Effect of Texture and Surface Chemistry on Deagglomeration and Powder Retention in Capsule-Based Dry Powder Inhaler.

Pulmonary delivery systems should administer a high dose of the required formulation with the designated dry powder inhaler (DPI) to achieve therapeutic success. While the effects of device geometry and individual components used on powder dispersion are described in literature, potential effects of DPI surface properties on powder retention within the device and deagglomeration have not been adequately studied, but could impact inhalation therapy by modifying the available dose. For this, inner parts of a model DPI were modified by plasma treatment using various processes. Since both the hydrophilic-hydrophobic and structural properties of the surface were altered, conclusions can be drawn for future optimization of devices. The results show that surface topography has a greater influence on powder deposition and deagglomeration than hydrophilic or hydrophobic surface modification. The most important modification was observed with an increased rough surface texture in the mouth piece, resulting in lower powder deposition in this part (from 5 to 1% quantified amount of powder), without any change in powder deagglomeration compared to an untreated device. In summary, increasing the surface roughness of DPI components in the size range of a few nanometers could be an approach for future optimization of DPIs to increase the delivered dose.

Process parameters of microsphere preparation based on propylene carbonate emulsion-precursors.

AIM: This study aimed for a detailed understanding of the impact of different process parameters involved during celecoxib-loaded microsphere preparation based on propylene carbonate emulsion-precursors. METHODS: Microspheres were prepared by a modified emulsification-solvent extraction method. Performed investigations included polymer solubility and viscosity, microsphere size, morphology and stability, propylene carbonate content as well as celecoxib solid state, content and release. RESULTS: Rough-walled round microspheres with sizes between 21 µm and 122 µm and an internal sponge-like structure filled with residual propylene carbonate (content between 1.9 ± 0.1% and 6.7 ± 0.5% w/w) were obtained. Encapsulation efficiencies varied between 28.3 ± 0.1% and 66.8 ± 1.0%. The release rates were affected by the polymer concentration, the emulsion phase ratio and the residual propylene carbonate content (t50% varied between 2.2 hours and 23.4 hours). CONCLUSIONS: This study identified the most relevant process parameters for this new preparation method for the model drug celecoxib.

Cyclodextrin Complex Formation with Water-Soluble Drugs: Conclusions from Isothermal Titration Calorimetry and Molecular Modeling.

Cyclodextrin (CD) complexes are frequently used for enhancing the solubility or absorption of poorly water-soluble drugs. On the contrary, little is known about their complex formation with water-soluble drugs. Here, we have studied the interaction between 2-hydroxypropyl ß-CD (HPßCD) and three water-soluble drugs, namely naloxone (NX), oxycodone (OC), and tramadol (TR), by isothermal titration calorimetry (ITC) combined with molecular modeling in view of the potential impact on drug release. The results showed that the complex formation of HPßCD with all three drugs occurs spontaneously. The complexes formed with NX and OC were found to be 2NX:1HPßCD and 3OC:2HPßCD, respectively. TR was found to form 2 complexes with HPßCD; of 1:2 and 1:1 complexation ratios. The binding of HPßCD to NX was greater than to OC due to the higher hydrophobicity of the structure of the former. Moreover, the binding affinity of HPßCD to TR was higher than to OC, which indicated the effect of the higher flexibility of the guest in increasing the binding affinity. In vitro drug release experiments from the various complexes revealed a significant impact of the stoichiometry of the complex on the release profiles. Accordingly, the co-administration of cyclodextrins with water-soluble drugs should be closely monitored, as it may result in unintentional complex formation that can potentially impact the drugs' gastrointestinal absorption.

Muscle Tissue as a Surrogate for In Vitro Drug Release Testing of Parenteral Depot Microspheres.

