Multidrug resistance (MDR) is one of the foremost causes of treatment failure in childhood malignancies. MDR is a multifactorial process, but classic resistance to cytotoxic drugs has most often been associated with over-expression of one or more MDR transporter proteins in malignant cells, conferring on them the ability to extrude an extraordinarily diverse array of endo- and xenobiotics out of the cell. The best characterized multidrug transporters, P-glycoprotein (Pgp) and the Multidrug Resistance-associated Protein (MRP), belong to the ATP-binding cassette (ABC) gene superfamily, and have been previously implicated in the development of drug resistance in the clinical context. The work described herein examined the various aspects of the MDR genotype and phenotype in the childhood malignancies acute lymphoblastic leukaemia (ALL) and neuroblastoma. The first series of studies tested the hypothesis that morphine, a potential Pgp substrate, might influence the efficacy and/or toxicity of clinically used chemotherapy agents which are substrates for Pgp. The results, however, provided no evidence in a variety of human tumour cell lines of morphine influencing response to selected chemotherapeutic drugs. This finding is particularly important as morphine remains to be the opioid of choice for the treatment of cancer pain in the clinic. The second series of studies examined the effect of single nucleotide polymorphisms (SNPs) in the MDR1 gene, encoding Pgp, and in the MRP1 gene, on patient outcome in childhood ALL or neuroblastoma, with a view to identifying novel prognostic markers for these malignancies. It was found that two of the examined SNPs in the MRP1 gene were associated with improved outcome in neuroblastoma, which had not previously been demonstrated in this disease. Moreover, each of the relevant MRP1 SNPs were associated with lower MRP1 gene expression in both patient samples and tumour cell lines, supporting previous studies indicating that low MRP1 expression in neuroblastoma is strongly associated with improved patient outcome. Importantly, the results of this study suggest a role for selected MRP1 polymorphisms in predicting clinical response in neuroblastoma. Finally, a series of studies were undertaken, using both in vitro and in vivo model systems, to test the efficacy of putative small molecule inhibitors of the MRP1 gene and its transcriptional regulator, the MYCN oncogene, in neuroblastoma. These studies demonstrated for the first time the efficacy of a novel compound, 4H10, at reversing multidrug resistance either in cultured neuroblastoma cells, or in the MYCN transgenic mice, which develop neuroblastoma that closely mirror the human disease. The results indicate that inhibition of MRP1 function has potential clinical importance in the treatment of neuroblastoma, and therefore warrant further research in this area. In contrast, the results failed to provide evidence of the in vivo efficacy of the novel putative small molecule MYCN inhibitors analysed in these studies. Collectively, the findings of these studies contribute to a better understanding of the mechanisms of clinical drug resistance, and may help in the development of new approaches for risk assessment and treatment of aggressive childhood malignancies and thereby improve the long-term outlook of children diagnosed with these debilitating diseases.

Thesis (Ph. D.)--Colorado State University, 2004. / Includes bibliographical references.

Until the recent introduction of short course regimens, treatment regimens for multidrug resistant TB (MDR-TB) were long and toxic. Consequently, only approximately half of MDRTB patients completed their treatment. TB dosing guidelines have historically been unrefined with little consideration for pharmacokinetic/pharmacodynamic relationships. Large knowledge gaps therefore exist in the understanding of pharmacokinetic/pharmacodynamic relationships for both efficacy and toxicity in MDR-TB. My PhD used clinical pharmacology approaches to improve the understanding of drug exposures, toxicity, and exposure-toxicity relationships during the first 12 weeks of MDR-TB therapy. Aims and methods 1. Using non-compartmental analyses, describe the pharmacokinetics of cycloserine and, using regression modelling, explore the association of covariates with cycloserine exposure. 2. Using validated screening tools, describe the incidence of neuropsychiatric toxicity in MDR-TB patients, and explore associations with cycloserine pharmacokinetics. 3. Using a validated pain-rating scale in a crossover study design, investigate whether the addition of a local anaesthetic reduces kanamycin-related injection pain, and explore effects on kanamycin pharmacokinetics. 4. Using geometric mean ratios, compare the exposures of crushed versus whole formulations of pyrazinamide, moxifloxacin, ethionamide, ethambutol, cycloserine, and isoniazid. Results and conclusions We found no measurable terizidone in plasma supporting the hypothesis that terizidone is hydrolysed pre-systemically to cycloserine. The cycloserine time-concentration profile supports once daily dosing of terizidone. We describe a high incidence of peripheral neuropathy in MDR-TB patients with both cycloserine clearance and high-dose pyridoxine significantly associated with neuropathy on multivariate analysis. The addition of a local anaesthetic reduced the pain experienced by MDR-TB patients in the first 15 minutes post intramuscular administration of kanamycin, which could improve adherence to MDR-TB treatment. We also found the bioavailability of crushed isoniazid to be approximately 42% less than the whole tablet formulation, and therefore recommend that the crushing of isoniazid be avoided. Although some recent treatment advances have improved MDR-TB outcomes, enhancing the understanding of drugs used to treat MDR-TB, which continues to have an unacceptably high mortality and treatment-related morbidity, is a public health priority. This thesis comprises four peer-reviewed publications, all of which made a pragmatic contribution to the fight against MDR-TB.

