Inhaled Amikacin to Prevent Ventilator-Associated Pneumonia.

BACKGROUND: Whether preventive inhaled antibiotics may reduce the incidence of ventilator-associated pneumonia is unclear. METHODS: In this investigator-initiated, multicenter, double-blind, randomized, controlled, superiority trial, we assigned critically ill adults who had been undergoing invasive mechanical ventilation for at least 72 hours to receive inhaled amikacin at a dose of 20 mg per kilogram of ideal body weight once daily or to receive placebo for 3 days. The primary outcome was a first episode of ventilator-associated pneumonia during 28 days of follow-up. Safety was assessed. RESULTS: A total of 850 patients underwent randomization, and 847 were included in the analyses (417 assigned to the amikacin group and 430 to the placebo group). All three daily nebulizations were received by 337 patients (81%) in the amikacin group and 355 patients (83%) in the placebo group. At 28 days, ventilator-associated pneumonia had developed in 62 patients (15%) in the amikacin group and in 95 patients (22%) in the placebo group (difference in restricted mean survival time to ventilator-associated pneumonia, 1.5 days; 95% confidence interval [CI], 0.6 to 2.5; P = 0.004). An infection-related ventilator-associated complication occurred in 74 patients (18%) in the amikacin group and in 111 patients (26%) in the placebo group (hazard ratio, 0.66; 95% CI, 0.50 to 0.89). Trial-related serious adverse effects were seen in 7 patients (1.7%) in the amikacin group and in 4 patients (0.9%) in the placebo group. CONCLUSIONS: Among patients who had undergone mechanical ventilation for at least 3 days, a subsequent 3-day course of inhaled amikacin reduced the burden of ventilator-associated pneumonia during 28 days of follow-up. (Funded by the French Ministry of Health; AMIKINHAL ClinicalTrials.gov number, NCT03149640; EUDRA Clinical Trials number, 2016-001054-17.).

Inhaled IgG1 antibodies: The buffering system is an important driver of stability during mesh-nebulization.

In the past decade, oral inhalation has been a thriving focus of research to administer antibody directly to the lungs as an aerosol, for local treatment of respiratory diseases. Formulation of inhaled antibodies is central for the stability of antibody, lung safety and to ensure inhaler performances. Surfactants have already been shown to prevent antibody degradation during aerosolization, but little is known about the impact of other components of liquid formulations on the structural stability of antibodies. Here, we report for the first time to the best of our knowledge, a significant effect of the buffering system on monoclonal antibodies stability, during mesh-nebulization. While the monoclonal antibody extensively aggregated in citrate buffer after nebulization and required high concentration of polysorbate 80 (PS80) to maintain protein integrity, acetate and histidine buffers resulted in a slight to moderate aggregation without PS80 and low concentration of PS80 was sufficient to stabilize antibody during mesh-nebulization.

Aggregates Associated with Instability of Antibodies during Aerosolization Induce Adverse Immunological Effects.

BACKGROUND: Immunogenicity refers to the inherent ability of a molecule to stimulate an immune response. Aggregates are one of the major risk factors for the undesired immunogenicity of therapeutic antibodies (Ab) and may ultimately result in immune-mediated adverse effects. For Ab delivered by inhalation, it is necessary to consider the interaction between aggregates resulting from the instability of the Ab during aerosolization and the lung mucosa. The aim of this study was to determine the impact of aggregates produced during aerosolization of therapeutic Ab on the immune system. METHODS: Human and murine immunoglobulin G (IgG) were aerosolized using a clinically-relevant nebulizer and their immunogenic potency was assessed, both in vitro using a standard human monocyte-derived dendritic cell (MoDC) reporter assay and in vivo in immune cells in the airway compartment, lung parenchyma and spleen of healthy C57BL/6 mice after pulmonary administration. RESULTS: IgG aggregates, produced during nebulization, induced a dose-dependent activation of MoDC characterized by the enhanced production of cytokines and expression of co-stimulatory markers. Interestingly, in vivo administration of high amounts of nebulization-mediated IgG aggregates resulted in a profound and sustained local and systemic depletion of immune cells, which was attributable to cell death. This cytotoxic effect was observed when nebulized IgG was administered locally in the airways as compared to a systemic administration but was mitigated by improving IgG stability during nebulization, through the addition of polysorbates to the formulation. CONCLUSION: Although inhalation delivery represents an attractive alternative route for delivering Ab to treat respiratory infections, our findings indicate that it is critical to prevent IgG aggregation during the nebulization process to avoid pro-inflammatory and cytotoxic effects. The optimization of Ab formulation can mitigate adverse effects induced by nebulization.

