Application of imaging MALDI-MS to the determination of hazardous compounds in skin.

The overall aim of this work described in this thesis was to apply MALDI-MS and imaging MALDI-MS (MALDI-MSI) to the analysis of compounds of interest in occupational hygiene monitoring.Isocyanates are highly reactive compounds with a wide variety of industrial uses. MALDI-MS and MS/MS were used to investigate diisocyanate stability and reactivity. A monoisocyanate intermediate product was observed from the hydrolysis of both an aromatic and also an aliphatic diisocyanate. The stability of this product was assessed over a 14 day experimental period. Ethanol was used to derivatise hexamethylene diisocyanate (HDI) and the resulting urethane was observed to be stable over 14 days. This derivatisation method was incorporated into a surface swab technique for sampling of HDI from work surfaces.Industrial diisocyanates have been reported to penetrate through skin and to be excreted as diamine metabolites in urine. A LC-MS method for the determination of free toluene diamine (TDA) monomer formed by biotransformation of toluene diisocyanate (TDI) when applied to HaCaT cells was developed. In the two experiments performed, TDA was only observed at low levels after spiking with high concentrations of TDI. This appeared to be due to most of the isocyanate becoming conjugated to proteins within the cell and thus not being extracted during the extraction procedure.A novel ethanol-saturated cellulose membrane blotting technique was developed for the extraction and ethanol-derivatisation of HDI from the surface of skin. However, not all of the HDI present on the membrane reacted with the ethanol. Increasing the amount of ethanol on the membrane did increase the amount of derivatised HDI monomer observed although this occurred at the expense of spatial information. The technique was also applied for analysis of the insecticide chlorpyrifos, for both skin surface sampling and permeation studies. From the images obtained, chlorpyrifos was observed to readily penetrate through the stratum corneum and reach a depth of 1.7mm. The highest amount was located in the dermis after the 1 hour exposure time.The dermal absorption of HDI was monitored after 1 hour exposure by mapping HDI monoamine penetration through the skin via indirect blotting and novel direct skin analysis methods. Similar profiles were observed from both methods. Penetration depths of 2.3 and 2.6 mm were observed for the direct skin and indirect blotting methods respectively. The highest level of HDI monoamine was located in the dermis

Application of imaging MALDI-MS to the determination of hazardous compounds in skin.

The overall aim of this work described in this thesis was to apply MALDI-MS and imaging MALDI-MS (MALDI-MSI) to the analysis of compounds of interest in occupational hygiene monitoring.Isocyanates are highly reactive compounds with a wide variety of industrial uses. MALDI-MS and MS/MS were used to investigate diisocyanate stability and reactivity. A monoisocyanate intermediate product was observed from the hydrolysis of both an aromatic and also an aliphatic diisocyanate. The stability of this product was assessed over a 14 day experimental period. Ethanol was used to derivatise hexamethylene diisocyanate (HDI) and the resulting urethane was observed to be stable over 14 days. This derivatisation method was incorporated into a surface swab technique for sampling of HDI from work surfaces.Industrial diisocyanates have been reported to penetrate through skin and to be excreted as diamine metabolites in urine. A LC-MS method for the determination of free toluene diamine (TDA) monomer formed by biotransformation of toluene diisocyanate (TDI) when applied to HaCaT cells was developed. In the two experiments performed, TDA was only observed at low levels after spiking with high concentrations of TDI. This appeared to be due to most of the isocyanate becoming conjugated to proteins within the cell and thus not being extracted during the extraction procedure.A novel ethanol-saturated cellulose membrane blotting technique was developed for the extraction and ethanol-derivatisation of HDI from the surface of skin. However, not all of the HDI present on the membrane reacted with the ethanol. Increasing the amount of ethanol on the membrane did increase the amount of derivatised HDI monomer observed although this occurred at the expense of spatial information. The technique was also applied for analysis of the insecticide chlorpyrifos, for both skin surface sampling and permeation studies. From the images obtained, chlorpyrifos was observed to readily penetrate through the stratum corneum and reach a depth of 1.7mm. The highest amount was located in the dermis after the 1 hour exposure time.The dermal absorption of HDI was monitored after 1 hour exposure by mapping HDI monoamine penetration through the skin via indirect blotting and novel direct skin analysis methods. Similar profiles were observed from both methods. Penetration depths of 2.3 and 2.6 mm were observed for the direct skin and indirect blotting methods respectively. The highest level of HDI monoamine was located in the dermis

High-resolution mapping of fluoroquinolones in TB rabbit lesions reveals specific distribution in immune cell types.

