Academic literature on the topic 'Pharmaceutical microbiology'

The exploitation of microorganisms has been part of humankind for millennia. Today this use has increased immensely as we re-purpose microorganisms in many novel ways to facilitate processes in food, pharmaceutical, detergent and mining industries. This issue of Microbiology Australia includes a brief look at the breadth of Industrial Microbiology and what it is offering us now and into the future.

Bacteria of the genus Streptomyces produce a very large number of secondary metabolites, many of which are of vital importance to modern medicine. There is great interest in the discovery of novel pharmaceutical compounds derived from strepomycetes, since novel antibiotics, anticancer and compounds for treating other conditions are urgently needed. Greece, as proven by recent research, possesses microbial reservoirs with a high diversity of Streptomyces populations, which provide a rich pool of strains with potential pharmaceutical value. This review examines the compounds of pharmaceutical interest that have been derived from Greek Streptomyces isolates. The compounds reported in the literature include antibiotics, antitumor compounds, biofilm inhibitors, antiparasitics, bacterial toxin production inhibitors and antioxidants. The streptomycete biodiversity of Greek environments remains relatively unexamined and is therefore a very promising resource for potential novel pharmaceuticals.

Biotherapeutics is a rapidly growing segment of the entire pharmaceutical industry that constitutes approximately one-quarter of ongoing new drug endorsements. Monoclonal antibodies are a major part of these endorsements every year (mAbs). MAbs' non-clinical pharmacology and toxicology research compare with substance components during progression, since these biotherapeutics are extracted from an organic source, and to inspire a pharmacological reaction, the creature models must also have similar epitopes (focuses) as individuals. Biotherapeutic items (BTPs) are the quickest developing drugs in the pharmaceutical market. Despite their clinical achievement, the immunogenicity of BTPs keeps on being a significant concern. The subcutaneous (SC) course is to cultivate a passion for the organisation of biotherapeutics. Both monoclonal antibodies and various biotherapeutics are discussed. Medicine has been disrupted by biotherapeutic drugs (BPs), altering the way we treat a few processes. Comparative BPs (SBPs) are discussed here, also called biosimilars, including the assembly process and administrative viewpoints used. Monoclonal antibodies can apply synergistic antitumour impacts in blend with other immunomodulatory approaches, for example, chemotherapy, radiotherapy, directed treatment specialists, immunisations, or different immunomodulators. Probiotics have gotten profoundly perceived as enhancements for people and specifically for creatures given their gainful result on wellbeing improvement and prosperity support.

To meet the demands of political, ethical and scientific pressures on animal testing, research into possible alternatives is required. Data obtained with animal models often cannot be related to humans. Testing with current cell-based assays, microdosing and pharmacokinetic models contribute to reducing animal testing and improving the drug development process. Micro-fabrication and rapid prototyping techniques offer potential solutions to reduce the need for animal toxicity testing. The aim of this research was to develop biological platforms for in vitro toxicity testing to provide physiologically relevant, high-throughput solutions to reduce animal testing. This was achieved by investigating and integrating microfabrication methods of microfluidics, dielectrophoresis and additive manufacturing. Three approaches were taken: (i) micro-pattern protein arrays for primary hepatocyte cell culture enclosed within microfluidics devices for high-throughput toxicity testing. It was observed that hepatocytes attached to the micro-pattern within microfluidics and maintained viability, however liver specific functions observed by florescence assays, the P450 enzymes, were observed to be reduced compared to Petri dish conditions. (ii) A biomimetic dielectrophoretic cell patterning technique to form liver lobule-like tissue structures within agar on a paper substrate was developed for toxicity testing. Observation of these biomimetic micro liver structures showed high viability (80-90%) and an increase in liver specific function marker albumin protein (20%) compared to control samples after 48 hours. (iii) Rapid prototyping methods were explored with regard to fabrication of microfluidic chips for the automated trapping, imaging and analysis of zebrafish embryos. Monolithic microfluidic chips for zebrafish were developed to be suitable for optical based toxicity assays. The biocompatibility of 3D printed materials was investigated. A method to render the photopolymer Dreve Fototec 7150 compatible with zebrafish culture was observed to provide 100% viability. Future development of this research will aim to (i) develop the liver lobule-like system to use layers of multiple cell types to form complex micro-liver models using additive manufactured microfluidic systems for toxicity testing. (ii) Automation of zebrafish handing using additive manufactured microfluidic devices for in-situ analysis of dechorionated zebrafish for high-throughput toxicity studies.

