Multimodal imaging of drug and excipients in rat lungs following an inhaled administration of controlled-release drug laden PLGA microparticles.

Controlled-release formulations, in the form of micro- or nanoparticles, are increasingly attractive to the pharmaceutical industry for drug delivery. For respiratory illnesses, controlled-release microparticle formulations provide an opportunity to deliver a higher percentage of an inhaled medicament dose to the lung, thus potentially reducing the therapeutic dose, frequency of dosing, and minimising side-effects. We describe the use of a multimodal approach consisting of MALDI MS imaging, 3D depth profiling TOF-SIMS analysis, and histopathology to monitor the distribution of drug and excipients in sections taken from excised rat lungs following an inhaled administration of drug-laden microparticles. Following a single dose, the administered drug was detected in the lung via both MALDI MS and TOF-SIMS over a range of time points. Both imaging techniques enabled the characterisation of the distribution and retention of drug particles and identified differences in the capabilities of both imaging modalities. Histochemical staining of consecutive sections was used to provide biological context to the findings and will also be discussed in this presentation. We demonstrate how this multimodal approach could be used to help increase our understanding of the use of controlled release microparticles.

Monitoring the three-dimensional distribution of endogenous species in the lungs by matrix-assisted laser desorption/ionization mass spectrometry imaging.

RATIONALE: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is routinely employed to monitor the distribution of compounds in tissue sections and generate two-dimensional (2D) images. Whilst informative the images do not represent the distribution of the analyte of interest through the entire organ. The generation of 3D images is an exciting field that can provide a deeper view of the analyte of interest throughout an entire organ. METHODS: Serial sections of mouse and rat lung tissue were obtained at 120 µm depth intervals and imaged individually. Homogenate registration markers were incorporated in order to aid the final 3D image construction. Using freely available software packages, the images were stacked together to generate a 3D image that showed the distribution of endogenous species throughout the lungs. RESULTS: Preliminary tests were performed on 16 serial tissue sections of mouse lungs. A 3D model showing the distribution of phosphocholine at m/z 184.09 was constructed, which defined the external structure of the lungs and trachea. Later, a second experiment was performed using 24 serial tissue sections of the left lung of a rat. Two molecular markers, identified as [PC (32:1) + K]+ at m/z 770.51 and [PC (36:4) + K]+ at m/z 820.52, were used to generate 3D models of the parenchyma and airways, respectively. CONCLUSIONS: A straightforward method to generate 3D MALDI-MS images of selected molecules in lung tissue has been presented. Using freely available imaging software, the 3D distributions of molecules related to different anatomical features were determined.

Evaluation of Swallow Function Post-Extubation: Is It Necessary to Wait 24 Hours?

BACKGROUND: Post-extubation dysphagia is associated with an increased incidence of nosocomial pneumonias, longer hospitalizations, and higher re-intubation rates. The purpose of this study was to determine if it is necessary to delay swallow evaluation for 24 hours post-extubation. METHODS: A prospective investigation of swallowing was conducted at 1, 4, and 24 hours post-extubation to determine if it is necessary to delay swallow evaluation following intubation. Participants were 202 adults from 5 different intensive care units (ICU). RESULTS: A total of 166 of 202 (82.2%) passed the Yale Swallow Protocol at 1 hour post-extubation, with an additional 11 (177/202; 87.6%) at 4 hours, and 8 more (185/202; 91.6%) at 24 hours. Only intubation duration ≥4 days was significantly associated with nonfunctional swallowing. CONCLUSIONS: We found it is not necessary to delay assessment of swallowing in individuals who are post-extubation. Specifically, the majority of patients in our study (82.2%) passed a swallow screening at 1 hour post-extubation.

Clinical Probability Tools for Deep Venous Thrombosis, Pulmonary Embolism, and Bleeding.

Overdiagnosis of venous thromboembolism is associated with increasing numbers of patient complications and health care burden. Multiple clinical tools exist to estimate the probability of pulmonary embolism and deep venous thrombosis. When used with d-dimer testing, these can further stratify venous thromboembolism risk to help inform the use of additional diagnostic testing. Although there are similar tools to estimate bleeding risk, these are not as well-validated and lack reliability.

