Photoactivated Antiviral & Antitumor Compositions

The core of the invention involves a novel composition for targeted photodynamic therapy (PDT), designed to inactivate viruses and destroy tumor cells. This composition comprises three key components: a photosensitizing chemical (e.g., hypericin, porphyrins, or analogs), an energy-donating chemical (e.g., luciferin, dioxetanes, or analogs), and a mechanism for regulating the energy transfer or light emission. The photosensitizing chemical, upon activation, generates cytotoxic species like singlet oxygen, leading to cell death. The energy-donating chemical provides the energy for this photoactivation. The optional use of a chemical tether connecting the two components is suggested for enhanced efficiency. The methods involve introducing this composition into cells, where the energy-donating chemical is activated, triggering photoactivation of the photosensitizer and subsequent destruction of viral or tumor cells. This innovative approach offers a promising strategy for targeted antiviral and antitumor therapy.

A critical aspect of this invention is the regulated activation of the energy-donating chemical, ensuring targeted action against virus-infected or tumor cells. This selectivity minimizes harm to healthy cells. The preferred method involves the use of an expression plasmid containing a gene for the activating chemical (e.g., luciferase) under the control of a promoter. This promoter is designed to be transactivated by viral replication (e.g., using HIV LTR or EIAV LTR promoters) or elevated levels of proteins expressed in tumor cells. This ensures that the activating chemical is produced predominantly within the target cells, thereby localizing the photoactivation of the photosensitizer. Alternatively, the use of dioxetanes as energy donors, triggered by proteases specific to infected or neoplastic cells, offers another means of achieving targeted delivery. The system addresses limitations of conventional PDT, where light penetration is restricted, by using an internally activated light source.

The patent highlights specific preferences for the chemical components. Hypericin is the preferred photosensitizing chemical due to its absorption of light in the 540-660 nm range and its high quantum yield for singlet oxygen production. However, other quinones, porphyrins, and hematoporphyrin derivatives are also mentioned as suitable alternatives. Luciferin is the preferred energy-donating chemical because of its high quantum yield in luciferase-catalyzed reactions, with its emission spectrum overlapping the absorption spectrum of hypericin. However, various alternatives such as dioxetanes, dioxetanones, and dioxetanediones are also considered. The synthesis of luciferin analogs is discussed and several tethered compounds, combining luciferin analogs with hypericin or its analogs are described, illustrating the flexibility of the system. The design of these tethered compounds allows for efficient intramolecular energy transfer, enhancing the efficiency of the therapy. The selection of tethers is based on the rate of energy transfer from the donor to acceptor and the substrate recognition by the enzyme for the light-producing reaction.

The document describes synthetic pathways for creating tethered compounds linking the photosensitizer and energy donor. Two general types of tethered compounds are detailed: one type uses a “caged” luciferin where the carboxylic acid group is protected as an activated ester, preventing premature reaction with luciferase until intracellular cleavage. The second type uses a “non-caged” luciferin analog, forming a stable link with the photosensitizer. This approach allows for efficient intramolecular energy transfer. The synthesis of various luciferin analogs is detailed, focusing on routes for creating allyl ether and nitrile modifications. The use of 1,2-dioxetane as an alternative energy-donating chemical is described, highlighting its oxygen-independent activation and suitability for hypoxic cells. The document emphasizes the flexibility of the tethering approach, allowing for the incorporation of a variety of photosensitizers and energy donors. Tether selection considerations include energy transfer rate and enzyme-substrate recognition, allowing for optimization of the system for different applications.

The mechanism of action relies on photoactivation of a photosensitizing chemical, primarily hypericin or a porphyrin analog. Upon activation (triggered by energy transfer from an energy-donating chemical), the photosensitizer generates cytotoxic singlet oxygen. This highly reactive species damages cellular components, leading to cell death—either viral inactivation or tumor cell destruction. The efficiency of this process is improved through the use of a chemical tether that links the photosensitizing and energy-donating chemicals, enabling efficient energy transfer between them. The light emission from the energy donor needs to overlap with the absorption of the photosensitizer for optimal activation. Luciferin, with its emission band centered at 560 nm and overlapping the absorption bands of hypericin, is one such preferred energy donor. The process is thus dependent on the photoactivation of the photosensitizer and the subsequent production of reactive oxygen species that are toxic to cells.

Targeted delivery is critical to minimize damage to healthy cells. This is achieved by regulating the expression of the activating chemical (e.g., luciferase) using an expression plasmid. This plasmid contains a gene encoding the activating chemical, under the control of a promoter which is specifically activated in virus-infected or tumor cells. The use of viral promoters like the HIV LTR or EIAV LTR provides a mechanism to achieve high levels of expression in infected cells only. For tumor cells, the promoter is selected to be responsive to proteins highly expressed in tumor cells. This targeted expression ensures that the energy-donating chemical is activated mainly within the targeted cells, triggering selective photoactivation and subsequent cell death, thereby maximizing therapeutic efficacy and minimizing side effects. The expression plasmid is introduced into the target cells by various methods, including liposome-mediated transfer and direct transfection.

