Pharmacokinetics of the 13C labeled anticancer agent temozolomide detected in vivo by selective cross-polarization transfer

The anticancer agent temozolomide labeled with ¹³C (8-Carbamoyl-3-13C-methylimidazo-[5,1-d]-1,2,3,5-tetrazin-4-(3H)-one), was noninvasively detected in subcutaneous RIF-1 tumors by a selective cross polarization ¹³C NMR method, at a field strength of 9.4T. Pharmacokinetics of the drug, at a dose of 150 mg/kg, were determined for intravenous and intraperitoneal modes of administration (three animals per mode). The half-life of the drug in the tumors was approximately 60 min. The uptake and clearance of the drug, however, varied significantly between individual hosts, for both modes of administration. These results demonstrate the feasibility of obtaining pharmacokinetics of anticancer agents for individual tumors without the need for a label that might modify drug activity (e.g., fluorine). The variability of the in vivo measurements, even within the same tumor model, demonstrates the necessity of directly monitoring the tumor to evaluate drug pharmacokinetics.     

Energy metabolism, pH changes, and lactate production in RIF-1 tumors following intratumoral injection of glucose.

The metabolic consequences of increased glucose availability were examined in subcutaneous RIF-1 tumors in vivo, using 13C and 31P NMR spectroscopy. Significant increases in the levels of nucleotide triphosphates and phosphocreatine relative to low energy phosphates and in tumor pH were observed within 30 min following injection of 1 g/kg of glucose directly into the tumor. These changes did not occur following an equivalent intratumoral dose of the non-metabolizable sugar alcohol, mannitol. When [1-13C]-glucose was administered, [3-13C]-lactate and [3-13C]-alanine were the only labeled metabolites detected in the in vivo 13C NMR spectra during the period of bioenergetic improvement. Biochemical analysis revealed a substantial increase in tumor and plasma glucose concentration, but no increase in either tumor or plasma lactate, consistent with the absence of acidosis. Evaluation of the distribution of glucose in the tumor by quantitative autoradiography of [1-14C]-2-deoxyglucose administered with the glucose indicated that, on average, 7 mM of the added glucose distributed over the entire tumor within 10 min. The significant improvement in overall metabolic status of the tumors following glucose administration is attributed to the existence of substrate limited regions within the tumor.     

Reduction of vascular and permeable regions in solid tumors detected by macromolecular contrast magnetic resonance imaging after treatment with antiangiogenic agent TNP-470.

PURPOSE: The availability of noninvasive techniques to detect the effects of antiangiogenic agents is critically important for optimizing treatment of cancer with these agents. Magnetic resonance imaging (MRI) is one such noninvasive technique that is routinely used clinically. EXPERIMENTAL DESIGN: In this study, we have evaluated the use of MRI of the intravascular contrast agent albumin-GdDTPA to detect the effects of the antiangiogenic agent TNP-470 on the vascular volume and permeability of the MatLyLu prostate cancer model. RESULTS: TNP-470-treated tumors demonstrated a significant decrease of vascular volume, as well as a significant reduction in vascular and permeable regions, compared with volume-matched control tumors. Although the fractional volume of permeable regions in the tumor decreased, the average value of tumor permeability did not decrease significantly. This was attributable to increase in permeability in some regions of the tumor. These regions were mostly associated with low vascular volume. ELISA assays of control and treated MatLyLu tumors also detected a significant increase of vascular endothelial growth factor in the TNP-470-treated tumors. CONCLUSION: MRI detected significant changes in tumor vascular characteristics after treatment with TNP-470.     

Targeting choline phospholipid metabolism: GDPD5 and GDPD6 silencing decrease breast cancer cell proliferation,migration, and invasion

Abnormal choline phospholipid metabolism is associated with oncogenesis and tumor progression. We have investigated the effects of targeting choline phospholipid metabolism by silencing two glycerophosphodiesterase genes, GDPD5 and GDPD6, using small interfering RNA (siRNA) in two breast cancer cell lines, MCF‐7 and MDA‐MB‐231. Treatment with GDPD5 and GDPD6 siRNA resulted in significant increases in glycerophosphocholine (GPC) levels, and no change in the levels of phosphocholine or free choline, which further supports their role as GPC‐specific regulators in breast cancer. The GPC levels were increased more than twofold during GDPD6 silencing, and marginally increased during GDPD5 silencing. DNA laddering was negative in both cell lines treated with GDPD5 and GDPD6 siRNA, indicating absence of apoptosis. Treatment with GDPD5 siRNA caused a decrease in cell viability in MCF‐7 cells, while GDPD6 siRNA treatment had no effect on cell viability in either cell line. Decreased cell migration and invasion were observed in MDA‐MB‐231 cells treated with GDPD5 or GDPD6 siRNA, where a more pronounced reduction in cell migration and invasion was observed under GDPD5 siRNA treatment as compared with GDPD6 siRNA treatment. In conclusion, GDPD6 silencing increased the GPC levels in breast cancer cells more profoundly than GDPD5 silencing, while the effects of GDPD5 silencing on cell viability/proliferation, migration, and invasion were more severe than those of GDPD6 silencing. Our results suggest that silencing GDPD5 and GDPD6 alone or in combination may have potential as a new molecular targeting strategy for breast cancer treatment. Copyright © 2016 John Wiley & Sons, Ltd.     

