Tracking Drugs and Lipids: Quantitative Mass Spectrometry Imaging of Liposomal Doxorubicin Delivery and Bilayer Fate in Three-Dimensional Tumor Models.

Targeted therapy to the tumor would greatly advance precision medicine. Many drug delivery vehicles have emerged, but liposomes are cited as the most successful to date. Recent efforts to develop liposomal drug delivery systems focus on drug distribution in tissues and ignore liposomal fate. In this study, we developed a novel method to elucidate both drug and liposomal bilayer distribution in a three-dimensional cell culture model using quantitative matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI qMSI) alongside fluorescence microscopy. Imaging liposomal distribution in a cell culture model is challenging, as lipids forming the bilayer are endogenous to the model system. To resolve this issue, we functionalized the bilayer by chemically cross-linking a fluorescent tag to the alkyne-containing lipid hexynoyl phosphoethanolamine (HPE). We synthesized liposomes incorporating the tagged HPE lipid and encapsulated within them doxorubicin, yielding a theranostic liposome capable of both drug delivery and monitoring liposomal uptake. We employed an "in-tissue" MALDI qMSI approach to generate a calibration curve with R2 = 0.9687, allowing for quantification of doxorubicin within spheroid sections at multiple time points. After 72 h of treatment with the theranostic liposomes, full doxorubicin penetration was observed. The metabolites doxorubicinone and 7-deoxydoxorubicinone were also detected after 48 h. Modification of the bilayer allowed for fluorescence microscopy tracking of liposomes, while MALDI MSI simultaneously permitted the imaging of drugs and metabolites. While we demonstrated the utility of our method with doxorubicin, this system could be applied to examine the uptake, release, and metabolism of many other liposome-encapsulated drugs.

Electroblotting through Enzymatic Membranes to Enhance Molecular Tissue Imaging.

MALDI-TOF mass spectrometry imaging (MSI) is a powerful tool for studying biomolecule localization in tissue. Protein distributions in tissue provide important histological information; however, large proteins exhibit a high limit of detection in MALDI-MS when compared to their corresponding smaller proteolytic peptides. As a result, several techniques have emerged to digest proteins into more detectable peptides for imaging. Digestion is typically accomplished through trypsin deposition on the tissue, but this technique increases the complexity of the tissue microenvironment, which can limit the number of detectable species. This proof-of-principle study explores tryptic tissue digestion during electroblotting through a trypsin-containing membrane. This approach actively extracts and enzymatically digests proteins from mouse brain tissue sections while simultaneously reducing the complexity of the tissue microenvironment (compared to trypsin deposition on the surface) to obtain an increased number of detectable peptide fragments. The method does not greatly compromise spatial location or require expensive devices to uniformly deposit trypsin on tissue. Using electrodigestion through membranes, we detected and tentatively identified several tryptic peptides that were not observed after on-tissue digestion. Moreover, the use of pepsin rather than trypsin in digestion membranes allows extraction and digestion at low pH to detect peptides from a complementary subset of tissue proteins. Future studies will aim to further improve the method, including changing the substrate membrane to increase spatial resolution and the number of detected peptides.

In situ metabolite and lipid analysis of GluN2D-/- and wild-type mice after ischemic stroke using MALDI MSI.

The N-methyl-D-aspartate (NMDA) receptor is a crucial mediator of pathological glutamate-driven excitotoxicity and subsequent neuronal death in acute ischemic stroke. Although the roles of the NMDAR's composite GluN2A-C subunits have been investigated in this phenomenon, the relative importance of the GluN2D subunit has yet to be evaluated. Herein, GluN2D-/- mice were studied in a model of ischemic stroke using MALDI FT-ICR mass spectrometry imaging to investigate the role of the GluN2D subunit of the NMDA receptor in brain ischemia. GluN2D-/- mice underwent middle cerebral artery occlusion (MCAO) and brain tissue was subsequently harvested, frozen, and cryosectioned. Tissue sections were analyzed via MALDI FT-ICR mass spectrometry imaging. MALDI analyses revealed increases in several calcium-related species, namely vitamin D metabolites, LysoPC, and several PS species, in wild-type mouse brain tissue when compared to wild type. In addition, GluN2D-/- mice also displayed an increase in PC, as well as a decrease in DG, suggesting reduced free fatty acid release from brain ischemia. These trends indicate that GluN2D-/- mice show enhanced rates of neurorecovery and neuroprotection from ischemic strokes compared to wild-type mice. The cause of neuroprotection may be the result of an increase in PGP in knockout mice, contributing to greater cardiolipin synthesis and decreased sensitivity to apoptotic signals. Graphical abstract.