Inhaled amikacin versus placebo to prevent ventilator-associated pneumonia: the AMIKINHAL double-blind multicentre randomised controlled trial protocol.

INTRODUCTION: Pre-emptive inhaled antibiotics may be effective to reduce the occurrence of ventilator-associated pneumonia among critically ill patients. Meta-analysis of small sample size trials showed a favourable signal. Inhaled antibiotics are associated with a reduced emergence of antibiotic resistant bacteria. The aim of this trial is to evaluate the benefit of a 3-day course of inhaled antibiotics among patients undergoing invasive mechanical ventilation for more than 3 days on the occurrence of ventilator-associated pneumonia. METHODS AND ANALYSIS: Academic, investigator-initiated, parallel two group arms, double-blind, multicentre superiority randomised controlled trial. Patients invasively ventilated more than 3 days will be randomised to receive 20 mg/kg inhaled amikacin daily for 3 days or inhaled placebo (0.9% Sodium Chloride). Occurrence of ventilator-associated pneumonia will be recorded based on a standardised diagnostic framework from randomisation to day 28 and adjudicated by a centralised blinded committee. ETHICS AND DISSEMINATION: The protocol and amendments have been approved by the regional ethics review board and French competent authorities (Comité de protection des personnes Ouest I, No.2016-R29). All patients will be included after informed consent according to French law. Results will be disseminated in international scientific journals. TRIAL REGISTRATION NUMBERS: EudraCT 2016-001054-17 and NCT03149640.

In situ Analytical Quality Control of chemotherapeutic solutions in infusion bags by Raman spectroscopy.

Analytical Quality Control (AQC) in centralised preparation units of oncology centers is a common procedure relying on the identification and quantification of the prepared chemotherapeutic solutions for safe intravenous administration to patients. Although the use of Raman spectroscopy for AQC has gained much interest, in most applications it remains coupled to a flow injection analyser (FIA) requiring withdrawal of the solution for analysis. In addition to current needs for more rapid and cost-effective analysis, the risk of exposure of clinical staff to the toxic molecules during daily handling is a serious concern to address. Raman spectroscopic analysis, for instance by Confocal Raman Microscopy (CRM), could enable direct analysis (non-invasive) for AQC directly in infusion bags. In this study, 3 anticancer drugs, methotrexate (MTX), 5-fluorouracil (5-FU) and gemcitabine (GEM) have been selected to highlight the potential of CRM for withdrawal free analysis. Solutions corresponding to the clinical range of each drug were prepared in 5% glucose and data was collected from infusion bags placed under the Raman microscope. Firstly, 100% discrimination has been obtained by Partial Least Squares Discriminant Analysis (PLS-DA) confirming that the identification of drugs can be performed. Secondly, using Partial Least Squares Regression (PLSR), quantitative analysis was performed with mean % error of predicted concentrations of respectively 3.31%, 5.54% and 8.60% for MTX, 5-FU and GEM. These results are in accordance with the 15% acceptance criteria used for the current clinical standard technique, FIA, and the Limits of Detection for all drugs were determined to be substantially lower than the administered range, thus highlighting the potential of confocal Raman spectroscopy for direct analysis of chemotherapeutic solutions.

