Peter Peschke

Simultaneous delivery of doxorubicin and gemcitabine to tumors in vivo using prototypic polymeric drug carriers

Copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) are prototypic and well-characterized polymeric drug carriers that have been broadly implemented in the delivery of anticancer therapeutics. To demonstrate that polymers, as liposomes, can be used for simultaneously delivering multiple chemotherapeutic agents to tumors in vivo, we have synthesized and evaluated an HPMA-based polymer-drug conjugate carrying 6.4wt% of gemcitabine, 5.7wt% of doxorubicin and 1.0mol% of tyrosinamide (to allow for radiolabeling). The resulting construct, i.e. poly(HPMA-co-MA-GFLG-gemcitabine-co-MA-GFLG-doxorubicin-co-MA-TyrNH(2)), was termed P-Gem-Dox, and was shown to effectively kill cancer cells in vitro, to circulate for prolonged period of time, to localize to tumors relatively selectively, and to inhibit tumor growth. As compared to control regimens, P-Gem-Dox increased the efficacy of the combination of gemcitabine and doxorubicin without increasing its toxicity, and it more strongly inhibited angiogenesis and induced apoptosis. These findings demonstrate that passively tumor-targeted polymeric drug carriers can be used for delivering two different chemotherapeutic agents to tumors simultaneously, and they thereby set the stage for more elaborate analyses on the potential of polymer-based multi-drug targeting.

Non-invasive determination of tumor oxygen tension and local variation with growth

PURPOSE The objective was to develop and demonstrate a novel noninvasive technique of measuring regional pO2 in tumors. The method is based on measuring 19F nuclear magnetic resonance spin-lattice relaxation rate (R1 = 1/T1) of perfluorocarbon (PFC) emulsion discretely sequestered in a tumor. METHODS AND MATERIALS We have examined pO2 in the Dunning prostate tumor R3327-AT1 implanted in a Copenhagen rat. Oxypherol blood substitute emulsion was administered intravenously and became sequestered in tissue. Proton magnetic resonance imaging (MRI) showed tumor anatomy and correlated 19F MRI indicated the distribution of perfluorocarbon. Fluorine-19 spectroscopic relaxometry was used to measure pO2 in the tumor and repeated measurements over a period of 3 weeks showed the variation in local pO2 during tumor growth. RESULTS Perfluorocarbon initially resided in the vascularized peripheral region of the tumor: 19F nuclear magnetic resonance R1 indicated pO2 approximately 75 torr in a small tumor (approximately 1 cm) in an anesthetized rat. As the tumor grew, the sequestered PFC retained its original distribution. When the tumor had doubled in size the residual PFC was predominantly in the core of the tumor and the pO2 of this region was approximately 1 torr indicating central tumor hypoxia. CONCLUSION We have demonstrated a novel noninvasive approach to monitoring regional tumor pO2. Given the critical role of oxygen tension in tumor response to therapy this may provide new insight into tumor physiology, the efficacy of various therapeutic approaches, and ultimately provide a clinical technique for assessing individual tumor oxygenation.

Image-guided and passively tumour-targeted polymeric nanomedicines for radiochemotherapy.

Drug targeting systems are nanometer-sized carrier materials designed for improving the biodistribution of systemically applied (chemo-) therapeutics. Reasoning that (I) the temporal and spatial interaction between systemically applied chemotherapy and clinically relevant fractionated radiotherapy is suboptimal, and that (II) drug targeting systems are able to improve the temporal and spatial parameters of this interaction, we have here set out to evaluate the potential of ‘carrier-based radiochemotherapy’. N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were used as a model drug targeting system, doxorubicin and gemcitabine as model drugs, and the syngeneic and radio- and chemoresistant Dunning AT1 rat prostate carcinoma as a model tumour model. Using magnetic resonance imaging and γ-scintigraphy, the polymeric drug carriers were first shown to circulate for prolonged periods of time, to localise to tumours both effectively and selectively, and to improve the tumour-directed delivery of low molecular weight agents. Subsequently, they were then shown to interact synergistically with radiotherapy, with radiotherapy increasing the tumour accumulation of the copolymers, and with the copolymers increasing the therapeutic index of radiochemotherapy (both for doxorubicin and for gemcitabine). Based on these findings, and on the fact that its principles are likely broadly applicable, we propose carrier-based radiochemotherapy as a novel concept for treating advanced solid malignancies.

