Periodic Reporting for period 2 - INFRADYNAMICS (Overcoming the Barriers of Brain Cancer Treatment: Targeted and Fully NIR Absorbing Photodynamic Therapy Agents with Extremely Low Molecular Weights and Controlled Lipophilicity)
Période du rapport: 2021-05-01 au 2022-10-31
Every other day we hear more good news about an advancement towards treating cancer, the truth however is quite a different story when it comes to brain cancers. 5-year survival rates have been almost unchanged for decades. Unfortunately, the state of the art clinically used agent temozolomide increases patient survival only by several months in average. Targeted therapies are emerging significantly towards differentiating between tumors and healthy tissue, thus increasing the potency, and decreasing the side-effects. For brain cancers however, up to now, there is only one FDA approved targeted drug in the market and effectiveness of this drug is yet to be clearly demonstrated. One key obstacle for many drugs is the Blood-Brain Barrier, one of the most controlled gateways in human body, which significantly restricts the size and lipophilicity of molecules that can be delivered into the brain. In Infradynamics we aim to change this pessimistic picture by developing scientific foundations towards making an early, targeted, and light-based treatment possible for brain cancer by combining unique drug design and targeting strategies with in-vitro and in-vivo testing. In the light induced treatment modality, namely photodynamic therapy (PDT), a drug called a photosensitizer (PS) is administered and a specific wavelength of light is applied to a region of interest. When photosensitizers are exposed to this light, they produce reactive forms of oxygen (ROS) that kills nearby cells. PDT has already been demonstrated an effective treatment modality for a group of cancers and has attained elevated attention as due to promising properties such as no long-term side effects, short treatment times, precise light targeting Being non-invasive character and potentially being cheaper. The PS drug for successful PDT action is required to be: 1) non-toxic, 2) water-soluble, 3) should have high ROS generation efficiency, 4) should effectively target tumor cells and, finally 5) it should be activated by near infrared (NIR) light as it has much better tissue penetration compared to visible light for treating internal tumors. For brain up to 5 cm of penetration was demonstrated with light at wavelength of 800 nm. The combination of all these functions is essential for a successful PDT treatment. Additionally, for brain cancer specifically low molecular weight and controlled lipophilicity is also required for effective penetration through BBB. It is important to note here that from a chemical structure perspective, NIR absorption and low molecular weights are quite contradictory. In INFRADYNAMICS advanced fluorophores based on a key insight are being pursued whereby we aim to create drugs that have strong absorption in NIR region (>700 nm) and that have a low Mw with synthetic flexibility to control lipophilicity to ensure they can get through the Bloor Brain Barrier. The elegant approach is to combine two separate “single atom” modifications that were shown to red-shift absorption maxima significantly in several fluorophore systems. The critical question here was if the effect from both modifications is additive? And, fortunately, the answer is yes. Our next key step is to decorate the fluorophores with heavy atoms to attain photosensitizers, which is essential for ROS generation. The final step in our strategy will be targeting brain cancer cells, specifically glioblastoma. Although PDT treatment modality has an intrinsic selectivity by delivering light directly to tumors, realization of PS drugs that are dormant in healthy tissue however gets activated by a tumor metabolite and/or over-expressed enzyme activity is the key to realize true selectivity. In InDy we aimed to target 3 different enzymes that has been demonstrated to be upregulated in glioblastomas. Finally, all pieces of the puzzle will be put together to realize NIR absorbing low molecular weight (Mw) PS with controlled lipophilicity, a traceless linker that enables safe approach to the enzyme pocket, a reaction-based cage group that will effectively target upregulated enzymes. Once the drug meets with the enzyme a sequence will be generated to release the active drug, and hopefully, the light will take care of the rest.
Our design principle in InDy was based on implementing two structural modifications on a common fluorophore which results in NIR absorbing materials with low molecular weights (MWs). Then, modification of these fluorophores with heavy atoms such as bromine or iodine which allows fluorophores, which is essential for ROS generation. We realized however, even though fluorophores with one of the modifications were known, none of these were modified into PSs. Hence, in the first part of the project we synthesized two PSs based on these fluorophores to make sure these cores can be utilized as PDT agents (RS1 and SFI). R1 not only marks the first example of a resorufin based photosensitizer but also marks the first ever example of a monoamine oxidase (MAO) activatable therapeutic agent (ACS Med. Chem. Lett. 2020, 11, 2491, cover article). On the other side, SFI was shown to be highly effective on colon and breast cancer cells upon light irradiation and proved to be a theranostic core (both treatment and imaging capabilities) thanks to its emissive nature (ACS Med. Chem. Lett. 2021, 12, 752).
