Theranostic ratings of bioreductively-activated prodrugs for the direction of hypoxic solid tumours
Summary:The undertaking pursues the translational geographic expeditions of glucose-tirapazamine ( TPZ ) -based conjugates to measure their possible in future clinical direction of solid hypoxic tumours in malignant neoplastic disease diseases.TPZ is a extremely powerful hypoxia-selective clinical drug that was used in several clinical tests, but was withdrawn from the clinic due to its terrible neurotoxic manifestations and hapless patient population choice. Our attack transforms TPZ into ‘next generation’ mutimodal ( diagnosing, radiosensitization, chemotherapy, molecular radiation therapy ) theranostics ( therapy+diagnostic ) , which would turn ‘hypoxia from job to advantage’.
Rationale for the undertaking:
Solid tumours often exhibit rapid growing and deviant vasculature which leads to oxygen depletion ( hypoxia ) and hapless drug and nutrient’s supply.1,2Hypoxia alters cellular metamorphosis, which can trip transcriptional responses, bring oning changes in cistron look, largely mediated by hypoxia-inducible factors 1 & A ; 2 ( HIF ) .3,4At low O degrees, HIFs trigger the activation of cistrons involved in glycolysis, cell endurance, reproduction, angiogenesis, tissue invasion and metastatis.5-7As a effect, the malignant potency of hypoxic tumours is altered which makes them resistant to conventional radio- and chemotherapy ( due to impaired drug bringing ) intercessions.10Therefore tumour hypoxia presents important human wellness challenges and contributes to hapless overall endurance of malignant neoplastic disease patients.
TPZ, is a hypoxia aiming bioreductive anticancer drug, but was withdrawn from clinic due to its terrible toxicity. What happens in TPZ is, it gets metabolized and lysed before making the targeted cells. Hence, conjugates of glucose and an parallel of TPZ, that is less toxic is being developed in our lab, in order to command these tumours. Glucose mediety will ease their conveyance ( through upregulated glucose transporters ) in hypoxic malignant neoplastic disease cells where TPZ will bioreductively trip in hypoxic atmosphere selectively and adhere to cytoplasmic supermolecules in this to leave quadruple theranostic characteristics.
Healthy cells are oxygenated therefore the drug will non be retained in this, and minimum toxicity will be experienced by them. Overall, the end of this survey, is to analyze the multimodal theranostic potency of TPZ-glucose conjugates and the corresponding radiohalogenated pharmaceuticals for the direction of hypoxic tumours.
Role of HIF1 in tumour hypoxia:
The phenomenon of tumour hypoxia has been studied since several decennaries and was foremost investigated and discussed by Grayet Al11,1953. Since so, tremendous research has been conducted in order to command the hypoxic effects in solid tumours thereby to better the efficaciousness of intervention given to malignant neoplastic disease patients. The estimated degree of oxygenation in normal tissues is said to be 40mmHg, whereas the degree reduces to 10mmHg in hypoxic status, and slowing to 1 % at anoxia ( at extreme hypoxia )12. Decrease in O degrees are chiefly due to hapless vascularisation in tumour tissues which renders it hard to fulfill the high demand of O created due to rapid and aggressive proliferation of cells13.
Response of GLUT1 at hypoxic conditions:
The important measure in the production of ATP in cells, is oxidative phosphorylation, where the H atoms and negatrons are transferred to oxygen. But of course in hypoxic ambiance, due to low O2, cells choose an alternate tract such as anaerobiotic glycolysis to let go of energy, and this tract is regulated by HIF-1. Basically hypoxia induces a displacement from aerophilic to anaerobic glycolysis, which leads to increase in look of cistrons encoding cardinal glycolytic enzymes and glucose transporters ( GLUT1 )8,9.
