Doctoral Degrees (Pharmaceutical Sciences)

Permanent URI for this collectionhttps://hdl.handle.net/10413/6746

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Design of advanced multifunctional biomaterial-based biomimetic and pH-responsive hybrid nanocarriers for antibiotic delivery against bacterial infections and sepsis.
(2023) Elhassan, Eman Hussain Elmubarak.; Govender, Thirumala.; Omolo, Calvin Andeve.
Despite the notable improvements in the management of bacterial infections and sepsis, the mounting threat of antibiotic resistance on a global scale is leading towards a post-antibiotic era. Nano-drug delivery systems have improved the delivery and efficacy of various antibiotics. Biomimicry and stimuli-responsiveness have recently been used to improve the targetability of these nanocarriers, and enhance their localization at infected sites, thus improving overall therapeutic outcomes and reducing toxicity. Strategies such as targeting bacterial biofilms and efflux pumps can further enhance the delivery and effectiveness of antibiotics. Developing smart biomaterials with multifunctional properties to confer biomimetic, stimuli-responsive and antivirulence properties to antibiotic nanocarriers is the focus of ongoing research. Therefore, the general aim of this study was to investigate the potential of various novel multifunctional biomaterial-based hybrid nanocarriers (HNs), including biomimetic and/or pH-responsive HNs in enhancing the targeted delivery of antibiotics and modulating the proinflammatory response against bacterial infections and sepsis. In this study, two biomaterials with multifunctional activities, hyaluronic acid-lysine conjugate (HA-Lys) and tannic acid (TA), were employed to design, formulate, and extensively characterize innovative biomimetic and pH-responsive HNs for efficient and targeted delivery of antibiotics. The novel HA-Lys was synthesized and fully characterized using proton nuclear magnetic resonance (1H NMR) spectroscopy and Fourier-transform infrared spectroscopy (FT-IR). Then it was successfully employed with tocopherol succinate (TS) and Oleylamine (OLA) to fabricate biomimetic pH-responsive vancomycin-loaded hybrid nanostructured lipid carriers (VCM-HNLCs). The prepared VCM-HNLCs were spherical and had average diameters, zeta potential, polydispersity index, drug encapsulation efficiency and loading capacity of 110.77  1.69 nm, 0.11  0.02, -2.92  0.21 mV, 76.27  1.20 % and 8.36  0.25 %, respectively. Both HA-Lys conjugate and its respective nanoformulations had excellent biosafety profiles (>70 % cell viability and ˂ 1 % hemolytic effect). Possible VCM-HNLCs competitive inhibition activity to toll-like receptors 2 and 4 (TLR2 and TLR4) was demonstrated via microscale thermophoresis (MST) analysis, which showed a 5-times and 16-times lower Kd values than their natural substrates peptidoglycan (PGN) and lipopolysaccharide (LPS), respectively. VCM-HNLCs exhibited a pH-responsive drug release profile under acidic conditions, higher bacterial killing kinetics, enhanced antibacterial, anti-biofilm, and efflux pump inhibition activities over bare VCM. Also, they showed an improved activity in neutralizing reactive oxygen species (ROS) and modulating the inflammatory response induced by LPS. On the other hand, tannic acid (TA) and Oleylamine (OLA) were successfully employed to formulate biomimetic ciprofloxacin-loaded tannic acid hybrid nanoparticles (CIP-loaded TAH-NPs) to enhance the efficacy of CIP against bacterial infections and sepsis. The prepared HNs had onion-shaped morphology, with average diameters, zeta potential, polydispersity index, drug encapsulation efficiency and loading capacity of 85.65 ± 0.89 nm, 0.126 ± 0.01, +16.3 ± 0.23 mV, 68.73 ± 0.54 % and 6.86 ± 0.09 %, respectively. The hemolysis and MTT assays confirmed the biosafety and non-hemolytic activity of CIP-loaded TAH-NPs formulations (>70 % cell viability and ˂ 1 % hemolytic effect). The results of MST investigations and in-silico simulations demonstrated that TA and its nanoformulation (CIP-loaded TAH-NPs) competitively inhibited TLR4 compared to its natural substrate LPS. CIP-loaded TAH-NPs showed a diffusion-based sustained release profile at physiological pH 7.4. Also, in comparison to bare CIP, the hybrid nanovesicles demonstrated improved antibacterial, anti-biofilm and efflux pump inhibition properties, as well as faster bacterial killing kinetics. Moreover, they showed a significant neutralization of ROS and the ability to control the inflammatory responses brought on by LPS. In summary, VCM-HNLCs and CIP-loaded TAH-NPs were successfully formulated and showed significant improvement in antibiotics efficacy and overall therapeutic outcomes. This study confirmed the potential of biomimetic stimuli-responsive antibiotic hybrid nanocarriers for enhancing antibiotic efficacy against bacterial sepsis and addressing the antimicrobial resistance crisis. The data from this study has resulted in one first-authored review article, two first-authored research publications and one co-authored review article.
