Ian D. Spiers

Intra nasal administration of poly-lactic acid microsphere co-encapsulated Yersinia pestis subunits confers protection from pneumonic plague in the mouse

Equivocal doses of soluble, or high molecular weight poly (lactic acid) microsphere co-encapsulated, F1 and V subunit antigens of Yersinia pestis were used to immunize mice intra-nasally. Animals were dosed on day 1 and 7 with 2.724 micrograms V plus 0.956 micrograms F1. Co-encapsulated antigens induced superior systemic and mucosal immunity in comparison with free F1 and V. All of the mice immunized with soluble antigens died shortly after an aerosol challenge consisting of 1 x 10(5) colony-forming units of plague bacteria. In contrast, 66% of the co-encapsulated subunit vaccinees survived this lethal challenge. Humoral immunity to plague was improved further, resulting in 80% protection from challenge, if a relatively high dose (10 micrograms) of cholera toxin B subunit was added to the microsphere suspension prior to intra-nasal delivery. Significantly, by adding 10 micrograms cholera toxin B subunit to the free antigen solution, a 100% post-challenge survival rate was attained. We conclude that in this animal model of pneumonic plague, intra-nasal administration of microgram quantities of Yersinia pestis subunits confers protective immunity, provided the vaccines are microencapsulated or admixed with a strong mucosal adjuvant, such as the cholera toxin B subunit.

Analysis of local and systemic immunological responses after intra-tracheal, intra-nasal and intra-muscular administration of microsphere co-encapsulated Yersinia pestis sub-unit vaccines

Intra-tracheal, intra-nasal and intra-muscular immunisation with admixed Y. pestis sub-units (3 micrograms V, 0.47 microgram F1) or equivalent doses of poly-L-lactide microsphere co-encapsulated antigens was done. Systemic and mucosal responses to F1 and V differed according to immunisation route, and encapsulated status of the sub-units. Irrespective of immunisation site, particulated sub-units stimulated statistically superior primary systemic reactions, with intra-tracheal and nasal microsphere immunisations eliciting superior serum anti-V IgG titres in comparison to intra-muscular injection of free vaccines (p < 0.001 beyond day 8). Pulmonary and nasal delivery of microspheres induced primary serum anti-V IgG titres which were greater (p < 0.039) or equal to (p > 0.056) those after intra-muscular injection of spheres. In terms of serum anti-F1 titres, mice responded best to intra-muscular, and comparatively poorly to intra-nasal immunisations. Intra-tracheal administration of microspheres induced strongest responses in the respiratory tract, dominated by the IgG rather than IgA isotype. An intra-nasal booster immunisation on day 63 potentiated strong local and circulating anti-V IgG titres in microsphere vaccinees. Priming and boosting with free vaccines induced significantly depressed secondary serum anti-F1 titres relative to microsphere immunisations (p < 0.024 at days 78 and 120). In contrast to other priming sites, intra-tracheal instillation of encapsulated vaccines facilitated the induction of IgG antibody to both F1 and V in day 146 broncho-alveolal washings. With the exception of primary responses to F1 in mice immunised intra-tracheally with microspheres, IgG1 was the dominant subclass of anti-F1/V IgG in serum. We conclude that introduction of biodegradable microspheres containing the F1 and V sub-units into to the upper or lower respiratory tract engenders immune responses of a magnitude comparable with that induced by parenteral immunisation, and may present a means of protecting individuals from plague.

