Design Considerations for Intratracheal Delivery Devices to Achieve Proof-of-Concept Dry Powder Biopharmaceutical Delivery in Mice.

PURPOSE: Intratracheal delivery and consistent dosing of dry powder vaccines is especially challenging in mice. To address this issue, device design of positive pressure dosators and actuation parameters were assessed for their impacts on powder flowability and in vivo dry powder delivery. METHODS: A chamber-loading dosator assembled with stainless-steel, polypropylene or polytetrafluoroethylene needle-tips was used to determine optimal actuation parameters. Powder loading methods including tamp-loading, chamber-loading and pipette tip-loading were compared to assess performance of the dosator delivery device in mice. RESULTS: Available dose was highest (45%) with a stainless-steel tip loaded with an optimal mass and syringe air volume, primarily due to the ability of this configuration to dissipate static charge. However, this tip encouraged more agglomeration along its flow path in the presence of humidity and was too rigid for intubation of mice compared to a more flexible polypropylene tip. Using optimized actuation parameters, the polypropylene pipette tip-loading dosator achieved an acceptable in vivo emitted dose of 50% in mice. After administering two doses of a spray dried adenovirus encapsulated in mannitol-dextran, high bioactivity was observed in excised mouse lung tissue three days post-infection. CONCLUSIONS: This proof-of-concept study demonstrates for the first time that intratracheal delivery of a thermally stable, viral-vectored dry powder can achieve equivalent bioactivity to the same powder, reconstituted and delivered intratracheally. This work may guide the design and device selection process for murine intratracheal delivery of dry powder vaccines to help progress this promising area of inhalable therapeutics.

Dextran Mass Ratio Controls Particle Drying Dynamics in a Thermally Stable Dry Powder Vaccine for Pulmonary Delivery.

PURPOSE: Thermally stable, spray dried vaccines targeting respiratory diseases are promising candidates for pulmonary delivery, requiring careful excipient formulation to effectively encapsulate and protect labile biologics. This study investigates the impact of dextran mass ratio and molecular weight on activity retention, thermal stability and aerosol behaviour of a labile adenoviral vector (AdHu5) encapsulated within a spray dried mannitol-dextran blend. METHODS: Comparing formulations using 40 kDa or 500 kDa dextran at mass ratios of 1:3 and 3:1 mannitol to dextran, in vitro quantification of activity losses and powder flowability was used to assess suitability for inhalation. RESULTS: Incorporating mannitol in a 1:3 ratio with 500 kDa dextran reduced viral titre processing losses below 0.5 log and displayed strong thermal stability under accelerated aging conditions. Moisture absorption and agglomeration was higher in dextran-rich formulations, but under low humidity the 1:3 ratio with 500 kDa dextran powder had the lowest mass median aerodynamic diameter (4.4 µm) and 84% emitted dose from an intratracheal dosator, indicating strong aerosol performance. CONCLUSIONS: Overall, dextran-rich formulations increased viscosity during drying which slowed self-diffusion and favorably hindered viral partitioning at the particle surface. Reducing mannitol content also minimized AdHu5 exclusion from crystalline regions that can force the vector to air-solid interfaces where deactivation occurs. Although increased dextran molecular weight improved activity retention at the 1:3 ratio, it was less influential than the ratio parameter. Improving encapsulation ultimately allows inhalable vaccines to be prepared at higher potency, requiring less powder mass per inhaled dose and higher delivery efficiency.

Whole-colon investigation vs. flexible sigmoidoscopy for suspected colorectal cancer based on presenting symptoms and signs: a multicentre cohort study.

