Visualization of Time-Dependent Distribution of Rifampicin in Rat Brain Using MALDI MSI and Quantitative LCMS/MS

Abstract

Rifampicin (RIF) is a major component for short-course chemotherapy against tuberculosis, since it is active against rapidly metabolizing as well as dormant bacteria. According to the Lipinski rules, RIF should not enter the blood–brain barrier. Visualization of tissue drug distribution is of major importance in pharmacological studies; thus, far imaging of RIF in the brain has been limited to positron emission tomography. We propose using matrix-assisted laser desorption/ionization mass spectrometry imaging techniques as a suitable alternative for the visualization and localization of drug tissue distribution. Using the liquid chromatography mass spectrometric (LCMS) technique, we were able to quantify the concentrations of RIF in the uninfected rat brain; we paired this with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to show the time-dependent manner in which RIF is able to enter the brain. Our results show that even at the minute concentrations measured with LCMS/MS we were able visualize the drug and show its exact distribution in the rat brain. Other available methods require nuclear labeling and the detection of gamma rays produced by labeled compounds to visualize the compound and its localization; MALDI MSI is a more recently developed technique, which can provide detailed information on drug distribution in tissues when compared to other imaging techniques. This study shows that without any requirement for complex preprocessing we are able to produce images with a relatively improved resolution and localization than those acquired using more complex imaging methods, showing MALDI MSI to be an invaluable tool in drug distribution studies.

Introduction

Short-course chemotherapy (SCC) is currently the only effective treatment available for pulmonary tuberculosis (TB) as well as extra pulmonary forms such as TB meningitis (TBM). TBM occurs more frequently in HIV-infected children¹ than adults, and lower CD4⁺ counts are associated with severe infection and poor outcomes.^2,3 Rifampicin (RIF) is the most important drug in SCC as it is the only anti-TB drug capable of effectively killing rapidly metabolizing as well as dormant bacteria.⁴ It inhibits RNA polymerase of the bacteria, which is the enzyme responsible for DNA transcription by forming a stable drug–enzyme complex at 37°C with a binding constant of 10⁻⁹ M.⁵ However, oral administration of RIF leads to low bioavailability due to the high first-pass effect and autoinduction.⁶ In vitro studies have even shown that orally administered RIF does not effectively reach TB granulomas in lung alveoli.⁷

RIF is a drug that one would typically not expect to enter the brain due to its size. Drugs that are able to diffuse through the blood–brain barrier are normally <400 Da and have less than eight hydrogen bonds.⁸ RIF is still able to penetrate the blood–brain barrier, although it only obeys one of the conditions of Lipinski's rule of five due to its lipophilicity.⁹ Several studies have shown the presence of RIF in the brain with five reporting on the quantification by measurements of this drug in the cerebrospinal fluid (CSF).^10

–14 After oral dosing, concentrations of RIF in the CSF, determined by fluorometric and microbiologic procedures, were reported to be only slightly above its minimum inhibitory concentration (MIC) for Mycobacterium tuberculosis (MTB).¹⁴ In contrast to oral dosing, human studies on intravenous administration of standard doses of RIF reported a more favorable penetration into the CSF and cerebral extracellular space.^11,13 However, CSF determination of drug concentration is not an accurate measure of the amount of the compound that actually enters the brain, as CSF is only a fluid produced by the choroid plexus and not an anatomical feature of the brain.¹⁵ It is, therefore, necessary to study the amount of drug directly in the brain as a true measure of its neuroprotective potential. Mindermann et al. reported the first direct measurement of RIF concentrations in various compartments of the human brain and this was achieved through a microdialysis probe inserted into the cortex and found that concentrations of this drug in a brain tumor is not a true reflection of its concentration in a normal brain.^11,12 Qosa et al. recently reported the first quantification of RIF directly from the rat brain homogenate, while investigating the clearance of amyloid-β, and found that RIF crossed the blood–brain barrier and had a protective effect against Alzheimer's disease. The concentration of RIF was comparable to patients receiving therapeutic doses.¹⁶

