Final Report Summary - METOXIA (Metastatic tumours facilitated by hypoxic tumour micro-environments)
Executive Summary:
The METOXIA consortium answered a call which was ambitious, aiming to translate knowledge on the molecular machinery responsible for survival and spread of metastatic tumour cells under hypoxic conditions into innovative and validated molecular targets with therapeutic applicability that target the cancer cell or tumour stroma. The wording here can hardly be understood in any other way than to accept that the consortium also would have to protect its IP with the aim to try and secure a potential drug development. The level of ambition was furthermore strengthened by the statement that the consortium should include participants with ample clinical expertise to guarantee a clinical proof-of-principle. The METOXIA-consortium met all these premises. It was a large consortium, involving expertise over such a broad range as clinical and experimental medicine, molecular biology, synthetic chemistry, biophysics and electronics.
As indicated above several product developments are successful as planned. The greatest challenge is however the aim for the research to result in a new drug introducing a new principle into cancer therapy.
As the project stands by the end of METOXIA there are patents in international phase which can be further developed. We now see the possibility that inhibition with small molecules of a mechanism in the molecular machinery causing hypoxic cancer cells to survive and spread may come as far as to clinical tests in the relatively near future. There are several challenges to face before one reaches this goal, but we hope we have laid an important basis for the development.
During the 3rd and 4th periods we sub-contracted the company Cyprotex Discovery Ltd, to perform professional preclinical testing on 8 selected compounds from our patented sulfamates and dual-activity compounds. We have used these data together with our various partner’s effect testing in animal- and in vitro-models to complete a report (denoted deliverable D5.18) which the patent owners can use for further development of the compounds in collaboration with large pharma industry. Our view is that the potential social-economic impact of a new low-toxicity cancer treatment, halting or even stopping metastasis and improving the effect of conventional therapy can hardly be over-estimated. In our clinical programme we have cancers of the lung, breast, prostate, colon-rectum and head-neck; altogether some of the dominating cancer types in the world. So far we have reason to expect, although this has not been proven, that at least some patients within all these groups may benefit from a hypoxia-specific drug. We expect that the new knowledge brought up by the METOXIA-consortium will be of utmost value for future pharmaceutical development within this field.
Project Context and Objectives:
Scientific background of METOXIA
The METOXIA project has an over-all focus on the increased understanding of the regulatory mechanisms which help cancer cells survive under unfavourable micro-environmental conditions characterized by low oxygenation (denoted hypoxic areas). Solid cancers generally contain areas with lower levels of oxygen than what is usual in normal tissues and thus, the cancer cells learn how to adapt to such conditions. The METOXIA project furthermore aims to use the increased knowledge of the protective regulatory mechanisms to develop new principles for cancer detection and treatment: Since only cancer cells and not normal cells experience hypoxia one can develop cancer-specific detection and treatment by attacking molecules specific to the protective regulatory mechanisms activated by the cells under hypoxic conditions.
It has long been known that the problems created by hypoxic areas in solid cancers are serious for the patient: In short, cancer cells in such hypoxic micro-environments represent a reduced chance for successful treatment. With respect to radiotherapy the problem relates to increased dose tolerance for individual cells under hypoxic as compared to well-oxygenated conditions while for chemotherapy there is in some cases both increased dose tolerance and reduced drug access to the hypoxic as compared to well-oxygenated areas. Even if the patient is treated with surgery alone there are indications that the amount of tumour tissue which is hypoxic correlates negatively with the outcome for the patient. Recent research has furthermore shown that variable hypoxia in tumours is one of the major drivers of metastatic spread of cancer, the major cause of death by the disease. Thus, hypoxia is responsible for a double effect of reducing the potential of a successful treatment of the cancer patient: Resistance to treatment and ability to spread to distant parts of the body. The positive side of this problem is that the very low level of oxygen found in solid tumours is specific to cancer. Therefore, if one could develop new methods to specifically detect and inactivate cells in hypoxic areas one might obtain a cancer-specific effect, selective for the most harmful of the cancer cells. In the original call which METOXIA answered it was made clear that the research should not be limited to pre-clinical biological models, but should include proof-of-principle clinical research. As part of the consortium some of the leading cancer clinics in Europe are partners.
Main Objectives of METOXIA:
On basis of the clinical problems raised by hypoxic tumour micro-environments the work within METOXIA encompasses increasing the knowledge concerning the metastatic process in the hypoxic micro-environment at the molecular level in order to develop novel strategies for modification of this micro-environment and thus improve the efficacy of chemotherapy and radiotherapy. Major objectives are:
• Gain new knowledge about molecular mechanisms behind hypoxia-driven metastasis in order to reduce metastatic spread and increase patient cure rate.
• Develop improved methods to detect the propensity of a cancer to metastasise before the metastatic spread has become manifest clinically.
• Develop new treatment management of metastatic disease.
• Develop new methods to increase the effectiveness of treatment in hypoxic areas of the primary tumour.
• Development of new methods for detection/imaging of hypoxic areas.
• Generate pre-clinical models for the study of the role of hypoxia in metastases.
• Development of new tools for monitoring and control of peri-cellular micro-environment in vitro.
The partner organisation:
There are at present 21 partners in the consortium. The work was partitioned in 8 Work Packages (WP) of which one (WP9) relates to management and one (WP6) is empty (the activity originally included in WP6 was during the 2nd period amendment moved into WP5, WP7 and WP8 after a decision made at the 2nd General Assembly in June 2010). This reorganization turned out to be a success and no further reorganization of the consortium has been done during the 3rd, 4th and 5th periods.
Largely the non-management WPs can be divided into 2 main groups (WP6 is now empty):
• Work involving cancer patients (WP1, WP5 and WP8).
• Work involving primarily fundamental research, divided into two sub-areas:
A) Studies of primarily biological nature (WP2, WP3, WP4)
B) Development of more technical nature (development of equipment for monitoring and control of micro-environmental parameters related to hypoxia) (WP7).
The WEB-site of METOXIA is on the following address: http://www.metoxia.uio.no/
In line with the consortium agreement some of the reported material is confidential. The WEB-site thus is divided into an open area accessible for all and a restricted area accessible for partners only.
The work performed since the beginning of the project and the main results achieved so far.
Over the 5.5 year total duration of the METOXIA project 105 scientific deliverable-reports have been completed and, in addition also 18 management deliverables. Thus, considering the amount of work here reported only some major findings can be dealt with in a summary.
The line of research which has led to the most optimistic results concerning the possibility to develop a new drug for cancer therapy has the following background: The hypoxic micro-environment in solid cancers influences several regulatory cascades in cells. Since cancer cells in solid tumours experience such conditions over a long time these regulatory mechanisms represent a selective pressure of cancer cells into more hypoxia-tolerant phenotypes. This development correlates with a higher degree of malignancy and furthermore, with higher metastatic potential. For development of new diagnostic and treatment concepts it is important that these findings are connected to knowledge of hypoxia-regulated molecular pathways with which we can interfere. Even a small reduction in oxygenation results in regulatory consequences for a large part of the genome of our cells. There are several key regulators of these cascades, but the one responding to the smallest reduction in oxygenation is the protein HIF (Hypoxia Inducing Factor). This protein is what we denote a transcription factor; meaning that it can regulate activation of certain genes on DNA. It turns out that HIF-regulation involves a large number of genes and several processes which are vital for the cells and tissues to survive hypoxic conditions: Cell metabolism, energy production, angiogenesis (i.e. formation of blood vessels), extracellular matrix properties (EMT-epithelial mesenchymal transition) and pH-control. All these have been thoroughly studied within the METOXIA programme and valuable new knowledge has been accumulated. Notice however, that since HIF itself regulates so many processes it can hardly be considered as a target for treatment. Attacking HIF might affect too many normal processes and might not be cancer-specific. The focus for development of new cancer therapy therefore is not primarily on this central regulator, but rather on several of the cascades it regulates. One such mechanism has been developed into METOXIA-patented products of potential value as new anti-cancer drugs. This relates to certain small-molecular inhibitors of the HIF-regulated protein Carbonic Anhydrase IX (CAIX) involved in cellular uptake of CO2 for bio-mass synthesis and pH-regulation. These CAIX-inhibitors have in pre-clinical experiments shown a great potential for specific treatment effects on reduction of the metastatic potential of the primary tumour and also to some extent seem to increase the effect of traditional radiotherapy and some chemotherapy. Two arms of development have been worked out:
a) Several METOXIA-partners collaborate on the development of small-molecular sulfamate compounds (patented). These include chemicals with high specificity to the active site of the CAIX-protein. By specifically inhibiting CAIX our findings indicate that we have not only new possibilities for cancer treatment, but also new diagnostic principles. Thus, the METOXIA results point to new possibilities for drug development. Still, we need to add that we have also experienced the threshold for such development through our contact with large pharma: The practical problems related to development of hypoxia-specific treatment seem to exceed those of more traditional drug development. The reason for this is that the killing of hypoxic cells alone by no means can be expected to have a major influence on the primary tumour. Hypoxic cells proliferate less than well-oxygenated cells (i.e. they do not give rise to more cells as long as they are hypoxic). The problem they create is resistance against traditional treatment and increased ability to form distant metastasis. Thus, they are expected to be responsible for relapses after completed treatment. Development of a new hypoxia-specific agent therefore will have to be done in combination with traditional treatment and in the short run it may become difficult to observe an extra effect on the primary tumour compared to the effect of the traditional treatment alone.
b) As a possible improvement of the combination treatment a sub-group of METOXIA partners have synthesized a group of chemicals (patented) having a dual effect: These chemicals both have the ability to inhibit CAIX and to increase radiosensitivity of hypoxic cells. To follow up the development of these chemicals a new company (an SME) was founded shortly before the end of METOXIA and the aim is to perform preclinical and clinical studies of the effect of these compounds given simultaneously with radiation therapy.
Targets other than the HIF-regulated pathways have also been investigated and some found to be of great potential interest as targets for hypoxia-specific markers and treatment. Two targets of interest related to pH-regulation and metabolism are the lactate transporter MCT4 and its subunit CD147/Basigin. Our partner 13/CNRS has tested an MCT4-inhibitor produced by AstraZeneca and confirmed its specificity for MCT4 and reports this compound to be ready for clinical trials in the near future. Also the two stress-related processes initiated under hypoxic conditions denoted unfolded protein response (UPR) and the mammalian Target of Rapamycin (mTOR) have been investigated by several partners with 20/GROW-UM as most central. Both processes act as hypoxia sensors in the cells. UPR is a process which leads to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum while mTOR is a protein kinase which regulates cell growth. In a collaboration between 20/GROW-UM, 2/MAASTRO and 5/UOXF.BP it was shown that hypoxia activation of UPR induces expression of the metastasis-associated gene LAMP3, thus identifying LAMP3 as a new candidate biomarker of UPR activation by hypoxia in tumours and a potential mediator of hypoxia-induced metastasis. Regarding mTOR-inhibitors, these have been introduced as anti-cancer drugs for renal cell carcinoma (tensirolimus and everolimus).
Another field which has led to filed patent applications is the induction (and also counteraction) of radiation-induced resistance to chemotherapeutic compounds by low dose-rate irradiation. During the 3rd and 4th periods this research at 1/UIO indicated that radiosensitivity of cells can be modified even several hours after the cell was irradiated and that the effect is easily induced also in animals. The relevance to METOXIA stems from our finding that the mechanism activated by low dose-rate irradiation is also activated by variable hypoxia. Thus, we have the possibility to attack mechanisms responsible for adaptation to chemotherapy resistance during hypoxic conditions. So far we do not have a patented compound for this action, but in the 5th period we have shown in animal experiments that the protein to inhibit is transforming growth factor beta3 (TGF-beta3), and we have shown that at least one commercial chemical, not developed into a drug can inhibit TGF-beta3 and counteract radio- and chemo-resistance.
Various new biological test models as well as technical measurement devices have been developed. New in vitro 3D-models as based on purified alginates for pre-clinical studies of efficiency of new modalities have been taken into use towards proof-of-principle testing of treatment benefit. Also new animal models for pre-clinical studies of efficiency of new modalities have been developed towards proof-of-principle testing of treatment benefit. Special emphasis has been placed on development of CAIX knock-down models so that the effect of CAIX-inhibitors can be assured to result from a specific effect on the CAIX-protein and not result from some unspecific toxicity. For the testing of effects on metastasis orthotopically implanted tumours have been used. Development of new sensor- and micro-fluidic technology and instrumentation for detection and control of oxygen and other substances in the cell and tissue micro-environments are being developed. A disposable cell culture flask with a sensors molded into the flask bottom has been developed.
A central clinical investigation is the study of micro-metastases (i.e. circulating cancer cells) as a means for individual evaluation of the metastatic potential of cancers in patients. Also our effort to develop a comprehensive classification of tumour hypoxia, anoxia and reoxygenation has been highly prioritized as it aims to allow clinicians to predict response to targeted agents which include those activated by hypoxia. In this connection detection and visualization of hypoxia in patient tumours are highly prioritized and a promising new method passed phase I study during the 3rd period and protocols have been completed during the 4th period for this to continue with 3 phase II studies which has been ongoing during the 5th period of METOXIA. This method involves non-invasive PET imaging of tumour hypoxia by use of a radioactive tracer denoted [18F]HX4, a member of the 2-nitroimidazole family of chemicals. The special quality of these chemicals, denoted bioreductive compounds, is that they are chemically modified under hypoxic conditions and thereby can represent both chemotherapeutic drugs specific to hypoxic cells and also offer PET-imaging of reactive activity. This particular study was not included in the plans of METOXIA from the start, but is an example of the importance of amendments of new possibilities coming up during the development of the project.
Although the idea of targeting a sub-set of hypoxic tumour cells to improve treatment outcome is intriguing, clinical studies proving that not only do hypoxic intervention work but it does so only in patients with hypoxic tumors has not been conducted until now. In this context, the development of a hypoxia gene signature at 6/AUH,AS which, when tested in material from a large randomized phase III study, was able to identify those HNSCC patients which benefit from hypoxic intervention with Nimorazole (radiation sensitizer) was an important step forward. As a direct spin-off, several multi-center clinical projects that will further clarify the full potential of this gene signature to identify patients suitable for hypoxic intervention has been launched.
Also development of bone-seeking alpha-particle-emitting radio-nuclides (Alpharadin®) for localized treatment of bone metastases has been involved in METOXIA. As alpha-particles deliver their energy concentrated (i.e. with high LET) this type of radiation is equally effective under hypoxic as under aerobic condition and is thus expected to abolish the radioprotective effect of hypoxia. This project was started before METOXIA by the Norwegian company Algeta. A global phase III clinical trial (ALSYMPCA) with Alpharadin® in patients with castration-resistant prostate cancer (CRPC) and bone metastases was successfully completed in June 2011. Algeta was recently bought by Bayer Pharma AG for more than 2 billion euros and Alpharadin is presently manufactured under the trade name Xofigo.