Inhaled antibodies: formulations require specific development to overcome instability due to nebulization.

Respiratory infections are life-threatening and therapeutic antibodies (Ab) have a tremendous opportunity to benefit to patients with pneumonia due to multidrug resistance bacteria or emergent virus, before a vaccine is manufactured. In respiratory infections, inhalation of anti-infectious Ab may be more relevant than intravenous (IV) injection-the standard route-to target the site of infection and improve Ab therapeutic index. One major challenge associated to Ab inhalation is to prevent protein instability during the aerosolization process. Ab drug development for IV injection aims to design a high-quality product, stable to different environment stress. In this study, we evaluated the suitability of Ab formulations developed for IV injection to be extended for inhalation delivery. We studied the aerosol characteristics and the aggregation profile of three Ab formulations developed for IV injection after nebulization, with two mesh nebulizers. Although the formulations for IV injection were compatible with mesh nebulization and deposition into the respiratory tract, the Ab were more unstable during nebulization than exposition to a vigorous shaking. Overall, our findings indicate that Ab formulations developed for IV delivery may not easily be repurposed for inhalation delivery and point to the requirement of a specific formulation development for inhaled Ab.

Understanding the discrimination and quantification of monoclonal antibodies preparations using Raman spectroscopy.

The use of Raman spectroscopy for analytical quality control of anticancer drug preparations in clinical pharmaceutical dispensing units is increasing in popularity, notably supported by commercially available, purpose designed instruments. Although not legislatively compulsory, analytical methods are frequently used post-preparation to verify the accuracy of a preparation in terms of identity and quantity of the drug in solution. However, while the rapid, cost effective and label free analysis achieved with Raman spectroscopy is appealing, it is important to understand the molecular origin of the spectral contributions collected from the solution of actives and excipients, to evaluate the strength and limitation for the technique, which can be used to identify and quantify either the prescribed commercial formulation, and/or the active drug itself, in personalised solutions. In the current study, four commercial formulations, Erbitux®, Truxima®, Ontruzant® and Avastin® of monoclonal antibodies (mAbs), corresponding respectively to cetuximab, rituximab, trastuzumab and bevacizumab have been used to highlight the key role of excipients in discrimination and quantification of the formulations. It is demonstrated that protein based anticancer drugs such as mAbs have a relatively weak Raman response, while excipients such as glycine, trehalose or histidine contribute significantly to the spectra. Multivariate analysis (partial least square regression and partial least square discriminant analysis) further demonstrates that the signatures of the mAbs themselves are not prominent in mathematical models and that those of the excipients are solely responsible for the differentiation of formulation and accurate determination of concentrations. While Raman spectroscopy can successfully validate the conformity of mAbs intravenous infusion solutions, the basis for the analysis should be considered, and special caution should be given to excipient compositions in commercial formulations to ensure reliability and reproducibility of the analysis.

Protein stability during nebulization: Mind the collection step!

Inhaled protein therapeutics meet a growing interest for the treatment of respiratory diseases. In liquid aerosols, proteins face stresses that may generate instabilities, such as physicochemical denaturations, aggregation and loss of activity. Monitoring protein stability is thus crucial but implies collection of aerosol droplets before analysis. Many aerosol collection methods may be used, still their interference on protein stability is unknown. In this study, we compared the impact of six aerosol samplers on the stability of a model monoclonal antibody (Ig1), aerosolized with a mesh nebulizer. Ig1 stability was assessed for aggregation and biological activity. The six aerosol samplers generated distinct aggregation profiles for Ig1 at all size scales; counts of micron-sized particles varied by a factor of 100. The heterogeneity did not impact Ig1 activity, which was not significantly changed after nebulization. To extrapolate these results, we evaluated the impact of two samplers on three other proteins. Depending on the protein, samplers gave discordant aggregation and/or activity profiles, sometimes in the reverse trend as compared to Ig1. In conclusion, aerosol samplers interfere with protein stability; this impact depends both on the samplers and the protein, highlighting the importance of using the same collection device throughout the aerosol development process.