Therefore, glucose conveyance regulated by hypoxia, is said to be triphasic. Initially, GLUT-1 gets activated on the preexistent plasma membrane due to reduced oxidative phosphorylation and ATP production, and if hypoxia still exists, GLUT-1 molecules are translocated from intracellular cysts to plasma membrane, and in chronic hypoxic cells,de novosynthesis of GLUT-1 is mediated by HIF-1 ( Fig 3 )10. This ordinance form, strongly renders GLUT-1 as a agency of aiming hypoxic tissue in tumor. High look of membranous Glut1 was observed in caput and cervix carcinomas after extract of pimonidazole, an exogenic hypoxic cell marker ( Fig 4 ) .22Hence, GLUT1 has been regarded as a diagnostic trademark of tumour hypoxia.
Use of parallels and conjugates of GLUT-1 to aim hypoxic cells:
Surveies have been carried out on the usage of parallel of glucose viz. , 2-deoxy-D-glucose ( 2-DG ) , that would suppress glycolysis23. Due to the enhanced consumption of glucose parallels in malignant neoplastic disease cells, high toxicity was observed in hypoxic cells. But, since these surveies, show non- specific suppression of glycolysis in tumours, more research was conducted on the footing of GLUT-1 look. Preclinical surveies were carried out on 2-halogen substituted parallels of glucose, 2-deoxy ( 2-DG ) , 2- chloro ( 2-CG ) and 2- bromo ( 2-BG ) which specifically target HIF- regulated enzyme hexokinase rendered better tumour response24( Fig 5 ) .
Offlate, a better alternate scheme, to utilize GLUT1 molecules as a tumour marker has been developed. Advanced structural betterments in the glucose conjugates by associating it with a cytotoxic species have been developed, so that the conjugate gets selectively transported into tumour cells by GLUT-1. In a earlier survey, 2- glu-SNAP, glucose molecule conjugated with a nitric- oxide giver, ( S-nitroso-N-acetyl-penicillamine ) SNAP on a spongioblastoma cell line demoing increased activity of tumour cytotoxicty when compared to unconjugated SNAP molecule25.
Another survey shows the usage of combined intervention of Glufosfamide, a conjugate of glucose and isophoramide mustard, a metabolite of ifosfamide ( which exhibits DNA- alkylating belongingss ) and gemcitabine ( anti-cancer agent ) in phase-I clinical tests in pancreatic malignant neoplastic disease26. Hence, this thought, marks the foundation of our survey to utilize GLUT1 conjugates in add-on with a hypoxia selective bioreductive pro-drug-TPZ ( Fig 6 ) .
Recent promotions in Tirapazamine, a hypoxia-dependent bioreductive agent:
Oxygen Acts of the Apostless as a radiosensitizer by heightening the efficiency of ionising radiation dosage during radiation therapy, by formation of DNA- damaging free groups in malignant neoplastic disease cells. Therefore hypoxic tumours are immune to radio and chemotherapy. Although, several hypoxia- aiming O mimetic drugs have been developed to do the hypoxic tumours sensitive to therapies, still several surveies are being carried out to place a better bioreductive agent, that possess high hypoxic derived function.
In our survey, Tirapazamine, a hypoxic cytotoxin, that gets bioreductively activated at hypoxic atmosphere is being used. TPZ, a heterocyclic di-N-oxide ( belongs to benzotriazine household ) undergoes enzymatic metamorphosis at hypoxic atmosphere from a dioxide to a mono-N-oxide metabolite, which exhibits O mimetic ( radiosensitizing ) belongingss.28.29The activation of TPZ is catalysed by cytochrome P450 enzymes which reduces the prodrug to cytotoxic extremist by exhibiting DNA-strand breakage belongingss25.
Phase-II clinical tests conducted on TPZ, in combination with cisplatin, etoposide and radiotherapy, in little cell-lung malignant neoplastic disease showed increased toxicity and decreased endurance benefit30. In phase-III clinical test of caput and cervix malignant neoplastic disease type, consequence of TPZ, cisplatin and radiation therapy were compared with consequence of cisplatin conjugated with fluorouracil, and radiation. Positron emanation imaging ( PET ) was used to analyze the hypoxic tumours before and after intervention and consequences suggest, that patients with hypoxic tumours who had received TPZ, found no survival benefit31.