El control microbiològic és essencial en la indústria farmacèutica ja que es tracta d’un indicador de la seguretat dels productes. Els mètodes microbiològics utilitzats a les companyies farmacèutiques per analitzar l’ambient del procés de fabricació així com els productes finals estan basats en mètodes de cultiu tradicional. La tecnologia d’aquests mètodes manca de precisió comparada amb les tecnologies més modernes de detecció i identificació microbiològica. Cada vegada, els requeriments dels controls microbiològics de productes i processos són més estrictes, el que fa que es necessiten mètodes cada vegada més ràpids i precisos. Dintre d’aquest context s’han desenvolupat els mètodes de microbiologia ràpida (RMM) per a la seva implantació a la industria farmacèutica, avalada per les diferents agències reguladores. Les diferents tecnologies en les quals es basen els RMM es coneixen a l’àmbit acadèmic des de fa dècades, no obstant la seva implantació i validació a les indústries farmacèutiques és relativament recent. La implementació de noves metodologies a l’àmbit farmacèutic està regulada per la normativa de bones practiques de fabricació (BPF), el qual va associat a una adequada validació de les tècniques. Aquesta tesi descriu la implementació de RMM al control microbiològic del procés de fabricació i productes a l’empresa Laboratoris Reig Jofré (RJF). El principal objectiu ha estat dividit en 3 blocs individuals: implementació d’un programa d’identificació microbiana per als aïllats del procés de fabricació i productes; implementació d’un mètode de detecció de microorganismes a l’aire de sales classificades per fabricació asèptica basat en fluorescència induïda per làser i l’ avaluació d’un sistema basat en citometria en fase sòlida per la detecció de microorganismes en productes filtrables. En general, els RMM implementats han contribuit a obtenir resultats de manera més ràpida, el qual permet mitigar el risc de contaminació en el mateix moment que ha sigut detectat. Aquest projecte assenta les bases per futures aplicacions de RMM en el procés de fabricació farmacèutica.
Microbiological testing plays an essential role in the Pharmaceutical Industry as it is an indicator of safety in drug products. The microbiological methods used in the pharmaceutical companies for testing the environment of the manufacturing process as well as the final products are based on traditional culture methods. These methods, although being appropriate for their intended use, rely on century-old technology that lacks accuracy when compared to most up-to-date methodologies for microbial detection and identification. Every time the requirement of microbiological testing of products and processes increases, arising the need of faster and more accurate methods. In this paradigm, rapid microbiological methods (RMM) are developed for their implementation in the pharmaceutical industry encouraged by regulatory agencies. The different technologies in which rapid microbiological methods are based have been known in the academic field for decades, however their implementation and validation in the pharmaceutical industries is relatively recent. Implementation of new methodologies in a pharmaceutical environment ruled under Good Manufacturing Practice (GMP) guidelines needs proper validation of the techniques involved. This thesis describes the implementation of RMM in the microbial monitoring of the pharmaceutical manufacturing process and products in Reig Jofré Laboratories (RJF). The main objective has been divided into three different blocks: implementation of a microbial identification program for the isolates found in the production process and products; implementation of a laser-induced fluorescence system for the detection of airborne microorganisms in cleanrooms for aseptic processing and evaluation of a solid-phase cytometry system for the detection of microorganisms in filterable products. In general, the RMM implemented have contributed to obtain faster results which allows to mitigate contamination risk at the moment it is detected. This projects lays the ground for further applications of RMM in the pharmaceutical manufacturing process.

Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Due to modern high-throughput technologies, large numbers of compounds are produced by parallel synthesis and combinatorial chemistry. The pharmaceutical industry therefore requires rapid and accurate methods to screen new drugs leads for membrane permeability potential in the early stages of drug discovery. Around 50 % of all investigational new drugs fail in pre-clinical and clinical phases of development due to inadequate absorption/permeation, distribution, metabolism, excretion and/or unacceptable toxicity. This may be decreased by applying in vitro screening methods early in the discovery process. Reliable in vitro models can be applied to determine permeation of the test compounds, which will help avoid the wasting of valuable resources for the development of drugs that are destined to fail in preclinical and clinical phases due to insufficient permeability properties. It is important to decide as early as possible on the most promising compound and physical formulation for the intended route of administration. With awareness of the increasing importance of in vitro models in the investigations of the permeability properties of drug compounds, this research project was specifically devoted to determine the suitability of our in vitro model to evaluate and predict drug permeability. A continuous flow-through diffusion system was employed to evaluate the permeability of nine different compounds/drugs with different chemical properties, across three biological membranes. The biological membranes chosen for the present study were human vaginal mucosa, human skin tissue and human small intestine mucosa. The continuous flow-through diffusion system was furthermore utilised to investigate the effects of de-epithelialisation of mucosal surfaces, chemical enhancers, temperature, permeant concentration and formulation on the permeability of the test compounds/drugs. The in vitro permeability information and data from the flow-through diffusion model were compared to in vitro and in vivo literature studies and drug profile. An in vitro model that is able to reliably predict in vivo data will shorten the drug development period, economise resources and may potentially lead to improved product quality. In this thesis research results are reported on the permeability of the mentioned biological membranes to the various chemical markers, including anti-HIV (human immunodeficiency virus) drugs. The permeability studies will be discussed in three sections: vaginal mucosa, skin tissue, small intestine mucosa. The results of the vaginal permeability studies showed that the three peptides (MEA- 5, MDY-19 and PCI) readily penetrated the vaginal mucosa. MDY-19 had a higher flux rate than MEA-5, commensurate with its smaller molecular size (weight). The surfactant enhanced the flux rate of MDY-19 approximately 1.3 times and decreased the lag time of the peptide. Removal of the vaginal epithelium increased the flux rates of the peptides across the mucosa and may have implications for a more rapid uptake of these and other microbicides in vivo. The permeability of 1 mM MDY-19 and PCI at 37 °C were significantly (p<0.05) higher than at 20 °C. At 37 °C the AUCs of the overall mean flux values of MDY-19 and PCI increased with concentration according to well-established diffusion theory. The experiments on the permeability of different terbinafine hydrochloride formulations through human skin demonstrated that the terbinafine hydrochloride formulations used in this study, readily diffused into the skin tissue. However, no flux values for any of the terbinafine hydrochloride formulations through the skin into the acceptor fluid were found. The mean terbinafine concentrations in the skin after 24 h exposure to the three commercial, terbinafine hydrochloride formulations were 3.589, 1.590 and 4.219 μg/ml respectively. The mean terbinafine concentration in the skin exposed to the 10 mg/ml PBS/Methanol solution was higher than those from the three commercial formulations. The results of the temperature study demonstrated that an increase of 5 ºC caused a significant increase in flux values of tritiated water across skin. The flux values for tritiated water across skin at 37 ºC were on average double those at a temperature of 32 ºC. The permeability of excised human small intestine mucosa to different oral dosage drugs was investigated over a 24 h period. The four drugs selected were zidovudine, propranolol hydrochloride, didanosine and enalapril maleate. They were selected as representative model compounds of drug classes 1 (high solubility, high permeability) and 3 (high solubility, low permeability) according to the Biopharmaceutics Classification System. The flux rates of the four chosen test drugs were influenced by the length of the experiment. Between the time periods 2-4 h and 4-6 h, zidovudine’s mean flux values across small intestine tissue were respectively 1.8 and 2.0 times higher than didanosine and 2.3 and 2.2 times higher than enalapril. Propranolol’s mean flux values were respectively 1.2 and 1.4 times higher than didanosine and 1.6 higher than enalapril during both the 2-4 and 4-6 h time periods. Between both the time periods 2-4 and 4-6 h AZT’s mean flux values were 1.4 times higher than propranolol and didanosine’s mean flux values were respectively 1.3 and 1.1 times higher than enalapril during the mentioned time periods. Class 1 drugs showed a significantly higher flux rate across the jejunal mucosa compared to the class 3 drugs and these results are in line with their Biopharmaceutics Classification System classification. The in vitro model has proved to be reliable to predict permeability of class 1 and 3 drugs and also showed correlation with human in vivo data. It seems that the in vitro flow-through diffusion model used in the present study have the potential to overcome some of the problems and limitations demonstrated by other in vitro techniques and may potentially serve as a future tool for pharmaceutical companies to predict the diffusion characteristics of new drugs and different formulations, across different biological membranes. Furthermore, it may serve as a prospective method for assessing the bioequivalence of alternative (generic) vehicles or formulations containing the same drug/compound.