METOXIA furthermore takes part in the large-scale randomized trial started several years ago in the Netherlands comparing Accelerated Radiotherapy (AR) with Accelerated Radiotherapy plus Carbogen and Nicotinamide (ARCON) in laryngeal cancer. In this large multi-centric trial METOXIA has taken part in the evaluation of consequences of Carbogen and Nicotinamide in relation to degree of tumour hypoxia. Despite lack of benefit in local tumour control for advanced laryngeal cancers the results are promising, indicating a significant gain in regional control rate, with equal levels of toxicity, and even indicating increased disease-free survival for the patients having the most hypoxic tumours.
The expected final results and their potential impacts and use (including socio-economic impact and the wider societal implications of the project so far).
The METOXIA consortium answered a call which was ambitious, aiming to translate knowledge on the molecular machinery responsible for survival and spread of metastatic tumour cells under hypoxic conditions into innovative and validated molecular targets with therapeutic applicability that target the cancer cell or tumour stroma. The wording here can hardly be understood in any other way than to accept that the consortium also would have to protect its IP with the aim to try and secure a potential drug development. The level of ambition was furthermore strengthened by the statement that the consortium should include participants with ample clinical expertise to guarantee a clinical proof-of-principle. The METOXIA-consortium met all these premises. It was a large consortium, involving expertise over such a broad range as clinical and experimental medicine, molecular biology, synthetic chemistry, biophysics and electronics. As indicated above several product developments are successful as planned. The greatest challenge is however the aim for the research to result in a new drug introducing a new principle into cancer therapy. As the project stands by the end of METOXIA there are patents in international phase which can be further developed. We now see the possibility that inhibition with small molecules of a mechanism in the molecular machinery causing hypoxic cancer cells to survive and spread may come as far as to clinical tests in the relatively near future. There are several challenges to face before one reaches this goal, but we hope we have laid an important basis for the development. During the 3rd and 4th periods we sub-contracted the company Cyprotex Discovery Ltd, to perform professional preclinical testing on 8 selected compounds from our patented sulfamates and dual-activity compounds. We have used these data together with our various partner’s effect testing in animal- and in vitro-models to complete a report which the patent owners can use for further development of the compounds in collaboration with large pharma industry. Our view is that the potential social-economic impact of a new low-toxicity cancer treatment, halting or even stopping metastasis and improving the effect of conventional therapy can hardly be over-estimated. In our clinical programme we have cancers of the lung, breast, prostate, colon-rectum and head-neck; altogether some of the dominating cancer types in the world. So far we have reason to expect, although this has not been proven, that at least some patients within all these groups may benefit from a hypoxia-specific drug. We expect that the new knowledge brought up by the METOXIA-consortium will be of utmost value for future pharmaceutical development within this field.
Project Results:
Main S & T results/foreground of METOXIA
A summary over the main findings of a large-scale project like METOXIA covering 5.5 years of study is complicated since the consortium covers almost every aspect of cancer research related to tumour micro-environments. The amount of results and findings to be analyzed are large. In the following, therefore, only brief references are made to the various main findings.
1 Hypoxia: Scientific background for translational cancer research.
Solid cancers generally contain areas with abnormally low levels of oxygen. Such areas are denoted hypoxic. It has long been known that cancer cells in such hypoxic micro-environments are resistant to treatment. Recent research has furthermore shown that variable hypoxia in tumours is one of the major drivers of metastatic spread of cancer, the major cause of death by the disease. Thus, hypoxia is responsible for a double effect of reducing the potential of a successful treatment of the cancer patient: Resistance to treatment and ability to spread. At the same time the very low level of oxygen found in solid tumours are specific to cancer. Therefore, if one could develop new methods to specifically detect and inactivate cells in hypoxic areas one might obtain a cancer-specific effect, selective for the most harmful of the cancer cells. This development is the over-all task of the METOXIA project.
2 Securing of IP and strategy to develop a cancer medicine (development during 3rd, 4th and 5th period):
Development of new compounds for inhibition of Carbonic Anhydrase IX (CAIX) is a major endeavour of METOXIA which has been given extensive effort by a sub-group (working across both the organizational structures mentioned above) for all the first 4 periods. This sub-group was flexible with respect to member organisations, those who were interested in this activity and had the capacity to contribute were accepted to join in. However, this success could naturally not be planned from the start and therefore some amendments to the plan have been worked out earlier. For a full description of the chemistry, see PCT patent application Number WO 2011/098610 A1.
During the 3rd period contact was made with Cancer-Research-UK (CR-UK) to seek advice for a possible step-in by them for further drug development. In the 4th period, according to their advice, the METOXIA management contacted a professional company, Cyprotec Discovery Ltd to perform necessary pre-clinical testing before an application to be worked out forCR-UK’s New Agents Committee (deliverable D5.18). During the spring-summer 2014 however, CR-UK together with Bayer AG sent to the METOXIA patent owners a conclusion that they will not take on the development of a compound which can only be used in combination treatment and which can not reduce the volume of the primary tumour alone. The problem with a hypoxia-specific drug is that the killing of hypoxic cells alone will hardly reduce the primary tumour (or any tumour) because hypoxic cells can hardly grow and divide. Such specific cell kill must be combined with radical radiotherapy (or chemotherapy) to kill aerobic cells and the benefit for the patient is expected to be reduced late relapse. Such drug development therefore is expected to be extra expensive and time-consuming.
Major findings and deliverables:
2.1 Selection of patents.
Although compounds from 4 different patents were originally included in the Cyprotex investigation their toxicology results as well as pharmacokinetics led to the determination by the METOXIA CAIX-sub-group to concentrate their investigations on compounds from only two patents for partner effect studies: Those two patents were:
o The sulfamate patent (WO 2011/098610 A1: Titled: “Carbonic Anhydrase Inhibitors”)
o The dual-activity patent (US 2013/0274305 A1: Titled: “Cancer Targeting Using Carbonic Anhydrase Isoform IX Inhibitors”).
2.2 Selection of compounds for effect studies.
From the sulfamate patent 3 compounds have been tested: S4, FC9-398A and FC9-403A. From the dual-activity patent the compound DH348 was tested. This compound was later denoted DTP348.
The selection of S4 was performed before the Cyprotex studies and was done on basis of data from 9/UNIFI showing that this compound had the strongest selectivity for the active site of CAIX protein among the tested sulfamate compounds. As shown by Figure 6 S4 was however shown to have a low bioavailability when given by oral administration, while FC9-398A had far better bioavailability. Thus FC9-398A was included in the effect studies. Early tests of the compound FC9-403A indicated positive effects also of this compound and it was decided to include also this one in some of the effect studies.
Compound DH348 was shown to have very high bioavailability, but short half-life. This compound was selected due to early findings of efficient combination effects with radiation.
2.3 Toxicology and pharmacokinetics.
• FC9-403A had a limited solubility with a lower bound of 3µM whereas all the remaining compounds had lower limits of solubility of 30µM or higher.
• None of the tested compounds were found to have any significant blood brain barrier permeability.
• Protein binding data indicated between 89 and 98% bound of the sulfamate compounds only between 55 and 70% of the dual activity compounds were bound.
• Regarding liver metabolism (by Cloe Screeen Microsomal Stability Test) none of the 10 compounds tested by Cyprotex have a high clearance (DCT-24a not measured). FC9-398A, and DH348, are metabolically stable in human and mouse. FC9-403A has moderate human clearance with low mouse clearance and S4 is metabolically stable in the human with moderate clearance in the mouse.
• The 11 tested compounds were found to have no or weak Cytochrome P450- inhibition.
• The data in Table VIII suggest that none of the tested compounds would have cardiac toxicity since there was no or weak hERG channel inhibition by all compounds.
• Mouse PK-studies:
a) For FC9-398A solubility was not a problem and bioavailabilty over the 0-8 hr time period tested was high (57.6%). FC9-398A also demonstrates the lowest rate of clearance following an IV dose (59.9 .l/min/kg) suggesting that the compound is more metabolically stable than the other sulfamate compounds tested. S4 and FC9-403A produced a bioavailability value of only 3% and 4.5% respectively over the 0-8 hr time course possibly making these less interesting for oral administration.
b) For DH348 there was no adverse effect observed in the animals from either dose route. Bioavailability was far above 100% indicating non-linear pharmacokinetic behaviour (either saturation of an efflux transporter or saturation of the metabolic clearance rate).
• In Silico prediction of PK in human and mouse:
a) FC9-398A and S4 were found to have the lowest clearance of about 0.3 and 0.8 mL/min/kg for human and mouse respectively. The corresponding values for DH348 were 2.5 and 4.5 mL/min/kg and for FC9-403A it was 1.2 and 8 mL/min/kg.
b) From Figures 27 to 38 a comparison is done regarding the two prioritized candidate compounds from the two selected patents: FC9-398A and DH348. It is concluded that peak drug levels in various tissues are 2 to 10 times higher with FC9-398A compared to DH348 and that that this difference is over-all higher regarding long-term levels due to lower clearance of FC9-398A as compared to DH348.
2.4 Effect studies performed by METOXIA partners.
8/UNIMAN concludes that S4 (and FC9-398A) work as an anti-tumour drug in cell lines that are highly dependent on CAIX (here SCLC). In contrast, in cell lines which are less CAIX dependent S4 has anti-metastatic effects (here MDA-MB-231, FaDu).
2/MAASTRO concludes that oral administration of FC9-398A in the H460 xenograft model is ineffective in reducing primary tumour growth. Furthermore, it might be suggested from these results that the metastatic potential of the H460 tumour cells is not significantly affected by FC9-398A treatment. Histological analysis of the lungs needs to give final conclusions.
3/UEDIN concludes that FC9-403A inhibits explant invasion at all concentrations used between 1 and 100 µM, and causes regression of invasion. S4 also strongly inhibits explant invasion at higher concentrations but this preliminary data indicates that it is less effective than FC9-403A at reversing invasion. Immunohisto-chemistry of S4-treated explants indicates that the mechanisms responsible for the inhibitory effects of these compounds on tumour explant growth and invasion involve an increase in levels of apoptosis and a decrease in levels of proliferation. The novel inhibitor DTP348 (DH348) showed little efficacy in in vitro 3D models, but significantly decreased growth, cell viability and cell proliferation in xenograft models. The explant model suggested that radiation resistance may be reversed by FC9-398A treatment. Unfortunately none of the explant materials used to examine DTP348 proved to be resistant to radiation, therefore the effect of this compound on radiation sensitivity could not be assessed in the present study.
4/RUNMC concludes that CAIX ectodomain shedding into the blood stream was decreased by S4. As the meaning of this observation is not clear, further studies are required to elucidate whether the CAIX ectodomain has a paracrine or autocrine signalling function in cancer biology. S4 did not influence the tumour micro-environment in a laryngeal tumour model in terms of the amount of proliferation, apoptosis, necrosis and hypoxia.
3 Studies directly related to patients:
3.1 WP1: Increasing the treatment effect on the primary tumour. (Including work performed in WP1 in the 1st, 2nd , 3rd, 4th and 5th periods.):
One early deliverable completed at Month 4 by Lambin, 2/MAASTRO (D1.1) (WP1: Obj2) was followed up with a second deliverable at month 18 as planned. The value of adding a new respiratory amplitude-based PET reconstruction method called Optimal Gating (OG) was tested. The aim was to provide accurate image quantification in lung cancer. The conclusion was that optimal gating PET is a better alternative to both 3D PET suffering from breathing averaging and the noisy images of a 4D PET acquisition. Partner 2/MAASTRO also completed an extensive deliverable (D1.3) (WP1: Obj1) on a planned randomized phase II study to be conducted in patients with inoperable stage II or III non-small cell lung cancer (NSCLC). The patients are randomized to receive the standard 66 Gy given in 24 fractions of 2.75 Gy with an integrated boost to the primary tumour as a whole (Arm A) or with an integrated boost to the 50% SUVmax area of the primary tumour (Arm B). The ultimate aim of the study is to increase the local progression-free survival (LPFS) after 1 year from 70 % to 85 %.
Our partner 4/RUNMC completed his deliverable (D1.4) on co-localization of hypoxia-markers and EGFR in the 2nd period and found a correlation between both these phenomena, a correlation with distance from blood vessels and, importantly; association with poor loco-regional clinical control. EGFR therefore could function to increase survival of cancer cells in the hypoxic tumour micro-environment. Furthermore, co-localization of hypoxic and metabolic markers (HIF-1α, CAIX, LDH-5, GLUT-1, MCT1 and MCT4) was, in a separate report shown to give additional clinical information compared to single protein analyses.
In the 3rd period partner 4/RUNMC completed his deliverable (D1.5) on the large-scale randomized trial started several years ago in the Netherlands comparing Accelerated Radiotherapy (AR) with Accelerated Radiotherapy plus Carbogen and Nicotinamide (ARCON) in laryngeal cancer. In this large multi-centric trial METOXIA takes part in the evaluation of consequences of Carbogen and Nicotinamide in relation to degree of tumour hypoxia. Despite lack of benefit in local tumour control for advanced laryngeal cancers the results are promising, indicating a significant gain in regional control rate, with equal levels of toxicity, and even indicating increased disease-free survival for the patients having the most hypoxic tumours.
In the 4th period the work in WP1 continued although all contracted deliverables had been completed at M36. Partner 4/RUNMC have continued their work on the boosting of the hypoxic radio-resistant areas in the tumour with high doses of radiotherapy. While the FDG-high uptake BTV boost is ongoing, they will soon also start the HX4-high uptake boost study which is a collaboration with partner 2/MAASTRO.
In the 5th period partner 2/MAASTRO continued the work on the possibility to boost radiation doses to hypoxic areas of the tumour. The efficiacy of using [18F]DG PET to select the tumour biological target volume and trying to validate the optimal dose delivery protocol has been prioritized. The conclusion is, however rather negative: The results suggest that no clinically meaningful differences may exist between dose redistribution based on high or low FDG-PET uptake regions compared to uniform irradiation. In the future, it will be important to test other biological features of the tumour microenvironment. Partner 2/MAASTRO also tested different PET tracers (i.e. [18F]FMISO, [18F]FAZA and [18F]HX4) but concluded that each tracer has its own strengths and depending on the question to be answered a different tracer can be put forward.
