Saudi Cultural Missions Theses & Dissertations
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Item Restricted PROTEIN NUTRITION IN PAEDIATRIC CROHN’S DISEASE(University of Nottingham, 2025) Aljilani, Bayan; Moran, Gordon; Tsintzas, KostasAltered nutritional status is well documented in Crohn’s disease patients, which may lead to low muscle mass and deteriorated function (sarcopenia) and adverse disease outcomes. Although a variety of experimental methods have assessed muscle mass and function in paediatric CD patients, there is no conclusive consensus on the definition of sarcopenia in those patients. However, most of the studies in the literature have showed reduction in muscle mass (MM), lean mass (LM) and fat-free mass (FFM) in paediatric CD compared with healthy controls or reference data. Historically, underweight was frequently linked with paediatric CD. However, recent evidence suggests a shift towards adiposity rather than underweight being prevalent in paediatric CD. Our knowledge of the relationship between adiposity and adverse disease outcomes in children with CD is limited. In addition, clinical characteristics of CD patients may vary depending on age at diagnosis. Moreover, adult CD patients exhibit altered nutritional status and eating behaviour that impact negatively on habitual dietary protein intake, protein metabolism and muscle mass. However, these relationships have never been investigated in paediatric CD. This PhD thesis used the gold standard technique, magnetic resonance imaging (MRI), to assess leg muscle volume in children with stable CD disease and compared it with matched healthy subjects. These findings were linked with dietary protein intake, fasting plasma amino acids (AAs) and eating behaviour traits. In addition, the MRI technique was used to compare psoas muscle cross sectional area (PCSA) between paediatric with active CD versus those with inactive CD. However, conclusive findings were limited due to small sample size in both studies. This thesis highlighted the need for standardisation of body composition and sarcopenia terminology, as well as the valid use of assessment tools in adequately powered populations with appropriately matched comparators, to establish a clear definition of sarcopenia and assess its prevalence in paediatric IBD. In addition, it revealed that adiposity is linked to poor clinical outcomes in a long-term follow-up cohort of paediatric-onset CD, and that the age at disease onset is also associated with negative clinical outcomes. Moreover, this thesis revealed comparable dietary protein and energy intake, and eating behaviour traits between stable CD paediatric patients and matched healthy controls, which may explain the lack of differences in leg muscle volume and handgrip strength (indices of sarcopenia) and circulating AAs between groups. Furthermore, there were no significant differences in PCSA assessed by MRI between active and inactive paediatric CD; however, age and gender were associated with muscle mass. These findings might have been affected by disease duration and further disease burden that need to be taken into consideration in future studies. Data collection in this thesis was significantly impacted by the COVID pandemic. Hence, most of the studies performed were either retrospective in nature or cross-sectional studies which require further prospective cohort studies with larger sample size to enhance generalisability and confirm findings. Therefore, the aims of this thesis were to a) systematically evaluate changes in skeletal muscle mass and function in paediatric IBD, b) investigate the relationship between adiposity and age of disease onset with adverse clinical outcomes in paediatric patients with CD, c) assess protein intake, circulating AAs and skeletal muscle mass and evaluate eating behaviour in paediatric CD and age, sex and BMI-matched healthy controls and d) describe the differences in PCSA assessed by MRI between active and inactive paediatric CD and examine the impact of age, gender, disease activity, and other disease-related variables on muscle mass. This research integrated several approaches to assess the impact of nutritional status, early age of disease onset on disease outcomes, and muscle size in paediatric CD patients. In addition, it compared protein intake, eating behaviour between children with stable CD and healthy matched peers to offer a holistic understanding of their impact on muscle mass and strength (as an index of sarcopenia) and circulating amino acids profiles in paediatric CD. While the studies presented in this thesis did not reveal significant differences in certain aspects of dietary protein intake, muscle size and strength, they did highlight several areas requiring further investigation. The complicated nature of disease burden and activity, alongside paediatric growth, necessitates further research to better understand the definition, management and prevention of sarcopenia in paediatric CD patients.11 0Item Restricted Development and Application of Chemical Sulfation Methods(University of Birmingham, 2024) Alshehri, Jaber; Jones, Alan; He, ShanSulfation is one of the most important modifications that occurs to a wide range of bioactive small molecules including carbohydrates, proteins, flavonoids, and steroids. In turn, these sulfated molecules have significant biological and pharmacological roles in diverse processes including cell signalling, modulation of immune and inflammation response, anti-coagulation, anti-atherosclerosis, and anti-adhesion. However, methods to incorporate sulfate and related sulfur functionality have drawbacks. This thesis is composed of seven individual chapters and contains the collective works of six separate investigations. Chapter 1 describes the approaches to the sulfation of small molecules: current progress and future directions. This chapter highlights the importance of sulfation in critical biological signalling cascades and a key phase II drug metabolism step. It also