Developing a clinically relevant radiosensitizer for temozolomide-resistant gliomas.

The prognosis for patients with glioblastoma (GB) remains grim. Concurrent temozolomide (TMZ) radiation-the cornerstone of glioma control-extends the overall median survival of GB patients by only a few months over radiotherapy alone. While these survival gains could be partly attributed to radiosensitization, this benefit is greatly minimized in tumors expressing O6-methylguanine DNA methyltransferase (MGMT), which specifically reverses O6-methylguanine lesions. Theoretically, non-O6-methylguanine lesions (i.e., the N-methylpurine adducts), which represent up to 90% of TMZ-generated DNA adducts, could also contribute to radiosensitization. Unfortunately, at concentrations attainable in clinical practice, the alkylation capacity of TMZ cannot overwhelm the repair of N-methylpurine adducts to efficiently exploit these lesions. The current therapeutic application of TMZ therefore faces two main obstacles: (i) the stochastic presence of MGMT and (ii) a blunted radiosensitization potential at physiologic concentrations. To circumvent these limitations, we are developing a novel molecule called NEO212-a derivatization of TMZ generated by coupling TMZ to perillyl alcohol. Based on gas chromatography/mass spectrometry and high-performance liquid chromatography analyses, we determined that NEO212 had greater tumor cell uptake than TMZ. In mouse models, NEO212 was more efficient than TMZ at crossing the blood-brain barrier, preferentially accumulating in tumoral over normal brain tissue. Moreover, in vitro analyses with GB cell lines, including TMZ-resistant isogenic variants, revealed more potent cytotoxic and radiosensitizing activities for NEO212 at physiologic concentrations. Mechanistically, these advantages of NEO212 over TMZ could be attributed to its enhanced tumor uptake presumably leading to more extensive DNA alkylation at equivalent dosages which, ultimately, allows for N-methylpurine lesions to be better exploited for radiosensitization. This effect cannot be achieved with TMZ at clinically relevant concentrations and is independent of MGMT. Our findings establish NEO212 as a superior radiosensitizer and a potentially better alternative to TMZ for newly diagnosed GB patients, irrespective of their MGMT status.

Fluorescent Radiosensitizing Gold Nanoparticles.

Ultrasmall polyaminocarboxylate-coated gold nanoparticles (NPs), Au@DTDTPA and Au@TADOTAGA, that have been recently developed exhibit a promising potential for image-guided radiotherapy. In order to render the radiosensitizing effect of these gold nanoparticles even more efficient, the study of their localization in cells is required to better understand the relation between the radiosensitizing properties of the agents and their localization in cells and in tumors. To achieve this goal, post-functionalization of Au@DTDTPA nanoparticles by near-infrared (NIF) organic dyes (aminated derivative of cyanine 5, Cy5-NH2) was performed. The immobilization of organic Cy5-NH2 dyes onto the gold nanoparticles confers to these radiosensitizers fluorescence properties which can be exploited for monitoring their internalization in cancerous cells, for determining their localization in cells by fluorescence microscopy (a common and powerful imaging tool in biology), and for following up on their accumulation in tumors after intravenous injection.

Efficient Protocol for the Identification of Hypoxic Cell Radiosensitisers.

An evolution in radiotherapy practice is leading to greater use of stereotactic body radiotherapy (SBRT), raising the prospect of increased hypoxic cell radioresistance. New clinical interest in nitroimidazole radiosensitisers, combined with appropriate biomarkers, signals a revival for radiosensitisers in the context of SBRT. Our interest in modifiers of radiation therapy led us to revisit this area and we have identified a new class of nitroimidazole radiosensitiser. We have developed an abbreviated screening protocol suitable for an academic drug discovery laboratory which allows expeditious triage of compounds with poor physicochemical and in vitro properties and combines in vitro radiosensitisation data with tumour pharmacokinetic data to efficiently select candidates for further evaluation.

Development of a novel noncapillary plasma microsampling device for ultra-low volume of blood collection.

The desire for serial microsampling in mice has led to extensive research in this field within the pharmaceutical industry. The ability to profile a compound's in vivo properties with less material and fewer mice has obvious advantages. A new device and workflow was developed at the Takeda Oncology site to allow scientists to isolate plasma from very low volumes of mouse blood (as low as 20 µl) collected using standard microsampling techniques. A side-by-side in vitro comparison of plasma concentrations was performed using this new device and conventional sampling methods with commercial and in-house molecules. The plasma concentrations of the molecules tested were very consistent between the conventional sampling techniques and this new device/workflow. In addition, several in-life studies have also been conducted to validate this new technique as a primary PK screening tool at the Takeda Boston. The new device is simple to use and very cost effective with the added benefit that no additional training is needed for the animal technicians and the same centrifuge equipment can be employed. This device can be used for blood volumes ranging from 20 to 100 µl enabling studies not just in rat and dog but more importantly in mice.