3.2 WP5: Clinical application of new drugs and treatment strategies involving both patient and animal studies (Including only work performed in WP5 during the 4th and 5th period):
A total of 8 deliverables were completed in WP5 during the two last periods in accordance with the plan and contract as described in amended AnnexI of May 2013.
A) One deliverable (D5.10) was completed by our partner 7/FMC who reported data on a clinical trial synopsis and design for evaluating the effect of alpha emitters in metastatic tumours within a hypoxic environment. This project was started before METOXIA by the Norwegian company Algeta. A global phase III clinical trial (ALSYMPCA) with Alpharadin® in patients with castration-resistant prostate cancer (CRPC) and bone metastases was successfully completed in June 2011. Algeta was recently bought by Bayer Pharma AG for more than 2 billion euros and Alpharadin is presently manufactured under the trade name Xofigo.
B) A second deliverable (D5.13) by our partner 3/UEDIN was originally planned to be performed on the established CA-inhibitory drug acetazolamide. This was in reality changed during earlier amendments because acetazolamide is hypoxia non-specific. The study instead was done on patented CAIX-inhibitors with high specificity. See text to deliverable 5.13 and 5.16II (below).
C) A third deliverable (D5.15) was completed by our partner 21/OU-Lyng on the Analysis of the micro-metastasis frequency in prostate cancer patients and of signal transduction and metabolic profile (MR) in patients with and without micro-metastases. The disseminated tumour cell (DTC)- and pimonidazole data suggest the use of the surgical cohort and the applied methods to further explore the relationship between hypoxia and DTCs. The HIF1 and apparent diffusion coefficient (ADC) data were consistent with a decrease in the hypoxic fraction, suggesting that HIF1 expression and ADC could be possible biomarkers of hypoxia in prostate cancer.
D) A fourth deliverable (D5.16II) was completed by our partner 3/UEDIN in collaboration with 9/UNIFI on assessment of a panel of selected, patented CAIX-inhibitors on cell migration and 3D assays (D5.16II; M48). This deliverable was added during amendment of Jan 2012 as a follow-up of the testing in 2D-assays of a larger selection of >20 compounds (deliverable 5.16I at M36). The deliverable represents a vital step in our effort to develop our patented compounds into a drug. The results reported in D5.16II forms a major basis for the selected compounds to be further tested in the 5th period by Cyprotex for PK and PD studies.
E) Also a fifth deliverable (D5.17) was completed by our partner 3/UEDIN on the assessment of novel CAIX and sodium-proton exchange inhibitors on the growth and invasion of breast cancer explants. Initial experiments focused on CAIX inhibitor S4. Again this represent a further follow-up of the effects of the patented compounds. The conclusion was that two compounds in particular was efficient in inhibition of explants invasion, the compounds encoded by S4 and FC903A. Immunohistochemistry of S4-treated explants indicateed that the mechanisms responsible for the inhibitory effects of these compounds on tumour explant growth and invasion involve an increase in levels of apoptosis and a decrease in the extent of cell proliferation.
F) One deliverable (D5.3) was completed by our partner 21/OU-Lyng who reported data on signal transduction in relation to tumour physiological and metabolic parameters in rectal and prostate cancer, and analysis of the above mentioned parameters against clinical outcome.
G) A second deliverable (D5.12) was completed by our partner 3/UEDIN on randomised trial of preoperative radiotherapy+/-CA9 inhibitor in early breast cancer (T1-T2
In addition to the 8 deliverables in WP5 partner 21/OU-Lyng has during the last period studied gene expression and signal transduction profiles associated with tumour hypoxia (by array slides) and the occurrence of micro-metastases. Several questions were raised regarding timing of tracers in this study. Partner 6/AUH,AS studied the clinical potential of non-invasive imaging methods (FMISO, FAZA, PET with CA9i, DCE-MRI) for identifying hypoxia. As in the study of 21/OU-Lyng several questions were raised and more research is needed in order to draw a clear conclusion. Partner 9/UNIFI has studied design and synthesis of carbonic anhydrase IX and -XII inhibitors as non-invasive imaging diagnostic tools as well as possible future drugs, patenting of new compounds for commercialization. Several structures are presented which may represent effective inhibitors as potential drugs. Partner 2/MAASTRO has studied design and synthesis of carbonic anhydrase IX and XII inhibitors as non-invasive imaging diagnostic tools as well as possible future drugs, patenting of new compounds for commercialization. They have patented a new group of CAIX-inhibitors which also act as hypoxic cell sensitizers in combination with radiotherapy and describe establishment of a new SME (DualTPharma) to develop the invention further into a drug. Partner 18/IVSAS have studied design and synthesis of carbonic anhydrase IX and XII inhibitors a non-invasive imaging diagnostic tool as well as possible future drugs. They conclude that interaction of carnosine with CAIX leads to conformational changes of CAIX and impaired formation of its metabolon, which in turn disrupts CAIX function. These findings suggest that carnosine could be a promising anticancer drug through its ability to attenuate the activity of CAIX. Partner 8
3.3 WP8: Integrated treatment protocols based on personalized and phenotyped data (Including only work performed in WP8 during the 5th period):
In this WP three deliverables have been completed during the 5th period, in accordance with the plan and contract as described in amended AnnexI of May 2013.
A) A first deliverable (D8.7) was completed by our partner 21/O-Lyng who reported data on the stratification of patients for therapy based on multivariate analysis including micro-metastasis. The conclusion was that the presence of micro-metastases at diagnosis as well as the angiogenesis and PI3K signatures are promising candidate biomarkers in rectal cancer and should be included in a larger study to assess their value in risk stratification. Moreover, the hypoxia related pimonidazole gene signature could have a potential value in risk stratification of patients with prostate cancer.
B) The second deliverable (D8.10) was completed by partner 6/AUH,AS on 3 different clinical trials on hypoxia-related molecular targets in patients with Head and Neck and Uterine cervix carcinomas, all showing beneficial effects of the hypoxic cell sensitized Nimorazole.
C) A third deliverable of WP8 (D8.11) was completed by our partner 2/MAASTRO on arguments to embark or not in a randomized trial boosting hypoxia with high dose radiation selecting patients based on the machine learning based predictive algorithm integrating imaging and non-imaging biomarkers. The general conclusion was that one found a reasonable voxelwise correlation for the two PET tracers, although also hypoxia outside high FDG uptake regions was observed. Hypoxia PET imaging gives complementary information to metabolic FDG imaging. But because at the moment we have more arguments, with pattern of relapse studies, that high FDG uptake areas are therapy resistant and because FDG uptake covers several cause of therapy resistance, we propose to focus the next dose painting trials on FDG rather than hypoxia. We suggest as well to systematically integrate hypoxia-imaging in the dose painting trials together with pattern of relapse studies to get more insights in the most therapy resistant voxels (hypoxic? Hypoxic and high FDG?
4 Fundamental research of biological nature:
4.1 WP2: Determination, visualization, modelling and characterization of the hypoxic tumour micro-environment:
Most deliverables within WP2 were completed during the first 3 years, which is natural since development of models had to be concentrated early in the program. Still the WP2-program was ongoing at high intensity in several of METOXIA’s partner organizations during the 4th and 5th periods and one deliverable was completed in accordance with the contract (D2.7 by 12/DHZM). Partner 20/GROW-UM reports an important finding related to the unfolded protein response (UPR) that proteins remain in a folding competent state during hypoxia, causing a burst of oxidation upon re-oxygenation. They also conclude that LAMP3 is a key mediator of hypoxia-driven nodal metastasis. 19/IBT reports that the expression level of both heterodimeric splicing factor U2AF subunits play an important role in hypoxia dependent pre-mRNA splicing regulation. 12/DHZM reports studies on the role of ROS in connection with reoxygenation events under WP2, Obj5. During the 5th period this partner completed an extensive deliverable D2.7 on the evaluation of bio-markers for tumour progression and/or metastatic risk and hypoxia/reoxygenation in animal models/patient samples. The conclusion was that NADPH oxidases are differentially expressed in distinct tumour entities. They are induced by hypoxia, and are present in hypoxic tumour areas in animal models and human tumour samples. In ovarian cancer, they are coexpressed with DNA damage proteins as markers of severe cellular stress such as hypoxia/reoxygenation and are more abundant in advanced stage tumours with adverse prognosis. Together with our findings that NADPH oxidases contribute to tumour growth, these results suggest that NADPH oxidases might act as biomarkers for hypoxia/reoxygenation, as well as for advanced stage in distinct tumours such as ovarian cancer. Also partner 17/UCL continued the studies on arginine depletion using both in vitro and in vivo models (WP2, Obj8).
4.2 WP3: Identifying the molecular mechanisms of hypoxia-driven metastasis:
There was no deliverable contracted for the 5th period in this WP, but several objectives were still worked on. Partners 10/KI-Cao and 5/UOXF.BP studied both the interplay between angiogenic as well as lymphangigenic factors and hypoxia. 10/KI-Cao report that TNF-α markedly promotes tumour lymphangiogenesis and lymphatic metastasis. The TNF-α-TNFR1 signalling pathway directly stimulates lymphatic endothelial cell (LEC) activity through a VEGFR3-independent mechanism. Partner 5/UOXF.BP concludes that RIOK3 is required for actin cytoskeletal organisation in both normoxia and hypoxia. Therefore, the development of RIOK3 inhibitors that prevent cell invasion is a promising anti-invasion strategy. Further biochemical studies are needed to define the kinase activity and substrates of RIOK3, and these efforts would aid in drug development projects. Partner 11/MAD also studied lymphangiogenesis in addition to the function of VHL in epithelial-mesenchymal transition (EMT). Their data indicate that when the PHD/VHL axis is operative and the mutated pVHL is still capable to bind HIF1α there is protection from the oxygen independent SART1 degradation. However under hypoxia the binding of HIF1α to a mutated VHL is compromised by the absence of PHD activity, which favours SART1 binding and subsequent proteasomal HIF1α degradation. Partner 18/IVSAS has studied involvement of CAIX in EMT and metastatic spread. This partner used an anti-CAIX antibody encapsulated into microspheres. They conclude that encapsulation of anti-CA IX antibody represent a novel strategy toward a treatment of cancer patients and could be valuable especially for those purposes where frequent administration of antibody is needed. To our knowledge, this was the first study establishing a model of anti-CAIX antibody encapsulation, which could be suitable for effective delivery and controllable release of antibody for CAIX-selective anticancer therapy.
4.3 WP4: Innovative new treatment management of metastasised tumours:
Three deliverables were reported completed during the 5th period in WP4 (D4.12 D4.18 D4.23.
A) A first deliverable (D4.12) was completed by our partner 3/UEDIN on xenograft studies evaluating the anticancer effects of candidate inhibitors in combination with RT and/or cytotoxics. The conclusion was that the initial studies with the METOXIA-patented CAIX inhibitors DH348 and FC3-98A indicate that modest but significant growth inhibition can be obtained in the subcutaneous MDA-MB-231 xenograft model when these agents are given at doses of 10 mg/kg/day or more. This result was confirmed for DH348 at a higher dose of 50mg/kg/day. The percentage of viable cells was decreased after treatment with DH348 but not with FC9-398A under the conditions tested.
B) A second deliverable (D4.18) was completed by our partner 20/Grow-UM on the theme: “Determination of the contribution of UPR and mTOR responsive pathways as mediators of hypoxia-induced metastasis”. The conclusion was that the data support accumulating evidence implicating ER stress responses as important contributors to aggressive hypoxic tumour biology. This potential importance of ER stress responses in cancer has led to some attempts to identify and incorporate agents aimed at disrupting ER homeostasis either alone or in combination with conventional agents. Our findings suggest that such agents are likely to be most effective when given in curative settings in combination with conventional therapy or with other agents that target well oxygenated cells. This approach would produce synergy not only by targeting distinct populations of cells in the tumour, but also by targeting intrinsic mechanisms that enable the hypoxic cells to escape and colonize distant nodes or other organs.
C) The third deliverable (D4.23) was completed by partner 8/UNIMAN on the theme: “Demonstration that bioreductive agents can target metastatic disease and could be used as adjuvants to standard therapy (M24), Novel agents.” The conclusion on this study was that bioreductive agents are effective in inhibiting metastatic growth when the metastatic growth is accompanied by the expression of abundant hypoxia.
In addition to the 3 deliverables in WP4 in the 5th period partner 10/Ki-Poellinger has studied hypoxia in relation to cell differentiation and EMT. Their findings indicate that in neuroblastoma cells there may be a link between hypoxia, Jmjd1a, G9a, epigenetic regulatory mechanisms and stem cell regulation. They conclude that the combined effect of dysregulated expression of Jmjd1a and G9a in hypoxia may determine the epigenetic landscape of target promoters and the selection of malignant, immature, stem cell like neuroblastoma cells. Partner 13/CNRS continued their study on the identification, development and validation of novel therapeutic targets induced by tumoural hypoxic stress (NHEs, MCTs, AEs, CA9/CA12, autophagy) and also on the design and validation of the best therapies combining anti-angiogenesis and blockers of pHi-regulating systems. They conclude that inhibition of the MCTs/BASIGIN complex, combined with phenformin provides a novel acute anticancer strategy to target highly glycolytic tumours. This genetic approach validates the anticancer potential of the MCT1 and MCT4 inhibitors in current development. Partner 6/AUH,AS has done pre-clinical testing of cycling and prolonged hypoxia as associated targets in animal models. They briefly conclude that they may be able to interfere with tumour metabolism directly when using a highly sensitive cell line and treating with a high phenformin dose (a biguanide). It remains to be determined however whether this translates into any antitumor effect. Whereas single treatment with 100 mg/kg phenformin was well-tolerated, long-term treatment (> 5 days) was associated with severe side effect. Partner 1/UIO has continued their studies on the effect of low dose-rate priming doses and how the factor produced by this irradiation (TGF-β3) is induced in mice after irradiation. The conclusion is that prior exposure to LDR irradiation may affect the response to subsequent radiation treatment or accidental radiation exposure through a mechanism involving TGF-β3 and iNOS activity. A similar protection can be induced 24 h post irradiation by a single injection of TGF-β3. The transient activation of TGF-β3 during hypoxia appears to be through a different mechanism involving HIF-1α. Partner 4/RUNMC has studied criteria for adding CAIX inhibitor to radiation therapy of larynx cancer and concludes that CAIX ectodomain shedding into the blood stream was decreased by the patented CAIX-inhibitor S4. The meaning of this finding is not clear yet. Partner 7/FMC has continued their work on 3D in vitro models based on alginate substrates (part of Obj13). This industrial partner now reports a method for predetermining if cells require the cell attachment peptide RDG for growth in alginate foam scaffolds. Additionally, they also report work towards commercialization of NovaMatrix-3D as a cell culture system.
