The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides

The drug efflux pump P-glycoprotein (P-gp) (ABCB1) confers multidrug resistance, a major cause of failure in the chemotherapy of tumours, exacerbated by a shortage of potent and selective inhibitors. A high throughput assay using purified P-gp to screen and characterise potential inhibitors would greatly accelerate their development. However, long-term stability of purified reconstituted ABCB1 can only be reliably achieved with storage at -80 °C. For example, at 20 °C, the activity of ABCB1 was abrogated with a half-life of <1 day. The aim of this investigation was to stabilise purified, reconstituted ABCB1 to enable storage at higher temperatures and thereby enable design of a high throughput assay system. The ABCB1 purification procedure was optimised to allow successful freeze drying by substitution of glycerol with the disaccharides trehalose or maltose. Addition of disaccharides resulted in ATPase activity being retained immediately following lyophilisation with no significant difference between the two disaccharides. However, during storage trehalose preserved ATPase activity for several months regardless of the temperature (e.g. 60% retention at 150 days), whereas ATPase activity in maltose purified P-gp was affected by both storage time and temperature. The data provide an effective mechanism for the production of resilient purified, reconstituted ABCB1.

Detergent-free purification of ABC (ATP-binding-cassette) transporters

ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up awide range of possibilities for the future study of their structure and function. © The Authors Journal compilation © 2014 Biochemical Society.

Genomic evaluation during permeability of indomethacin and its solid dispersion

Drug resistance was first identified in cancer cells that express proteins known as multidrug resistance proteins that extrude the therapeutic agents out of the cells resulting in alteration of pharmacokinetics, tissue distribution, and pharmacodynamics of drugs. To this end studies were carried out to investigate the role of pharmacological inhibitors and pharmaceutical excipients with a primary focus on P-glycoprotein (P-gp). The aim of this study was to investigate holistic changes in transporter gene expression during permeability upon formulation of indomethacin as solid dispersion. Initial characterization studies of solid dispersion of indomethacin showed that the drug was dispersed within the carrier in amorphous form. Analysis of permeability data across Caco-2 monolayers revealed that drug absorption increased by 4-fold when reformulated as solid dispersion. The last phase of the work involved investigation of gene expression changes of transporter genes during permeability. The results showed that there were significant differences in the expression of both ATP-binding cassette (ABC) transporter genes as well as solute carrier transporter (SLC) genes suggesting that the inclusion of polyethylene glycol as well as changes in molecular form of drug from crystalline to amorphous have a significant bearing on the expression of transporter network genes resulting in differences in drug permeability. © 2011 Informa UK, Ltd.

Frequência e significado clínico da expressão da glicoproteína P e da proteína relacionada a resistência a múltiplas drogas na leucemia mielóide aguda

Despite the advances in the cure rate for acute myeloid leukemia, a considerable number of patients die from their disease due to the occurrence of multidrug resistance (MDR). Overexpression of the transporter proteins P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP) confer resistance to the treatment these leukemias. OBJECTIVE: To analyze the expression of the Gpp and MRP1 in patients with AML by flow cytometry (FC) and to determine the correlation between expression and demographic and also clinical and laboratorial variables. METHODS: Bone marrow and peripheral blood samples from 346 patients with a diagnosis of AML were assessed for the expression of Pgp and MRP1 by FC. RESULTS: The expression of Pgp and MRP1 was found in 111 (32.1%) and 133 (38.4%) patients, respectively, with greater prevalence in older patients and lower in adolescents, observing also a high incidence in patients with refractory disease, recurrence and secondary in comparison with the cases of de novo AML. Regarding the laboratory findings, we observed a higher correlation statistically significant between the expression of Pgp and MRP1 in AML CD34+ and FAB AML M7, M5A and M2 and lower the M3 subtype, not observed statistically significant correlation between the phenotype MDR and other laboratory data such with hemoglobin, leukocyte count, platelet count, aberrant expression of lymphoid antigens (CD2, CD7 and CD19) and clinical signs related to the disease. CONCLUSIONS: The results showed that the detection of MDR phenotype by flow cytometry can be a molecular marker for prognosis independent patients diagnosed with AML.

Mathematical Modeling to Assess the Drivers of the Recent Emergence of Typhoid Fever in Blantyre, Malawi

BACKGROUND: Multiyear epidemics of Salmonella enterica serovar Typhi have been reported from countries across eastern and southern Africa in recent years. In Blantyre, Malawi, a dramatic increase in typhoid fever cases has recently occurred, and may be linked to the emergence of the H58 haplotype. Strains belonging to the H58 haplotype often exhibit multidrug resistance and may have a fitness advantage relative to other Salmonella Typhi strains.

