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Bisphosphonate drugs and myo-inositol trispyrophosphate are of concern to the racing industry and have been listed as prohibited substances in equine sports. The current bisphosphonate plasma screening analysis employed at the Australian Racing Forensic Laboratory involves the use of sequential solid-phase extraction procedures, passing the samples through a mixed mode cartridge, followed by a weak anion exchange cartridge. The eluates collected following the second extraction are then methylated and analysed by liquid chromatography-mass spectrometry. Under these extraction conditions, some bisphosphonates have shown poor recovery. To improve the extraction efficacy, the effects of cartridge chemistry were evaluated. In particular, the weak anion exchange cartridges used for screening were compared to an affinisep AttractSPE polymeric phase cartridge. The effectiveness of each extraction approach was assessed through both a visual comparison of signal to noise in extracted chromatograms and recovery measurements to determine the best approach for routine screening.
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Bisphosphonates and myo-inositol trispyrophosphate (ITPP) are two classes of difficult-to-detect polar drugs that are prohibited under the rules of racing. ITPP is a drug capable of increasing the amount of oxygen in hypoxic tissues, and studies have shown that administration of ITPP increases the maximal exercise capacity in mice. The properties of ITPP make it an ideal candidate as a doping agent to enhance performance in racehorses. In recent years, ITPP had indeed been detected in racehorses and confiscated items. As for bisphosphonates, it is especially critical to control their use as since February 2019, the International Agreement on Breeding, Racing and Wagering (IABRW) by the International Federation of Horseracing Authorities (IFHA) had identified specific conditions on which bisphosphonates should not be administered to a racehorse. A recent review of literature shows that there is yet a simultaneous screening method for detecting ITPP and bisphosphonates in equine samples. This paper describes an efficient ion chromatography high-resolution mass spectrometry (IC-HRMS) method for the simultaneous detection of ITPP and 10 bisphosphonates at sub-parts-per-billion (ppb) to low-ppb levels in equine plasma after solid-phase extraction (SPE) and its application to an administration study of clodronic acid in horses.
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Tumour evolution and efficacy of treatments are controlled by the microenvironment, the composition of which is primarily dependent on the angiogenic reaction to hypoxic stress. Tumour angiogenesis normalization is a challenge for adjuvant therapy strategies to chemo-, radio- and immunotherapeutics. Myo-inositol trispyrophosphate (ITPP) appears to provide the means to alleviate hypoxia in the tumour site by a double molecular mechanism. First, it modifies the properties of red blood cells (RBC) to release oxygen (O2 ) in the hypoxic sites more easily, leading to a rapid and stable increase in the partial pressure of oxygen (pO2 ). And second, it activates the endothelial phosphatase and tensin homologue deleted on Chromosome 10 (PTEN). The hypothesis that stable normalization of the vascular system is due to the PTEN, a tumour suppressor and phosphatase which controls the proper angiogenic reaction was ascertained. Here, by direct biochemical measurements of PTEN competitive activity in relation to PIP2 production, we show that the kinetics are complex in terms of the activation/inhibition effects of ITPP with an inverted consequence towards the kinase PI3K. The use of the surface plasmon resonance (SPR) technique allowed us to demonstrate that PTEN binds inositol derivatives differently but weakly. This method permitted us to reveal that PTEN is highly sensitive to the local concentration conditions, especially that ITPP increases the PTEN activity towards PIP3, and importantly, that PTEN affinity for ITPP is considerably increased by the presence of PIP3, as occurs in vivo. Our approach demonstrates the validity of using ITPP to activate PTEN for stable vessel normalization strategies.
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Fosfatos de Inositol , Oxígeno , Humanos , Oxígeno/metabolismo , Fosfatos de Inositol/farmacología , Hipoxia/metabolismo , Monoéster Fosfórico Hidrolasas , Fosfohidrolasa PTENRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, and it is a disease of dismal prognosis. While immunotherapy has revolutionized the treatment of various solid tumors, it has achieved little success in PDAC. Hypoxia within the stroma-rich tumor microenvironment is associated with resistance to therapies and promotes angiogenesis, giving rise to a chaotic and leaky vasculature that is inefficient at shuttling oxygen and nutrients. Hypoxia and its downstream effectors have been implicated in immune resistance and could be contributing to the lack of response to immunotherapy experienced by patients with PDAC. Paradoxically, increasing evidence has shown hypoxia to augment genomic instability and mutagenesis in cancer, suggesting that hypoxic tumor cells could have increased production of neoantigens that can potentially enable their clearance by cytotoxic immune cells. Strategies aimed at relieving this condition have been on the rise, and one such approach opts for normalizing the tumor vasculature to reverse hypoxia and its downstream support of tumor pathogenesis. An important consideration for the successful implementation of such strategies in the clinic is that not all PDACs are equally hypoxic, therefore hypoxia-detection approaches should be integrated to enable optimal patient selection for achieving improved patient outcomes.