Designed, synthesis and antibacterial evolution of piperazine hybrids.
(2023) Girase, Pankaj Sanjay.; Karpoormath, Rajshekhar.
Piperazine is a kind of azacycloalkane that has two nitrogen atoms at1-4 places on a six-membered ring. It is well known that molecules with the piperazine ring, a key component of the N-heterocyclicfamily of bioactive natural products, are often prevalent in biologically active substances. There are many antitumor, antibacterial, antiinflammatory, antipsychotic, antifungal, and anti-diabetic compounds based on the piperazine scaffold, which has been recognized as an active structure in drug discovery. Piperazine hybrids with different moieties such as isoniazid, coumarin, benzothiazinones, isoquinoline, triazole,pyrrole, and oxazolidinone showed good activity against mycobacterium tuberculosis and microbial strains. In this thesis we have demonstrated the synthesis of piperazine hybrid with hydrazides, hydrazines, and coumarines and tested their activity against mycobacterium tuberculosis, gram positive and gram negative microbial strains. In Chapter 2, we have covered topics related to the analogues of piperazine that have anti-tubercular efficacy. This chapter we have published as a review article in European Journal of Medicinal Chemistry. In this review, we have made a concerted effort to trace the development of anti-mycobacterial compounds during the past 50 years (1971-2019), focusing on instances where piperazine has been utilized as a key building block. In depth discussion of the design, rationale, and structure-activity relationship (SAR) of the reported potent piperazine-based anti-TB molecules will help medicinal chemists fill in the blanks, capitalize on the reported strategies, and create more effective, selective, and less hazardous anti-mycobacterial drugs. In chapter 3, we have developed and synthesized a new class of hybrids between phenylpiperazine and hydrazides (c1-c15). During the synthesis of phenyl piperazines, the formylation of piperazine was observed, a phenomenon on which we have developed a different methodology discussed in chapter 5. All of the derivatives have been tested in vitro against H37Rv, a strain of mycobacterium. In addition, we have analysed the zone of inhibition against eight different bacterial strains, including both gram-positive (methicillin resistant staphylococcus aureus (MRSA), Streptococcus pyrogens, Bacillus subtilis, Enterococcus faecium, and Staphylococcus aureus), and gram-negative (Enterobacter hormaechei, Pseudomonas aeruginosa, and Escherichia coli) bacteria. Among the derivatives tested, only compound c8 showed action against the mycobacterium strain H37Rv (MIC value of 0.39-0.78 g/ml). No zone of inhibition was seen for any of the microbiological strains when exposed to any of the synthesized compounds. The hybrids between phenylpiperazine sulphonamide and phenyl hydrazide (E1-E6) and phenylpiperazine sulphonamide and phenyl hydrazine (F7-F19) were proposed and synthesized in chapter 4. All substances were evaluated against mycobacterium tuberculosis, five gram-positive and three gram-negative bacterial strains in vitro. Derivatives E1 and E2 with an isoniazid moiety were the most effective in inhibiting the growth of the H37Rv strain of tuberculosis, with an IC50 value of 3.125 M. Of the derivatives tested, F10 showed significant action against the gram-positive bacteria Enterococcus faecium (7.81 μg/mL), whereas the others (E2, E6, F7, F9, F14) were only moderately active (250-62.5 μg/mL). Using a the molecular hybridization strategy, we were enabled to create novel analogues of coumarin-(phenylsulfonyl)piperazine and 4-methyl coumarin-(phenylsulfonyl)piperazine in chapter 5.All synthesised compounds were evaluated for their in vitro anti-mycobacterial and antimicrobial activity against H37Rvand a variety of antimicrobial gram-positive and gram-negative strains.The Compounds 6G, 6H, 10D and 10E displayed moderate inhibition against gram positive and gram negative strains with MIC values in the range of 62.5-250 (table 1) against MRSA, Bacillus subtilis, and Enterococcus faecium, and gram negative strains Enterobacter hormaechei, Pseudomonas aeruginosa, and Escherichia coli.In addition, the Structure-Activity Relationship (SAR) analysis showed that phenyl ring substituents could enhance antibacterial activity. Chapter 6 came from the process of synthesizing phenyl piperazine in chapter 1. This chapter disclosed a method for efficient synthesis of transamidation in the presence of Iodine and NH2OH.HCl which published in Chemistry Select. This method is efficient for a broad range of primary, secondary, and tertiary amides, and it enables the formylation, acylation, and benzoylation of a number of different amines. The key benefits of the present technique are that it is easy to follow, quick, does not need a metal catalyst, uses a starting material that is inexpensive, and has a low effect on the environment when the synthesis process is carried out. All of the chapters in this thesis are written in thesis by publication style, rather than the conventional style.
