Synthesis and Solid State Pharmaceutical Centre (SSPC) - Doctoral Theses

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Application of fluorescence spectroscopy for the in-vial investigation of protein solutions
(University College Cork, 2024) ElKassas, Khaled; Crean, Abina; Vucen, Sonja; Science Foundation Ireland; Engineering and Physical Sciences Research Council
Therapeutic protein formulations are subjected to various stressors during manufacturing, transport, and storage, causing destabilisation, in turn leading to deleterious effects such as immunogenic reactions or inefficacy upon administration. An essential part of this production and supply process is establishing protein formulation stability at all stages. Spectroscopy is a common laboratory tool for the characterisation of therapeutic proteins. Conventional techniques for determining protein denaturation are lab-based and require sample removal from the sealed primary packaging vial and sample destruction. To overcome the limitations of traditional techniques, the development of a fluorescence-based spectroscopic technique for “in-vial” protein analysis was a key thesis aim. A fluorescence excitation wavelength of 365 nm was found and confirmed to be transmissible through borosilicate glass vial walls. The excitation resulted in an intrinsic emission at 462 nm for a model protein, bovine serum albumin (BSA). Using a bespoke apparatus, the change in fluorescence signal with time was monitored over the reconstitution period of lyophilised protein formulations, without breach of the vial seal. First, we demonstrated how analysis of changes in fluorescence signal with time during reconstitution, using principal component analysis, could be used to determine an instrumentally quantified reconstitution time. At high protein concentrations (10% w/v BSA) the variability of the reconstitution time measurements was reduced from 80.4% relative standard deviation obtained via the pharmacopeial visual method to 8.2% for the instrumental method. Spectroscopic measurements resulted in longer measured reconstitution time compared to the visual method, possibly owing to the detection of subvisible particulates undergoing dissolution. In the second part of this work, the bespoke spectroscopic apparatus was modified with polarising filters to monitor protein aggregation and denaturation in-vial. Detector measurement of the emission wavelength (365 nm) allowed measurement of protein aggregation via light scattering. Fluorescence anisotropy (a measurement of optical polarizability at different angles) at 462 nm was used to determine protein conformational changes. Fluorescence scattering and anisotropy measurements for freshly prepared, aggregated, and denatured BSA solutions were compared to reference circular dichroism (CD) and size-exclusion chromatography (SEC) measurements. Anisotropy analysis of thermal unfolding showed strong correlations with secondary structure as quantified through a CD-neural networking model. Scattering correlated with large molecular weight aggregate measurements via SEC. A study was conducted to find the relationship between the fluorescent amino acids and the overall protein fluorescence for 4 different proteins. Using a multivariate technique, multivariate curve resolution to resolve 3D emission excitation spectra, the fluorescence of individual amino acid solutions and the total fluorescence of the proteins was correlated. Finally, a partial least squares regression (PLSR) model relating the anisotropy and scattering measurements to reference CD and SEC measurements was established from a sample set of standardised and stored samples. A total of 72 spectra were collected for the batch standardised protein solutions and the stored stability study samples. Samples were stored under real-world-like stress conditions (temperature, shaking, ambient light exposure). The fluorescence anisotropy and scattering results were included as the predictors vs. CD and SEC measurements as the responses. The PLSR showed strong correlations with an average R2 of 0.87 and a root mean PRESS of 0.487 at 9 latent variables. In conclusion, the combination of bespoke apparatus and 365 nm excitation was capable of monitoring protein stability in-vial successfully. The spectroscopic measurement of reconstitution time resulted in more precise measurements. The method overcomes the challenges present in the current pharmacopeial standard measurement as it can detect subvisible particulates and is independent of analyst capabilities and subjectivity. The protein solution stability monitoring provided a holistic non-destructive alternative to multi-instrument analysis while maintaining reasonable correlations with established reference stability indicators. This research provides a platform for a cost-effective portable solution to provide a top-level overview of biopharmaceutical product stability in-vial, from manufacture to the point of administration.