5 Fundamental research within sensor development:
5.1 WP7: Development of technology for imitation of tumour micro-environment in model systems:
Seven deliverables were reported completed during the 4th and 5th periosd (D7.8 D7.9 D7.10 D7.13 D7.14 D7.17 and 7.18).
A) A first deliverable of the 4th period (D7.10) was completed by our partners 21/OU-Kragh and 15/Jobst on the establishment of a small scale culture system using micro fluidics to deliver cytokines and micro-dialysis for continuous change of culture medium. This work represent practical usage of the micro-fluidic system developed by partner 15/Jobst in an attempt to improve in vitro proliferation of CD34+ haematopoietic stem cells isolated from mobilized peripheral blood from adults. The system was integrated into the mini-laboratory developed by partner 16/VX. It turned out that the collaborating team run into several technical problems with the pump´s head. At the same time no significant expansion of CD34+ cells compared to the control was seen, except from the first experiment. Partner 21/OU-Kragh concluded that they would terminate the attemts to expand CD34+ cells further with the pump.
B) A second deliverable of the 4th period (D7.13) was completed by our partner 14/ALU-FR on integration of the micro fluidic and micro dialysis systems with sensor systems. In that work the aim is to integrate liquid transport into and out of cell cultures with the technology and platform of the Sensing Cell Culture Flask (SCCF) by on-chip microfluidic structures. The application of 14/ALU-FR’s surface micromachining process with photoresist as sacrificial layer was used for fabrication of microfluidic structures. Silicon nitride was used as material for the micro-channels. Therewith micro-channels in the height range of 2 to 30 µm could be fabricated. In order to allow higher channel widths and to obtain more robust structures the concept of pillar-like structures was successfully demonstrated. However, to achieve a stable process, silicon nitride formed by plasma-enhanced chemical vapour deposition is not the optimal material choice. Therefore on-going work is targeting Parylene as cover material.
C) A third deliverable of the 4th period (D7.17) was completed by partner 15/Jobst on the application development and peri-cellular oxygen tension control aiming to supplying ready-to-use tools for different applications to METOXIA partners. This work represent an attempt to meet with the partner requirements that a traditional clinical/biochemical analyzer function is widely missed. To provide this, the former fluid handling concept of 15/Jobst (using commercially available syringe pumps; see problems reported in deliverable 7.10 above) was replaced by small size-and-cost peristaltic pumps developed by 15/Jobst during the 4th period, and the range of applications extended by a “personal analyzer”. All other applications also (can) make use of the novel peristaltic pump. The conclusion was: “Utilizing the high content hardware toolkit presented in Deliverable 7.16 (M36) along with newly added further enabling components, four applications, including per-cellular oxygen control, were elaborated. The applications are already partially used by METOXIA partners. Application of these tools both in vitro and in vivo is planned to be further distribute amongst METOXIA partners and also amongst customers and partners outside METOXIA. Significant potential for product developments and economic exploitation by Jobst Technologies is foreseen.
D) A first deliverable of the 5th period (D7.8) was completed by our SME partner 16/VX on “upgrade from disinfection to sterilization methods not affecting sensitive equipment.” This is a description of the advanced sterilization procedures built into the 3rd generation of the ViVox TC-BiosystemTM. It was concluded that a sterile atmosphere in both the airlock and workspace was achieved in accordance to ISO class 3. The aim of the ViVoX Biosystem has been to achieve a level of containment where the risk of contamination biological material is as low as possible. Though, the ViVoX TC biosystem can not be used as an alternative to an autoclave, the cleanness in the workspace and airlock is extremely high and suitable for handling cellular material for reinserting in humans.
E) Also a second deliverable (D7.9) was completed by our partner 16/VX on “connecting/binding together monitoring and control of Biosystems placed at different institutions.” It has been demonstrated that protocols can be saved and used on several units. In the activity report (WP7, obj6) partner 16/VX shows plans to install several ViVox TC-BiosystemsTM in Denmark.
F) A third deliverable (D7.14) was completed by partner 14/ALU-FR on “Optimisation of ROS sensors, initiation of the first test phase.” The conclusion was that Superoxide sensors based on the technological platform of the SCCF have been produced and characterized with respect to sensor performance like sensitivity, selectivity and stability. It was shown that the electrochemical sensing approach can be used on bare gold electrodes utilizing the direct oxidation of superoxide anions on the working electrode. Sensor stability can be enhanced by covering the working electrodes with linear polyethyleneimine by dispensing from aqueous solution. Artificial superoxide production system xanthine/xanthine oxidase was characterised and utilised for sensor calibration. This characterisation was done using spectrophotometry and electron spin resonance spectroscopy as complementary techniques to electrochemical sensing approach.
G) A fourth deliverable (D7.18) was completed by partner 21/OU-Kragh-Kvalheim on “Role of Valporic acid, notch ligand and dimethoxyflavone on the stabilization of HIF 1α.” They conclude that the study suggests a positive effect of 3′4′--dimethoxyflavone (DMF) on human hematopoietic stem cells. However, it warrants further studies to validate the findings as well as to further explore the potential benefits of 3′4′-DMF, either alone or in synergy with other agents, in HSC biology.
In addition to the 4 deliverables in WP7 of the 5th period our SME partner 15/Jobst has worked on further development of minianalyzers in connection with obj1. The conclusion is that the analytical performance of the mini-analyser was found highly adequate for the purpose. A second generation of the mini-analyser was developed. Standard connectivity (including wireless), the pocket sized dimensions, and possible affordable pricing should allow successful marketing of such device after the product development phase. This product development after the expiration of METOXIA majorly encompasses software implementation and usability studies – and of course probing of the envisaged markets. It is therefore expected that the results generated during the METOXIA project will prevail and migrate in a variety of Biotech and Medtech applications.
Potential Impact:
The public health impact of the present project is potentially high since the activities have increased understanding of the basic mechanisms underlying metastatic spread of cancer. This includes mechanisms such as Notch/Hypoxia-Inducible Factor (HIF); CAIX-inhibitors; the Unfolded Protein Response (UPR); the mammalian Target of Rapamycin (mTOR); cellular-internal pH regulators (pHi regulators); hypoxic stress, autophagy and reactive oxygen species (ROS). Particularly important is the implicit aim to translate this increased understanding to the patient within the same consortium. As a consequence of the METOXIA-results the most important impact will hopefully be felt by patients with metastatic hypoxic cancers, whose quality of life is commonly impaired by pain and other distressing symptoms of advanced disease. The treatment-resistant hypoxic cells are probably one of the most important reasons for relapse after seemingly successful radical treatment and our ultimate goal is to reduce relapses as well as improve the effect of radical treatment.
Impacts in relation to the topics in question:
• We have determined the role of activated forms of Notch and HIF transcription factors to regulate EMT under hypoxic conditions, and define strategies to inhibit these effects. This has indicated an attack mechanism to directly influence the propensity of a cancer to spread and may as such represent a new treatment modality with a great impact on cancer treatment as a whole.
• CAIX inhibitors; novel drugs as based on METOXIA-inventions: CAIX inhibitors are potentially important both as hypoxia-specific biomarkers and as a stromal component of hypoxic tumours. The function of CAIX is important for the epithelial-mesenchymal transition (EMT) (which represent mechanisms for the cancer cells to migrate in general) and cancer-metastatic spread to distant parts of the body. CAIX-inhibitors therefore represent possible new anticancer drugs particularly in combination with radiotherapy or established chemotherapy which are known to be effective against the well-oxygenated regions of the tumour. These possibilities have been tested pre-clinically with newly-synthesized and patented CAIX-inhibitors and the basis has been laid for pursuing this principle into clinics in the present project. CAIX-inhibitors may thus have impact both on future diagnosis/imaging, on radical systemic cancer treatment and as potential radiation-modifying compounds.
• We have performed studies to largely clarify the role of the UPR and mTOR pathways as mediators of metastasis and the role of reoxygenation events. These molecular mechanisms are activated at specific levels of hypoxia (i.e. at more severe hypoxia than the HIF/CAIX-mechanism) and/or under particular conditions. Therefore they represent potential possibilities to attack conditions of tumour hypoxia that are different from those related to HIF. They are involved in cellular protein translation and thus influence cell-growth as such. This is a process which is to a small degree attacked directly by other treatment modalities and therefore could represent a valuable additional treatment load in combination therapy with traditional cancer treatment modalities.
• pHi-novel treatment strategies: We have combined anti-angiogenesis and blockers of pHi-regulating systems and the modulation of the angiogenesis inhibitor thrombospondin 1 (TSP1) by hypoxia. Two targets of interest related to pH-regulation and metabolism are the lactate transporter MCT4 and its subunit CD147/Basigin. Our partner 13/CNRS has tested an MCT4-inhibitor produced by AstraZeneca and confirmed its specificity for MCT4 and reports this compound to be ready for clinical trials in the near future.
• Effectors of autophagy (beclin): Identification, development and validation of novel therapeutic targets induced by tumoural hypoxic stress (NHEs, MCTs, AEs, CA9/CA12, autophagy). The negative aspects of hypoxia for cancer cells are so far not much turned to account in cancer therapy. We expect that increased knowledge in this field could have a considerable impact on cancer treatment and plan to see these mechanisms as potentials for exploitation in relation to the pHi strategies.
• We have contributed to clarify the role of reoxygenation events and reactive oxygen species (ROS) at the molecular and cellular level in tumour cells, vascular cells and stromal cells as well as in the in vivo tumour setting with regard to tumour growth and progression including EMT, invasion and metastasis. Together with the role of angiogenic factors and lymphangiogenesis which we believe may all be vital to metastatic spread this new knowledge may potentially give rise to new treatment modalities in the future.
Generally, the increased understanding of these mechanisms addressed above will have great impact on the possibility of development of new drugs and treatment strategies since they will present new targets for treatment of cancer. This will open up new areas for pharmaceutical companies to develop new drugs as well as new possibilities for clinicians to choose new strategies for cancer treatment. One new SME company named DualTPharma has been started up as a spin-off of METOXIA, working on the development of patented CAIX-inhibitors.
The present consortium has itself translated the new information into the clinical setting. The new knowledge has been exploited to develop:
A) Novel drugs (WP4, 5 and 8). i.e. CAIXi and pHi-regulators. In order to get a full overview of this result of METOXIA one needs to see the deliverable report D5.18 which summarizes the studies completed over the last 2.5 years of METOXIA leading up to clinical testing of our own new, patented compounds. A brief conclusion is that we have develop lead compounds of 2 different patents which show beneficial preclinical toxicology (i.e. largely no toxicity for relevant dosing), positive pharmacokinetics opening up for potential oral administered drugs and good effect results in CAIX-expressing cells in hypoxic areas in preclinical models both in vitro and in vivo.
B) New treatment strategies. One example is our plan to pursue the method of blocking the proton export of metastasizing cancer cells by combination of different modalities (NHE, CA9i etc). If this proves to have a therapeutic potential it will have opened up a whole new avenue for targeted cancer treatment with a major impact on clinical oncology. Another example is the newly patented alpha irradiating and bone seeking drug [223Ra] which has a palliative effect on metastases from prostate cancer where we expect to develop this treatment into life prolonging effects for these patients. A similar potential is also present for breast cancer since these also frequently develop life-threatening metastases. This project is now fully commercialized since Bayer pharma AG bought the Norwegian pharma company Algeta ASA which owned the patent to this drug.
C) Validated biomarkers and prevention research should lead to better strategies in both primary and secondary cancer prevention and clinical research will improve patient treatment and care. In the long run, the impact of the investment in this area will contribute to reaching the aims of reducing cancer incidence and mortality and improving quality of life for patients.
Our work on diagnosing/imaging and stratification likewise has a high potential impact on treatment outcome. The improved 3-dimensional imaging of hypoxic areas by PET-scanning usin will improve the focusing of enhanced irradiation to specifically hypoxic tumour areas and thus improve the clinical outcome, most likely without increased side-effects.
Our very recent pre-clinical observations of an unexpected high tumour cell-killing effect of low dose-rate beta irradiation and our observation of the possibility of inducing a transferable radio-protection factor needs more pre-clinical work, but could in the end lead to development of a new radiation treatment modality and even to better protection of the surrounding normal tissue.
Confirmation of our understanding that it is not moderate hypoxia as dominated by HIF regulated pathways, but rather variations in tumour-oxygenation between deeper and more moderate hypoxia that is most relevant to both metastasing and direct tumour cell response to therapy will have a major intellecutal impact on the whole field of research and its teaching.
A commercial impact has been achieved with our development of equipment for measuring and controlling the micro-environment, and for imaging. Furthermore, newly synthesised drugs such as CAIXi’s may become of interest to the pharmacological industry. Taken together the above is likely to create new employment.
Dissemination activities and exploitation of results:
The METOXIA project has contributed to dissemination largely through two different channels: a) Publications in international research journals with peer review, many of which are among the highest-ranked journals. b) Participation at international scientific conferences.
The number of published papers in scientific journals (with acknowledgement for METOXIA funding) is:
1st period: 30
2nd period: 72
3rd period 81
4th period 120
5th period: 185
The number of international conference presentations where METOXIA-findings and –funding has been reported are at least of the same order of magnitude as the number of scientific publications.
Final review article:
At the last METOXIA General Assembly meeting it was agreed that all partners should be invited to collaborate and together write an extensive review article where the background of METIXIA related to the general hypoxia-problem should be described. During the 5th period that endeavour was completed. In that review paper the main results of METOXIA have been set into context. On the 15th September 2014 the article was accepted for publication in the Journal of Enzyme Inhibition and Medicinal Chemistry. The title is: “Targeting Tumour Hypoxia to Prevent Cancer Metastasis. From Biology, Biosensing and Technology to Drug Development: the METOXIA Consortium”. This article sums up the work performed during the 5.5 years of the METOXIA project for the international scientific community. It also gives an updated over-view of the problem of hypoxia and cancer.
Patenting and secrecy strategy:
Regarding the inventions and patenting work it has been necessary to keep many results back from publication for a period of time. However, by the end of METOXIA it can be stated that all results can be published since all patents that will be protected in the future are now open.
List of Websites:
The public website address of METOXIA: http://www.metoxia.uio.no/
The website has a restricted area accessible for METOXIA partners.
Contact details:
Co-ordinator Professor Erik O. Pettersen,
Address: Department of Physics, The University of Oslo, B.O.Box 1048 Blindern, 0316 Oslo, Norway.