The conformation of an inhibitor bound to the gastric proton pump

Substituted imidazo[1,2‐a]pyridines are pharmaceutically important small molecule inhibitors of the gastric H+/K+‐ATPase, the membrane‐bound therapeutic target for peptic ulcer disease. A non‐perturbing analytical technique, rotational resonance NMR spectroscopy, was used to measure a precise (to ±0.2 Å) distance between atomic sites in a substituted imidazo[1,2‐a]pyridine, TMPIP, bound to H+/K+‐ATPase at its high‐affinity site in the intact, native membrane. The structural analysis of the enzyme–inhibitor complex revealed that the flexible moiety of TMPIP adopts a ‘syn‐type’ conformation at its site of action. Hence, the conformation of an inhibitor has been resolved directly under near‐physiological conditions, providing a sound experimental basis for rational design of many active compounds of pharmaceutical interest. Chemically restraining the flexible moiety of compounds like TMPIP in the syn‐type binding conformation was found to increase activity by over 2 orders of magnitude. Such information is normally only available after extensive synthesis of related compounds and multiple screening approaches.

Tissue distribution of radioactivity following intranasal administration of radioactive microspheres

The aim of this study was to increase understanding of the kinetics of microparticle distribution and elimination following intranasal application. To do this we investigated the in‐vivo distribution of radioactivity following intranasal instillation of scandium‐46 labelled styrene‐divinyl benzene 7‐μm‐diameter microspheres. Groups of BALB/c mice received 0.250 mg (47.5 kBq) particles suspended in either 50‐μl or 10‐μl volumes of phosphate buffered saline. The in‐vivo distribution of radioactivity was influenced by the volume of liquid that was used to instil the microsphere suspension. Comparatively large (50 μl) administration vehicle volumes resulted in substantial bronchopulmonary deposition (∼ 50% of administered dose). Intranasal instillation of microspheres suspended in 10‐μl volumes tended to restrict particle deposition initially to the nasal cavity. For both administration vehicle volumes tested, the radioactivity per unit mass of excised nasal‐associated lymphoid tissue (NALT) was found to be consistently elevated relative to other tissues. This corroborates the findings of other workers who have previously identified NALT as an active site of microparticle accumulation following intranasal application. Elimination via the alimentary canal was the principal fate of intranasally applied radiolabeled material. No significant concentration of radioactivity within excised gut‐associated lymphoid tissue (GALT) (Peyers patches) was noted. At latter time points we observed, in mice that received the 50‐μl volume particle suspension nasally, accumulation of potentially relevant quantities of radioactivity in the liver (0.3% after 576 h) and spleen (0.04% after 576 h). Thus, our data corroborate the notion that epithelial membranes in the lung are probably less exclusive to the entry of microparticulates into systemic compartments than are those mucosae in the gastrointestinal tract or nasopharynx. This effect may contribute to the effectiveness of pulmonary delivered antigen‐loaded microparticles as humoral immunogens.

Biodegradable microparticles with different release profiles: effect on the immune response after a single administration via intranasal and intramuscular routes.

In the development of single‐dose microparticulate vaccines, identification of the type of protein release profile required to elicit high and sustainable immune responses is important. Microparticles exhibiting different protein release profiles (continuous, pulsatile and plateau) were made by solvent evaporation or solvent extraction methods from biodegradable polymers encapsulating the model antigen, bovine serum albumin (BSA). The immune responses obtained after a single intranasal or intramuscular administration of microparticles were determined, and also after a subcutaneous boost after 11 months.

Synthesis and iron binding studies of myo-inositol 1,2,3-trisphosphate and (±)-myo-inositol 1,2-bisphosphate, and iron binding studies of all myo-inositol tetrakisphosphates☆

The first syntheses of the natural products myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate are described. The protected key intermediates 4,5,6-tri-O-benzoyl-myo-inositol and (+/-)-3,4,5,6-tetra-O-benzyl-myo-inositol were phosphorylated with dibenzyl N,N-di-isopropylphosphoramidite in the presence of 1H-tetrazole and subsequent oxidation of the phosphite. The crystal structures of the synthetic intermediates (+/-)-1-O-(tert-butyldiphenylsilyl)-2,3,O-cyclohexylidene-myo-inos itol and (+/-)-4,5,6-tri-O-benzoyl-1-O-(tert-butyldiphenylsilyl)-2,3-O-cycl ohexylidene- myo-inositol are reported. myo-Inositol 1,2,3-trisphosphate, (+/-)-myo-inositol 1,2-bisphosphate, and all isomeric myo-inositol tetrakisphosphates were evaluated for their ability to alter HO. production in the iron-catalysed Haber-Weiss reaction. The results demonstrated that a 1,2,3-grouping of phosphates in myo-inositol was necessary for inhibition, also that (+/-)-myo-inositol 1,2-bisphosphate potentiated HO. production. myo-Inositol 1,2,3-trisphosphate resembled myo-inositol hexakisphosphate (phytic acid) in its ability to act as a siderophore by promoting iron-uptake into Pseudomonas aeruginosa.