Visualizing the exact localization of drugs in tissue is an essential tool in drug distribution studies, as this will allow researchers to investigate whether or not the drug reaches its desired site of action and be a determining factor in its neuroprotective efficacy. This is especially important for drugs such as RIF, since it has a high degree of plasma protein binding (∼80%), leaving only about 20% to diffuse into CSF.¹⁷ The most common methods for tissue distribution employ radiolabeled drugs for imaging through positron emission tomography (PET), magnetic resonance imaging, and X-ray computerized tomography.¹⁸ Liu et al. reported the use of PET-quantified radiolabeled RIF in the brain; this study, which compared the permeability of the three frontline TB chemotherapeutics, concluded that they entered through the blood–brain barrier of baboons with RIF showing the least penetration.¹⁹ The study concluded that the predicted concentration of RIF in the infected baboons was three to four times above the MTB MIC, which confirms the suitability of the drug for treating central nervous system (CNS) TB infections. MALDI MSI is an attractive research tool, which serves as an alternative to the use of radiolabeled compounds that are less widely available.²⁰ This method can precisely depict the anatomical distribution of a compound with a lateral resolution of a few tens of microns and is currently used for the development of drugs to treat CNS conditions such as Parkinson's.²¹

This present study is aimed to investigate the penetration and distribution of RIF in the brain of healthy rats using MALDI MSI upon intraperitoneal application of the drug.

Materials and Methods

Animals

All animal experiments were undertaken with the approval of the Institutional Animal Ethics Committee of the University of KwaZulu-Natal (UKZN), Durban, with the ethics number 067/14/Animal. Healthy male Sprague Dawley rats with an average weight of 95±10 g were obtained from the Biomedical Resource Unit (BRU), University of KwaZulu-Natal, Durban, South Africa. Animals were housed at the BRU in polycarbonate cages in air-conditioned rooms (50%–70% humidity and between 21°C and 24°C) with a 12-h light/12-h dark cycle. Animals were allowed ad libitum access to drinking water and standard rat chow.

Drug Administration and Tissue Sampling

Animals (95±10 g) were dosed with RIF (Sigma Aldrich) of 10 mg/kg, respectively, prepared in 10% DMSO and 90% ultrapure water; this dose was calculated based on the average body weight. RIF was administered intraperitoneally (i.p.) to six groups of animals (n=3). Animals were anesthetized with halothane before blood was collected by cardiac puncture at 0, 20, 40, 60, 120, and 240 min post-RIF administration in heparinized tubes. Blood samples were immediately centrifuged at 12,000×g for 10 min and aliquots of plasma (1.5 mL) and immediately stored at −80°C before analysis.

Dissection and Storage

The rats were euthanized with halothane, termination was confirmed by the absence of breathing, and dissection was performed immediately. The lungs and brain were surgically removed and snap-frozen in liquid nitrogen vapor before storage at −80°C.

Sample Preparation for MALDI-MSI

Serial sections (10 μm thick) were sectioned from each biopsy and freeze–thaw mounted onto an indium titanium oxide (ITO)-coated glass slide (Bruker) with a cryostat (Leica Microsystems CM1100) using an optimum cutting temperature embedding medium and kept at −80°C till needed for analysis. Glass slides were removed from the deep freeze and immediately transferred to a desiccator. Following 30-min desiccation time, the slides were scanned using a flatbed scanner (HP LaserJet 3055). Matrix preparation was done by preparing 7 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) (Bruker) matrix solution in 50% acetonitrile (ACN) containing 0.2% trifluoroacetic acid (TFA). The matrix solution was sonicated for 5 min and centrifuged at 9,390×g for 10 min before being transferred to the ImagePrep (Bruker Daltonics). This is a spraying device that automatically deposits the matrix solution onto the tissue in a consistent manner. In short, aerosol is generated by the vibrating spray generator producing tiny droplets (average droplet size of ≈20 μm) under controlled conditions.

