Determining the solubility of haematin and haem-drug complexes at relevant pH in aqueous medium via spectrophotometric titration
| dc.contributor.advisor | Wicht, Kathryn | |
| dc.contributor.author | Soogary, Ghannish | |
| dc.date.accessioned | 2024-07-02T10:14:21Z | |
| dc.date.available | 2024-07-02T10:14:21Z | |
| dc.date.issued | 2023 | |
| dc.date.updated | 2024-06-04T13:52:30Z | |
| dc.description.abstract | For centuries, quinoline-based compounds have formed the mainstay of treatment against the pervasive and deadly infectious disease of malaria. While the ongoing emergence of resistance to existing chemotherapies has driven efforts to discover new antimalarial chemotypes, interest in the quinoline drugs has not waned. The two most important classes of quinoline-based drugs, namely the 4-aminoquinolines and the quinoline methanols — represented in this study by chloroquine (CQ) and quinidine (QD), respectively — are widely accepted to act against the most virulent malaria parasite, Plasmodium falciparum, via the haemozoin inhibition pathway within the acidic digestive vacuole (DV) of the pathogen. However, mechanistic details of this mode of action are still unresolved. Following the release of toxic ferriprotoporphyrin IX (Fe(III)PPIX, or ferrihaem) in the DV upon haemoglobin degradation by the parasite, the formation of a drug– ferrihaem complex has been hypothesised to be responsible for the parasiticidal effects of various haemozoin inhibitors. Information on the solubilities of drug–ferrihaem complexes and of ferrihaem itself is scarce, and would enrich the arsenal of knowledge toward resolving the intricate mechanism of parasite death caused by these complexes. As the concentration of the drug−ferrihaem complex relies on the solubility of ferrihaem itself, the latter was investigated in this research at pH values approximating those of the parasitic DV, in exclusively aqueous medium. Based on the methodology employed in a previous study, UV– visible spectrophotometric titration experiments of haematin with sodium hydroxide were performed. The optimal incubation time periods allowing for complete sample equilibration were determined to be 16 days at both 10°C and 20°C, 12 days at 25°C and 8 days at both 37°C and 50°C. The recrystallisation of haematin was determined to be unnecessary since indistinguishable titration and solubility data were obtained from the use of recrystallised haematin versus commercially-sourced haematin without further purification. However, at each of the five temperatures, erratic data points were observed at low pH values in the spectrophotometric titration data, possibly due to solid haematin not dissolving. As a result, reliable and reproducible thermodynamic parameters for ferrihaem solubility could not be determined under these conditions. Nevertheless, the solubility of π–π dimeric ferrihaem at the parasitic DV pH of 5.0 was predicted to be 2.6 ± 1.3 µM at 37°C via extrapolation from the remaining data points. In an alternative approach, spectrophotometric titrations of haematin with perchloric acid were conducted to directly investigate ferrihaem solubility at a more relevant pH range. The optimal incubation time to ensure sample equilibration was determined to be 24 hours. However, as pH was lowered, the UV–vis spectra exhibited the collapse of the ferrihaem Q band and the concomitant appearance of a charge-transfer band due to porphyrin aggregation, making it impossible to obtain solubility data. Consequently, a pyridine–ferrihaemochrome method was employed to enable the quantification of free ferrihaem, yielding a reliable solubility value for dimeric ferrihaem of 0.24 ± 0.08 µM at a pH of 5.0 and a temperature of 37°C. The solubility product constant for the dimer was computed to be (1.9 ± 0.4) × 10-42. The pyridine-based acid titration methodology was also utilised to determine the solubilities of two drug–ferrihaem complexes. First, the optimal sample incubation time was determined to be 48 hours for each of the CQ–ferrihaem and QD–ferrihaem complexes, however, consistent scatter in the UV–vis spectrophotometric titration data was observed due to extremely poor solubilities of the complexes at pH values close to 5.0. Consequently, ranges of values were reported within which the true solubility of the complexes would most likely lie: for the dimeric CQ–ferrihaem complex, lower and upper bounds of 0.5 nM and 1.8 nM with a 95% confidence interval of 1.0 nM – 1.1 nM, and for the QD–ferrihaem complex, lower and upper bounds of 0.2 nM and 18.0 nM with a 95% confidence interval of 2.0 nM – 2.9 nM. Overall, this dissertation has employed spectrophotometric tools to provide physiologicallyrelevant solubility data for ferrihaem and, for the first time, the drug–ferrihaem complexes of the clinically-relevant antimalarials CQ and QD. These represent a crucial set of information to bolster an in silico model aiming at elucidating the haemozoin inhibition pathway and the modes of action of existing and experimental antimalarials — paving the way for the development of new, life-saving medicines against this devastating infectious disease. | |
| dc.identifier.apacitation | Soogary, G. (2023). <i>Determining the solubility of haematin and haem-drug complexes at relevant pH in aqueous medium via spectrophotometric titration</i>. (). ,Faculty of Science ,Department of Chemistry. Retrieved from http://hdl.handle.net/11427/40203 | en_ZA |
| dc.identifier.chicagocitation | Soogary, Ghannish. <i>"Determining the solubility of haematin and haem-drug complexes at relevant pH in aqueous medium via spectrophotometric titration."</i> ., ,Faculty of Science ,Department of Chemistry, 2023. http://hdl.handle.net/11427/40203 | en_ZA |
| dc.identifier.citation | Soogary, G. 2023. Determining the solubility of haematin and haem-drug complexes at relevant pH in aqueous medium via spectrophotometric titration. . ,Faculty of Science ,Department of Chemistry. http://hdl.handle.net/11427/40203 | en_ZA |
