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In the pharmaceutical development pipeline, most drugs are poorly soluble; therefore, it is important to tackle this problem by applying novel drug delivery systems or alter the drug substances with either chemical or physical modification such as amorphous formation, co-crystal formation or solid dispersion system to yield better dissolution, absorption and thus therapeutic efficacy. Applying technologies from other fields to develop such systems could be advantageous. Carbon dioxide (CO2) laser irradiation causes rapid melting and vapourization of materials through the absorption of infrared energy from a laser beam. The aim of this work was to investigate the potential pharmaceutical applications of CO2 laser irradiation in several areas including drug delivery systems, drug substances and mixtures of drug and polymer. CO2 laser irradiation was successfully utilized for three main pharmaceutical systems. Firstly, modifying drug release of irradiated enteric coated tablets (Eudragit®L100-55, L30D-55 and HPMCAS-MF) targeting to the duodenum gave rise to both fast and delayed release in pH 5.6 phosphate buffer resembling the duodenum. These changes were caused by pore formation and the change in wettability on the surface of the irradiated coat. Cast films were used to examine the physicochemical and mechanical changes and were shown to have different mechanical properties, contact angles and wettability upon high irradiation. Secondly, a proof-of-concept study of pharmaceutical co-crystal production upon irradiation at certain energy (S50P50) from a mixture of specific stoichiometric ratio of dry powders was presented through rapid melting, sublimation and vapour crystallization. Furthermore, the CO2 laser can induce the physical conversion to amorphous state of the drug-alone system (classic indomethacin) and to many physical states of the various ratios of the drug (paracetamol) and polymer (polyvinylpyrrolidone K 30) mixtures upon rapid heating process. These were clearly confirmed by a range of analytical techniques and physical stability studies. The effect of underlying process settings, namely, laser power, scanning speed, irradiation area, resolution, working distance and scale-up on production were performed.

Palatability is crucial for patient acceptability and compliance with prescribed medicines, in particular with paediatric patients. However, palatability studies with human taste panels can only be carried out during advanced drug development phases when toxicology data of the drug are available. Thus, there is a great need to develop a method to assess the taste of pharmaceutical compounds and formulations at early stages of drug development. This could enable to identify taste aversive compounds at screening stages of pharmaceutical development and optimise taste-masking approaches for ensuring patient compliance. The feasibility of using the rat Brief-Access Taste Aversion (BATA) model for taste assessment of pharmaceutical compounds and formulations was investigated. The rat BATA model is a taste assessment technique where mildly water-deprived rats are presented with several solutions for few seconds; the taste is assessed by the number of licks recorded with a lickometer. Preliminary experiments conducted with the rat BATA model showed that the model was capable to detect the aversive taste of structurally different compounds having different physico-chemical properties and several levels of aversiveness. They also enabled to identify experimental parameters that were optimised with the help of a Design of Experiments. The analysis of the data provided by the BATA experiments was explored and a new Emax model was developed to determine the IC50 value enabling the quantification of the oral aversiveness of each test compound. Five model drugs were assessed by a human taste panel; a strong correlation was found between the two in vivo taste assessment methods. The levels at which several sweeteners commonly used in paediatric formulations could be assessed with the applied model. Finally, it was found that juvenile rats could be a model predicting more naïve taste in children.