Bioinspired hyaluronic acid and polyarginine nanoparticles for DACHPt delivery.

This work here presented provides insights over a novel biodegradable polymeric nanosystem made of hyaluronic acid and polyarginine for diaminocyclohexane-platinum (DACHPt) encapsulation. Using mild conditions based on ionic gelation technique, monodispersed blank and DACHPt-loaded nanoparticles (NP) with a size of around 200 nm and negative ζ potential (-35 mV) were obtained. The freeze-drying process was optimized to improve the stability and shelf-life of the developed nanoparticles. After reconstitution, nanoparticles maintained their size showing an association efficiency of around 70% and a high drug loading (8%). In vitro cytotoxicity studies revealed that DACHPt-loaded nanoparticles had a superior anticancer activity compared with oxaliplatin solution. The IC50 was reduced by a factor of two in HT-29 cells (IC50 39 µM vs 74 µM, respectively), and resulted almost 1.3 fold lower in B6KPC3 cells (18 µM vs 23 µM respectively). Whereas toxic effects of both drug and DACHPt-loaded nanoparticles were comparable in the A549 cell line (IC50 11 µM vs 12 µM). DACHPt-loaded nanoparticles were also able to modulate immunogenic cell death (ICD) in vitro. After incubation with B6KPC3 cells, an increase in HMGB1 (high-mobility group box 1) production associated with ATP release occurred. Then, in vivo pharmacokinetic studies were performed after intravenous injection (IV) of DACHPt-loaded nanoparticles and oxaliplatin solution in healthy mice (35.9 µg of platinum equivalent/mouse). An AUC six times higher (24 h * mg/L) than the value obtained following the administration of oxaliplatin solution (3.76 h * mg/L) was found. Cmax was almost five times higher than the control (11.4 mg/L for NP vs 2.48 mg/L). Moreover, the reduction in volume of distribution and clearance clearly indicated a more limited tissue distribution. A simulated repeated IV regimen was performed in silico and showed no accumulation of platinum from the nanoparticles. Overall, the proposed approach discloses a novel nano-oncological treatment based on platinum derivative with improved antitumor activity in vitro and in vivo stability as compared to the free drug.

In Vitro Characterization and Stability Profiles of Antibody-Fluorophore Conjugates Derived from Interchain Cysteine Cross-Linking or Lysine Bioconjugation.

Fluorescent labelling of monoclonal antibodies (mAbs) is classically performed by chemical bioconjugation methods. The most frequent labelling technique to generate antibody-fluorophore conjugates (AFCs) involves the bioconjugation onto the mAb lysines of a dye bearing an N-hydroxysuccinimide ester or an isothiocyanate group. However, discrepancies between labelling experiments or kits can be observed, related to reproducibility issues, alteration of antigen binding, or mAb properties. The lack of information on labelling kits and the incomplete characterization of the obtained labelled mAbs largely contribute to these issues. In this work, we generated eight AFCs through either lysine or interchain cysteine cross-linking bioconjugation of green-emitting fluorophores (fluorescein or BODIPY) onto either trastuzumab or rituximab. This strategy allowed us to study the influence of fluorophore solubility, bioconjugation technology, and antibody nature on two known labelling procedures. The structures of these AFCs were thoroughly analyzed by mass spectroscopy, and their antigen binding properties were studied. We then compared these AFCs in vitro by studying their respective spectral properties and stabilities. The shelf stability profiles and sensibility to pH variation of these AFCs prove to be dye-, antibody- and labelling-technology-dependent. Fluorescence emission in AFCs was higher when lysine labelling was used, but cross-linked AFCs were revealed to be more stable. This must be taken into account for the design of any biological study involving antibody labelling.

Qualitative and quantitative analysis of therapeutic solutions using Raman and infrared spectroscopy.