Aggressive Treatment of Intermediate-Risk Patients with Acute Symptomatic Pulmonary Embolism.

Contemporary studies of acute pulmonary embolism (PE) have evaluated the role of thrombolytics in intermediate-risk PE. Significant findings are that thrombolytic therapy may prevent hemodynamic deterioration and all-cause mortality but increases major bleeding. Benefits and harms are finely balanced with no convincing net benefit from thrombolytic therapy among unselected patients. Among patients with intermediate risk PE, additional prognostic factors or subtle hemodynamic changes might alter the risk-benefit assessment in favor of thrombolytic therapy before obvious hemodynamic instability.

The 3D OrbiSIMS-label-free metabolic imaging with subcellular lateral resolution and high mass-resolving power.

We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 µm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters-gamma-aminobutyric acid, dopamine and serotonin-with high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone.

Intracellular Drug Uptake-A Comparison of Single Cell Measurements Using ToF-SIMS Imaging and Quantification from Cell Populations with LC/MS/MS.

ToF-SIMS is a label-free imaging method that has been shown to enable imaging of amiodarone in single rat macrophage (NR8383) cells. In this study, we show that the method extends to three other cell lines relevant to drug discovery: human embryonic kidney (HEK293), cervical cancer (HeLa), and liver cancer (HepG2). There is significant interest in the variation of drug uptake at the single cell level, and we use ToF-SIMS to show that there is great diversity between individual cells and when comparing each of the cell types. These single cell measurements are compared to quantitative measurements of cell-associated amiodarone for the population using LC/MS/MS and cell counting with flow cytometry. NR8383 and HepG2 cells uptake the greatest amount of amiodarone with an average of 2.38 and 2.60 pg per cell, respectively, and HeLa and Hek 293 have a significantly lower amount of amiodarone at 0.43 and 0.36 pg per cell, respectively. The amount of cell-associated drug for the ensemble population measurement (LC/MS/MS) is compared with the ToF-SIMS single cell data: a similar amount of drug was detected per cell for the NR8383, and HepG2 cells at a greater level than that for the HEK293 cells. However, the two techniques did not agree for the HeLa cells, and we postulate potential reasons for this.

Semiempirical Rules To Determine Drug Sensitivity and Ionization Efficiency in Secondary Ion Mass Spectrometry Using a Model Tissue Sample.

There is an increasing need in the pharmaceutical industry to reduce drug failure at late stage and thus reduce the cost of developing a new medicine. Since most drug targets are intracellular, this requires a better understanding of the drug disposition within a cell. Secondary ion mass spectrometry has been identified as a potentially important technique to do this, as it is label-free and allows imaging in 3D with subcellular resolution and recent studies have shown promise for amiodarone. An important analytical parameter is sensitivity, and we measure this in a bovine liver homogenate reference sample for 20 drugs representing important class types relevant to the pharmaceutical industry. We also measure the sensitivity for pure drug and show, for the first time, that the secondary ion mass spectrometry (SIMS) positive ionization efficiency for small molecules is a simple power-law relationship to the log P value. This discovery will be important for advancing the understanding of the SIMS ionization process in small molecules that has, until now, been elusive. This simple relationship is found to hold true for drug doped in the bovine liver homogenate reference sample, except for fluticasone, nicardipine, and sorafenib which suffer from severe matrix suppression. This relationship provides a simple semiempirical method to determine drug sensitivity for positive secondary ions. Furthermore, we show, on chosen models, how the use of different solvents during sample preparation can affect the ionization of analytes.

Obesity Treatment at HealthPartners: Adaptation of Clinical Guidelines into Systems for Practice Operations.

The purpose of this paper is to describe how HealthPartners health system in Minneapolis, MN, has translated a clinical guideline for obesity among adults into an efficient care delivery practice operations system. Based on a foundation provided by the physician-led Institute of Clinical Systems Improvement (ICSI)-developed Prevention and Management of Obesity for Adults Health Care Guideline, HealthPartners adapted the guideline into an electronic health record-based "Smart Set" that provides frontline physicians with the information, treatment options, and referral steps necessary to care for their patients with obesity. Additional context is provided in terms of insurance coverage and systems-based resources designed to prevent and treat obesity for adults.