AFRIKAANSE OPSOMMING: As gevolg van moderne hoë spoed tegnologie kan groot hoeveelhede middels vervaardig word deur ooreenkomende sintese en kombinasieleer chemie. Die farmaseutiese industrie benodig dus vinnige en akkurate metodes om nuwe geneesmiddels te evalueer t.o.v. membraan deurlaatbaarheid. Hierdie evaluasie moet verkieslik so vroeg moontlik in die geneesmiddel se ontwikkelingsproses geskied. Ongeveer 50 % van alle potensiële geneesmiddels misluk in pre-kliniese en kliniese fases van geneesmiddelontwikkeling. Die mislukte pogings kan toegskryf word aan onvoldoende absorbsie/deurlaatbaarheid, distribusie, metabolisme, ekskresie en/of onaanvaarbare middel toksisiteit. Dit is daarom belangrik om so vroeg moontlik in die geneesmiddelontwikkelingsproses te besluit op die mees belowende middel, asook die geskikte formulasie vir die spesifieke roete van toediening van die middel. Die farmaseutiese industrie benodig tans in vitro modelle met die potensiaal om die deurlaatbaarheid van geneesmiddels te bepaal en te voorspel. Betroubare in vitro modelle kan aangewend word om die deurlaatbaarheid van potensiële geneesmiddels te toets. Sodoende sal die onnodige uitgawes op die ontwikkkeling van geneesmiddels wat in elk geval later gaan faal in pre-kliniese en kliniese fases van geneesmiddelproewe a.g.v. deurlaatbaarheidseienskappe, vermy word. Hierdie navorsingsprojek was dus spesifiek onderneem om die waarde en toepaslikheid van ‘n in vitro deurlopende-vloei perfusie model te ondersoek. Die model se potensiaal om geneesmiddels se deurlaatbaarheid en absorpsie te voorspel was geëvalueer. Die deurlopende-vloei perfusie apparaat was gebruik om die deurlaatbaarheidsvloede van drie verskillende biologiese membrane t.o.v. nege chemiese stowwe (MEA-5, MDY-19, PCI, terbinafien hidrochloried, getritieerde water, zidovudien, propranolol, hidrochloried, didanosien, enalapril maleaat) te bepaal. Die drie biologiese membrane wat gebruik was, was vaginale weefsel, vel en klein intestinale weefsel. Al drie weefsel tipes was van menslike oorsprong. Die deurlopende-vloei perfusie apparaat was ook gebruik om die effek wat verwydering van die mukosa se epiteellaag op deurlaatbaarheidsvloede het, te ondersoek. Verder was navorsing gedoen op die effek van temperatuur en die konsentrasie en formulasie van die toetsmiddels op hulle diffusie vloedwaardes. Daar was ook gekyk na die invloed van ander chemiese stowwe op die toetsmiddels se diffusie vloedwaardes. Die in vitro deurlaatbaarheidsinformasie en -gegewens was vergelyk met ander in vitro en in vivo literatuurstudies en geneesmiddel databasisse. ‘n In vitro model wat in staat is om in vivo resultate betroubaar te voorspel, het die potensiaal om die tyd wat dit neem om geneesmiddels te ontwikkel, te verkort, finansiële uitgawes te besnoei en om geneesmiddelkwaliteit te verseker. In die tesis word dan die resultate gerapporteer van die deurlaatbaarheidsvloede van die verskillende tipes weefsel ten op sigte van verskeie chemiese stowwe, insluitende anti-MIV (menslike immuniteitsgebreksvirus) middels. Die deurlaatbaarheidstudies word bespreek in drie afdelings: vaginale mukosa, vel en klein intestinale mukosa. Die resultate van die deurlaatbaarheidstudies op die vaginale weefsel dui daarop dat die drie peptiede (MEA-5, MDY-19 and PCI) die vaginale mukosa goed penetreer. Soos verwag, het MDY-19 hoër diffusie vloedwaardes as MEA-5 gehad. Dit kan toegeskryf word aan MDY-19 se kleiner molekulere grootte (gewig). Surfaktant het die diffusie vloedwaardes van MDY-19 1.3 keer vergroot en het ook die tyd na vaste vlak verminder. Die verwydering van die vaginale epiteel het die diffusie vloedwaardes van die peptiede verhoog en mag dus dui op die vinniger opname van peptiede en moontlike ander mikrobisiede in vivo, wanneer die belyning van die epiteel onderbreek. Die deurlaatbaarheid van 1 mM MDY-19 en PCI by 37 °C was satisties beduidend (p<0.05) hoer as teem 20 °C. Die area onder die kurwe (AOK) van die gemiddelde vloedwaardes van MDY-19 en PCI by 37 °C, het toegeneem met ‘n toename in die konsentrasie van hierdie peptiede. Die toename vloedwaardes ondersteun dus die alombekende diffusie teorie. Die transdermale diffusie eksperimente van verskillende terbinafien formulasies het getoon dat terbinafien geredelik vrygestel word vanuit hierdie formulasies na die