BACKGROUND: Patients with suspected colorectal cancer (CRC) usually undergo colonoscopy. Flexible sigmoidoscopy (FS) may be preferred if proximal cancer risk is low. We investigated which patients could undergo FS alone. METHODS: Cohort study of 7375 patients (≥55 years) referred with suspected CRC to 21 English hospitals (2004-2007), followed using hospital records and cancer registries. We calculated yields and number of needed whole-colon examinations (NNE) to diagnose one cancer by symptoms/signs and subsite. We considered narrow (haemoglobin <11 g/dL men; <10 g/dL women) and broad (<13 g/dL men; <12 g/dL women) anaemia definitions and iron-deficiency anaemia (IDA). RESULTS: One hundred and twenty-seven proximal and 429 distal CRCs were diagnosed. A broad anaemia definition identified 80% of proximal cancers; a narrow definition with IDA identified 39%. In patients with broad definition anaemia and/or abdominal mass, proximal cancer yield and NNE were 4.8% (97/2022) and 21. In patients without broad definition anaemia and/or abdominal mass, with rectal bleeding or increased stool frequency (41% of cohort), proximal cancer yield and NNE were 0.4% (13/3031) and 234. CONCLUSION: Most proximal cancers are accompanied by broad definition anaemia. In patients without broad definition anaemia and/or abdominal mass, with rectal bleeding or increased stool frequency, proximal cancer is rare and FS should suffice.

Optimization of Spray Drying Conditions for Yield, Particle Size and Biological Activity of Thermally Stable Viral Vectors.

PURPOSE: This work examines the relevance of viral activity in the optimization of spray drying process parameters for the development of thermally stable vaccine powders. In some instances, the actual active pharmaceutical ingredient (API) is not included in the process optimization as it is deemed too costly to use until the final selection of operating conditions, however, that approach is inappropriate for highly labile biopharmaceutics. We investigate the effects of spray drying parameters on i) yield, ii) particle size and iii) viral vector activity of a mannitol/dextran encapsulated recombinant human type 5 adenoviral vector vaccine, to demonstrate the effects and magnitude of each effect on the three responses, and further show that the API must be included earlier in the optimization. METHODS: A design of experiments approach was used with response surface methodology (RSM) to optimize parameters including inlet temperature, spray gas flow rate, liquid feed rate and solute concentration in the feed. RESULTS: In general, good conditions for maintaining viral activity led to reduced yield and fewer particles of the desired size. Within the range of parameters tested, the yield varied from 50 to 90%, the percentage of ideally size particles was 10-50%, and the viral vector titre loss was 0.25-4.0 log loss. CONCLUSIONS: RSM indicates that the most significant spray drying parameters are the inlet temperature and spray gas flow rate. It was not possible to optimize all three output variables with one set of parameters, indicating that there will only be one dominant criteria for processing which in the case of viral vaccines will likely be viral vector activity.

Corrigendum: Differential biodistribution of adenoviral-vectored vaccine following intranasal and endotracheal deliveries leads to different immune outcomes.

[This corrects the article DOI: 10.3389/fimmu.2022.860399.].

Internal microstructure of spray dried particles affects viral vector activity in dry vaccines.

To maintain the activity of sensitive biologics during encapsulation by spray drying, a better understanding of deactivation pathways in dried particles is necessary. The effect of solid-air interfaces within dried particles on viral deactivation was examined with three binary excipient blends, mannitol/dextran (MD), xylitol/dextran (XD), and lactose/trehalose (LT). Particles encapsulating human serotype 5 adenovirus viral vector (AdHu5) were produced via both spray drying and acoustic levitation. The particles' internal microstructure was directly visualized, and the location of a viral vector analogue was spatially mapped within the particles by volume imaging using focused ion beam sectioning and scanning electron microscopy. The majority of the viral vector analogue was found at, or near, the solid-air interfaces. Peclet number and crystallization kinetics governed the internal microstructure of the particles: XD particles with minimal internal voids retained the highest viral activity, followed by MD particles with a few large voids, and finally LT particles with numerous internal voids exhibited the lowest viral activity. Overall, AdHu5 activity decreased as the total solid-air interfacial area increased (as quantified by nitrogen sorption). Along with processing losses, this work highlights the importance of surface area within particles as an indicator of activity losses for dried biologics.

Cryoprotective agents influence viral dosage and thermal stability of inhalable dry powder vaccines.

