Marcus Gutmann

Biocompatible Azide–Alkyne “Click” Reactions for Surface Decoration of Glyco-Engineered Cells

Bio‐orthogonal copper (I)‐catalyzed azide–alkyne cycloaddition (CuAAC) has been widely used to modify azide‐ or alkyne‐bearing monosaccharides on metabolic glyco‐engineered mammalian cells. Here, we present a systematic study to elucidate the design space for the cytotoxic effects of the copper catalyst on NIH 3T3 fibroblasts and on HEK 293‐F cells. Monitoring membrane integrity by flow cytometry and RT‐PCR analysis with apoptotic and anti‐apoptotic markers elucidated the general feasibility of CuAAC, with exposure time of the CuAAC reaction mixture having the major influence on biocompatibility. A high labeling efficiency of HEK 293‐F cells with a fluorescent alkyne dye was rapidly achieved by CuAAC in comparison to copper free strain‐promoted azide–alkyne cycloaddition (SPAAC). The study details effective and biocompatible conditions for CuAAC‐based modification of glyco‐engineered cells in comparison to its copper free alternative.

Prevention of colitis by controlled oral drug delivery of carbon monoxide

Carbon monoxide (CO) is an endogenous signal transmitter involved in numerous physiological processes including the gastrointestinal (GI) homeostasis. CO has been recognized as potential new therapeutic agent for motility related and inflammatory disorders of the GI tract. A therapeutic use, however, is challenged by inappropriate drug delivery modes. Here we describe a micro scale Oral Carbon Monoxide Release System (M-OCORS) designed for localized and controlled exposure of the GI tract with in situ generated CO. M-OCORS allowed for controlled release profiles lasting for several minutes or up to almost one day. These in vitro release profiles translated into a large pharmacokinetic design space following oral administration in mice and measured as CO-hemoglobin (CO-Hb) formation. M-OCORS with a release profile featuring exposure of the intestine was profiled in two independently performed studies demonstrating preventive effects in chemically induced colitis. M-OCORS significantly reduced damage scores and prevented upregulation of colitis biomarkers.

Matrix Metalloproteinase Responsive Delivery of Myostatin Inhibitors

PurposeThe inhibition of myostatin - a member of the transforming growth factor (TGF–β) family - drives regeneration of functional skeletal muscle tissue. We developed a bioresponsive drug delivery system (DDS) linking release of a myostatin inhibitor (MI) to inflammatory flares of myositis to provide self-regulated MI concentration gradients within tissues of need.MethodsA protease cleavable linker (PCL) – responding to MMP upregulation – is attached to the MI and site-specifically immobilized on microparticle surfaces.ResultsThe PCL disintegrated in a matrix metalloproteinase (MMP) 1, 8, and particularly MMP-9 concentration dependent manner, with MMP-9 being an effective surrogate biomarker correlating with the activity of myositis. The bioactivity of particle-surface bound as well as released MI was confirmed by luciferase suppression in stably transfected HEK293 cells responding to myostatin induced SMAD phosphorylation.ConclusionsWe developed a MMP-responsive DDS for MI delivery responding to inflammatory flare of a diseased muscle matching the kinetics of MMP-9 upregulation, with MMP-9 kinetics matching (patho-) physiological myostatin levels.ᅟGraphical AbstractSchematic illustration of the matrix metalloproteinase responsive delivery system responding to inflammatory flares of muscle disease. The protease cleavable linker readily disintegrates upon entry into the diseased tissue, therby releasing the mystatin inhibitor.

Controlled therapeutic gas delivery systems for quality-improved transplants.

Therapeutic gases enriched into perfusion solutions have been effectively used for the improvement of organ transplant quality. At present, the enrichment of perfusion solutions with gases requires complex machinery/containers and handling precautions. Alternatively, the gas is generated within the perfusion solution by supplemented carbonylated transition metal complexes with associated toxicological concerns when these metals contact the transplant. Therefore, we developed therapeutic gas releasing systems (TGRSs) allowing for the controlled generation and release of therapeutic gases (carbon monoxide and hydrogen sulfide) from otherwise hermetically sealed containers, such that the perfusion solution for the transplant is saturated with the gas but no other components from the TGRS are liberated in the solution. The release from the TGRS into the perfusion solution can be tailored as a function of the number and thickness of gas permeable membranes leading to release patterns having been linked to therapeutic success in previous trials. Furthermore, the surrogate biomarker HMGB1 was significantly downregulated in ischemic rat liver transplants perfused with enriched CO solution as compared to control. In conclusion, the TGRS allows for easy, reliable, and controlled generation and release of therapeutic gases while removing safety concerns of current approaches, thereby positively impacting the risk benefit profile of using therapeutic gases for transplant quality improvement in the future.

Bioinspired co-crystals of Imatinib providing enhanced kinetic solubility

Graphical abstract Figure. No caption available. Abstract Realizing the full potential of co‐crystals enhanced kinetic solubility demands a comprehensive understanding of the mechanisms of dissolution, phase conversion, nucleation and crystal growth, and of the complex interplay between the active pharmaceutical ingredient (API), the coformer and co‐existing forms in aqueous media. One blueprint provided by nature to keep poorly water‐soluble bases in solution is the complexation with phenolic acids. Consequently, we followed a bioinspired strategy for the engineering of co‐crystals of a poorly water‐soluble molecule – Imatinib – with a phenolic acid, syringic acid (SYA). The dynamics of dissolution and solution‐mediated phase transformations were monitored by Nuclear Magnetic Resonance (NMR) spectroscopy, providing mechanistic insights into the 60 fold‐increased long lasting concentrations achieved by the syringate co‐crystals as compared to Imatinib base and Imatinib mesylate. This lasting effect was linked to SYA’s ability to delay the formation and nucleation of Imatinib hydrate – the thermodynamically stable form in aqueous media – through a metastable association of SYA with Imatinib in solution. Results from permeability studies evidenced that SYA did not impact Imatinib’s permeability across membranes while suggesting improved bioavailability through higher kinetic solubility at the biological barriers. These results reflect that some degree of hydrophobicity of the coformer might be key to extend the kinetic solubility of co‐crystals with hydrophobic APIs. Understanding how kinetic supersaturation can be shaped by the selection of an interactive coformer may help achieving the needed performance of new forms of poorly water‐soluble, slowly dissolving APIs.