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Electron paramagnetic resonance (EPR) spectroscopy has long been established across various scientific disciplines for characterizing organic radicals, organometallic complexes, protein structures and dynamics, polymerization processes, and radical degradation phenomena. Despite its extensive utility in these areas, EPR spectroscopy’s application within pharmaceutical science has historically been constrained, primarily due to factors such as high equipment costs, a steep learning curve, complex spectral deconvolution and analysis, and a traditional lack of emphasis on single-electron chemistry in pharmaceutical research. This review aims to provide a thorough examination of EPR spectroscopy’s applications in analyzing a wide array of para-magnetic species relevant to pharmaceutical research. We detail how EPR spectroscopy can be employed to assess free radical scavenging properties in pharmaceutical compounds, elucidate drug mechanisms of action, and explore pharmacokinetics. Additionally, we investigate the role of free radicals in drug-induced toxicity and drug-membrane interactions, while also covering the application of EPR spectroscopy in drug delivery research, advanced studies of metallodrugs, and monitoring of oxygen levels in biological systems through EPR oximetry. The recent advancements in the miniaturization of EPR spectro meters have paved the way for their application in and mo nitoring during the manufacturing process and quality control of pharmaceutical substances and final drug formulations due to being the only direct and non-invasive detection technique for radical detection. Through these discussions, we highlight the substantial contributions of EPR spectroscopy to the advancement of pharmaceutical sciences.

Background: Glioblastoma multiforme (GBM) is the most common highly aggressive, primary malignant brain tumor in adults. Current experimental strategies include photodynamic therapy (PDT) and new drug delivery technologies such as nanoparticles, which could play a key role in the treatment, diagnosis, and imaging of brain tumors. Objectives: The purpose of this study was to test the efficacy of PDT using AGuIX-TPP, a polysiloxane-based nanoparticle (AGuIX) that contains TPP (5,10,15,20-tetraphenyl-21H,23H-porphine), in biological models of glioblastoma multiforme and to investigate the vascular mechanisms of action at multiple complexity levels. Methods: PDT effects were studied in monolayer and spheroid cell culture, as well as tumors in chicken chorioallantoic membranes (CAMs) and in mice were studied. Results: Treatment was effective in both endothelial ECRF and glioma U87 cells, as well as in the inhibition of growth of the glioma spheroids. PDT using AGuIX-TPP inhibited U87 tumors growing in CAM and destroyed their vascularization. The U87 tumors were also grown in nude mice. Their vascular network, as well as oxygen partial pressure, were assessed using ultrasound and EPR oximetry. The treatment damaged tumor vessels and slightly decreased oxygen levels. Conclusions: PDT with AGuIX-TPP was effective against glioma cells, spheroids, and tumors; however, in mice, its efficacy appeared to be strongly related to the presence of blood vessels in the tumor before the treatment.

Tumour hypoxia is a well‐established factor of resistance in radiation therapy (RT). Myo‐inositol trispyrophosphate (ITPP) is an allosteric effector that reduces the oxygen‐binding affinity of haemoglobin and facilitates the release of oxygen by red blood cells. We investigated herein the oxygenation effect of ITPP in six tumour models and its radiosensitizing effect in two of these models. The evolution of tumour pO2 upon ITPP administration was monitored on six models using 1.2 GHz Electron Paramagnetic Resonance (EPR) oximetry. The effect of ITPP on tumour perfusion was assessed by Hoechst staining and the oxygen consumption rate (OCR) in vitro was measured using 9.5 GHz EPR. The therapeutic effect of ITPP with and without RT was evaluated on rhabdomyosarcoma and 9L‐glioma rat models. ITPP enhanced tumour oxygenation in six models. The administration of 2 g/kg ITPP once daily for 2 days led to a tumour reoxygenation for at least 4 days. ITPP reduced the OCR in six cell lines but had no effect on tumour perfusion when tested on 9L‐gliomas. ITPP plus RT did not improve the outcome in rhabdomyosarcomas. In 9L‐gliomas, some of tumours receiving the combined treatment were cured while other tumours did not benefit from the treatment. ITPP increased oxygenation in six tumour models. A decrease in OCR could contribute to the decrease in tumour hypoxia. The association of RT with ITPP was beneficial for a few 9L‐gliomas but was absent in the rhabdomyosarcomas.

Small vessel disease is associated with white-matter (WM) magnetic resonance imaging (MRI) hyperintensities (WMHs) in patients with vascular cognitive impairment (VCI) and subsequent damage to the WM. Although WM is vulnerable to hypoxic-ischemic injury and O2 is critical in brain physiology, tissue O2 level in the WM has not been measured and explored in vivo. We hypothesized that spontaneously hypertensive stroke-prone rat (SHR/SP) fed a Japanese permissive diet (JPD) and subjected to unilateral carotid artery occlusion (UCAO), a model to study VCI, would lead to reduced tissue oxygen (pO2) in the deep WM. We tested this hypothesis by monitoring WM tissue pO2 using in vivo electron paramagnetic resonance (EPR) oximetry in SHR/SP rats over weeks before and after JPD/UCAO. The SHR/SP rats experienced an increase in WM pO2 from 9 to 12 weeks with a maximal 32% increase at week 12, followed by a dramatic decrease in WM pO2 to near hypoxic conditions during weeks 13 to 16 after JPD/UCAO. The decreased WM pO2 was accompanied with WM damage and hemorrhages surrounding microvessels. Our findings suggest that changes in WM pO2 may contribute to WM damage in SHR/SP rat model, and that EPR oximetry can monitor brain pO2 in the WM of small animals.

