Optimizing the Bioavailability of Subcutaneously Administered Biotherapeutics Through Mechanochemical Drivers.

The subcutaneous route offers myriad benefits for the administration of biotherapeutics in both acute and chronic diseases, including convenience, cost effectiveness and the potential for automation through closed-loop systems. Recent advances in parenteral administration devices and the use of additives which enhance drug dispersion have generated substantial additional interest in IV to SQ switching studies. Designing pre-clinical and clinical studies using SQ mediated delivery however requires deep understanding of complex inter-related physiologies and transport pathways governing the interstitial matrix, vascular system and lymphatic channels. This expert review will highlight key structural features which contribute to transport and biodistribution in the subcutaneous space and also assess the impact of drug formulations. Based on the rapidly growing interest in the SQ delivery route, a number of potential areas for future development are highlighted, which are likely to allow continued evolution and innovation in this important area.

Eosinophil survival activity identified as interleukin-5 is associated with eosinophil recruitment and degranulation and lung injury twenty-four hours after segmental antigen lung challenge.

BACKGROUND: Segmental antigen challenge in allergic volunteer subjects leads to the recruitment of inflammatory cells, including eosinophils, to the lung and to lung injury as shown by albumin influx into the alveolar air space. The goal of this study was to determine whether eosinophil-active cytokines, including IL-3, IL-5, or granulocyte-macrophage colony-stimulating factor, are released into the lung 24 hours after segmental antigen challenge of ragweed allergic subjects with allergic rhinitis and to determine whether the presence of the cytokine or cytokines is correlated with markers of lung inflammation and lung injury. METHODS: Volunteers underwent challenge with a wide variety of antigen doses, which resulted in the recruitment of inflammatory cell mixtures both with and without eosinophils. RESULTS: Eosinophil survival activity (ESA), the ability of the cytokine to prolong blood eosinophil survival in culture, was found in 5 of 17 ragweed allergic subjects and only in subjects challenged with relatively high doses of ragweed antigen (0.2 ragweed antigen units/ml or more). No ESA was found in bronchoalveolar lavage (BAL) fluid in any of eight nonragweed allergic subjects. This activity could be almost completely neutralized by preincubating BAL fluid with specific antibody to IL-5, although a small contribution by granulocyte-macrophage colony-stimulating factor may also have been present. ESA correlated with eosinophil recruitment (r = 0.72, p < 0.001) and degranulation in the lung (r = 0.63 to 0.81, p < 0.01, for eosinophil granule constituents in BAL fluid) and lung injury as shown by albumin influx into the alveolar air spaces (r = 0.83, p < 0.001). ESA was unrelated to the presence of other inflammatory cells in the lung. Subjects who had IL-5 in BAL fluid appeared to undergo more severe initial reactions to antigen challenge. CONCLUSIONS: We conclude that IL-5 is the most important constituent in ESA in the lung 24 hours after antigen challenge and that it correlates with eosinophil recruitment, degranulation, and lung injury.

Immunoglobulin E-mediated increase in vascular permeability correlates with eosinophilic inflammation.

An increase in bronchovascular permeability is thought to play an important role in the pathogenesis of allergic asthma. We sought to determine whether the increase in permeability observed 24 h after segmental antigen challenge in ragweed-allergic human volunteers was associated with the infiltration and degranulation of a specific cell type. A 20,000-fold range of antigen concentrations was used to alter the number and type of inflammatory cells recruited to the lung by challenge. Although large numbers of inflammatory cells were recruited to lung air spaces over a large range of antigen concentrations, significant numbers of eosinophils (731.3 +/- 232.9 x 10(3)/ml) were recruited only when the concentration of antigen used for segmental challenge was > or = 100-fold higher than the concentration needed to produce an 8 to 10 mm wheal 20 min after intradermal skin testing. In addition, large increases in bronchoalveolar lavage (BAL) albumin concentration (636.3 +/- 170.5 micrograms/ml) were observed only in this same group of subjects. The correlation coefficient between the logarithms of the BAL eosinophil concentration and albumin concentration was +0.82 (p < 0.001), and between eosinophil-derived neurotoxin and albumin it was +0.88 (p < 0.001). In a stepwise, multiple regression analysis, eosinophils accounted for 67% of the variance in BAL albumin concentration, whereas no other cell type was a significant predictor of albumin flux into BAL fluid. We conclude that eosinophil recruitment and degranulation are associated with large increases in bronchovascular permeability after segmental antigen challenge in humans.