Tel +47 2285 5644 (Mobile: +47 9093 0743)
Email: e.o.pettersen@fys.uio.no
The METOXIA consortium answered a call which was ambitious, aiming to translate knowledge on the molecular machinery responsible for survival and spread of metastatic tumour cells under hypoxic conditions into innovative and validated molecular targets with therapeutic applicability that target the cancer cell or tumour stroma. The wording here can hardly be understood in any other way than to accept that the consortium also would have to protect its IP with the aim to try and secure a potential drug development. The level of ambition was furthermore strengthened by the statement that the consortium should include participants with ample clinical expertise to guarantee a clinical proof-of-principle. The METOXIA-consortium met all these premises. It was a large consortium, involving expertise over such a broad range as clinical and experimental medicine, molecular biology, synthetic chemistry, biophysics and electronics.
As indicated above several product developments are successful as planned. The greatest challenge is however the aim for the research to result in a new drug introducing a new principle into cancer therapy.
As the project stands by the end of METOXIA there are patents in international phase which can be further developed. We now see the possibility that inhibition with small molecules of a mechanism in the molecular machinery causing hypoxic cancer cells to survive and spread may come as far as to clinical tests in the relatively near future. There are several challenges to face before one reaches this goal, but we hope we have laid an important basis for the development.
During the 3rd and 4th periods we sub-contracted the company Cyprotex Discovery Ltd, to perform professional preclinical testing on 8 selected compounds from our patented sulfamates and dual-activity compounds. We have used these data together with our various partner’s effect testing in animal- and in vitro-models to complete a report (denoted deliverable D5.18) which the patent owners can use for further development of the compounds in collaboration with large pharma industry. Our view is that the potential social-economic impact of a new low-toxicity cancer treatment, halting or even stopping metastasis and improving the effect of conventional therapy can hardly be over-estimated. In our clinical programme we have cancers of the lung, breast, prostate, colon-rectum and head-neck; altogether some of the dominating cancer types in the world. So far we have reason to expect, although this has not been proven, that at least some patients within all these groups may benefit from a hypoxia-specific drug. We expect that the new knowledge brought up by the METOXIA-consortium will be of utmost value for future pharmaceutical development within this field.
Project Context and Objectives:
Scientific background of METOXIA
The METOXIA project has an over-all focus on the increased understanding of the regulatory mechanisms which help cancer cells survive under unfavourable micro-environmental conditions characterized by low oxygenation (denoted hypoxic areas). Solid cancers generally contain areas with lower levels of oxygen than what is usual in normal tissues and thus, the cancer cells learn how to adapt to such conditions. The METOXIA project furthermore aims to use the increased knowledge of the protective regulatory mechanisms to develop new principles for cancer detection and treatment: Since only cancer cells and not normal cells experience hypoxia one can develop cancer-specific detection and treatment by attacking molecules specific to the protective regulatory mechanisms activated by the cells under hypoxic conditions.
It has long been known that the problems created by hypoxic areas in solid cancers are serious for the patient: In short, cancer cells in such hypoxic micro-environments represent a reduced chance for successful treatment. With respect to radiotherapy the problem relates to increased dose tolerance for individual cells under hypoxic as compared to well-oxygenated conditions while for chemotherapy there is in some cases both increased dose tolerance and reduced drug access to the hypoxic as compared to well-oxygenated areas. Even if the patient is treated with surgery alone there are indications that the amount of tumour tissue which is hypoxic correlates negatively with the outcome for the patient. Recent research has furthermore shown that variable hypoxia in tumours is one of the major drivers of metastatic spread of cancer, the major cause of death by the disease. Thus, hypoxia is responsible for a double effect of reducing the potential of a successful treatment of the cancer patient: Resistance to treatment and ability to spread to distant parts of the body. The positive side of this problem is that the very low level of oxygen found in solid tumours is specific to cancer. Therefore, if one could develop new methods to specifically detect and inactivate cells in hypoxic areas one might obtain a cancer-specific effect, selective for the most harmful of the cancer cells. In the original call which METOXIA answered it was made clear that the research should not be limited to pre-clinical biological models, but should include proof-of-principle clinical research. As part of the consortium some of the leading cancer clinics in Europe are partners.
Main Objectives of METOXIA:
On basis of the clinical problems raised by hypoxic tumour micro-environments the work within METOXIA encompasses increasing the knowledge concerning the metastatic process in the hypoxic micro-environment at the molecular level in order to develop novel strategies for modification of this micro-environment and thus improve the efficacy of chemotherapy and radiotherapy. Major objectives are:
• Gain new knowledge about molecular mechanisms behind hypoxia-driven metastasis in order to reduce metastatic spread and increase patient cure rate.
• Develop improved methods to detect the propensity of a cancer to metastasise before the metastatic spread has become manifest clinically.
• Develop new treatment management of metastatic disease.
• Develop new methods to increase the effectiveness of treatment in hypoxic areas of the primary tumour.
• Development of new methods for detection/imaging of hypoxic areas.
• Generate pre-clinical models for the study of the role of hypoxia in metastases.
• Development of new tools for monitoring and control of peri-cellular micro-environment in vitro.
The partner organisation:
There are at present 21 partners in the consortium. The work was partitioned in 8 Work Packages (WP) of which one (WP9) relates to management and one (WP6) is empty (the activity originally included in WP6 was during the 2nd period amendment moved into WP5, WP7 and WP8 after a decision made at the 2nd General Assembly in June 2010). This reorganization turned out to be a success and no further reorganization of the consortium has been done during the 3rd, 4th and 5th periods.
Largely the non-management WPs can be divided into 2 main groups (WP6 is now empty):
• Work involving cancer patients (WP1, WP5 and WP8).
• Work involving primarily fundamental research, divided into two sub-areas:
A) Studies of primarily biological nature (WP2, WP3, WP4)
B) Development of more technical nature (development of equipment for monitoring and control of micro-environmental parameters related to hypoxia) (WP7).
The WEB-site of METOXIA is on the following address: http://www.metoxia.uio.no/
In line with the consortium agreement some of the reported material is confidential. The WEB-site thus is divided into an open area accessible for all and a restricted area accessible for partners only.
The work performed since the beginning of the project and the main results achieved so far.
Over the 5.5 year total duration of the METOXIA project 105 scientific deliverable-reports have been completed and, in addition also 18 management deliverables. Thus, considering the amount of work here reported only some major findings can be dealt with in a summary.
The line of research which has led to the most optimistic results concerning the possibility to develop a new drug for cancer therapy has the following background: The hypoxic micro-environment in solid cancers influences several regulatory cascades in cells. Since cancer cells in solid tumours experience such conditions over a long time these regulatory mechanisms represent a selective pressure of cancer cells into more hypoxia-tolerant phenotypes. This development correlates with a higher degree of malignancy and furthermore, with higher metastatic potential. For development of new diagnostic and treatment concepts it is important that these findings are connected to knowledge of hypoxia-regulated molecular pathways with which we can interfere. Even a small reduction in oxygenation results in regulatory consequences for a large part of the genome of our cells. There are several key regulators of these cascades, but the one responding to the smallest reduction in oxygenation is the protein HIF (Hypoxia Inducing Factor). This protein is what we denote a transcription factor; meaning that it can regulate activation of certain genes on DNA. It turns out that HIF-regulation involves a large number of genes and several processes which are vital for the cells and tissues to survive hypoxic conditions: Cell metabolism, energy production, angiogenesis (i.e. formation of blood vessels), extracellular matrix properties (EMT-epithelial mesenchymal transition) and pH-control. All these have been thoroughly studied within the METOXIA programme and valuable new knowledge has been accumulated. Notice however, that since HIF itself regulates so many processes it can hardly be considered as a target for treatment. Attacking HIF might affect too many normal processes and might not be cancer-specific. The focus for development of new cancer therapy therefore is not primarily on this central regulator, but rather on several of the cascades it regulates. One such mechanism has been developed into METOXIA-patented products of potential value as new anti-cancer drugs. This relates to certain small-molecular inhibitors of the HIF-regulated protein Carbonic Anhydrase IX (CAIX) involved in cellular uptake of CO2 for bio-mass synthesis and pH-regulation. These CAIX-inhibitors have in pre-clinical experiments shown a great potential for specific treatment effects on reduction of the metastatic potential of the primary tumour and also to some extent seem to increase the effect of traditional radiotherapy and some chemotherapy. Two arms of development have been worked out:
a) Several METOXIA-partners collaborate on the development of small-molecular sulfamate compounds (patented). These include chemicals with high specificity to the active site of the CAIX-protein. By specifically inhibiting CAIX our findings indicate that we have not only new possibilities for cancer treatment, but also new diagnostic principles. Thus, the METOXIA results point to new possibilities for drug development. Still, we need to add that we have also experienced the threshold for such development through our contact with large pharma: The practical problems related to development of hypoxia-specific treatment seem to exceed those of more traditional drug development. The reason for this is that the killing of hypoxic cells alone by no means can be expected to have a major influence on the primary tumour. Hypoxic cells proliferate less than well-oxygenated cells (i.e. they do not give rise to more cells as long as they are hypoxic). The problem they create is resistance against traditional treatment and increased ability to form distant metastasis. Thus, they are expected to be responsible for relapses after completed treatment. Development of a new hypoxia-specific agent therefore will have to be done in combination with traditional treatment and in the short run it may become difficult to observe an extra effect on the primary tumour compared to the effect of the traditional treatment alone.
b) As a possible improvement of the combination treatment a sub-group of METOXIA partners have synthesized a group of chemicals (patented) having a dual effect: These chemicals both have the ability to inhibit CAIX and to increase radiosensitivity of hypoxic cells. To follow up the development of these chemicals a new company (an SME) was founded shortly before the end of METOXIA and the aim is to perform preclinical and clinical studies of the effect of these compounds given simultaneously with radiation therapy.
Targets other than the HIF-regulated pathways have also been investigated and some found to be of great potential interest as targets for hypoxia-specific markers and treatment. Two targets of interest related to pH-regulation and metabolism are the lactate transporter MCT4 and its subunit CD147/Basigin. Our partner 13/CNRS has tested an MCT4-inhibitor produced by AstraZeneca and confirmed its specificity for MCT4 and reports this compound to be ready for clinical trials in the near future. Also the two stress-related processes initiated under hypoxic conditions denoted unfolded protein response (UPR) and the mammalian Target of Rapamycin (mTOR) have been investigated by several partners with 20/GROW-UM as most central. Both processes act as hypoxia sensors in the cells. UPR is a process which leads to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum while mTOR is a protein kinase which regulates cell growth. In a collaboration between 20/GROW-UM, 2/MAASTRO and 5/UOXF.BP it was shown that hypoxia activation of UPR induces expression of the metastasis-associated gene LAMP3, thus identifying LAMP3 as a new candidate biomarker of UPR activation by hypoxia in tumours and a potential mediator of hypoxia-induced metastasis. Regarding mTOR-inhibitors, these have been introduced as anti-cancer drugs for renal cell carcinoma (tensirolimus and everolimus).
Another field which has led to filed patent applications is the induction (and also counteraction) of radiation-induced resistance to chemotherapeutic compounds by low dose-rate irradiation. During the 3rd and 4th periods this research at 1/UIO indicated that radiosensitivity of cells can be modified even several hours after the cell was irradiated and that the effect is easily induced also in animals. The relevance to METOXIA stems from our finding that the mechanism activated by low dose-rate irradiation is also activated by variable hypoxia. Thus, we have the possibility to attack mechanisms responsible for adaptation to chemotherapy resistance during hypoxic conditions. So far we do not have a patented compound for this action, but in the 5th period we have shown in animal experiments that the protein to inhibit is transforming growth factor beta3 (TGF-beta3), and we have shown that at least one commercial chemical, not developed into a drug can inhibit TGF-beta3 and counteract radio- and chemo-resistance.
Various new biological test models as well as technical measurement devices have been developed. New in vitro 3D-models as based on purified alginates for pre-clinical studies of efficiency of new modalities have been taken into use towards proof-of-principle testing of treatment benefit. Also new animal models for pre-clinical studies of efficiency of new modalities have been developed towards proof-of-principle testing of treatment benefit. Special emphasis has been placed on development of CAIX knock-down models so that the effect of CAIX-inhibitors can be assured to result from a specific effect on the CAIX-protein and not result from some unspecific toxicity. For the testing of effects on metastasis orthotopically implanted tumours have been used. Development of new sensor- and micro-fluidic technology and instrumentation for detection and control of oxygen and other substances in the cell and tissue micro-environments are being developed. A disposable cell culture flask with a sensors molded into the flask bottom has been developed.
A central clinical investigation is the study of micro-metastases (i.e. circulating cancer cells) as a means for individual evaluation of the metastatic potential of cancers in patients. Also our effort to develop a comprehensive classification of tumour hypoxia, anoxia and reoxygenation has been highly prioritized as it aims to allow clinicians to predict response to targeted agents which include those activated by hypoxia. In this connection detection and visualization of hypoxia in patient tumours are highly prioritized and a promising new method passed phase I study during the 3rd period and protocols have been completed during the 4th period for this to continue with 3 phase II studies which has been ongoing during the 5th period of METOXIA. This method involves non-invasive PET imaging of tumour hypoxia by use of a radioactive tracer denoted [18F]HX4, a member of the 2-nitroimidazole family of chemicals. The special quality of these chemicals, denoted bioreductive compounds, is that they are chemically modified under hypoxic conditions and thereby can represent both chemotherapeutic drugs specific to hypoxic cells and also offer PET-imaging of reactive activity. This particular study was not included in the plans of METOXIA from the start, but is an example of the importance of amendments of new possibilities coming up during the development of the project.
Although the idea of targeting a sub-set of hypoxic tumour cells to improve treatment outcome is intriguing, clinical studies proving that not only do hypoxic intervention work but it does so only in patients with hypoxic tumors has not been conducted until now. In this context, the development of a hypoxia gene signature at 6/AUH,AS which, when tested in material from a large randomized phase III study, was able to identify those HNSCC patients which benefit from hypoxic intervention with Nimorazole (radiation sensitizer) was an important step forward. As a direct spin-off, several multi-center clinical projects that will further clarify the full potential of this gene signature to identify patients suitable for hypoxic intervention has been launched.
Also development of bone-seeking alpha-particle-emitting radio-nuclides (Alpharadin®) for localized treatment of bone metastases has been involved in METOXIA. As alpha-particles deliver their energy concentrated (i.e. with high LET) this type of radiation is equally effective under hypoxic as under aerobic condition and is thus expected to abolish the radioprotective effect of hypoxia. This project was started before METOXIA by the Norwegian company Algeta. A global phase III clinical trial (ALSYMPCA) with Alpharadin® in patients with castration-resistant prostate cancer (CRPC) and bone metastases was successfully completed in June 2011. Algeta was recently bought by Bayer Pharma AG for more than 2 billion euros and Alpharadin is presently manufactured under the trade name Xofigo.