Our first main target based on our design strategy in InDy was F1, which was successfully synthesized using a novel synthetic approach. We were pleased to see the material showed NIR absorption, however, photophysical characterizations revealed limited fluorescence for F1 which hindered its further utilization. Next, synthetically challenging, elusive fluorophore F2 recently attained. The absorption band is quite wide extending all the way to 700 nm which results in significant overlapping with the therapeutic window (range of wavelengths appropriate for PDT treatment). More importantly F2 showed strong emission nm showing that the fluorophore can be modified into a photosensitizer. A new selenium containing PS was also realized (PS3) and photophysical studies showed extremely high ROS generation efficiency (73%) with strong NIR absorption. Initial in-vitro studies showed promising results and we are currently working on a glioma targeted derivative for further in-vitro and in-vivo studies. As InDy evolved, we realized several additional cores with our design principle could be realized and we concentrated our efforts for their synthesis. We have made significant progress on several cores with very exciting preliminary results. Significant progress was also demonstrated for the design and synthesis of masking groups, which will render our PSs inactive until they reach tumor sites. We are currently working on modification of several fluorophore cores towards translating them into targeted, reaction-based PSs.
In addition to these studies, our detailed analyses showed that glioblastoma cells have significantly increased B-galactosidase (B-gal) activity. To investigate the possible utilization of this over-expression we modified of our PS, RS1, with the corresponding masking unit. This work marks the first ever example of a B-gal responsive PS for selective treatment and imaging of glioblastoma cells which demonstrated that high B-gal level in gliomas can be utilized as a promoter in the design of therapeutics (under review).
In InDy several novel synthetic methodologies and approaches have been and are being developed for targeted, reaction-based PSs. While keeping this approach as our main motivation we are investigating possible carrier options in case the overall stability of target PSs in in-vivo conditions do not meet the expected criteria. Targeting/crossing of BBB by surface-functionalized nano-carriers makes them ideal candidates for drug delivery in brain tumors. Accordingly, IR780 (commercial NIR PS) nanostructures were prepared, their surfaces were modified, and preliminary in vitro experiments were performed which yielded promising results in terms of cellular toxicity upon glioblastoma cell lines (T98G, U118 MG) with higher cellular uptake and duration capacity compared to unmodified IR780. With further surface modification, decoration with functionalized systems and targeting components we are aiming to realize facile nanocarrier systems that can avoid potential side effects on healthy cells and enhancing laser irradiation response in gliomas. We are also investigating zeolites as carries towards a dual-mode pH-responsive gate-opening concept to develop a more efficient targeted delivery system for encapsulating PS candidates and improve PDT efficiency through an intelligent delivery concept. Additionally, via utilization of targeted and activatable PSs of InDy, an additional layer of selectivity between tumor and healthy cells will be realized, resulting in extremely sensitive brain cancer treatment systems.
Our first main target based on our design strategy in InDy was F1, which was successfully synthesized using a novel synthetic approach. We were pleased to see the material showed NIR absorption, however, photophysical characterizations revealed limited fluorescence for F1 which hindered its further utilization. Next, synthetically challenging, elusive fluorophore F2 recently attained. The absorption band is quite wide extending all the way to 700 nm which results in significant overlapping with the therapeutic window (range of wavelengths appropriate for PDT treatment). More importantly F2 showed strong emission nm showing that the fluorophore can be modified into a photosensitizer. A new selenium containing PS was also realized (PS3) and photophysical studies showed extremely high ROS generation efficiency (73%) with strong NIR absorption. Initial in-vitro studies showed promising results and we are currently working on a glioma targeted derivative for further in-vitro and in-vivo studies. As InDy evolved, we realized several additional cores with our design principle could be realized and we concentrated our efforts for their synthesis. We have made significant progress on several cores with very exciting preliminary results. Significant progress was also demonstrated for the design and synthesis of masking groups, which will render our PSs inactive until they reach tumor sites. We are currently working on modification of several fluorophore cores towards translating them into targeted, reaction-based PSs.
In addition to these studies, our detailed analyses showed that glioblastoma cells have significantly increased B-galactosidase (B-gal) activity. To investigate the possible utilization of this over-expression we modified of our PS, RS1, with the corresponding masking unit. This work marks the first ever example of a B-gal responsive PS for selective treatment and imaging of glioblastoma cells which demonstrated that high B-gal level in gliomas can be utilized as a promoter in the design of therapeutics (under review).
