Crossing the Uncrossable Barrier
By Mohamed El-Abtah
Glioblastoma multiforme (GBM) is one of the most aggressive forms of brain cancer, claiming the lives of approximately 14,000 people per year in the United States alone. Despite GBM’s very poor prognosis, the standard of care for the treatment of GBM has been unchanged for many decades and consists of aggressive surgical resection of as much tumor as possible, followed by radiation and chemotherapy. Even in the best circumstances, this procedure only extends the mean survival from two to three months to less than a year. 1
Despite having several confirmed targets and associated therapeutics, there is virtually 0% success rate in GBM clinical trials. This phenomenon is primarily due to the poor delivery of drugs across the Blood Brain Barrier (BBB). 2 The Blood Brain Barrier (BBB) refers to the tight junctions between endothelial cells in the brain that restrict the diffusion of molecules between the blood and the central nervous system to prevent toxins from infiltrating the brain. 2 However, this mechanism prevents the delivery of many potentially useful drugs into the brain tissue which results in the poor pharmacokinetic and pharmacodynamic properties associated with GBM drugs.
Currently, the mechanism of resistance for any given patient’s tumor to a therapy cannot be predicted a priori and cannot be determined in a clinical trial.3 For this reason, identification of the drugs’ failure mechanism would provide crucial insight into various strategies to improve clinical success.
Metabolic pathways may provide information on tumor phenotypes, apoptosis, cellular proliferation, migration rates, and invasive capacity of the tumor cells. Since these tumor phenotypes regulate tumor density and blood flow, they may very well affect how well a drug operates in modulating tumor response to treatment.
The metabolic characteristics of each tumor could provide insight into the adaptive resistance mechanism of tumors, which could in turn bring improvements to patient prognosis in a clinical setting. This hypothesis was tested by characterizing the metabolic profiles of patient derived glioblastoma xenograft tumors in rats with and without treatment of Erlotinib. Erlotinib is a FDA approved drug that is currently used to treat lung, pancreatic, and liver cancer. Erlotinib works by inhibiting a signalling pathway—the epidermal growth factor pathway (EGF)—which leads to uncontrollable cell growth.3
Using mass spectrometry, it becomes possible to image drug distribution and the tumor response to therapeutic treatments of Erlotinib. This approach can provide crucial insight into the dynamics of the blood brain barrier and glioblastoma.
Although the concentration of drug administered to the animal model is known, the amount actually delivered to the brain in unknown. The overall aim of this analysis was to determine the absolute concentration of drug in the dosed animal tissues, hinting at the relationship between the blood brain barrier and drug penetrance.
Samples of the different Erlotinib concentrations and sent for mass spectrometry analysis for detection of the drug (m/z 394176). The absolute intensity of the drug in each of the different concentrations was recorded and used for the generation of a calibration curve, a graph used to determine the concentration of the drug in unknown samples.
Mass spectrometry was then used to determine if the types of metabolic products produced by the brain cells differ among different drug doses and between normal and cancerous tissue.
It was observed that for all the rat tumors, the normal tissue had very low traces of fatty acyl carnitine. However, for the tumor tissue, there was an approximately five-fold increase in the intensity of the fatty acyl carnitine. This is a hint that increased fatty acid oxidation is taking place within tumor cells, a sign that cells that are constantly proliferating. This result makes sense, given the highly proliferative nature of glioblastoma.
It was observed that there was an approximately five-fold increase in the intensity of this metabolite in tumor samples, which may be indicative of increased metabolism within the GBM tumor due to the metabolite’s role in energy production.
In addition, fatty acyl carnitine represents a clinically relevant molecule of interest. Fatty acyl carnitines are critical components of a biochemical pathway that transports fatty acids across the mitochondrial membrane to be used for energy production. This, too, indicates the high proliferation rates of cells, which require larger quantities of ATP—a small, molecular store of energy produced by the mitochondria—which needs to be generated in large quantities for tumor cells to divide.3
Overall, this research presents a quantitative method to measure both drug concentration and metabolic profiles of brain cells. Traditionally, this process would require highly expensive and time-intensive procedures, such as liquid extraction, separation, purification and liquid chromatography-mass spectrometry (LCMS). The hope is that streamlined procedures such as the ones described in this project can be used to more quickly and accurately characterize glioblastoma and other cancers, eventually making it possible to design more effective drug therapies against these maladies.
Works Cited
[1] Cecchelli, R. et al. Modelling of the blood-brain barrier in drug discovery and development. Nat. Rev. Drug Discov. 6, 650–661 (2007).
[2] Jeffrey, P. & Summerfield, S. Assessment of the blood-brain barrier in CNS drug discovery. Neurobiol. Dis. 37, 33–37 (2010).
[3] Ling, J. et al. Metabolism and excretion of erlotinib, a small molecule inhibitor of epidermal growth factor receptor tyrosine kinase, in healthy male volunteers. Drug Metab. Dispos. 34, 420– 426 (2006).