Magnetic bead-based peptide extraction methodology for tissue imaging.

MALDI-TOF imaging mass spectrometry (IMS) is a common technique used for analyzing tissue samples, as it allows the user to detect multiple different analytes simultaneously. However, the detection and analysis of these analytes may sometimes be hampered due to the presence of contaminants in the tissue microenvironment, which leads to ion suppression. This challenge necessitates the development of an active extraction technique to selectively isolate analytes of interest without compromising their spatial localization within a tissue sample. This study proposes a magnetic bead-based active extraction approach to selectively sequester peptides of interest from tissue samples. The technique utilizes a heterobifunctional cross-linker to covalently bind peptides with free primary amine groups to functionalized magnetic beads. The cross-linked peptides can then be collected using a transfer magnet and imaged using MALDI-TOF IMS. We have established that this technique not only successfully isolates peptides both in-solution and on a solid surface, but also extracts peptides from a tissue section without significantly compromising their spatial localization. This novel method provides the means to detect a unique subset of peptides from tissue sections when compared to unextracted tryptically digested tissue, all while minimizing the presence of contaminants and maintaining spatial localization.

Burden of Friedreich's Ataxia to the Patients and Healthcare Systems in the United States and Canada.

OBJECTIVE: The study intended to substantiate healthcare resource utilization, costs, and funding patterns of US and Canadian Friedreich's Ataxia (FRDA) populations, to assess compliance with treatment guidance and to identify areas where novel healthcare measures or improved access to existing care may improve patients' functional and social capabilities and reduce the financial impact on the healthcare systems. METHODS: Healthcare resource utilization and costs were collected in a cross-sectional study in the US (N = 197) and Canada (N = 43) and analyzed across severity of disease categories. Descriptive statistics, correlation analysis, and hypothesis testing were applied. RESULTS: In the US, healthcare costs of FRDA patients were higher than those of "adults with two and more chronic conditions." Significantly higher costs were incurred in advanced stages of the disease, with paid homecare being the main driver. This pattern was also observed in Canada. Compliance with the recommended annual neurological and cardiological follow-up was high, but was low for the recommended regular speech therapy. In the US public and private funding ratios were similar for the FRDA and the general populations. In Canada the private funding ratio for FRDA was higher than average. CONCLUSION: The variety of healthcare measures addressing the broad range of symptoms of FRDA, and the increasing use of paid home care as disease progresses made total US healthcare costs of FRDA exceed the costs of US adults with two and more chronic conditions. Therefore, measures delaying disease progression will allow patients to maintain their independence longer and may reduce costs to the healthcare system. Novel measures to address dysarthria and to ensure access to them should be further investigated. The higher than average private funding ratio in Canada was due to the relatively high cost of the pharmacological treatment of FRDA.

Comparison of the efficacy and safety of arformoterol 15 microg twice daily and arformoterol 30 microg once daily in COPD: a single-dose, multicenter, randomized, modified-blind, two-way crossover study.