Compared to two-dimensional (2D) cell culture, cellular aggregates or spheroids (3D) offer a more appropriate alternative system where individual cell-cell communication and micro-environment more closely represent the organ; yet we understand little of the physiological conditions at this scale. The relationship between spheroid size and oxygen microenvironment, an important factor influencing the metabolic capacity of cells, was first established using the fish intestine derived RTgutGC cell line. Subsequently, pharmaceutical metabolism (Propranolol), as determined by high performance liquid chromatography, in this intestinal model was examined as a function of spheroid size. Co-efficient of variation between spheroid size was below 12% using the gyratory platform method, with the least variation observed in the highest cell seeding density. The viable, high oxygen micro-environment of the outer rim of the spheroid, as determined by electron paramagnetic resonance (EPR) oximetry, decreased over time, and the hypoxic zone increased as a function of spheroid size. Despite a trend of higher metabolism in smaller spheroids, the formation of micro-environments (quiescent, hypoxic or anoxic) did not significantly affect metabolism or function of an environmentally relevant pharmaceutical in this spheroid model.

Oxygenation is one of the most important physiological parameters of biological systems. Low oxygen concentration (hypoxia) is associated with various pathophysiological processes in different organs. Hypoxia is of special importance in tumor therapy, causing poor response to treatment. Triaryl methyl (TAM) derivative radicals are commonly used in electron paramagnetic resonance (EPR) as sensors for quantitative spatial tissue oxygen mapping. They are also known as magnetic resonance imaging (MRI) contrast agents and fluorescence imaging compounds. We report the properties of the TAM radical tris(2,3,5,6-tetrachloro-4-carboxy-phenyl)methyl, (PTMTC), a potential multimodal (EPR/fluorescence) marker. PTMTC was spectrally analyzed using EPR and characterized by estimation of its sensitivity to the oxygen in liquid environment suitable for intravenous injection (1 mM PBS, pH = 7.4). Further, fluorescent emission of the radical was measured using the same solvent and its quantum yield was estimated. An in vitro cytotoxicity examination was conducted in two cancer cell lines, HT-29 (colorectal adenocarcinoma) and FaDu (squamous cell carcinoma) and followed by uptake studies. The stability of the radical in different solutions (PBS pH = 7.4, cell media used for HT-29 and FaDu cells culturing and cytotoxicity procedure, full rat blood and blood plasma) was determined. Finally, a primary toxicity test of PTMTC was carried out in mice. Results of spectral studies confirmed the multimodal properties of PTMTC. PTMTC was demonstrated to be not absorbed by cancer cells and did not interfere with luciferin-luciferase based assays. Also in vitro and in vivo tests showed that it was non-toxic and can be freely administrated till doses of 250 mg/kg BW via both i.v. and i.p. injections. This work illustrated that PTMTC is a perfect candidate for multimodal (EPR/fluorescence) contrast agent in preclinical studies.

Cerebral tissue oxygenation (oxygen tension, pO 2 ) is a critical parameter that is closely linked to brain metabolism, function, and pathophysiology. In this work, we have used electron paramagnetic resonance oximetry with a deep-tissue multi-site oxygen-sensing probe, called implantable resonator, to monitor temporal changes in cerebral pO 2 simultaneously at four sites in a rabbit model of ischemic stroke induced by embolic clot. The pO 2 values in healthy brain were not significantly different among the four sites measured over a period of 4 weeks. During exposure to 15% O 2 (hypoxia), a sudden and significant decrease in pO 2 was observed in all four sites. On the other hand, brief exposure to breathing carbogen gas (95% O 2  + 5% CO 2 ) showed a significant increase in the cerebral pO 2 from baseline value. During ischemic stroke, induced by embolic clot in the left brain, a significant decline in the pO 2 of the left cortex (ischemic core) was observed without any change in the contralateral sites. While the pO 2 in the non-infarct regions returned to baseline at 24-h post-stroke, pO 2 in the infarct core was consistently lower compared to the baseline and other regions of the brain. The results demonstrated that electron paramagnetic resonance oximetry with the implantable resonator can repeatedly and simultaneously report temporal changes in cerebral pO 2 at multiple sites. This oximetry approach can be used to develop interventions to rescue hypoxic/ischemic tissue by modulating cerebral pO 2 during hypoxic and stroke injury.

