Construction of a Photoactivated Insulin Depot

Insulin is a primary tool for the treatment of type I diabetes. However, multiple challenges accompany its administration. Because of low oral bioavailability, it is typically injected multiple times per day, a significant lifetime burden on patients. Moreover, because of fluctuating blood sugar levels, the required amounts of insulin continuously vary over the course of a day. A current solution to the challenge of multiple daily injections is the use of an insulin pump, which delivers insulin transdermally through a cannula, an awkward and limiting solution. We have conceived of a less invasive approach, in which light is used to activate the release of insulin from a covalently linked depot (Figure 1). The elements of the system are a polymer, a photocleavable linker, and the therapeutic, insulin. Ideally, the polymer should be insoluble, so that it remains at the site of injection, and biodegradable, so that after the majority of the insulin has been released, the polymer can be cleared from the system. Insulin has features that make it particularly amenable to the photoactivated depot (PAD) approach. Because blood sugar levels vary greatly, the required concentrations of insulin in the blood also vary greatly from hour to hour. Moreover, the total volume of insulin required in a day is small, on the order of 1 mg, the equivalent of 1 mL. Even with a 10:1 ratio of carriers to insulin in a PAD, a typical 250 mL injection of insulin would contain the equivalent of 25 days insulin. The principle challenge is to engineer a material in which insulin maintains its integrity during the synthetic process, and is retained effectively until light releases it in a precisely metered fashion and in its native form. We have successfully made this material by using a new azide derivative of the 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE) group that reacts with insulin. The final link to the insoluble resin is made through a “click”-type reaction with a strained cyclooctyne (DBCO) that is resin-bound. We demonstrate that insulin modified with both one and two DMNPE–azide moieties is photoreleased in a similar fashion, with the dimodified species being photolyzed in two sequential reactions, each of which has identical kinetics. This suggests chemically similar sites of modification. Furthermore, we demonstrate that the insulin modified with DMNPE–azide efficiently reacts with resin-bound DBCO, and that this final material shows efficient and metered photorelease of native insulin when using a 365 nm lightemitting diode (LED). We are pursuing multiple ways of making the photocleavable link between insulin and an insoluble resin. One of the most direct ways of accomplishing this is using a “click” approach to make the final link between resin and insulin (Figure 2). We started from the known DMNPE derivative 2, which contains both carboxy and ketone groups. This was modified with an amino azide 1 to make the corresponding amide 3 in good yield. The ketone group was then transformed into the hydrazone 4 by using hydrazine, and finally converted to the diazo derivative 5 by using MnO2, a process based on previously developed DMNPE chemistry in our group. We found that a parallel approach, in which propargyl amine was condensed with 2, was not effective. The diazo–DMNPE–azide 5 was reacted with insulin directly in DMSO, using a 2:1 mole ratio. HPLC analysis of the reaction mixture shows two main, well-resolved peaks as well as a minor one (Figure 3a). The compounds in these peaks were isolated and identified by using ESI–MS as unreacted insulin (58%), monomodified insulin (“insulin monoazide” 6, 32%), and dimodified insulin (“insulin diazide” 7, 9%). A less highly modified sample was prepared for the resin modification experiments using a 1:1 ratio of insulin to 5. This reaction product showed 75% unreacted insulin, 23% insulin monoazide 6, and 2% insulin diazide 7. For the resin linking, this mixture was used directly, because Figure 1. Overall approach to the photoactivated depot (PAD). A drug, insulin in this case, is linked to an insoluble but biodegradable resin, through a photocleavable linker. The conjugate is injected in a shallow depot cutaneously or subcutaneously. Irradiation breaks the link of insulin from the resin, thereby allowing it to diffuse away from the resin and be absorbed by the systemic circulation. Ultimately the resin is biodegraded.