Increasing viral dosage within dry powder vaccines reduces the powder mass required to elicit an immune response through pulmonary delivery. This work analyzes how cryoprotective agents affect viral activity, particle properties and thermal stability of a spray dried, inhalable vaccine vector under high viral loading. Stock suspensions of a human serotype 5 adenovirus (AdHu5) vector in either neat phosphate buffered saline (PBS), 10% glycerol in PBS, or 5% trehalose in PBS were added to a mannitol-dextran formulation prior to spray drying. At high viral loading, spray dried powder containing glycerol had a viral titre log loss of 2.8 compared to 0.7 log loss using neat PBS. Powders containing glycerol had a lower glass transition temperature (Tg) compared to all other formulations, permitting greater viral mobility and exposure to heat damage. Inclusion of glycerol also promoted particle cohesion during spray drying and lower yields. Using 5% trehalose as a cryogenic alternative, viral powders had a viral log loss of 1.5 and the highest displayed thermal stability over time. Additionally, trehalose-containing powders had smaller particles with lower water moisture content and higher powder yield compared to glycerol-containing powders. These findings demonstrate the importance of cryoprotective agent selection when developing thermostable vaccine powders.

Differential Biodistribution of Adenoviral-Vectored Vaccine Following Intranasal and Endotracheal Deliveries Leads to Different Immune Outcomes.

Infectious diseases of the respiratory tract are one of the top causes of global morbidity and mortality with lower respiratory tract infections being the fourth leading cause of death. The respiratory mucosal (RM) route of vaccine delivery represents a promising strategy against respiratory infections. Although both intranasal and inhaled aerosol methods have been established for human application, there is a considerable knowledge gap in the relationship of vaccine biodistribution to immune efficacy in the lung. Here, by using a murine model and an adenovirus-vectored model vaccine, we have compared the intranasal and endotracheal delivery methods in their biodistribution, immunogenicity and protective efficacy. We find that compared to intranasal delivery, the deepened and widened biodistribution in the lung following endotracheal delivery is associated with much improved vaccine-mediated immunogenicity and protection against the target pathogen. Our findings thus support further development of inhaled aerosol delivery of vaccines over intranasal delivery for human application.

Aerosol delivery, but not intramuscular injection, of adenovirus-vectored tuberculosis vaccine induces respiratory-mucosal immunity in humans.

BackgroundAdenovirus-vectored (Ad-vectored) vaccines are typically administered via i.m. injection to humans and are incapable of inducing respiratory mucosal immunity. However, aerosol delivery of Ad-vectored vaccines remains poorly characterized, and its ability to induce mucosal immunity in humans is unknown. This phase Ib trial evaluated the safety and immunogenicity of human serotype-5 Ad-vectored tuberculosis (TB) vaccine (AdHu5Ag85A) delivered to humans via inhaled aerosol or i.m. injection.MethodsThirty-one healthy, previously BCG-vaccinated adults were enrolled. AdHu5Ag85A was administered by single-dose aerosol using Aeroneb Solo Nebulizer or by i.m. injection. The study consisted of the low-dose (LD) aerosol, high-dose (HD) aerosol, and i.m. groups. The adverse events were assessed at various times after vaccination. Immunogenicity data were collected from the peripheral blood and bronchoalveolar lavage samples at baseline, as well as at select time points after vaccination.ResultsThe nebulized aerosol droplets were < 5.39 µm in size. Both LD and HD of AdHu5Ag85A administered by aerosol inhalation and i.m. injection were safe and well tolerated. Both aerosol doses, particularly LD, but not i.m., vaccination markedly induced airway tissue-resident memory CD4+ and CD8+ T cells of polyfunctionality. While as expected, i.m. vaccination induced Ag85A-specific T cell responses in the blood, the LD aerosol vaccination also elicited such T cells in the blood. Furthermore, the LD aerosol vaccination induced persisting transcriptional changes in alveolar macrophages.ConclusionInhaled aerosol delivery of Ad-vectored vaccine is a safe and superior way to elicit respiratory mucosal immunity. This study warrants further development of aerosol vaccine strategies against respiratory pathogens, including TB and COVID-19.Trial registrationClinicalTrial.gov, NCT02337270.FundingThe Canadian Institutes for Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada funded this work.

Validation of a diffusion-based single droplet drying model for encapsulation of a viral-vectored vaccine using an acoustic levitator.