Therefore, TPZ should be structurally altered to bring on low toxicity to render them as executable marks for curative response. In our lab, an parallels of TPZ ( ie. , less toxic ) are being synthesized in order to research the potencity of this prodrug. Recent survey conducted by Johnsonet Al, 2014, suggests that add-on of N mustards, cytotoxic chemotherapeutic agents to TPZ, might trip a DNA-alkylating species selectively in hypoxic tissues, which in order will bring on DNA harm to hypoxic tumour cells11( Fig 7 ) .
Theranostic attack of Tirapazamine:
Several converting consequences have demonstrated the usage of PET (18F [ FDG ] as radiotracer ) , in naming hypoxic tumours after intervention with TPZ. Many research workers have besides examined the function of TPZ as a chemotherapeutic agent. When combined with radiation therapy, TPZ exhibits increased anti-tumor consequence. A combined survey of x-ray radiation therapy ( XRT ) and TPZ was found to be uneffective, due to the inactivation of TPZ before making the mark. Hence, in this survey, the potencity of glucose- TPZ based conjugates will be explored farther ( Fig 8 ) .
Hypothesis:We hypothesize that our prodrugs, when incorporating a diagnostic radionuclide ( e.g. , F-18, I-124 ) , will let diagnosing of hypoxic tumours by PET and, when labelled with a curative radioiodine ( e.g. , I-131 ) , will leave hypoxia-targetedin situmolecular radiation therapy ( MRT ) . The drug can besides be used as a radiosensitizer and possible chemotherapy agent. The purpose is to supply better theranostic options for malignant neoplastic disease patients diagnosed with therapy-resistant solid tumours.
1.Determine the pharmacodynamics and dynamicss of the GLUT-1 look in selected malignant neoplastic disease cell lines followed by look intoing the cytotoxicity of TPZin vitro.
2.Determine the consequence of cardinal proteins involved in the synthesized glucose-TPZ conjugates.
3.Investigate the cytotoxic consequence and theranostic characteristics of glucose-TPZ conjugates in hypoxic tumor- bearing animate being theoretical accounts.
Basically, proof of a series of theranostic surveies will find the anti-cancer potency of the conjugates, thereby supplying a footing for future clinical tests with healing response.
Aim 1. Determine the pharmacodynamics and dynamicss of the GLUT-1 look in selected malignant neoplastic disease cell lines followed by look intoing cytotoxicity of TPZin vitro.
Initially, to find the kineticss and dynamicss of GLUT1 look, two cell lines of caput and cervix malignant neoplastic disease cell types- unwritten squamous carcinoma cells ( SCC ) and human hypopharyngeal squamous carcinoma cells ( FaDu ) are considered. The cells will be incubated at different O2conditions, viz. normoxic – 20 % O2and hypoxic – 5 % , 2 % , 1 % and 0.5 % O2for 24 hours. Confluence and sourness will be taken into history so that they don’t exhibit a negative impact on the experimental procedures.
Protein lysates and RNA will be collected from the incubated cells. Western blotting will be carried out to find the degrees of GLUT-1 and HIF-1 comparative to ?-actin. Real clip RT-PCR will be used to find the degrees of look of GLUT-1 and LOX when compared to housekeeping cistron RPLPO. After finding of an O2status that best upregulates the GLUT-1 look, a time-course will be run by incubating cells in selected Oxygen2status ( near anoxia ) at different clip points such as 0 min, 6hrs, 12 hour, 24 hour and 48 hour.
Western blotting and Real clip RT-PCR will be carried out from the extracted protein cell lystaes and RNA as described above. In order to analyze the cytotoxicity of the drug ( TPZ ) , cells will be incubated at hypoxic/anoxic status at clip established in dynamicss, and will be treated with different concentrations of drug ( TPZ ) . Output will be studied by the live/dead check by analyzing the settlement organizing ability of cells.
Aim 2. Determine the consequence of cardinal proteins involved in the synthesized glucose-TPZ conjugates.
Radiation induced cell decease normally consequences from DNA harm.17Inhibition of DNA harm fix proteins will be studied to look into the importance of cardinal proteins known to act upon DNA responses to radiation and hypoxia after intervention with drug. Key proteins such as p53, a tumour suppresser is known to play a major function in ordinance of DNA fix in irradiated cells. Consequence of p53 inhibitor, pfithirin on normal and hypoxic cells before and after the intervention of prodrugs will be studied.18Consequence and look of HIF1-? will besides be investigated under normoxic and hypoxic conditions.