MALDI-MSI Analysis

Standard solutions of RIF were analyzed using the matrix-assisted laser desorption/ionization time of flight (MALDI TOF) MS, operated both at reflectron and LIFT modes, respectively, with the aid of the Autoflex III MALDI TOF/TOF 1 KHz smartbeam laser (Bruker Daltonics) and FlexControl acquisition software run in the positive ion mode. The instrument was calibrated using standard peptides bradykinin, angiotensin II, angiotensin I, and substance P spotted with the CHCA matrix on the ground steel target (Bruker Daltonics) for m/z 700–1,400. Each spectrum was acquired from 200 laser shots. Tissue sections were imaged with spatial resolution from 25 to 100 μm. The MALDI images were displayed using the software FlexImaging 3.0 (Bruker Daltonics). Spotting different concentrations (0.5, 1, 5, 10, 100, and 500 ng/mL, respectively) of RIF with the matrix onto a brain section of an untreated animal was used to evaluate background signals interfering with the analyte. The chosen matrix (CHCA) would not promote the detection of lipids.²² The drug was mixed (1:2) with a matrix solution, and the different concentrations of RIF were spotted on the tissue in a total volume of 3 μL. The spots were analyzed in the reflectron mode by collecting 200 shots per spot. The tandem mass spectroscopy (MS/MS) experiments were performed using a LIFT method optimized for the drug by specific tuning of the timing of the LIFT cell and of the precursor ion selector. The LIFT method was calibrated with peptide calibration standards as mentioned above. The same method was used to perform MS/MS directly on the RIF-matrix spotted tissue section, and spectra were compared to the MS/MS spectra generated from the drug standard only. For the initial analysis of the manually deposited spots, spectra consisting of 1,000 laser shots were acquired in bundles of 5×200 shots and data were collected in the range between m/z 700 and 1,000. A product ion scan of the sodium adduct precursor ions (m/z 845) revealed a clear fragmentation pattern with the transition m/z 845→791 dominating the spectra with a laser power of 60%. This transition represents the loss of CH₃OH and sodium ion, respectively. An increase in the laser power led to production of other product ions, but with less sensitivity. The software Flex Imaging 3.0 was used to set up the acquisition of the imaging experiments. The imaging MS/MS experiments were performed by collecting spectra with a raster width of 200 μm in the same m/z range as above. The spectra were baseline subtracted (Convex hull) and smoothed (Savitzski golay) in the processing software during acquisition. In the LIFT mode, all spectra were normalized against the total ion count to reduce potential suppression by, that is, matrix hot spots. We define the total ion current as the sum of all intensities in the mass range analyzed. For this analysis, both fragment and parent spectra were acquired from each spot and 500 laser shots were summed up in a random walk pattern from each position. The RIF parent ion of m/z 845 served as a qualifier ion, while m/z 791, a major fragment of RIF, was used to visualize the brain sections in triplicate and m/z 773 further confirmed the fragment to be due to the loss of water from the fragment.

Quantification in Biological Samples

The methods used for the extraction and detection of RIF followed those of Srivastava et al. (2012) with minor modifications.²³ Drug-free tissues stored at −80°C to be used as matrix for calibration were homogenized in water at a ratio of 3 mL of water for every 1 g of tissue using a manual tissue homogenizer. Tissue concentrations of RIF were measured using LCMS/MS after protein precipitation with a 100% methanol ratio of 9:1 for plasma and lung, while a ratio of 2:1 for the brain tissue. This was followed by refrigeration for 10 min and centrifugation at 15,870×g for 15 min; supernatants were filtered through the Supelco hybrid solid phase extraction 30 mg phospholipid cartridges. Matrices were spiked with standard drug concentrations to obtain quality control samples and a calibration curve for quantification. Recovery of the analyte and the internal standard were determined on the quality control samples of the three matrices.

The samples were separated using an Ultimate 3000 LC system (Dionex) coupled to an amaZon speed ion trap (IT) equipped with an electrospray ionization (ESI) ion source from Bruker Daltonics. The IT-MS was operated in the ultrascan mode. The high-performance liquid chromatography (HPLC) system was a Dionex UltiMate 3000, consisting of a binary pump, connected to an autosampler equipped with a 20-μL injection loop and the temperature of the column was maintained at 25°C. The flow rate was 0.4 mL/min. The analytical column was a 150×3.0 mm (internal diameter) stainless steel column YMC Triart C₁₈, 3 μm from YMC Europe Gmbh. The mobile phases were Milli-Q water (0.1% v/v formic acid) and ACN (0.1% v/v formic acid). The gradient profile was initially from 35% to 65% ACN in 7 min, then in the next 3 min increased to 90% ACN (held 2 min), after which time the mobile phase was returned to the initial conditions (35% ACN) in 3 min.

The MS was operated in the ESI-positive mode, and auto MSn spectra collection was applied. The MS settings were as follows: capillary outlet 4,500 V and end plate voltage 500 V. The nebulizer was set to 1.5 bar, and desolvation gas temperature was 210°C at a flow-rate of 8 L/min. Positive ion mode MS spectra were obtained within a mass range of 100–1,200 m/z. The smart parameter setting was set to 823.5 m/z. The ion charge control was set to 200,000 with a maximum accumulation time of 200 ms. Collision-induced ionization was performed using helium as collision gas.