METHODS: To explore hypotheses for the increased number of typhoid fever cases in Blantyre, we fit a mathematical model to culture-confirmed cases of Salmonella enterica infections at Queen Elizabeth Central Hospital, Blantyre. We explored 4 hypotheses: (1) an increase in the basic reproductive number (R0) in response to increasing population density; (2) a decrease in the incidence of cross-immunizing infection with Salmonella Enteritidis; (3) an increase in the duration of infectiousness due to failure to respond to first-line antibiotics; and (4) an increase in the transmission rate following the emergence of the H58 haplotype.

RESULTS: Increasing population density or decreasing cross-immunity could not fully explain the observed pattern of typhoid emergence in Blantyre, whereas models allowing for an increase in the duration of infectiousness and/or the transmission rate of typhoid following the emergence of the H58 haplotype provided a good fit to the data.

CONCLUSIONS: Our results suggest that an increase in the transmissibility of typhoid due to the emergence of drug resistance associated with the H58 haplotype may help to explain recent outbreaks of typhoid in Malawi and similar settings in Africa.

Renal Drug Transporters – Clinical Significance and Role in Drug-Drug Interactions

Thesis (Ph.D.)--University of Washington, 2016-07

Anti-MDR and antitumoral action of acetylsalicylic acid on leukaemic cells

ASA (acetylsalicylic acid) is an NSAID (non-steroidal anti-inflammatory drug). ASA has gained attention as a potential chemopreventive and chemotherapeutic agent for several neoplasms. The aim of this study was to analyse the possible antitumoural effects of ASA in two erythroleukaemic cell lines, with or without the MDR (multidrug resistance) phenotype. The mechanism of action of different concentrations of ASA were compared in K562 (non-MDR) and Lucena (MDR) cells by analysing cell viability, apoptosis and necrosis, intracellular ROS (reactive oxygen species) formation and bcl-2, p53 and cox-2 gene expression. ASA inhibited the cellular proliferation or induced toxicity in K562 and Lucena cell lines, irrespective of the MDR phenotype. The ASA treatment provoked death by apoptosis and necrosis in K562 cells and only by necrosis in Lucena cells. ASA also showed antioxidant activity in both cell lines. The bcl-2, p53 and cox-2 genes in both cell lines treated with ASA seem to exhibit different patterns of expression. However, normal lymphocytes treated with the same ASA concentrations were more resistant than tumoral cells. The results of this work show that both cell lines responded to treatment with ASA, demonstrating a possible antitumoral and anti-MDR role for this drug.

Ação fotodinâmica da C-Ficocianina, pigmento extraído da Spirulina platensis, nas linhagens tumorais humanas K562 (não MDR) e K562 – Lucena 1 (MDR)

