Simon H. Friedman

In this work, we demonstrate that the photoactivated depot (PAD) approach can be used to produce pulsatile release of human gonadotropin releasing hormone (GnRH) using green light. Pulsatile release is critical for a range of hormones and produces distinct physiological responses in comparison with continuous release. In particular, this has been observed with the use of GnRH for the treatment of cognitive impairment in Down syndrome. We have synthesized a GnRH PAD material utilizing a green light activated nonpolar tag based on the thiocoumarin photocleavable group. This material is essentially insoluble, as required of depots in the PAD approach. Upon photolysis, it releases native soluble GnRH. A major synthetic challenge for the synthesis of this PAD was that GnRH has neither of the standard functional groups previously utilized (carboxyl or amine) to link to the light-activated, solubility controlling moiety. Using proteolysis and de novo peptide sequencing, we demonstrate that GnRH is modified via the guanidino group of arginine, making an unusual linkage. We demonstrate that this material is highly stable toward chemical hydrolysis and releases pulses of soluble GnRH in response to pulses of 500 nm light.

Protein self‐interaction leading to aggregation is a major challenge facing protein pharmaceuticals. It leads to a range of problems, including increases in immunogenicity and loss of activity. In this work, we describe an approach for blocking or antagonizing the quaternary interactions that drive self‐association. We applied the approach to glucagon, a therapeutic peptide known for its propensity to form fibrils due to self‐interaction. We synthesized a regio‐pure common feedstock that allowed easy modification with potential blocking peptides that represented a range of chemical types (anionic, cationic, polar, and nonpolar). From these synthesized materials, we identified two modified glucagons that showed significant stabilization against fibril formation compared with unmodified glucagon. This was confirmed by three complementary biophysical techniques. Both successful modifications introduced excess net charge to glucagon, consistent with overall electrostatic repulsion being at the root of the observed fibrillation resistance. This approach can potentially be applied to other therapeutic proteins that suffer from the problems associated with self‐association.

ConspectusMany therapeutic proteins can benefit from controlling the timing and amount of their release. This is especially true for signaling molecules such as insulin, whose requirements vary continually throughout the day. Currently, the only way to provide this variable delivery is through a pump. Pumps, and their required cannulas/needles, introduce a wide range of problems, including cannula occlusion, infection, and biofouling. We have instead pursued the photoactivated depot or PAD approach, in which therapeutic proteins are released into the body through light activation of shallow, skin-based depots that are activated by small LED light sources ( Angew. Chem. 2013, 125(5), 1444-1449, Mol. Pharmaceutics 2016, 13(11), 3835-3841, J. Am. Chem. Soc. 2017, 139(49), 17861-17869, ACS Biomater Sci. Eng. 2021, 7(4), 1506-1514, and ACS Biomater Sci. Eng. 2024, 10(6), 3806-3812). By linking protein release to transcutaneous irradiation, we can control the amount and timing of therapeutic release by varying the amount and timing of irradiation. At the heart of this approach are PAD materials that contain three key elements: the therapeutic protein, a photocleavable (PC) group, and a solubility reducing moiety. This latter element is needed to allow the PAD material to stay at the site of injection, so that light can be effectively directed to it. The light causes the PC group to break its bond with the therapeutic protein, which can then diffuse into the capillary bed and be absorbed into systemic circulation. We have pursued four distinct methods of achieving solubility reduction prior to irradiation. The first approach is to use a highly insoluble polymer that is linked to the therapeutic protein via the PC group. This was the approach we used in our first attempt at making a PAD material and proved to be effective in both in vitro and in vivo settings. The main challenge with this first approach is that the polymer is left in the body after the protein is released, necessitating additional optimization to clear it, using biodegradation. In addition, it is very inefficient, with only a minority of the material being the therapeutic. In the second approach, we created polymers/oligomers out of the protein, using small light-cleaved links. The simplest of these, a trimer of proteins linked to a central core, is 90% therapeutic, and retains the preirradiation insolubility required of the PAD approach. In the third approach, we link charged groups to the protein to shift its iso-electric point, such that the material will be insoluble (and hence able to form a depot) at pH 7, but will release native, active protein after photolysis cleaves off the charged groups. Finally, in the fourth approach, we confer insolubility by attaching highly nonpolar groups to the therapeutic protein via a PC linkage. In this article, the challenges, strengths and weaknesses of each of these approaches will be described, and guidance will be given for the application of the PAD approach to other systems that can benefit from the controlled release that it enables.