A biological transporter for the delivery of peptide nucleic acids (PNAs) to the nuclear compartment of living cells.

To facilitate nuclear delivery of biomolecules we describe the synthesis of a modular transporter bearing a cellular membrane transport peptide (pAntp) and, as a cargo, a 16-mer peptide nucleic acid (PNA) covalently linked to a nuclear localisation signal (NLS[SV40-T]). Transport peptide and PNA are connected via N-terminal activated cysteine to form cleavable disulphide bonds. Internalization and subsequent delivery of PNA to the nucleus was verified in living and fixed cells by confocal laser scanning microscopy (CLSM) and fluorescence correlation spectroscopy (FCS). Double-labelling experiments indicate the cytoplasmic cleavage of the two modules and the effective nuclear import of the chromophore-tagged cargo. A non-degradable linker between transport module and cargo as well as a construct without NLS did not enable nuclear PNA import under the described experimental conditions. FCS-measurements revealed that most of the PNAs delivered into the cytoplasm by the modular transporter are anchored or encapsulated, indicating that intracellular transport of these compounds is not governed by molecular diffusion. Our results clearly demonstrate efficient compartment-directed transport using a synthetic, non-toxic modular transporter in living cells.

4-amino-1,8-naphthalimide : a novel inhibitor of poly(ADP-ribose) polymerase and radiation sensitizer

PURPOSE Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a chromatin-bound enzyme which is known to regulate chromatin structure by poly(ADP-ribosyl)ation of nuclear proteins, to facilitate DNA base excision repair, and to contribute to cellular recovery following DNA damage. Because inhibitors of PARP are able to potentiate the cell-killing effects of some DNA-damaging agents and to inhibit the repair of induced DNA strand breaks, such compounds may enhance the anti-tumour efficacy of radiotherapy or cytotoxic drug treatment. The PARP-inhibitory effects and radiosensitization of a new compound, 4-amino-1,8-naphthalimide (ANI), were examined. MATERIALS AND METHODS The inhibition of radiation-induced poly(ADP-ribosyl)ation (50 Gy; 60Co gamma-radiation) was evaluated by immunofluorescence assay using MoAb 10H directed against poly(ADP-ribose). Cell survival was assessed by colony forming assay (CFA) to determine the cytotoxicity of radiosensitization potential in exponentially growing hamster lung fibroblasts (V79), rat prostate carcinoma (R3327-AT1) and human prostate carcinoma (DU145) cells. RESULTS At concentrations above 30 nmol x dm(-3) ANI, radiation-induced poly(ADP-ribose) was not detectable by immunofluorescence in V79, AT1 and DU145 cells. At the highest concentration tested for chronic exposure (20 micromol x dm(-3)), ANI was not cytotoxic and significantly potentiates the cytotoxicity of gamma-irradiation. The level of radiation enhancement was directly proportional to drug concentration. Survival curves for the three cell lines using 20 micromol x dm(-3) ANI revealed sensitizer enhancement ratios of 1.3 for V79, 1.5 for AT1 and 1.3 for DU145. CONCLUSIONS In living cells, ANI is about 1000-fold more potent at inhibiting PARP activity compared with 3-aminobenzamide (3-ABA). CFA studies demonstrated that ANI is a radiation sensitizer at non-toxic and lower concentrations (20 micromol x dm(-3)) than 3-ABA (10 mmol x dm(-3)).

Correlations of noninvasive BOLD and TOLD MRI with pO2 and relevance to tumor radiation response

To examine the potential use of blood oxygenation level dependent (BOLD) and tissue oxygenation level dependent (TOLD) contrast MRI to assess tumor oxygenation and predict radiation response.