Development of thermally stable spray dried viral-vectored vaccine powders is dependent on the selection of a proper excipient or excipient blend for encapsulation, which can be a time and resource intensive process. In this work, a diffusion-based droplet drying model was developed to compute droplet drying time, size, and component distribution. The model predictions were validated using an acoustic levitator to dry droplets containing protein-coated or fluorescently labelled silica nanoparticles (as adenoviral vector analogues) and a range of excipient blends. Surface morphology of the dried particles was characterized by atomic force microscopy and the distribution of silica nanoparticles was quantified by confocal microscopy. The modelled distributions of adenovirus agreed with the microscopy results for three mannitol/dextran excipient blends with varying molecular weight dextrans, verifying the equations and assumptions of the model. Viral vector activity data for adenovirus in a range of (poly)saccharide/sugar alcohol formulations were also compared to the model outputs, suggesting that viral activity decreases when the model predicts increasing adenovirus concentrations near the air-solid interface. Using a validated model with excipient property inputs that are readily available in the literature can facilitate the development of viral-vectored vaccines by identifying promising excipients without the need for experimentation.

Solvent-free production of thermoplastic lignocellulose from wood pulp by reactive extrusion.

A novel acylation approach suited to rapid bulk thermoplasticization of lignocellulose without solvents was previously demonstrated by the authors in benchtop batch studies. The method relies upon a benzethonium chloride/sulfuric acid functionalizing agent at low concentrations to act as a wetting agent for the wood pulp, similar to an ionic liquid, yet binds to the lignocellulose ester as a flow aid in the final thermoplastic. The present investigation evaluates the approach in a residence time-limited (45-90 s) continuous twin-screw reactor, where intensive mixing and heat were found to yield high acylation. The modified lignocellulose exhibited desired thermoplasticity by being melt moldable without the need for plasticizers and maintained much of the excellent stiffness of cellulose, demonstrating a maximum flexural modulus of 5.4 GPa and tensile modulus of 1.8 GPa. The influence of extrusion conditions on thermoplasticity was examined by a Design of Experiments (DOE) analysis.

Spray dried VSV-vectored vaccine is thermally stable and immunologically active in vivo.

Effective vaccine delivery and coverage to rural and resource-poor countries is hindered by the dependence on cold chain storage. As such, developments of cold chain-free technologies are highly sought. Although spray dried adenoviral vectors have shown long term stability at ambient temperatures and relatively low humidity, it remains to be determined whether similar excipient formulations are applicable to other viral vectors. To address this, we have spray dried vesicular stomatitis virus (VSV)-vectors with a panel of well-characterized sugar excipients to determine the optimal formulation for vector stabilization. Upon reconstitution, we show that trehalose conferred superior stability of VSV both in vitro and in vivo. Importantly, following cold chain-free storage at elevated temperatures at 37 °C for 15 days, we show that a VSV-vectored vaccine retains its in vivo immunogenicity, whereas a liquid control completely lost its immune-stimulating ability. Our results provide foundational evidence that spray drying with properly tested excipients can stabilize viral vectors such as VSV, allowing them to be stored long-term at elevated temperatures without dependency on cold chain conditions.

Effect of Shear Stresses on Adenovirus Activity and Aggregation during Atomization To Produce Thermally Stable Vaccines by Spray Drying.

Considering the substantive potential benefits of thermally stable dry powder vaccines to public health, causes for inactivation of their sensitive viral vectors during preparation require intensive study. The focus of this work was atomization of suspensions containing encapsulating excipients and a human type 5 adenovirus, involving a detailed investigation of shear stresses in the nozzle of a spray dryer. Samples were sprayed at 25 °C into falcon tubes and immediately evaluated for viral activity by in vitro testing, minimizing the confounding of thermal effects on the deactivation of the virus, although interfacial stresses could not be decoupled from shear stresses. Despite the expectations of only virus deactivation with ever-increasing shear stresses in the spray nozzle, some conditions were found to show better activity than the positive control, leading to investigations of viral aggregation. It was found that the adenovirus experienced minor aggregation when mixed with the excipient solutions, which was reversed by subjecting samples to moderate shear conditions in the spray nozzle. At very high shear rates, the activity diminished again because of damage to the viral capsid fibers, which also led to the production of new aggregates after atomization. Despite these findings, activity losses caused by shear were small compared to the overall spray drying process loss. However, formulation composition, solution viscosity, and process conditions should be considered carefully for optimization because of their impact on aggregation. This is the first known report comparing shear, aggregation, and biological activity loss during the atomization step of spray drying viral vaccines.

