Fine mapping the spatial distribution and concentration of unlabeled drugs within tissue micro-compartments using imaging mass spectrometry

Abstract

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 microm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.

Figure 1. The distribution of tiotropium on two central lung tissue sections from a rat after inhalation dosing.

The arrows indicate the route of drug delivery. (A) Photographic image of a rat lung tissue section analyzed by MALDI IMS with (B) the obtained distribution of tiotropium represented by the parent ion m/z 392.1. (C) A typical MS spectra obtained by MALDI MS analysis on rat lung tissue. (D) Photographic image of a rat lung tissue section analyzed by MALDI IMS/MS with (E) the obtained distribution of tiotropium represented by the daughter ion m/z 152.1. (F) MS/MS spectra of tiotropium. The distribution of tiotropium obtained in MS mode (B) correlate very well with the distribution pattern obtained by rastering over the tissue in MS/MS mode (E). The MALDI matrix CHCA was applied by an automatic sprayer device.

Figure 2. Estimation of tiotropium levels in lung tissue from rats dosed with the drug (a, b) by comparison to drug standard samples spotted on control tissue (c, d).

Control lung tissue was spotted with different amounts of tiotropium. In total, 20 fmol*, 40 fmol*, 80 fmol, 160 fmol, 320 fmol, 640 fmol, 1.3 pmol, 2.6 pmol, 5 pmol, or 10 pmol were placed on the control tissue (c, d). The spotted controls were coated with matrix using an automatic spraying device and analyzed on the same MALDI target as the tissue sections from the animals dosed with tiotropium. One pair of samples (spotted control and dosed tissue) was analyzed in MS mode (a) and one pair was analyzed in MS/MS mode (b). Average intensities from the tiotropium spiked regions were calculated and a standard curve was constructed from these values for the MS and MS/MS experiments, respectively (e, f). Three different regions, representing low, medium and high intensity areas, respectively, were selected on the dosed tissue sections (a, b) and the average intensities of these were matched to the standard curves. The color intensity scales on the controls (c, d) were set to reflect 0–100% of the maximal peak intensity of the sections from the dosed animals. Hence, this saturated the pixels on the controls with a higher intensity than the maxima of the dosed tissues. Calculated tiotropium levels from the high, medium, and low intensity regions on the dosed lung tissue sections (f). The levels obtained in MS mode correlate very well with the levels measured by MS/MS. (*only in MS/MS mode).

Figure 3. The spatial distribution of inhaled tiotropium in serially sectioned rat lung tissue.

Raw MS images (left column) showing MS localization of inhaled tiotropium (TTP) ion (m/z 392) by pixel location in serial sections of whole lung moving anatomically in lung volume from pleural to central to pleural. The arrow indicates approximate carinal entry point of the drug into the central conducting airways. Contour plots (right) of the relative concentration gradients of TPP found in the various lung segments showed a rapid and homogeneous transport of the drug from airways into the parenchyma within 15 minutes after exposure. The MALDI matrix CHCA was applied by an automatic sprayer device.

Figure 4. The distribution of endogenous molecules in lung tissue compartments by MS and identification by MS/MS.

Photograph of rat lung tissue (A). Panel B–E displays the distribution of four different endogenous molecules present in (A). These molecules were identified as different phosphatidylcholines by MS/MS directly on the tissue section. (F) MS/MS spectrum of PC(12∶0/0∶0)K⁺, (G) MS/MS spectrum of PC(18∶0/0∶0), (H) MS/MS spectrum of PC(16∶1/10∶0), (I) MS/MS spectrum of PC(32∶0)K⁺. The MALDI matrix CHCA was applied by an automatic sprayer device.

Figure 5. Localization of endogenous phosphatidylcholines (PC) ions in specific histological compartments.

We detected a variety of PC ion species in the global MALDI IMS scans of lung tissue. Shown here is the distribution profile of PC m/z 674.6 detected as a raw image by pixel location (A) and as contours of the ion intensity (color code: aqua/high intensity, blue/medium intensity) that have been re-plotted onto scanned images of the original sample after histological processing and staining (B). PC m/z 674.6 was closely associated with the location of small bronchioles throughout the lung section. Deconvolution of the H&E stained image (C) allowed clearer visualization of these bronchioles (indicated by arrows) supporting this observation. The MALDI matrix CHCA was applied by an automatic sprayer device.