Despite the importance of drug release testing of parenteral depot formulations, the current in vitro methods still require ameliorations in biorelevance. We have investigated here the use of muscle tissue components to better mimic the intramuscular administration. For convenient handling, muscle tissue was used in form of a freeze-dried powder, and a reproducible process of incorporation of tested microspheres to an assembly of muscle tissue of standardized dimensions was successfully developed. Microspheres were prepared from various grades of poly(lactic-co-glycolic acid) (PLGA) or ethyl cellulose, entrapping flurbiprofen, lidocaine, or risperidone. The deposition of microspheres in the muscle tissue or addition of only isolated lipids into the medium accelerated the release rate of all model drugs from microspheres prepared from ester-terminated PLGA grades and ethyl cellulose, however, not from the acid-terminated PLGA grades. The addition of lipids into the release medium increased the solubility of all model drugs; nonetheless, also interactions of the lipids with the polymer matrix (ad- and absorption) might be responsible for the faster drug release. As the in vivo drug release from implants is also often faster than in simple buffers in vitro, these findings suggest that interactions with the tissue lipids may play an important role in these still unexplained observations.

Elevated level of cerebrospinal fluid and systemic chemokine CCL5 is a predictive biomarker of clinical outcome after aneurysmal subarachnoid hemorrhage (aSAH).

BACKGROUND: Pathophysiology of aneurysmal subarachnoid hemorrhage (aSAH) is highly complex. Bleeding from ruptured aneurysm causes increase in intracranial pressure that disrupts blood-brain barrier leading to infiltration of peripheral immune cells. Interactions between the infiltrated leukocytes and the resident brain cells in the injured tissue mainly determine the delayed tissue damage. Recruitment of leukocytes in the injured brain is mainly mediated by the chemokines. Chemokine C-C motif ligand 5 (CCL5) is a potent pro-inflammatory chemokine shown to be upregulated in preclinical SAH studies. However, detailed clinical investigations exploring the association of cerebrospinal fluid (CSF) and systemic CCL5 and post-aSAH complications and clinical outcome are still lacking. This study investigated CSF and systemic CCL5 after aSAH and its association with clinical outcome and post-aSAH complications. METHODS: CSF and serum from control and aSAH patients were obtained after centrifugation of the CSF and peripheral blood, and were preserved at -80 °C until quantification by an enzyme-linked immunoassay. Patient pertinent data, post-aSAH complications and clinical outcome (modified Rankin scale [mRS] and Glasgow outcome scale [GOS]) were retrieved from patient records. RESULTS: A significant increase in CSF and serum CCL5 levels was observed on post-aSAH day 1 and day 7 compared to control patients. Dichotomization of patients to poor (mRS 3-6 or GOS 1-3) and good (mRS 0-2 or GOS 4-5) clinical outcomes showed significantly higher serum CCL5 levels in patients with good clinical outcome at discharge, but lower CSF CCL5 levels. Interestingly, significantly lower serum CCL5 levels were observed on post-aSAH day 7 in patients who have additional intracerebral bleeding or the patients who developed chronic hydrocephalus or pneumonia. Whereas, CSF CCL5 levels significantly increased on post-aSAH day 1 in patients developing chronic hydrocephalus, delayed ischemic neurological deficits and intraventricular hemorrhage. CSF CCL5 levels on post-aSAH day 1 were correlated with poor clinical outcome, however, serum CCL5 levels on post-aSAH day 7 were correlated with good clinical outcome. CONCLUSION: Systemic and CSF CCL5 levels were elevated after aSAH and levels of serum CCL5 on day 7 were associated independently with clinical outcome (GOS and mRS) at discharge. Therapeutic approaches targeting CCL5 might be beneficial in aSAH.

Cascade Impactor Performance of Commercial pMDI Formulations Using Modified Induction Ports.