OBJECTIVE: The aim of this study was to compare the efficacy and safety of nebulized arformoterol 15 microg/2 mL twice daily (ARF15 BID) and 30 microg/4 mL once daily (ARF30 QD) in subjects with moderate to severe chronic obstructive pulmonary disease (COPD). METHODS: In this single-dose, multicenter, randomized, modified-blind, 2-way crossover study, subjects aged > or =45 years with moderate to severe COPD, a forced expiratory volume in 1 second (FEV(1)) > or =0.7 L, and < or =65% predicted FEV1, and a FEV(1):forced vital capacity ratio < or =70% were randomly assigned to receive single-day treatment with ARF15 BID or ARF30 QD in random order, separated by a 5 +/- 2-day washout period. The primary efficacy end point was time-normalized AUC of FEV(1) from baseline (hour 0) to 24 hours (FEV(1)AUC(0-24)). Secondary efficacy end points were time-normalized AUC of FEV(1) from baseline to 12 hours (FEV(1)AUC(0-12)) and from 12 to 24 hours (FEV(1)AUC(12-24)), and FEV(1) at 24 hours after administration of the morning dose (trough FEV(1)). Equivalence of the 2 therapies was assessed by comparing the 90% CI value for the difference of the least squares mean (LSM) to a study-specific predefined equivalence range for change in FEV(1)AUC(0-24) of -0.07 to 0.07 L. RESULTS: A total of 33 subjects were enrolled (20 men, 13 women; mean [SD] age, 64.5 [8.8] years; 15 subjects received ARF15 BID first; 18 received ARF30 QD first). ARF15 BID and ARF30 QD were associated with similar improvements from baseline in (FEV(1)AUC(0-24), LSM 0.15 and 0.16 L, respectively; Delta, 0.01 L; 90% CI, -0.02 to 0.04) and trough FEV(1) (LSM, 0.15 and 0.12 L, respectively; Delta, -0.03 L; 90% CI, -0.09 to 0.03). FEV(1)AUC(0-12) was improved more with ARF30 QD than ARF15 BID (Delta, 0.06 L; 90% CI, 0.04 to 0.09), and FEV(1)AUC(12-24) was improved more with ARF15 BID than ARF30 Qd (Delta, -0.04 L; 90% CI, -0.08 to 0.01). The 90% CI for FEV(1)AUC(0-24) for the treatment difference between ARF15 BID and ARF30 QD was within the prespecified range of -0.07 to 0.07 L, indicating that both treatments resulted in equivalent FEV(1)AUC(0-24) values. CONCLUSIONS: In these subjects with moderate to severe COPD, single-day administrations of ARF15 BID or ARF30 QD were associated with FEV(1) responses over a period of 24 hours that were considered equivalent per the protocol definition employed in the present study. FEV1 improvement over 12 hours was greater for ARF30 QD after the morning dose and for ARF15 BID after the evening dose. ClinicalTrials.gov Identifier: NCT00571428.

Comparison of levalbuterol and racemic albuterol in hospitalized patients with acute asthma or COPD: a 2-week, multicenter, randomized, open-label study.