| dc.identifier.ris | TY - Thesis / Dissertation AU - Soogary, Ghannish AB - For centuries, quinoline-based compounds have formed the mainstay of treatment against the pervasive and deadly infectious disease of malaria. While the ongoing emergence of resistance to existing chemotherapies has driven efforts to discover new antimalarial chemotypes, interest in the quinoline drugs has not waned. The two most important classes of quinoline-based drugs, namely the 4-aminoquinolines and the quinoline methanols — represented in this study by chloroquine (CQ) and quinidine (QD), respectively — are widely accepted to act against the most virulent malaria parasite, Plasmodium falciparum, via the haemozoin inhibition pathway within the acidic digestive vacuole (DV) of the pathogen. However, mechanistic details of this mode of action are still unresolved. Following the release of toxic ferriprotoporphyrin IX (Fe(III)PPIX, or ferrihaem) in the DV upon haemoglobin degradation by the parasite, the formation of a drug– ferrihaem complex has been hypothesised to be responsible for the parasiticidal effects of various haemozoin inhibitors. Information on the solubilities of drug–ferrihaem complexes and of ferrihaem itself is scarce, and would enrich the arsenal of knowledge toward resolving the intricate mechanism of parasite death caused by these complexes. As the concentration of the drug−ferrihaem complex relies on the solubility of ferrihaem itself, the latter was investigated in this research at pH values approximating those of the parasitic DV, in exclusively aqueous medium. Based on the methodology employed in a previous study, UV– visible spectrophotometric titration experiments of haematin with sodium hydroxide were performed. The optimal incubation time periods allowing for complete sample equilibration were determined to be 16 days at both 10°C and 20°C, 12 days at 25°C and 8 days at both 37°C and 50°C. The recrystallisation of haematin was determined to be unnecessary since indistinguishable titration and solubility data were obtained from the use of recrystallised haematin versus commercially-sourced haematin without further purification. However, at each of the five temperatures, erratic data points were observed at low pH values in the spectrophotometric titration data, possibly due to solid haematin not dissolving. As a result, reliable and reproducible thermodynamic parameters for ferrihaem solubility could not be determined under these conditions. Nevertheless, the solubility of π–π dimeric ferrihaem at the parasitic DV pH of 5.0 was predicted to be 2.6 ± 1.3 µM at 37°C via extrapolation from the remaining data points. In an alternative approach, spectrophotometric titrations of haematin with perchloric acid were conducted to directly investigate ferrihaem solubility at a more relevant pH range. The optimal incubation time to ensure sample equilibration was determined to be 24 hours. However, as pH was lowered, the UV–vis spectra exhibited the collapse of the ferrihaem Q band and the concomitant appearance of a charge-transfer band due to porphyrin aggregation, making it impossible to obtain solubility data. Consequently, a pyridine–ferrihaemochrome method was employed to enable the quantification of free ferrihaem, yielding a reliable solubility value for dimeric ferrihaem of 0.24 ± 0.08 µM at a pH of 5.0 and a temperature of 37°C. The solubility product constant for the dimer was computed to be (1.9 ± 0.4) × 10-42. The pyridine-based acid titration methodology was also utilised to determine the solubilities of two drug–ferrihaem complexes. First, the optimal sample incubation time was determined to be 48 hours for each of the CQ–ferrihaem and QD–ferrihaem complexes, however, consistent scatter in the UV–vis spectrophotometric titration data was observed due to extremely poor solubilities of the complexes at pH values close to 5.0. Consequently, ranges of values were reported within which the true solubility of the complexes would most likely lie: for the dimeric CQ–ferrihaem complex, lower and upper bounds of 0.5 nM and 1.8 nM with a 95% confidence interval of 1.0 nM – 1.1 nM, and for the QD–ferrihaem complex, lower and upper bounds of 0.2 nM and 18.0 nM with a 95% confidence interval of 2.0 nM – 2.9 nM. Overall, this dissertation has employed spectrophotometric tools to provide physiologicallyrelevant solubility data for ferrihaem and, for the first time, the drug–ferrihaem complexes of the clinically-relevant antimalarials CQ and QD. These represent a crucial set of information to bolster an in silico model aiming at elucidating the haemozoin inhibition pathway and the modes of action of existing and experimental antimalarials — paving the way for the development of new, life-saving medicines against this devastating infectious disease. DA - 2023 DB - OpenUCT DP - University of Cape Town KW - Chemistry LK - https://open.uct.ac.za PY - 2023 T1 - Determining the solubility of haematin and haem-drug complexes at relevant pH in aqueous medium via spectrophotometric titration TI - Determining the solubility of haematin and haem-drug complexes at relevant pH in aqueous medium via spectrophotometric titration UR - http://hdl.handle.net/11427/40203 ER - | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11427/40203 | |
| dc.identifier.vancouvercitation | Soogary G. Determining the solubility of haematin and haem-drug complexes at relevant pH in aqueous medium via spectrophotometric titration. []. ,Faculty of Science ,Department of Chemistry, 2023 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/40203 | en_ZA |
| dc.language.rfc3066 | Eng | |
| dc.publisher.department | Department of Chemistry | |
| dc.publisher.faculty | Faculty of Science | |
| dc.subject | Chemistry | |
| dc.title | Determining the solubility of haematin and haem-drug complexes at relevant pH in aqueous medium via spectrophotometric titration | |
| dc.type | Thesis / Dissertation | |
| dc.type.qualificationlevel | Masters | |
| dc.type.qualificationlevel | MSc |