The induction port (IP) for aerosol analysis with the Next Generation Pharmaceutical Impactor as monographed in the United States and European pharmacopoeia (USPIP) lacks physiological relevance, which, amongst other reasons, has been identified as critical for the predictability of in vitro aerosol data to lung deposition observed in vivo. In this publication, we report the impact of replacing the USPIP with two modified induction ports, which were designed based around geometries derived from a computer tomographic scan of a human trachea and the distal section of the USPIP. Test formulations were selected on the basis of availability of in vivo lung deposition data so that results obtained in vitro could be evaluated for their predictability. All formulations assessed showed increased deposition in the modified induction ports, and different mechanisms of particle deposition have been identified. In vitro predictions of the lung deposition were found to correlate well with the in vivo observations reported using the modified induction ports. Furthermore, the quality of the correlation was found superior to the one achieved with the USPIP with an average deviation of the predicted from observed values (n = 10) of 6 ± 4, 12 ± 6, and 16 ± 6% for the modified induction ports (mIP and mIPext) and the USPIP, respectively, when using a fine particle fraction (FPF) cutoff value of 5 µm. Using a FPF cutoff value of 3 µm yielded a more accurate in vitro-in vivo correlation with an average deviation of the predicted from observed values of 5 ± 4, 7 ± 5, and 8 ± 4% for the mIP, mIPext, and USPIP, respectively. For both FPF size cutoff values, the mIP yielded the most accurate in vitro-in vivo correlation.

Nanoparticles' properties modify cell type-dependent distribution in immune cells.

Polymeric nanoparticles can passively target inflamed tissues. How their physicochemical properties affect their distribution pattern among the infiltrating immune cells is unknown. Polyvinyl acetate nanoparticles with different particle size (100 and 300 nm) and surface charge (cationic, non-ionic, and anionic) were prepared and incubated with either LPS-activated or unactivated murine splenocytes. Nanoparticle association with macrophages, dendritic cells, neutrophils, B and T cells was investigated using flow cytometry. Cells associated with nanoparticles as follows: cationic>anionic>non-ionic and 300 nm > 100 nm. 40% of ionic nanoparticles were distributed among unactivated macrophages, reduced to 25% for activated macrophages. 60% of 100 nm and 40% of 300 nm non-ionic nanoparticles were distributed among unactivated and LPS-activated macrophages. This study highlights that particles' physicochemical properties impact the number of nanoparticles associating with immune cells more than their distribution pattern, which is principally determined by the cell activation state. This suggests a disease-dependent distribution pattern for therapeutic nanoparticles.

Elevated Systemic IL-10 Levels Indicate Immunodepression Leading to Nosocomial Infections after Aneurysmal Subarachnoid Hemorrhage (SAH) in Patients.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (SAH) is a highly complex disease with very high mortality and morbidity. About one-third of SAH patients suffer from systemic infections, predominantly pneumonia, that can contribute to excess mortality after SAH. Immunodepression is probably the most important mechanism leading to infections. Interleukin-10 (IL-10) is a master regulator of immunodepression, but it is still not clear if systemic IL-10 levels contribute to immunodepression, occurrence of infections and clinical outcome after SAH. METHODS: This explorative study included 76 patients with SAH admitted to our neurointensive care unit within 24 h after ictus. A group of 24 patients without any known intracranial pathology were included as controls. Peripheral venous blood was withdrawn on day 1 and day 7 after SAH. Serum was isolated by centrifugation and stored at -80 °C until analysis. Serum IL-10 levels were determined by enzyme-linked immunoassay (ELISA). Patient characteristics, post-SAH complications and clinical outcome at discharge were retrieved from patients' record files. RESULTS: Serum IL-10 levels were significantly higher on day 1 and day 7 in SAH patients compared to controls. Serum IL-10 levels were significantly higher on day 7 in patients who developed any kind of infection, cerebral vasospasm (CVS) or chronic hydrocephalus. Serum IL-10 levels were significantly higher in SAH patients discharged with poor clinical outcome (modified Rankin Scale (mRS) 3-6 or Glasgow Outcome Scale (GOS) 1-3). CONCLUSION: Serum IL-10 might be an additional useful parameter along with other biomarkers to predict post-SAH infections.

Formulation of Ketoconazole Nanocrystal-Based Cryopellets.

