An evaluation of a high-pressure (11)CO carbonylation apparatus.

[(11)C]Carbon monoxide ((11)CO) is a versatile building block for the synthesis of Positron Emission Tomography (PET) radioligands. However, the difficulty of trapping (11)CO in a small solvent volume has limited its utility. We here report an evaluation of a simple, fully automated high-pressure synthesizer prototype for the use in (11)C-carbonylation reactions. [(11)C]Carbon monoxide was easily prepared by online reduction of [(11)C]carbon dioxide using either Mo(s) or Zn(s) as the reducing agent. The conversion yield of (11)CO was >99% when zinc was used as the reducing agent, and the corresponding value for Mo was approximately 71%. When the Zn or Mo column was constantly kept under inert atmosphere, no significant decrease in reducing properties was observed for more than 100 (11)CO productions. However, in our hands, Mo reductant was much easier to service. A total of nine functional groups were successfully radiolabeled using the (11)CO synthesizer prototype. All measured radiochemical yields exceeded 37%, and the (11)CO trapping efficiency was generally above 90%, except for the Suzuki coupling where the trapping efficiency was 80%. This high-pressure synthesizer using [(11)C]carbon monoxide as the labeling precursor is easy to operate allowing for (11)C-carbonylation reactions to be performed in a high yield and in a routinely fashion.

Regional lung targeting with a fluticasone/salmeterol aerosol using a bolus breath hold method of the PreciseInhale® system: A first evaluation in humans.

BACKGROUND: In development of inhaled drugs- and formulations the measured concentration in the systemic circulation is often used as a surrogate for local dosimetry in the lungs. To further elucidate regional differences in the fate of drugs in the lungs, different aerodynamic sizes of aerosols have been used to target major airway regions. An alternative approach to achieve regional targeting of aerosols, is to use a defined aerosol bolus together with a bolus breath hold strategy. A small volume of test aerosol is intercalated and stopped at different penetration depths, to achieve increased drug deposition at chosen lung locations. Drug permeation from the lung regions is then investigated by repeatedly sampling venous blood from the systemic circulation. The PreciseInhale® (PI) exposure platform was developed to allow generation of aerosols from different sources, including clinical inhalers, into a holding chamber, for subsequent use with alternative exposure modules in vitro and in vivo. In the current first-in-human study was investigated the feasibility of a new clinical exposure module added to the PI system. By extracting aerosol puffs from a medical inhaler for subsequent delivery to volunteers, it was possible to administer whole lung exposures, as well as regional targeting exposures. METHODS: Aerosols containing 250 µg/25 µg fluticasone propionate (FP)/salmeterol xinafoate (SMX) were automatically actuated and extracted from the pressurized Metered Dose Inhaler (pMDI) Evohaler Seretide forte into the PI system's holding chamber, then administered to the healthy volunteers using controlled flowrate and volume exposure cycles. Two main comparisons were made by measuring the systemic PK response: I. One label dose directly from the inhaler to the subject was compared to the same dose extracted from the pMDI into the PI system and then administered to the subject. II A small aerosol bolus at a penetration level in the central airways was compared to a small aerosol bolus at a penetration level in the peripheral lung. RESULTS AND CONCLUSIONS: When one inhaler dose was administered via the PI system, the absorbed dose, expressed as AUC24, was approximately twice as high and the CV was less than half, compared to direct inhalation from the same pMDI. Bolus breath hold targeting of drugs from the same aerosol mixture to the peripheral lung and the central airways showed a difference in their appearance in the systemic circulation. Normalized to the same deposited dose, SMX had a 57 % higher Cmax in the peripheral lung compared to the central airways. However, from 6 to 24 h after dosing the systemic concentrations of SMX from both regions were quite similar. FP had parallel concentrations curves with a 23 % higher AUC24 in the peripheral lung with no noticeable elevation around Cmax. The permeability of these two substances from similar sized aerosols was indeed higher in the thinner air/blood barriers of the peripheral lung compared to the central airways, but differences as measured on the venous side of the circulation were not dramatic. In conclusion, the PI system provided better control of actuation, aspiration, and dispensation of aerosols from the clinical inhaler and thereby delivered higher quality read outs of pharmacokinetic parameters such as tmax, Cmax, and AUC. Improved performance, using PI system, can likely also be employed for studying regional selectivity of other responses in the lungs, for use in drug development.