Anticancer drugs are prescribed and administrated to an increasing number of patients on a daily basis. As a consequence, a number of concerns have been raised about the patient health and safety in the case that the drugs administered are not at the required concentration or even worse not the correct ones. Quality control of therapeutic solutions has therefore been extensively implemented in hospital environments, in order to avoid any failure in the intense workflow faced by administering pharmacists. In the present study, infrared (IR) and Raman spectroscopy have been employed for the analysis of 3 commercially available therapeutic solutions TEVA®, MYLAN®, CERUBIDINE®, respectively containing doxorubicin, epirubicin and daunorubicin. They perfectly illustrate the analytical difficulties encountered, as these 3 chemotherapeutic drugs are isomers, hardly distinguishable with conventional approaches such as UV/VIS spectrometry. Any analytical failure to identify these molecules can lead to delays in patient treatment. While Partial Least Squares Regression analysis demonstrates that both Raman and IR can deliver satisfactory quantitative analysis in the clinical range, with respective Root Mean Square Error of Cross Validation (RMSECV) between 0.0127 - 0.0220 g·L-1 and 0.0573 - 0.0759 g·L-1, the identification rate between the 2 techniques differs substantially. Indeed, Principal Component Analysis - Factorial Discriminant Analysis (PCA-FDA) highlights that, depending on the data preprocessing applied to Raman spectra, the discrimination between the 3 drugs is decreased, with in some cases specificity and sensitivity below 50%. However, IR analysis displays encouraging results with an overall specificity and sensitivity between 99 and 100%, suggesting that reliable validation of the therapeutic solution for administration to patients can be achieved. IR and Raman spectroscopy could assist and support quality control of chemotherapeutic solutions prepared in personalised concentrations for each patient. The effective and reliable characterisation of therapeutic solutions could have a lot to offer to improve current practices in a near future.

Exploring the fate of inhaled monoclonal antibody in the lung parenchyma by microdialysis.

Therapeutic antibodies (Abs) are emerging as major drugs to treat respiratory diseases, and inhalation may provide substantial benefits for their delivery. Understanding the behavior of Abs after pulmonary deposition is critical for their development. We investigated the pharmacokinetics of a nebulized Ab by continuous sampling in lung parenchyma using microdialysis in non-human primates. We defined the optimal conditions for microdialysis of Ab and demonstrated that lung microdialysis of Ab is feasible over a period of several days. The concentration-profile indicated a two-phase non-linear elimination and/or distribution of inhaled mAbX. Lung exposition was higher than the systemic one over a period of 33 hours and above MabX affinity for its target. The microdialysis results were supported by an excellent relationship with dosages from lung extracts.

[Biosimilar monoclonal antibodies: comparative study of analytical and functional quality]. / Anticorps monoclonaux biosimilaires - Étude comparative des qualités analytique et fonctionnelle.

Biosimilars marketing authorization requires a strict demonstration of similarity with the reference antibody, through preclinical and clinical studies. This article reviews the panel of in vitro physicochemical and functional analyses, which are performed prior to clinical studies. For each critical attribute of the antibody, we detail the commonly used analytical techniques, their working principle and the type of information they may give.

TITLE: Anticorps monoclonaux biosimilaires - Étude comparative des qualités analytique et fonctionnelle. ABSTRACT: La mise sur le marché de biosimilaires requiert une démonstration stricte de la similarité avec l'anticorps de référence, au travers d'études précliniques et cliniques. Cet article synthétise l'ensemble des analyses physicochimiques et fonctionnelles mises en œuvre in vitro, préalables à la réalisation d'études cliniques. Pour chaque caractéristique critique de l'anticorps, nous avons détaillé les techniques analytiques communément employées, leur principe de fonctionnement, ainsi que le type d'informations que ces techniques permettent d'obtenir.

MAbDelivery: Administration routes for antibody therapy Third LabEx MAbImprove industrial workshop, July 2, 2015 Tours, France.