Studies on the co-encapsulation, release and integrity of two subunit antigens: rV and rF1 from Yersinia pestis.

In the development of combination or multiple sub‐unit vaccines, determination of the encapsulation, release and integrity of two or more proteins co‐encapsulated within microspheres is an important issue. A new extraction method, which exhibits excellent protein recovery, has been developed which enables samples to be used for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and subsequent measurement of individual antigens encapsulated within microspheres. Using the new method, the protein loading of poly‐(L‐lactide) microspheres co‐encapsulating two plague sub‐unit antigens was found to be 1.22% (w/w) for recombinant V antigen (rV) and 1.24% (w/w) for recombinant F1 (rF1) by SDS‐PAGE. The total protein loading was 2.49% (w/w) by bicinchoninic acid assay. The individual release of the two subunit antigens from the co‐encapsulated microspheres was determined by SDS‐PAGE analysis and rF1 was found to have a higher burst release than rV. The integrity and immunological activity of both rF1 and rV antigens was shown to be unaffected by the microencapsulation process.

The first synthesis and iron binding studies of the natural product, myo-inositol 1,2,3-trisphosphate

Abstract The natural product myo -inositol 1,2,3-trisphosphate 1 has been prepared and shown to inhibit Fe 3+ catalysed hydroxyl radical formation.

Protection against plague following immunisation with microencapsulated V antigen is reduced by co-encapsulation with IFN-γ or IL-4, but not IL-6

We have investigated intranasal delivery of novel vaccines for plague, based on poly-L-lactide (PLLA) microencapsulated recombinant V antigen (rV) of Yersinia pestis. Microspheres containing rV alone or co-encapsulated with the cytokines IFN-gamma, IL-4 or IL-6 were administered in a two-dose regimen and antibody responses and protective efficacy were monitored. All treatment groups stimulated high rV-specific antibody titres in serum, predominantly of the IgG1 isotype, which were maintained over several months. There was evidence of both IgG and IgA responses in lung samples from all groups. Formulations based on rV antigen alone or rV co-encapsulated with IL-6 provided complete protection against systemic challenge with Y. pestis strain GB; however protective efficacy was impaired by co-encapsulating either IFN-gamma or IL-4 with rV.

Inositol polyphosphate-mediated iron transport in Pseudomonas aeruginosa

It has previously been shown that myo‐inositol hexakisphosphate (myo‐InsP6) mediates iron transport into Pseudomonas aeruginosa and overcomes iron‐dependent growth inhibition. In this study, the iron transport properties of myo‐inositol trisphosphate and tetrakisphosphate regio‐isomers were studied. Pseudomonas aeruginosa accumulated iron (III) at similar rates whether complexed with myo‐Ins(1,2,3)P3 or myo‐InsP6. Iron accumulation from other compounds, notably d/lmyo‐Ins(1,2,4,5)P4 and another inositol trisphosphate regio‐isomer, d‐myo‐Ins(1,4,5)P3, was dramatically increased. Iron transport profiles from myo‐InsP6 into mutants lacking the outer membrane porins oprF, oprD and oprP were similar to the wild‐type, indicating that these porins are not involved in the transport process. The rates of reduction of iron (III) to iron (II) complexed to any of the compounds by a Ps. aeruginosa cell lysate were similar, suggesting that a reductive mechanism is not the rate‐determining step.