Anal. Chem. 2010, 82, 7787–7794
Correlation of Skin Blanching and Percutaneous Absorption for Glucocorticoid Receptor Agonists by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging and Liquid Extraction Surface Analysis with Nanoelectrospray Ionization Mass Spectrometry Peter Marshall,*,† Valerie Toteu-Djomte,† Philippe Bareille,‡ Hayley Perry,§ Gillian Brown,| Mark Baumert,⊥ and Keith BiggadikeX BioMolecular Analysis, Computational and Structural Chemistry, Discovery Medicine, Respiratory CEDD, Histology, Computational and Structural Chemistry, Department of Medicinal Chemistry, Respiratory CEDD, GlaxoSmithKline, Gunnels Wood Road, Stevenage, U.K., Discovery Biometrics, GlaxoSmithKline, Greenford Road, Greenford, U.K., and Advion Biosciences Ltd., Queens Road, Hethersett, Norwich, U.K. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) and liquid extraction surface analysis (LESA) with nanoelectrospray ionization mass spectrometry (nESI-MS) have both been successfully employed to determine the degree of percutaneous absorption of three novel nonsteroid glucocorticoid receptor (GR) agonists in porcine ear sections. Historically, the ability of a glucocorticoid to elicit a skin blanching response when applied at low dose in ethanol solution to the forearms of healthy human volunteers has been a reliable predictor of their topical anti-inflammatory activity. While all three nonsteroidal GR agonists under investigation caused a skin blanching effect, the responses did not correlate with in vitro GR agonist potencies and different time courses were also observed for the skin blanching responses. MALDI MSI and LESA with nESIMS were used to investigate and understand these different responses. The findings of the investigation was that the depth of porcine skin penetration correlates to the degree of skin blanching obtained for the same three compounds in human volunteers. Glucocorticoids, when applied topically on human skin, elicit a pharmacological response resulting in skin blanching.1 While the precise mechanism of action is not fully understood, the glucocorticoids undergo percutaneous absorption leading to the vasoconstriction of any superficial vasculature resulting in the skin blanching response. This skin blanching assay or vasoconstriction
test in which a single dose of a drug formulation is applied to the ventral forearm of human subjects for 16 h, removed, and the resulting skin blanching response of the treated skin assessed 2 h later (see Figure 1), was first devised by McKenzie and Stoughton.2 An arbitrary scoring of 0, 1, 2, or 3 is assigned on the basis of the intensity of blanching, 0 indicating no effect and 3 indicating maximum blanching. The approach has been used to assess the potency of novel glucocorticoids and has been correlated with clinical anti-inflammatory efficacy.3,4 In order to see skin blanching, the compound needs to reach the blood vessels lying beneath the epidermis. The lack of a skin blanching response can be attributed to many factors, often the likely cause is the lack of uptake into the rate-limiting barrier to percutaneous absorption, namely, the epidermis. A technique was developed by Pershing et al.5 which simultaneously compared a skin blanching bioassay with drug content in human stratum corneum (outer epidermis). The quantification of the degree of drug uptake was achieved using a simple tapestripping procedure and liquid chromatography analysis; this method demonstrated that an increase in the amount of drug in the tape-stripped epidermis correlated with an increased skin blanching score. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI), pioneered by Caprioli et al. in 1997,6 was first used to image the distribution of pharmaceutical compounds in animal tissue in 2003,7 although the first demonstration of the use of MALDI to directly study pharmaceutical compounds in
* To whom correspondence should be addressed. E-mail: Peter.S.Marshall@ gsk.com. † BioMolecular Analysis, Computational and Structural Chemistry, GlaxoSmithKline. ‡ Discovery Medicine, Respiratory CEDD, GlaxoSmithKline. § Discovery Biometrics, GlaxoSmithKline. | Histology, Computational and Structural Chemistry, GlaxoSmithKline. ⊥ Advion Biosciences Ltd. X Department of Medicinal Chemistry, Respiratory CEDD, GlaxoSmithKline. (1) Shah, V. P.; Peek, C. C.; Skelly, J. P. Arch. Dermatol. 1989, 125, 1558– 1561.