BACKGROUND: The National Heart, Lung, and Blood Institute guideline recommends that dosing racemic albuterol be administered every 1 to 4 hours for treating patients with asthma or chronic obstructive pulmonary disease (COPD) in the hospital. Previously published preliminary and retrospective studies suggested that levalbuterol can be administered every 8 hours for the treatment of bronchoconstriction in hospitalized patients. However, it is unclear how the different dosing regimens affect the total number of nebulizations (scheduled plus as-needed treatments) and the costs of treatment of bronchoconstriction in a hospital setting. Moreover, it is not clear how the different dosing regimens affect symptom outcomes and health status in hospitalized patients with asthma or COPD. OBJECTIVE: The aim of this study was to evaluate these issues in hospitalized patients with acute asthma or COPD. METHODS: In this prospective, multicenter, randomized, open-label study, hospitalized patients aged > or = 18 years were randomly assigned to receive 14-day treatment with levalbuterol 1.25 mg q6-8h or racemic albuterol 2.5 mg q1-4h, administered per routine hospital practice at each institution. The primary efficacy end point was total number of nebulizations during hospitalization. Pulmonary function, symptom evaluation (subject general well-being score [SGWB], disease symptom assessment [DSA], and beta-mediated adverse effect scores), hospital costs (excluding medication costs) and hospital length of stay (LOS) were also evaluated. RESULTS: In the intent-to-treat population (n = 479; levalbuterol, 241;racemic albuterol, 238), the mean (SE) age was 55.3 (16.9) years, the majority of patients were white (57.8%), and the mean (SE) weight was 80.9 (24.5) kg. Demographic characteristics were similar between the 2 treatment groups, except that there were more females with COPD in the levalbuterol treatment group (63.88%) compared with the racemic albuterol treatment group (45.5%) (P = 0.005). Patients treated with levalbuterol required significantly fewer median total nebulizations (10 vs 12; P = 0.031) and scheduled nebulizations (9 vs 11; P = 0.009) compared with those in the racemic albuterol group. The 2 treatment groups required 0 rescue nebulizations. Mean (SD) forced expiratory volume in 1 second improved from baseline with both levalbuterol and racemic albuterol (0.06 [0.43] and 0.10 [0.37] L, respectively); these improvements were maintained throughout the hospital stay (0.11 [0.48] and 0.16 [0.52] L). DSA and SGWB scores improved significantly from baseline in both treatment groups, and beta-mediated adverse effects mean scores were significantly greater with levalbuterol versus racemic albuterol (P < 0.001). In the levalbuterol and racemic albuterol treatment groups, hospital LOS (70.6 and 65.7 hours, respectively), time to discharge (66.0 and 62.8 hours), and total hospital costs (least squares mean [SE], US $4869.30 [$343.58] and $4899.41 [$343.20]) were similar. CONCLUSIONS: In these hospitalized patients with acute asthma or COPD treated with levalbuterol every 6 to 8 hours or racemic albuterol every 1 to 4 hours, significantly fewer total nebulizations were required with levalbuterol, without an increased need for rescue nebulizations during 14 days of hospitalization. Both treatments were associated with improvements from baseline in symptoms and health status. The costs of treating bronchoconstriction in hospitalized patients were similar between the levalbuterol and racemic albuterol groups.

An evaluation of levalbuterol HFA in the prevention of exercise-induced bronchospasm.

BACKGROUND: Exercise-induced bronchospasm (EIB) affects up to 90% of all patients with asthma. Objective. This study evaluated the ability of levalbuterol hydrofluoroalkane (HFA) 90 mug (two actuations of 45 microg) administered via metered dose inhaler (MDI) to protect against EIB in mild-to-moderate asthmatics. METHODS: This was a randomized, double-blind, placebo-controlled, two-way cross-over study. Patients with asthma (n = 15) were > or =18 years, had a > or =6-month history of EIB, > or = 70% baseline predicted forced expiratory volume in 1 second (FEV1), and a 20% to 50% decrease in FEV(1) after treadmill exercise challenge using single-blind placebo MDI. Levalbuterol or placebo was self-administered 30 minutes before exercise. Treatment sequences were separated by a 3-to 7-day washout period. Spirometry was performed predose, 20 minutes postdose/pre-exercise, and 5, 10, 15, 30, and 60 minutes post-exercise. The primary endpoint was the maximum percent decrease in FEV1 from baseline (postdose/pre-exercise). The percentage of protected (< or = 20% decrease in post-exercise FEV1) patients was also assessed. RESULTS: Levalbuterol had significantly smaller maximum percent post-exercise decrease in FEV1 compared with placebo (LS mean +/- SE; -4.8% +/- 2.8% versus -22.5% +/- 2.8%, respectively). For levalbuterol, 14/15 (93.3%) patients had < 20% decrease in post-exercise FEV1 compared with 8/15 (53.3%) for placebo (p = 0.0143). Treatment was well tolerated. CONCLUSION: Levalbuterol HFA MDI (90 microg) administered 30 minutes before exercise was significantly more effective than placebo in protecting against EIB after a single exercise challenge and was well tolerated. CLINICAL IMPLICATIONS: Levalbuterol HFA MDI when administered before exercise was effective in protecting adults with asthma from EIB.