The multiple reaction monitoring settings were as follows: isolation width for RIF was set at 4.0 and percentage amplification was set at 25%, while for the internal standard (rifapentine), the isolation width was set at 1.0 and the percentage amplification was also set at 25%. Collision energy enhanced the fragmentation mode ramping from 80%–120%, fragmentation time 20 ms, isolation width 4 m/z. For MS/MS precursor selection, the most intense ions were isolated. The transitions were chosen to be m/z 823.5→791.5 for RIF, m/z 877.5→845.5 for the internal standard, and this represents the loss of CH₃OH in each case.

All results were stored and analyzed with Data Analysis 4.0 SP 5 (Bruker Daltonics) and Quant analysis.

Results and Discussion

MALDI MSI is not a quantitative method of evaluation, and with only a single report on the direct measurement of the concentration of RIF in the rat brain,¹² we thought it would be necessary to create an effective and verified quantitative method for the determination of this drug in this tissue. We developed an LCMS/MS method to quantify the amount of RIF in the plasma, lung, and brain that had a relatively short run time of 15 min. The retention times for RIF and its internal standard (rifapentine) were 8.5 and 9.6 min, respectively, as shown in Figure 1. The limit of detection was evaluated as the lowest concentration that gave a signal-to-noise ratio of three for the analyte, while the lower limit of quantification (LLOQ) was determined in six replicates based on the criterion that the analyte response at LLOQ is at least five times baseline noise and it is within the acceptable limit of accuracy and precision. The method demonstrated good sensitivity with a limit of detection for the analyte in plasma, lung, and brain homogenates of 0.5 ng/mL. We chose 50 ng/mL as the limit of quantification for the plasma and 1 ng/mL for lung and brain homogenates, respectively. The calibration curves were linear with at least 0.995 in each case, a linear range of 50–1,200 ng/mL for plasma samples and 1–150 ng/mL for lung and brain homogenates were obtained in each case. The extraction method for the analyte and internal standard from the matrices proved to be efficient with mean recoveries of at least 87% for each compound. Plasma, lung, and brain concentration–time curves of RIF are shown in Figure 2. The relevant pharmacokinetic parameters, including T_max, C_max, T_1/2, and area under the curve (AUC), are listed in Table 1. In plasma, RIF reached a C_max of 7,952.90 ng/mL after a T_max of 60 min, which is in accordance with lower values of several studies.^24

–27 In the lung, the C_max was 3,110.38 ng/mL with a similar T_max of 60 min. Curves for both the plasma and lung concentrations showed similar trends, where there were steady initial increases in drug concentrations followed by a gradual decrease until trough concentrations were reached at 240 min. Compared to the lung and brain concentrations, the plasma had greater amounts of RIF with AUCs that were significantly higher. The observed results for plasma concentrations of RIF are in agreement with that of data reported.^16,28 The AUC and the C_max values in the lung homogenates were about half of that in the plasma. The T_max (120 min) observed in the brain homogenate was twice that observed in both the plasma and lung matrices, respectively. Poor brain permeability was also observed for RIF after its intraperitoneal administration of 20 mg/kg to healthy mice, which was twice the dose administered in our research, but yet with relatively close plasma and brain concentrations.¹⁶ This result is not strange as it has been earlier reported¹² that only about 0.3%–1% of the RIF plasma concentration penetrates into the brain. The lower availability of RIF in the brain is also associated with the rapid penetration of this drug into other tissues as observed in the mean lung concentrations in our study. It readily diffuses into most organs, tissues, bones, and body fluids, including exudates into TB-infected lung cavities.²⁹ About 20% of serum concentration has been observed in the saliva and other high-concentration therapeutic concentrations appeared in the lachrymal glands, tears, and urine.³⁰

Fig. 1.

Extracted ion chromatogram and mass spectra of rifampicin (RIF) (823.5→791.5) and rifapentine (877.6→845.6), respectively.

Fig. 2.

The concentration–time curve for plasma, lung, and brain, respectively, after a 10 mg/kg intraperitoneal dose of RIF. This represents the data obtained from plasma, lung, and brain homogenates of a single animal used for the study. Data acquired with LC/MS. Color images available online at www-liebertpub-com.web.bisu.edu.cn/adt

Table 1.