In addition to the luciferin-luciferase system, the patent proposes the use of dioxetanes as an alternative energy-donating chemical. Dioxetanes offer advantages, including oxygen-independent activation which makes them suitable for use in hypoxic (low-oxygen) environments such as those often found in solid tumors. These dioxetanes are designed as tethered compounds containing both the photosensitizing chemical and a trigger moiety (component three). The trigger, preferably a polypeptide sequence, is cleaved by a protease produced by the virus or tumor cell. This cleavage destabilizes the dioxetane, leading to the production of an excited singlet and the subsequent photoactivation of the photosensitizer. The choice of trigger polypeptide can be tailored to specific viral or tumor proteases, enhancing target specificity. The reagent to trigger the dioxetane can also be a strong base, or in the case of a carbohydrate trigger, beta-galactosidase.

The patent details the chemical synthesis of key components. A preferred route for synthesizing luciferin analogs is described, starting from a benzothiazolehydroxy nitrile intermediate. The synthesis involves standard organic reactions such as allyl ether formation, Claisen rearrangement, and hydroboration, ultimately incorporating alkenyl groups with 1–15 carbon atoms. The synthesis of tethered compounds, crucial for efficient energy transfer, is also outlined. These compounds are formed by linking luciferin or its analogs to photosensitizing chemicals like hypericin or its octahydroxy analog. Two main types of tethered compounds are described: one uses a “caged” luciferin derivative, where the carboxylic acid is protected as an activated ester, which is cleaved by esterases within the cell to activate the luciferin. The second type uses a “non-caged” luciferin analog, remaining chemically linked to the photosensitizer for highly efficient energy transfer. The synthesis of these tethered molecules is described in detail, highlighting their flexibility and potential for various modifications.

1,2-Dioxetane is presented as a preferred alternative energy-donating chemical. The patent describes the formation of stable dioxetane molecules containing both a light acceptor (the photosensitizer) and a trigger moiety. This integrated approach simplifies administration since all three components are contained within a single molecule. A significant advantage of the dioxetane system is its oxygen independence; it functions effectively in hypoxic environments, unlike some other systems. This makes it particularly suitable for treating solid tumors where oxygen levels are low. The synthesis of dioxetanes is not detailed, but the document suggests it is achievable using known techniques. The trigger moiety in the dioxetane can be a polypeptide cleavable by a viral or tumor protease, further enhancing target specificity and allowing for a single-capsule or injection approach to the therapy.

The patent discusses pharmaceutical formulations for administering the compositions. These formulations include both solid and liquid forms. Solid formulations might employ carriers such as starch, sugar, bentonite, or silica. Liquid formulations can utilize carriers or diluents like propylene glycol, benzyl alcohol, isopropanol, ethanol, DMSO, or dimethylacetamide. Aqueous solutions with a pH above 7 are also considered. The document mentions examples for parenteral administration (e.g., sterile isotonic saline solutions containing specified concentrations of the active compounds). The choice of formulation depends on the desired route of administration and is subject to optimization based on factors such as tissue accumulation properties of the compounds. The concentration of active ingredients can range widely, from 0.001 micrograms to 100,000 micrograms depending on the dosage form and therapeutic strategy.

A crucial aspect of the invention is the use of gene therapy for targeted delivery of the activating chemical. This is accomplished through the construction of an expression plasmid. The plasmid incorporates a gene encoding the activating chemical (preferably luciferase) under the control of a specific promoter. The promoter's key role is to regulate the expression of the activating chemical, ensuring it is predominantly produced in virus-infected or tumor cells. For antiviral applications, promoters responsive to viral replication are used, such as the HIV long terminal repeat (LTR), the EIAV LTR, or the Herpes Simplex Virus alpha gene promoter. These promoters are transactivated during viral replication, leading to increased expression of the activating chemical and subsequent activation of the energy-donating chemical. The inclusion of a selectable marker like neomycin resistance allows selection for cells that have successfully integrated the plasmid.

The expression plasmid, containing the activating chemical gene and, potentially, a selectable marker, is delivered into cells via various methods. These include incorporating the plasmid into a liposome for delivery, direct naked DNA transfer, microinjection, or calcium phosphate precipitation. Following transfection, cells expressing the activating chemical are selected—for example, by using a neomycin resistance gene as the selectable marker to identify cells successfully expressing the gene. The selected cells which are expressing the gene can then be used for further experiments or reintroduced into the patient. This allows for the creation of producer cells that generate retroviral particles encoding the activating chemical under the control of a regulated promoter. In some embodiments, this involves the use of retroviral vectors capable of integrating the expression plasmid stably into the host cell genome. The duration of expression from the inserted DNA and the survival time of these cells will impact the frequency of administering the third component.

The use of viral vectors, particularly retroviral vectors, is explored for the delivery of the expression plasmid. Retroviral vectors offer advantages for stable integration of the therapeutic gene into the host cell's genome, resulting in long-term expression of the activating chemical. The system employs packaging cells—cells that harbor proviral sequences for expressing retroviral structural proteins but lack sequences necessary for packaging and replication of RNA. The plasmid, introduced into the packaging cells, gets encapsidated by retroviral structural proteins and generates retroviral particles that carry the genetic material of interest. These retroviral vectors containing the regulated promoter driving expression of the activating chemical are then used to infect target cells, ensuring that gene expression and subsequent activation of the photodynamic therapy components are localized to the infected or tumor cells. The efficiency of this delivery method is important in determining the overall efficacy and duration of the therapeutic effect.