METOXIA furthermore takes part in the large-scale randomized trial started several years ago in the Netherlands comparing Accelerated Radiotherapy (AR) with Accelerated Radiotherapy plus Carbogen and Nicotinamide (ARCON) in laryngeal cancer. In this large multi-centric trial METOXIA has taken part in the evaluation of consequences of Carbogen and Nicotinamide in relation to degree of tumour hypoxia. Despite lack of benefit in local tumour control for advanced laryngeal cancers the results are promising, indicating a significant gain in regional control rate, with equal levels of toxicity, and even indicating increased disease-free survival for the patients having the most hypoxic tumours.
The expected final results and their potential impacts and use (including socio-economic impact and the wider societal implications of the project so far).
The METOXIA consortium answered a call which was ambitious, aiming to translate knowledge on the molecular machinery responsible for survival and spread of metastatic tumour cells under hypoxic conditions into innovative and validated molecular targets with therapeutic applicability that target the cancer cell or tumour stroma. The wording here can hardly be understood in any other way than to accept that the consortium also would have to protect its IP with the aim to try and secure a potential drug development. The level of ambition was furthermore strengthened by the statement that the consortium should include participants with ample clinical expertise to guarantee a clinical proof-of-principle. The METOXIA-consortium met all these premises. It was a large consortium, involving expertise over such a broad range as clinical and experimental medicine, molecular biology, synthetic chemistry, biophysics and electronics. As indicated above several product developments are successful as planned. The greatest challenge is however the aim for the research to result in a new drug introducing a new principle into cancer therapy. As the project stands by the end of METOXIA there are patents in international phase which can be further developed. We now see the possibility that inhibition with small molecules of a mechanism in the molecular machinery causing hypoxic cancer cells to survive and spread may come as far as to clinical tests in the relatively near future. There are several challenges to face before one reaches this goal, but we hope we have laid an important basis for the development. During the 3rd and 4th periods we sub-contracted the company Cyprotex Discovery Ltd, to perform professional preclinical testing on 8 selected compounds from our patented sulfamates and dual-activity compounds. We have used these data together with our various partner’s effect testing in animal- and in vitro-models to complete a report which the patent owners can use for further development of the compounds in collaboration with large pharma industry. Our view is that the potential social-economic impact of a new low-toxicity cancer treatment, halting or even stopping metastasis and improving the effect of conventional therapy can hardly be over-estimated. In our clinical programme we have cancers of the lung, breast, prostate, colon-rectum and head-neck; altogether some of the dominating cancer types in the world. So far we have reason to expect, although this has not been proven, that at least some patients within all these groups may benefit from a hypoxia-specific drug. We expect that the new knowledge brought up by the METOXIA-consortium will be of utmost value for future pharmaceutical development within this field.
Project Results:
Main S & T results/foreground of METOXIA
A summary over the main findings of a large-scale project like METOXIA covering 5.5 years of study is complicated since the consortium covers almost every aspect of cancer research related to tumour micro-environments. The amount of results and findings to be analyzed are large. In the following, therefore, only brief references are made to the various main findings.
1 Hypoxia: Scientific background for translational cancer research.
Solid cancers generally contain areas with abnormally low levels of oxygen. Such areas are denoted hypoxic. It has long been known that cancer cells in such hypoxic micro-environments are resistant to treatment. Recent research has furthermore shown that variable hypoxia in tumours is one of the major drivers of metastatic spread of cancer, the major cause of death by the disease. Thus, hypoxia is responsible for a double effect of reducing the potential of a successful treatment of the cancer patient: Resistance to treatment and ability to spread. At the same time the very low level of oxygen found in solid tumours are specific to cancer. Therefore, if one could develop new methods to specifically detect and inactivate cells in hypoxic areas one might obtain a cancer-specific effect, selective for the most harmful of the cancer cells. This development is the over-all task of the METOXIA project.
2 Securing of IP and strategy to develop a cancer medicine (development during 3rd, 4th and 5th period):
Development of new compounds for inhibition of Carbonic Anhydrase IX (CAIX) is a major endeavour of METOXIA which has been given extensive effort by a sub-group (working across both the organizational structures mentioned above) for all the first 4 periods. This sub-group was flexible with respect to member organisations, those who were interested in this activity and had the capacity to contribute were accepted to join in. However, this success could naturally not be planned from the start and therefore some amendments to the plan have been worked out earlier. For a full description of the chemistry, see PCT patent application Number WO 2011/098610 A1.
During the 3rd period contact was made with Cancer-Research-UK (CR-UK) to seek advice for a possible step-in by them for further drug development. In the 4th period, according to their advice, the METOXIA management contacted a professional company, Cyprotec Discovery Ltd to perform necessary pre-clinical testing before an application to be worked out forCR-UK’s New Agents Committee (deliverable D5.18). During the spring-summer 2014 however, CR-UK together with Bayer AG sent to the METOXIA patent owners a conclusion that they will not take on the development of a compound which can only be used in combination treatment and which can not reduce the volume of the primary tumour alone. The problem with a hypoxia-specific drug is that the killing of hypoxic cells alone will hardly reduce the primary tumour (or any tumour) because hypoxic cells can hardly grow and divide. Such specific cell kill must be combined with radical radiotherapy (or chemotherapy) to kill aerobic cells and the benefit for the patient is expected to be reduced late relapse. Such drug development therefore is expected to be extra expensive and time-consuming.
Major findings and deliverables:
2.1 Selection of patents.
Although compounds from 4 different patents were originally included in the Cyprotex investigation their toxicology results as well as pharmacokinetics led to the determination by the METOXIA CAIX-sub-group to concentrate their investigations on compounds from only two patents for partner effect studies: Those two patents were:
o The sulfamate patent (WO 2011/098610 A1: Titled: “Carbonic Anhydrase Inhibitors”)
o The dual-activity patent (US 2013/0274305 A1: Titled: “Cancer Targeting Using Carbonic Anhydrase Isoform IX Inhibitors”).
2.2 Selection of compounds for effect studies.
From the sulfamate patent 3 compounds have been tested: S4, FC9-398A and FC9-403A. From the dual-activity patent the compound DH348 was tested. This compound was later denoted DTP348.
The selection of S4 was performed before the Cyprotex studies and was done on basis of data from 9/UNIFI showing that this compound had the strongest selectivity for the active site of CAIX protein among the tested sulfamate compounds. As shown by Figure 6 S4 was however shown to have a low bioavailability when given by oral administration, while FC9-398A had far better bioavailability. Thus FC9-398A was included in the effect studies. Early tests of the compound FC9-403A indicated positive effects also of this compound and it was decided to include also this one in some of the effect studies.
Compound DH348 was shown to have very high bioavailability, but short half-life. This compound was selected due to early findings of efficient combination effects with radiation.
2.3 Toxicology and pharmacokinetics.
• FC9-403A had a limited solubility with a lower bound of 3µM whereas all the remaining compounds had lower limits of solubility of 30µM or higher.
• None of the tested compounds were found to have any significant blood brain barrier permeability.
• Protein binding data indicated between 89 and 98% bound of the sulfamate compounds only between 55 and 70% of the dual activity compounds were bound.
• Regarding liver metabolism (by Cloe Screeen Microsomal Stability Test) none of the 10 compounds tested by Cyprotex have a high clearance (DCT-24a not measured). FC9-398A, and DH348, are metabolically stable in human and mouse. FC9-403A has moderate human clearance with low mouse clearance and S4 is metabolically stable in the human with moderate clearance in the mouse.
• The 11 tested compounds were found to have no or weak Cytochrome P450- inhibition.
• The data in Table VIII suggest that none of the tested compounds would have cardiac toxicity since there was no or weak hERG channel inhibition by all compounds.
• Mouse PK-studies:
a) For FC9-398A solubility was not a problem and bioavailabilty over the 0-8 hr time period tested was high (57.6%). FC9-398A also demonstrates the lowest rate of clearance following an IV dose (59.9 .l/min/kg) suggesting that the compound is more metabolically stable than the other sulfamate compounds tested. S4 and FC9-403A produced a bioavailability value of only 3% and 4.5% respectively over the 0-8 hr time course possibly making these less interesting for oral administration.
b) For DH348 there was no adverse effect observed in the animals from either dose route. Bioavailability was far above 100% indicating non-linear pharmacokinetic behaviour (either saturation of an efflux transporter or saturation of the metabolic clearance rate).
• In Silico prediction of PK in human and mouse:
a) FC9-398A and S4 were found to have the lowest clearance of about 0.3 and 0.8 mL/min/kg for human and mouse respectively. The corresponding values for DH348 were 2.5 and 4.5 mL/min/kg and for FC9-403A it was 1.2 and 8 mL/min/kg.
b) From Figures 27 to 38 a comparison is done regarding the two prioritized candidate compounds from the two selected patents: FC9-398A and DH348. It is concluded that peak drug levels in various tissues are 2 to 10 times higher with FC9-398A compared to DH348 and that that this difference is over-all higher regarding long-term levels due to lower clearance of FC9-398A as compared to DH348.
2.4 Effect studies performed by METOXIA partners.
8/UNIMAN concludes that S4 (and FC9-398A) work as an anti-tumour drug in cell lines that are highly dependent on CAIX (here SCLC). In contrast, in cell lines which are less CAIX dependent S4 has anti-metastatic effects (here MDA-MB-231, FaDu).
2/MAASTRO concludes that oral administration of FC9-398A in the H460 xenograft model is ineffective in reducing primary tumour growth. Furthermore, it might be suggested from these results that the metastatic potential of the H460 tumour cells is not significantly affected by FC9-398A treatment. Histological analysis of the lungs needs to give final conclusions.
3/UEDIN concludes that FC9-403A inhibits explant invasion at all concentrations used between 1 and 100 µM, and causes regression of invasion. S4 also strongly inhibits explant invasion at higher concentrations but this preliminary data indicates that it is less effective than FC9-403A at reversing invasion. Immunohisto-chemistry of S4-treated explants indicates that the mechanisms responsible for the inhibitory effects of these compounds on tumour explant growth and invasion involve an increase in levels of apoptosis and a decrease in levels of proliferation. The novel inhibitor DTP348 (DH348) showed little efficacy in in vitro 3D models, but significantly decreased growth, cell viability and cell proliferation in xenograft models. The explant model suggested that radiation resistance may be reversed by FC9-398A treatment. Unfortunately none of the explant materials used to examine DTP348 proved to be resistant to radiation, therefore the effect of this compound on radiation sensitivity could not be assessed in the present study.
4/RUNMC concludes that CAIX ectodomain shedding into the blood stream was decreased by S4. As the meaning of this observation is not clear, further studies are required to elucidate whether the CAIX ectodomain has a paracrine or autocrine signalling function in cancer biology. S4 did not influence the tumour micro-environment in a laryngeal tumour model in terms of the amount of proliferation, apoptosis, necrosis and hypoxia.
3 Studies directly related to patients:
3.1 WP1: Increasing the treatment effect on the primary tumour. (Including work performed in WP1 in the 1st, 2nd , 3rd, 4th and 5th periods.):
One early deliverable completed at Month 4 by Lambin, 2/MAASTRO (D1.1) (WP1: Obj2) was followed up with a second deliverable at month 18 as planned. The value of adding a new respiratory amplitude-based PET reconstruction method called Optimal Gating (OG) was tested. The aim was to provide accurate image quantification in lung cancer. The conclusion was that optimal gating PET is a better alternative to both 3D PET suffering from breathing averaging and the noisy images of a 4D PET acquisition. Partner 2/MAASTRO also completed an extensive deliverable (D1.3) (WP1: Obj1) on a planned randomized phase II study to be conducted in patients with inoperable stage II or III non-small cell lung cancer (NSCLC). The patients are randomized to receive the standard 66 Gy given in 24 fractions of 2.75 Gy with an integrated boost to the primary tumour as a whole (Arm A) or with an integrated boost to the 50% SUVmax area of the primary tumour (Arm B). The ultimate aim of the study is to increase the local progression-free survival (LPFS) after 1 year from 70 % to 85 %.
Our partner 4/RUNMC completed his deliverable (D1.4) on co-localization of hypoxia-markers and EGFR in the 2nd period and found a correlation between both these phenomena, a correlation with distance from blood vessels and, importantly; association with poor loco-regional clinical control. EGFR therefore could function to increase survival of cancer cells in the hypoxic tumour micro-environment. Furthermore, co-localization of hypoxic and metabolic markers (HIF-1α, CAIX, LDH-5, GLUT-1, MCT1 and MCT4) was, in a separate report shown to give additional clinical information compared to single protein analyses.
In the 3rd period partner 4/RUNMC completed his deliverable (D1.5) on the large-scale randomized trial started several years ago in the Netherlands comparing Accelerated Radiotherapy (AR) with Accelerated Radiotherapy plus Carbogen and Nicotinamide (ARCON) in laryngeal cancer. In this large multi-centric trial METOXIA takes part in the evaluation of consequences of Carbogen and Nicotinamide in relation to degree of tumour hypoxia. Despite lack of benefit in local tumour control for advanced laryngeal cancers the results are promising, indicating a significant gain in regional control rate, with equal levels of toxicity, and even indicating increased disease-free survival for the patients having the most hypoxic tumours.
In the 4th period the work in WP1 continued although all contracted deliverables had been completed at M36. Partner 4/RUNMC have continued their work on the boosting of the hypoxic radio-resistant areas in the tumour with high doses of radiotherapy. While the FDG-high uptake BTV boost is ongoing, they will soon also start the HX4-high uptake boost study which is a collaboration with partner 2/MAASTRO.
In the 5th period partner 2/MAASTRO continued the work on the possibility to boost radiation doses to hypoxic areas of the tumour. The efficiacy of using [18F]DG PET to select the tumour biological target volume and trying to validate the optimal dose delivery protocol has been prioritized. The conclusion is, however rather negative: The results suggest that no clinically meaningful differences may exist between dose redistribution based on high or low FDG-PET uptake regions compared to uniform irradiation. In the future, it will be important to test other biological features of the tumour microenvironment. Partner 2/MAASTRO also tested different PET tracers (i.e. [18F]FMISO, [18F]FAZA and [18F]HX4) but concluded that each tracer has its own strengths and depending on the question to be answered a different tracer can be put forward.
