Photopharmacology is an emerging multidisciplinary field that combines photochemistry and pharmacology. 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. The light-based modulation is achieved by incorporating molecular photoswitches such as azobenzene and diarylethenes or photocages such as o-nitrobenzyl, coumarin, and BODIPY compounds into the pharmacophore. 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.
History Though photopharmacology is a relatively new field, the concept of using light in therapeutic applications came into practice a few decades ago. Photodynamic therapy (PDT) is a well-established clinically practiced protocol in which photosensitizers are used to produce singlet oxygen for destroying diseased or damaged cells or tissues. Optogenetics is another method that relies on light for dynamically controlling biological functions especially brain and neural. 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.
Figure 1. Schematic representation of the mechanism of (a) photopharmacology (b) photodynamic therapy, and (c) optogenetics.
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. 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.Lakowicz, Joseph R. Principles of Fluorescence Spectroscopy, 3rd Edition (2006), Page 5, Springer Science+Business Media, LLC. ISBN 978-0 387-31278-1.
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.Qiao, Zhi., et al. Azobenzene-isoxazoline as photopharmacological ligand for optical control of insect GABA receptor and behavior. Pest Manag. Sci. 78, 467–474 (2022). 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. There are a number of similar photoswitches which isomerize between E and Z configurations across an azo group (for instance, azobenzene and azopyrazole) or an ethylene bridge (for instance, stilbene and hemithioindigo).
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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).Andrew W., Lincoln, Per., Tuite, Eimer M. Environmental effects on the photophysics of transition metal complexes with dipyrido[2,3-a:3,2-c]phenazine (dppz) and related ligands. Coordination Chemistry Reviews 255, 2676–2692 (2011). 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. They are introduced into the structure of bioactive compounds via insertion, extension, or bioisosteric replacement. These incorporations can be supported by structural considerations of the molecule or SAR (structure-activity relationship) analysis to determine the optimal position. Some examples of photoswitchable molecules commonly used in photopharmacology are azobenzenes, diarylethenes, and photocages.
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. When in its photoactive cis form, phototrexate has been shown to be a potent antifolate and is relatively inactive when in the trans form. 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.
Diarylethenes Diarylethene photoswitches have reversible cyclization and cycloreversion reactions that are photoinduced. 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).
Figure 4. Figure showing stilbene isomerizations under light from E to 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. 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.
Donor-acceptor Stenhouse adducts (DASAs) DASAs are a relatively new class of molecular photoswitches (invented in 2014) that unlike conventional compounds requiring UV light, are characterized by activation by visible and near-infrared light and negative photochromism. Negative or T-type photoswitches are colored in their stable state and become colorless (less absorbing) upon light irradiation, which allows photoisomerization to propagate deep in a material or tissue. Their complex mechanism of isomerization (cyclization from a linear, polar, colored form to a non-polar, colorless, closed-ring form) has been studied in detail. Their application to photopharmacology in aqueous solution has been explored recently.
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". The photosensitive groups physically and chemically protect the target from being released until the molecule undergoes photoreaction. 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. Some well-known photocages include "o-nitrobenzyl derivatives, coumarin derivatives, BODIPY, xanthene derivatives, quinone and diarylenes derivatives". 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.
Figure 5. Example of a photocage release system activated by NIR.
Application