(2) McKenzie, A. W.; Stoughton, R. B. Arch. Dermatol. 1962, 86, 608–610. (3) Gibson, J. R.; Kirsch, J. M.; Darley, C. R.; Harvey, S. G.; Burke, C. A.; Hanson, M. E. Br. J. Dermatol. 1984, 111 (27), 204–212. (4) Barry, B. W.; Woodford, R. J. Clin. Pharmacol. 1978, 3, 43–65. (5) Pershing, L. K.; Corlet, J. L.; Lambert, L. D.; Poncelet, C. E. J. Invest. Dermatol. 1994, 102 (5), 734–739. (6) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751–4760. (7) Reyzer, M. L.; Hsieh, Y.; Ng, K.; Korfmacher, W. A.; Caprioli, R. M. J. Mass Spectrom. 2003, 38, 1081–1092.
10.1021/ac1017524 2010 American Chemical Society Published on Web 08/17/2010
Analytical Chemistry, Vol. 82, No. 18, September 15, 2010
7787
Figure 1. A typical skin blanching anti-inflammatory response on the forearm of a healthy volunteer.
animal tissue had occurred a few years earlier by Troendle et al.8 Both groups produced thin sections of tissue and in preparation for the MALDI analysis coated or spotted the tissue with an organic matrix solution. Since these publications there have been several others on the use of MALDI-MS and imaging for determining the distribution of pharmaceutical compounds and their metabolites in animal tissue. These include the study by Wang et al.,9 which while not MALDI imaging demonstrated the localization and analysis of chlorisondamine and cocaine in rat brain following either intracranial or intraperitoneal injection. Also using rat tissue sections, Hsieh et al.10 reported the detection and imaging of the distribution of the antipsychotic drug, clozapine, in the brain, and Signor et al.11 reported the distribution of the drug, erlotinib, and its metabolites in rat liver and spleen sections. The first publication of the use of MALDI-MSI to study drug distribution in a whole body mouse section was made by Rohner et al.12 in 2005. This was followed in 2007 by a more comprehensive study by the same group,13 which described the distribution of the compound of interest and its metabolites and compared these findings with those obtained from whole-body autoradioluminography. Drug distribution and individual metabolite distributions within whole-body tissue sections were shown for the first time by Khatib-Shahidi et al.14 Most recently, Trim et al.15 describe the use of MALDI-ion mobility separation-MS for the imaging of the anticancer drug vinblastine in whole-body sections. A more (8) Troendle, F. T.; Reddick, C. D.; Yost, R. A. J. Am. Soc. Mass Spectrom. 1999, 10, 1315–1321. (9) Wang, H.-Y. J.; Jackson, S. N.; McEuen, J.; Woods, A. S. Anal. Chem. 2005, 77, 6682–6686. (10) Hsieh, Y.; Casale, R.; Fukuda, E.; Chen, J.; Knemeyer, I.; Wingate, J.; Morrison, R.; Korfmacher, W. Rapid. Commun. Mass Spectrom. 2006, 20, 965–972. (11) Signor, L.; Varesio, E.; Staack, R. F.; Starke, V.; Richter, W. F.; Hopfgartner, G. J. Mass Spectrom. 2007, 42, 900–909. (12) Rohner, T. C.; Staab, D.; Stoeckli, M. Mech. Ageing Dev. 2005, 126, 177– 185. (13) Stoeckli, M.; Staab, D.; Schweitzer, A. Int. J. Mass Spectrom. 2007, 260, 195–202. (14) Khatib-Shahidi, S.; Andersen, M.; Herman, J. L.; Gillespie, T. A.; Caprioli, R. M. Anal. Chem. 2006, 78, 6448–6456. (15) Trim, P. J.; Henson, C. M.; Avery, J. L.; McEwen, A.; Snel, M. F.; Claude, E.; Marshall, P. S.; West, A.; Princivalle, A. P.; Clench, M. R. Anal. Chem. 2008, 80, 8628–8634.