vel. Geen terbinafien vloedwaardes, van enige van die formulasies, was egter gevind in die ontvangselle van die deurlopende-vloei perfusie apparaat nie. Die gemiddelde terbinafien konsentrasies in die vel na 24 h se blootstelling aan drie kommersiële terbinafien hidrochloried formulasies was onderskeidelik 3.589, 1.590 en 4.219 μg/ml. Die gemiddelde terbinafien konsentrasie in die vel wat aan 10 mg/ml PBS/metanol blootgestel was, was hoër as die konsentrasies in die vel wat aan die drie kommersiële formulasies blootgestel was. Die resultate van die temperatuurstudie op vel het aangetoon dat ‘n temperatuur toename van 5 ºC ‘n statisties beduidende toename in vloedwaardes van getritieerde water oor vel veroorsaak. Die vloedwaardes van die getritieerde water oor vel teen ‘n temperatuur van 37 ºC was gemiddeld dubbeld so veel as teen 32 ºC. Die deurlaatbaarheidsvloede van klein intestinale mukosa ten opsigte van verskillende geneesmiddels (wat oraal toegedien word) was ondersoek gedurende ‘n 24 h eksperiment. Die vier geneesmiddels wat gebruik was, was zidovudine, propranolol hidrochloried, didanosien en enalapril maleaat. Hierdie geneesmiddels is verteenwoordigers van die Biofarmaseutiese Klassifikasie Sisteem se klas 1 (hoë oplosbaarheid, hoë deurlaatbaarheid) en klas 3 (hoë oplosbaarheid, lae deurlaatbaarheid) geneesmiddels. Die vloedwaardes van die vier geneesmiddels het gewissel na aanleiding van die tydsverloop in die eksperiment. Zidovudien se gemiddelde vloedwaardes tussen 2-4 en 4-6 h was onderskeidelik 1.8 en 2.0 keer hoër as didanosien se gemiddelde vloedwaardes vir hierdie tyd periodes en onderskeidelik 2.3 en 2.2 keer hoër as enalapril se gemiddelde vloedwaardes. Tydens hierdie selfde periodes was propranolol se gemiddelde vloedwaardes 1.2 en 1.4 keer hoër as didanosien en vir beide periods 1.6 keer hoër as enalapril se gemiddelde vloedwaardes. Gedurende beide genoemde tyd periodes was zidovudien se gemiddelde vloedwaardes 1.4 keer hoer as propranolol en didanosien se gemiddelde vloedwaardes was onderskeidelik 1.3 en 1.1 keer hoër as enalapril tydens 2-4 en 4-6 h. Die klas 1 geneesmiddels het statisties beduidende hoër vloedwaardes gehad as die klas 3 geneesmiddels. Hierdie resultate stem ooreen met die geneesmiddels se Biofarmaseutiese Klassifikasie Sisteem klassifikasie. Dit wil dus voorkom asof die in vitro model wat gebruik was in die studie, gebruik kan word om die deurlaatbaarheidsvloede van klas 1 en 3 te voorspel. Die resultate van hierdie studie stem ooreen met ander in vivo studies. Dit wil voorkom asof die in vitro deurlopende-vloei perfusie apparaat die potensiaal het om sommige van die probleme en tekortkominge van ander in vitro modelle te oorkom en dat dit moontlik die potensiaal het om die diffusie-eienskappe van nuwe geneesmiddels en verskillende formulasies oor verskillende biologiese membrane te voorspel. Die model kan verder moontlik dien as ‘n potensiële toestel om biogelykbaarheid van alternatiewe (generiese) formulasies, wat dieselfde geneesmiddel/chemiese stof bevat, te bepaal.

Title: Treatment of CMV Vitritis in a Preterm Newborn Author’s Section: Remil Simon1, Darshan Shah1, Peter Blosser1, Demetrio Macariola1, Jeffrey Carlsen2 1.Department of Pediatrics, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 2.Johnson City Eye Clinic, Johnson City, TN Body: Cytomegalovirus (CMV) infection in the neonate is an infrequent occurrence in the developing world, and observing the symptoms of ocular CMV infection such as vitritis is rare. Treating CMV infection promptly is necessary to prevent mortality and potential neurological deficits including blindness and hearing loss. We encountered a preterm infant presenting with CMV sepsis immediately after birth. Our question was: will the current standard of treatment for CMV sepsis prevent CMV ocular infection? With our method of treatment, we followed the current standard of treatment for CMV infection by administering intravenous Gancyclovir for 6 weeks and oral Valgancyclovir for 6 months. Despite using the standard treatment to prevent neurological sequelae, the patient developed CMV vitritis and retinitis bilaterally. Although the treatment did not prevent CMV ocular infection, the severity of CMV retinitis and vitritis improved with treatment, and full resolution of vitritis was noted by day of life 61.

