molecular species across the ear cross section (see Table S1 in Supporting Information for specific ion peaks used to represent different species; CN− + CNO− represents protein fragments). Numbers below each image denote the maximum number of detected ions per pixel (MC) and the total number of detected ions in the entire image (TC). Field of view is 500 × 500 μm2. (Middle row, fourth panel) Overlay image shows PE (red), cholesterol sulfate (green), and C24:0 (blue); (bottom row, fourth panel) overlay image shows SM16:0 (red), PI36:1 (green), and C22:0 (blue).

histological features of the ear tissue are readily recognized, including the epidermis, dermis, and dermal cartilage (Figure 1c). Stratum corneum, which consists of multiple corneocyte layers, can also be easily identified in the SEM images. In addition, the SEM images reveal substantial cutting artifacts from the sample preparation, resulting in asymmetric and uneven tissue distribution on either side of the central cartilage and morphological distortions caused by the drying of the tissue. Identification and Localization of Lipids in Skin Cross Sections by Time-of-Flight Secondary Ion Mass Spectrometry. TOF-SIMS analysis revealed the presence of a variety of lipids in the mouse ear cross sections, including phospholipids, sphingolipids (sphingomyelin and ceramides), cholesterol, cholesterol sulfate, fatty acids, and triglycerides.41 See Tables S1 and S2 in Supporting Information for assignment of peaks used to represent the different compounds. Ion images showing the spatial distributions of different lipid components in a control mouse ear cross section (no API) are shown in Figures 2 and 3. The negative-ion images (Figure 2) demonstrate relatively homogeneous distributions of proteins, phosphatidylethanolamine (PE), C18:0 fatty acid, sphingomyelin (SM), and phosphatidylinositol (PI) 38:4, whereas the C24:0 fatty acid and cholesterol sulfate are strongly localized to the stratum corneum region of the cross section. C22:0 fatty acid is mainly localized to the stratum corneum but does also show a strong signal from several rounded structures, likely hair follicles within the dermal region. These structures are also

visible in the PI36:1 image and (to a lesser extent) in the cholesterol sulfate images as areas of increased intensity (bright areas). Interestingly, the two different PI images indicate complementary distributions, with PI36:1 localized mainly to the putative hair follicles and PI38:4 homogeneously distributed around these structures. The differences in lipid composition throughout the skin cross section are further demonstrated in the positive-ion images (Figure 3). Whereas phosphatidylcholine (PC) and potassium (K) are relatively homogeneously distributed (similarly to, for example, PE in Figure 2), ceramide is exclusively localized to stratum corneum. The almost identical distributions of the peaks assigned to ceramide fragments and to ceramide molecular ions confirm the assignments of these peaks to the same compound. The cholesterol image shows a relatively strong signal from the putative hair follicles and only low signal from the stratum corneum region. Diacylglycerol (DAG) displays a significant signal in the dermis region, but the main proportion derives from the glass support peripheral to the tissue section. We assign the DAG signal mainly to represent triacylglycerol (TAG), since DAG is a major fragment of TAG in TOF-SIMS42 and TAG is expected to be present at higher concentrations than DAG in the skin. The strong DAG signal outside the tissue section indicates that TAG was present on the skin surface and has deposited onto the glass surface during sample preparation. A similar effect was observed in the case of cholesterol and the APIs. In the case of cholesterol, it is known that it is mobile in vacuum at room temperature,43 3446



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Figure 3. Positive-ion TOF-SIMS images of a control mouse ear cross section from the same analysis area as in Figure 2. The imaged molecular species is indicated below each image, and the specific ions used are given in Table S2 in Supporting Information. MC denotes the maximum number of detected ions per pixel, and TC is the total number of detected ions in the entire image. Field of view is 500 × 500 μm2. (Middle row, third panel) Overlay image shows PC (red), cholesterol (green), and ceramide fragments (blue); (bottom row, third panel) overlay image shows K (red), DAG (green), and ceramide fragments (blue).