Artificial plasmid labeled with 5-bromo-2'-deoxyuridine: a universal molecular system for strand break detection.

DNA strand breaks (SBs) are among the most cytotoxic forms of DNA damage, and their residual levels correlate directly with cell death. Hence, the type and amount of SBs is directly related to the efficacy of a given anticancer therapy. In this study, we describe a molecular tool that can differentiate between single (SSBs) and double (DSBs) strand breaks and also assess them quantitatively. Our method involves PCR amplification of a linear DNA fragment labeled with a sensitizing nucleotide, circularization of that fragment, and enzymatic introduction of supercoils to transform the circular relaxed form of the synthesized plasmid into a supercoiled one. After exposure of the molecule to a damaging factor, SSB and DSB levels can be easily assayed with gel electrophoresis. We applied this method to prepare an artificial plasmid labeled with 5-bromo-2'-deoxyuridine and to assay SBs photoinduced in the synthesized plasmid.

A surgical loupe system for observing protoporphyrin IX fluorescence in high-grade gliomas after administering 5-aminolevulinic acid.

BACKGROUND: We recently developed a surgical loupe system for observing the fluorescence emitted by protoporphyrin IX (PpIX), a metabolite of 5-aminolevulinic acid. METHODS: This system used a semiconductor laser as the excitation light source. A compact, transparent, and ultraviolet cut-off filter was mounted on an eyepiece lens, which did not require filter on-off manipulation. RESULTS: Good quality protoporphyrin IX fluorescence was acquired using the surgical loupe system during glioblastoma resection, which was nearly identical to that acquired by fluorescent microscopy. In addition, surgeons can perform ordinary surgical procedures using this surgical loupe system under white light. CONCLUSION: This surgical loupe system enables the detection of PpIX fluorescence during resection of high-grade glioma. Further evaluations of this system are required to determine the extent of surgical resection before its practical application.

Synthesis of analogs of the radiation mitigator JP4-039 and visualization of BODIPY derivatives in mitochondria.

JP4-039 is a lead structure in a series of nitroxide conjugates that are capable of accumulating in mitochondria and scavenging reactive oxygen species (ROS). To explore structure-activity relationships (SAR), new analogs with variable nitroxide moieties were prepared. Furthermore, fluorophore-tagged analogs were synthesized and provided the opportunity for visualization in mitochondria. All analogs were tested for radioprotective and radiomitigative effects in 32Dcl3 cells.

γ-H2AX kinetics as a novel approach to high content screening for small molecule radiosensitizers.

BACKGROUND: Persistence of γ-H2AX after ionizing radiation (IR) or drug therapy is a robust reporter of unrepaired DNA double strand breaks in treated cells. METHODS: DU-145 prostate cancer cells were treated with a chemical library ±IR and assayed for persistence of γ-H2AX using an automated 96-well immunocytochemistry assay at 4 hours after treatment. Hits that resulted in persistence of γ-H2AX foci were tested for effects on cell survival. The molecular targets of hits were validated by molecular, genetic and biochemical assays and in vivo activity was tested in a validated Drosophila cancer model. RESULTS: We identified 2 compounds, MS0019266 and MS0017509, which markedly increased persistence of γ-H2AX, apoptosis and radiosensitization in DU-145 cells. Chemical evaluation demonstrated that both compounds exhibited structurally similar and biochemical assays confirmed that these compounds inhibit ribonucleotide reductase. DNA microarray analysis and immunoblotting demonstrates that MS0019266 significantly decreased polo-like kinase 1 gene and protein expression. MS0019266 demonstrated in vivo antitumor activity without significant whole organism toxicity. CONCLUSIONS: MS0019266 and MS0017509 are promising compounds that may be candidates for further development as radiosensitizing compounds as inhibitors of ribonucleotide reductase.

Dynamic contrast-enhanced-MRI of tumor hypoxia.