summarises the most encountered chemical sulfation approaches of small molecules that have been applied to a wide range of molecules, including carbohydrates, proteins, steroids, amongst many others. Chapter 2 describes the design and synthesis of novel sulfur trioxide complexes for sulfation chemistry. This includes the synthesis of tertiary amine sulfur trioxide complexes such as tripropylamine and bulky tertiary amines like triisobutylamine and isopropyl-N-methyl-tert-butylamine. This chapter also provides a general preparation of novel hydrophilic N-substituted morpholine sulfur trioxide (4-methylmorpholine SO3 and 4-ethylmorpholine SO3) which is suitable for sulfating hydrophilic substrates, including amino acids. These reagents were investigated on a simple benzyl alcohol and benzylamine to test whether they could be used as a sulfation source. The results of this investigation led to the preparation of a series of novel sulfating reagents including tripropylamine sulfur trioxide, isopropyl-N-methyl-N-tert-butylamine sulfur trioxide and 4-methyl and 4-ethylmorpholine sulfur trioxide complexes. Chapter 3 describes the sulfation of selected amino acids using 4-methylmorpholine SO3 and 4-ethylmorpholine SO3. This chapter describes the optimised conditions for the sulfation of selected amino acids using a lower temperature strategy and H2O/MeCN solvent system without the need for column chromatography techniques. Chapter 4 describes a novel exchange method to access sulfated molecules using Py·SO3 and Me3N·SO3 complexes. This chapter reports a low-cost in-situ version of Bu3N•SO3 using Py•SO3 and Me3N•SO3 followed by a lipophilic exchange with tributylamine (Bu3N). This method provides an alternative sulfation method based on a cheap, molecularly efficient and solubilising cation exchanging method. This method is amenable to a range of differentially substituted benzyl alcohols, benzylamines and aniline and can also be performed at low temperature for sensitive substrates in good to excellent isolated yields. Chapter 5 provides a convenient chemoselective conversion of the steroidal alcohol and phenol moieties to their corresponding organosulfate using tributylsulfoammonium betaine (TBSAB). This method can be conducted on a millimolar scale and the corresponding steroid sulfates isolated as their biologically relevant sodium salts without the need for ion-exchange chromatography. Furthermore, this chapter reports a convenient method to install an isotopic label, deuterium (2H), combined with estrone sulfation which could be exploited for mass-spectrometric quantification in biological studies. The results of this investigation have demonstrated a suitable method for the preparation of mono- or di-sulfated steroidal skeletons of importance to the fields of biology and spectroscopy. A simplified deuterium labelling-sulfation strategy for estrone is also reported. This isotopically labelled estrone could be used for the detection of substances of abuse through to cancer diagnosis applications as well as pharmacokinetics studies. Chapter 6 describes the investigation in to whether the biologically active heparan sulfate-glycomimetic could act as a source of the sulfation pathway in the body. This hypothesis of in situ sulfur transfer was tested on a simple benzyl alcohol using a biologically active heparan sulfate-glycomimetic. Unfortunately, this attempt was not successful as confirmed by 1H NMR spectroscopic data. Additionally, several attempts were made to sulfate other benzyl alcohol analogues using synthesised sulfation sources such as sodium 4-methylbenzyl sulfate and sodium 4-chlorobenzyl sulfate but also there was no robust evidence of the formation of the desired molecule as confirmed by 1H NMR spectroscopic data. However, for the first time, robust evidence for the stability of benzylic sulfates was found a major anticipated issue with sulfation chemistry more generally. Chapter 7 discusses the biological role of hydrogen sulfide (H2S) and the synthesis of cysteine trisulfide (Cys-SSS-Cys). This chapter describes the synthesis of cysteine trisulfide and highlights the proposed reaction mechanism as well as purification and isolation strategies. The gram-scale synthesis of cysteine trisulfide was submitted to our biological collaborator; Dr Madhani (Institute of Cardiovascular Sciences, University of Birmingham, UK) and screened for biological activity. Treatment of HEK293T (Human embryonic kidney) cells with the cysteine trisulfide resulted in high intracellular levels of hydropersulfides and thus, protection from electrophilic stress.11 0Item Restricted Studies of class II drugs-amino acid hydrotrope and dissolution of freeze dried co-amorphous formulations(2023-05-01) Alsalhi, Mohammed Suleiman; Chan, Ka Lung AndrewBackground The aqueous solubility and dissolution of active pharmaceutical ingredients (APIs) are defined as the most critical issues plaguing the development pipeline of solid formulations in the pharmaceutical industry. An estimated 40% of market approval drugs and 90% of the new API suffer from poor water solubility. Amino acids have shown promising abilities to form complexes with poorly water-soluble drugs (PWSDs) and improve their physicochemical properties. Amino acids have the advantages of being generally regarded as safe, organic, natural and low molecular weight excipients with varying in size, hydrophobicity and polarity, and the ability to produce various non-ionic or/and ionic interactions or as hydrotropes with drug molecules to improve the solubility and dissolution of PWSDs. However, there is still a lack of understanding of amino acids at the molecular level in their ability to enhance the aqueous solubility of PWSDs. A freeze drying method also has not been investigated yet for its ability to successfully produce a CAM system that has non-ionic interactions between PWSD and amino acid. Aim The main aim of this thesis is to demonstrate the potential to enhance the aqueous solubility and dissolution