Leucine improves the aerosol performance of dry powder inhaler formulations of siRNA-loaded nanoparticles.

Thermostable dry powder inhaler (DPI) formulations with high aerosol performance are attractive inhalable solid dosage forms for local treatment of inflammatory lung diseases. We recently demonstrated that lipidoid-polymer hybrid nanoparticles (LPNs) loaded with small interfering RNA (siRNA) directed against tumor necrosis factor alpha (TNF-α) mediate efficient intracellular siRNA delivery and reduce inflammation in vivo. Here, we show that mixtures of the stabilizing excipients trehalose (Tre) and dextran (Dex), in combination with the shell-forming dispersion enhancer leucine (Leu), stabilize TNF-α siRNA-loaded LPNs during spray drying into nanocomposite microparticles, and result in DPI formulations with high aerosol performance. At low Leu content (0 to 10%, w/w), the DPI formulations were amorphous, and exhibited poor aerosol performance. When the Leu content was increased from 20 to 60% (w/w), the surface content of Leu increased from 39.2 to 68.1 mol%, and the flowability was significantly improved. Microscopy analysis suggest that the improved powder dispersibility is the result of a wrinkled surface morphology, which reduces the surface area available for interparticle interactions. Increasing the Leu content further (to above 10%, w/w) did not influence the aerosol performance, and the aerosol yield was maximal at 30-40% Leu (w/w). Formulations containing 40% Leu and a Tre:Dex ratio of 10:90 (w/w) displayed a high fine particle fraction and aerosol properties suitable for inhalation. The chemical integrity of TNF-α siRNA was preserved in the solid state, and biodistribution studies in mice showed that pulmonary administration of DPI formulations with high aerosol performance resulted in homogenous deep lung deposition. Our results demonstrate that at optimal ratios, ternary excipient mixtures of Leu, Tre and Dex protect TNF-α siRNA-loaded LPNs during spray drying. Hence, this study shows that microparticles with an amorphous Tre/Dex matrix and a crystalline Leu shell efficiently stabilize the nanocomposite LPNs in the solid state, and ensure aerosol properties suitable for inhalation.

A Reperfusion BOLD-MRI Tissue Perfusion Protocol Reliably Differentiate Patients with Peripheral Arterial Occlusive Disease from Healthy Controls.

There is no established technique that directly quantifies lower limb tissue perfusion. Blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI) is an MRI technique that can determine skeletal muscle perfusion. BOLD-MRI relies on magnetic differences of oxygenated and deoxygenated hemoglobin, and regional changes in oxy/deoxyhemoglobin ratio can be recorded by T2* weighted MRI sequences. We aimed to test whether BOLD-MRI can differentiate lower limb tissue perfusion in peripheral arterial occlusive disease (PAOD) patients and healthy controls. Twenty-two PAOD patients and ten healthy elderly volunteers underwent lower limb BOLD-MRI. Reactive hyperemia was provoked by transient cuff compression and images of the gastrocnemius and soleus muscles were continuously acquired at rest, during ischemia and reperfusion. Key BOLD parameters were baseline T2* absolute value and time to T2* peak value after cuff deflation (TTP). Correlations between imaging parameters and ankle-brachial index (ABI) was investigated. The mean TTP was considerably prolonged in PAOD patients compared to healthy controls (m. gastrocnemius: 111 ± 46 versus 48 ± 22 s, p = 0.000253; m. soleus: 100 ± 42 versus 41 ± 30 s, p = 0.000216). Both gastrocnemius and soleus TTP values correlated strongly with ABI (-0.82 and -0.78, p < 0.01). BOLD-MRI during reactive hyperemia differentiated most PAOD patients from healthy controls. TTP was the most decisive parameter and strongly correlated with the ABI.