Commercial development of nanosuspensions for oral drug delivery generally involves a drying step which aims to generate a stable product that rapidly releases the nanocrystals once rehydrated and can be easily processed into a final dosage form (e.g., filled into hard capsule). Cryopelletisation is a freeze drying technique allowing the production of lyophilised micrometric spheres with good flowability. In the current work, the possibility to formulate redispersible ketoconazole nanocrystal-based cryopellets able to withstand intensive handling was investigated. Cryopellets were generated by first freezing regular droplets of nanosuspension using liquid nitrogen followed by water removal by sublimation in a standard freeze dryer. Low-friable cryopellets (< 1%) were produced by embedding the nanocrystals in a stabilizing hydroxypropyl cellulose SSL grade matrix, thus proving that these structures can withstand intensive handling. A threshold quantity of hydroxypropyl cellulose SSL grade (5/20 hydroxypropyl cellulose SSL grade-to-drug mass ratio) was required in combination with D-α-tocopherol polyethylene glycol 1000 succinate (vitamin E TPGS) to successfully recover the nanocrystals over storage. A further addition of micronised crospovidone has shown a positive effect on the dissolution performance of cryopellets. Altogether, this study demonstrated that the design of cryopellets combining the strengths of freeze-dried powders (porous internal structure, low residual humidity) and pellets (free-flowing units, mechanical resistance during handling) can potentially improve the nanocrystal's redispersibility compared with other drying techniques while facilitating the downstream processing.

Modulation of Nanostructure-Based Lipopolysaccharide Active Immunotherapy in Cancer: Size and Composition Determine Short- and Long-Term Tolerability.

Despite holding promise for cancer immunotherapy, the strong pro-inflammatory properties of lipopolysaccharide (LPS) also account for severe localized and systemic side effects, restricting its administrable dosage and the possibility of chronic dosing. Herein, we exploited the surface-active properties of LPS molecules to develop pathogen-mimicking LPS-decorated nanostructures with different compositions (lipid nanoemulsion vs polymeric nanospheres) and sizes (volumetric mean diameters of 100 nm vs 700 nm). The formulations were tested in cell culture for their immunostimulatory properties and in vivo against a murine subcutaneous colorectal cancer model. While all nanostructures resulted in similar levels of apoptotic cell death in tumor cells cultured with splenocytes, both the size and the composition of the nanostructures were found to govern the short- and long-term tolerability of LPS-based immunotherapy in vivo. The toxicity-related end point of the animal trials was decided upon in the case of a body condition score (BCS) of 1 and poor hair coat, or more than 15% loss of the original body weight, while in the absence of long-term intolerability, the experiments were terminated in the case of full remission or once the tumor surpassed a volume of 1000 mm3. Size was an important determinant of short-term tolerability, with larger particles being associated with higher incidence and extent of localized necrosis (3-6% necrotic surface area). Nanostructure composition, on the other hand, predominantly governed the long-term systemic tolerability. Within this context, the higher affinity of LPS molecules to the triglyceride core of the nanoemulsion compared to the polymeric matrix significantly improved the tolerability of the former over time. In fact, the mean survival estimate of the animals treated with small LPS nanoemulsion (LPS-NE (small)) was at least 42 days longer than that of the LPS and the LPS-decorated polymeric nanoparticle (LPS-NP) groups. Unlike other treatment groups, the experiments on 80% of the animals in LPS-NE (small) were terminated due to complete remission or tumor volume >1000 mm3. While a better understanding of these findings requires a larger scale, mechanistic-oriented trial on larger animal models, they indicate the role of nanostructures as beyond the carriers of the incorporated immunotherapeutic cargos. This highlights the importance of a wise selection of nanoparticle composition and a purposeful tuning of their physicochemical properties to enhance the safety profile and improve the eventual immunotherapeutic outcome.

Silk Fibroin as a Novel Alcohol-Resistant Excipient for Sustained-Release Tablet Formulation.