Multidrug-resistance is a global health concern and in bacteria it may be conferred by the acquisition of multiple-drug resistance genes and/or by the action of multidrug-efflux pumps. The current study targeted these processes as a means to combat the spread of multidrug resistance genes among bacteria, and reinstate the efficacy of antibiotics against efflux-mediated drug-resistant strains. Our aim was therefore to isolate and characterise natural products that function by either inhibiting bacterial conjugation and/or by potentiating antibiotic activity against efflux-related multidrug-resistant (MDR) strains. Selected medicinal plants, some of which have antibacterial properties (Amoracia rusticana, Borago officinalis, Brassica oleracea, Lepidium sativum, Myristica Iowiana, Sinapis alba, Uncaria tomentosa and Zingiber officinale) were extracted with solvents of increasing polarity. The extracts were then screened against Escherichia coli conjugation pairs of donors (pKM101, IncN; TP114, IncI2; pUB307, IncP; and R7K, IncW), and recipients (ER1793 and JM109), and Staphylococcus aureus strains expressing distinct efflux-related multidrug-resistance pumps; SA-1199B (NorA) and XU212 (TetK). The active extracts were further fractionated using various chromatographic techniques (Thin Layer Chromatography, Solid Phase Extraction, Vacuum Liquid Chromatography, Column Chromatography and High Performance Liquid Chromatography). The compounds, which were isolated from the bioactive fractions, were then characterized by the use of spectroscopic techniques (NMR, MS, IR and UV) and re-assessed for anti-conjugation and antibiotic potentiation activity. The isolated glucosinolates from the Brassica plants showed moderate activity (10 - 50% reduction) against the conjugal transfer of the tested plasmids while the isothiocyanates, which are degradation products of the glucosinolates, showed better broad-range anti-conjugal activity. An amide, isolated from M. lowiana, showed significant anti-conjugal inhibitory activity (16.7 ± 2.0%) against the R7K plasmid. Its anti-conjugal activity was plasmid specific and non-toxic to human dermal fibroblasts, adult cells. In addition, a gingerol compound isolated from Z. officinale, the isolated amide from M. lowiana, and benzyl isothiocyanate, significantly potentiated the activity of norfloxacin and tetracycline against SA-1199B (NorA) and XU212 (TetK), respectively. Their potentiation activity ranged from 2 to 512-fold. In conclusion, the study identified natural product inhibitors of the type-IV secretion-related processes and efflux pump systems. Compounds with such anti-conjugative and antibiotic potentiation activity could help decrease the spread of multidrug resistance genes via conjugation and prolonging the efficacy of existing antibiotics.

Prolonging therapeutic levels of a drug within the vitreous to treat blinding diseases is one of the most important goals in ophthalmic drug development. Intravitreal (IVT) injections of therapeutic proteins and the use of steroid implants in the vitreous cavity are currently the best clinical methods to achieve prolonged exposure in the back of the eye. Therapeutic biologics registered for ophthalmic use by IVT injection comprise a PEGylated-aptamer (Pegaptanib), antibody fragment (ranibizumab), and an Fc fusion (alfibercept). The monoclonal antibody (mAb), bevacizumab is also widely used as an unlicensed medicine to treat age-related macular degeneration (AMD). It is anticipated that ophthalmic protein-based medicines, which tend to be potent and have a rapid onset of action, will continue to be developed as molecular mechanisms involved in blinding diseases become better understood. Mass exchange within the eye is dominated by aqueous flow, which is secreted at 2.0–2.5 μL/min into the vitreous from the ciliary body. There are two main drug elimination pathways from the vitreous: (a) the aqueous outflow into the anterior chamber and (b) permeation through the retina via retinal-choroid-sclera (RCS) pathways. Therapeutic proteins are high molecular weight (MW) and charged molecules, and they clear predominantly by the anterior route. Proteins consequently have longer half-lives (t1/2) i.e. days in the vitreous cavity than low MW drugs i.e. hours, many of which are lipophilic and permeable. There is much research focused on developing new strategies to increase the vitreous residence times of macromolecular drugs to decrease the frequency of IVT injections. A two-compartment, aqueous outflow model scaled to the human eye called the PK-Eye was created to aid the development of new ocular drug formulations and delivery systems. The PK-Eye was shown to be particularly useful in evaluating protein PK and stability properties, and for the preclinical evaluation of novel, long-acting formulations, which are particularly difficult to evaluate in animal models because of the generation of anti-drug antibodies (ADAs). Ranibizumab and bevacizumab were used to validate the PK-Eye using simulated vitreous to give clearance t1/2s that were comparable to human values. The model was also used to determine the outflow clearance t1/2 of triamcinolone acetonide (TA, Kenalog®),which is clinically