Multiple drug resistance (MDR) has been a severe issue in treatment and recovery from infection.Gram-negative bacteria intrinsically exhibit higher drug tolerance than Gram-positive microbes. In this thesis, two proteins involved in Gram-negative bacterial MDR were studied, AcrB and SurA. Resistance-nodulation-cell division pump AcrAB-TolC is the major MDR efflux system in Gram-negative bacteria and efficiently extrudes a broad range of substances from the cells. To study subtle conformational changes of AcrB in vivo, a reporter platform was designed. Cysteine pairs were introduced into different regions in the periplasmic domain of the protein, and the extents of disulfide bond formation were examined. Using this platform, an inactive mutant, AcrB∆loop, was created that existed as a well-folded monomer in vivo. Next, random mutageneses were performed on a functionally compromised mutant, AcrBP223G, to identify residues that restored the function loss. The mechanism of function restoration was examined. SurA is a periplasmic molecular chaperone for outer membrane biogenesis. Deletion of SurA decreased outer membrane density and bacterial drug resistance. The dependence of SurA function on structural flexibility and stability was examined. In addition, the effect of molecular crowding on SurA interaction with its outer membrane protein substrates was examined.

Cancer remains the most cause death disease in Taiwan at least ten years. Liver cancer, which consists predominantly of hepatocellular carcinoma (HCC), is the most common cause of cancer mortality in men and the second most in women. Not only liver section and liver transplantation are used in HCC therapy but also local ablation therapy and transarterial therapy. Transarterial chemoembolization (TACE) is one of the local therapies that inject chemotherapeutic drugs directly into liver tumor. However, drug resistance is the mainly restriction in patient after chemotherapy. Moreover, it is known that drug resistance was associated to over-expression of certain ABC transporter genes, especially ABCB1, ABCG2, and ABCC family in cancer cell and those ABC transporters were also expressed in liver. Base on clinical study, they use 5-fluororuacil, cisplatin and mitomycin-C for liver cancer treatment. In this study, we hypothesized that cancer therapies may be augmented through blocked the drug efflux ABC channels with the ABC transporter inhibitors such as verapamil. The associations among drug treatments, inhibitor incorporation and the expression of ABC transporters were evaluated in HepG2 and Hep3B cells. MTT assay demonstrated that the cell viability was considerable decreased by treating triple drugs with verapamil. RT-PCR data showed that ABC transporters mRNA expression has no significantly change. However, membrane ABCB1 and ABCG2 were induced after drugs and inhibitors treatment either 1 or 24 hours by flow cytometry analysis. P-glycoprotein functional assay also showed p-glycoprotein was inhibited by verapamil, and hence Rhodamine 123 retention was increased. Taken together, there are different response of ABC transporters in HepG2 and Hep3B after drugs and inhibitors treatment. Membrane ABCB1 and ABCG2 were induced by drugs and inhibitors treatment. However, p-glycoprotein¡¦s function was restrained simultaneously by inhibitors treatment. Therefore, verapamil can enhance cell death by inhibiting ABC transporters and its cytotoxic effect rather than the increased expression of ABC transporters. This finding might provide a better way in liver cancer therapy.

