History

Photopharmacology is an emerging multidisciplinary field that combines photochemistry and pharmacology.^[1] Built upon the ability of light to change the pharmacokinetics and pharmacodynamics of bioactive molecules, it aims at regulating the activity of drugs in vivo by using light.^[2] This selective activation of the biomolecules helps prevent or minimize off-target activity and systemic side effects. Moreover, light being the regulatory element offers additional advantages such as the ability to be delivered with high spatiotemporal precision, low to negligible toxicity, and the ability to be controlled both qualitatively and quantitatively by tuning its wavelength and intensity. ^[3]

Though photopharmacology is a relatively new field, the concept of using light in therapeutic applications came into practice a few decades ago. Photodynamic therapy (PTD) is a well-established clinically practiced protocol in which photosensitizers are used to produce singlet oxygen for destroying diseased or damaged cells or tissues.^[2] Optogenetics is another method that relies on light for dynamically controlling biological functions especially brain and neural.^[3] Though this approach has proven useful as a research tool, its clinical implementation is limited by the requirement for genetic manipulation. Mainly, these two techniques laid the foundation for photopharmacology. Today, it is a rapidly evolving field with diverse applications in both basic research and clinical medicine which has the potential to overcome some of the challenges limiting the range of applications of the other light-guided therapies.

The discovery of natural photoreceptors such as rhodopsins in the eye inspired the biomedical and pharmacology research community to engineer light-sensitive proteins for therapeutic applications.^[2] The development of synthetic photoswitchable molecules is the most significant milestone in the history of light-delivery systems. Scientists are continuing with their efforts to explore new photoswitches and delivery strategies with enhanced performance to target different biological molecules such as ion channels, nucleic acid, and enzyme receptors. Photopharmacology research progressed from in vitro to in vivo studies in a significantly short period of time yielding promising results in both forms. Clinical trials are underway to assess the safety and efficacy of these photopharmacological therapies further and validate their potential as an innovative drug delivery approach.

Mechanism of Action

Molecular photoswitches are utilized in the field of photopharmacology, where the energetics of a molecule can be reversibly controlled with light to achieve spatial and temporal resolution of a particular effect. Photoswitches may function by undergoing photoisomerization through which light is used to conformationally adapt a molecule to a biological site, or through an environmental effect where an external factor such as a solvent effect or hydrogen bonding can selectively allow or quench an emissive state within a molecule. To visualize photophysical processes, a useful depiction is the Jablonski diagram. This is a diagram which depicts electronic and vibrational energy levels within a molecule as vertical levels and shows the possible relaxation pathways from excited states. Typically, the ground state is referred to as S0, and is drawn at the bottom of the figure with nearby vibrational excitations just above it. An absorption will promote an electron into the S1 state at any vibrational energy level, or into a higher order excited state if the absorbed energy has enough magnitude. The excited state can then undergo internal conversion which is the electronic relaxation to a lower state with the same vibrational energetics or vibrational relaxation within a state. This may be followed by an intersystem crossing wherein the electron undergoes a spin flip, or a radiative or nonradiative decay back to the ground state.^[4]

One example of an organic compound that undergoes photoisomerization is azobenzene. The structure is two phenyl rings joined with a N=N double bond and is the simplest aryl azo compound. Azobenzene and its derivatives have two accessible absorbance bands: the S1 state from a n-π* transition which can be excited into using blue light, and the S2 state from a π-π* transition that can be excited into using ultraviolet light.^[5] Azobenzene and its derivatives have two isomers, trans and cis. The trans isomer, having the phenyl rings on opposite sides of the azo double bond, is the thermally preferred isomer as there is less stereoelectronic distortion and more delocalization present. However, excitation of the trans isomer to the S2 state facilitates a shift to the cis isomer. The S1 absorption is associated with a conversion back to the trans isomer. In this way, azobenzene and its derivatives can act as reversible stores of energy by maintaining a strained configuration in the cis isomer. Modifications of the substituents on azobenzene allow the energetics of these absorptions to be tuned, and if they are engineered such that the two absorption bands overlap a single wavelength of light can be used to flip between them.

