The intra- and extracellular activities of 2 novel tetramethylpiperidine (TMP)-substituted phenazines, B4121 and B4128 against Mycobacterium tuberculosis H37R (ATCC 27294) were determined and compared with those of clofazimine (B663). Clofazimine, together with B4121 and B4128, were also tested for their activities against drug-resistant strains of M.tuberculosis. Both B4121 and B4128 were significantly more active than clofazimine against M.tuberculosis, including multidrug-resistant clinical strains of this microbial pathogen, demonstrating a lack of cross resistance between the riminophenazines and standard anti-tuberculous drugs. Using M.tuberculosis-infected monocyte-derived macrophages both B4121 and B4128 were found to possess intracellular activity, which was superior to that of both clofazimine and rifampicin. The relationship between anti mycobacterial action of the TMP-subsitituted phenazines and clofazimine and the effects of these agents on microbial PLA2 activity, cation (K+, Ca2+) fluxes and energy metabolism (ATP) was also investigated. PLA2 and cation fluxes were measured by radiometric procedures, while microbial ATP was assayed using a luciferin/luciferase chemiluminescence method. All 3 riminophenazines, particularly B4128 caused dose-related enhancement of microbial PLA2 activity, which was associated with inhibition of K+-influx and enhancement of uptake of Ca2+. The results of kinetics studies demonstrated that riminophenazine-mediated enhancement of PLA2 activity and inhibition of K+ uptake in mycobacteria are rapidly-occurring and probably related events that precede, by several minutes, any detectable effects on microbial ATP concentrations and uptake of Ca2+. Inclusion of the extracellular and intracellular Ca2+-chelating agents EGTA and BAPTA, respectively, individually or in combination, did not prevent the effects of the riminophenazines on mycobacterial PLA2 (enhancement) or K+ transport (inhibition), whereas α-tocopherol, which neutralizes PLA2 primary hydrolysis products, antagonized the inhibitory effects of the riminophenazines on microbial K+ uptake. These results demonstrated that the riminophenazine-mediated enhancement of PLA2 is a Ca2+-independent event. The involvement of PLA2 in the antimicrobial activity of the riminophenazines was supported by the observation that added, exogenous Iysophosphotidylcholine (a primary hydrolysis product of PLA2 action on membrane phospholipids) also inhibited K+ transport and growth of mycobacteria. Enhancement of endogenous PLA2 as a mechanism of riminophenazine-mediated disruption of cation transport and antimycobacterial activity was further investigated using the conventional calcium-mobilizing stimuli, calcium ionophore A23187 and thapsigargin. Both agents, but A23187 in particular caused in dose-related enhancement of microbial PLA2 activity, which was associated with inhibition of K+ influx and growth. Influx of Ca2+ into A23187- and thapsigargin-treated mycobacteria was observed using both radiometric and FURA-2-based spectrofluorimetric procedures. Exposure of the mycobacteria to these agents resulted in an immediate increase in uptake of Ca2+, which implies that enhancement of PLA2 activity in calcium-mobilizing stimuli-treated mycobacteria is Ca2+ dependent. In conclusion, the TMP-substituted phenazines possess anti mycobacterial properties which are superior to those of clofazimine, particularly against intraphagocytic M.tuberculosis. The superior anti mycobacterial properties of these agents is paralleled by their potentiating effects on microbial PLA2 and consequent inhibitory action on uptake of K+, particularly in the case of B4128. Mycobacterial PLA2 and K+ transporters may therefore represent novel targets for antimicrobial chemotherapy. / Thesis (DPhil (Medical Immunology))--University of Pretoria, 2007. / Immunology / unrestricted

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The prevalence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis has been increasing to alarming levels globally. This has been exacerbated by tuberculosis (TB) co-infection with HIV where the epidemic is endemic. South Africa as a developing country is hit hard by TB and efforts to develop TB drugs that are compatible with anti-retroviral medication and also effective against MDR/XDR, could help shorten the treatment duration of the current TB treatment regimens. This thesis presents the identification and characterisation of a novel furanone based compound (F1082) and its derivatives as leads for anti-TB drug development. Furanones are generally known for an array of biological activities ranging from antibacterial, antifungal and antitumor. F1082 has an aromatic benzene structure and was identified from screening synthetic compounds against M. tuberculosis. It is potent against M. tuberculosis at minimum