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PURPOSE: The Bohr effect describes hemoglobin's affinity for oxygen dependent on solution pH. Within pH range 6.0-8.5, hemoglobin's oxygen affinity decreases with decreasing pH. This results in increased oxygen delivery to metabolically active, acidic tissues and improved oxygen uptake in basic regions including lung tissue. Myo-Inositol tripyrophosphate (ITPP) translocates the erythrocyte membrane and allosterically modifies hemoglobin (Hb). We tested the hypothesis that ITPP does not abrogate the Bohr effect. METHODS: Experiments were conducted to determine the effect of increasing concentrations of ITPP on P50 with varying pH. We incubated 10 mL red blood cells at 37 °C for 1 h with ITPP concentrations from 0 to 240 mM. The Clark oxygen electrode (Hemox-Analyzer; TCS Scientific, New Hope, PA) determined oxygen affinity of each sample, in triplicate, using buffers pH 6.8, 7.4, and 7.6. A mixed linear regression model with fixed effects for ITPP concentration and pH was used. RESULTS: Increasing ITPP concentration and decreasing pH increased P50 (p < 0.0001 for ITPP concentration, p < 0.0001 for pH). ITPP modulated increased P50 in normal pH (7.4) and acidic condition pH (6.8); with no effect at alkaline pH (7.6). CONCLUSION: The Bohr effect is conserved, with ITPP augmenting the decreased oxygen affinity seen with tissue acidosis, while not affecting oxygen affinity in conditions similar to a pulmonary microenvironment.
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Eritrocitos/metabolismo , Hemoglobinas/metabolismo , Oxígeno/metabolismo , Recuento de Eritrocitos , Humanos , Concentración de Iones de Hidrógeno , Fosfatos de Inositol/metabolismoRESUMEN
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.
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Fosfatos de Inositol/farmacología , Oxígeno/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Glioma/tratamiento farmacológico , Glioma/metabolismo , Hemoglobinas/metabolismo , Humanos , Hipoxia/tratamiento farmacológico , Hipoxia/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Desnudos , Oximetría/métodos , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Endogámicas F344 , RoedoresRESUMEN
INTRODUCTION: A defining feature of human hemoglobin is its oxygen binding affinity, quantified by the partial pressure of oxygen at which hemoglobin is 50% saturated (p50), and the variability of this parameter over a range of physiological and environmental states. Modulation of this property of hemoglobin can directly affect the degree of peripheral oxygen offloading and tissue oxygenation. Areas covered: This review summarizes the role of hemoglobin oxygen affinity in normal and abnormal physiology and discusses the current state of the literature regarding artificial modulation of p50. Hypoxic tumors, sickle cell disease, heart failure, and transfusion medicine are discussed in the context of recent advances in hemoglobin oxygen affinity manipulation. Expert commentary: Of particular clinical interest is the possibility of maintaining adequate end-organ oxygen availability in patients with anemia or compromised cardiac function via an increase in systemic p50. This increase in systemic p50 can be achieved with small molecule drugs or a packed red blood cell unit processing variant called rejuvenation, and human trials are needed to better understand the potential clinical benefits to modulating p50.
