Final Report Summary - TDCCPCS (Targeted delivery of charged membrane impermeant compounds to pain-sensing and cancer cells)
This report sums up 4 years of extensive research. We have focused on development of new platforms for introducing agents (cytotoxic, inhibitors of cellular transduction pathways and antagonists of specific channels) into specific cell types (excitable, non-excitable and cancerous) via large pore cationic channels that are expressed with a deferential expression profile on different cell types. Only the cells that harbor these channels will undergo the effect of the specific agent. Using this approach we have succeeded in affecting specific neuronal populations such as itch sensing and pain sensing sensory afferents. We also expended this platform to target specifically, cancer cells, but nor "healthy" neighboring cells. We have shown very promicing in-vitro results and have initiated in-vivo experiments with this platform. The opening of such channels was performed actively, using agonists but we have also investigated physiological and patho-physiological conditions which lead to their opening. This will and already has shown reduction in non specificity of effect and increased in efficacy index.
The impact of our studies goes beyond the basic research of signal transduction pathways and intracellular mechanisms. These newly developed platforms will impact the way we look at disease states and how we relate to them. But more importantly the way we treat such states, in terms of effectiveness and well being to patients.
There are cancer treatments that used lowered, less effective dose of chemotherapy drugs in attempt to reduce unwanted "off target" side effects. Moreover, there are many cancer states which are untreatable due to severe side effects of the current drugs. Our approach, by limiting the effect of the drugs only to the cancer cells, may improve significantly the treatment prognosis. These platforms might also be cost effective and therefore decrease significantly general health costs and disease timelines dramatically. Such states as skin conditions which entail itching to the point of damage may very well eradicated in a simple non invasive manner therefore revolutionizing the conditions and reducing them to an imbalance in immune response, relieving both the patient but also medical institution from having to also care for that.
We foresee that the development of the above mentioned platforms will definitely make the transition from bench to bedside.
This projects aims to develop a platform for targeted delivery of membrane inpermeant compounds, selectively into neuronal and cancer cells, thereby affecting them in specific manner. Aim 1 of this project suggested to selectively silence peripheral afferent C-fibers, to block selectively pain or itch without affecting other neuronal populations. To this end, we suggested to target the charged sodium channel blocker QX-314 into either pain or itch peripheral fibers which selectively express large-pore cation non-selective channels TRPV1 (noxious-heat sensitive) and TRPA1 (activated by noxious chemical irritants) through which the agent would pass. Here we hypothesize that during pathological conditions such as itch, the above-mentioned channels are endogenously activated and open we could and therefore can use them to insert charged sodium channel blockers into the neurons.
Itch is a complex unpleasant cutaneous sensation that in some respects resembles pain, yet is different in terms of its intrinsic sensory quality and the urge to scratch. At a cellular level there is a considerable overlapping responsiveness of trigeminal (TG) and dorsal root ganglia (DRG) neurons to multiple pain-producing algogens and itch-producing pruritogens, although the G-protein coupled receptors (GPCRs) responsive to different specific pruritogens are quite distinct. Histamine-sensitive H1 receptors (H1Rs) generate histamine itch and are expressed by TRPV1+/phospholipase-β-3 (PLCβ3)+ fibers, while the itch evoked by chloroquine (CQ) is mediated by the Mas-related G-protein-coupled receptors A3 (MrgprA3) and C11 (MrgprC11), respectively. Interestingly, co-activation of TRPV1 and H1R is required to produce histamine-dependent itch, and that of TRPA1 and MrgprA3 or C11 for non-histamine-mediated itch. We have demonstrated that this endogenous pruritogen-mediated activation of TRPV1 and TRPA1 channels is sufficient to shuttle QX-314 selectively into itch-generating neurons. We showed that application of QX-314 together with the pruritogens, but not alone, lead to complete blockade of sodium currents in-vitro and blockade of scratching in-vivo. There results suggest that targeting silencing of activated sensory fibers may represent a clinically useful anti-pruritic therapeutic approach. This approach also allowed us to gain deeper understanding of the physiology of itch sensation.
In-vitro calcium imaging experiments indicate that neurons expressing MrgprA3 also respond to histamine, which has been interpreted as indicating the presence of a single neuronal path for producing both histaminergic and non-histaminergic itch. Supporting this, ablation of neurons expressing MrgprA3 has recently been shown to reduce the scratching evoked by both CQ and histamine. However, other investigators report that while a responsiveness of primary sensory neurons to multiple itch mediators occurs in juvenile mice, this decreases with age suggesting that in the adult, activation of afferents that respond only to histamine or only to non-histamine pruritogens may be sufficient to independently generate the two types of itch. This distinction is clinically important since therapies targeting neuronal activity exclusively of histaminergic itch fibers might be therapeutically ineffective for the treatment of non-histaminergic itch if the neurons mediating the two itches are functionally distinct in the mature nervous system.