negative polarities. In general, distributions of the APIs were found to be strongly inhomogeneous along the mouse ear sections and only detectable within certain regions. Furthermore, all three APIs were almost exclusively localized to the stratum corneum and outside the skin. Roflumilast alone displayed a weak but distinctive signal in the interior skin layers, indicating penetration through the stratum corneum (see Figures S4−S6 in Supporting Information). Spatial distributions of roflumilast in the mouse ear cross section treated with this API are shown in Figure 4, together with distributions of selected lipids. Similar images for tofacitinib and ruxolitinibin can be seen in Figures S2 and S3 in Supporting Information. In the case of roflumilast (Figure 4), the drug is inhomogeneously distributed along the same linear region that also shows strong ceramide signal, indicating that roflumilast is mainly localized to the stratum corneum. PC is homogeneously distributed in the skin tissue, however, with a dark region at stratum corneum, in agreement with the expected low concentration of phospholipids in the stratum

suggesting that the cholesterol images may not accurately reflect the original cholesterol distributions in the tissue samples. The lipid distributions presented in Figures 2 and 3 are in good agreement with the reported lipid composition in the various skin layers, particularly stratum corneum,3,5 thus demonstrating the capability of TOF-SIMS to localize specific lipid components to different regions and structures in the skin cross section. In addition, spectra obtained separately from the stratum corneum and dermis further demonstrate the different lipid compositions in these regions of the cross section (see Figure S1 in Supporting Information). In conclusion, signals from the C24:0 and ceramide fragments can be used to identify and localize the stratum corneum layer. Localization of API Substances by TOF-SIMS and SEM. Spatial distributions of the three drug molecules in mouse ear tissue were analyzed with TOF-SIMS using the molecular ion peaks of each API (see Table 1). Reference spectra acquired from the pure substances showed that all APIs produce strong peaks corresponding to the molecular ions, in both positive and 3447



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Figure 4. Positive-ion images showing distribution of roflumilast in the cross section of a mouse ear treated with roflumilast dissolved in acetone. Field of view is 500 × 500 μm2. Overlay images show roflumilast (green) and ceramide (blue), together with PC (bottom row, third panel) or DAG (bottom row, fourth panel) in red.

Figure 5. Positive-ion overlay images showing distributions of (left) roflumilast, (center) tofacitinib, and (right) roflumilast in green, together with ceramide (blue) and PC (red). Field of view is 500 × 500 μm2.

corneum.44 The PC signal from the area outside stratum corneum is most likely caused by deposition of lipid onto the glass substrate during sample preparation. The DAG image indicates the presence of TAG in the stratum corneum region. However, the overlay image shows that the elevated DAG signal is primarily located outside the ceramide layer, suggesting an increased abundance of TAG on the surface of stratum corneum. Cholesterol shows only weak signal in association with stratum corneum but a distinct localization to rounded structures likely to represent hair follicles inside the dermal region. Tofacitinib displays a similar distribution as roflumilast, that is, an inhomogeneous distribution along the ceramidecontaining stratum corneum region (Figure 5). Also here, the DAG image indicates the presence of TAG on the corneal surface (Figure S2, Supporting Information). Cholesterol is primarily localized to the stratum corneum, but some signal outside and inside stratum corneum can also be observed. Finally, ruxolitinib is also mainly localized to the stratum corneum (Figure 5); however, a considerable part of the API is positioned outside the ceramide-rich region, indicating that ruxolitinib is present on the surface of, rather than within, the

stratum corneum. The cholesterol image shows strong signal from a slightly elongated structure in the dermal region, and the DAG image indicates extensive deposition of TAG onto the glass substrate (Figure S3, Supporting Information). In order to study the localization of APIs in more detail, cross-section samples of mouse ear tissue were analyzed by TOF-SIMS at optimized image resolution and subsequently by SEM for the same sample areas. Combined high-resolution TOF-SIMS and SEM images are presented in Figures 6 and 7 for roflumilast and ruxolitinib, respectively. The roflumilast TOF-SIMS image (Figure 6) shows a highly inhomogeneous patchlike distribution of the API within or on the surface of the stratum corneum. Here, the SEM image of the stratum corneum region is superimposed on the TOF-SIMS images of the C24:0 fatty acid, which previously was shown to colocalize with ceramide in stratum corneum (see Figures 2 and 3), and PO2−, representing the distribution of phospholipids. The overlay image clearly demonstrates that the C24:0 fatty acid distribution closely matches the structure in the SEM image that can be identified as stratum corneum. Note that although a perfect match between the SEM and TOF-SIMS images is not possible due to the topography of the tissue 3448



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Figure 6. High-resolution TOF-SIMS images and SEM images showing the distribution of roflumilast in stratum corneum of a roflumilast-treated mouse ear. The PO2− image represents the distribution of phospholipids, and the C24:0 image indicates the stratum corneum area. Field of view is 150 × 150 μm2. In the overlay images, roflumilast is shown in green, PO2− in red, and C24:0 in blue.