3.2 WP5: Clinical application of new drugs and treatment strategies involving both patient and animal studies (Including only work performed in WP5 during the 4th and 5th period):
A total of 8 deliverables were completed in WP5 during the two last periods in accordance with the plan and contract as described in amended AnnexI of May 2013.
A) One deliverable (D5.10) was completed by our partner 7/FMC who reported data on a clinical trial synopsis and design for evaluating the effect of alpha emitters in metastatic tumours within a hypoxic environment. This project was started before METOXIA by the Norwegian company Algeta. A global phase III clinical trial (ALSYMPCA) with Alpharadin® in patients with castration-resistant prostate cancer (CRPC) and bone metastases was successfully completed in June 2011. Algeta was recently bought by Bayer Pharma AG for more than 2 billion euros and Alpharadin is presently manufactured under the trade name Xofigo.
B) A second deliverable (D5.13) by our partner 3/UEDIN was originally planned to be performed on the established CA-inhibitory drug acetazolamide. This was in reality changed during earlier amendments because acetazolamide is hypoxia non-specific. The study instead was done on patented CAIX-inhibitors with high specificity. See text to deliverable 5.13 and 5.16II (below).
C) A third deliverable (D5.15) was completed by our partner 21/OU-Lyng on the Analysis of the micro-metastasis frequency in prostate cancer patients and of signal transduction and metabolic profile (MR) in patients with and without micro-metastases. The disseminated tumour cell (DTC)- and pimonidazole data suggest the use of the surgical cohort and the applied methods to further explore the relationship between hypoxia and DTCs. The HIF1 and apparent diffusion coefficient (ADC) data were consistent with a decrease in the hypoxic fraction, suggesting that HIF1 expression and ADC could be possible biomarkers of hypoxia in prostate cancer.
D) A fourth deliverable (D5.16II) was completed by our partner 3/UEDIN in collaboration with 9/UNIFI on assessment of a panel of selected, patented CAIX-inhibitors on cell migration and 3D assays (D5.16II; M48). This deliverable was added during amendment of Jan 2012 as a follow-up of the testing in 2D-assays of a larger selection of >20 compounds (deliverable 5.16I at M36). The deliverable represents a vital step in our effort to develop our patented compounds into a drug. The results reported in D5.16II forms a major basis for the selected compounds to be further tested in the 5th period by Cyprotex for PK and PD studies.
E) Also a fifth deliverable (D5.17) was completed by our partner 3/UEDIN on the assessment of novel CAIX and sodium-proton exchange inhibitors on the growth and invasion of breast cancer explants. Initial experiments focused on CAIX inhibitor S4. Again this represent a further follow-up of the effects of the patented compounds. The conclusion was that two compounds in particular was efficient in inhibition of explants invasion, the compounds encoded by S4 and FC903A. Immunohistochemistry of S4-treated explants indicateed that the mechanisms responsible for the inhibitory effects of these compounds on tumour explant growth and invasion involve an increase in levels of apoptosis and a decrease in the extent of cell proliferation.
F) One deliverable (D5.3) was completed by our partner 21/OU-Lyng who reported data on signal transduction in relation to tumour physiological and metabolic parameters in rectal and prostate cancer, and analysis of the above mentioned parameters against clinical outcome.
G) A second deliverable (D5.12) was completed by our partner 3/UEDIN on randomised trial of preoperative radiotherapy+/-CA9 inhibitor in early breast cancer (T1-T2
In addition to the 8 deliverables in WP5 partner 21/OU-Lyng has during the last period studied gene expression and signal transduction profiles associated with tumour hypoxia (by array slides) and the occurrence of micro-metastases. Several questions were raised regarding timing of tracers in this study. Partner 6/AUH,AS studied the clinical potential of non-invasive imaging methods (FMISO, FAZA, PET with CA9i, DCE-MRI) for identifying hypoxia. As in the study of 21/OU-Lyng several questions were raised and more research is needed in order to draw a clear conclusion. Partner 9/UNIFI has studied design and synthesis of carbonic anhydrase IX and -XII inhibitors as non-invasive imaging diagnostic tools as well as possible future drugs, patenting of new compounds for commercialization. Several structures are presented which may represent effective inhibitors as potential drugs. Partner 2/MAASTRO has studied design and synthesis of carbonic anhydrase IX and XII inhibitors as non-invasive imaging diagnostic tools as well as possible future drugs, patenting of new compounds for commercialization. They have patented a new group of CAIX-inhibitors which also act as hypoxic cell sensitizers in combination with radiotherapy and describe establishment of a new SME (DualTPharma) to develop the invention further into a drug. Partner 18/IVSAS have studied design and synthesis of carbonic anhydrase IX and XII inhibitors a non-invasive imaging diagnostic tool as well as possible future drugs. They conclude that interaction of carnosine with CAIX leads to conformational changes of CAIX and impaired formation of its metabolon, which in turn disrupts CAIX function. These findings suggest that carnosine could be a promising anticancer drug through its ability to attenuate the activity of CAIX. Partner 8
3.3 WP8: Integrated treatment protocols based on personalized and phenotyped data (Including only work performed in WP8 during the 5th period):
In this WP three deliverables have been completed during the 5th period, in accordance with the plan and contract as described in amended AnnexI of May 2013.
A) A first deliverable (D8.7) was completed by our partner 21/O-Lyng who reported data on the stratification of patients for therapy based on multivariate analysis including micro-metastasis. The conclusion was that the presence of micro-metastases at diagnosis as well as the angiogenesis and PI3K signatures are promising candidate biomarkers in rectal cancer and should be included in a larger study to assess their value in risk stratification. Moreover, the hypoxia related pimonidazole gene signature could have a potential value in risk stratification of patients with prostate cancer.
B) The second deliverable (D8.10) was completed by partner 6/AUH,AS on 3 different clinical trials on hypoxia-related molecular targets in patients with Head and Neck and Uterine cervix carcinomas, all showing beneficial effects of the hypoxic cell sensitized Nimorazole.
C) A third deliverable of WP8 (D8.11) was completed by our partner 2/MAASTRO on arguments to embark or not in a randomized trial boosting hypoxia with high dose radiation selecting patients based on the machine learning based predictive algorithm integrating imaging and non-imaging biomarkers. The general conclusion was that one found a reasonable voxelwise correlation for the two PET tracers, although also hypoxia outside high FDG uptake regions was observed. Hypoxia PET imaging gives complementary information to metabolic FDG imaging. But because at the moment we have more arguments, with pattern of relapse studies, that high FDG uptake areas are therapy resistant and because FDG uptake covers several cause of therapy resistance, we propose to focus the next dose painting trials on FDG rather than hypoxia. We suggest as well to systematically integrate hypoxia-imaging in the dose painting trials together with pattern of relapse studies to get more insights in the most therapy resistant voxels (hypoxic? Hypoxic and high FDG?
4 Fundamental research of biological nature:
4.1 WP2: Determination, visualization, modelling and characterization of the hypoxic tumour micro-environment:
Most deliverables within WP2 were completed during the first 3 years, which is natural since development of models had to be concentrated early in the program. Still the WP2-program was ongoing at high intensity in several of METOXIA’s partner organizations during the 4th and 5th periods and one deliverable was completed in accordance with the contract (D2.7 by 12/DHZM). Partner 20/GROW-UM reports an important finding related to the unfolded protein response (UPR) that proteins remain in a folding competent state during hypoxia, causing a burst of oxidation upon re-oxygenation. They also conclude that LAMP3 is a key mediator of hypoxia-driven nodal metastasis. 19/IBT reports that the expression level of both heterodimeric splicing factor U2AF subunits play an important role in hypoxia dependent pre-mRNA splicing regulation. 12/DHZM reports studies on the role of ROS in connection with reoxygenation events under WP2, Obj5. During the 5th period this partner completed an extensive deliverable D2.7 on the evaluation of bio-markers for tumour progression and/or metastatic risk and hypoxia/reoxygenation in animal models/patient samples. The conclusion was that NADPH oxidases are differentially expressed in distinct tumour entities. They are induced by hypoxia, and are present in hypoxic tumour areas in animal models and human tumour samples. In ovarian cancer, they are coexpressed with DNA damage proteins as markers of severe cellular stress such as hypoxia/reoxygenation and are more abundant in advanced stage tumours with adverse prognosis. Together with our findings that NADPH oxidases contribute to tumour growth, these results suggest that NADPH oxidases might act as biomarkers for hypoxia/reoxygenation, as well as for advanced stage in distinct tumours such as ovarian cancer. Also partner 17/UCL continued the studies on arginine depletion using both in vitro and in vivo models (WP2, Obj8).
4.2 WP3: Identifying the molecular mechanisms of hypoxia-driven metastasis:
There was no deliverable contracted for the 5th period in this WP, but several objectives were still worked on. Partners 10/KI-Cao and 5/UOXF.BP studied both the interplay between angiogenic as well as lymphangigenic factors and hypoxia. 10/KI-Cao report that TNF-α markedly promotes tumour lymphangiogenesis and lymphatic metastasis. The TNF-α-TNFR1 signalling pathway directly stimulates lymphatic endothelial cell (LEC) activity through a VEGFR3-independent mechanism. Partner 5/UOXF.BP concludes that RIOK3 is required for actin cytoskeletal organisation in both normoxia and hypoxia. Therefore, the development of RIOK3 inhibitors that prevent cell invasion is a promising anti-invasion strategy. Further biochemical studies are needed to define the kinase activity and substrates of RIOK3, and these efforts would aid in drug development projects. Partner 11/MAD also studied lymphangiogenesis in addition to the function of VHL in epithelial-mesenchymal transition (EMT). Their data indicate that when the PHD/VHL axis is operative and the mutated pVHL is still capable to bind HIF1α there is protection from the oxygen independent SART1 degradation. However under hypoxia the binding of HIF1α to a mutated VHL is compromised by the absence of PHD activity, which favours SART1 binding and subsequent proteasomal HIF1α degradation. Partner 18/IVSAS has studied involvement of CAIX in EMT and metastatic spread. This partner used an anti-CAIX antibody encapsulated into microspheres. They conclude that encapsulation of anti-CA IX antibody represent a novel strategy toward a treatment of cancer patients and could be valuable especially for those purposes where frequent administration of antibody is needed. To our knowledge, this was the first study establishing a model of anti-CAIX antibody encapsulation, which could be suitable for effective delivery and controllable release of antibody for CAIX-selective anticancer therapy.
4.3 WP4: Innovative new treatment management of metastasised tumours:
Three deliverables were reported completed during the 5th period in WP4 (D4.12 D4.18 D4.23.
A) A first deliverable (D4.12) was completed by our partner 3/UEDIN on xenograft studies evaluating the anticancer effects of candidate inhibitors in combination with RT and/or cytotoxics. The conclusion was that the initial studies with the METOXIA-patented CAIX inhibitors DH348 and FC3-98A indicate that modest but significant growth inhibition can be obtained in the subcutaneous MDA-MB-231 xenograft model when these agents are given at doses of 10 mg/kg/day or more. This result was confirmed for DH348 at a higher dose of 50mg/kg/day. The percentage of viable cells was decreased after treatment with DH348 but not with FC9-398A under the conditions tested.
B) A second deliverable (D4.18) was completed by our partner 20/Grow-UM on the theme: “Determination of the contribution of UPR and mTOR responsive pathways as mediators of hypoxia-induced metastasis”. The conclusion was that the data support accumulating evidence implicating ER stress responses as important contributors to aggressive hypoxic tumour biology. This potential importance of ER stress responses in cancer has led to some attempts to identify and incorporate agents aimed at disrupting ER homeostasis either alone or in combination with conventional agents. Our findings suggest that such agents are likely to be most effective when given in curative settings in combination with conventional therapy or with other agents that target well oxygenated cells. This approach would produce synergy not only by targeting distinct populations of cells in the tumour, but also by targeting intrinsic mechanisms that enable the hypoxic cells to escape and colonize distant nodes or other organs.
C) The third deliverable (D4.23) was completed by partner 8/UNIMAN on the theme: “Demonstration that bioreductive agents can target metastatic disease and could be used as adjuvants to standard therapy (M24), Novel agents.” The conclusion on this study was that bioreductive agents are effective in inhibiting metastatic growth when the metastatic growth is accompanied by the expression of abundant hypoxia.
In addition to the 3 deliverables in WP4 in the 5th period partner 10/Ki-Poellinger has studied hypoxia in relation to cell differentiation and EMT. Their findings indicate that in neuroblastoma cells there may be a link between hypoxia, Jmjd1a, G9a, epigenetic regulatory mechanisms and stem cell regulation. They conclude that the combined effect of dysregulated expression of Jmjd1a and G9a in hypoxia may determine the epigenetic landscape of target promoters and the selection of malignant, immature, stem cell like neuroblastoma cells. Partner 13/CNRS continued their study on the identification, development and validation of novel therapeutic targets induced by tumoural hypoxic stress (NHEs, MCTs, AEs, CA9/CA12, autophagy) and also on the design and validation of the best therapies combining anti-angiogenesis and blockers of pHi-regulating systems. They conclude that inhibition of the MCTs/BASIGIN complex, combined with phenformin provides a novel acute anticancer strategy to target highly glycolytic tumours. This genetic approach validates the anticancer potential of the MCT1 and MCT4 inhibitors in current development. Partner 6/AUH,AS has done pre-clinical testing of cycling and prolonged hypoxia as associated targets in animal models. They briefly conclude that they may be able to interfere with tumour metabolism directly when using a highly sensitive cell line and treating with a high phenformin dose (a biguanide). It remains to be determined however whether this translates into any antitumor effect. Whereas single treatment with 100 mg/kg phenformin was well-tolerated, long-term treatment (> 5 days) was associated with severe side effect. Partner 1/UIO has continued their studies on the effect of low dose-rate priming doses and how the factor produced by this irradiation (TGF-β3) is induced in mice after irradiation. The conclusion is that prior exposure to LDR irradiation may affect the response to subsequent radiation treatment or accidental radiation exposure through a mechanism involving TGF-β3 and iNOS activity. A similar protection can be induced 24 h post irradiation by a single injection of TGF-β3. The transient activation of TGF-β3 during hypoxia appears to be through a different mechanism involving HIF-1α. Partner 4/RUNMC has studied criteria for adding CAIX inhibitor to radiation therapy of larynx cancer and concludes that CAIX ectodomain shedding into the blood stream was decreased by the patented CAIX-inhibitor S4. The meaning of this finding is not clear yet. Partner 7/FMC has continued their work on 3D in vitro models based on alginate substrates (part of Obj13). This industrial partner now reports a method for predetermining if cells require the cell attachment peptide RDG for growth in alginate foam scaffolds. Additionally, they also report work towards commercialization of NovaMatrix-3D as a cell culture system.