Glioblastoma multiforme (GBM) is one of the most aggressive forms of brain cancer, claiming the lives of approximately 14,000 people per year in the United States alone. Despite GBM’s very poor prognosis, the standard of care for the treatment of GBM has been unchanged for many decades and consists of aggressive surgical resection of as much tumor as possible, followed by radiation and chemotherapy. Even in the best circumstances, this procedure only extends the mean survival from two to three months to less than a year. 1
Despite having several confirmed targets and associated therapeutics, there is virtually 0% success rate in GBM clinical trials. This phenomenon is primarily due to the poor delivery of drugs across the Blood Brain Barrier (BBB). 2 The Blood Brain Barrier (BBB) refers to the tight junctions between endothelial cells in the brain that restrict the diffusion of molecules between the blood and the central nervous system to prevent toxins from infiltrating the brain. 2 However, this mechanism prevents the delivery of many potentially useful drugs into the brain tissue which results in the poor pharmacokinetic and pharmacodynamic properties associated with GBM drugs.
Currently, the mechanism of resistance for any given patient’s tumor to a therapy cannot be predicted a priori and cannot be determined in a clinical trial.3 For this reason, identification of the drugs’ failure mechanism would provide crucial insight into various strategies to improve clinical success.
Metabolic pathways may provide information on tumor phenotypes, apoptosis, cellular proliferation, migration rates, and invasive capacity of the tumor cells. Since these tumor phenotypes regulate tumor density and blood flow, they may very well affect how well a drug operates in modulating tumor response to treatment.
The metabolic characteristics of each tumor could provide insight into the adaptive resistance mechanism of tumors, which could in turn bring improvements to patient prognosis in a clinical setting. This hypothesis was tested by characterizing the metabolic profiles of patient derived glioblastoma xenograft tumors in rats with and without treatment of Erlotinib. Erlotinib is a FDA approved drug that is currently used to treat lung, pancreatic, and liver cancer. Erlotinib works by inhibiting a signalling pathway—the epidermal growth factor pathway (EGF)—which leads to uncontrollable cell growth.3
Using mass spectrometry, it becomes possible to image drug distribution and the tumor response to therapeutic treatments of Erlotinib. This approach can provide crucial insight into the dynamics of the blood brain barrier and glioblastoma.
Although the concentration of drug administered to the animal model is known, the amount actually delivered to the brain in unknown. The overall aim of this analysis was to determine the absolute concentration of drug in the dosed animal tissues, hinting at the relationship between the blood brain barrier and drug penetrance.
Samples of the different Erlotinib concentrations and sent for mass spectrometry analysis for detection of the drug (m/z 394176). The absolute intensity of the drug in each of the different concentrations was recorded and used for the generation of a calibration curve, a graph used to determine the concentration of the drug in unknown samples.
Mass spectrometry was then used to determine if the types of metabolic products produced by the brain cells differ among different drug doses and between normal and cancerous tissue.
It was observed that for all the rat tumors, the normal tissue had very low traces of fatty acyl carnitine. However, for the tumor tissue, there was an approximately five-fold increase in the intensity of the fatty acyl carnitine. This is a hint that increased fatty acid oxidation is taking place within tumor cells, a sign that cells that are constantly proliferating. This result makes sense, given the highly proliferative nature of glioblastoma.
It was observed that there was an approximately five-fold increase in the intensity of this metabolite in tumor samples, which may be indicative of increased metabolism within the GBM tumor due to the metabolite’s role in energy production.
In addition, fatty acyl carnitine represents a clinically relevant molecule of interest. Fatty acyl carnitines are critical components of a biochemical pathway that transports fatty acids across the mitochondrial membrane to be used for energy production. This, too, indicates the high proliferation rates of cells, which require larger quantities of ATP—a small, molecular store of energy produced by the mitochondria—which needs to be generated in large quantities for tumor cells to divide.3
Overall, this research presents a quantitative method to measure both drug concentration and metabolic profiles of brain cells. Traditionally, this process would require highly expensive and time-intensive procedures, such as liquid extraction, separation, purification and liquid chromatography-mass spectrometry (LCMS). The hope is that streamlined procedures such as the ones described in this project can be used to more quickly and accurately characterize glioblastoma and other cancers, eventually making it possible to design more effective drug therapies against these maladies.
Works Cited
[1] Cecchelli, R. et al. Modelling of the blood-brain barrier in drug discovery and development. Nat. Rev. Drug Discov. 6, 650–661 (2007).
[2] Jeffrey, P. & Summerfield, S. Assessment of the blood-brain barrier in CNS drug discovery. Neurobiol. Dis. 37, 33–37 (2010).
[3] Ling, J. et al. Metabolism and excretion of erlotinib, a small molecule inhibitor of epidermal growth factor receptor tyrosine kinase, in healthy male volunteers. Drug Metab. Dispos. 34, 420– 426 (2006).