Emerging multidrug-resistant tuberculosis (MDR-TB) is one of the most urgent global public health issues. Recent advances in molecular techniques should enable the development of different rapid detection tests for drug-resistant TB. Large-scale comparative studies on the diagnostic accuracy and turn-around-time (TAT) of these novel assays may promote their smooth implementation as routine tests for TB in diagnostic laboratories. In a pilot evaluation of 30 clinical isolates and 202 sputum specimens, diagnostic performance of a novel in-house assay for MTB identification (IS6110 qPCR) was compared to a commercial COBAS TaqMan MTB test (Roche Diagnostics). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IS6110 qPCR were 100%, 94.6%, 85.2% and 100%, respectively, compared to 94.7%, 100%, 100% and 98.6% for COBAS TaqMan MTB. Large-scale validation using 2,350 sputum specimens revealed the optimal cut-off crossing point (Cp) value of IS6110 qPCR was 29.61 with 97.3% sensitivity and 98.3% specificity determined by receiver operating characteristics (ROC) curve analysis. The median TAT for IS6110 qPCR and COBAS TaqMan MTB test to the reporting of results was 0.9 and 1.2 days, respectively. Among the IS6110 qPCR-positive specimens in the large-scale validation, 287 samples were tested in-house by katG MAS-PCR and rpoB PCR sequencing assays and 159 samples were tested by GenoType® MTBDRplus assay (Hain LifeScience). The sensitivity and specificity of katG MAS-PCR for isoniazid (INH) resistance detection were 71.4% and 99.5%, respectively. The sensitivity and specificity of rpoB PCR sequencing for rifampicin (RIF) resistance detection were 100% and 99.6%, respectively. Commercial GenoType® MTBDRplus assay reached 100% sensitivity for both INH and RIF resistance detection at a specificity of 99.3% and 100%, respectively. The median TAT for the in-house assays and GenoType® MTBDRplus assay to the reporting of the results was 4.7 and 1.4 days, respectively. The findings from this study provide different implementation strategies for diagnostic test combinations. The most cost-effective drug-resistant TB diagnosis cascade was IS6110 qPCR followed by GenoType® MTBDRplus assay. The TAT for results is 2.3 days at a cost of US$49.7. Despite an additional cost of US$24.6, COBAS TaqMan MTB test should replace IS6110 qPCR in populations with high prevalence of IS6110-negative strains. The in-house katG MAS-PCR and rpoB PCR sequencing assays should be used in developing countries instead of the expensive GenoType® MTBDRplus assay. Subsequently, accurate diagnosis of drug-resistant tuberculosis can be achieved in 4.5 days with a reasonable reagent cost of US$9.3. In conclusion, excellent diagnostic accuracy and shorter TAT of the molecular diagnostic cascade for drug-resistant TB, in particular IS6110 qPCR, can serve to guide physicians in the prompt choice of chemotherapy. This leads to timely delivery of anti-TB treatments to patients and holds the promise of easing the MDR-TB burden. / published_or_final_version / Microbiology / Master / Master of Philosophy

The ATP-binding cassette (ABC) transporter superfamily consists of 49 members, to which both Multidrug Resistance Protein 1 (MRP1/gene symbol: ABCC1) and MRP4 (ABCC4) belong. Single nucleotide polymorphisms (SNPs) in drug metabolizing genes have been shown to affect individual responses to drugs and toxins. However, the role of SNPs in modulating the activity of drug transporters, such as MRP1 and MRP4, is poorly characterized. The overall goal of my thesis was to determine the effects of SNPs in the promoter regions of human ABCC1 and ABCC4. For MRP1/ABCC1, two proximal promoter SNPs (-275A>G, -260G>C) were identified in the literature and recreated in vitro, and the activity of the mutant ABCC1 promoter constructs was measured in five human cell lines using a dual luciferase assay. The activity of the -275A>G promoter was comparable to the wild-type ABCC1 promoter. On the other hand, the -260G>C substitution decreased ABCC1 promoter activity in HepG2, MCF-7 and HeLa (40 - 60%) cells. A 1706 bp fragment containing the 5’-flanking and untranslated regions of ABCC4 were isolated from two bacterial artificial chromosome clones and six serially deleted ABCC4 promoter reporter constructs generated. Luciferase assays of the basal promoter constructs of ABCC4 in HEK293T cells revealed the presence of one or more negative regulatory regions between -1706 and -876, between -876 and -641, and one or more positive regulatory regions between -641 and -356, and between -356 and -17. Also, the ABCC4 promoter displayed differential activity in MDCKI and LLC-PK1 cells than in HEK293T cells. One SNP (-523G>C) was identified from an online database and its activity tested. However, -523G>C SNP did not cause any significant change in the ABCC4 promoter activity in both HEK293T and HepG2 cells (80 – 130%). In summary, the data obtained suggest that the promoter SNPs studied may affect the transcriptional activity of ABCC1 or ABCC4, but it seems likely that this is not true in all cell types. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2007-09-28 10:03:16.119