Concomitant intake of alcoholic beverages with sustained-release oral formulations may potentially lead to dose dumping. Alcohol-resistance testing is currently a requirement for the manufacturers by regulatory authorities. Silk fibroin produced by silkworm Bombyx mori is suggested in this work as a potential alternative to a narrow spectrum of alcohol-resistant excipients. Oxycodone HCl, tramadol HCl, and flurbiprofen were selected as model drugs and formulated with regenerated silk fibroin either in the form of an amorphous solid dispersion or as a physical mixture and compressed into tablets. Preliminary compactability and tampering-resistance studies were performed. The ethanol-resistance was tested in media containing 5%, 10%, 20%, or 40% (v/v) ethanol concentration. Drug release profiles were compared using f2 similarity factor. Good mechanical tampering-resistance (tensile strength of 14.6 MPa at 400 MPa compression pressure) was obtained for tablets compressed from physical mixture. Tablets compressed from amorphous solid dispersion had lower tensile strength (2.2 MPa) but showed chemical tampering-resistance to extraction by pure ethanol (7.1% of oxycodone HCl after 24 h). Drug release is controlled predominantly by swelling and diffusion. With an increasing ethanol concentration in release medium, the tablets swelled less, resulting in a slower release. This trend was similar for all tested drugs and for both physical states formulations. No dose dumping occurred in the presence of ethanol; therefore, silk fibroin could be considered as an alternative alcohol-resistant excipient for sustained release application.

Systemic High-Mobility Group Box-1: A Novel Predictive Biomarker for Cerebral Vasospasm in Aneurysmal Subarachnoid Hemorrhage.

OBJECTIVES: To investigate the release of proinflammatory damage-associated molecular pattern molecule "high-mobility group box-1" in the serum of patients after aneurysmal subarachnoid hemorrhage and its association with cerebral vasospasm. DESIGN: Retrospective observational study. SETTING: University hospital. PATIENTS: Aneurysmal subarachnoid hemorrhage patients admitted within 24 hours of ictus. INTERVENTIONS: Standard subarachnoid hemorrhage treatment after clipping or coiling of aneurysm. MEASUREMENTS AND MAIN RESULTS: We enrolled 53 aneurysmal subarachnoid hemorrhage patients from which peripheral venous blood was withdrawn on days 1, 3, 5, 7, 9, 11, and 13 and once from the controls to obtain the serum. Serum high-mobility group box-1 concentration was quantified by enzyme-linked immunosorbent assay. Serum interleukin-6 and peripheral blood leukocytes were also determined over the first 2 weeks after subarachnoid hemorrhage. Patients' data were recorded prospectively. Serum high-mobility group box-1 was significantly elevated in subarachnoid hemorrhage patients from day 1 to day 13 when compared with nonsubarachnoid hemorrhage patients (p < 0.05). Patients with cerebral vasospasm showed significantly higher high-mobility group box-1 starting from day 1 to day 13 when compared with patients without cerebral vasospasm. Cumulative levels of high-mobility group box-1 showed significant correlation with peripheral blood leukocytes and interleukin-6 levels (p < 0.05). Receiver operating characteristic curve analysis showed that serum high-mobility group box-1 level at admission may be a predictive biomarker for cerebral vasospasm with a sensitivity of 59% and a specificity of 82% at a cutoff value of 5.6 ng/mL. CONCLUSIONS: Serum high-mobility group box-1 is differentially elevated after subarachnoid hemorrhage. Serum high-mobility group box-1 levels were elevated early after subarachnoid hemorrhage (day 1) and remained significantly high until day 13 in patients who developed cerebral vasospasm. Our data suggest that serum high-mobility group box-1 may be a predictive biomarker for the detection of CVS.

TLR4-Based Immunotherapeutics in Cancer: A Review of the Achievements and Shortcomings.