7788
Analytical Chemistry, Vol. 82, No. 18, September 15, 2010
comprehensive review of imaging mass spectrometry has been authored by McDonnell and Heeren.16 MALDI-MSI methodology for skin absorption experiments have been reported17,18 using porcine ear skin as a model of human skin.19 In these studies, an indirect (tissue blotting) procedure was utilized to demonstrate the absorption of the antifungal compound ketoconazole into porcine skin. In the work presented in this paper, MALDI-MSI was used to directly analyze the distributions of three GR agonists in porcine skin tissue in order to understand the different responses obtained from the skin blanching responses and in vitro GR agonist potency assays. We also utilized liquid extraction sampling to determine the depth of percutaneous absorption of the three compounds in porcine ear sections. Atmospheric pressure surface sampling/ ionization techniques for mass spectrometry has been thoroughly reviewed by Van Berkel et al.20 There are two basic forms of liquid extraction sampling probes available, the “liquid microjunction” surface sampling probe (LMJ-SSP) first described by Henion et al.21,22 and the “sealing” surface sampling probe (SSSP) introduced by Luftmann.23 Kertesz and Van Berkel24 first reported the use of the Advion Nanomate chip-based infusion nanoelectrospray ionization system as a fully automated liquid extraction-based surface sampling device. Here we report the use of a similar approach for the determination of the depth of percutaneous absorption of the three compounds in the porcine ear sections. EXPERIMENTAL SECTION Chemicals. HPLC grade ethanol and acetonitrile were purchased from Fisher Scientific (Fisher Scientific UK Ltd., Loughborough, U.K.). Ultrapure water was produced from an Elga water (16) McDonnell, L. A.; Heeren, R. M. A. Mass Spectrom. Rev. 2007, 26, 606– 643. (17) Bunch, J.; Clench, M.; Richards, D. S. Rapid Commun. Mass Spectrom. 2004, 18, 3051–3060. (18) Prideaux, B.; Atkinson, S. J.; Carolan, V. A.; Morton, J.; Clench, M. R. Int. J. Mass Spectrom. 2007, 260, 243–251. (19) Dick, I. P.; Scott, R. C. J. Pharm. Pharmacol. 1992, 44, 640–645. (20) Van Berkel, G. J.; Pasilis, S. P.; Ovchinnikova, O. J. Mass Spectrom. 2008, 43, 1161–1180. (21) Lee, E. D.; Muck, W.; Covey, T. R.; Henion, J. D. Biomed. Environ. Mass Spectrom. 1989, 18, 844–850. (22) Wachs, T.; Henion, J. D. Anal. Chem. 2001, 73, 632–638. (23) Luftmann, H. Anal. Bioanal. Chem. 2004, 378, 964–968. (24) Kertesz, V.; Van Berkel, G. J. J. Mass Spectrom. 2010, 45, 252–260.
following the addition of 0.35 volumes of assay buffer (2 mg/mL p-nitrophenylphosphate dissolved in 1 M diethanolamine, 0.28 M NaCl, 0.5 mM MgCl2). Dose response curves were constructed from which pIC50 values were estimated. Measurements of Chromatographic Hydrophobicity Indices (CHI) at pH 7.4. The CHI lipophilicity on the Luna C-18 stationary phase using a gradient of acetonitrile and 50 mM ammonium acetate at pH 7.4 was obtained as described by Valko et al.29,30 The gradient retention times of the compounds were converted to CHI values using the obtained slope and intercept values of the calibration plots (data not shown). The CHI values then were converted to the octanol/water partition coefficient scale (log D) using the formula: CHI log D ) (0.0525CHI) - 1.467
Figure 2. The skin blanching molecules and assay results.
