A national centre of pharmaceutical drug research

Abstract

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common forms of neurodegenerative disorders affecting approximately 50 million people worldwide. The underlying neuropathological processes leading to AD and PD share many similarities, i.e. aberrant protein aggregation of tau and alpha-synuclein (αSyn) in the brain. Monitoring tau and αSyn aggregation is challenging, due to morphological heterogeneity of the aggregating species and problems in preserving the antigen conformation ex vivo.

In paper-I, we validated the usefulness of proximity ligation assay (PLA), a technique that enabled us to visualize previously undetected early αSyn pathology in the A30P-tg mouse model of PD. We observed an age-progressive increase in the levels of phosphorylated αSyn (pSyn^S129) and the compactness of aggregates in the brain. Although loss of dopaminergic neurons was not found, a subtle dysregulation of other catecholamines was recorded in the older mice.

In paper-II, we revealed a wide distribution of pSyn^S129 aggregates in alpha-synucleinopathy-patient brains. By using a PLA setup with certain antibody pair combinations on brain sections, we observed unique staining patterns, which could not be visualized using regular immunohistochemistry (IHC). In A30P-tg mice, the morphological pattern of the PLA signals indicated an intracellular shift of pSyn^S129  from the periphery towards the neuronal soma.

In Paper-III, we demonstrated that multiplex pTau^S202,T205-pTau^T231, singleplex pTau^T231 and singleplex pSyn^S129 PLAs can recognize an extensive tau and αSyn pathology compared to regular IHC. We found that using our PLA approach we could differentiate between pTau^S202,T205 and pTau^T231 pathology in AD brains, whereas IHC could not. Similarly, in the PD brain, singleplex pSyn^S129 PLA detected novel structures, i.e. apparent thick intercellular tunnelling nanotubes and early aggregates; whereas pSyn^S129 IHC was limited to the detection of mature pathology. Lastly, we demonstrated that our multiplex PLA approach detected co-aggregates of pSyn^S129-pTau.

In Paper-IV, in an αSyn seeding mouse model we observed pSyn^S129 immunoreactivity close to the striatal injection site one day post-injection (dpi). Intriguingly, this type of staining disappeared with the concurrent formation of peri-nuclear pSyn^S129 inclusions in specific brain regions after 14 dpi. In parallel, astrocytic activation prior to pSyn^S129 inclusion formation was observed.

In conclusion, we have developed several novel PLAs that detect both tau and αSyn pathology with a higher ex vivo sensitivity and specificity than currently used immunostaining methods. This thesis work provides valuable insights that potentially could be used for the development of future biomarkers for tauopathies and synucleinopathies.

Abstract

Pulmonary route has been used as a source of drug delivery to lungs for centuries. Drugformulation decides the type of inhaler devices such as pressurized metered-dose inhalers(pMDIs), nebulizers and dry powder inhalers (DPIs). The most commonly used formulationin DPI consists of an ordered unit consisting of smaller drug particles (3 to 5 μm) (activepharmaceutical ingredient, API) attached to larger inert particles (carrier particles, 100 μm)called adhesive mixtures. APIs are highly cohesive in nature due to high surface to volume ratio.The adhesive mixture prevents self-agglomeration of APIs and helps deliver it to the deep lungs.In the manufacturing of adhesive mixtures, the focus has been on mixing and release insideinhaler. The research work in this thesis focuses on unaddressed issues on mixture stability andsegregation using modeling and experimental techniques. The Discrete Element Method (DEM)was used to investigate the mechanics of adhesive units, formed by randomized distributionof APIs on the surface of carrier particle. Binary collisions (head on and oblique) betweenadhesive units were simulated for different number density of fines on the carrier (surfacecoverage ratio, SCR), surface energy (interfacial adhesion/cohesion energy), shape of APIs(spherical, triangular bipyramidal and tetrahedral), size of carrier particles (50, 100, 200 μm),type of carrier particle (lactose and mannitol) and the angle of impact. To account for variationthree different initial randomized distributions of API on the carrier were considered. The dataobtained was analysed in terms of effective mechanical properties (coefficient of restitution),effective friction, physical stability of adhesive unit and redistribution of fines on the carriersurface. The coefficient of restitution follows a Kawakita type equation for higher velocity andfor different surface energy. The effect of the fine particle shape was predominant for low SCRs,and adhesive units formed from tetrahedral fines exhibited the largest physical stability andlargest friction during oblique collisions. In terms of carrier size and properties it was observedthat mannitol particles are more stable than lactose with similar dispersion performance andthe200 μm carrier is the most stable among the sizes investigated. To complement the modeling,segregation of adhesive mixture (consisting of budesonide and salbutamol sulphate as APIand Inhalac 70 as carrier) was studied. Experiments showed significant loss of APIs and selfagglomeration at higher SCR. The micro mechanical models and experiments lay a foundationtowards a better understanding of the adhesive mixture dynamics.