We noninvasively monitored the partial pressure of oxygen (pO 2 ) in rat’s small intestine using a model of chronic mesenteric ischemia by electron paramagnetic resonance oximetry over a 7-day period. The particulate probe lithium octa- n -butoxynaphthalocyanine (LiNc-BuO) was embedded into the oxygen permeable material polydimethyl siloxane by cast-molding and polymerization (Oxy-Chip). A one-time surgical procedure was performed to place the Oxy-Chip on the outer wall of the small intestine (SI). The superior mesenteric artery (SMA) was banded to ~30 % of blood flow for experimental rats. Noninvasive measurement of pO 2 was performed at the baseline for control rats or immediate post-banding and on days 1, 3, and 7. The SI pO 2 for control rats remained stable over the 7-day period. The pO 2 on day-7 was 54.5 ± 0.9 mmHg (mean ± SE). SMA-banded rats were significantly different from controls with a noted reduction in pO 2 post banding with a progressive decline to a final pO 2 of 20.9 ± 4.5 mmHg (mean ± SE; p  = 0.02). All SMA-banded rats developed adhesions around the Oxy-Chip, yet remained asymptomatic. The hypoxia marker Hypoxyprobe™ was used to validate the low tissue pO 2 . Brown cytoplasmic staining was consistent with hypoxia. Mild brown staining was noted predominantly on the villus tips in control animals. SMA-banded rats had an extended region of hypoxic involvement in the villus with a higher intensity of cytoplasmic staining. Deep brown stainings of the enteric nervous system neurons and connective tissue both within layers and in the mesentery were noted. SMA-banded rats with lower pO 2 values had a higher intensity of staining. Thus, monitoring SI pO 2 using the probe Oxy-Chip provides a valid measure of tissue oxygenation. Tracking pO 2 in conditions that produce chronic mesenteric ischemia will contribute to our understanding of intestinal tissue oxygenation and how changes impact symptom evolution and the trajectory of chronic disease.

Head and neck squamous cell carcinoma (HNSCC) tumours are associated with high mortality despite advances in therapy. The monoclonal antibody cetuximab (Erbitux ® ) has been approved for the treatment of advanced HNSCC. However, only a subset of HNSC patients receiving cetuximab actually responds to treatment, underlining the need for a means to tailor treatments of individual patients. The aim of the present study was to investigate the effect of cetuximab treatment on tumour growth, on tumour partial oxygen pressure as measured by LiPc electron paramagnetic resonance oximetry and on the expression of proteins involved in tumour growth, metabolism and hypoxia. Two HNSCC cell lines, UT-SCC-2 and UT-SCC-14, were used to generate xenografts on female BALB/c (nu/nu) nude mice. Mice with xenografts were given three injections of intraperitoneal cetuximab or phosphate-buffered saline, and the tumour volume was recorded continuously. After treatment the tumour partial oxygen pressure was measured by LiPc electron paramagnetic resonance oximetry and the expression of epidermal growth factor receptor (EGFR), phosphorylated EGFR, Ki-67, MCT1, MCT4, GLUT1, CAIX and HIF-1α were investigated by immunohistochemistry. In xenografts from both cell lines (UT-SCC-2 and UT-SCC-14) cetuximab had effect on the tumour volume but the effect was more pronounced on UT-SCC-14 xenografts. A higher tumour oxygenation was measured in cetuximab-treated tumours from both cell lines compared to untreated controls. Immunocytochemical staining after cetuximab treatment shows a significantly decreased expression of EGFR, pEGFR, Ki67, CAIX and nuclear HIF-1α in UT-SCC-14 tumours compared to untreated controls. MCT1 and GLUT1 were significantly decreased in tumours from both cell lines but more pronounced in UT-SCC-14 tumours. Taken together, our results show that cetuximab treatment decreases the tumour growth and increases the tumour partial oxygen pressure of HNSCC xenografts. Furthermore we found a potential connection between the partial oxygen pressure of the tumours and the expression of proteins involved in tumour growth, metabolism and hypoxia.

The use of oxygen-sensing water-insoluble paramagnetic probes, such as lithium octa- n -butoxynaphthalocyanine (LiNc-BuO), enables repeated measurements of pO 2 from the same location in tissue by electron paramagnetic resonance (EPR) spectroscopy. In order to facilitate direct in vivo application, and hence eventual clinical applicability, of LiNc-BuO, we encapsulated LiNc-BuO microcrystals in polydimethylsiloxane (PDMS), an oxygen-permeable and bioinert polymer, and developed an implantable chip. In vitro evaluation of the chip, performed under conditions of sterilization, high-energy irradiation, and exposure to cultured cells, revealed that it is biostable and biocompatible. Implantation of the chip in the gastrocnemius muscle tissue of mice showed that it is capable of repeated and real-time measurements of tissue oxygenation for an extended period. Functional evaluation using a murine tumor model established the suitability and applicability of the chip for monitoring tumor oxygenation. This study establishes PDMS-encapsulated LiNc-BuO as a promising choice of probe for clinical EPR oximetry.