Smart drug delivery systems for siRNA therapeutics in prostate cancer immunotherapy
(University College Cork, 2023) Sun, Yao; O'Driscoll, Caitriona M.; Synthesis and Solid State Pharmaceutical Centre; Science Foundation Ireland
Prostate cancer (PCa) as an immunosuppressive cancer remains a serious condition threatening the health of men due to the complicated nature of the tumour microenvironment (TME). Conventional treatments of PCa face challenges including poor prognosis and tumour resistance, therefore new therapeutic strategies are urgently needed. Tumour-associated macrophages (TAMs) are a potential therapeutic target in cancer immunotherapy. Colony stimulating factor-1/colony stimulating factor-1 receptor (CSF-1/CSF-1R) pathway plays a crucial role in the polarization of the immunosuppressive TAMs, M2 macrophages. Downregulation of CSF-1R expression by small interfering RNA (siRNA), a double-stranded non-coding RNA, is known to reprogram the immunosuppressive TAMs, M2 macrophages, to the immunostimulatory phenotype, M1 macrophages. Sialic acid (SA) is a ligand for Siglec-1 (CD169) which is overexpressed on M2 macrophages with little expression in other phenotypes. M2 macrophage-targeting peptide (M2pep) is an artificially designed peptide and could specifically target M2 macrophages, the sequence of which was selected by subtractive phage biopanning. Beta-cyclodextrins (CDs) are cyclic oligosaccharides derived from starch, consisting of at 7 glucose subunits linked by α-1,4 glycosidic bonds, the primary and secondary sides of the ring structure are hydrophilic and the cavity is hydrophobic. Cationic CDs have been used for delivery of therapeutic nucleic acids including siRNA as non-viral vectors. Therefore, in this thesis, 2 different CD-based nanoparticles (NPs) with PEGylated SA or PEGylated M2pep as the ligand was designed and characterized for the delivery of CSF-1R to M2 macrophages in both human and mouse cells to study the macrophage reprogramming efficacy. In the meanwhile, a Transwell model was built for co-culture of M2 macrophages and PCa cells to test the anti-PCa effectiveness in vitro. In vitro results show that the sizes of three NPs were around 250 nm, and the surface charges were 11 ~ 29 mV, the poly dispersity index (PDI) was lower than 0.30. All CD-based formulations were stable in both salt- and serum-containing media, and did not present any cytotoxicity in either human or mouse macrophages. Both two NPs achieved M2 macrophage reprogramming, and post-transfected cells expressed M1 macrophage markers. The co-culture results show that the formulations caused the apoptosis of PCa cells due to the M2 macrophage reprogramming. In the in vitro studies, the CD.siRNA.DSPE-PEG-M2pep formulation presented the best CSF-1R gene silencing efficacy, superior M2 macrophage reprogramming and anti-tumour efficacy. Therefore, the CD.siRNA.DSPE-PEG-M2pep formulation was selected for assessment in vivo. A PCa-bearing mouse model was built in C57 BL/6 mice by subcutaneous injection of PCa cells and used to study the targeting and anti-PCa efficacy of CD.siRNA.DSPE-PEG-M2pep in vivo. The NPs increased M2 macrophage targeting in vivo, promoting the release of M1 factors and simultaneously downregulating the levels of M2 factors through TAM reprogramming. The subsequent remodelling of the TME resulted in a reduction in tumour growth in the PCa mouse model, mainly mediated through the recruitment of cytotoxic T cells. In summary, the CD-based targeted siRNA NPs successfully reprogrammed the immunosuppressive M2 macrophages thus promoting PCa immunotherapy and providing an alternative strategy for PCa treatment worthy of further investigation.
Cocrystallization of organic compounds
(University College Cork, 2023) Huang, Shan; Lawrence, Simon; Science Foundation Ireland
This thesis discusses the synthesis, characterization, and properties of multi-component crystalline materials of active pharmaceutical ingredients. A special emphasis is placed on cocrystallization, which is the supramolecular phenomenon of aggregation of two or more different chemical entities in a crystalline lattice through non-covalent interactions. This research has been divided into seven chapters. Chapter 1 gives an overview of the concept of multi-component crystalline materials and cocrystallization, where the design, methodology, characterization and application of cocrystals are also included. Chapter 2 discusses the synthesis of multi-component crystal forms of a sulfonamide compound, sulfasalazine, through cocrystallization and explores the crystal structure landscape of sulfasalazine. Furthermore, the differences are illustrated between cocrystals and salts of sulfasalazine via structural analysis, Hirshfeld surface analysis and frontier molecular orbitals analysis. Chapter 3 investigates the hydrogen bonding interactions in cocrystals of a frequently used sulfonamide compound, sulfaguanidine, by both experimental methods and theoretical calculations including the analysis of Hirshfeld surface, molecular electrostatic potential surfaces and quantum theory of atoms in molecules. Chapter 4 focuses on pharmaceutical salts of piroxicam and meloxicam with three basic organic counterions, respectively. The solubility of six salts and two parent drugs in sodium phosphate solution were conducted. Furthermore, piroxicam and its salts exhibited different luminescent properties, thus, the different luminescent mechanisms were discussed. Chapter 5 explores cocrystallization of 19 natural L-amino acids and both enantiomers of four pharmaceutically relevant chiral compounds. The formation of diastereomeric or enantiospecific systems were explored using an examination of their hydrogen bonding motifs. Chapter 6 investigates the formation of diastereomeric cocrystal pairs of S mandelamide with both enantiomers of mandelic acid and proline, respectively. In addition, the crystal structures of (±)-mandelamide, S-mandelamide and enantio-enriched mandelamide (94 S:6 R) were determined. Detailed crystal structural analyses together with Hirshfeld surface analysis were carried out. Chapter 7 summarizes the main findings of the entire work and examines future work, such as the use of ternary phase diagrams to assist in developing chiral separation processes.