Regional tumor oximetry : 19F NMR spectroscopy of hexafluorobenzene

PURPOSE An accurate method for monitoring oxygen tension (pO2) of individual tumors could be valuable for optimizing treatment plans. We have recently shown that 19F nuclear magnetic resonance (NMR) spin-lattice relaxometry of hexafluorobenzene (HFB) provides a highly sensitive indicator of tumor oxygenation. We have now refined the methodology to provide enhanced precision, and applied the method to investigate dynamic changes in tumor oxygenation. METHODS AND MATERIALS Dunning prostate adenocarcinoma R3327-AT1 was grown in the form of pedicles on the foreback of male Copenhagen rats. When the tumors reached approximately equal to 1 cm diameter, HFB (20 microl) was administered, either centrally or peripherally, by direct intratumoral (i.T) injection. Local pO2 was determined using pulse-burst saturation recovery (PBSR) 19F NMR spectroscopy on the basis of the spin-lattice relaxation rate, R1. RESULTS Interrogation of the central region of tumors provided typical values in the range pO2 = 1.4-6.4 mmHg, with a typical stability of +/-2 mmHg over a period of 20 min, when rats breathed 33% O2. Altering the inhaled gas to oxygen or carbogen (95% O2/5% CO2) produced no significant change. In contrast, interrogation of tumor periphery indicated baseline pO2 in the range 7.9-78.9 mmHg. Altering inspired gas produced significant changes (p < 0.0001) with O2 or carbogen, although the change was generally greater with carbogen. In each case, pO2 returned to baseline within 16 min of returning the inhaled gas to baseline. CONCLUSION We believe this method provides a valuable new approach with the requisite precision and accuracy to investigate tumor pO2.

High-resolution three-dimensional MR angiography of rodent tumors: morphologic characterization of intratumoral vasculature.

To evaluate high‐resolution three‐dimensional MR angiography (MRA) for the visualization and morphologic characterization of intratumoral vasculature.

Enhanced iodide transport after transfer of the human sodium iodide symporter gene is associated with lack of retention and low absorbed dose

Transfer of the sodium iodide symporter (hNIS) has been proposed as a new principle of cancer gene therapy. Using clinically relevant doses of 131I for the treatment of NIS-expressing prostate carcinoma cells, we investigated the kinetics and the absorbed doses obtained in these tumors. hNIS-expressing cell lines accumulated up to 200 times more iodide when compared to wild-type cells. However, a rapid efflux of the radioactivity (80%) occurred during the first 20 min after replacement of the medium. In rats, the hNIS-expressing tumors accumulated up to 20 times more iodide when compared to contralateral transplanted wild-type tumors. After 24 h and doses of 550, 1200 or 2400 MBq/m2 hNIS-expressing tumors lost 89, 89 and 91% of the initial activity, respectively. Dosimetric calculations showed that 1200 MBq/m2 resulted in 3±0.5 Gy (wild-type tumor 0.15±0.1 Gy) and 2400 MBq/m2 resulted in 3.1±0.9 Gy (wild-type tumor 0.26±0.02 Gy). Although transduction of the hNIS gene induces iodide transport in rat prostate adenocarcinoma a rapid efflux occurs, which leads to a low absorbed dose in genetically modified tumors. With regard to a therapeutic application additional conditions need to be defined leading to iodide trapping.

Regional Tumor Oxygenation and Measurement of Dynamic Changes

We recently described a novel approach to measuring regional tumor oxygen tension using (19)F pulse burst saturation recovery (PBSR) nuclear magnetic resonance (NMR) echo planar imaging (EPI) relaxometry of hexafluorobenzene. We now compare oxygen tension measurements in a group of size-matched R3327-AT1 Dunning prostate rat tumors made using this new method with those using a traditional polarographic method: the Eppendorf histograph. Similar oxygen tension distributions were found using the two methods, and both techniques showed that tumors with volume greater than 3.5 cm(3) were significantly (P < 0.0001) less well oxygenated than smaller tumors (volume less than 2 cm(3)). Using the (19)F EPI approach, we also examined the response to respiratory challenge. Increasing the concentration of inspired oxygen from 33% to 100% O(2) produced a significant increase (P < 0.0001) in tumor oxygenation for a group of small tumors. In contrast, no change was observed in the mean pO(2) for a group of large tumors. Consideration of individual tumor regions irrespective of tumor size showed a strong correlation between the maximum pO(2) observed when breathing 100% O(2) compared with mean baseline pO(2). These results further demonstrate the usefulness of (19)F EPI to assess changes in regional tumor oxygenation.