A C-ficocianina (C-FC), um pigmento comum nas cianobctérias e um dos mais abundantes constituintes da Spirulina platensis, vem sendo estudada por possuir várias propriedades como antioxidante, hepatoprotetora, antiinflamatória e inibidora da enzima COX-2. Alguns autores atribuem também a C-FC um efeito oxidante quando ela é o agente fotossensibilizante utilizado na terapia fotodinâmica (TFD), podendo ser um importante agente no tratamento do câncer. Entretanto ainda pouco se sabe sobre a ação da C-FC, como substância fotosensibilizante, no tratamento de ação fotodinâmica (AFD) em modelos biológicos. A AFD provoca a fotooxidação de substratos biológicos através da geração de espécies reativas de oxigênio produzidas pela associação entre um determinado comprimento de onda, uma substância fotosensível e oxigênio. Existem dois caminhos que levam a morte celular pelo processo de fotooxidação conhecidos como mecanismo do tipo I e tipo II. No mecanismo tipo I são gerados radicais como o radical ânion superóxido e radical hidroxila, enquanto no mecanismo do tipo II a espécie reativa de oxigênio gerada é o oxigênio singlete (1O2). A TFD da C-PC possui muitas vantagens em relação ao uso das hematoporfirinas e seus derivados, como rápida preparação e fácil purificação, ampla faixa de absorção do UV e visível, nenhum efeito local, e significativa redução da fotosensibilidade em tecidos normais por ter uma rápida metabolização em vivo. As pesquisas que avaliam os possíveis efeitos celulares da AFD têm sido também estendidas para as células tumorais que adquirem fenótipo de resistência a múltiplas drogas (MDR). A MDR é um fenômeno no qual células tumorais, selecionadas resistentes a um agente quimioterápico, adquirem resistência a outras drogas, 5 aparentemente não relacionadas. O fenótipo MDR é multifatorial, mas o mecanismo melhor estudado é a super expressão da glicoproteína-P, que é uma proteína de membrana capaz de fazer a extrusão de quimioterápicos para fora de célula. Com isso o objetivo deste estudo é avaliar a sensibilidade das linhagens celulares que expressem (Lucena) ou não (K562) o fenótipo MDR à AFD do pigmento C-FC, extraído da cianobactéria S. platensis, e propor um possível mecanismo de ação. A extração da C-PC foi feita no Laboratorio de Microbiologia e Engenharia de Bioprocesos (FURG). Diferentes concentrações de C-PC (0.025, 0.05, 0.10, 0.20 e 0.40 mg/ml para os testes de PDA da C-PC e 0.05, 0.10, 0.20, 0.40 e 0.60 mg/ml para os testes no escuro) foram usadas. O número de células viáveis foi avaliada imediatamente, 24 h e 48 h após o tratamento com C-PC ou PDA da C-PC através de exclusão por azul de trypan. A concentração de 0.05 mg/ml foi utilizada para determinar o possível papel da Pgp na resposta da linhagem Lucena e a concentração de 0.10 mg/ml foi utilizada nos testes de peroxidação lipídica (LPO), de produção de espécies reativas de oxigênio (ROS) e quantificação de apoptose/necrose. A PDA da CPC causou uma diminuição no número de células viáveis em ambas linhagens K562 (não MDR) e Lucena (MDR), sendo que a linhagem MDR foi menos sensível que a não MDR. Já nos testes realizados no escuro, nenhuma toxicidade foi encontrada para as duas linhagens. Nenhuma alteração na resistência da linhagem Lucena foi encontrada quando o modulador verapamil foi colocado durante o tratamento de APD com C-PC e até às 48h de acompanhamento após o tratamento. Também não foi encontrada diferença significativa de lipoperoxidação (LPO) mas houve uma tendência de aumento na produção de ROS, que foi mais evidente na linhagem K562. Além disso a linhagem Lucena apresentou uma produção basal de ROS significativamente maior que a K562. Nos testes de apoptose/necrose nenhuma diferença foi encontrada entre as células controle e tratadas em ambas linhagens. Os resultados encontrados neste estudo sugerem que a C-PC possa ser um potente agente fotosensibilizante, tanto para linhagens não MDR quanto para linhagens MDR e também que o mecanismo tipo II esteja envolvido em maior parte no efeito observado na PDA da C-PC, mas uma menor participação do mecanismo tipo I não pode ser descartada. 

Safety and Efficacy of Intravenous Colistin in Neonates With Culture Proven Sepsis

Background: Although it is well described among adults, intravenous colistin use and its associated toxicities in newborns are poorly understood. Objectives: We present our experience of efficacy and safety of intravenous colistin in the treatment of sepsis in term and preterm neonates. Patients and Methods: The records of neonates who received colistin between January 2013 and February 2014 were retrospectively reviewed. All neonates with culture proven nosocomial infections due to multidrug resistant organisms and treated continuously with colistin for more than 72 hours were included in the study. Results: Patients were evaluated for clinical and microbiological response to the drug and its and side effects. Twelve newborn infants with mean 31.8 ± 3.5 weeks gestational age and median 1482 (810 - 3200) gram birth weight were included. 11/12 (91.7%) patients showed microbiological clearance with intravenous colistin. One patient who had recurrent cerebrospinal fluid positive culture was treated with intraventricular colistin. The major side effects observed was hyponatremia and hypokalemia in 2 (16.6%) patients, all infants required magnesium supplementation. Conclusions: Intravenous colistin administration appears to be safe and efficacious for multidrug-resistant gram-negative infections in neonates, including preterm infants. However, we believe that large prospective controlled studies are needed to confirm its efficacy and safety in neonates.

Enteropatógenos y antibióticos

La gastroenteritis infecciosa continúa siendo un problema de salud pública. La etiología bacteriana es la responsable de la mayoría de los casos graves. En nuestro país, Campylobacter y Salmonella son los géneros bacterianos más prevalentes, mientras que Yersinia y Shigella son mucho menos frecuentes. La mayoría de los casos suelen ser autolimitados y, en general, el tratamiento antibiótico no está indicado, salvo en pacientes con factores de riesgo de infección grave y en shigelosis. Ciprofloxacino, cefalosporinas de tercera generación, azitromicina, ampicilina, cotrimoxazol y doxiciclina son los fármacos más recomendados. El patrón de sensibilidad de las diferentes bacterias determina la elección del tratamiento antibiótico más adecuado. El objetivo de esta revisión es analizar la situación, las novedades y la evolución de la resistencia y la multirresistencia en estos 4 enteropatógenos.