Figure 7. High-resolution TOF-SIMS images and SEM images showing the distribution of ruxolitinib in stratum corneum of a ruxolitinib-treated mouse ear. The ceramide fragment image indicates the stratum corneum area. Field of view is 150 × 150 μm2. In the overlay images, ruxolitinib is shown in green, PC fragments in red, and ceramide fragments in blue.

has deposited on the glass substrate, likely due to relatively limited penetration of ruxolitinib into the stratum corneum upon topical application. The intense ruxolitinib signal from the glass substrate suggests that the particular area presented in Figure 7 represents a local accumulation of this particular API.

surface (see Experimental Section), the alignment of the images has been optimized to display a maximum match at the stratum corneum. In the case of ruxolitinib (Figure 7), the API is partially localized to the stratum corneum, with an inhomogeneous patchlike distribution. However, most of the ruxolitinib 3449



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Table 2. Measured Secondary Ion Yields and Estimated Molar Concentrations of Roflumilast, Tofacitinib, and Ruxolitiniba ion

mass m/z

SI yield (SC tissue)

SI yield (ref)

yield ratio

est concn in SC (mM)

2.5 × 10−2 1.4 × 10−2

1.7 × 10−3

4.2

3.2 × 10−2 5.5 × 10−3

1.4 × 10−2

45

5.5 × 10−2 1.4 × 10−2

3.8 × 10−3

12

Roflumilast pos neg

(M + H)+ (M − H)−

403.03 401.01

pos neg

(M + H)+ (M − H)−

313.18 311.16

pos neg

(M + H)+ (M − H)−

307.18 305.16

4.3 × 10−5 Tofacitinib 4.4 × 10−4 Ruxolitinib 2.1 × 10−4

a

Measured secondary ion (SI) yields were measured in the stratum corneum (SC) region of the corresponding skin cross section and in pure reference samples (ref). Estimated molar concentrations in the stratum corneum region are based on measured yield ratios (without taking matrix effects into account and assuming densities of the pure substances of 1 kg/L).

“signal” and average API signal intensity in nontreated samples as “background”. On the basis of these signal-to-background ratios, and with the assumption of a minimum signal-tobackground ratio of 3 for detection, the lowest detectable concentrations for these APIs could be estimated as 1.5, 1.8, and 1.3 mM for roflumilast, toficitinib, and ruxolitinib, respectively. It should be emphasized that these are the local limits of detection, that is, the lowest detectable concentration in a certain area, while the overall concentration may be considerably smaller. However, the area with the required concentration (above LOD) needs to be large enough to provide a sufficiently high signal-to-noise ratio, which in the present case, with relatively high secondary ion yields of API molecular peaks, can be assumed to be in the range of 10−100 μm2. Because pharmacologically relevant API concentrations are in the micromolar range, these LODs would in many cases be insufficient for detecting penetration through the stratum corneum layer. Further technical development is therefore warranted to increase the detection sensitivity of TOF-SIMS for this type of analysis. Possible strategies for increasing the sensitivity may include, for example, use of deuterated APIs and/or extending the analysis beyond the static limit by using giant argon cluster ions to remove damaged material between high-resolution imaging analysis cycles.45 Alternatively, a threedimensional analysis starting from the skin surface and inward, instead of the cross-section approach used in the present study, with giant argon cluster ions for sputter removal of tissue material may provide a more sensitive strategy for detecting low API concentrations inside and below the stratum corneum layer.