Nanotechnology has become the most advanced type of drug delivery system within the last decade. This advancement shifted the focus on small carriers to increase the efficiency of the drugs. Among these, gold nanoparticles (GNPs) were found to have profound biomedical applications. In current research, kanamycin embedded GNPs were prepared in a single step, single phase, and bio-friendly (green synthesis) procedure. The synthesized Kanamycin-GNPs (Kan-GNPs) were spherical in shape and had a size range of 15 ± 3 nm. The chosen kanamycin is an aminoglycosidic antibiotic that is isolated from Streptomyces kanamyceticus. These special antibiotic GNPs are further characterized using several analytical methods like Transmission Electron Microscopy (TEM), Energy Dispersive Spectroscopy (EDS), Fourier Transform Infrared Spectroscopy (FTIR), and Ultra-Voilet/Visible spectroscopy (UV/Vis spectroscopy). The following research is a direct bio-friendly embedment of an antibiotic agent on the surface of the GNPs without any secondary capping agent or surface modification procedures.

Natural product drug discovery has traditionally been the corner stone of medicine having provided cures to many of today's most common diseases. In particular, antibiotics have revolutionised healthcare and extended human lifespan. However, since their introduction into the clinic, resistance to these drugs has arisen. With the number of new antibiotics being discovered in recent years declining, and fewer drugs making it past clinical trials, we have reached the point where antibiotic resistant infections have become common place and a serious threat to health and society. There is now an urgent requirement for the discovery of new antibiotics and in particular those with unexploited mode of action. This thesis details the different areas of natural product drug development from discovery through to analogue generation. In Chapter one, the history of natural products as therapeutics is explored with a particular focus on antibiotics and how resistance arises against these agents. It outlines why the discovery of new antibiotics is so important and new methods used to facilitate this search. Chapter two follows with the development of a screening platform for antibiotic induction, using the model Streptomyces; Streptomyces coleiolor M145. A variety of culture additives are explored for their ability to induce secondary metabolism production. Chapter three then details the sampling and identification of microbes from a pseudo-marine environment and their screening for their ability to produce secondary metabolites with antibiotic properties. The second half of this thesis centres on the non-ribosomal peptide echinomycin. Collaborators Aquapharm supplied the marine derived strain AQP-4895, capable of producing echinomycin. Chapter four details the establishment of AQP-4895 culturing conditions and the shift observed in production profile. Next Chapter five looks at producing echinomycin analogues through precursor directed biosynthesis. A range of halogenated quinoxaline carboxylic acids are synthesised and fed to AQP-4895, and the respective echinomycin analogues monitored by LC-MS. Chapter Six then aims to direct biosynthesis of the halogenated analogues, using mutasynthesis. Due to the lack of genetic data available surrounding the strain, an unusual approach was taken, using iPCR to create a template for homologous recombination.

The extensive and sometimes incorrect and noncompliant use of various types of antimicrobial agents has accelerated the development of antimicrobial resistance (AMR). In fact, AMR has become one of the greatest global threat to human health in this era. The broad-spectrum antibiotics aminoglycosides (AGs) display excellent potency against most Gram-negative bacteria, mycobacteria, and some Gram-positive bacteria, such as Staphylococcus aureus. The AG antibiotics amikacin, gentamicin, kanamycin, and tobramycin are still commonly prescribed in the U.S.A. for the treatment of serious infections. Unfortunately, bacteria evolve to acquire resistance to AGs via four different mechanisms: i) changing in membrane permeability to resist drugs from entering, ii) upregulating efflux pumps for active removal of intracellular AGs, iii) modifying the antimicrobial target(s) to prevent drugs binding to their targets, and iv) acquiring resistance enzymes to chemically inactivate the compounds. Amongst all, the acquisition of resistance enzymes, AG-modifying enzymes (AMEs), is the most common resistance mechanism identified. Depending on the chemistry each enzyme catalyzes, AMEs can be further divided into AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), and AG O-nucleotidyltransferases. To overcome AME-related resistance, we need to better understand these resistance enzymes and further seek ways to either escape or inhibit their actions. In this dissertation, I summarized my efforts to characterize the AAC(6') domain and its mutant enzymes from a bifunctional AME, AAC(6')-Ie/APH(2")-Ia as well as another common AME, APH(3')-IIa. I also explained my attempt to inhibit the action of various AAC enzymes using metal salts. In an effort to explore the current resistance epidemic, I evaluated the resistance against carbapenem and AG antibiotics and the correlation between the resistance profiles and the AME genes in a collection of 122 Pseudomonas aeruginosa clinical isolates obtained from the University of Kentucky Hospital System. Besides tackling the resistance mechanisms in bacteria, I have also attempted to explore a new antifungal option by repurposing an existing antipsychotic drug, bromperidol, and a panel of its derivatives into a combination therapy with the azole antifungals against a variety of pathogenic yeasts and filamentous fungi.