Processing of exogenous liposome-encapsulated antigens in vivo generates class I MHC-restricted T cell responses.

Acid-sensitive liposomes have been developed for cytosolic delivery of encapsulated substances. We now demonstrate delivery of liposome-encapsulated Ag into the class I MHC Ag processing pathway in peritoneal macrophages in vitro using several types of acid-sensitive liposomes, including those composed of dioleoylphosphatidylethanolamine (DOPE)/palmitoylhomocysteine, DOPE/cholesterol hemisuccinate, DOPE/dioleoylsuccinylglycerol, and DOPE/dipalmitoylsuccinylglycerol. Our previous studies showed that acid-resistant liposomes (dioleoylphosphatidylcholine/dioleoylphosphatidylserine) did not engender class I-mediated presentation in vitro. However, in vivo immunization with OVA encapsulated in acid-resistant as well as acid-sensitive liposomes generated class I MHC-restricted T cell responses, as determined by subsequent in vitro cytotoxicity assays using OVA-transfected target cells. Target lysis by these cells was OVA- and class I MHC (Kb)-specific. This response was not generated by immunization with equivalent amounts of soluble OVA. Thus, a pathway for in vivo class I processing of Ag encapsulated in acid-resistant liposomes has been missed in vitro, perhaps because it is dependent on specific populations of APC or interactions between cells that have not been reconstituted in vitro. This pathway may explain the ability of many exogenous particulate Ag (liposomes, bacteria, parasites, and mammalian cells) to generate class I MHC-restricted T cell responses.

Effect of antigen dose on the recruitment of inflammatory cells to the lung by segmental antigen challenge.

Bronchoscopic antigen challenge of atopic volunteers results in an immediate release of inflammatory mediators and, after a number of hours, the recruitment of inflammatory cells to the lung. The purpose of this work was to investigate the effect of antigen dose on the subsequent recruitment of inflammatory cells to the lung. Twenty-two volunteers without asthma, eight nonatopic control subjects, and 14 ragweed-allergic subjects underwent 25 local antigen-challenge procedures that consisted of a baseline lavage of a control segment, antigen challenge of another segment in the contralateral lung, and lavage of the challenged segment 24 hours later. A 25,000-fold range of antigen doses was used from 0.004 to 100 PNU/ml (0.02 to 500 ng/ml of ragweed antigen E [Amb a I]). Challenge of nonatopic control subjects resulted in the recruitment of only a small number of inflammatory cells, less than a twofold increase in comparison with the cells of control lavage; this increase was primarily due to an increase in neutrophils. Challenge of atopic subjects, in contrast, resulted in approximately a threefold to ninefold increase in inflammatory cells with more cells recruited at larger doses of antigen. Only subjects challenged with a "high" dose of antigen (greater than or equal to 1 PNU/ml) recruited significant quantities of eosinophils to the lung. In these subjects, a twofold increase in macrophages, a fourfold increase in lymphocytes, a 90-fold increase in neutrophils, and an 800-fold increase in eosinophils were observed; the number of neutrophils and eosinophils recruited averaged between 30 and 60 million.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of disulfide bonds within lysosomes is a key step in antigen processing.