Quantitative Analysis (LCMS/MS) of RIF in Plasma, Lung, and Brain Homogenates

Parameters	Plasma (ng/mL)	Lung (ng/mL)	Brain (ng/mL)
20 min	5,926.90±0.61	1,701.00±2.12	11.27±0.40
40 min	6,665.80±0.98	2,969.89±9.51	60.49±1.14
60 min	7,952.90±7.80	3,110.38±9.66	73.52±1.21
120 min	4,925.50±5.21	2,293.06±5.56	89.27±0.91
240 min	2,413.10±0.64	900.97±1.18	30.24±0.6
T_max (min)	60	60	120
C_max (ng/mL)	7,952.90±7.80	3,110.38±9.66	89.27±0.91
AUC_0–4 (ng·min/mL)	1,158,042	748,269.6	14,224.70
T_½ (min)	165	172	208

Presented data originate from one animal only and n=3 corresponds to three injections to the high-performance liquid chromatography, values are expressed as mean±SD.

RIF, rifampicin.

The lateral ventricles house the choroid plexus, which is responsible for producing the CSF found in the brain. In our study, we have found the lateral ventricles to be devoid of RIF (Fig. 3), thus implying that RIF may not be entering or leaving the brain through the CSF; this has not been previously demonstrated. There is ∼40 μL of CSF in the rat brain and the entire volume is turned over every 2 h³¹; this could produce a washout effect and be responsible for the high concentrations of RIF observed at the brain stem. A previous report that demonstrated the use of PET imaging on baboon brain,¹⁹ which observed the penetration of RIF through the blood–brain barrier of diseased and healthy animals, however, did not comment of the availability of RIF in the brain CSF. The axial and coronal views (Fig. 3) support this theory with the observed presence of RIF concentration at the brain stem in the time-dependent images. The high density of the drug in the cortex and striatum in our findings is in keeping with those found in mice.³² The detected RIF in the isocortex region of the brain supports the point that administration of RIF reduces brain injury after both permanent and transient focal cerebral ischemia in mice.^32
–34 The mechanisms expediting such effects of RIF may involve fortification against oxidative stress and or the activation of glucocorticoid receptors in areas of the hippocampal formation.^32
–34

Fig. 3.

Quantitative analysis of brain tissue homogenates together with the images of the mass spectrometry imaging (MSI). The tissue sections represent several time points as well as axial (a) and coronal (b) sections shown at 100 μm resolution. Using the images at 40 min as an example, we have shown the cortex (indicated by the red arrow) and the lateral ventricles (indicated by the red oval) in the axial section. In the coronal section, the cortex is also shown (indicated by the red arrow). The results show the time-dependent penetration of RIF into brain tissue, as shown in the shape of the curve, but also as demonstrated by the steady development of the bright pink to white color in the tissue sections representing the presence of the drug as visualized by m/z 791±0.01. Color images available online at www-liebertpub-com.web.bisu.edu.cn/adt

In conclusion, the novelty of this study is twofold. First, we were able to show that MALDI imaging is a sensitive and powerful tool for evaluating drug distribution, even at low concentrations, in the brain as in the case of RIF when compared to other anti-TB drugs such as isoniazid or pyrazinamide, which have better penetration into brain tissue¹⁹; hence the exploration of the sensitivity of MALDI imaging. Second, we were able to demonstrate the time-dependent manner in which RIF penetrates into the brain. These data, despite being collected in an uninfected model, are highly significant as it allows us to determine the minimum doses reaching the brain in the early course of an infection and its distribution. This will enable us to evaluate the drug's location and whether the drug is reaching the therapeutic doses in the brain required to treat TB-associated meningeal infections.³³ The MALDI-TOF/TOF method developed for this study had a relatively reasonable run time. The main advantage of this LIFT method is that both parent and fragments of the analyte can be determined consecutively, providing adequate information of adducts formed in the process. The transition of RIF from its sodium adduct m/z 845→791 made its mapping successful on the brain sections.

Footnotes

Acknowledgments

The authors would like to thank the National Research Foundation, SA; Aspen Pharmacare, SA; and the University of KwaZulu-Natal, Durban, SA, for having funded this project.

Disclosure Statement

No competing financial interests exist.

Abbreviations Used

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