Airway Epithelial Lining Fluid and Plasma Pharmacokinetics of Inhaled Fluticasone Propionate and Salmeterol Xinafoate in Mechanically Ventilated Pigs.

Background: The lower respiratory tract of the landrace pig has close anatomical and physiological similarities with that of the human, and hence, for inhalation studies this species is well suited for biopharmaceutical research. Methods: The objective of this study was to evaluate pharmacokinetics in pigs following one dose of Diskus™ Seretide™ forte device, labeled 500/50 fluticasone propionate (FP) and salmeterol xinafoate (SX), respectively. The PreciseInhale™ (PI) instrument was used to actuate the inhaler for in vitro testing and aerosol dosing to pigs. In vitro, the aerosol was characterized with a cascade impactor with respect to mass median aerodynamic diameter, geometric standard deviation, and fine particle dose. In vivo, dry powder inhalation exposure was delivered as a short bolus dose, to anesthetized and mechanically ventilated landrace pigs. In addition to plasma PK, PK assessment of airway epithelial lining fluid (ELF) was used in this study. ELF of the depth of three to fourth airway generation of the right lung was accessed using standard bronchoscopy and a synthetic absorptive matrix. Results and Conclusions: Dry powder inhalation exposures with good consistency and well characterized aerosols to the pig lung were achieved by the use of the PreciseInhale™ instrument. Drug concentrations of ELF for both FP and SX were demonstrated to be four to five orders of magnitude higher than its corresponding systemic plasma drug concentrations. Clinical PK following inhalation of the same dose was used as benchmark, and the clinical study did demonstrate similar plasma PK profiles and drug exposures of both FP and SX as the current pig study. Two factors explain the close similarity of PK (1) similiar physiology between species and (2) the consistency of dosing to animals. To conclude, our study demonstrated the utility and translational potential of conducting PK studies in pigs in the development of inhaled pharmaceuticals.

Vasoconstriction after inhalation of budesonide: a study in the isolated and perfused rat lung.

INTRODUCTION: Clinical studies have shown that inhaled corticosteroids can induce rapid vasoconstriction in the airways, leading to decreased mucosal blood flow. The aim of this study was to investigate whether vasoconstriction of the pulmonary circulation after short inhalation of a corticosteroid can be detected in the isolated and perfused rat lung (IPL) - a model which could serve as a substitute or a complement to clinical models. METHODS: IPLs were briefly exposed to dry powder aerosol of budesonide. The pulmonary perfusate flow rate was assessed during 100min post-exposure. A reduction in perfusion flow rate was interpreted as vasoconstriction. MAIN RESULTS: Vasoconstriction was more pronounced after brief inhalation of 10 and 50microg budesonide than 2microg. The onset of vasoconstriction became statistically significant within 10-40min after inhalation. Co-administration of a selective alpha(1)-adrenoceptor antagonist (prazosin 50nM added to the perfusate) reduced vasoconstriction by approximately 50% during 100min of perfusion (p=0.003). CONCLUSIONS: Inhaled budesonide rapidly induces pulmonary vasoconstriction suggesting a nongenomic mechanism probably related to disposition of noradrenaline at the neuro-muscular junction. This ex vivo model could serve as a substitute or a complement to clinical models for investigating rapid effects of glucocorticoid receptor agonists on the pulmonary/bronchial circulation.

Adapting the Aerogen Mesh Nebulizer for Dried Aerosol Exposures Using the PreciseInhale Platform.