inhibitory concentration (MIC) of 8 μg/ml. It is selective for mycobacteria since it did not inhibit the growth of Gram-positive and Gram-negative bacteria at concentrations five times the MIC for M. tuberculosis. F1082 is generally bacteriostatic around MIC concentrations in its effects against M. tuberculosis however; it may be bactericidal at higher concentrations. It is as effective against MDR, XDR and clinical isolates of M. tuberculosis at the same concentration as the M. tuberculosis H37Rv reference strain. This suggests that F1082 may have a different mechanism of action compared to current TB drugs. It has been shown to have no antagonistic effect with the first-line anti-TB drugs and it has been shown to synergize with rifampicin by reducing the MIC of rifampicin. A drawback of F1082 is that it is cytotoxic to human cell lines, but this is presently being addressed through the synthesis of analogues that have shown improved activity and less cytotoxicity. The synthesis of more than 40 analogues has led to identification of 4 compounds that have more than five times higher activity and more than 100 times less cytotoxicity against human cell-lines. Microarray analyses have identified possible metabolic pathway/s in M. tuberculosis that is/are affected by F1082. One subset of genes which showed the most prominent alteration encodes the siderophores, which are involved with iron homeostasis in the M. tuberculosis bacillus. Of these genes, 7 were of interest (mbtB, mbtC, mbtD, mbtE, mbtF, mbtH and bfrB) as they all fall in the same cluster and are involved in iron acquisition. Due to the involvement of iron we also show that F1082 generates oxidative stress that is metal (iron) dependent. From the results we conclude that F1082 is a promising antituberculosis lead compound with unique target properties and also specificity against mycobacteria. / AFRIKAANSE OPSOMMING: Die voorkoms van veelvuldige middelweerstandige M.tuberculosis (MDR) en uiters middelweerstandige M.tuberculosis (XDR) is besig om toe te neem teen ‘n kommerwekkende tempo wêreldwyd. Hierdie situasie word vererger met die ko-infektering van M.tuberculosis en HIV. Suid- Afrika, as ontwikkelende land, word sleg benadeel met tuberkulose siekte. Antituberkulose middels wat kan saamwerk met bestaande antiretrovirale middels en ook effektief is teen MDR en XDR stamme, kan alles meewerk om die behandelingstyd van tuberkulose te verkort. In hierdie tesis identifiseer en karakteriseer ons ‘n furanoon-gebaseerde verbinding (F1082) en derivate daarvan as voorloper-middels vir anti-tuberkulose middelontwikkeling. Furanone is algemeen bekend vir ‘n verskeidenheid van biologiese aktiwiteite insluitende antibakteriële-, antifungale- en antitumor aktiwiteite. F1082 bevat ‘n aromatiese benseenstruktuur en is oorspronklik geïdentifiseer gedurende die skandering van sintetiese middels teen M.tuberculosis. Dit het ‘n sterk werking teen M.tuberculosis met ‘n minimum inhibitoriese konsentrasie (MIC) van 8ug/ml. Dit is baie selektief vir mikobakterieë aangesien dit nie gram-positiewe of gram-negatiewe bakterieë teen 5 maal die MIC, soos vir M.tuberculosis, geïnhibeer het nie. F1082 is bevind om, by laer konsentrasies, bakteriostaties te wees in sy aktiwiteit teen M.tuberculosis maar by hoër konsentrasies word ‘n meer bakteriosidiese effek waargeneem. F1082 is effektief teen MDR, XDR en kliniese isolate van M.tuberculosis en teen dieselfde konsentrasie soos vir die M. tuberculosis H37Rv verwysingstam waargeneem is. Dit impliseer dat F1082 dalk ‘n alternatiewe meganisme van werking het in vergelyking met die van die huidige TB teenmiddels. F1082 toon geen antagonistiese werking in kombinasie met die voorste anti- TB middels nie, maar toon wel sinergistiese werking in kombinasie met rifampisien. F1082 toon nog sitotoksiese aktiwiteit teenoor menslike sellyne, maar die sintese van derivate van F1082 toon tot dusvêr groter anti-TB aktiwiteit en verminderde sitotoksisiteit. Die sintese van meer as 40 homoloë het gelei tot die identifisering van vier verbindings met vyf keer hoër anti-TB aktiwiteit en honderd keer verminderde sitotoksisiteit teen menslike sellyne as F1082 self. “Microarray” ontledings het ‘n aantal metabolise paaie geïdentifiseer waar F1082 ‘n effek kan uitoefen. Een stel gene wat die mees uitstaande effek toon kodeer vir siderofore wat betrokke is by yster homeostase in M.tuberculosis. Van hierdie gene was daar sewe van belang omdat hulle in dieselfde groep voorkom en almal betrokke is by ysteropname (mbtB, mbtC, mbtD, mbtE, mbtF, mbtH, bfrB). Weens die rol wat F1082 in ysterhomeostase speel, toon ons ook dat F1082 intrasellulêre oksidatiewe stres bevorder wat yster afhanklik is. Al ons resultate dui daarop dat F1082 ‘n belowende ant-TB voorloper verbinding is met spesifisiteit teen M.tb en unieke teikeneienskappe in M. tuberculosis.