In InDy several novel synthetic methodologies and approaches have been and are being developed for targeted, reaction-based PSs. While keeping this approach as our main motivation we are investigating possible carrier options in case the overall stability of target PSs in in-vivo conditions do not meet the expected criteria. Targeting/crossing of BBB by surface-functionalized nano-carriers makes them ideal candidates for drug delivery in brain tumors. Accordingly, IR780 (commercial NIR PS) nanostructures were prepared, their surfaces were modified, and preliminary in vitro experiments were performed which yielded promising results in terms of cellular toxicity upon glioblastoma cell lines (T98G, U118 MG) with higher cellular uptake and duration capacity compared to unmodified IR780. With further surface modification, decoration with functionalized systems and targeting components we are aiming to realize facile nanocarrier systems that can avoid potential side effects on healthy cells and enhancing laser irradiation response in gliomas. We are also investigating zeolites as carries towards a dual-mode pH-responsive gate-opening concept to develop a more efficient targeted delivery system for encapsulating PS candidates and improve PDT efficiency through an intelligent delivery concept. Additionally, via utilization of targeted and activatable PSs of InDy, an additional layer of selectivity between tumor and healthy cells will be realized, resulting in extremely sensitive brain cancer treatment systems.
Progress beyond the state of the art:
Demonstration of high B-galactosidase (B-gal) activity in glioblastoma to be utilized as important activation mode for PDT agents in selective cyto-toxicity brain tumors (Figure 1):
We developed the first ever example of a B-gal responsive phototheranostic agent (PTA) for selective treatment and imaging of glioblastoma cells. PTA was tested in cell culture studies and selective photocytotoxicity was detected in U87 cancer cell (glioblastoma) with a negligible dark toxicity even at high doses. PS was also used to monitor lysosomal B-gal activity since the material retains relatively strong emissive character even with over %50 ROS generation efficiency. These results demonstrated, for the first time in literature, that high B-gal level in gliomas can be utilized as an effective targeting strategy for development of next generation PDT agents for glioblastoma treatment with selective cyto-toxicity. This proof-of-concept study now paved the way for utilization of B-gal activity in a range of glioblastoma cell lines utilizing NIR absorbing PDT agents of InDy for detection and treatment deep tumors in animal models.
Demonstration of iodinated resorufin to be an effective PDT agent that induce selective cyto-toxicity for neuroblastoma cells (Figure 2):
We realized the first ever example of a resorufin-based PDT agent as well as the first anti-cancer drug that can be activated by a monoamine oxidase, MAO, (an upregulated enzyme in prostate cancers, gliomas, and neuroblastomas) enzyme selectively in cancer cells. Recent studies have also shown that abnormal expression of MAO triggers tumor progression and metastasis, which makes MAO an attractive target for cancer research. This important enzyme however has not been used in the scope of PDT or in any drug design. Besides being first anti-cancer drug that can be activatable with a MAO enzyme, our agent also showed highly promising properties as a PS such as high singlet oxygen generation yield in aqueous solutions, red-shifted absorption signal and negligible dark toxicity. In addition to these valuable characteristics, our PS was shown to selectively treat neuroblastoma cells via in-vitro cell culture studies. This work introduces a new activatable PS platform, which holds a great promise towards realization of highly effective and cancer cell selective new generation PDT drugs.
Demonstration of brominated SiMe2 substituted fluoresceins as effective PDT agents, as well as their utilization in fluorescence imaging (Figure 3):
We developed the first ever example of a silicon fluorescein-based photosensitizer (SFI), which is also highly emissive to yield a theranostic agent. SFI is an easily accessible compound that shows highly promising properties as therapeutic and imaging agent such as water solubility, high ROS quantum yield in aqueous solutions (up to 45%), red-shifted absorption/emission signals and negligible dark toxicity. Thus, it directly addresses the chronic problems of the current small molecule based theranostic agents. SF-I is shown to induce cytotoxic singlet oxygen generation and consequent effective cell death in two different cancer cells with limited chemotherapy options and is used to image cells under confocal microscopy at the same time. SFI is a novel theranostic core, which holds a great promise towards realization of highly effective and cancer cell selective new generation organic theranostic agents as the core structure offers various modification sites to design cancer cell activatable and/or targeted agents.