To study if histamine- and non histamine-mediated itch are functionally distinct we have utilized a strategy of silencing specific subsets of pruritogen-sensitive primary sensory fibers by delivery of a charged sodium channel blocker through active large pore channels to demonstrate that functional blockade of the sensory fibers that mediate histamine itch does not affect the histamine-independent itch evoked by chloroquine or SLIGRL-NH2, and vice versa. Moreover, we demonstrate that a targeted silencing of itch-generating fibers does not reduce pain-associated behavior. However, we found that silencing TRPV1+ or TRPA1+ neurons allows capsaicin or AITC respectively to evoke itch, implying that certain peripheral afferents may normally indirectly inhibit algogens from eliciting itch. These findings both support the presence of functionally distinct sets of fibers for producing histaminergic and non-histaminergic itch behavior in adult mice, and suggest that the targeted silencing of activated sensory fibers may represent a clinically useful anti-pruritic therapeutic approach.
Aim 2 of this proposal suggest to use the abovementioned approach for selective ablation of cancer cells.
The ultimate goal of developing an anticancer drug is to only target cancer cells leaving normal cells untouched. Several approaches are used to emphasize the effect of anticancer drugs on tumor cells. Some of these strategies are tuned to target cancer specific cellular machineries. Others, by using polymeric drug carriers such as liposomes and nanoparticles restricting the delivery of non-specific chemotherapeutics primarily to the tumor cells. In this proposal we have developed a different method for the selective elimination of tumor cells by the targeting of otherwise membrane impermeable hydrophilic chemotherapy agents into cancer cells via the pore of cation non-selective transient receptor potential (TRP) channels. The members of TRP channel family, which overexpressed by tumor cells, plays a critical role in tumorogenesis, tumor vascularization and the ability of tumor cell to proliferate and migrate. Here, we exploited the TRP channels as cell-specific “natural” drug delivery system for targeted application of charged molecules that are cytotoxic or antiproliferative when inside the cells, but relatively innocuous outside, to cancer cells, minimizing unwanted effects on other non-TRP expressing cells. As discussed above we have shown previosly that activation of TRPV1 and TRPA1 channels provides a pathway for selective entry of QX-314 into pain and itch generating neurons and therefore inhibition of pain and itch signals without effecting non-painful sensory and motor neurons Here we show that by applying a compound that activates and opens a large-pore cationic channel receptor, TRPV2, which is present to a large extent in mouse hepatocellular carcinoma BNL.1M/luc and sets the expression profile to be different between cancer and non-cancerous liver and heart cells, it allows entry of the of a positively-charged chemotherapeutic compound Doxorubicin into BNL.1M/luc cells specifically. We also showed that application of Doxorubicin in subeffective doses ceased proliferation of BNL.1M/luc cells and lead to cell death only when co-applied with the activator of TRPV2 channels. Such facilitated entry will minimize the off-target effect of Doxorubicin and therefore will substantially reduce adverse side effects.
We believe that the results of our work will produce a novel platform for the selective targeting of compounds which are able to modulate intracellular signal transduction and metabolism pathways and thereby not only treat cancer but also other pathological conditions where the selective activation or disruption of a cellular signal transduction or metabolism pathway is beneficial. This approach will assure minimization of adverse side effects and yet create a confined temporal spatial deployment of an effector at the exact target site.
The impact of our studies goes beyond the basic research of signal transduction pathways and intracellular mechanisms. These newly developed platforms will impact the way we look at disease states and how we relate to them. But more importantly the way we treat such states, in terms of effectiveness and well being to patients.
There are cancer treatments that used lowered, less effective dose of chemotherapy drugs in attempt to reduce unwanted "off target" side effects. Moreover, there are many cancer states which are untreatable due to severe side effects of the current drugs. Our approach, by limiting the effect of the drugs only to the cancer cells, may improve significantly the treatment prognosis. These platforms might also be cost effective and therefore decrease significantly general health costs and disease timelines dramatically. Such states as skin conditions which entail itching to the point of damage may very well eradicated in a simple non invasive manner therefore revolutionizing the conditions and reducing them to an imbalance in immune response, relieving both the patient but also medical institution from having to also care for that.
We foresee that the development of the above mentioned platforms will definitely make the transition from bench to bedside.
This projects aims to develop a platform for targeted delivery of membrane inpermeant compounds, selectively into neuronal and cancer cells, thereby affecting them in specific manner. Aim 1 of this project suggested to selectively silence peripheral afferent C-fibers, to block selectively pain or itch without affecting other neuronal populations. To this end, we suggested to target the charged sodium channel blocker QX-314 into either pain or itch peripheral fibers which selectively express large-pore cation non-selective channels TRPV1 (noxious-heat sensitive) and TRPA1 (activated by noxious chemical irritants) through which the agent would pass. Here we hypothesize that during pathological conditions such as itch, the above-mentioned channels are endogenously activated and open we could and therefore can use them to insert charged sodium channel blockers into the neurons.