Reliable quantification with TOF-SIMS is generally not possible without extensive use of calibration standards, mainly due to matrix effects. Matrix effects in TOF-SIMS is a collective term for the fact that the signal intensity for a given analyte concentration can vary considerably depending on the chemical environment. However, under the consideration that the results will be uncertain due to unknown matrix effects, an indication of the API concentrations in the mouse ear cross sections can be obtained by comparing the measured secondary ion yields (i.e., number of detected ions per incident primary ion) from the stratum corneum region of the tissue section with that of the pure API reference sample. Table 2 presents secondary ion yields of molecular ion peaks of the three APIs as measured in pure reference samples and (for positive ions) stratum corneum region of the corresponding skin section samples (see Figure S7 in Supporting Information for spectra). The results show that the secondary ion yield in the stratum corneum is 0.17 % of that in the reference sample for roflumilast, 1.3% for tofacitinib, and 0.38% for ruxolitinib. Without matrix effects taken into account, relative concentrations of the APIs in stratum corneum, in relation to concentration in pure substances, will be given by the secondary yield ratios, that is, 0.17%, 1.3%, and 0.38% for roflumilast, tofacitinib, and ruxolitinib, respectively. Furthermore, if it is assumed that the densities of the APIs are roughly 1 kg/L, the molar concentrations of the pure substances are 2.5, 3.2, and 3.3 M for roflumilast, tofacitinib, and ruxolitinib, respectively, which means that the molar concentrations in stratum corneum can be estimated as 4.2, 45, and 12 mM, respectively. When it is taken into account that the API is mainly localized to the stratum corneum and that stratum corneum (on the API-treated side) constitutes only ∼10−20% of the cross section on each side of the center cartilage, these numbers are in relatively good agreement with the API concentrations determined by bioanalytical analyses. However, it should be emphasized that these rough estimates need to be corrected for matrix effects in order to provide reliable values and that they can be considered correct only to the extent to which matrix effects can be neglected (which is normally not the case). The limit of detection (LOD) for the analysis of APIs in mouse ear tissue by TOF-SIMS can be estimated by considering the signal-to-background ratio of API peak intensities in the present data. For the spectra presented in Figure S7 in Supporting Information, the signal-to-background ratios were determined as 8, 74, and 28 for roflumilast, tofacitinib, and ruxolitinib, respectively, by use of signal intensity of the API molecular peak in the treated sample as

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CONCLUSIONS The spatial distributions of three topical drugs in histological skin sections were characterized by TOF-SIMS and SEM after topical application to a mouse ear. The combination of TOFSIMS and SEM turned out to be beneficial, since SEM easily and clearly was able to identify and localize the different skin layers, whereas TOF-SIMS was able to identify and localize the different APIs and skin components. API substances were found to be inhomogeneously distributed on the skin surface and within the stratum corneum. Only in the case of roflumilast were small amounts of API observed to have penetrated into the epidermis beyond the corneal layer. In addition, imaging of a number of endogenous lipids in the skin cross sections demonstrated spatial distributions well in accordance with lipid compositions in various skin compartments reported in the 3450



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literature. In particular, ceramide, cholesterol sulfate, and longchain fatty acids were found to localize strongly in the stratum corneum. Hence, the combination of TOF-SIMS and SEM differs from standard analytical techniques such as tapestripping or heat separation, where the stratum corneum, epidermis and dermis concentrations are determined but not localized to specific depths. Furthermore, inhomogeneous distribution issues are not addressed in these techniques. Visualization of the inhomogeneous distribution of API dosed in acetone on mouse ears has been highly valuable for understanding the large variation in pharmacokinetics/ pharmacodynamics relationships for our in vivo models. These new results have encouraged us to further investigate how to measure pharmacokinetics and pharmacodynamics in the same tissue sample. A simplistic, semiquantitative calculation estimated the API concentrations in the stratum corneum region of different mouse ear cross section samples to values in good agreement with results from global concentration measurements using LC/MS. Furthermore, the limit of detection (LOD) was estimated to be in the 1−2 mM range for analysis of these APIs in tissue by TOF-SIMS. It can be concluded that the TOFSIMS technique needs further development in order to be able to detect APIs at pharmacologically relevant concentrations in the micromolar range.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Additional text, two tables, and seven figures with assignment of peaks in measured TOF-SIMS spectra, positive-ion images showing distribution of tofacitinib and ruxolitinib, TOF-SIMS spectra, and positive-ion TOF-SIMS spectra of stratum corneum. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Authors

*E-mail [email protected]. *E-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Maja Wichmann for her expert technical assistance with the in vivo work. Financial support for this work was provided by VINNOVA Swedish Governmental Agency for Innovation Systems (P.S.).

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REFERENCES

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