Reduction of disulfide bonds is a key step in antigen processing both to allow the unfolding of protein antigens, increasing the access of proteolytic processing enzymes, and to expose free Cys residues within linear peptide epitopes recognized by T cells. We show here that reduction and alkylation of Ag (hen egg lysozyme and ribonuclease A) vastly increased their proteolysis (by specific enzymes or lysosomal fractions) and the production of specific immunogenic peptides that bound to class II MHC molecules recognized by T hybridoma cells. We also show that the lysosome is the vesicular compartment that mediates protein disulfide reduction. We coupled [125I]tyrosine to 131I-alpha 2-macroglobulin or [131I] transferrin via a reducible disulfide linker. Removal of [125I]tyrosine from the alpha 2-macroglobulin conjugate was initiated only after 15 to 20 min of uptake by macrophages, suggesting that reduction occurred late in the endocytic pathway. No reduction of transferrin conjugates was seen, indicating that early, recycling endosomes did not contain reducing activity. Subcellular fractionation showed that the disulfide bonds were reduced only in heavy density (lysosome) fractions and remained intact in fractions of light density (endosomes and plasma membrane). These results indicate the importance of lysosomes in the biochemical processing of protein Ag presented to T cells.

Liposome-encapsulated antigens engender lysosomal processing for class II MHC presentation and cytosolic processing for class I presentation.

Liposome-encapsulated protein Ag were used to dissect the roles of various subcellular compartments in Ag processing for class I and class II MHC-restricted presentation. Macrophages exhibited efficient processing of Ag encapsulated in acid-resistant dioleoylphosphatidylcholine/dioleoylphosphatidylserine liposomes, which sequester their contents from potential endosomal processing events and release them only after delivery to lysosomes. Lysosomal processing was demonstrated for all four Ag studied (OVA, murine hemoglobin, bovine ribonuclease A, and hen egg lysozyme), establishing the recycling of immunogenic peptides from lysosomes after Ag processing. These acid-resistant liposomes did not engender class I processing. Ag encapsulated within acid-sensitive dioleoylphosphatidylethanolamine/palmitoylhomocysteine liposomes were also processed via the class II pathway. Of the four Ag encapsulated in liposomes, one, OVA, was tested for ability to stimulate a class I-specific response. OVA in acid-resistant liposomes did not engender a class I-specific response. In contrast, OVA encapsulated in acid-sensitive liposomes was presented by class I molecules, albeit less efficiently than it was presented by class II molecules. We interpret this to be the result of the release of a minor portion of the encapsulated Ag into the cytosol.

Comparative bioavailability of two flurbiprofen products: stereospecific versus conventional approach.

In this randomized, crossover study comparing the bioavailability of a film-coated (Ansaid) with a sugar-coated (Froben) 100 mg tablets of racemic flurbiprofen in 23 healthy young men, no significant differences were found for Cmax, tmax or AUC, using a nonstereoisomeric assay for flurbiprofen. Minor differences in the appearance of flurbiprofen in serum during the first 30-min post-dosing period were noted, with Ansaid appearing earlier than Froben. These differences likely reflect dissolution rate dissimilarity between the two products. Stereospecific determinations demonstrate a small (7.8 per cent) but significant difference in AUC of the active S-configuration (Froben greater than Ansaid). No significant differences between Ansaid and Froben were found for tmax or Cmax for the S-flurbiprofen. In bioequivalency studies of chiral drugs, stereospecific approaches provide a more accurate assessment of products.

Liposome-encapsulated antigens are processed in lysosomes, recycled, and presented to T cells.

Antigen processing requires intracellular antigen catabolism to generate immunogenic peptides that bind to class II MHC molecules (MHC-II) for presentation to T-cells. We now provide direct evidence that these peptides are produced within dense lysosomes, as opposed to earlier endocytic compartments. The protein antigen hen egg lysozyme was targeted to endosomes or lysosomes by encapsulating it in liposomes of different membrane composition. Acid-sensitive liposomes released their contents in early endosomes, whereas acid-resistant liposomes sequestered their contents from potential endosomal processing events and released their contents only after delivery to lysosomes. Antigen encapsulated in acid-resistant liposomes was processed in a chloroquine-sensitive manner and presented more efficiently than soluble antigen or antigen encapsulated in acid-sensitive liposomes. Thus, peptides may be recycled from lysosomes, transported to endosomes to bind MHC-II, and then expressed at the cell surface.