purification system (Elga, High Wycombe, U.K.). Trifluoroacetic acid was obtained from Acros (Acros, New Jersey). Glucocorticoids. The arylpyrazole (M) (Figure 2) was prepared using the method described by Clackers et al.,25 the aryl pyrazolopyrimidine (E) and the aryl indazole (L) were prepared as described in the patent literature.26,27 Fluticasone propionate was purchased from Sigma-Aldrich (Sigma Aldrich, Poole, U.K.) In Vitro Assay: Glucocorticoid Mediated Transrepression of NFKB Activity. Human A549 lung epithelial cells were engineered to contain a secreted placental alkaline phosphatase gene under the control of the distal region of the NFκB dependent ELAM promoter as previously described by Ray et al.28 Compounds were solvated and diluted in DMSO and transferred directly into assay plates such that the final concentration of DMSO was 0.7%. Following the addition of cells (10K per well), 384-well plates were incubated for 1 h prior to the addition of 3.2 ng/mL human recombinant TNFR. Following continued incubation for 16 h, alkaline phosphatase activity was determined by measuring the change in optical density at 405 nM with time (25) Clackers, M.; Coe, D. M.; Demaine, D. A.; Hardy, G. W.; Humphreys, D.; Inglis, G. G.; Johnston, M. J.; Jones, H. T.; House, D; Loiseau, R.; Minick, D. J.; Skone, P. A.; Uings, I.; Mclay, I. M.; Macdonald, S. J. F. Bioorg. Med. Chem. Lett. 2007, 17 (17), 4737–4745. (26) Biggadike, K.; House, D.; Inglis, G. G.; Macdonald, S. J. F.; McLay, I. M.; Skone, P. A. PCT Int. Appl. WO 2007054294, 2007. (27) Eldred, C. D.; House, D.; Inglis, G. G.; Macdonald, S. J. F.; Skone, P. A. PCT Int. Appl. WO 2006108699, 2006. (28) Ray, K.; Farrow, S.; Daly, M.; Talbot, F.; Searle, N. Biochem. J. 1997, 28, 707–715.
Using this protocol, compound E was determined as being the most lipophilic and compound M being the least lipophilic compound (Figure 2). Skin Blanching Study. The skin blanching response of three novel nonsteroidal glucocorticoid receptor agonists (compound M, compound E, and compound L) was assessed in 24 healthy male volunteers aged 18-55 years, with pale skin and no known dermatological conditions, at doses of 40, 200, 1000, and 4000 ng. Fluticasone propionate (40 ng) was used as a positive control. Compounds were dosed as solutions in 95% ethanol/5% water. This was a single center, randomized, double-blind, placebo-controlled, three-way crossover study. Subjects received all treatments at least twice over the three treatment periods, in accordance with an individual randomization schedule for each subject. Preparation of Tissue Sections for MALDI MS Imaging and LESA with nESI-MS. All animal studies were subject to ethical approval, and all the animal use and handling in this work abides by UK Home Office Regulations and Guidelines. Pigs ears provided from in-house experiments were washed thoroughly with water and air-dried prior to use. Approximately 2 cm2 pieces were cut and placed in small Petri dishes for dosing with a mixture of the three compounds of interest. Our initial experiments (data not shown) were performed using individual doses of each compound rather than as a mixture and achieved very similar results, but to eliminate any variability introduced by the different ear sections used and to enable a direct comparison, it was considered more appropriate to dose the sections with a mixture of all three compounds. The dosing was achieved by placing a ring or barrier of silicon grease onto the upper surface of the piece of ear. A cocktail of compounds M, E, and L, each at a final concentration of 200 ng/µL, was prepared in 95% ethanol and 5% water. Into the center of the ring of silicon grease (∼5 mm diameter), 20 µL of compound in solution was added slowly and dropwise. The purpose of the silicon grease was to prevent “run-off” of the solution. Two time points were taken, t ) 0 and t ) 16 h. The t ) 16 h dosed porcine ear pieces were incubated at 37 °C in a humid environment. At each time point, the porcine ear pieces were frozen and sectioned vertical to the surface in a Leica CM3050 (29) Valko, K. In Proceedings of AAPS Meeting, Borchardt, R. T., Kerns, E. H., Lipinski, C. A., Thakker, D. R., Wang, B., Eds.; 2003; pp 127-182. (30) Valko, K.; Du, C. M.; Bevan, C.; Reynolds, D. P.; Abraham, M. H. Curr. Med. Chem. 2001, 8 (9), 1137–1146.
