Dynamic Bioluminescence Imaging: Development of a Physiological Pharmacokinetic Model of Tumor Metabolism

poster abstractBioluminescent imaging (BLI) has proven to be a valuable tool for the study of cellular biology and therapeutic response in a wide array of tumor types. Several BLI analytical approaches have been developed to assess tumor function and growth, all with the primary assumption that substrate concentrations saturate the luciferase enzyme. Recent work suggests that when D-luciferin is administered over the range from 75-600mg/kg, target tissue concentrations of D-luciferin are well below the Km of luciferase for the reaction, and, that the pharmacokinetics of D-luciferin significantly impact observed emission rates. To address the concentration and PK concerns, we developed a three compartment physiologically based pharmacokinetic (PhPK) model for D-luciferin including oxidation by luciferase via Michaelis-Menten kinetics. The model was applied to dynamically acquired BLI in NOD/SCID mice with ectopic luciferase-transfected SF767 tumors. The current PhPK model estimates tumor volume, tumor substrate metabolism (M ̅), tumor blood flow (Vb) and substrate extraction from the blood (Er). Studies were conducted using intraperitoneal, subcutaneous and intravenous routes of administration of 150 mg/kg of D-luciferin, where dynamic BLI was conducted weekly for four weeks. The D-luciferin concentration in tumor tissue, determined immediately after the last imaging session, was found to be approximately 8-fold below the reported Km for the reaction across all routes of administration, supporting the need for a PhPK modeling approach for analyzing BLI data. The model-predicted tumor volumes increased over time and were highly correlated with caliper-measured tumor volumes (y=1.984x, R2=0.980, p<0.0001). Tumor D-luciferin metabolism was found to increase exponentially over the 4 weeks, while blood flow decreased over this same interval, a finding which is consistent with the interpretation of a Warburg effect. When tumor M ̅ was compared with the traditional measures of peak emission (Cmax) and area under the curve (AUC), it was found that metabolism increased exponentially with increases in either Cmax (y=92.7exp(8E-11x), R2= 0.997) or AUC ( y=86.4exp(5E-14x), R2= 0.989), suggesting that Cmax and AUC may substantially underestimate the magnitude of tumor metabolism. The present PhPK model of D-luciferin distribution and metabolism overcomes limitations in the Cmax and AUC approaches caused by incorrect substrate: enzyme concentration assumptions, and thus provides a more reliable estimate of tumor burden, growth, and therapeutic response

Bcr/Abl Interferes with the Fanconi Anemia/BRCA Pathway: Implications in the Chromosomal Instability of Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. Although it is well known that CML cells are genetically unstable, the mechanisms accounting for this genomic instability are still poorly understood. Because the Fanconi anemia (FA) pathway is believed to control several mechanisms of DNA repair, we investigated whether this pathway was disrupted in CML cells. Our data show that CML cells have a defective capacity to generate FANCD2 nuclear foci, either in dividing cells or after DNA damage. Similarly, human cord blood CD34+ cells transduced with BCR/ABL retroviral vectors showed impaired FANCD2 foci formation, whereas FANCD2 monoubiquitination in these cells was unaffected. Soon after the transduction of CD34+ cells with BCR/ABL retroviral vectors a high proportion of cells with supernumerary centrosomes was observed. Similarly, BCR/ABL induced a high proportion of chromosomal abnormalities, while mediated a cell survival advantage after exposure to DNA cross-linking agents. Significantly, both the impaired formation of FANCD2 nuclear foci, and also the predisposition of BCR/ABL cells to develop centrosomal and chromosomal aberrations were reverted by the ectopic expression of BRCA1. Taken together, our data show for the first time a disruption of the FA/BRCA pathway in BCR/ABL cells, suggesting that this defective pathway should play an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies

Are Thai MSM willing to take PrEP for HIV prevention? An analysis of attitudes, preferences and acceptance.

OBJECTIVE: We aimed to understand the attitudes, preferences and acceptance of oral and parenteral PrEP among men who have sex with men (MSM) in Thailand. BACKGROUND: Pre-exposure prophylaxis (PrEP), the use of antiretrovirals to prevent HIV acquisition, has shown promising results in recent trials. To assess the potential impact of this new HIV prevention method, in addition to efficacy data, we need to understand which psychosocial factors are likely to determine its uptake among members of potential user groups. METHODS AND FINDINGS: Surveys of willingness to use PrEP products were administered to MSM. Spearman's rank tests were used to uncover associations between questionnaire items. Mann-Whitney tests were performed to ascertain differences between groups. Conjoint analysis was used to examine the attitudes and preferences of MSM towards PrEP attributes. Most participants were willing to consider taking PrEP (39.2% "yes, definitely" and 49.2% "yes, probably") and perceived PrEP as giving them new possibilities in their lives (38.5% "a lot of hope" and 55.8% "some hope"), even after being instructed of potential side effects and costs. HIV testing was considered the most important attribute and a daily pill and longer lasting injection in the arm were the preferred routes of administration. CONCLUSIONS: Despite its multiple challenges, MSM in Thailand would be willing to take PrEP, even if they had to experience inconvenience and expense. If PrEP were to be implemented in Thailand, our findings show that its uptake could be considerable