Isolation of rotaviruses from turkeys and chickens: demonstration of distinct serotypes and RNA electropherotypes.

Six isolates of rotavirus were made from turkeys and two from chickens. Three of these required trypsin treatment for isolation and serial passage in cell cultures. The remainder were isolated without trypsin treatment. Most of these viruses were isolated in chick embryo liver cell cultures from the faeces of birds aged under 1 week. In six of the eight instances, rotavirus isolation was associated with enteric disturbance, characterised by signs such as diarrhoea, poor or abnormal appetite, abnormally fluid or gaseous intestinal contents or increased mortality. Cross immunofluorescence tests showed that while avian and mammalian rotaviruses shared a common group antigen, the avian viruses were more closely related to each other than to the Nebraska calf rotavirus isolate. On the basis of serum neutralisation tests seven of the eight avian rotaviruses were grouped into three serotypes, with two turkey isolates (Ty1 and Ty3) and a chicken (Ch1) virus being the prototype strains. The remaining virus, Ty2, was intermediate in type between Ty1 and Ch1. Analysis of the RNA of these viruses by polyacrylamide gel electrophoresis showed that they could also be grouped into a number of electropherotypes. The isolates which were serologically distinct were also electrophoretically distinct. Similarly the five isolates which belonged to the Ty3 sero-type were electrophoretically identical. Analysis of the serological and electrophoretic differences suggested that RNA segment 5 may code for a type-specific antigen.

Isolation and serological studies with infectious bursal disease viruses from fowl, turkeys and ducks: demonstration of a second serotype.

The isolation of a number of strains of infectious bursal disease (IBD) virus from fowl, turkeys and ducks is described. These isolates could be grouped into two serotypes using the neutralisation test. It is proposed that the cell culture adapted vaccine strain from fowl should be the prototype virus for serotype 1 and that the TY89 isolate from a turkey should be the prototype for serotype 2. The isolates in serotype 2 consisted of an antigenically homogeneous group of viruses from turkeys and fowl. However, within serotype 1, which represented isolates from fowl and ducks, some isolates showed only a 30% cross reaction with the vaccine strain. If cross protection mirrors cross neutralisation, then infection with viruses belonging to serotype 2 or with antigenically distant strains from serotype 1 provides one explanation for the apparent failure of the vaccine on certain sites. However, if cross protection does not mirror cross neutralisation, then a virus from serotype 2 could be used as a heterotypic vaccine for young birds with high levels of maternally derived antibody to serotype 1.

Studies on a depressed egg production syndrome in Northern Ireland.

A syndrome causing depressed egg production is described. It is characterised either by a failure to attain predicted production targets or by a fall in egg numbers. The depression in production can reach 30% and it may or may not return to normal. For a short period the eggs produced are smaller, lose colour, have poor egg shell strength and many soft shelled eggs are laid. The birds remain apparently healthy and there is a marked age incidence, with most flocks starting this depression in egg production at 29-31 weeks of age. This syndrome has been recently recorded in the Netherlands, but has not been seen before in Northern Ireland. Viruses which agglutinated fowl erythrocytes to very high titres were isolated in chick embryo liver cells from six affected flocks. Three of these isolates were from the oviduct, two from the upper respiratory tract and one from the faeces. These agents are similar to adenoviruses, but were not neutralised by antisera to 11 prototype fowl adenoviruses. In addition, 17 adenoviruses were also isolated from the flocks showing the syndrome described. These isolates fell into five serological types, in addition to nine which could not be typed using antisera to 11 prototype adenoviruses. Investigations of flocks with falls in egg production not conforming to this syndrome yielded five isolates. Six adenoviruses were also isolated from birds with diarrhoea.

Bovine leptospirosis: infection by the Hebdomadis serogroup and mastitis.

An outbreak of mastitis involving approximately 70 out of 140 cows over a two-month period is described. Common mastitis pathogens were not incriminated. Leptospires belonging to the Hebdomadis serogroup were isolated from the milk of three out of five cows and the blood of two of those cows.