Pseudo‐gout is caused by the deposition of highly insoluble calcium pyrophosphate dihydrate (CPPD) crystals in the joints of sufferers. This leads to inflammation and ultimately joint damage. The insolubility of CPPD is driven by the strong attraction of di‐cationic calcium ions with tetra‐anionic pyrophosphate ions. One of the challenges of dissolving CPPD is that a related mineral, hydroxy apatite (HA) is present in larger amounts in the form of bone and also contains strongly interacting calcium and phosphate ions. Our aim in this work was to selectively dissolve CPPD in preference to HA. To accomplish this, we used a known receptor for pyrophosphate that contains two complexed zinc ions that are ideally spaced to interact with the tetra‐anion of pyrophosphate. We hypothesized that such a molecule could act as a preorganized tetra‐cation that would be able to outcompete the two calcium ions present in the crystal lattice of CPPD. We demonstrate both visually and through analysis of released phosphorous that this molecule is able to preferentially dissolve CPPD over the closely related HA and thus can form the basis for a possible approach for the treatment of pseudo‐gout.

In this work, for the first time, we demonstrate light control of a therapeutic protein's release from a depot in the subcutaneous layer of the skin. The subcutaneous layer is a standard location for therapeutic protein depots due to its large size and ease of access, but prior attempts to utilize this space failed because insufficient light can reach this deeper layer. An analysis of existing biophysical literature suggested that an increase of photoactivation wavelength from 365 to 500 nm could allow an increase of depot irradiation in the subcutaneous by >100-fold. We therefore used a green light-activated thio-coumarin-based material and demonstrated robust release of a therapeutic, insulin, in response to skin illumination with an LED light source. We further demonstrated that this release is ultrafast, as fast or faster than any commercially used insulin, while maintaining the native insulin sequence. This release of insulin was then accompanied by a robust reduction in blood glucose, demonstrating the retention of bioactivity despite the synthetic processing required to generate the material. In addition, we observed that the material exhibits slow basal release of insulin, even in the absence of light, potentially through biochemical or photochemical unmasking of insulin. Thus, these materials can act much like the healthy pancreas does: releasing insulin at a slow basal rate and then, upon skin irradiation, releasing an ultrafast bolus of native insulin to reduce postprandial blood glucose excursions.

The front cover artwork is provided by the group of Professor Simon H. Friedman at the School of Pharmacy, University of Missouri–Kansas City (USA). The image depicts, in an abstract and minimalistic way, a central challenge for photopharmacology: the attenuation of light as it passes through tissue. Read the full text of the Minireview at 10.1002/cptc.202100001.

In this work, we have brought the release of glucagon under the control of light. The aim of this approach is to allow minimally invasive, two-hormone control of blood glucose. Glucagon has two major challenges associated with its therapeutic application: (1) the required amount and timing of glucagon release is highly variable, and (2) glucagon rapidly fibrillates in solution, forming aggregates that are inactive. We have developed a light activated glucagon trimer, in which we have joined three glucagon molecules via light cleaved linkers. We demonstrated that this material can be stimulated by light to release glucagon in a predictable manner. In addition, we demonstrated that in the absence of light, the trimer does not form fibrils and thus releases normal unfibrillated glucagon upon irradiation. These qualities make this material ideal for incorporation into a two hormone light-activated artificial pancreas system.