Analytical Chemistry, Vol. 82, No. 18, September 15, 2010
7789
Figure 3. Skin blanching results in human volunteers.
cryostat (Leica Microsystems, Wetzlar, Germany) to produce 12 µm thick sections through the tissue to enable the depth of compound penetration to be determined. The sections were transferred and thaw-mounted onto indium tin oxide coated glass slides (Bruker Daltonics, Bremen, Germany). The glass mounted tissue sections were optically scanned in a Nikon Super CoolScan 5000ED scanner fitted with a MA-21 slidemount adapter (Nikon Corporation, Tokyo, Japan) to produce a digital image for future reference. For MALDI MS imaging analysis, the tissue sections were coated with matrix, consisting of 5 mL of 7 mg/mL sinapinic acid in 50:50 (v/v) acetonitrile/ water containing 0.1% trifluoroacetic acid, using a Bruker ImagePrep matrix application device (Bruker Daltonics, Bremen, Germany). MALDI MS Imaging. The analysis was performed on an Ultraflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics) equipped with smart beam laser technology.31 The digital image of the sections was imported into the FlexImaging software (Bruker Daltonics), and through the software the instrument was taught the dimensions of the sections. The instrument was operated in the LIFT MS/MS mode configuration with a potential of 19.14 kV. The spatial resolution of the images was 200 µm, with typical laser spot diameters of 75-80 µm. The MS/MS data were acquired in the m/z range between 20 and 640. The acquisition method was calibrated using a mixture of standards (Bruker Daltonics) before the start of each experiment. MS/MS data were automatically acquired using the FlexImaging software from a total of 500 spectra acquired at each spot position using the proprietary random walk mode in 50-shot increments at a laser frequency of 100 Hz. It is the use of this acquisition mode and the large spatial resolution that contributes to the production of the appearance of the analyte spreading off the tissue. The limits of detection studies have previously been performed (data not shown) for all three compounds spotted onto porcine ear sections at the same compound concentrations, and the results obtained showed a very similar response for all three compounds. 7790
Analytical Chemistry, Vol. 82, No. 18, September 15, 2010
Liquid Extraction Surface Analysis with Nanoelectrospray Ionization Mass Spectrometry. The samples were analyzed using an Advion TriVersa NanoMate system (Advion BioSciences, Inc. Ithaca, NY) coupled to a Waters QTOF Premier mass spectrometer (Waters Corporation, Manchester, U.K.). A nanoelectrospray voltage of 1.49 kV and gas pressure of 0.4 psi were applied to all the experiments. The operation of the NanoMate platform for surface sampling has been described previously.24 In our experiments, customized robotic arm step movements (in multiples of seven steps, each single step equating to a distance of 90 µm) were performed and set up in the sequence panel of the ChipSoftManager software that controlled the NanoMate. The solvent extraction volumes (microliters) were as follows: aspirated volume ) 1.0 and dispensed volume was 0.5. The QTOF mass spectrometer was run in positive electrospray mode, V mode reflectron. The MS/MS data were acquired in the range 100-1500, using the selected precursor ions, M ) 513, E ) 554, and L ) 538, with a collision energy of 45 eV and a scan speed of 3.0 s. Histology of Porcine Ear Sections. Sections of 5 µm thickness were cut vertical to the surface using a Leica CM3050 cryostat (Leica Microsystems, Wetzlar, Germany). The sections were stained with hematoxylin and eosin (H&E) following standard histological procedures.32 Images were taken on a Hamamatsu nanozoomer (Hamamatsu Photonics, Hamamatsu City, Japan), scanned at 20× magnification. RESULTS AND DISCUSSION The data obtained for the skin blanching studies in human volunteers for the three compounds at different dosage levels is summarized in graphical form in Figure 3. The three novel glucocorticoid receptor agonists showed a dose-dependent increase in the derived weighted mean blanching response. For compound M at 200, 1000, and 4000 ng, compound E at 1000 (31) Holle, A.; Haase, A.; Kayser, M.; Hohndorf, J. J. Mass Spectrom. 2006, 41, 705–716. (32) Lillie, R. D.; Pizzolato, P.; Donaldson, P. T. Histochemistry 1976, 49, 23– 35.