Cocrystallising acids and amides: towards pharmaceutical cocrystals
(University College Cork, 2021-07-20) Stokes, Stephen P.; Lawrence, Simon E.; Maguire, Anita; Science Foundation Ireland
The work presented in this dissertation focuses on the molecular features of small molecules and their interactions with pharmaceutically relevant molecules. Emphasis is placed on cocrystallisation; a method that facilitates the formation of multicomponent forms of molecules with non-ionisable or weakly ionisable functional groups. This research has been divided into nine chapters. Chapter 1 describes the concept of crystallisation and introduces supramolecular chemistry. Through the explanation of hydrogen and halogen bonding, which is prominent throughout this work, the concept of supramolecular synthons is unveiled for multicomponent compounds. Finally, applications of cocrystals involving pharmaceutically industrially relevant compounds amongst others are discussed. Chapter 2 gives an overview of the general procedures carried out in this work. Furthermore, a list of the analytical equipment employed is described. Chapter 3 initially describes the crystal landscape of the secondary amide γ-lactam, 2-pyrrolidone, 1, with extension to other lactam systems. This compound is of relevance as it is used routinely as a high boiling solvent in the pharmaceutical industry. A cocrystal screen of 1 was carried out with a range of amide and carboxylic acid based coformers, and fenamic acid active pharmaceutical ingredients (APIs). The common structural motifs of the successfully resolved single crystals were grouped and discussed, and the robustness of the cocrystal forming abilities of 1 demonstrated. Chapter 4 discusses the benzene fused γ-lactam, 2-oxindole, 2, which is the benzene fused analogue of 1. An initial polymorphic screen was employed with a subsequent cocrystal screen of 2 with a range of coformers similar to 1. From this, an investigation into the solid-state similarities between cocrystals of 1 and 2 was conducted, revealing both similarities and variations in the observed motifs. Chapter 5 focuses on a molecule of pharmaceutical relevance, namely modafinil, 3, an anti-narcoleptic drug. In an attempt to utilise the halogen bonding potential of both 3 and 1,4-diiodotetraflourobenzene, the discovery of a dihydrate of 3 is described with characterisation of the resultant solid-state structure. Chapter 6 focuses on 6-propyl-2-thiouracil, 4, an API used in the treatment of Graves’ disease. A cocrystal screening involving acids, amides, fenamic acids and other API molecules was conducted. The motifs of successful single crystal structures are discussed and the structural properties rationalised. Chapter 7 describes the synthetic strategies to synthesise four fenamic acid based molecules, two of which are novel molecules containing methylene linkers. Chapter 8 Conclusion summarising the main findings of the entire work. Chapter 9 Appendix
Telescoping of transition metal catalysed and biocatalysed reactions
(University College Cork, 2020-11-10) Kelly, Áine; Moynihan, Humphrey A.; Science Foundation Ireland
This thesis describes the telescoping reactions which exploit the diverse catalytic approaches of transition metal catalysis and biocatalysed processes. These processes would normally have conditions that are incompatible. Telescoping these processes into a single process will provide powerful enantioselectivity which exploits the efficiency of transition metal catalysis and the stereoselectivity of biocatalysis. Chapter one provides an overview of asymmetric synthesis, both –diazocarbonyl compound synthesis and reactions, and biotransformations. It also gives an overview of telescoping systems from the literature. The results of this research are discussed in both chapter two and three. Chapter two describes the initial work undertaken during this project including attempting intramolecular X–H insertion reactions and intramolecular C–H insertion and then the Baker’s yeast reduction of those products. The batchwise step–by–step transformations were undertaken initially and then telescoped together overcoming problems associated with the process. Chapter three describes the C–H intramolecular reactions with both rhodium (II) acetate and copper (II) triflate to produce a range of 2–benzenesulfonyl substituted cyclopentanone compounds. These compounds were then kinetically resolved via a Baker’s yeast mediated reduction. These individual steps were optimised before they were telescoped together. The range of 2–benzenesulfonyl substituted cyclopentanone compounds also underwent ring cleavage to give simple carboxylic acids which could then be desulfonylated to simple alcohol compounds with a stereocentre in the middle of the chain. Chapter four contains the full experimental details and spectral characterisation of all the compounds synthesised in this project, while the details of the chiral stationary phase HPLC analysis is included in the appendix.

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