administered as a suspension. The clearance t1/2 for TA was overestimated by the PK-Eye compared to the human t1/2 since the model does not measure RCS clearance. Prolonging the vitreous residence time of low MW molecules relies on at least a 2-step process: (i) dissolution or release from an implant and (ii) clearance of solubilised drug from the vitreous. The first step will often be the rate-limiting step that defines the overall clearance time of a candidate preparation. Once the active is in the vitreous solution, the model has the capacity to provide a good estimate of clearance by the anterior outflow route. To model RCS clearance, invitro in vivo correlations (IVIVCs)can be developed using the PK-Eye data for outflow clearance times obtained from suspensions implants. The PK -Eye clearance t1/2for Kenalog ® and was used with TA permeability data in the literature to better estimate the human clearance time for TA. This was an important first step for developing IVIVCs that would be needed for ocular DDS of small molecules. Other DDS using electrospun (ES) fibres and microparticles were investigated using small MW drugs to examine the utility of the PK-Eye. Permeability and clearance data from the PK-Eye for dexamethasone release from microparticles were used to estimate the possible human clearance t1/2. The utility of the PK-Eye was also examined to evaluate therapeutic proteins. If more stable protein formulations are developed, higher molar concentrated IVT doses may become more routine where a therapeutic tail can be exploited to extend duration of action. Increasing doses of bevacizumab were examined in the PK-Eye where it was possible to observe a therapeutic tail. Hydrogels and in-situ forming gels have been extensively investigated for a wide range of applications. The highly hydrated state of hydrogels could be a good environment for maintaining protein stability. PEGylated-ranibizumab (PEG10-Fabrani) when encapsulated within a crosslinked NIPAAM in-situforming gel displayed an extended clearance time in the PK-Eye compared to non-encapsulated PEG10-Fabrani. In contrast, no difference in clearance times were observed between NIPAAM hydrogel encapsulated and non-encapsulated bevacizumab. Since PEG is a random coil, PEG10-Fabraniappeared to be more entangled within the hydrogel compared to bevacizumab, which may explain the prolonged clearance times that were observed. Considering that an in situ forming gel collapses on injection, controlling the burst release of the encapsulated protein is a challenge. To maintain the protein in a highly hydrated state, affinity based drug delivery was examined in the hope that the vitreous could be made into a depot that could extend the duration of action. Both endogeneous and exogeneous affinity anchors were examined. Much effort was focused on developing a bispecific antibody mimetic with one Fab targeted to collagen II as an endogenous depot anchoring site and the other Fab targeted to VEGF to inhibit angiogenesis. Implantable exogeneous anchors were also examined. Proof of concept experiments using the PK-Eye to anchor albumin as a model anchor suggest that more work is justified to develop an affinity based drug delivery strategy in the vitreous.

The assessment of skin permeation is critically important in many fields and a suitable and robust model that may be used to quantify and predict percutaneous penetration is necessary. Currently available models include in vitro models using human skin, animal skin, synthetic membranes and cell culture models. Recently, the Skin Parallel Artificial Membrane Permeation Assay (PAMPA) has been proposed as a simple but high throughput screening system that may be suitable to study skin permeation. In the present study, a lipophilic active, ibuprofen and a hydrophilic active, caffeine were selected to conduct in vitro permeation studies in the conventional Franz cell models using silicone membrane, porcine skin and human skin, and the novel Skin PAMPA model. The overarching aim was to determine the utility of Skin PAMPA for routine in vitro skin permeation testing with reference to topical formulations. The in vitro permeation studies conducted in Franz cell models using silicone membrane and porcine skin showed that as a lipophilic active, ibuprofen permeated rapidly though silicone membrane and porcine skin. Compared with ibuprofen, caffeine went through the skin more slowly. In vitro permeation studies in Franz cell models using human skin indicated that the ibuprofen percentage permeation values for human skin were much lower than corresponding values in porcine skin as expected. Various in vitro permeation studies were conducted in the novel skin PAMPA model for different ibuprofen and caffeine formulations. In general, the Skin PAMPA model did discriminate between different formulation types and different solvent systems compared with other models, with low variability in the permeation data. The more permeable nature of the PAMPA, silicone membrane and porcine tissue models to ibuprofen compared with human skin was also demonstrated, while the permeation of caffeine, a hydrophilic compound, in the PAMPA model was comparable to that in human skin.