Toll-like Receptor 4 (TLR4) agonists have had a long journey in the field of cancer immunotherapy. Nevertheless, despite the remarkable number of the TLR4 ligands that have gone through various preclinical and clinical stages, only two (Bacillus Calmette-Guérin (BCG) and monophosphoryl lipid A (MPLA)) have hitherto obtained the FDA approval for clinical application in cancer treatment. This paper provides a comprehensive review of the TLR4 agonists' journey as cancer active immunotherapeutics. Following a brief discussion of the rationale behind the use of TLR ligands in cancer immunotherapy, we will initially focus on the forerunner of the TLR4 agonists, bacterial lipopolysaccharide (LPS). Within this context, the potentials and shortcomings of immunotherapy with this agent will be addressed, the strategies that have been devised to enhance the associated therapeutic outcome will be discussed, and the consequent achievements and shortcomings will be summarized. Subsequently, further and perhaps less well-known, molecular, bacterial, and viral TLR4 agonists with potential for cancer immunotherapy will be introduced, and if present, the outcome of the preclinical and clinical investigations of these agents will be reviewed. Finally, a look will be cast upon the promising souvenirs of the relatively new arena of nanotechnology, where TLR4 activating nanoparticulate systems will be proposed as potential candidates for the future development of this field.

Role of Damage Associated Molecular Pattern Molecules (DAMPs) in Aneurysmal Subarachnoid Hemorrhage (aSAH).

Aneurysmal subarachnoid hemorrhage (aSAH) represents only a small portion of all strokes, but accounts for almost half of the deaths caused by stroke worldwide. Neurosurgical clipping and endovascular coiling can successfully obliterate the bleeding aneurysms, but ensuing complications such as cerebral vasospasm, acute and chronic hydrocephalus, seizures, cortical spreading depression, delayed ischemic neurological deficits, and delayed cerebral ischemia lead to poor clinical outcomes. The mechanisms leading to these complications are complex and poorly understood. Early brain injury resulting from transient global ischemia can release molecules that may be critical to initiate and sustain inflammatory response. Hence, the events during early brain injury can influence the occurrence of delayed brain injury. Since the damage associated molecular pattern molecules (DAMPs) might be the initiators of inflammation in the pathophysiology of aSAH, so the aim of this review is to highlight their role in the context of aSAH from diagnostic, prognostic, therapeutic, and drug therapy monitoring perspectives. DAMPs represent a diverse and a heterogenous group of molecules derived from different compartments of cells upon injury. Here, we have reviewed the most important DAMPs molecules including high mobility group box-1 (HMGB1), S100B, hemoglobin and its derivatives, extracellular matrix components, IL-1α, IL-33, and mitochondrial DNA in the context of aSAH and their role in post-aSAH complications and clinical outcome after aSAH.

Aneurysmal subarachnoid hemorrhage lead to systemic upregulation of IL-23/IL-17 inflammatory axis.

IL-23 and IL-17 are pro-inflammatory cytokines. IL-23 is secreted by activated macrophages and dendritic cells, while IL-17 by Th17 cells. Serum IL-23 and IL-17 are known to be elevated in numerous inflammatory diseases including neurodegenerative diseases. The role of serum IL-23 and IL-17 in aneurysmal subarachnoid hemorrhage (aSAH) has still not been investigated. The present work investigates the serum IL-23 and IL-17 levels and their association with post hemorrhagic complications and clinical outcome in patients with aSAH. METHODS: In this study, 80 patients with aSAH (Hunt and Hess grade I-V) were prospectively recruited. We enrolled 24 control patients with lumbar spinal stenosis. Peripheral venous blood was withdrawn from controls and from aSAH patients at day 1 and day 7, allowed to clot and centrifuged to obtain serum. Enzyme linked immunoassay kits were employed to quantify the serum levels of IL-23 and IL-17 by applying 50µL of serum samples. Post hemorrhagic complications and clinical outcome were documented prospectively from patient's hospital record. RESULTS: Serum IL-23 and IL-17 levels were significantly elevated in aSAH patients at day 1 and day 7 (n=80) as compared to control patients (n=24). Further analysis after dichotomy of patients who suffered from post hemorrhagic complications including cerebral vasospasm, chronic hydrocephalus, seizures, cerebral ischemia, delayed neurological deficits showed differential correlations with different post hemorrhagic complications (Table 1). Serum IL-23 and IL-17 levels did not correlate with clinical outcome. CONCLUSION: Serum IL-23 and IL-17 levels were elevated in patients with aSAH showing upregulation of IL-23/IL-17 inflammatory axis after aSAH. Serum IL-23 and IL-17 showed differential correlations with post hemorrhagic complications and no correlation with clinical outcome.