properties of class II BCS drugs using amino acids either as hydrotrope or as the low molecular weight (LMW) hydrophilic excipients via formulating co-amorphous (CAM) system by freeze drying method using TBA-water solvent system. Methods The molecular basis of hydrotropic interactions was demonstrated by investigating two model class II BCS drugs (carbamazepine and indomethacin) combined with 12 amino acids including phenylalanine, tryptophan, isoleucine, proline, valine, glycine, serine, threonine, arginine, lysine, histidine and aspartic acid in water by UV-Vis and NMR spectroscopies at 25 ◦C, 30 ◦C and 45 ◦C. The amino acids were chosen based on their different side chains (neutral aromatic, aliphatic, polar charged or uncharged) to investigate their hydrotropic performance. II The mechanism of solubility enhancement study has evaluated the contribution of non-ionic interactions in salt-based CAM systems between a model acidic drug, indomethacin, and basic amino acids, arginine, lysine and histidine in a water solvent system using UV-Vis and FTIR spectroscopies. Freeze drying was used to produce the salt drug-amino acid CAM for further characterization of their interactions in the solid phase using thermal analysis (DSC and TGA) and spectroscopy. The non-salt-based CAM system study has explored the potential of freeze drying as an efficient manufacturing process to produce a CAM system that has weak non-ionic interactions between the amino acid and the drug using the tert-butyl alcohol (TBA)-water cosolvent system. The liquid, frozen solutions and freeze-dried materials were systemically characterised for their thermal properties or physical cake appearance, residual solvent, amorphous formation, molecular interactions and drug content using a number of techniques (e.g., DSC, TGA, XRPD, FTIR and UV Vis). The optimal drug: co-former ratio for dissolution profile and long-term storage stability was also evaluated. Results In the hydrotropic study, a linear solubility curve was observed between indomethacin and mono-neutral hydrophobic amino acids (phenylalanine, tryptophan, isoleucine, proline and valine) at a molar ratio of well beyond 1:1 indicating that the interaction is predominantly non ionic between the drug and the hydrotropes. Interestingly, the aqueous solubility of carbamazepine (a neutral compound) was enhanced by neutral, charged basic or acidic amino acids, confirming the presence of hydrophobic interactions that involve H-bonds, H/π and π/π stacking. The results were confirmed by UV-Vis and NMR spectroscopies. The combination of multiple neutral amino acids has shown an additive hydrotropic effect in the indomethacin solubility study with up to 7-fold increase being observed. In the mechanism of solubility enhancement study, at low concentrations of amino acids, indomethacin-arginine or lysine complexes have shown a linear relationship (AL-type phase solubility diagram) between indomethacin solubility and amino acid concentrations, producing III 0.92:1 or 0.97:1 (near stoichiometry) molar ratio of drug-arginine or lysine complexes, respectively as expected due to the strong electrostatic interactions. However, indomethacin histidine complexes have shown a nonlinear relationship with lower improvement in indomethacin solubility due to the weaker electrostatic interactions when compared to arginine and lysine. Interestingly, the results have also shown that at high arginine concentrations, the linearity was lost between indomethacin solubility and amino acid concentration with a negative diversion from linearity, following the type-AN phase solubility. This is indicative of that the electrostatic interaction is being interrupted by non-electrostatic interactions, as seen with histidine. The indomethacin-lysine complex, on the other hand, has shown a complex curved phase solubility diagram (type BS) as lysine self-assembles and polymerizes at higher concentrations with results showing the involvement of weak non-ionic interactions. Non-ionic interactions including H-bonds, H/π and π/π stacking were confirmed to be involved in the salt based CAM systems between acidic drugs and basic amino acids using FTIR spectroscopy. In the non-salt-based CAM system study, freeze-drying appears to be able to successfully produce non-salt CAM with a uniform and elegant cake appearance using the cosolvent system. The molecular interactions involved H-bonds, H/π and π–π between compounds have been confirmed to be involved. Interestingly, the drug release rate of 70% w/w drug loading and below formulations were superior compared to the pure crystalline drug. Further, formulations with below 80% w/w drug loading have shown to be physically stable over 9 months at dry condition/25 °C. The optimal ratio between indomethacin and tryptophan, based on the long term storage physical stability result and dissolution profile is higher than the 1:1 molar ratio (1:0.53 weight ratio), although a 1:1 ratio is often used in producing the CAM system. Conclusion This research demonstrates for the first time the potential of amino acids as hydrotropes to improve aqueous solubility of PWSDs. It also confirms the solubility improvement of the insoluble acidic drug in the presence of basic amino acids was due to not just the ionic interactions but also has some contribution from non-ionic interactions. The gentle freeze drying method has also shown to be a feasible technique for producing non-salt CAM poorly class II drug to amino acid IV with the TBA-water cosolvent system, with an improved dissolution rates and physical stability upon long-term storage. The non-ionic CAM system is important as these interactions do not alter the structural or functional properties of drugs, and also are less dependent on the pH, and thus may help to overcome issues such as disproportionation on storage and dissolution. An understanding, at the molecular level, of the CAM systems is important to achieve their full performance and optimum use in the oral delivery route12 0