El problema de la resistència bacteriana ha esdevingut un problema de salut pública global. Cada cop apareixen més soques bacterianes resistents a dos o més fàrmacs i és especialment preocupant la manca d'antibiòtics en el mercat per a combatre les infeccions que aquests provoquen. És per això que un dels grans objectius del descobriment de nous fàrmacs antibiòtics és la recerca de nous compostos capaços de combatre aquests microorganismes. Els pèptids antimicrobians són una classe d'antibiòtics que en el darrers anys han despertat interès degut al seu particular mecanisme d'acció: actuen principalment sobre la membrana bacteriana, i són menys susceptibles de generar resistència per part del bacteris. Destaquem entre aquests pèptids antimicrobians, la polimixina, antibiòtic natural produït pel Bacillus polymyxa. És un compost antibacterià actiu contra bacteris Gram-negatius, però que presenta l'inconvenient de ser tòxic a nivell renal i neuronal. En aquesta tesi es descriu el disseny, la síntesi i l'activitat d'anàlegs de polimixina. Es va simplificar l'esquelet peptídic del producte natural, de manera que fos mes assequible la síntesi química dels anàlegs, la preparació a gran escala, i una potencial reducció de toxicitat. Les modificacions que es van dur a terme van ser substituïr l'enllaç lactàmic que tanca el cicle, per un enllaç disulfur, l'àcid gras natural ramificat, es va substituir per un de lineal, i es van fer substitucions d'alguns aminoàcids per observar l'efecte que provocaven en l'activitat bacteriana. També es van sintetitzar anàlegs retroenantiòmers, per tal de veure la implicació de l'esquelet peptídic en la interacció amb les membranes, i finalment es va preparar un compost conjugat d'un anàleg de polimixina i l'espermina, una poliamina de cadena llarga. Es va estudiar la seva activitat antibiòtica in vitro amb assajos de concentració mínima inhibitòria (CMI). I posteriorment es van realitzar estudis d'activitat biofísica, realitzant assajos amb models de membrana bacteriana, com són els liposomes i les monocapes lipídiques. Aquesta tesi demostra que és possible simplificar l'estructura de la polimixina, obtenint-se compostos antibiòtics actius in vitro que mostren propietats biofísiques similars a les del producte natural.
The problem of bacterial resistance has become a global public health issue. Bacterial strains are becoming resistant to existing drugs and it is particularly worrying lack of antibiotics on the market to fight the infections they cause. That is why one of the major goals of antibiotic drug discovery today is the search for new compounds capable of combating these microorganisms. Antimicrobial peptides are a class of antibiotics that have raised particular interest these recent years due to its mechanism of action: they act primarily on the bacterial membrane, and are less likely to generate resistance by the bacteria. Among these antimicrobial peptides, the polymyxins, natural antibiotic produced by Bacillus polymyxa takes special interest for us. This antibacterial compound is active against Gram-negative bacteria, but it is nephrotoxic an neurotoxic. This thesis describes the design, synthesis and activity of polymyxin analogues. We report a simplified peptide structure, with a chemically accessible scaffold and upscalable, that might show potential toxicity reduction. The modifications that were carried out were substitution of the lactam bond that closes the heptapeptide cycle by a disulphide bond; branched natural fatty acid, was replaced by a linear one, and they were made some amino acid substitutions in order to observe the effect in the bacterial activity. We also synthesize analogues retroenantiomers, to see the involvement of the peptide scaffold when binding to bacterial membranes, and finally it was prepared a conjugate compound of an analogue of polymyxin and spermine, a long chain polyamine. We carried out assays of minimum inhibitory concentration (MIC) in vitro and also studies of biophysics activity, conducting tests with bacterial membrane models, such as liposomes and lipid monolayers. This thesis shows that it is possible to simplify the structure of polymyxin, obtaining active compounds in vitro with biophysical properties that are similar to the natural product.