The annual "LabEx MAbImprove Industrial Workshops" are primarily intended to provide a comprehensive view about topics of interest for the pharmaceutical industry to scientists involved in research on therapeutic antibodies. The third workshop in this series, held July 2, 2015 in Tours, was dedicated to the optimization of delivery, namely all processes leading monoclonal antibodies to reach their target site. The commonly used intravenous (IV) route, although advantageous in terms of pharmacokinetics and pharmacodynamics, presents some disadvantages in terms of patients' convenience, therapeutic target access or treatment cost. Such problems led pharmaceutical companies to consider more straightforward and patient-friendly administration routes, bringing the need for specific formulations adapted to the specific inherent physicochemical challenges. In this context, the workshop provided an overview of these advances and opened discussion on new administration routes and formulation development. In the first session, the opportunities and challenges of 3 main routes of administration (IV, subcutaneous (SC), and pulmonary) were discussed, integrating protein stability issues. The next session was dedicated to medical devices intended for SC and pulmonary administration. The last session focused on specific formulations for monoclonal antibodies, particularly to successfully protect antibodies upon aerosolization, to develop highly concentrated formulations for SC administration, and to use formulation as a mean to overcome the barriers to oral protein delivery. As in the previous editions, this workshop gathered people from the academic and industrial spheres and allowed rich debates and discussions.

Ultra-filtration of human serum for improved quantitative analysis of low molecular weight biomarkers using ATR-IR spectroscopy.

Infrared spectroscopy is a reliable, rapid and cost effective characterisation technique, delivering a molecular finger print of the sample. It is expected that its sensitivity would enable detection of small chemical variations in biological samples associated with disease. ATR-IR is particularly suitable for liquid sample analysis and, although air drying is commonly performed before data collection, just a drop of human serum is enough for screening and early diagnosis. However, the dynamic range of constituent biochemical concentrations in the serum composition remains a limiting factor to the reliability of the technique. Using glucose as a model spike in human serum, it has been demonstrated in the present study that fractionating the serum prior to spectroscopic analysis can considerably improve the precision and accuracy of quantitative models based on the partial least squares regression algorithm. By depleting the abundant high molecular weight proteins, which otherwise dominate the spectral signatures collected, the ability to monitor changes in the concentrations of the low molecular weight constituents is enhanced. The Root Mean Square Error for the Validation set (RMSEV) has been improved by a factor of 5 following human serum processing with an average relative error in the predictive values below 1% being achieved. Moreover, the approach is easily transferable to different bodily fluids, which would support the development of more efficient and suitable clinical protocols for exploration of vibrational spectroscopy based ex vivo diagnostic tools.

Inhaled phage therapy: a promising and challenging approach to treat bacterial respiratory infections.

INTRODUCTION: Bacterial respiratory tract infections (RTIs) are increasingly difficult to treat due to evolving antibiotic resistance. In this context, bacteriophages (or phages) are part of the foreseen alternatives or combination therapies. Delivering phages through the airways seems more relevant to accumulate these natural antibacterial viruses in proximity to their bacterial host, within the infectious site. Areas covered: This review addresses the potential of phage therapy to treat RTIs and discusses preclinical and clinical results of phages administration in this context. Recent phage formulation and aerosolization attempts are also reviewed, raising technical challenges to achieve efficient pulmonary deposition via inhalation. Expert opinion: Overall, the inhalation of phages as antibacterial treatment seems both clinically relevant and technically feasible. Several crucial points still need to be investigated, such as phage product pharmacokinetics and immunogenicity. Furthermore, given phage-specific features, appropriate regulatory and manufacturing guidelines will need to be defined. Finally, randomized controlled clinical trials should be carried out to establish phage therapy's clinical positioning in the antimicrobial arsenal against RTIs.

Transcriptome of Cultured Lung Fibroblasts in Idiopathic Pulmonary Fibrosis: Meta-Analysis of Publically Available Microarray Datasets Reveals Repression of Inflammation and Immunity Pathways.