Background: Many substances used in inhalation research are water soluble and can be administered as nebulized solutions. Typical examples are therapeutic, small-molecular agents, or macromolecules. Another category is a number of water-soluble agents used for airway diagnostics or disease modeling. Mesh nebulizers have facilitated well-controlled liquid aerosol exposures. Meanwhile, a benchtop inhalation platform, PreciseInhale, was developed for providing small-scale, well-controlled aerosol exposures in preclinical configurations. The purpose of the current research was to adapt the Aerogen mesh nebulizer to work within the PreciseInhale system for both cell culture and rodent exposures. Methods: The wet aerosols produced with the Aerogen Pro nebulizer were dried out in an aerosol holding chamber by supplying dry carrier air, which was provided by passing the incoming ambient air through a column with silica gel. The nebulizer was installed in an aerosol holding chamber between an upstream flow-rate pneumotach and a downstream aerosol monitor. By pulsing, the nebulizer output was reduced to 1%-10% of continuous operation to better match the exposure ventilation requirements. Additional drying was obtained by mantling the holding chamber with dried paper. Results and Conclusions: The nebulizer output was reduced to 3-30 µL/min and dried out before reaching the in vitro or in vivo exposure modules. Using solute concentrations in the range of 0.5%-2% (w/w), dried aerosols were produced with a mass median aerodynamic diameter of 1.5-2.0 µm, compared to the 4-5 µm droplets emitted by the nebulizer. Controlling the Aerogen nebulizer under a reduced output scheme within the PreciseInhale platform gave two major advantages: (i) by reducing aerosol output to better match exposure flow rates of single rodents, increased airway deposition yields were obtained in a range of 1%-10% relative to the nebulized amount of test substance and (ii) shrinking aerosol particle sizes through drying improved the peripheral lung deposition of test aerosols.

DissolvIt: An In Vitro Method for Simulating the Dissolution and Absorption of Inhaled Dry Powder Drugs in the Lungs.

The main purpose of this work was to develop an in vitro method for simulating the dissolution and absorption of inhaled dry powder drugs that also mimics systemic pharmacokinetic data. A second purpose was to evaluate this method. DissolvIt® was developed as a simulation of the air-blood barrier of the upper airways, constituting: "airborne" particles deposited on a glass cover slip, a mucus simulant, a polycarbonate (basal) membrane, and a pumped albumin buffer simulating the pulmonary blood flow. The PreciseInhale® exposure system was used to aerosolize and deposit test formulations onto cover slips. The particle dissolution was observed by optical microscopy as particle disappearance, and it was started directly when the particles came into contact with the mucus simulant. Solute from the dissolving particles diffused through the barrier and was absorbed into the perfusate. The drug concentration in the perfusate over time and the remaining drug in the barrier at the end of the experiment were quantitated by using liquid chromatography-tandem mass spectrometry. Budesonide and fluticasone propionate generated different pharmacokinetic dissolution/absorption profiles in DissolvIt. This study indicates that DissolvIt simulates dissolution and absorption of drugs in the lung, and that DissolvIt also mimics pharmacokinetic profiles and parameters.

How much will linked deformable registrations decrease the quality of multi-atlas segmentation fusions?

BACKGROUND AND PURPOSE: Multi-atlas segmentation can yield better results than single atlas segmentation, but practical applications are limited by long calculation times for deformable registration. To shorten the calculation time pre-calculated registrations of atlases could be linked via a single atlas registered in runtime to the current patient. The primary purpose of this work is to investigate and quantify segmentation quality changes introduced by such linked registrations. We also determine the optimal parameters for fusing linked multi-atlas labels using probabilistic weighted fusion. MATERIAL AND METHODS: Computed tomography images of 10 head and neck cancer patients were used as atlases, with parotid glands, submandibular glands, the mandible and lymph node levels II-IV segmented by an experienced radiation oncologist following published consensus guidelines. The change in segmentation quality scored by Dice similarity coefficient (DSC) for linking free-form deformable registrations, modeled by B-splines, was investigated for both single- and multi-atlas label fusion by using a leave-one-out approach. RESULTS: The median decrease of the DSC was in the range 2.8% to 8.4% compared to direct registrations for all structures while reducing the computer calculation time to that of a single deformable registration. Linking several registrations showed a DSC decrease almost linear to the number of links, suggesting that extrapolation to zero links provides an observer independent measure of the inherent precision with which the segmentation guidelines can be applied. CONCLUSIONS: Linking pre-made registrations of multiple atlases via a runtime registration of a single atlas provides a feasible method for reducing computation time in multi-atlas registration.