Itch is a complex unpleasant cutaneous sensation that in some respects resembles pain, yet is different in terms of its intrinsic sensory quality and the urge to scratch. At a cellular level there is a considerable overlapping responsiveness of trigeminal (TG) and dorsal root ganglia (DRG) neurons to multiple pain-producing algogens and itch-producing pruritogens, although the G-protein coupled receptors (GPCRs) responsive to different specific pruritogens are quite distinct. Histamine-sensitive H1 receptors (H1Rs) generate histamine itch and are expressed by TRPV1+/phospholipase-β-3 (PLCβ3)+ fibers, while the itch evoked by chloroquine (CQ) is mediated by the Mas-related G-protein-coupled receptors A3 (MrgprA3) and C11 (MrgprC11), respectively. Interestingly, co-activation of TRPV1 and H1R is required to produce histamine-dependent itch, and that of TRPA1 and MrgprA3 or C11 for non-histamine-mediated itch. We have demonstrated that this endogenous pruritogen-mediated activation of TRPV1 and TRPA1 channels is sufficient to shuttle QX-314 selectively into itch-generating neurons. We showed that application of QX-314 together with the pruritogens, but not alone, lead to complete blockade of sodium currents in-vitro and blockade of scratching in-vivo. There results suggest that targeting silencing of activated sensory fibers may represent a clinically useful anti-pruritic therapeutic approach. This approach also allowed us to gain deeper understanding of the physiology of itch sensation.
In-vitro calcium imaging experiments indicate that neurons expressing MrgprA3 also respond to histamine, which has been interpreted as indicating the presence of a single neuronal path for producing both histaminergic and non-histaminergic itch. Supporting this, ablation of neurons expressing MrgprA3 has recently been shown to reduce the scratching evoked by both CQ and histamine. However, other investigators report that while a responsiveness of primary sensory neurons to multiple itch mediators occurs in juvenile mice, this decreases with age suggesting that in the adult, activation of afferents that respond only to histamine or only to non-histamine pruritogens may be sufficient to independently generate the two types of itch. This distinction is clinically important since therapies targeting neuronal activity exclusively of histaminergic itch fibers might be therapeutically ineffective for the treatment of non-histaminergic itch if the neurons mediating the two itches are functionally distinct in the mature nervous system.
To study if histamine- and non histamine-mediated itch are functionally distinct we have utilized a strategy of silencing specific subsets of pruritogen-sensitive primary sensory fibers by delivery of a charged sodium channel blocker through active large pore channels to demonstrate that functional blockade of the sensory fibers that mediate histamine itch does not affect the histamine-independent itch evoked by chloroquine or SLIGRL-NH2, and vice versa. Moreover, we demonstrate that a targeted silencing of itch-generating fibers does not reduce pain-associated behavior. However, we found that silencing TRPV1+ or TRPA1+ neurons allows capsaicin or AITC respectively to evoke itch, implying that certain peripheral afferents may normally indirectly inhibit algogens from eliciting itch. These findings both support the presence of functionally distinct sets of fibers for producing histaminergic and non-histaminergic itch behavior in adult mice, and suggest that the targeted silencing of activated sensory fibers may represent a clinically useful anti-pruritic therapeutic approach.
Aim 2 of this proposal suggest to use the abovementioned approach for selective ablation of cancer cells.
The ultimate goal of developing an anticancer drug is to only target cancer cells leaving normal cells untouched. Several approaches are used to emphasize the effect of anticancer drugs on tumor cells. Some of these strategies are tuned to target cancer specific cellular machineries. Others, by using polymeric drug carriers such as liposomes and nanoparticles restricting the delivery of non-specific chemotherapeutics primarily to the tumor cells. In this proposal we have developed a different method for the selective elimination of tumor cells by the targeting of otherwise membrane impermeable hydrophilic chemotherapy agents into cancer cells via the pore of cation non-selective transient receptor potential (TRP) channels. The members of TRP channel family, which overexpressed by tumor cells, plays a critical role in tumorogenesis, tumor vascularization and the ability of tumor cell to proliferate and migrate. Here, we exploited the TRP channels as cell-specific “natural” drug delivery system for targeted application of charged molecules that are cytotoxic or antiproliferative when inside the cells, but relatively innocuous outside, to cancer cells, minimizing unwanted effects on other non-TRP expressing cells. As discussed above we have shown previosly that activation of TRPV1 and TRPA1 channels provides a pathway for selective entry of QX-314 into pain and itch generating neurons and therefore inhibition of pain and itch signals without effecting non-painful sensory and motor neurons Here we show that by applying a compound that activates and opens a large-pore cationic channel receptor, TRPV2, which is present to a large extent in mouse hepatocellular carcinoma BNL.1M/luc and sets the expression profile to be different between cancer and non-cancerous liver and heart cells, it allows entry of the of a positively-charged chemotherapeutic compound Doxorubicin into BNL.1M/luc cells specifically. We also showed that application of Doxorubicin in subeffective doses ceased proliferation of BNL.1M/luc cells and lead to cell death only when co-applied with the activator of TRPV2 channels. Such facilitated entry will minimize the off-target effect of Doxorubicin and therefore will substantially reduce adverse side effects.
We believe that the results of our work will produce a novel platform for the selective targeting of compounds which are able to modulate intracellular signal transduction and metabolism pathways and thereby not only treat cancer but also other pathological conditions where the selective activation or disruption of a cellular signal transduction or metabolism pathway is beneficial. This approach will assure minimization of adverse side effects and yet create a confined temporal spatial deployment of an effector at the exact target site.