A new variant of type II von Willebrand disease with aberrant multimeric structure of plasma but not platelet von Willebrand factor (type IIF)

A patient with a lifelong bleeding disorder was diagnosed as having Type II von Willebrand disease. The larger multimers of von Willebrand factor were absent from her plasma but present in platelets. A high- resolution electrophoretic technique was used to study the complex structure of individual von Willebrand factor multimers. In normal plasma, each multimer could be resolved into five bands: a more intense central one and four less intense, two moving faster and two slower than the central band. In normal platelets, each multimer could also be resolved into five bands. The central one had a mobility similar to that in plasma, whereas the four satellite bands had a mobility that differed from that of the corresponding plasma bands. In the patient, platelet von Willebrand factor antigen content and ristocetin cofactor activity were normal, and von Willebrand factor showed the same structure of individual multimers as seen in normal platelets. On the other hand, plasma von Willebrand factor antigen and ristocetin cofactor activity were decreased, and the structure of individual von Willebrand factor multimers was different from that of normal plasma and similar to that seen in normal and patient's platelets. After infusion of 1-deamino-8-D-arginine vasopressin, the largest von Willebrand factor multimers, as well as new satellite bands with a mobility similar to those in normal plasma, appeared in the patient plasma, and the levels of von Willebrand factor antigen and ristocetin cofactor activity became normal. Yet no relevant change in the prolonged bleeding time was observed. This new variant of von Willebrand disease, therefore, is characterized by the presence of a dysfunctional von Willebrand factor molecule that exhibits unique structural abnormalities in plasma but appears to be normal in platelets. The designation of Type IIF is proposed for this type of von Willebrand disease in accordance with the terminology that has been previously used.     

Effects of blood flow modifiers on tumor metabolism observed in vivo by proton magnetic resonance spectroscopic imaging

Perfusion plays a key role in tumor proliferation and therapeutic response. Tumor heterogeneity necessitates use of the highest spatial resolution to monitor metabolic correlates of blood flow changes. This is best achieved with ¹H NMR spectroscopy, which permits noninvasive acquisition of high resolution spectroscopic images (SI) of subcutaneous tumors in a relatively short scan time (e.g., 12-25 μl voxels with signal-to-noise ratio 7:1 in 30 min at 4.7 T). This study seeks to identify ¹H spectroscopic indices of tumor blood flow. Proton SI of subcutaneous murine RIF-1 tumors were recorded (a) before and after administration of nicotinamide (1 g/kg) to increase blood flow, and (b) before and after hydralazine (10 mg/kg) to decrease flow. Nicotinamide produced a significant decrease in the total choline peak amplitudes, which subsequent high resolution NMR spectroscopy of tumor extracts revealed to be due to decreases in phosphocholine and glycerophosphocholine. The deamidation of nicotinamide to nicotinic acid, which is known to have hypolipidemic effects and to stimulate the formation of prostaglandins, may have sufficiently altered lipid metabolism to affect the in vivo concentration of the NMR-visible choline-containing compounds. The main effect of hydralazine was a significant increase of lactate, which is consistent with a reduction of tumor blood flow.     

Image-guided enzyme/prodrug cancer therapy.

PURPOSE: The success of enzyme/prodrug cancer therapy is limited by the uncertainty in the delivery of the enzyme in vivo. This study shows the use of noninvasive magnetic resonance (MR) and optical imaging to image the delivery of a prodrug enzyme. With this capability, prodrug administration can be timed so that the enzyme concentration is high in the tumor and low in systemic circulation and normal tissue, thereby minimizing systemic toxicity without compromising therapeutic efficiency. EXPERIMENTAL DESIGN: The delivery of a multimodal imaging reporter functionalized prodrug enzyme, cytosine deaminase, was detected by MR and optical imaging in MDA-MB-231 breast cancer xenografts. Stability of the enzyme in the tumor was verified by (19)F MR spectroscopy, which detected conversion of 5-fluorocytosine to 5-flurouracil. The optimal time window for prodrug injection determined by imaging was validated by immunohistochemical, biodistribution, and high-performance liquid chromatographic studies. The therapeutic effect and systemic toxicity of this treatment strategy were investigated by histologic studies and tumor/body weight growth curves. RESULTS: The delivery of the functionalized enzyme in tumors was successfully imaged in vivo. The optimal time window for prodrug administration was determined to be 24 h, at which time the enzyme continued to show high enzymatic stability in tumors but was biodegraded in the liver. Significant tumor growth delay with tolerable systemic toxicity was observed when the prodrug was injected 24 h after the enzyme. CONCLUSION: These preclinical studies show the feasibility of using a MR-detectable prodrug enzyme to time prodrug administration in enzyme/prodrug cancer therapy.