Figure 4. (a) MS/MS spectrum of compound M, (b) MS/MS spectrum of compound E, and (c) MS/MS spectrum of compound L.
and 4000 ng, compound L at 1000 and 4000 ng, and fluticasone propionate at 40 ng, the derived weighted mean was significantly greater than the placebo. From these results, it was evident that compound M (depicted as orange colored curves) demonstrated the greatest skin blanching response, peaking at 2-3 h after dressing removal, and was deemed to be the most potent compound with respect to blanching response. Interestingly, while compounds M and E showed a similar blanching profile over time to fluticasone propionate, compound L showed a delayed and prolonged response. However, these data were not consistent with the in vitro NFκB assay data (Figure 2) that showed the compounds to have a similar range of potencies with compound L showing the highest activity in the assay. The skin blanching profiles were also not consistent with the lipophilicity measurements (CHI values) (Figure 2) that showed that compound E was the most lipophilic and compound M the least. This lack of correlation with the in vitro potency and also
lipophilicity (Figure 2) prompted us to investigate MALDI MS imaging and LESA with nESI-MS to compare the skin penetration of these three compounds. The positive ion MALDI and nanoelectrospray mass spectra of compounds M, E, and L spotted on tissue both showed that, as expected, the protonated molecule of compound M has an m/z of 513, compound E has an m/z of 554, and compound L has an m/z of 538 (data not shown). The MS/MS spectra obtained for the three compounds by both MALDI (Figure 4a-c) and nanoelectrospray MS indicated the highest intensity fragment ions were m/z 204 for compound M, m/z 275 for compound E, and m/z 272 for compound L. For the MALDI MS imaging experiments, ion density maps (Figure 5a-c), corresponding to the plot of an ion intensity versus (x,y) position, were obtained for compounds M, E, and L by monitoring the selected fragment ion signal across all regions of Analytical Chemistry, Vol. 82, No. 18, September 15, 2010
7791
Figure 5. The ion density maps obtained: (a) The distribution of the m/z 204 fragment ion, derived from the m/z 513 precursor ion, depicted as orange pixels (representing the distribution of compound M) overlaid onto the optical image of the porcine ear section, Time zero (left) and 16 h incubation (right). (b) The distribution of the m/z 275 fragment ion, derived from the m/z 554 precursor ion, depicted as sky blue pixels (representing the distribution of compound E) overlaid onto the optical image of the porcine ear section, Time zero (left) and 16 h incubation (right). (c) The distribution of the m/z 272 fragment ion, derived from the m/z 538 precursor ion, depicted as pink pixels (representing the distribution of compound L) overlaid onto the optical image of the porcine ear section, Time zero (left) and 16 h incubation (right). (d) Image of a 5 µm thick cross-section of a porcine ear section stained by conventional H&E. Inset shows the full section for orientation.
the respective porcine ear sections. The ion density images obtained were overlaid onto the corresponding optical image in order to display the localization of the compounds within the 7792
Analytical Chemistry, Vol. 82, No. 18, September 15, 2010
tissue. Figure 5a illustrates the distribution of the m/z 204 fragment ion for compound M overlaid onto the optical image of the porcine ear section; the orange spots represent the presence
Figure 6. (a) Analysis of dosed porcine ears by LESA. (b) The depth of penetration of the three compounds measured by LESA.