5 Fundamental research within sensor development:
5.1 WP7: Development of technology for imitation of tumour micro-environment in model systems:
Seven deliverables were reported completed during the 4th and 5th periosd (D7.8 D7.9 D7.10 D7.13 D7.14 D7.17 and 7.18).
A) A first deliverable of the 4th period (D7.10) was completed by our partners 21/OU-Kragh and 15/Jobst on the establishment of a small scale culture system using micro fluidics to deliver cytokines and micro-dialysis for continuous change of culture medium. This work represent practical usage of the micro-fluidic system developed by partner 15/Jobst in an attempt to improve in vitro proliferation of CD34+ haematopoietic stem cells isolated from mobilized peripheral blood from adults. The system was integrated into the mini-laboratory developed by partner 16/VX. It turned out that the collaborating team run into several technical problems with the pump´s head. At the same time no significant expansion of CD34+ cells compared to the control was seen, except from the first experiment. Partner 21/OU-Kragh concluded that they would terminate the attemts to expand CD34+ cells further with the pump.
B) A second deliverable of the 4th period (D7.13) was completed by our partner 14/ALU-FR on integration of the micro fluidic and micro dialysis systems with sensor systems. In that work the aim is to integrate liquid transport into and out of cell cultures with the technology and platform of the Sensing Cell Culture Flask (SCCF) by on-chip microfluidic structures. The application of 14/ALU-FR’s surface micromachining process with photoresist as sacrificial layer was used for fabrication of microfluidic structures. Silicon nitride was used as material for the micro-channels. Therewith micro-channels in the height range of 2 to 30 µm could be fabricated. In order to allow higher channel widths and to obtain more robust structures the concept of pillar-like structures was successfully demonstrated. However, to achieve a stable process, silicon nitride formed by plasma-enhanced chemical vapour deposition is not the optimal material choice. Therefore on-going work is targeting Parylene as cover material.
C) A third deliverable of the 4th period (D7.17) was completed by partner 15/Jobst on the application development and peri-cellular oxygen tension control aiming to supplying ready-to-use tools for different applications to METOXIA partners. This work represent an attempt to meet with the partner requirements that a traditional clinical/biochemical analyzer function is widely missed. To provide this, the former fluid handling concept of 15/Jobst (using commercially available syringe pumps; see problems reported in deliverable 7.10 above) was replaced by small size-and-cost peristaltic pumps developed by 15/Jobst during the 4th period, and the range of applications extended by a “personal analyzer”. All other applications also (can) make use of the novel peristaltic pump. The conclusion was: “Utilizing the high content hardware toolkit presented in Deliverable 7.16 (M36) along with newly added further enabling components, four applications, including per-cellular oxygen control, were elaborated. The applications are already partially used by METOXIA partners. Application of these tools both in vitro and in vivo is planned to be further distribute amongst METOXIA partners and also amongst customers and partners outside METOXIA. Significant potential for product developments and economic exploitation by Jobst Technologies is foreseen.
D) A first deliverable of the 5th period (D7.8) was completed by our SME partner 16/VX on “upgrade from disinfection to sterilization methods not affecting sensitive equipment.” This is a description of the advanced sterilization procedures built into the 3rd generation of the ViVox TC-BiosystemTM. It was concluded that a sterile atmosphere in both the airlock and workspace was achieved in accordance to ISO class 3. The aim of the ViVoX Biosystem has been to achieve a level of containment where the risk of contamination biological material is as low as possible. Though, the ViVoX TC biosystem can not be used as an alternative to an autoclave, the cleanness in the workspace and airlock is extremely high and suitable for handling cellular material for reinserting in humans.
E) Also a second deliverable (D7.9) was completed by our partner 16/VX on “connecting/binding together monitoring and control of Biosystems placed at different institutions.” It has been demonstrated that protocols can be saved and used on several units. In the activity report (WP7, obj6) partner 16/VX shows plans to install several ViVox TC-BiosystemsTM in Denmark.
F) A third deliverable (D7.14) was completed by partner 14/ALU-FR on “Optimisation of ROS sensors, initiation of the first test phase.” The conclusion was that Superoxide sensors based on the technological platform of the SCCF have been produced and characterized with respect to sensor performance like sensitivity, selectivity and stability. It was shown that the electrochemical sensing approach can be used on bare gold electrodes utilizing the direct oxidation of superoxide anions on the working electrode. Sensor stability can be enhanced by covering the working electrodes with linear polyethyleneimine by dispensing from aqueous solution. Artificial superoxide production system xanthine/xanthine oxidase was characterised and utilised for sensor calibration. This characterisation was done using spectrophotometry and electron spin resonance spectroscopy as complementary techniques to electrochemical sensing approach.
G) A fourth deliverable (D7.18) was completed by partner 21/OU-Kragh-Kvalheim on “Role of Valporic acid, notch ligand and dimethoxyflavone on the stabilization of HIF 1α.” They conclude that the study suggests a positive effect of 3′4′--dimethoxyflavone (DMF) on human hematopoietic stem cells. However, it warrants further studies to validate the findings as well as to further explore the potential benefits of 3′4′-DMF, either alone or in synergy with other agents, in HSC biology.
In addition to the 4 deliverables in WP7 of the 5th period our SME partner 15/Jobst has worked on further development of minianalyzers in connection with obj1. The conclusion is that the analytical performance of the mini-analyser was found highly adequate for the purpose. A second generation of the mini-analyser was developed. Standard connectivity (including wireless), the pocket sized dimensions, and possible affordable pricing should allow successful marketing of such device after the product development phase. This product development after the expiration of METOXIA majorly encompasses software implementation and usability studies – and of course probing of the envisaged markets. It is therefore expected that the results generated during the METOXIA project will prevail and migrate in a variety of Biotech and Medtech applications.
Potential Impact:
The public health impact of the present project is potentially high since the activities have increased understanding of the basic mechanisms underlying metastatic spread of cancer. This includes mechanisms such as Notch/Hypoxia-Inducible Factor (HIF); CAIX-inhibitors; the Unfolded Protein Response (UPR); the mammalian Target of Rapamycin (mTOR); cellular-internal pH regulators (pHi regulators); hypoxic stress, autophagy and reactive oxygen species (ROS). Particularly important is the implicit aim to translate this increased understanding to the patient within the same consortium. As a consequence of the METOXIA-results the most important impact will hopefully be felt by patients with metastatic hypoxic cancers, whose quality of life is commonly impaired by pain and other distressing symptoms of advanced disease. The treatment-resistant hypoxic cells are probably one of the most important reasons for relapse after seemingly successful radical treatment and our ultimate goal is to reduce relapses as well as improve the effect of radical treatment.
Impacts in relation to the topics in question:
• We have determined the role of activated forms of Notch and HIF transcription factors to regulate EMT under hypoxic conditions, and define strategies to inhibit these effects. This has indicated an attack mechanism to directly influence the propensity of a cancer to spread and may as such represent a new treatment modality with a great impact on cancer treatment as a whole.
• CAIX inhibitors; novel drugs as based on METOXIA-inventions: CAIX inhibitors are potentially important both as hypoxia-specific biomarkers and as a stromal component of hypoxic tumours. The function of CAIX is important for the epithelial-mesenchymal transition (EMT) (which represent mechanisms for the cancer cells to migrate in general) and cancer-metastatic spread to distant parts of the body. CAIX-inhibitors therefore represent possible new anticancer drugs particularly in combination with radiotherapy or established chemotherapy which are known to be effective against the well-oxygenated regions of the tumour. These possibilities have been tested pre-clinically with newly-synthesized and patented CAIX-inhibitors and the basis has been laid for pursuing this principle into clinics in the present project. CAIX-inhibitors may thus have impact both on future diagnosis/imaging, on radical systemic cancer treatment and as potential radiation-modifying compounds.
• We have performed studies to largely clarify the role of the UPR and mTOR pathways as mediators of metastasis and the role of reoxygenation events. These molecular mechanisms are activated at specific levels of hypoxia (i.e. at more severe hypoxia than the HIF/CAIX-mechanism) and/or under particular conditions. Therefore they represent potential possibilities to attack conditions of tumour hypoxia that are different from those related to HIF. They are involved in cellular protein translation and thus influence cell-growth as such. This is a process which is to a small degree attacked directly by other treatment modalities and therefore could represent a valuable additional treatment load in combination therapy with traditional cancer treatment modalities.
• pHi-novel treatment strategies: We have combined anti-angiogenesis and blockers of pHi-regulating systems and the modulation of the angiogenesis inhibitor thrombospondin 1 (TSP1) by hypoxia. Two targets of interest related to pH-regulation and metabolism are the lactate transporter MCT4 and its subunit CD147/Basigin. Our partner 13/CNRS has tested an MCT4-inhibitor produced by AstraZeneca and confirmed its specificity for MCT4 and reports this compound to be ready for clinical trials in the near future.
• Effectors of autophagy (beclin): Identification, development and validation of novel therapeutic targets induced by tumoural hypoxic stress (NHEs, MCTs, AEs, CA9/CA12, autophagy). The negative aspects of hypoxia for cancer cells are so far not much turned to account in cancer therapy. We expect that increased knowledge in this field could have a considerable impact on cancer treatment and plan to see these mechanisms as potentials for exploitation in relation to the pHi strategies.
• We have contributed to clarify the role of reoxygenation events and reactive oxygen species (ROS) at the molecular and cellular level in tumour cells, vascular cells and stromal cells as well as in the in vivo tumour setting with regard to tumour growth and progression including EMT, invasion and metastasis. Together with the role of angiogenic factors and lymphangiogenesis which we believe may all be vital to metastatic spread this new knowledge may potentially give rise to new treatment modalities in the future.
Generally, the increased understanding of these mechanisms addressed above will have great impact on the possibility of development of new drugs and treatment strategies since they will present new targets for treatment of cancer. This will open up new areas for pharmaceutical companies to develop new drugs as well as new possibilities for clinicians to choose new strategies for cancer treatment. One new SME company named DualTPharma has been started up as a spin-off of METOXIA, working on the development of patented CAIX-inhibitors.
The present consortium has itself translated the new information into the clinical setting. The new knowledge has been exploited to develop:
A) Novel drugs (WP4, 5 and 8). i.e. CAIXi and pHi-regulators. In order to get a full overview of this result of METOXIA one needs to see the deliverable report D5.18 which summarizes the studies completed over the last 2.5 years of METOXIA leading up to clinical testing of our own new, patented compounds. A brief conclusion is that we have develop lead compounds of 2 different patents which show beneficial preclinical toxicology (i.e. largely no toxicity for relevant dosing), positive pharmacokinetics opening up for potential oral administered drugs and good effect results in CAIX-expressing cells in hypoxic areas in preclinical models both in vitro and in vivo.
B) New treatment strategies. One example is our plan to pursue the method of blocking the proton export of metastasizing cancer cells by combination of different modalities (NHE, CA9i etc). If this proves to have a therapeutic potential it will have opened up a whole new avenue for targeted cancer treatment with a major impact on clinical oncology. Another example is the newly patented alpha irradiating and bone seeking drug [223Ra] which has a palliative effect on metastases from prostate cancer where we expect to develop this treatment into life prolonging effects for these patients. A similar potential is also present for breast cancer since these also frequently develop life-threatening metastases. This project is now fully commercialized since Bayer pharma AG bought the Norwegian pharma company Algeta ASA which owned the patent to this drug.
C) Validated biomarkers and prevention research should lead to better strategies in both primary and secondary cancer prevention and clinical research will improve patient treatment and care. In the long run, the impact of the investment in this area will contribute to reaching the aims of reducing cancer incidence and mortality and improving quality of life for patients.
Our work on diagnosing/imaging and stratification likewise has a high potential impact on treatment outcome. The improved 3-dimensional imaging of hypoxic areas by PET-scanning usin will improve the focusing of enhanced irradiation to specifically hypoxic tumour areas and thus improve the clinical outcome, most likely without increased side-effects.
Our very recent pre-clinical observations of an unexpected high tumour cell-killing effect of low dose-rate beta irradiation and our observation of the possibility of inducing a transferable radio-protection factor needs more pre-clinical work, but could in the end lead to development of a new radiation treatment modality and even to better protection of the surrounding normal tissue.
Confirmation of our understanding that it is not moderate hypoxia as dominated by HIF regulated pathways, but rather variations in tumour-oxygenation between deeper and more moderate hypoxia that is most relevant to both metastasing and direct tumour cell response to therapy will have a major intellecutal impact on the whole field of research and its teaching.
A commercial impact has been achieved with our development of equipment for measuring and controlling the micro-environment, and for imaging. Furthermore, newly synthesised drugs such as CAIXi’s may become of interest to the pharmacological industry. Taken together the above is likely to create new employment.
Dissemination activities and exploitation of results:
The METOXIA project has contributed to dissemination largely through two different channels: a) Publications in international research journals with peer review, many of which are among the highest-ranked journals. b) Participation at international scientific conferences.
The number of published papers in scientific journals (with acknowledgement for METOXIA funding) is:
1st period: 30
2nd period: 72
3rd period 81
4th period 120
5th period: 185
The number of international conference presentations where METOXIA-findings and –funding has been reported are at least of the same order of magnitude as the number of scientific publications.
Final review article:
At the last METOXIA General Assembly meeting it was agreed that all partners should be invited to collaborate and together write an extensive review article where the background of METIXIA related to the general hypoxia-problem should be described. During the 5th period that endeavour was completed. In that review paper the main results of METOXIA have been set into context. On the 15th September 2014 the article was accepted for publication in the Journal of Enzyme Inhibition and Medicinal Chemistry. The title is: “Targeting Tumour Hypoxia to Prevent Cancer Metastasis. From Biology, Biosensing and Technology to Drug Development: the METOXIA Consortium”. This article sums up the work performed during the 5.5 years of the METOXIA project for the international scientific community. It also gives an updated over-view of the problem of hypoxia and cancer.
Patenting and secrecy strategy:
Regarding the inventions and patenting work it has been necessary to keep many results back from publication for a period of time. However, by the end of METOXIA it can be stated that all results can be published since all patents that will be protected in the future are now open.
List of Websites:
The public website address of METOXIA: http://www.metoxia.uio.no/
The website has a restricted area accessible for METOXIA partners.
Contact details:
Co-ordinator Professor Erik O. Pettersen,
Address: Department of Physics, The University of Oslo, B.O.Box 1048 Blindern, 0316 Oslo, Norway.
Tel +47 2285 5644 (Mobile: +47 9093 0743)
Email: e.o.pettersen@fys.uio.no