Cancer is responsible for upward of 13% of human deaths. Contemporary chemotherapy of disseminated cancer is often thwarted by dose limiting systemic toxicity and by multi-drug resistance (MDR). Riminophenazines are a novel class of potential anticancer agents that possess a potent multi-mechanistic antineoplastic action. Apart from their broad action against intrinsic, non-classical resistance, Riminophenazines inhibit the action of Pgp and hypothetically all ABC transporters demonstrating their great utility against classical MDR. Considering that combination chemotherapy is the norm, the vision directing R&D efforts was that Riminophenazines could be used with benefit within many standard chemotherapeutic regimes. The strategic intent of this project was to attain improved therapeutic benefit for patients through gains in both pharmaco dynamic and pharmacokinetic specificity for cancer cells over what is currently available. Tactically, this was driven through the use of synergistic Fixed-Ratio Drug Combinations (FRDC) encapsulated within tumour-targeting Nanoparticulate Drug Delivery Systems (NDDS). Long-term aims of this R&D project were to: 1) Screen FRDC of clofazimine (B663) and the lead derivative (B4125) with etoposide, paclitaxel and vinblastine for synergistic drug interactions in vitro. 2) Design, assemble and characterize a novel nanoparticulate, synergistic, anticancer co-formulation. 3) Evaluate the in vivo safety and efficacy of the developed product/s in accordance with international regulatory guidelines. Using the median effect and combination index equations, impressive in vitro synergistic drug interactions (CI<1) were shown for various FRDC of the three standard chemotherapeutics tested (etoposide, paclitaxel and vinblastine) in combination with either B663 or B4125 against MDR neoplastic cell cultures. Considering in vitro results and with the view to advance quickly to clinical studies, the already approved clofazimine (B663) was elected as the combination partner for paclitaxel (PTX). Considering the potency and wide action of PTX, a novel coformulation (designed to circumvent drug resistance) has the potential to greatly impact upon virtually all cancer types, particularly if selectively delivered through innovative delivery systems and loco-regional administration. A passively tumour targeting, micellular NDDS system called Riminocelles™ that encapsulates a synergistic FRDC of B663 and PTX has been designed, assembled using thin film hydration methods and characterized in terms of drug loading, particle size, zeta potential, CMC and drug retention under sink conditions. An acute toxicity and a GLP repeat dose toxicity study confirmed Riminocelles to be well tolerated and safe at clinically relevant dosages whilst Taxol® (QDx7) produced statistically significant (P<0.05) weight loss within 14 days. The same study demonstrated statistically significant (P<0.05) tumour growth delays superior to that of Taxol at an equivalent PTX dosage of 10 mg/kg. Importantly, all components (amphiphiles and drugs) used in assembly of Riminocelles are already individually approved for medicinal use - this promotes accelerated development towards advanced clinical trials and successful registration. Although these results are very promising (outperforming Taxol), this system was however found in a pharmacokinetic study to suffer from in vivo thermodynamic instability due to the high concentration (abundance) of albumin present in plasma. For this reason, in vivo longevity within circulation, permitting passive tumour accumulation was not fully realized. A second NDDS called the RiminoPLUS™ imaging system was additionally developed. This lipopolymeric nanoemulsion system has successfully entrapped Lipiodol® Ultra fluid (an oil based contrast agent) within the hydrophobic core of a monodisperse particle population with a size of roughly 100 nm and a stability of one week. This formulation is therefore thought capable of CT imaging of tumour tissue and drug targeting after either intravenous or loco-regional injection. In vivo proof of the imaging concept is warranted. The results of this study serve to highlight the great potential of in vitro optimized synergistic FRDC against drug resistant cancers. Lipopolymeric micelles are an effective way to formulate multiple hydrophobic drugs for intravenous administration and present a means by which cancer can be readily targeted; provided that the delivery system possess the prerequisite in vivo stability and surface attributes. Further experiments exploring synergistic drug and biological combinations as well as “intelligent” NDDS actively guided through specific molecular recognition are called for. / Thesis (PhD)--University of Pretoria, 2012. / Pharmacology / unrestricted