The in vitro and in vivo efficacy of novel metallo-β- lactamase inhibitors co-administered with meropenem to target CREs.
(2022) Reddy, Nakita.; Naicker, Tricia.
The evolution and phenotypic expression of metallo β-lactamase genes across the world has led to the escalated transmission rates of carbapenem resistance. The effect has crippled the already impaired healthcare system, with the emergence of COVID-19 exacerbating the crisis further. Our plight for a solution to combat antimicrobial resistance has not been greater. One strategy to tackle this non-susceptibility is the development of metallo-β-lactamase inhibitors that can neutralize the metallo-β-lactamase enzyme, thereby allowing the carbapenem antibiotic to elicit its function on the microorganism. Currently, there is no FDA-approved metallo-β-lactamase inhibitor to meet the clinical challenges of drug resistance. In a desperate need to find a candidate drug, research has been initiated into the discovery and development of biologically active inhibitors. Therefore, this thesis focuses on the advances made by our research group, the Catalysis and Peptide Research Unit, in developing novel β-lactam derived inhibitors; NOTA, NO3PY, BP- 1, 6,10 and 14, that re-sensitize the microbe to the efficacy of meropenem. The in vitro and in vivo activities of the initial chelators, NOTA and NO3PY, were evaluated as potential metallo-β-lactamase inhibitors (MBLIs) against metallo-β-lactamase (MBL) resistant bacteria. Time-kill studies showed that NOTA and NO3PY restored the efficacy of meropenem against all bacterial strains tested. A murine infection model was then used to study both metal chelators’ in vivo pharmacokinetics and efficacy. NO3PY displayed poor bioavailability at the selected doses using a validated LC-MS/MS method, therefore discouraging the in vivo efficacy evaluation. NOTA showed good bioavailability; hence, the in vivo efficacy was determined in a murine thigh infection model. The co-administration of meropenem and NOTA (100 mg/kg.bw each) significantly decreased the colony-forming units of K. pneumoniae NDM over an eight-hour treatment period. The findings suggested that chelators, such as NOTA, hold strong potential for use as an MBLI in treating CRE infections; however, further preclinical development was needed to improve the pharmacokinetic properties of these agents to increase their bioavailability and tissue distribution. With this information, our group derivatized NOTA by coupling it to a β-lactam to create the BP series of novel MBLIs. The results generated by the BP compounds have proven to interact synergistically with meropenem, by restoring the MIC of meropenem to therapeutically acceptable concentrations (< 2 mg/L) that concur with the breakpoints outlined by CLSI. In addition, the bactericidal activity of the re-sensitized meropenem was evident in the time-kill study over 24 hours. Cytotoxicity assays were further conducted to study the inhibitors, with an outcome in favor of safe administration in vivo. The metallo-β-lactamase inhibitors reported herein have demonstrated good potency against NDM-1 and VIM-2 metallo-β-lactamases with a Ki of 25-97μM. Since the BP compounds are metal chelators that function as metallo-β- lactamase inhibitors, it was important to determine the binding specificity of the BP compounds to a physiologically relevant zinc-harboring enzyme, glyoxylase II. At concentrations of up to 500 μM of BP, the activity of glyoxylase II remained unhindered. This confirmed the hypothesis of BP specificity to be exclusive to NDM-1 and VIM-2 metallo-β-lactamases. These findings prompted further interest in the binding exhibited by BP and led to additional studies to address the binding interactions of BP with the metallo-β-lactamases through quenching and computational experiments. Fluorescent quenching experiments investigating the Ka of BP indicated that a higher binding affinity was noted for NDM-1 compared to VIM-2 MBLs, thus implying a stronger interaction with NDM-1. Molecular docking and dynamic simulation experiments shed light on the BPs’ mode of action, showing the interaction of the chelators’ carboxylic moiety with the Zn 2+ ions in the MBLs structure. In favor of this BP series as functional inhibitors, in vivo efficacy was explored in a murine infection model (BP1 and BP10). In Klebsiella pneumoniae NDM infected mice, BP co-administered with meropenem was efficacious in reducing the bacterial load by > 3 log10 units’ post-infection, compared to meropenem monotherapy. These findings validate our strategy for derivatizing NOTA into the series of the BPs, as the bioavailability of NOTA, when coupled to a cephalosporin, improved the overall in vivo efficacy, and allowed the drug to be quantified in plasma under the same conditions previously used. This study clearly indicated the influence of the BP compounds in reducing the bacterial burden and the success of employing combination therapy as a treatment alternative. Moreover, the outcome of this preclinical development represents a solid foundation, whereby we can build on our existing knowledge. In aligning with our research goals of alleviating the threat of antimicrobial resistance, coupling β-lactams to a cyclic zinc chelator offers a safe and efficacious solution to meet the calamity that plagues our healthcare system.