MDR in cancer: Addressing the underlying cellular alterations with the use of nanocarriers.

Multidrug resistance (MDR) is associated with a wide range of pathological changes at different cellular and intracellular levels. Nanoparticles (NPs) have been extensively exploited as the carriers of MDR reversing payloads to resistant tumor cells. However, when properly formulated in terms of chemical composition and physicochemical properties, NPs can serve as beyond delivery systems and help overcome MDR even without carrying a load of chemosensitizers or MDR reversing molecular cargos. Whether serving as drug carriers or beyond, a wise design of the nanoparticulate systems to overcome the cellular and intracellular alterations underlying the resistance is imperative. Within the current review, we will initially discuss the cellular changes occurring in resistant cells and how such changes lead to chemotherapy failure and cancer cell survival. We will then focus on different mechanisms through which nanosystems with appropriate chemical composition and physicochemical properties can serve as MDR reversing units at different cellular and intracellular levels according to the changes that underlie the resistance. Finally, we will conclude by discussing logical grounds for a wise and rational design of MDR reversing nanoparticulate systems to improve the cancer therapeutic approaches.

Elevated Systemic IL-6 Levels in Patients with Aneurysmal Subarachnoid Hemorrhage Is an Unspecific Marker for Post-SAH Complications.

Background: Aneurysmal subarachnoid hemorrhage (aSAH) is still a fatal and morbid disease, although bleeding aneurysms can be secured in almost all cases. Occurrence of post-SAH complications including cerebral vasospasm, delayed cerebral ischemia, hydrocephalus, epilepsy, and infections are the main determinants of clinical outcome. Hence, it is important to search for early predictors for specific post-SAH complications to treat these complications properly. Both cellular and molecular (cytokines) inflammation play a key role after aSAH during the phase of occurrence of post-SAH complications. Interleukin-6 (IL-6) is a well-known cytokine that has been extensively analyzed in cerebrospinal fluid (CSF) of patients after aSAH, but detailed studies exploring the role of systemic IL-6 in aSAH associated complications and its impact on early clinical outcome prediction are lacking. The current study aims to analyze the systemic IL-6 levels over two weeks after bleeding and its role in post-SAH complications. Methods: We recruited 80 aSAH patients prospectively who underwent peripheral venous blood withdrawal in serum gel tubes. The blood was centrifuged to harvest the serum, which was immediately frozen at -80 °C until analysis. Serum IL-6 levels were quantified using Immulite immunoassay system. Patient records including age, gender, post-SAH complications, aneurysm treatment, and clinical outcome (modified Rankin scale and Glasgow outcome scale) were retrieved to allow different subgroup analysis. Results: Serum IL-6 levels were significantly raised after aSAH compared to healthy controls over the first two weeks after hemorrhage. Serum IL-6 levels were found to be significantly elevated in aSAH patients presenting with higher Hunt and Hess grades, increasing age, and both intraventricular and intracerebral hemorrhage. Interestingly, serum IL-6 was also significantly raised in aSAH patients who developed seizures, cerebral vasospasm (CVS), and chronic hydrocephalus. IL-6 levels were sensitive to the development of infections and showed an increase in patients who developed pneumoniae. Intriguingly, we found a delayed increase in serum IL-6 in patients developing cerebral infarction. Finally, IL-6 levels were significantly higher in patients presenting with poor clinical outcome in comparison to good clinical outcome at discharge from hospital. Conclusion: Serum IL-6 levels were elevated early after aSAH and remained high over the two weeks after initial bleeding. Serum IL-6 was elevated in different aSAH associated complications, acting as a non-specific marker for post-SAH complications and an important biomarker for clinical outcome at discharge.

Aerodynamic Droplet Stream Expansion for the Production of Spray Freeze-Dried Powders.