Expected results until the end of the project:
The main breakthrough of the InDy is yet to come: successful demonstration of targeted, activatable photodynamic action in in-vivo studies for effective brain cancer treatment. Towards this aim number of PSs designs have been / are being developed with great promise. Synthetic organic chemistry is full of surprises and even though we overcame several challenges up to so far, new ones will be waiting for us as we progress through InDy. With immense amount of experience gained in the last two years in this novel PS platform however, we are confident that remaining final designs will be realized in the near future. Once the entire palette of PSs in our hands with their photophysical and in-vitro studies completed, we will be moving on to the in-vivo studies for determining BBB penetration ability and PDT performance of our PSs for brain cancers. Several PSs have been realized and with several more to come, screening of these candidates in in-vivo studies will not be practical from time, cost and ethical perspectives. Hence a new strong collaboration started with expert scientist on the organoid research. Here, we are going to build BBB organoids for investigation permeability of InDy PSs and most promising candidates will be utilized further for in-vivo studies.
Demonstration of high B-galactosidase (B-gal) activity in glioblastoma to be utilized as important activation mode for PDT agents in selective cyto-toxicity brain tumors (Figure 1):
We developed the first ever example of a B-gal responsive phototheranostic agent (PTA) for selective treatment and imaging of glioblastoma cells. PTA was tested in cell culture studies and selective photocytotoxicity was detected in U87 cancer cell (glioblastoma) with a negligible dark toxicity even at high doses. PS was also used to monitor lysosomal B-gal activity since the material retains relatively strong emissive character even with over %50 ROS generation efficiency. These results demonstrated, for the first time in literature, that high B-gal level in gliomas can be utilized as an effective targeting strategy for development of next generation PDT agents for glioblastoma treatment with selective cyto-toxicity. This proof-of-concept study now paved the way for utilization of B-gal activity in a range of glioblastoma cell lines utilizing NIR absorbing PDT agents of InDy for detection and treatment deep tumors in animal models.
Demonstration of iodinated resorufin to be an effective PDT agent that induce selective cyto-toxicity for neuroblastoma cells (Figure 2):
We realized the first ever example of a resorufin-based PDT agent as well as the first anti-cancer drug that can be activated by a monoamine oxidase, MAO, (an upregulated enzyme in prostate cancers, gliomas, and neuroblastomas) enzyme selectively in cancer cells. Recent studies have also shown that abnormal expression of MAO triggers tumor progression and metastasis, which makes MAO an attractive target for cancer research. This important enzyme however has not been used in the scope of PDT or in any drug design. Besides being first anti-cancer drug that can be activatable with a MAO enzyme, our agent also showed highly promising properties as a PS such as high singlet oxygen generation yield in aqueous solutions, red-shifted absorption signal and negligible dark toxicity. In addition to these valuable characteristics, our PS was shown to selectively treat neuroblastoma cells via in-vitro cell culture studies. This work introduces a new activatable PS platform, which holds a great promise towards realization of highly effective and cancer cell selective new generation PDT drugs.
Demonstration of brominated SiMe2 substituted fluoresceins as effective PDT agents, as well as their utilization in fluorescence imaging (Figure 3):
We developed the first ever example of a silicon fluorescein-based photosensitizer (SFI), which is also highly emissive to yield a theranostic agent. SFI is an easily accessible compound that shows highly promising properties as therapeutic and imaging agent such as water solubility, high ROS quantum yield in aqueous solutions (up to 45%), red-shifted absorption/emission signals and negligible dark toxicity. Thus, it directly addresses the chronic problems of the current small molecule based theranostic agents. SF-I is shown to induce cytotoxic singlet oxygen generation and consequent effective cell death in two different cancer cells with limited chemotherapy options and is used to image cells under confocal microscopy at the same time. SFI is a novel theranostic core, which holds a great promise towards realization of highly effective and cancer cell selective new generation organic theranostic agents as the core structure offers various modification sites to design cancer cell activatable and/or targeted agents.
Expected results until the end of the project:
The main breakthrough of the InDy is yet to come: successful demonstration of targeted, activatable photodynamic action in in-vivo studies for effective brain cancer treatment. Towards this aim number of PSs designs have been / are being developed with great promise. Synthetic organic chemistry is full of surprises and even though we overcame several challenges up to so far, new ones will be waiting for us as we progress through InDy. With immense amount of experience gained in the last two years in this novel PS platform however, we are confident that remaining final designs will be realized in the near future. Once the entire palette of PSs in our hands with their photophysical and in-vitro studies completed, we will be moving on to the in-vivo studies for determining BBB penetration ability and PDT performance of our PSs for brain cancers. Several PSs have been realized and with several more to come, screening of these candidates in in-vivo studies will not be practical from time, cost and ethical perspectives. Hence a new strong collaboration started with expert scientist on the organoid research. Here, we are going to build BBB organoids for investigation permeability of InDy PSs and most promising candidates will be utilized further for in-vivo studies.