Controversies in the Management of Life-Threatening Pulmonary Embolism.

Patients with life-threatening pulmonary embolism (PE) offer clinicians a unique opportunity to intervene effectively on the patient's behalf. Hemodynamic status remains the most important short-term prognostic factor for patients with acute PE. Although the evidence is limited, the use of thrombolytic therapy is recommended for patients with acute symptomatic PE and associated hypotension or shock (i.e., high-risk PE) because these patients have a high short-term mortality risk (i.e., >15%) even when receiving anticoagulant treatment. In this setting, the hemodynamic benefits of thrombolytic treatment far outweigh its bleeding risk. For hemodynamically stable patients with PE, the identification of a subgroup of patients with a risk of PE-related complications similar to patients with PE and cardiovascular instability (i.e., intermediate-risk group) may assist with decision making regarding therapy. Given the lack of clear mortality benefit and increased bleeding risk, guidelines do not recommend routine use of systemic thrombolysis for this subgroup of patients. Careful monitoring and rescue fibrinolysis for intermediate-risk PE patients who experience hemodynamic compromise or deterioration while receiving standard anticoagulant therapy can minimize deaths from PE. For patients with life-threatening PE at high risk of bleeding, clinicians might consider the use of low-dose thrombolytic therapy, catheter-directed thrombolysis, or surgical embolectomy, if they have access to the required expertise and resources. The evidence does not support the use of inferior vena cava filters in patients with life-threatening PE unless there is a contraindication to anticoagulation. Since various medical and surgical specialties offer different perspectives and expertise, a multidisciplinary approach to patients with intermediate- and high-risk PE might improve patient outcomes.

Single-Cell Analysis: Visualizing Pharmaceutical and Metabolite Uptake in Cells with Label-Free 3D Mass Spectrometry Imaging.

Detecting metabolites and parent compound within a cell type is now a priority for pharmaceutical development. In this context, three-dimensional secondary ion mass spectrometry (SIMS) imaging was used to investigate the cellular uptake of the antiarrhythmic agent amiodarone, a phospholipidosis-inducing pharmaceutical compound. The high lateral resolution and 3D imaging capabilities of SIMS combined with the multiplex capabilities of ToF mass spectrometric detection allows for the visualization of pharmaceutical compound and metabolites in single cells. The intact, unlabeled drug compound was successfully detected at therapeutic dosages in macrophages (cell line: NR8383). Chemical information from endogenous biomolecules was used to correlate drug distributions with morphological features. From this spatial analysis, amiodarone was detected throughout the cell, with the majority of the compound found in the membrane and subsurface regions and absent in the nuclear regions. Similar results were obtained when the macrophages were doped with amiodarone metabolite, desethylamiodarone. The fwhm lateral resolution measured across an intracellular interface in high lateral resolution ion images was approximately 550 nm. Overall, this approach provides the basis for studying cellular uptake of pharmaceutical compounds and their metabolites on the single cell level.

Alternative surfactants for improved efficiency of in situ tryptic proteolysis of fingermarks.

Despite recent improvements to in situ proteolysis strategies, a higher efficiency is still needed to increase both the number of peptides detected and the associated ion intensity, leading to a complete and reliable set of biomarkers for diagnostic or prognostic purposes. In the study presented here, an extract of a systematic study is illustrated investigating a range of surfactants assisting trypsin proteolytic activity. Method development was trialled on fingermarks; this specimen results from a transfer of sweat from an individual's fingertip to a surface upon contact. As sweat carries a plethora of biomolecules, including peptides and proteins, fingermarks are, potentially, a very valuable specimen for non-invasive prognostic or diagnostic screening. A recent study has demonstrated the opportunity to quickly detect peptides and small proteins in fingermarks using Matrix Assisted Laser Desorption Ionization Mass Spectrometry Profiling (MALDI MSP). However, intact detection bears low sensitivity and does not allow species identification; therefore, a shotgun proteomic approach was employed involving in situ proteolysis. Data demonstrate that in fingermarks, further improvements to the existing method can be achieved using MEGA-8 as surfactant in higher percentages as well as combinations of different detergents. Also, for the first time, Rapigest SF, normally used in solution digestions, has been shown to successfully work also for in situ proteolysis.