The National Bowel Cancer Project: social deprivation is an independent predictor of nonrestorative rectal cancer surgery.

PURPOSE: This study was designed to assess the impact of social deprivation on rates of abdominoperineal excision of the rectum in the United Kingdom. METHODS: Data were extracted from the Association of Coloproctology of Great Britain and Ireland Colorectal Cancer Database (2000-2005). Social deprivation was assessed by using the Index of Multiple Deprivation (2004) score. Logistic regression was performed to identify independent predictors of nonrestorative surgery. RESULTS: A total of 12,128 patients underwent anterior resection or abdominoperineal excision for Dukes A-C cancer in 101 centers; 2,625 patients (21.6 percent) underwent abdominoperineal excision (median, 20.8 (interquartile range, 16.5-27.9) percent per unit). Abdominoperineal excision rates decreased from 24.3 to 18.2 percent (P < 0.001) and varied between the least and most deprived groups from 18 to 26.4 percent, respectively (P < 0.001). Independent predictors of abdominoperineal excision were: year of surgery (odds ratio = 0.855 per year increase, P < 0.001), female vs. male gender (odds ratio = 0.82, P < 0.001), use of neoadjuvant radiotherapy (odds ratio = 2.4, P < 0.001), and social deprivation (most vs. least deprived: odds ratio = 1.638, P < 0.001). CONCLUSIONS: Abdominoperineal excision rates vary considerably between centers. Gender and deprivation status independently predict formation of a permanent stoma. These results have important implications for intercenter comparisons of surgical quality and may suggest inequalities in health care provision.

Acoustic levitation as a screening method for excipient selection in the development of dry powder vaccines.

Spray drying is emerging as a promising technique to produce thermally stable powder vaccines containing viral vectors. One of the most important factors in developing dry powder vaccines is the selection of the excipient carrier, however this process is time intensive, and uses large amounts of costly viral material. In this work, an acoustic levitator modified with a hot air delivery system was evaluated for its ability to mimic spray drying and acts as a screening method for excipient selection. The ability of three binary excipient blends to stabilize a human type 5 adenovirus was evaluated through an in vitro activity assay based on the expression of green fluorescent protein. Dried particle size and shape, glass transition temperature, moisture content, and crystallinity of powders produced by levitation and spray drying were compared. The particles created in the acoustic levitator under moderate heat can be considered representative of the powders that would be produced via spray drying. Viral vector titre losses differ between the methods, however, the trends with respect to excipient performance remain the same. Key material characteristics such as particle morphology and thermal properties are conserved when using the levitator. The acoustic levitator is a good starting point for dry powder vaccine development, and can be used to identify promising excipients while consuming minimal amounts of the viral vector.

Stabilization of HSV-2 viral vaccine candidate by spray drying.

This work demonstrates that an HSV-2 candidate vaccine can be thermostabilized by spray drying to reduce cold chain demands. This work is also to optimize the process responses by varying spray dry parameters for pre-screened suitable excipients; and to determine the validity of current prescreening techniques. Vaccine activity losses were measured by in vitro plaque forming assay with Vero cell line. An accelerated storage condition of 45 °C for 10 days was used to determine spray dried sample stability. Prescreening studies demonstrated that trehalose and sucrose were superior to other tested excipients spray dry thermal stabilization of HSV-2. Subsequent optimization by design of experiments (DOE) of activity responses to spray dry parameter changes demonstrated significant differences between trehalose and sucrose for stability of the viral vaccine. Model parameters included the drying conditions inlet temperature, spray gas flow rate, and solids concentration for the model responses of vaccine stabilization. Trehalose was an effective and robust stabilizing excipient for spray drying HSV-2 vaccine. In contrast, stabilization by sucrose was greatly dependent on the spray dry process parameters. These DOE differences indicated inadequate excipient selection by prescreening methods and the variability demonstrated current prescreening techniques may not be adequate for determining optimal excipients.

Crystal structure and chemistry of tricadmium digermanium tetra-arsenide, Cd3Ge2As4.