Understanding the distribution patterns of antibiotics at the site of infection is paramount to selecting adequate drug regimens and developing new antibiotics. Tuberculosis (TB) lung lesions are made of various immune cell types, some of which harbor persistent forms of the pathogen, Mycobacterium tuberculosis. By combining high resolution MALDI MSI with histology staining and quantitative image analysis in rabbits with active TB, we have mapped the distribution of a fluoroquinolone at high resolution, and identified the immune-pathological factors driving its heterogeneous penetration within TB lesions, in relation to where bacteria reside. We find that macrophage content, distance from lesion border and extent of necrosis drive the uneven fluoroquinolone penetration. Preferential uptake in macrophages and foamy macrophages, where persistent bacilli reside, compared to other immune cells present in TB granulomas, was recapitulated in vitro using primary human cells. A nonlinear modeling approach was developed to help predict the observed drug behavior in TB lesions. This work constitutes a methodological advance for the co-localization of drugs and infectious agents at high spatial resolution in diseased tissues, which can be applied to other diseases with complex immunopathology

Mass spectrometry imaging of levofloxacin distribution in TB-infected pulmonary lesions by MALDI-MSI and continuous liquid microjunction surface sampling

A multi-modal mass spectrometry imaging (MSI) and profiling approach has been applied to assess the partitioning of the anti-TB fluoroquinolone levofloxacin into pulmonary lesions. Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and a commercial liquid microjunction surface sampling technology (LMJ-SSP), or flowprobe, have been used to both spatially profile and image drug distributions in lung tissue sections from TB-infected rabbits following oral administration of a single human-equivalent dose., Levofloxacin levels were highest at 6 h post-dose in normal lung, cellular granuloma, and necrotic caseum compartments. The drug accumulated in the cellular granuloma regions with lower amounts partitioning into central caseous compartments. Flowprobe imaging at 630 μm (limited by the probe tip diameter) enabled visualization of drug distribution into lesion compartments, including limited differentiation of relative drug abundance in cellular versus caseous regions of the lesions., MALDI-MSI analysis at 75 μm provided more detailed drug distribution, which clearly accumulated in the cellular region immediately surrounding the central caseum core. Imaging and profiling data acquired by flowprobe and MALDI-MSI were validated by quantitative LC/MS/MS analysis of lung and granuloma homogenates taken from the same animals., The results of the investigation show flowprobe imaging and sampling as a rapid and sensitive alternative to MALDI-MSI for profiling drug distributions into tissues when spatial resolution of data below the threshold of the probe diameter is not required

Fluoroquinolone Efficacy against Tuberculosis Is Driven by Penetration into Lesions and Activity against Resident Bacterial Populations.

Fluoroquinolones represent the pillar of multidrug-resistant tuberculosis (MDR-TB) treatment, with moxifloxacin, levofloxacin, or gatifloxacin being prescribed to MDR-TB patients. Recently, several clinical trials of "universal" drug regimens, aiming to treat drug-susceptible and drug-resistant TB, have included a fluoroquinolone. In the absence of clinical data comparing their side-by-side efficacies in controlled MDR-TB trials, a pharmacological rationale is needed to guide the selection of the most efficacious fluoroquinolone. The present studies were designed to test the hypothesis that fluoroquinolone concentrations (pharmacokinetics) and activity (pharmacodynamics) at the site of infection are better predictors of efficacy than the plasma concentrations and potency measured in standard growth inhibition assays and are better suited to determinations of whether one of the fluoroquinolones outperforms the others in rabbits with active TB. We first measured the penetration of these fluoroquinolones in lung lesion compartments, and their potency against bacterial populations that reside in each compartment, to compute lesion-centric pharmacokinetic-pharmacodynamic (PK/PD) parameters. PK modeling methods were used to quantify drug penetration from plasma to tissues at human-equivalent doses. On the basis of these metrics, moxifloxacin emerged with a clear advantage, whereas plasma-based PK/PD favored levofloxacin (the ranges of the plasma AUC/MIC ratio [i.e., the area under the concentration-time curve over 24 h in the steady state divided by the MIC] are 46 to 86 for moxifloxacin and 74 to 258 for levofloxacin). A comparative efficacy trial in the rabbit model of active TB demonstrated the superiority of moxifloxacin in reducing bacterial burden at the lesion level and in sterilizing cellular and necrotic lesions. Collectively, these results show that PK/PD data obtained at the site of infection represent an adequate predictor of drug efficacy against TB and constitute the baseline required to explore synergies, antagonism, and drug-drug interactions in fluoroquinolone-containing regimens

Tumor Drug Penetration Measurements Could Be the Neglected Piece of the Personalized Cancer Treatment Puzzle

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150514/1/cpt1211.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150514/2/cpt1211_am.pd