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Hemoglobinas/metabolismo , Hipoxia/sangre , Hipoxia/terapia , Neoplasias/sangre , Neoplasias/terapia , Oxígeno/sangre , Anemia de Células Falciformes , Enfermedad Crítica , HumanosRESUMEN
BACKGROUND: Solid tumors, such as hepato-pancreato-biliary cancer, develop tumor hypoxia with tumor growth. Despite advances in surgery, a majority of these patients are in an unresectable condition. At this stage standard cytotoxic chemotherapy regimens are applied with limited success. Novel biological treatment options based on an antiangiogenic mechanism of action neglect other hypoxia mediated mechanisms (e.g. epithelial-mesenchymal transition, Warburg effect, and immunological response) leading to an increased invasiveness with a poor outcome. The novel antihypoxic molecule myo-inositoltrispyrophosphate (ITPP, OXY111A) acts as an allosteric effector of hemoglobin and promotes normoxia in hypoxic tumors. In preclinical studies, tumor growth was reduced and survival prolonged. Additionally, a beneficial side effect profile was observed. METHODS: In this first Ib/IIa clinical trial we will assess safety and tolerability of OXY111A as well as a proof of concept regarding efficacy in patients with non-resectable primary and secondary tumors of the liver, pancreas, and biliary tract. The study design is exploratory, prospective, open-labelled and mono-centric. The study is divided in a dose escalation part with a maximum of 48 subjects and an extension part, in which 21 subjects will be included. DISCUSSION: The novel antihypoxic compound OXY111A has been tested in several cancer animal models showing beneficial effects for both survival and low side effect profiles. This first in patient application of OXY111A will reveal potential beneficial outcomes if anti-hypoxic therapy is added to standard cytotoxic treatment in patients with primary and secondary hepatopancreatobiliary tumors. TRIAL REGISTRATION: Institution Ethical Board Approval ID: KEK-ZH-Nr. 2014-0374; Swiss regulatory authority Swissmedic (2015DR1009); ClinicalTrials.gov Identifier: NCT02528526 , prospectively registered on November 11th, 2014.
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Neoplasias del Sistema Biliar/tratamiento farmacológico , Protocolos Clínicos , Fosfatos de Inositol/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias del Sistema Biliar/metabolismo , Neoplasias del Sistema Biliar/patología , Humanos , Hipoxia/metabolismo , Fosfatos de Inositol/administración & dosificación , Fosfatos de Inositol/efectos adversos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologíaRESUMEN
BACKGROUND: Genetically induced hepatocellular carcinoma (HCC) models are generally used to investigate carcinogenesis pathways, but very few attempts were made to valorize them for pharmacological testing. This study describes a micro-computed tomography (micro-CT) - based methodology for the diagnostic and lifelong follow-up of HCC in the hepatocyte-specific Trim24-null mouse line. Myo-inositol trispyrophosphate (ITPP) was tested as anti-cancer drug. METHODS: Partial hepatectomy was performed in 2 months-old Trim24-null mice, in order to accelerate the carcinogenesis process. HCC diagnosis was obtained by micro-CT scan with double contrast agent: 10 µl/g Fenestra™ LC was injected intraperitoneally 6 h prior to imaging and 10 µl/g Fenestra™ VC was injected intravenously 15 min prior to imaging. Twenty three hepatocyte-specific Trim24-null mice were considered for ITPP testing (3 mg/g/week intraperitoneally during 10 months in 12 mice, versus 11 controls). Lifelong follow-up was performed using micro-CT. Comparative analysis was performed using unpaired t test with Welch correction and survival curves were compared by log-rank test. Gene expression analysis was performed using the RT q-PCR technique. RESULTS: Double contrast micro-CT scan allowed HCC diagnosis as hypodense, isodense or hyperdense nodules. Positive predictive value was 81.3 %. Negative predictive value was 83.3 %. Tumor growth could be objectified by micro-CT scan before the ITPP treatment was started, and at 3 and 9 months follow-up. Significant progression of tumor volume was demonstrated in the both groups, with no difference between groups (p > 0.05). In the ITPP group, a mild decrease in tumor doubling time was first observed (31.9 +/- 12 days, p > 0.05) followed by a significant increase (59.8 +/- 18.3 days, p = 0.008). However, tumor doubling time was not different between groups (p > 0.05). Median survival after treatment initiation was 223 days (controls) versus 296 days (ITPP group, p = 0.0027). HIF1α, VEGF, glutamine synthase, osteopontin expression levels were not significantly modified at the end of follow-up. In the ITPP group, the p53 expression profile was inversed as compared to the control group, higher in non-tumor livers than in tumors. CONCLUSION: ITPP treatment allowed for a two-month survival improvement, with better tolerance of tumor burden and apoptosis increase in non-tumor, pathological livers.