Preclinical evaluation of a MYCN-specific inhibitor for the treatment of small cell lung cancer

Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, characterized by rapid growth, early metastasis and acquired drug resistance. SCLC is usually sensitive to initial treatment, however, most patients relapse within few months; thus more effective therapies are urgently needed. Key genetic alterations very frequently observed in SCLC include loss of TP53 and RB1 and mutations in the MYC family genes (MYC, MYCL or MYCN). One of them is amplified and overexpressed in a mutually exclusive manner and represents the most prominent activating oncogene alteration in this malignancy. In particular, MYCN amplification is associated with tumor progression, treatment failure and poor prognosis. Given the role of MYCN in SCLC and its restricted expression profile, MYCN represents a promising therapeutic target; although it is considered undruggable by traditional approaches. An innovative approach to target the oncogene concerns specific MYCN expression inhibition, acting directly at the level of DNA, through an antigene peptide nucleic acid (agPNA) oligonucleotide, called BGA002. This thesis focused on the study of BGA002, as a possible targeted therapeutic strategy for the treatment of MYCN-related SCLC. In this context, BGA002 proved to be a specific and highly effective inhibitor. Furthermore, MYCN silencing induced alterations in many downstream pathways and led to apoptosis, in concomitance with autophagy reactivation. Moreover, systemic administration of BGA002 was effective in vivo as well, significantly increasing survival in MNA mouse models, even in the scenario of multidrug-resistance. In addition, BGA002 treatment successfully reduced N-Myc protein expression and, more importantly, caused a massive diminishment in tumor vascularization in the multidrug-resistant model. Overall, these results proved that MYCN inhibition by BGA002 may represent a new promising precision medicine approach, to treat MYCN-related SCLC.

Epidemic multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport strains contain three phage regions and a MDR resistance plasmid

Multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport has caused serious disease in animals and humans in North America, whereas in the UK S. enterica serovar Newport is not associated with severe disease and usually sensitive to antibiotics; MDR S. Newport (not AmpC) strains have only been isolated from poultry. We found that UK poultry strains belonged to MLST type ST166 and were distinct from cattle isolates for being able to utilize D-tagotose and when compared by pulsed-field gel electrophoresis (PFGE), comparative genomic hybridization (CGH) and diversity arrays technology (DArT). Cattle strains belonged to the ST45 complex differing from ST166 at all seven loci. PFGE showed that 19 out of 27 cattle isolates were more than 85% similar to each other and some UK and US strains were indistinguishable. Both CGH and DArT identified genes (including phage-related ones) that were uniquely present in the US isolates and two such genes identified by DArT showed sequence similarities with the pertussis-like (artAB) toxin. This work demonstrates that MDR-AmpC S. Newport from the USA are genetically closely related to pan-susceptible strains from the UK, but contained three extra phage regions and a MDR plasmid.

High prevalence of multidrug-tolerant bacteria and associated antimicrobial resistance genes isolated from ornamental fish and their carriage water

Background: Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely added to the water these fish are shipped in to suppress the growth of potential pathogens during transport. Methodology/Principal Findings: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to >= 15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR). Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, blaTEM21, tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates. Conclusions: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes.

Antibiotic resistance in Lactococcus species from bovine milk: presence of a mutated multidrug transporter mdt(A) gene in susceptible Lactococcus garvieae strains

A total of 72 Lactococcus strains (41 Lactococcus lactis and 31 Lactococcus garvieae) isolated from bovine milk were tested for susceptibility to 17 antibiotics and screened for the presence of antibiotic resistance genes using a microarray. Resistance to tetracycline, clindamycin, erythromycin, streptomycin, nitrofurantoin were found. The tetracycline-resistant L. garvieae and L. lactis harbored tet(M) and tet(S). L. lactis that were resistant to clindamycin were also resistant to erythromycin and possessed the erm(B) gene. The multidrug transporter mdt(A), originally described in L. lactis, was detected for the first time in L. garvieae and does not confer decreased susceptibility to erythromycin nor tetracycline in this species. Mdt(A) of L. garvieae contains one mutation in each antiporter motif C, which is known to play an essential role in drug efflux antiporters. This suggests that the mutations found in the C-motifs of Mdt(A) from L. garvieae may be responsible for susceptibility. The study revealed the presence of antibiotic resistance genes in non-pathogenic and pathogenic lactococci from bovine milk, including a mutated multidrug transporter in L. garvieae.