Patients with highly hypoxic primary tumors show increased frequency of locoregional treatment failure and poor survival rates and may benefit from particularly aggressive treatment. The potential of gadolinium diethylene-triamine penta-acetic acid-based dynamic contrast-enhanced-MRI in assessing tumor hypoxia was investigated in this preclinical study. Xenografted tumors of eight human melanoma lines were subjected to dynamic contrast-enhanced-MRI and measurement of the fraction of radiobiologically hypoxic cells and the fraction of pimonidazole-positive hypoxic cells. Tumor images of K(trans) (the volume transfer constant of gadolinium diethylene-triamine penta-acetic acid) and v(e) (the fractional distribution volume of gadolinium diethylene-triamine penta-acetic acid) were produced by pharmacokinetic analysis of the dynamic contrast-enhanced-MRI data, and K(trans) and v(e) frequency distributions of the non-necrotic tumor tissue were established and related to the extent of hypoxia. Tumors showing high K(trans) values and high v(e) values had low fractions of hypoxic cells, whereas tumors showing both low K(trans) values and low v(e) values had high hypoxic fractions. K(trans) differentiated better between tumors with low and high hypoxic fractions than did v(e). This study supports the current attempts to establish dynamic contrast-enhanced-MRI as a method for assessing the extent of hypoxia in human tumors, and it provides guidelines for the clinical development of valid assays.

Synthesis, in vitro anticancer screening and radiosensitizing evaluation of some new 4-[3-(substituted)thioureido]-N-(quinoxalin-2-yl)-benzenesulfonamide derivatives.

Sulfonamides and quinoxaline derivatives possess many types of biological activities and have been recently reported to show substantial antitumor activity. This paper reports the synthesis of novel thioureido sulfaquinoxaline derivatives. All the newly synthesized compounds were evaluated for their in vitro anticancer activity against a human liver cell line (HEPG2) and showed higher activity than the reference drug doxorubicin. 4-(3-(4-Ethylbenzoate) thioureido)-N-(quinoxalin-2-yl)benzenesulfonamide (9) (IC50 = 15.6 µmol L⁻¹), N-(pyridin-2-yl)-4-(3-(4-(N-quinoxalin-2-yl-sulfamoyl)phenyl)thioureido)benzenesulfonamide (10) (IC50 = 26.8 µmol L⁻¹) and N-(quinoxalin-2-yl)-4-(3-(4-(N-thiazol-2-ylsulfamoyl)phenyl)thioureido)benzenesulfonamide (11) (IC50 = 24.4 µmol L⁻¹) were the most potent compared to doxorubicin (IC50 = 71.8 µmol L⁻¹). The most potent compounds 9, 10 and 11 were evaluated as radiosensitizing agents by subjecting the compounds to γ-irradiation (8 kGy).

Supramolecular zinc phthalocyanine-imidazolyl perylenediimide dyad and triad: synthesis, complexation, and photophysical studies.

Two new supramolecular architectures based on zinc phthalocyanine (Pc) and imidazolyl-substituted perylenediimide (PDI), ZnPc/DImPDI/ZnPc 1 and ZnPc/ImPDI 2, have been prepared. A strong electron-donor, ZnPc-8, which contained eight tert-octylphenoxy groups was synthesized to ensure high solubility, thereby reducing aggregation in solution and providing σ-donor features while avoiding regioisomeric mixtures. Also, PDI units were functionalized with tert-octylphenoxy groups at the bay positions, which provide solubility to avoid aggregation in solution, together with one and two imidazole moieties in the amide position, PDI-6 and PDI-4, respectively, to be able to strongly coordinate with the ZnPc complex. Supramolecular complexation studies by (1)H NMR spectroscopy and ESI-MS demonstrate a high coordinative binding constant between imidazole-substituted PDI-4 or PDI-6 and ZnPc-8. The same results were confirmed by UV/Vis and fluorescence titration studies. UV/Vis titration studies revealed the formation of a 1:1 complex ZnPc/ImPDI 2 for the systems ZnPc-8 and PDI-6 and a 2:1 complex ZnPc/DImPDI/ZnPc 1 for the interaction of ZnPc-8 and PDI-4. The binding constant in both cases was determined to be on the order of 10(5) M(-1). Femtosecond laser flash photolysis measurements provided a direct proof of the charge-separated state within both supramolecular assemblies by observing the transient absorption band at 820 nm due to the zinc phthalocyanine radical cation. The lifetimes of charge-separated states are (9.8±3) ns for triad 1 and (3±1) ns for dyad 2. As far as we know, this is the first time that a radical ion pair has been detected in a supramolecular assembled ZnPc-PDI system and has obtained the longest lifetime of a charge-separated state published for ZnPc-PDI assemblies.

Pharmacokinetic study of a novel sonosensitizer chlorin-e6 and its sonodynamic anti-cancer activity in hepatoma-22 tumor-bearing mice.