Antibiotic multidrug resistant infections are increasingly challenging to treat. To avoid entering a post antibiotic era novel antibiotics acting via new or underexploited mechanisms are required. In the human body bacteria are exposed to numerous stresses, e.g. methylglyoxal, and detoxification is essential for bacterial survival and infection progression. Glutathione and the glyoxalase (Glx) system are essential to this detoxification process. We hypothesise that inhibitors of bacterial GlxI and/or GlxII will disrupt this protective pathway leading to bactericidal activity, and may act alone or synergistically with existing antibiotics and the host immune system. To identify potent and selective chemical tools to investigate our hypothesis kinetic target guided synthesis (kTGS), where the enzyme generates its own inhibitors by acting as a catalyst to ‘click’ fragments together within the binding site, was employed in 2 ways; 1) a biased and 2) an unbiased approach. The chemistry used was a Huisgen [3+2] cycloaddition between azides and alkynes to form triazoles and hit compounds were identified by LC-MS/MS. In the biased approach an azido derivative of glutathione (GAz) was designed and synthesised. kTGS with GlxI and GlxII using 96 alkyne fragments to probe the hydrophobic pocket resulted in hit rates up to 28%. The unbiased approach used azide and alkyne fragment libraries to identify non-peptidic inhibitors and to investigate the potential of kTGS as a tool for fragment based drug discovery. Using 2 azide and 2 alkyne fragments and the corresponding fragment library resulted in hit rates up to 11%. Identified peptidic and non-peptidic hits were synthesised and biophysical evaluation demonstrated binding to and inhibition of the glyoxalase enzymes, thereby demonstrating the potential of kTGS for lead discovery and the opportunity to develop these hits into novel antibacterial agents.

Background and purpose: Malignant melanoma is the most aggressive form of skin cancer and natural products are an important source of anticancer lead compounds. The aim of this study is to search for agents with antimelanoma activity by studying 20 Saudi plants on human melanoma cells (A375). Methods: Bioguided isolation of active principles was performed utilising chromatographic methods and their characterisation was performed by NMR, MS and IR. Cell viability, proliferation and further mechanistic studies were conducted, including caspase-3/7, LDH, cell cycle analysis, AnnexinV/PI staining, cell morphology and Bax/Bcl-2 expression. Furthermore, the effect of drug combinations on melanoma migration was evaluated using B16-F10 cell lines in scratch and Transwell chamber assays. Results: Nine out of twenty plants showed significant cytotoxicity. Four cytotoxic lignans; justicidin A, justicidin B, tuberculatin and tuberculatin acetate were isolated from H. tuberculatum. They induced apoptosis due to the elevation of sub-G1 and AnnexinV positive populations and the activation of caspase-3/7. All lignans upregulated the Bax expression, but interestingly, justicidin A only downregulated the expression of Bcl-2 protein, suggesting the role of methyl substitution in this effect. Ursolic acid acetate, ursolic acid and quercetin were obtained from C. edulis. Ursolic acid acetate induced apoptosis (activating caspase-3/7, elevating AnnexinV positive population and Bax/Bcl-2 ratio), and caused cell cycle arrest at S phase. Morphological changes were stronger than those caused by ursolic acid, indicating that the acetyl group may potentiate the cytotoxicity. Combination studies showed synergistic antimigratory effects of ursolic acid and quercetin in melanoma migration at very low concentrations. Conclusion: This is the first time that cytotoxicity for most of the selected plants has been reported. This study sheds light on the mechanisms by which these extracts affect melanoma cells. Bioguided isolation did not afford new cytotoxic secondary metabolites, but allowed the study of the structure-activity relationship of important cytotoxic compounds like pentacyclic triterpenoids and arylnaphatalene lignans.