In spray freeze-srying (SFD), a solution is sprayed into a refrigerant medium, frozen, and subsequently sublimation dried, which allows the production of flowable lyophilized powders. SFD allows commonly freeze-dried active pharmaceutical ingredients (e.g., proteins and peptides) to be delivered using new applications such as needle-free injection and nasal or pulmonary drug delivery. In this study, a droplet stream was injected into a vortex of cold gas in order to reduce the risk of droplet collisions and therefore droplet growth before congelation, which adversely affects the particle size distribution. Droplets with initial diameters of about 40-50 µm were frozen quickly in a swirl tube at temperatures around -75°C and volumetric gas flow rates between 17 and 34 L/min. Preliminary studies that were focused on the evaluation of spray cone footprints were performed prior to SFD. A 23 factorial design with a model solution of mannitol (1.5% m/V) and maltodextrin (1.5% m/V) was used to create flowable, low density (0.01-0.03 g/cm3) spherical lyophilisate powders. Mean particle diameter sizes of the highly porous particles ranged between 49.8 ± 6.6 and 88.3 ± 5.5 µm. Under optimal conditions, the mean particle size was reduced from 160 to 50 µm (decrease of volume by 96%) compared to non-expanded streams, whereas the SPAN value did not change significantly. This method is suitable for the production of lyophilized powders with small particle sizes and narrow particle size distributions, which is highly interesting for needle-free injection or nasal delivery of proteins and peptides.

Hot Melt Extrusion for Sustained Protein Release: Matrix Erosion and In Vitro Release of PLGA-Based Implants.

The design of biodegradable implants for sustained release of proteins is a complex challenge optimizing protein polymer interaction in combination with a mini-scale process which is predictive for production. The process of hot melt extrusion (HME) was therefore conducted on 5- and 9-mm mini-scale twin screw extruders. Poly(lactic-co-glycolic acid) (PLGA) implants were characterized for their erosion properties and the in vitro release of the embedded protein (bovine serum albumin, BSA). The release of acidic monomers as well as other parameters (pH value, mass loss) during 16 weeks indicated a delayed onset of matrix erosion in week 3. BSA-loaded implants released 17.0% glycolic and 5.9% lactic acid after a 2-week lag time. Following a low burst release (3.7% BSA), sustained protein release started in week 4. Storage under stress conditions (30°C, 75% rH) revealed a shift of erosion onset of 1 week (BSA-loaded implants: 26.9% glycolic and 9.3% lactic acid). Coherent with the changed erosion profiles, an influence on the protein release was observed. Confocal laser scanning and Raman microscopy showed a homogenous protein distribution throughout the matrix after extrusion and during release studies. Raman spectra indicated a conformational change of the protein structure which could be one reason for incomplete protein release. The study underlined the suitability of the HME process to obtain a solid dispersion of protein inside a polymeric matrix providing sustained protein release. However, the incomplete protein release and the impact by storage conditions require thorough characterization and understanding of erosion and release mechanisms.

Triterpenoid Saponins from the Caryophyllaceae Family Modulate the Efflux Activity of the P-Glycoprotein in an In Vitro Model of Intestinal Barrier.

The oral bioavailability of drugs is often limited due to the presence of the P-glycoprotein, an efflux pump strongly expressed on the luminal side of the intestinal barrier. In an attempt to circumvent drug efflux, strategies consisting in the coadministration of drugs with surface-active agents have been found to be promising. In this context, the role of saponins on the intestinal permeability of a P-glycoprotein substrate was investigated. The P-glycoprotein inhibition activity of three triterpenoid saponins extracted from several plants of the Caryophyllaceae family was evaluated using an intestinal barrier model comprised of Caco-2 cell lines. The results showed a strong effect of two saponins on P-glycoprotein-mediated transport. At a concentration of 15 µM, the efflux ratio was close to 1 for both saponins, thus suggesting a total inhibition of the efflux pump in contrast to verapamil HCl, a conventional P-glycoprotein inhibitor. In addition, measurements of the transepithelial electrical resistance revealed that the integrity of the monolayers was not altered at such concentrations, thereby reducing potential adverse effects. The presence of acetylated sugars in the saponin structure could possibly facilitate interactions with the efflux pump by an ATP-dependent mechanism or by fluidization of cell membranes.