of compound M in the tissue section. Clearly, from Figure 5a, significant levels and depth of skin penetration of compound M has occurred. Compound M has penetrated through the epidermis layer into the dermis layer. Similarly, Figure 5b depicts the distribution of the m/z 275 ion for compound E overlaid onto the optical image of the porcine section. The distribution of compound E is represented by the light blue spots, and again there has been some penetration of the compound through the epidermis into the dermis layer. The distribution of the m/z 272 ion for compound L is represented in pink in Figure 5c and again is overlaid onto the optical image. Figure 5c shows that only limited penetration has occurred for compound L compared to compounds M and E. Both compounds M and E penetrated the epidermis layer into the dermis layer, while compound L failed to penetrate through
the epidermis layer to the dermis layer. None of the compounds were detected in the connective tissue. A histological image of a 5 µm thick cross-section of a porcine ear section stained by conventional H&E is shown in Figure 5d to highlight the relevant skin layers and their thickness within the ear sections. A similar distribution profile for the percutaneous absorption of compounds M, E, and L was also observed from the LESA with nESI-MS analysis (Figure 6). Although, the LESA with nESIMS analysis lacked the spatial resolution achieved using the MALDI MS imaging approach, it served as an additional confirmation step. With the use of the stepping function described earlier, and working from left to right (i.e., undosed to dosed side), several discrete positions (each 0.63 mm apart) across the porcine ear section were analyzed by the liquid microjunction surface sampling Analytical Chemistry, Vol. 82, No. 18, September 15, 2010
7793
Figure 7. Pictorial representation of the correlation between the depth of porcine skin penetration and the degree of skin blanching obtained for the same three compounds in human volunteers.
probe, see Figure 6a. For each discrete position, three discrete MS/MS experiments were performed and the signal intensity for the major fragment ion for each of the test compounds was determined, see Figure 6b. From Figure 6, compound M gives the greatest degree of percutaneous absorption and very limited penetration occurred with compound L. Figure 7 displays the skin blanching data from the human volunteers given a 4000 ng dose of each of the three compounds, and the MALDI mass spectrometry images showing the depth of porcine skin penetration of the three compounds following a 4000 ng administration. Compound M shows the greatest skin blanching response and from the MALDI image the greatest skin penetration of the three compounds. Similarly, compound E exhibited a greater skin blanching response than compound L, and this was also mirrored in the MALDI images of their distribution in the porcine ear sections. The delayed onset of skin blanching observed for compound L (10-12 h) followed by a slower decrease, with still a significant blanching response at 24 h, is probably explained by slower skin penetration as shown in the MALDI image (Figure 7). These results suggest that differences in skin penetration may be a key factor in the different responses seen in the skin blanching study. CONCLUSIONS The novel nonsteroidal glucocorticoids M, E, and L showed a dose-dependent increase in the derived weighted mean blanching response when applied to the forearms of healthy volunteers. Skin blanching is a well-known property of traditional glucocorticosteroids when applied to skin. MALDI imaging mass spectrometry
7794
Analytical Chemistry, Vol. 82, No. 18, September 15, 2010
and LESA with nESI-MS has been used to determine the degree of skin penetration in porcine ear sections for the three novel nonsteroid glucocorticoid receptor (GR) agonists, and the findings correlated well to the extent of skin blanching obtained for the same three compounds in human volunteers. The study demonstrates the potential of MALDI imaging mass spectrometry and LESA with nESI-MS for supporting clinical studies and for determining the degree of skin penetration for topically applied drugs. ACKNOWLEDGMENT The authors would like to thank Dr. Daniel Bradford (Study Director), staff, and volunteers at Northwick Park Hospital, U.K., who took part in the skin blanching studies. Also, to Klara Valko (GSK) for obtaining the CHI values, Margaret Clackers (GSK) for the in vitro assay results, to Dr. Peter Francis (GSK) for technical support on the Waters QToF mass spectrometer, and Dr. Klaus Schneider for his support to the MALDI mass spectrometry imaging work. Dr. Soeren-Oliver Deininger and Dr. Michael Becker of Bruker Daltonics, Bremen, Germany, and David Keightley of Bruker UK are thanked for their invaluable technical assistance. Equipment upgrade and software supplied to us by Advion Biosciences Ltd. and the ongoing support for our work given by Mark Allen of that company is valued and acknowledged. Received for review July 2, 2010. Accepted August 6, 2010. AC1017524