The use of hydrazine-based derivatization reagents for improved sensitivity and detection of carbonyl containing compounds using MALDI-MSI.

Hydrazine-based derivatization reagents have been used to detect the presence of the carbonyl containing glucocorticoid fluticasone proprionate in rat lung tissue by MALDI-MSI. Such reagents also act as a matrix for analysis by MALDI-MS and have been termed "reactive matrices". Cryosections of rat lung tissue (12 µm), spotted with a range of concentrations of fluticasone proprionate, were derivatized in situ with 2,4-dinitrophenylhydrazine (DNPH) and 4-dimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-hydrazine (DMNTH) by the use of an acoustic reagent spotter. It has been demonstrated that DMNTH gave superior results compared to DNPH and that analysis of samples immediately after application of DMNTH resulted in the detection of the protonated hydrazone derivative ([MD + H](+)) of fluticasone propionate at a concentration of 500 ng/µL. It has been further shown that a prolonged reaction time (~48 h) improves the detection limit of the protonated hydrazone derivative to 50 ng/µL and that improvements in sensitivity and limits of detection are obtained when a conventional MALDI matrix CHCA is employed in conjunction with the DNPH/DMNTH reactive matrix.

Using a single, high mass resolution mass spectrometry platform to investigate ion suppression effects observed during tissue imaging.

RATIONALE: The signal intensity of a given molecule across a tissue section when measured using mass spectrometry imaging (MSI) is prone to changes caused by the molecular heterogeneity across the surface of the tissue. Here we propose a strategy to investigate these effects using electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) on a single high-resolution mass spectrometry (HRMS) platform. METHODS: A rat was administered with a single inhaled dose of a compound and sacrificed 1 h after dosing. Sections were prepared from the excised frozen lung and analysed using MALDI, liquid extraction surface analysis (LESA) nano-ESI-MS and nano-ESI liquid chromatography (LC)/MS. The ESI and MALDI ion sources were mounted either side of the ion transfer system of the same Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. RESULTS: MALDI MSI clearly demonstrated widespread distribution of the dosed molecule throughout the lung, with the exception of a non-lung section of tissue on the same sample surface. Comparison of the lipid signals across the sample indicated a change in signal between the lung and the adipose tissue present on the same section. Use of ESI and MALDI, with and without an internal standard, supported the evaluation of changes in the signal of the dosed molecule across the tissue section. CONCLUSIONS: The results demonstrate the successful application of a dual ion source HRMS system to the systematic evaluation of data from MALDI MSI, used to determine the distribution of an inhaled drug in the lung. The system discussed is of great utility in investigating the effects of ion suppression and evaluating the quantitative and qualitative nature of the MSI data.

Chronic thromboembolic pulmonary hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a disease with high mortality and few treatment options. This article reviews the epidemiology of CTEPH and identifies risk factors for its development. The pathobiology and the progression from thromboembolic events to chronically increased right-sided pressures are discussed. The diagnosis and assessment of CTEPH requires several modalities and the role of these is detailed. The pre-operative evaluation assesses peri-operative risk and determines the likelihood of benefit from PTE. Pulmonary thromboendarterectomy (PTE) remains the treatment of choice in appropriate patients. Nonsurgical therapies for CTEPH may provide benefit in patients who cannot be offered surgery.

Diagnosis and management of life-threatening pulmonary embolism.