Extraction of proteins followed by western smudge analysis will be performed. Cells will be exposed to increasing doses of radiosensitizer drugs followed by X- beam irradiation ( as described by Barrancoet Al19) with and without prodrug intervention. In order to analyze the DNA fix caused by radiation, ? H2A-X check ( as described by Lucaet Al20) will be carried out with and without intervention of pfithirin. Cell sensitiveness to irradiation will be determined utilizing clonogenic cell endurance assay. Immunohistochemical staining of GLUT-1, HIF1-? and p-53 protein look will be performed in prodrugs after extract of pimonidazole, tumour hypoxic marker.
Aim 3. Investigate the cytotoxic consequence and theranostic characteristics of glucose-TPZ conjugates in hypoxic tumor- bearing animate being theoretical accounts.
Cytotoxicity and theranostic characteristics ( diagnosing, radiosensitization, chemotherapy, molecular radiation therapy ) will be examined separately on the glucose-TPZ based conjugatesin-vivo. Murine theoretical account of FaDu hypoxic tumour heterografts will be established in bare mice, as described by Chowdhuryet Al15and different doses of prodrugs will be injected into the tail-vein of heterografts. Histopathological surveies of the hypoxic tumour tissues will be studied to analyze the cytotoxicity of the administered pro drugs and the highest dosage with no seeable side-effect will be selected.
Radioactive- isotopes ( F-18, I-124, I-127, I-131 ) will be produced through specific atomic reactions in a cyclotron at MICF ( Medical Isotope and Cyclotron Facility, South campus ) . Then, these isotopes will be labelled to the synthesized Glucose-TPZ based conjugates. Initially, good known- diagnostic radionuclide,18F labelled with the synthesized prodrugs, will be injected into the heterografts.
Diagnosis of radiotherapy- immune hypoxic tumours will be carried out by PET due to the high consumption of glucose metamorphosis in hypoxic malignant neoplastic disease countries. Radionuclide124I, has been regarded as a good radiotracer, since they possess long half life ( 4.2 yearss ) , high clearance of non-specific radiation, high antielectron emanation energy and its labelling chemical science have been studied in item.16So, our prodrugs will be labelled with124I, to look into whether they can exhibit better tumour imaging features in PET when compared to18F. Radiation dosimetry estimations of our pro-drugs will be carried out by our group, to find theirin situMRT potency in hypoxic tumours.
Our prodrugs, labelled with curative radioiodine (131I ) will be administered into hypoxic tumour heterografts and PET surveies will be carried on to analyze their authority to leave hypoxia-targetedin situMRT. A radiotherapeutic agent177Lu DOTA-octreotate, will besides be labelled with the synthesized conjugates to analyze whether it can easein situMRT potency. To research the ability of our prodrugs as a radiosensitizer for XRT, clip of the consumption of prodrugs ( maximum dosage ) will be established by PET.
Surveies suggest that19F ( non-radioactive isotope ) and127I is a radiosensitizer of hypoxic cells to external beam XRT. Hence different doses of X ray will be given to tumor heterografts incorporating different doses of19F labelled prodrugs. Maximal accretion of radiosensitizer will be determined utilizing PET imaging. Comparative surveies will be carried on by labelling the conjugates with127I, in order to find the best radiosensitizer.
Freshness:No hypoxia aiming drug has been investigated in a multimodal theranostic attack for the direction of hypoxic tumours. Hence, our research is extremely advanced.
Collaborative Structure:Our multidisciplinary squad ( basic, translational and clinical scientific disciplines ) farther strengthens the research patterned advance, and provides excellence in all facets to successfully accomplish current aims.
Potential Outcome and Value: Our survey contributes to the diagnosing and intervention of hypoxic solid tumours taking to improved patient attention, reduced morbidity and mortality, significant wellness attention cost nest eggs, and thereby accordingly bettering the quality of life of malignant neoplastic disease patients.