PURPOSE: The sonodynamically induced anti-tumor effect of chlorin-e6 (Ce6) was studied in mice bearing hepatoma-22 solid tumors. METHODS: In order to determine the optimum timing of ultrasound exposure after administration of Ce6, the Ce6 concentrations in plasma, skin, muscle and tumor were estimated by measuring the fluorescence intensity of tissue extractions with a fluorescence photometer based on the standard curve. A three-dimensional optical imaging system (IVIS spectrum) was used further to characterize the distribution of Ce6 in H-22 tumor. The anti-tumor effects were estimated by measuring tumor size after sonodynamic therapy. RESULTS: Similar pharmacokinetic trends of Ce6 in mice were observed either by fluorescence spectrophotometry or by bio-optical imaging. The results also demonstrated that Ce6 has a preferential localization in tumors, but low accumulation and rapid clearance in normal tissues. The results of anti-tumor effects revealed that at an ultrasound intensity of 4 W/cm(2) and a Ce6 dose of ≥10 mg/kg, a significant synergistic effect of ultrasound combined with Ce6 was observed, reducing the tumor volume significantly. CONCLUSION: Chlorin-e6 is a potential sonosensitizer for fluorescence imaging as well as for sonodynamic therapy for cancer. The anti-tumor effect of ultrasound could be enhanced in the presence of Ce6, which might be involved in a sonochemical mechanism.

Radiosensitization of Salmonella spp. and Listeria spp. in ready-to-eat baby spinach leaves.

The FDA recently approved irradiation treatment of leafy greens such as spinach up to 1 kGy; however, it is important to reduce the dose required to decontaminate the produce while maintaining its quality. Thus, the objectives of this study were: (1) to assess the radiation sensitivities of Salmonella spp. and Listeria spp. inoculated in ready-to-eat baby spinach leaves under modified atmosphere packaging (MAP) and irradiated using a 1.35-MeV Van de Graff accelerator (the leaves were irradiated both at room temperature and at -5 °C); and (2) to understand and optimize the synergistic effect of MAP and irradiation by studying the radiolysis of ozone formation under different temperatures, the effect of dose rate on its formation, and its decomposition. Results showed that increased concentrations of oxygen in the packaging significantly increased the radiation sensitivity of the test organisms, ranging from 7% up to 25% reduction in D(10)-values. In particular, radiosensitization could be effected (P < 0.05) by production of ozone, which increases with increasing dose-rate and oxygen concentration, and reducing temperatures. Radiosensitization was demonstrated for both microorganisms with irradiation of either fresh or frozen (-5 °C) baby spinach. These results suggest that low-dose (below 1 kGy) e-beam radiation under modified atmosphere packaging (100% O(2) and N(2):O(2)[1:1]) may be a viable tool for reducing microbial populations or eliminating Salmonella spp. and Listeria spp. from baby spinach. A suggested treatment to achieve a 5-log reduction of the test organisms would be irradiation at room temperature under 100% O(2) atmosphere at a dose level of 0.7 kGy. Practical Application: Decontamination of minimally processed fruits and vegetables from food-borne pathogens presents technical and economical challenges to the produce industry. Internalized microorganisms cannot be eliminated by the current procedure (water-washed or treated with 200-ppm chlorine). The only technology available commercially is ionizing radiation; however, the actual radiation dose required to inactivate pathogens is too high to be tolerated by the product without unwanted changes. This study shows a new approach in using MAP with 100% O(2), which is converted to ozone to radiosensitize pathogens while improving the shelf life of minimally processed fruits and vegetables. The process results in a high level of microorganism inactivation using lower doses than the conventional irradiation treatments.

Validated spectrophotometric and spectrofluorimetric methods for determination of chloroaluminum phthalocyanine in nanocarriers.

UV-VIS-Spectrophotometric and spectrofluorimetric methods have been developed and validated allowing the quantification of chloroaluminum phthalocyanine (CIAIPc) in nanocarriers. In order to validate the methods, the linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and selectivity were examined according to USP 30 and ICH guidelines. Linearities range were found between 0.50-3.00 microg x mL(-1) (Y = 0.3829 X [CIAIPc, microg x mL(-1)] + 0.0126; r = 0.9992) for spectrophotometry, and 0.05-1.00 microg x mL(-1) (Y = 2.24 x 10(6) X [CIAIPc, microg x mL(-1)] + 9.74 x 10(4); r = 0.9978) for spectrofluorimetry. In addition, ANOVA and Lack-of-fit tests demonstrated that the regression equations were statistically significant (p<0.05), and the resulting linear model is fully adequate for both analytical methods. The LOD values were 0.09 and 0.01 microg x mL(-1), while the LOQ were 0.27 and 0.04 microg x mL(-1) for spectrophotometric and spectrofluorimetric methods, respectively. Repeatability and intermediate precision for proposed methods showed relative standard deviation (RSD) between 0.58% to 4.80%. The percent recovery ranged from 98.9% to 102.7% for spectrophotometric analyses and from 94.2% to 101.2% for spectrofluorimetry. No interferences from common excipients were detected and both methods were considered specific. Therefore, the methods are accurate, precise, specific, and reproducible and hence can be applied for quantification of CIAIPc in nanoemulsions (NE) and nanocapsules (NC).