MRPs mediate the ATP-dependent efflux of a structurally diverse array of compounds. Certain MRPs, including MRP1, MRP2 and MRP4, are involved in multidrug resistance in tumour cells, while in non-malignant cells these MRPs can influence the distribution and/or elimination of many clinically important drugs. In addition, these MRPs mediate the efflux of physiological metabolites, many of which are conjugated organic anions. Modulation of the drug transporting activity of MRP1 (and to a lesser extent MRP2 and MRP4) has been a long sought after therapeutic objective. In this study, the modulatory abilities of five structurally distinct classes (I-V) of chalcogenopyrylium dyes (CGPs) were examined utilizing an in vitro assay which measures inhibition of radiolabeled estradiol glucuronide ([3H]E217βG) (a prototypical MRP substrate) uptake into inside-out membrane vesicles prepared from MRP-transfected human embryonic kidney (HEK) cells. Additionally, some CGPs were tested in a calcein efflux assay using intact MRP1-transfected HEK cells. Sixteen of 34 CGPs initially tested at a single concentration (≤30 μM) inhibited MRP1-mediated uptake by >50%, with IC50’s ranging from 0.7-7.6 μM. Of the 9 CGPs with IC50’s ≤2 μM, five belonged to Class I, two to Class II, and two to Class IV. When tested in the calcein efflux assay, only 4 of 16 CGPs inhibited MRP1-mediated cellular efflux by >50% (I-3, -4, -6, IV-1) while a fifth (II-5) inhibited efflux by 23%. These five CGPs were then tested as modulators of [3H]E217βG uptake by MRP2 and MRP4. Their effects on MRP2 transport activity were differential with two (I-4, I-6) inhibiting transport (IC50’s 2.0, 7.1 μM), two (I-3, IV-1) stimulating transport (>2-fold), while II-5 had no effect. On the other hand, all five CGPs inhibited [3H]E217βG uptake by MRP4, but less effectively than by MRP1. Finally, five analogs of CGP IV-1 were tested for their effects on MRP1, MRP2 and MRP4 [3H]E217βG uptake, but none were more efficacious than CGP IV-1. The CGPs tested here represent novel MRP1, MRP2 and MRP4 modulators with variable effects on transport activities. These CGPs may represent a new avenue for the development of clinically applicable modulators of MRP proteins involved in multidrug resistance. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2011-11-28 17:50:49.432

Dissertation ( PhD)--University of Stellenbsoch, 2007. / Bibliography. Also available via the Internet.

Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2010 / The World Health Organisation estimates that nine million people are infected with tuberculosis (TB) every year of which ninety-five percent live in developing countries. Africa has one of the highest incidences of TB in the world. but few of its countries are equipped to diagnose drug-resistant TB. This study aimed to develop a robust. yet simple and cost-effective assay. which would require minimal sophisticated instrumentation and specialised personnel that would make drug sensitivity screening for multi-drug resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) accessible to resource-poor high-burden settings. A four-quadrant colorimetric agar plate method was developed which showed good specificity (97.3%-100%) and sensitivity (77.8%-100%) compared to the polymerase chain reaction (PCR) method used as gold standard. Agreement between the methods. using Simple Kappa Coefficients. ranged between very good and excellent. all with high statistical significance (P < 0.0001). The currently used BACTEC MGIT SIREN sensitivity assay coupled with the E-test® strip method. as routinely used in the TB reference laboratory. was compared and showed excellent comparison with the newlydeveloped plate method. for each antibiotic tested. as well as the resultant monoresistant, MDR- or XDR-TB diagnoses. Moreover. the new method was found to be extremely cost-effective. priced at half the cost of a peR assay. These four quadrant plates. with a colorimetric indicator and selected antibiotics. can be considered as an economic altemative or a complimentary method for laboratories wishing to reduce the cost and complexity for TB drug sensitivity testing. Routine diagnostic testing would thus be made more accessible and affordable to laboratories that are not presently diagnosing drug resistant TB. therefore enhancing case detection and treatment in the resource-poor settings hardest hit by this curable disease.