Heritable profibrotic differentiation of lung fibroblasts is a key mechanism of idiopathic pulmonary fibrosis (IPF). Its mechanisms are yet to be fully understood. In this study, individual data from four independent microarray studies comparing the transcriptome of fibroblasts cultured in vitro from normal (total n = 20) and IPF (total n = 20) human lung were compiled for meta-analysis following normalization to z-scores. One hundred and thirteen transcripts were upregulated and 115 were downregulated in IPF fibroblasts using the Significance Analysis of Microrrays algorithm with a false discovery rate of 5%. Downregulated genes were highly enriched for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional classes related to inflammation and immunity such as Defense response to virus, Influenza A, tumor necrosis factor (TNF) mediated signaling pathway, interferon-inducible absent in melanoma2 (AIM2) inflammasome as well as Apoptosis. Although upregulated genes were not enriched for any functional class, select factors known to play key roles in lung fibrogenesis were overexpressed in IPF fibroblasts, most notably connective tissue growth factor (CTGF) and serum response factor (SRF), supporting their role as drivers of IPF. The full data table is available as a supplement.

Screening the low molecular weight fraction of human serum using ATR-IR spectroscopy.

Vibrational spectroscopic techniques can detect small variations in molecular content, linked with disease, showing promise for screening and early diagnosis. Biological fluids, particularly blood serum, are potentially valuable for diagnosis purposes. The so-called Low Molecular Weight Fraction (LMWF) contains the associated peptidome and metabolome and has been identified as potentially the most relevant molecular population for disease-associated biomarker research. Although vibrational spectroscopy can deliver a specific chemical fingerprint of the samples, the High Molecular Weight Fraction (HMWF), composed of the most abundant serum proteins, strongly dominates the response and ultimately makes the detection of minor spectral variations a challenging task. Spectroscopic detection of potential serum biomarkers present at relatively low concentrations can be improved using pre-analytical depletion of the HMWF. In the present study, human serum fractionation by centrifugal filtration was used prior to analysis by Attenuated Total Reflection infrared spectroscopy. Using a model sample based on glycine spiked serum, it is demonstrated that the screening of the LMWF can be applied to quantify blinded concentrations up to 50 times lower. Moreover, the approach is easily transferable to different bodily fluids which would support the development of more efficient and suitable clinical protocols exploring vibrational spectroscopy based ex-vivo diagnostic tools. Revealing serum LMWF for spectral serological diagnostic applications.

Development of a drug delivery system for efficient alveolar delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin.

The high toxicity of ricin and its ease of production have made it a major bioterrorism threat worldwide. There is however no efficient and approved treatment for poisoning by ricin inhalation, although there have been major improvements in diagnosis and therapeutic strategies. We describe the development of an anti-ricin neutralizing monoclonal antibody (IgG 43RCA-G1) and a device for its rapid and effective delivery into the lungs for an application in humans. The antibody is a full-length IgG and binds to the ricin A-chain subunit with a high affinity (KD=53pM). Local administration of the antibody into the respiratory tract of mice 6h after pulmonary ricin intoxication allowed the rescue of 100% of intoxicated animals. Specific operational constraints and aerosolization stresses, resulting in protein aggregation and loss of activity, were overcome by formulating the drug as a dry-powder that is solubilized extemporaneously in a stabilizing solution to be nebulized. Inhalation studies in mice showed that this formulation of IgG 43RCA-G1 did not induce pulmonary inflammation. A mesh nebulizer was customized to improve IgG 43RCA-G1 deposition into the alveolar region of human lungs, where ricin aerosol particles mostly accumulate. The drug delivery system also comprises a semi-automatic reconstitution system to facilitate its use and a specific holding chamber to maximize aerosol delivery deep into the lung. In vivo studies in monkeys showed that drug delivery with the device resulted in a high concentration of IgG 43RCA-G1 in the airways for at least 6h after local deposition, which is consistent with the therapeutic window and limited passage into the bloodstream.