A cadmium germanium arsenide compound, Cd3Ge2As4, was synthesized using a double-containment fused quartz ampoule method within a rocking furnace and a melt-quench technique. The crystal structure was determined from single-crystal X-ray diffraction (SC-XRD), scanning and transmission electron microscopies (i.e. SEM, STEM, and TEM), and selected area diffraction (SAD) and confirmed with electron backscatter diffraction (EBSD). The chemistry was verified with electron energy loss spectroscopy (EELS).

Excipient selection for thermally stable enveloped and non-enveloped viral vaccine platforms in dry powders.

Two enveloped viral vectors, vesicular stomatitis virus and influenza virus, and a non-enveloped viral vector, human adenovirus type 5, were encapsulated by spray drying to enhance thermal stability.Results with these candidates led to the hypothesis that stability performance of chosen excipients may be less virus-specific, as previously postulated in the literature, and more differentiated based on whether the virus has a lipid envelope. Spray dried samples were characterized for their thermal properties, RNA viability and in vitro viral activity after storage at 37 °C for up to 30 days or at 45 °C for up to 3 days. The enveloped viral vectors, as a group, were more thermally stable in trehalose while the non-enveloped viral vector showed higher activity with mannitol as the primary excipient in blends. Trehalose shows strong hydrogen bonds with the envelope's lipid membrane than the other carbohydrates, more effectively replacing water molecules while maintaining the fluidity of the membrane. Conversely, the small size of mannitol molecules was attributed to the more effective hydrogen bonding between water and the protein capsid of non-enveloped viral vectors. In all cases, a matrix with high glass transition temperature contributed to thermal stabilization through vitrification. This work suggests that carbohydrate stabilizer selection may be more dependent on the envelope rather than the specific viral vector, which, if universally true, will provide a guideline for future formulation development.

Consecutive Spray Drying to Produce Coated Dry Powder Vaccines Suitable for Oral Administration.

Current global vaccination programs are challenged by costs associated with vaccine cold chain storage and administration. A solid, thermally stable oral dosage form for vaccines would alleviate these costs but is difficult to produce due to general vaccine instability and the complication of bypassing the gastric barrier. We developed a novel consecutive spray drying method that is suitable for use with biologics and employs Eudragit L100 polymer as the enteric coating. More specifically, in step 1, recombinant replication deficient human type-5 adenovirus and vesicular stomatitis virus were encapsulated by spray drying with sugars from a water solution, and in step 2, the microparticles from step 1 were suspended in ethanol with Eudragit and spray dried again. Up to 25% of the starting material was fully encapsulated within the enteric coating, and encapsulation efficiency was largely dependent on spray gas flow rate and the solids concentration in the feed. After step 2, the coated vaccine-sugar particles maintained their thermostability and were slightly larger in size with a rugous surface morphology compared to the particles produced in step 1. The coated particles retained viral vector activity in vitro after 15 min incubation in 1 M HCl (simulating the stomach environment) and anhydrous ethanol (to dissolve the Eudragit outer shell). The production of dry, orally administered vaccine particles from consecutive spray drying offers the potential to remedy a number of vaccine storage, transportation, and administration limitations.

Spray dried human and chimpanzee adenoviral-vectored vaccines are thermally stable and immunogenic in vivo.

Cold chain-free vaccine technologies are needed to ensure effective vaccine delivery and coverage, particularly in resource-poor countries. However, the immunogenicity and thermostability of spray dried live viral vector-based vaccines such as recombinant adenoviral-vectored vaccines remain to be investigated. To address this issue, we have spray dried human adenoviral (AdHu5)- and chimpanzee adenoviral (AdCh68)-vectored tuberculosis vaccines in a mannitol and dextran matrix. Spray dried powders containing these two vaccines display the morphologic and chemical properties desired for long-term thermostability and vaccination. Upon reconstitution, they effectively transfected the cells in vitro with relatively small losses in viral infectivity related to the spray drying process. Following in vivo vaccination, AdHu5- and AdCh68-vectored vaccines were as immunogenic as the conventional fresh, cryopreserved liquid vaccine samples. Of importance, even after cold chain-free storage, at ambient temperatures and relatively low humidity for 30 and 90days, the vaccines retained their in vivo immunogenicity, while the liquid vaccine samples stored under the same conditions lost their immune-activating capability almost entirely. Our results support further development of our spray drying technologies for generating thermally stable adenoviral-vectored and other viral-vectored vaccines.