Plant-derived medicines have played a major role in the history of man, especially as anti-infective agents. In the wake of the emergence of multidrug-resistant microbial infections, natural products are still a potential source of drug leads since they contain a wide array of structurally diverse compounds. Alongside the classical approach of finding anti-infective agents, this study also sought to investigate the potential of Swazi medicinal plants as efflux inhibitors in bacterial cell systems. This is because bacteria utilise the efflux system to avert the inhibitory action of antibiotics which may lead to multidrug resistance. The development of efflux inhibitors is one of the methods being employed to counter multidrug resistance in bacteria. Seven plant species used as traditional medicines in Swaziland for the treatment of infections were identified and collected with the assistance of traditional healers or local people who had knowledge of medicinal plants. The plants were Breonadia salicina, Ozoroa sphaerocarpa, Dioscorea sylvatica, Dioscorea cotinifolia, Syzygium cordatum, Ehretia rigida and Helichrysum acutatum. After preliminary screening of the plant extracts for antimicrobial activity using various Staphylococcus aureus strains and Escherichia coli; as well as for antibiotic potentiating activity using S. aureus strains over-expressing MDR efflux proteins (SA-1199B (NorA) and XU212 (TetK) pump), a diverse range of phytochemicals were isolated and characterised from the plants using chromatographic and spectroscopic (IR, UV, MS, NMR) techniques. Some cardanols and anacardic acids from Ozoroa sphaerocarpa exhibited antimicrobial activity against a range of microorganisms (MIC 8 – 512 μg/mL) whilst dioscin, from Dioscorea sylvatica, displayed anti-Candida activity (MIC 1 μg/mL). None of the plant extracts, nor the isolated compounds, had antimycobacterial activity against Mycobacterium smegmatis and M. aurum. At a sub-inhibitory concentration, (Z)-3-(heptadec-8-en-1-yl)phenol, from O. sphaerocarpa and ursolic acid, from B. salicina, potentiated the activity of tetracycline and norfloxacin against the S. aureus XU212 and SA-1199B strains, respectively. Their activity resulted in a 512-fold reduction in the minimum inhibitory concentration (MIC) of both antibiotics on the S. aureus strains. Some cinnamic acid derivatives isolated from D. sylvatica and D. cotinifolia also potentiated the activity of tetracycline and norfloxacin. The efflux pump inhibitory activity of the compounds was investigated using the ethidium bromide accumulation assay and a bibenzyl, from D. sylvatica and some diaryl nonadiones and a ω-hydroxy acid ester from D. cotinifolia, resulted in an increase in intracellular ethidium bromide accumulation in the SA-1199B strain, suggesting NorA pump inhibition. Ethidium bromide was not found to be a strong substrate for the TetK pump in the XU212 strain. Apart from moronic acid, isolated from O. sphaerocarpa, which showed some comparable efflux pump inhibition to reserpine, all the compounds which exhibited tetracycline-potentiating activity did not show a corresponding TetK pump inhibition activity. This suggested that some other mechanism of antibiotic potentiation activity was a possibility. Of the isolated compounds, dioscin was the most selective towards fungal cells compared to the human dermal cells; whilst ursolic acid was the least selective, showing toxicity to the human dermal cells at its active concentration. The anacardic acids and moronic acid also showed some degree of selectivity. In conclusion, the results of the study showed the potential of Swazi medicinal plants as a source of antimicrobial drug leads; as well as their potential use in combination therapy for the management of bacterial resistance.