En 1940, le biochimiste anglais Donald Woods propose une explication du mode d'action des nouveaux sulfamides antibactériens : l'inhibition compétitive. Son collègue Paul Fildes fonde sur cette base une nouvelle approche de la chimiothérapie, revendiquée comme rationnelle, un programme pour la recherche de nouveaux médicaments. Cette thèse explore l'impact de la théorie des antimétabolites, comme elle sera appelée, dans la recherche biochimique et pharmaceutique. La première partie retrace son élaboration dans le contexte de l'école de biochimie anglaise, puis sa reprise aux États-Unis à la suite de travaux menés en parallèle sur les vitamines. La seconde partie est consacrée au développement de deux programmes de recherche distincts dédiés aux antimétabolites, illustrant les modalités et fortunes divergentes d'appropriation de cette approche rationnelle. Le premier est une collaboration modeste entre le biochimiste Henry McIlwain et la firme pharmaceutique Glaxo pendant la guerre au Royaume-Uni. Le second consiste en la mise en place du programme de George Hitchings et Gertrude Elion chez Burroughs Wellcome aux États-Unis, souvent considéré comme l'origine du rational drug design actuel. La théorie des antimétabolites correspond aussi bien à l'ambition d'obtenir des chimiothérapies spécifiques qu'à un ensemble de pratiques dans le quotidien du laboratoire
In 1940, the British biochemist Donald Woods put forward an explanation of the mode of action of the new antibacterial sulfa drugs, competitive inhibition. His colleague, Paul Fildes, developed this work into a new approach to chemotherapy, which he qualified as a rational programme for drug discovery. This dissertation explores the impact of the theory of antimetabolites, as it came to be known, in biochemical and pharmaceutical research. The first part traces its development in the context of the British school of biochemistry and its further expansion in the United States following parallel research on vitamins. The second part deals with the construction of two distinct research programmes dedicated to antimetabolites, each one illustrating a different way of following this rational approach and their varying consequences. The first one is a modest collaboration between the biochemist Henry McIlwain and the Glaxo pharmaceutical company during the war in the United Kingdom. The second one corresponds to the establishment of George Hitchings' and Gertrude Elion's programme at Burroughs Wellcome in the United States, often considered as the origin of today's rational drug design. The theory of antimetabolites simultaneously embodied both the ambition of attaining specific chemotherapies, and a set of practices in day-to-day laboratory work

Mycobacterium tuberculosis isolates from multiple drug resistant or extensively drug resistant patients show a particular set of mutations in drug targets conferring resistance. However, the selection of drug-resistant strains in vitro yields an alternative set of mutations, thought to result from the cost-benefit associated with drug resistance. Mutations allowing for survival under antibiotic may not be beneficial when presented with the host environment or with a drug-free environment. These fitness effects drive the natural evolution of this bacterium. Using recombineering a large cohort of mutations was generated within two drug targets, inhA and gyrA, to study in vitro the variability of mutations allowable under either isoniazid or ofloxacin, respectively. As a proof of concept this process was carried out in Mycobacterium smegmatis. Analysis of survivors allowed for identification of novel mutations and substitutions, as well as showing mutations previously found only in clinical isolates can be present in laboratory isolates.

Good Manufacturing Practices (GMP), a component of Pharmaceutical Quality Systems, is aimed primarily at managing and minimizing the risks inherent in pharmaceutical manufacture to ensure the quality, safety and efficacy of products. Provision of adequate number of personnel with the necessary qualifications/practical experience and their continuous training and evaluation of effectiveness of the training is the responsibility of the manufacturer. (World Health Organization [WHO], 2014; International Organization for Standardization [ISO], 2015). The classroom method of training that has been used for GMP capacity building in the pharmaceutical manufacturing industry in Nigeria over the years, delivered by experts from stringently regulated markets, have not yielded commensurate improvement in the Quality Management Systems (QMS) in the industry. It is necessary and long over-due to explore an alternative training method that has a track record of success in other sectors. A lot of studies carried out on mentoring as a development tool in several fields such as academia, medicine, business, research etc., reported positive outcomes. The aim of this study was to explore mentoring as an alternative GMP training method in the pharmaceutical manufacturing industry in Nigeria. Specifically, the aim of this study was to evaluate the impact of mentoring as a GMP capability building tool in the pharmaceutical manufacturing industry in Nigeria, with focus on GMP documentations in XYZ pharmaceutical manufacturing company located in South-Western region of Nigeria. The methodology comprised gap assessment of GMP documentation of XYZ company to generate current state data, development of training materials based on the identified gaps and use of the training materials for the mentoring sessions. The outcome of the study was outstanding as gap assessment identified the areas of need that enabled development efforts to be targeted at these areas, unlike generic classroom training. The mentees’ acceptance of the mentoring support was evident by their request for additional training in some other areas related to the microbiology operations that were not covered in the gap assessment. This result portrays mentoring as a promising tool for GMP capacity building, but more structured studies need to be conducted in this area to generate results that can be generalized.