Figure 1. Isomerization of Azobenzene

Alternatively, photoswitches may themselves be emissive and exhibit environmental control over their properties. One such example is a class of ruthenium polypyridyl coordination complexes. Typically they contain two bidentate bipyridine or phenanthroline ligands and an extended phenanthroline-phenazine bidentate ligand such as dipyrido[3,2-a:2,3-c]phenazine (dppz).^[6]These complexes have an accessible metal to ligand charge transfer excited state (1MLCT) which undergoes rapid intersystem crossing to a 3MLCT state due to the strong spin-orbit coupling of the ruthenium center. These excited states are localized on the extended ligand phenazine nitrogens, and emission occurs from the 3MLCT state. Hydrogen bonding interactions such as the presence of water around these nitrogen atoms stabilizes the 3MLCT state, quenching the emission process. Thus, by controlling whether an aqueous or otherwise protic polar solvent is present, emissive behaviors can be “turned on/off”, and alternation between “bright states” and “dark states” is facilitated. This light switch behavior makes these and similar complexes of recent interest in photopharmacological applications such as photodynamic therapy.

Molecules

As previously mentioned, photopharmacology relies on the use of molecular photoswitches being incorporated into the structure of biologically active molecules which allows their potency to be controlled optically.^[7] They are introduced into the structure of bioactive compounds via insertion, extension, or bioisosteric replacement.^[7] These incorporations can be supported by structural considerations of the molecule or SAR (structure-activity relationship) analysis to determine the optimal position.^[7] Some examples of photoswitchable molecules commonly used in photopharmacology are azobenzenes, diarylethenes, and photocages.^[8]

Azobenzenes

Azobenzenes are a class of photoswitchable molecules and are used in photopharmalogical applications for their reversible photoisomerization, as described in the previous section. An example of a photoswitchable molecule that uses azobenzene is phototrexate. Phototrexate is an inhibitor of human dihydrofolate reductase and is an analogue of methotrexate, a chemotherapy agent.^[9] When in its photoactive cis form, phototrexate has been shown to be a potent antifolate and is relatively inactive when in the trans form.^[9] The azologization, or incorporation of azobenzene, of methotrexate allows for control of cytotoxic activity and is considered a step forward in developing targeted anticancer drugs with localized efficacy.^[9]

Diarylethenes

Diarylethene photoswitches have reversible cyclization and cycloreversion reactions that are photoinduced.^[10] They are a class of compounds that have aromatic functional groups bonded to each end of a carbon-carbon double bond. An example of this class of molecule that is used in photopharmacology is stilbene. Under the influence of light, stilbene switches between its two isomers (E and Z).

Diarylethenes have been shown to have some advantages over the more researched azobenzenes switches, such as thermal irreversibility, high photoswitching efficiency, favorable cellular stability, and low toxicity.^[10] Diarylethenes have been shown to have promise in fields other than photopharmacology as well. These fields include optical data storage, optoelectronic devices, supramolecular self-assembly and anti-counterfeiting.^[10]

Photocages

A class of substances known as photocages contain “photosensitive groups, also known as ‘photoremovable protecting groups’, from which target substances are released upon exposure to specific wavelengths of light”.^[11] The photosensitive groups physically and chemically protect the target from being released until the molecule undergoes photoreaction.^[11] Due to these interactions with light, they are commonly used molecules in photopharmacology. More recently, they have played an important role in photoactivated chemotherapy (PACT). In PACT, photocages utilize a photoremovable protecting group that protects cytotoxic drugs until the bond is cleaved via light interaction and the cytotoxic drug is released.^[12] Some well-known photocages include “o-nitrobenzyl derivatives, coumarin derivatives, BODIPY, xanthene derivatives, quinone and diarylenes derivatives”.^[11] However, there are limitations with using photocages in clinical applications as there are not many PPGs that can be used in vivo. This is due to PPG-payload conjugates needing to have acceptable solubility and biological inertness for biocompatibility and the need for efficient uncaging above 600 nm.^[12] Figure 5. Example of a photocage release system activated by NIR^[13]