Dry powder inhalation exposures of the endotracheally intubated rat lung, ex vivo and in vivo: the pulmonary pharmacokinetics of fluticasone furoate.

BACKGROUND: The isolated perfused rat lung (IPL) is a suitable model for studying lung-specific pharmacokinetics (PK) of inhaled drugs. So far, little has been known, however, whether the PK measured in the ex vivo organ corresponds to the PK measured in similarly exposed animals in vivo, in particular the endotracheally intubated rat (EIR). The purpose of the current research was to compare the PK of inhaled corticosteroid fluticasone furoate (FF) in the IPL and the EIR. METHOD: Aerosols of FF with mass median aerodynamic diameters ranging from 2.2 to 3.2 µm were generated with the DustGun aerosol generator. The IPL, perfused in the single-pass mode, was exposed via inhalation to 5.6 and 46 µg of FF. Following inhalation, the perfusate was repeatedly sampled for 100 min, after which the lungs were recovered for quantitation of remaining FF. Two groups of EIR were also exposed via inhalation to 7 µg of FF. One group was immediately euthanized for determination of the initial deposition of FF in the lungs. From the second group, four venous blood samples were drawn up to 4 hr after exposure. The animals were then sacrificed for determination of FF remaining in the lungs. RESULTS: Following inhalation, FF was slowly disappearing from both the IPL and the lungs of the EIR, with a half-life of pulmonary retention of 4.3-4.9 hr for all three exposure series. For the low exposure levels, the concentration curve of FF in the IPL perfusate was similar in shape to that in venous blood of the EIR, with a Cmax of 1.0 and 0.8 nM for the IPL and the EIR, respectively. CONCLUSIONS: The results indicate that the IPL and the EIR, when used jointly in PK studies, can provide a detailed characterization of inhaled drugs or toxicants.

Clinical evaluation of multi-atlas based segmentation of lymph node regions in head and neck and prostate cancer patients.

BACKGROUND: Semi-automated segmentation using deformable registration of selected atlas cases consisting of expert segmented patient images has been proposed to facilitate the delineation of lymph node regions for three-dimensional conformal and intensity-modulated radiotherapy planning of head and neck and prostate tumours. Our aim is to investigate if fusion of multiple atlases will lead to clinical workload reductions and more accurate segmentation proposals compared to the use of a single atlas segmentation, due to a more complete representation of the anatomical variations. METHODS: Atlases for lymph node regions were constructed using 11 head and neck patients and 15 prostate patients based on published recommendations for segmentations. A commercial registration software (Velocity AI) was used to create individual segmentations through deformable registration. Ten head and neck patients, and ten prostate patients, all different from the atlas patients, were randomly chosen for the study from retrospective data. Each patient was first delineated three times, (a) manually by a radiation oncologist, (b) automatically using a single atlas segmentation proposal from a chosen atlas and (c) automatically by fusing the atlas proposals from all cases in the database using the probabilistic weighting fusion algorithm. In a subsequent step a radiation oncologist corrected the segmentation proposals achieved from step (b) and (c) without using the result from method (a) as reference. The time spent for editing the segmentations was recorded separately for each method and for each individual structure. Finally, the Dice Similarity Coefficient and the volume of the structures were used to evaluate the similarity between the structures delineated with the different methods. RESULTS: For the single atlas method, the time reduction compared to manual segmentation was 29% and 23% for head and neck and pelvis lymph nodes, respectively, while editing the fused atlas proposal resulted in time reductions of 49% and 34%. The average volume of the fused atlas proposals was only 74% of the manual segmentation for the head and neck cases and 82% for the prostate cases due to a blurring effect from the fusion process. After editing of the proposals the resulting volume differences were no longer statistically significant, although a slight influence by the proposals could be noticed since the average edited volume was still slightly smaller than the manual segmentation, 9% and 5%, respectively. CONCLUSIONS: Segmentation based on fusion of multiple atlases reduces the time needed for delineation of lymph node regions compared to the use of a single atlas segmentation. Even though the time saving is large, the quality of the segmentation is maintained compared to manual segmentation.