T2 corrected quantification method of L-p-boronophenylalanine using proton magnetic resonance spectroscopy for boron neutron capture therapy.

In the present study, we aimed to evaluate a T2 corrected quantification method of l-p-boronophenylalanine (BPA) concentration using proton magnetic resonance spectroscopy (MRS). We used five phantoms containing BPA (1.5, 3.0, 5.0, 7.5, and 10 mmol/kg=15, 30, 50, 75, and 100 microg(10)B/g), N-acetyl-aspartic acid (NAA: 3.0 mmol/kg), creatine (Cr: 5.0 mmol/kg), and choline (Cho: 3.0 mmol/kg). The signal intensities of BPA and internal water were corrected by T2 relaxation time. The absolute concentrations of BPA were calculated by proton MRS using an internal water signal as a standard. The major BPA peaks were detected between 7.1 and 7.6 ppm. Mean T2 relaxation time was 314.3+/-10.8 ms in BPA, 885.1+/-39.7 ms in internal water. The calculated BPA concentrations were almost same as the actual concentration of BPA and the correlation coefficient was 0.99. Our BPA quantification method was very simple and non-invasive, also it had high accuracy. Therefore, our results indicate that proton MRS can be potentially useful technique for in vivo BPA quantification in boron neutron capture therapy (BNCT).

Assessment of chemical purity of 10B-enriched p-boronophenylalanine by high-performance liquid chromatography coupled on-line with inductively coupled plasma optical emission spectrometry.

A dual-detection technique, consisting of a combination of reversed-phase high-performance liquid chromatography and on-line detection of elemental boron in the column effluents by inductively coupled plasma optical emission spectrometry, was tested for drug analysis. The method was applied to assessing the chemical purity of p-boronophenylalanine (BPA), isotopically enriched in 10B. This compound is employed as a fructose complex solution for biodistribution studies in laboratory and clinical trials of boron neutron capture therapy. Besides the determination of the content of BPA, required for chemical quality controls of solutions of the complex used for infusions, resolution of mixtures of BPA and two usually accompanying residual impurities (phenylalanine and tyrosine) was achieved with UV detection. The limits of detection (in solution) were 1.5 and 0.6 ng ml-1, respectively. In addition, by monitoring a sensitive-element emission wavelength it was possible to jointly observe the elution of boron-containing compounds that may be transparent to UV radiation or to confirm the presence of boron in potential impurities accompanying the drug. Those impurities may arise from the BPA synthesis or may be produced by degradation during the aging of the solutions. Chromatographic peaks corresponding to the amino acids and also to a related inorganic compound were detected in BPA-fructose complex solutions that were stored for different times and under different conditions. An increase in the areas of the peaks attributed to tyrosine and phenylalanine was observed for BPA-fructose solutions stored refrigerated for 1 month to 1 year, suggesting that degradation processes able to reduce the amount of bioavailable BPA could be active.

Can dietary furanocoumarin ingestion enhance the erythemal response during high-dose UVA1 therapy?

As phototoxic skin reactions caused by psoralen are induced by wavelengths within the UVA1 spectrum, we assessed the potential of the small amount of psoralen in a normal diet to provoke phototoxicity in volunteers with skin types I and II. Threshold erythema was unaffected by ingestion of a 200-g portion of parsnip.

In vivo absorption spectroscopy of tumor sensitizers with femtosecond white light.

A system based on a femtosecond white-light continuum and a streak camera was used for recordings of the in vivo absorption spectra of the tumor-seeking agent disulphonated aluminum phthalocyanine. Measurements for different drug doses were performed on tumor tissue (muscle-implanted adenocarcinoma) and normal muscle tissue in rats. It was found that the shape of the spectrum is tissue dependent. The peak of the absorption spectrum is blueshifted in tumor tissue as compared with the muscle. Thus the contrast in the drug-related absorption can be altered by up to a factor of 2 from the primary drug molecular-concentration contrast between normal muscle and tumor by the proper selection of the illumination wavelength.