Pulmonary embolus (PE) is estimated to cause 200 000 to 300 000 deaths annually. Many deaths occur in hemodynamically unstable patients and the estimated mortality for inpatients with hemodynamic instability is between 15% and 25%. The diagnosis of PE in the critically ill is often challenging because the presentation is nonspecific. Computed tomographic pulmonary angiography appears to be the most useful study for diagnosis of PE in the critically ill. For patients with renal insufficiency and contrast allergy, the ventilation perfusion scan provides an alternative. For patients too unstable to travel, echocardiography (especially transesophageal echocardiography) is another option. A positive result on lower extremity Doppler ultrasound can also aid in the decision to treat. The choice of treatment in PE depends on the estimated risk of poor outcome. The presence of hypotension is the most significant predictor of poor outcome and defines those with massive PE. Normotensive patients with evidence of right ventricular (RV) dysfunction, as assessed by echocardiography, comprise the sub-massive category and are at intermediate risk of poor outcomes. Clinically, those with sub-massive PE are difficult to distinguish from those with low-risk PE. Cardiac troponin, brain natriuretic peptide, and computed tomographic pulmonary angiography can raise the suspicion that a patient has sub-massive PE, but the echocardiogram remains the primary means of identifying RV dysfunction. The initial therapy for patients with PE is anticoagulation. Use of vasopressors, inotropes, pulmonary artery (PA) vasodilators and mechanical ventilation can stabilize critically ill patients. The recommended definitive treatment for patients with massive PE is thrombolysis (in addition to anticoagulation). In massive PE, thrombolytics reduce the risk of recurrent PE, cause rapid improvement in hemodynamics, and probably reduce mortality compared with anticoagulation alone. For patients with a contraindication to anticoagulation and thrombolytic therapy, surgical embolectomy and catheter-based therapies are options. Thrombolytic therapy in sub-massive PE results in improved pulmonary perfusion, reduced PA pressures, and a less complicated hospital course. No survival benefit has been documented, however. If one is considering the use of thrombolytic therapy in sub-massive PE, the limited documented benefit must be weighed against the increased risk of life-threatening hemorrhage. The role of surgical embolectomy and catheter-based therapies in this population is unclear. Evidence suggests that sub-massive PE is a heterogeneous group with respect to risk. It is possible that those at highest risk may benefit from thrombolysis, but existing studies do not identify subgroups within the sub-massive category. The role of inferior vena cava (IVC) filters, catheter-based interventions, and surgical embolectomy in life-threatening PE has yet to be completely defined.

Upper extremity deep vein thrombosis.

Upper extremity deep vein thrombosis (UEDVT) is associated with significant morbidity and mortality. The susceptible populations and risk factors for UEDVT are well-known. The presenting symptoms can be subtle, and therefore objective testing is necessary for diagnosis. The optimal diagnostic strategy has not been determined, and more than one test may be required to exclude the diagnosis. Proper treatment reduces the occurrence of complications, and treatment should include long-term anticoagulation if the patient has no contraindications. This article discusses the risk factors, pathogenesis, diagnosis, complications, and management of UEDVT.

Matrix-assisted laser desorption/ionization-ion mobility separation-mass spectrometry imaging of vinblastine in whole body tissue sections.

During early-stage drug development, drug and metabolite distribution studies are carried out in animal tissues using a range of techniques, particularly whole body autoradiography (WBA). While widely employed, WBA has a number of limitations, including the following: expensive synthesis of radiolabeled drugs and analyte specificity and identification. WBA only images the radiolabel. MALDI MSI has been shown previously to be advantageous for imaging the distribution of a range of drugs and metabolites in whole body sections. Ion mobility separation (IMS) adds a further separation step to imaging experiments; demonstrated here is MALDI-IMS-MS whole body imaging of rats dosed at 6 mg/kg i.v. with an anticancer drug, vinblastine and shown is the distribution of the precursor ion m/z 811.4 and several product ions including m/z 793, 751, 733, 719, 691, 649, 524, and 355. The distribution of vinblastine within the ventricles of the brain is also depicted. Clearly demonstrated in these data are the removal of interfering isobaric ions within the images of m/z 811.4 and also of the transition m/z 811-751, resulting in a higher confidence in the imaging data. Within this work, IMS has shown to be advantageous in both MS and MS/MS imaging experiments by separating vinblastine from an endogenous isobaric lipid.