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Myo-inositol trispyrophosphate (ITPP) is a novel allosteric effector of haemoglobin with high permeation selectivity across the red blood cell plasma membrane. Due to its potential to reduce the oxygen affinity of haemoglobin, ITPP application results in an enhanced oxygen release in hypoxic tissues. Therefore, ITPP is being examined for the treatment of numerous illnesses that involve hypoxia, such as cardiovascular diseases, cancer or Alzheimer's disease. Similar to the prohibited substance Efaproxiral®, ITPP increases maximal exercise capacity in mice, providing high potential to be misused in sports. To keep up with cheating athletes, a fast and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for screening and confirmation of ITPP in human urine for doping control purposes was developed. According to the molecule's distinct hydrophilic properties, extraction from complex biological matrices is challenging and conventional reversed phase liquid chromatography (RPLC) separations are not suitable for its detection. Therefore an approach based on hydrophilic interaction liquid chromatography (HILIC) Orbitrap mass spectrometry was established. The methodology was fully validated for qualitative purposes. Screening and confirmation assay are characterized by satisfactory specificity and robustness, adequate intra-day (screening: 4.9-8.1%; confirmation: 2.0-6.7%) and inter-day precision (screening: 4.6-9.1%; confirmation: 1.8-6.6%), excellent linear correlations (>0.99) with sufficient LLOD in the sub ng/mL range (screening: 15 ng/mL; confirmation: 1 ng/mL). In addition it could be shown that ITPP is stable in human urine under the mandatory storage period and conditions for doping control laboratories. To our knowledge, this is the first validated 'dilute-and-inject' LC-MS/MS method for the reliable detection of ITPP in human urine.
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Doping en los Deportes/métodos , Fosfatos de Inositol/orina , Sustancias para Mejorar el Rendimiento/orina , Cromatografía Líquida de Alta Presión , Hemoglobinas/análisis , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Reproducibilidad de los Resultados , Espectrometría de Masas en TándemRESUMEN
Myo-Inositol tris pyrophosphate (ITPP) is a powerful allosteric modulator of haemoglobin that increases oxygen-releasing capacity of red blood cells. It is capable of crossing the red blood cell membrane unlike its open polyphosphate analog myo-inositol hexakisphosphate (IHP). Systemic administration of ITPP enhanced the exercise capacity in mice. There have been rumours of its abuse in the horse racing industry to enhance the performance of racing horses. In this paper, the detection of ITPP in equine plasma and urine after an administration of ITPP is reported. A Standardbred mare was administered 200 mg of ITPP intravenously. Urine and plasma samples were collected up to 120 h post administration and analyzed for ITPP by liquid chromatography-tandem mass spectrometry. ITPP was detected in post administration plasma samples up to 6 hours. The peak concentration was detected at 5 min post administration. In urine, ITPP was detected up to 24 h post administration. The peak concentration was detected at 1.5 h post administration.
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Caballos/sangre , Caballos/orina , Fosfatos de Inositol/sangre , Fosfatos de Inositol/orina , Detección de Abuso de Sustancias/métodos , Animales , Cromatografía Líquida de Alta Presión/métodos , Fosfatos de Inositol/administración & dosificación , Límite de Detección , Espectrometría de Masas en Tándem/métodosRESUMEN
Hypoxia and dysfunctional tumor vessels represent a prominent feature of pancreatic cancer, being, at least in part, responsible for chemotherapy resistance and immune suppression in these tumors. We tested whether the increase of oxygen delivery induced in vivo by myo-inositol trispyrophosphate (ITPP) can reverse hypoxia, control tumor growth and improve chemotherapy response. Tumor size, metastatic development (microcomputed tomography scan follow-up) and the survival of rats and nude or NOD.SCID mice, (bearing syngenic rat and MiaPaCa2- or patient-derived pancreatic tumors), were determined on ITPP and/or gemcitabine treatment. Partial oxygen pressure, expression of angiogenic factors and tumor histology were evaluated. Infiltration and oxidative status of immune cells, as well as chemotherapy penetration in tumors, were determined by fluorescence-activated cell sorting, fluorometry, nitric oxide release assays, Western blot and confocal microscopy. Weekly intravenous ITPP application resulted in the inhibition of metastasis development and restricted primary tumor growth, showing a superior effect on the rats' survival compared with gemcitabine. ITPP treatment restored tumor normoxia and caused a reduction in hypoxia inducible factor-1α levels, with subsequent VEGF and Lox downregulation, resulting in improved vessel structure and decreased desmoplasia. The latter effects translated into elevated immune cells influx and improved susceptibility to gemcitabine treatment. Growth of human pancreatic tumor xenografts was strongly inhibited by administration of ITPP. ITPP exploits a two-stage mechanism causing rapid, early and sustainable late stage normoxia. This is due to the angiogenic factor modulation and vascular normalization, leading to enhanced chemotherapy delivery and synergistic life prolongation, on combination with low doses of gemcitabine.