Molecular characterization of multi-drug resistant (MDR) gram-negative bacterial pathogens from environments, patients and staff in a teaching hospital in Ghana.
(2023) Yeboah, Esther Eyram Asare.; Essack, Sabiha Yusuf.; Owusu-Ofori, Alexander.; Agyepong, Nicholas.; Abia, Akebe Luther King.; Amoako, Daniel Gyamfi.; Mbanga, Joshua.
Multidrug resistant Gram-negative bacteria (MDR GNB) are implicated in serious infections both of community and nosocomial origin and may be disseminated in the hospital in the absence of efficient infection prevention and control (IPC) practices. The prevalence and risk factors for rectal colonization of MDR GNB among patients, the carriage of MDR GNB on healthcare workers’ (HCWs’) hands and the contamination patients’ environments with MDR GNB were investigated in a teaching hospital in Ghana. In this prospective study, conducted between April 2021 to July 2021, the phenotypic profiles of the MDR GNB isolates were determined using the VITEK 2 system. Risk factors for colonization with MDR GNB were assessed using univariate and multivariate analysis of associated data. The resistome, virulome, mobilome and genetic relatedness of MDR extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and ESBL-producing or carbapenem resistant Klebsiella pneumoniae isolates from patients and their environment were also determined using whole genome sequencing performed on the Nextseq 550 (2 x 150 bp) and bioinformatics analysis. A total of 585 samples were collected from patients, HCWs’ hands and the hospital environment within the study period. The prevalence of MDR GNB rectal colonization among patients was 50.62% on admission and 44.44% after 48 hours. MDR GNB, frequently E. coli and K. pneumoniae were isolated from 6 (5.26%) and 24 (11.54%) of HCW’s hand swabs and environmental swabs, respectively. Previous hospitalization (p-value = 0.021, OR,95% CI= 7.170 (1.345-38.214) was significantly associated with colonization by MDR GNB after 48 hours of admission while age (21-30 years) (p-value =0.022, OR, 95% CI =0.103(0.015-0.716) was significantly identified as a protective factor associated with a reduced risk of rectal MDR GNB colonization. Rectal carriage and acquisition of ESBL-producing E. coli among patients was 13.65% and 11.32% respectively. blaTEM-1B and blaCTX-M-15 were commonly associated with IncFIB plasmid replicons and co-occurred with aminoglycoside, macrolide, and sulfamethoxazole/trimethoprim resistance. Multiple virulence genes, predominantly, terC were detected in the ESBL E. coli isolates. Sequence types (STs) were diverse and included one novel ST (ST13846) present in two isolates. Phylogenetic analysis grouped the ESBL E. coli isolates into four main clusters. High genetic relatedness was observed between two carriage isolates of ST940 and between a carriage isolate and an environmental isolate of ST648. Isolates with different STs, collected at different times and locations, also showed genetic similarities. Of the ten selected MDR K. pneumoniae isolates, the β-lactamase gene, blaCTX-M-15 was observed in six isolates. Mutations were found in both ompK36 and ompK37 in all isolates (both carriage isolates and isolates from hospital environments). Genes encoding resistance to fluoroquinolone (qnrB), aminoglycosides (aadA1, aadA2, aac(3)-IIa, aac(6')-Ib-cr,aph(3'')-Ib , aph(6)-Id) sulphamethoxazole/trimethoprim (sul1, sul2, dfrA14, dfrA15) were also detected. The K. pneumoniae isolates belonged to seventeen different STs with ST39 most commonly observed and common to both carriage isolates and isolates from hospital environments. A myriad of virulence genes, including irp1, irp2, iutA, gndA, ompA, fes, fep, mrkD and fimH were detected in both carriage and isolates from the hospital environment. IncFIB was the most abundant plasmid replicon occurring in nine (four carriage isolates and five isolates from hospital environments). ESBL-producing K. pneumoniae isolates appeared to be introduced into the hospital from the community. The high colonization of MDR GNB in patients, the carriage of MDR GNB on HCW’s hands, the contamination of hospital environments and the circulation of ESBL-producing E. coli and K. pneumoniae isolates with diverse genomic characteristics, highlights the need for patient screening, and stringent infection prevention and control practices to prevent the spread of MDR GNB in hospitals. The observed clonal relatedness among isolates from patients and the hospital environment, as well as between different patients, suggests a possible transmission within and between sources, hence infection prevention and control practices need to be enhanced to prevent the dissemination and transmission of these resistant strains in the hospital. This study further highlights the usefulness of whole genome sequencing as an effective tool in AMR surveillance.

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Browsing Doctoral Degrees (Pharmaceutical Sciences) by Subject "Antibiotic resistance."