Applications

Photopharmacology, the use of light to control the activity of drugs, has emerged as a promising approach to drug delivery and therapy. By harnessing the power of light, researchers can achieve precise control over drug release and activation, offering new possibilities for targeted and personalized treatments. In this subsection, we explore the application of photopharmacology in drug delivery, focusing on recent advancements and potential clinical applications. In this paper^[14] they’ve used Photopharmacology to control drug activity; multifunctional fibers in the study deliver light and drugs to specific body areas. Implanted fibers activate light-responsive drugs, altering their structure, and offering precise drug delivery for conditions needing exact timing or dosage. In another application^[15] researchers use photopharmacology to control drug release and focus on a drug interacting with tubulin, visualizing its release in real time with time-resolved serial crystallography. This technique offers insights into drug-tubulin interactions and demonstrates the potential for designing drugs with precise actions. In this paper,^[16] an in vitro protocol to test different light wavelengths on human cancer cell lines is developed, finding that blue light most effectively inhibited cell growth. This suggests that photopharmacology could offer new cancer treatment options by targeting specific light wavelengths to modulate drug activity in tumor cells. In this study,^[17] researchers designed HDAC inhibitors which can be activated or deactivated with light, providing precise therapeutic control. This approach could reduce the side effects of traditional chemotherapy by targeting inhibitors to specific body areas, potentially leading to more effective and personalized cancer treatments. In another study,^[18] ligands were designed to switch their binding mode to G-quadruplex DNA upon exposure to visible light. This method could potentially modulate the activity of G-quadruplex DNA, crucial in gene expression and telomere maintenance, offering new therapeutic avenues, particularly in cancer treatment. The study underscores photopharmacology's promise in targeting specific DNA structures, suggesting G-quadruplex DNA as a viable target for future photopharmacological interventions. Another application of photopharmacology^[19] is developing a luminescent photoCORM grafted on carboxymethyl chitosan, which, when exposed to light, releases carbon monoxide (CO) to induce apoptotic death in colorectal cancer cells, demonstrating precise control over CO release for targeted cancer therapy. This study^[20] developed photoactivatable antibody-photoCORM conjugates targeting human ovarian cancer cells, releasing CO upon light exposure to diminish cell viability. This approach offers precise cancer cell targeting while minimizing harm to healthy tissue, showcasing the potential of photopharmacology in cancer therapy. In another application^[21] by using light to control the assembly of nanopores, researchers can potentially regulate the flow of ions or molecules through these nanopores. This approach could have applications in various fields, including sensing, drug delivery, and nanotechnology. In this paper,^[22] a photoactivatable compound that binds to and modulates the activity of the CRY1 protein, regulating the mammalian circadian clock has been developed. By using light to control the compound's activity, researchers can potentially treat circadian rhythm disorders and related health conditions by modulating the function of CRY1. Photopharmacology involves using light to control the activity of drugs. In this study,^[23] the researchers developed a strategy to attach a photoswitchable group to a common antibiotic; ciprofloxacin. By attaching the photoswitchable group, researchers can control the activity of ciprofloxacin with light. This approach could potentially lead to new ways of treating bacterial infections, with the ability to switch the antibiotic's activity on and off as needed. Researchers developed a toolbox of photoswitchable antagonists that can interact with GPCRs, a class of proteins involved in various cellular processes.^[24] By using light to switch the activity of these antagonists, researchers can control the interaction between the antagonists and GPCRs in real-time. This approach allows for precise modulation of GPCR activity, which could lead to new insights into cellular signaling pathways and potential therapeutic applications.

Future Directions

The future of photopharmacology holds immense promise. It has the potential to revolutionize conventional drug therapy offering new avenues for precision medicine, treating neurological disorders, and in the field of oncology and ophthalmology.^[1] Additionally, it holds promise for the field of regenerative medicine where photoswitches can be used to modulate the activity of signaling pathways for targeted tissue repair and regeneration.^[3] Photopharmacology will continue to grow and expand with the new discoveries and advances happening in other related fields such as synthetic chemistry, biology, nanotechnology, pharmacology, and bioengineering. While the potential of photopharmacology is vast, there are some challenges that need to be addressed to make it a clinical reality. One such challenge is the development of stable and biocompatible photoswitches that are selective for their target receptors without cross-activity.^[2] It is particularly important that these photoswitchables have their absorbance bands fall within the wavelength range of 650 nm to 900 nm.^[2] Hence, optimum molecular designing of photoswitches is required to achieve the aforementioned characteristics and desired level of performance. At present, photopharmacology uses a rational drug design approach based on studying the structure-activity relationship, however, a phenotypic screening for photoswitchable drugs could also be beneficial. Another hurdle is finding appropriate and effective ways to deliver light to deep tissues in the body and tissues avoiding issues such as scattering and absorption. Although, external delivery of light is the most preferred method, the use of internal exogenous light sources such as luminescent compounds where light would be delivered directly at the site of action. This could avoid the issues related to light penetration and also enhance the degree of selectivity. In addition, this creates the opportunity to use photopharmacology as a theranostic approach that combines targeted drug delivery and molecular imaging.^[2]