Matrix Metalloproteinase-Responsive Delivery of PEGylated Fibroblast Growth Factor 2.

Attachment of polyethylene glycol (PEG) chains is a common, well-studied, and Food and Drug Administration-approved method to address the pharmacokinetic challenges of therapeutic proteins. Occasionally, PEGylation impairs the activity of pharmacodynamics (PD). To overcome this problem, disease-relevant cleavable linkers between the polymer and the therapeutic protein can unleash full PD by de-PEGylating the protein at its target site. In this study, we engineered a matrix metalloproteinase (MMP)-responsive fibroblast growth factor 2 (FGF-2) mutant that was site-specifically extended with a PEG polymer chain. Using bioinspired strategies, the bioconjugate was designed to release the native protein at the desired structure/environment with preservation of the proliferative capacity in vitro on NIH3T3 cells. In vivo, hepatic exposure was diminished but not its renal distribution over time compared to unconjugated FGF-2. By releasing the growth factor from the PEG polymer in response to MMP cleavage, restored FGF-2 may enter hard-to-reach tissues and activate cell surface receptors or nuclear targets.

Oral Use of Therapeutic Carbon Monoxide for Anyone, Anywhere, and Anytime.

Carbon monoxide (CO) is a therapeutic gas with therapeutic potential in intestinal bowel disease. Therapeutic efficacy in the gastrointestinal tract (GIT) must be paired with safe and convenient use. Therefore, we designed an oral CO releasing system (OCORS) pairing tunable CO release into the GIT while preventing the release of any other molecule from within the device, causing safety concerns. The dimensions of the device, which is manufactured from 3D printed components, are within compendial limits. This is achieved by controlling CO decarbonylation from a molybdenum complex with a FeCl3 solution. OCORS' surrounding silicon membranes control release rates, as does the loading with carbonylated molybdenum complex and FeCl3 solution. Herein we describe the development of the system, the characterization of the CO releasing molecule (CORM), and the CO release kinetics of the overall system. Neither the CORM nor isocyanoacetate as a potential reaction byproduct were cytotoxic. Finally, we demonstrated by design validation in an in vivo porcine model that, except for the release of the therapeutic CO, OCORS isolates all components during transit through the stomach. We could show that OCORS generated and released CO locally into the stomach of the animals without systemic exposure, measured as the carboxyhemoglobin content in the blood of the pigs. In conclusion, OCORS derisks oral development by limiting patient exposure to (desirable) CO while preventing contact with any further (undesirable) chemical, by-, or degradation products. CO generating devices come in reach, which now can be used by anyone, anywhere, and anytime.

Azobenzene derivatives with activity against drug-resistant Candida albicans and Candida auris.

Increasing resistance against antimycotic drugs challenges anti-infective therapies today and contributes to the mortality of infections by drug-resistant Candida species and strains. Therefore, novel antifungal agents are needed. A promising approach in developing new drugs is using naturally occurring molecules as lead structures. In this work, 4,4'-dihydroxyazobenzene, a compound structurally related to antifungal stilbene derivatives and present in Agaricus xanthodermus (yellow stainer), served as a starting point for the synthesis of five azobenzene derivatives. These compounds prevented the growth of both fluconazole-susceptible and fluconazole-resistant Candida albicans and Candida auris strains. Further in vivo studies are required to confirm the potential therapeutic value of these compounds.

Amber Light Control of Peptide Secondary Structure by a Perfluoroaromatic Azobenzene Photoswitch.

The incorporation of photoswitches into the molecular structure of peptides and proteins enables their dynamic photocontrol in complex biological systems. Here, a perfluorinated azobenzene derivative triggered by amber light was site-specifically conjugated to cysteines in a helical peptide by perfluoroarylation chemistry. In response to the photoisomerization (trans→cis) of the conjugated azobenzene with amber light, the secondary structure of the peptide was modulated from a disorganized into an amphiphilic helical structure.

Bile and excipient interactions directing drug pharmacokinetics in rats.

Bile solubilization plays a major role in the absorption of poorly water-soluble drugs. Excipients used in oral drug formulations impact bile-colloidal properties and their molecular interactions. Polymer-induced changes of bile colloids, e.g., by Eudragit E, reduced the flux of the bile interacting drug Perphenazine whereas bile non-interacting Metoprolol was not impacted. This study corroborates these in vitro findings in rats. Eudragit E significantly reduced systemic availability of Perphenazine but not Metoprolol compared to the oral administrations without polymer. This study confirms the necessity to carefully select polymers for bile interacting drugs whereas non-bile interacting drugs are more robust in terms of excipient choice for formulation. The perspective of bile interaction may introduce interesting biopharmaceutical leverage for better performing oral formulations of tomorrow.

PEtOxylated Interferon-α2a Bioconjugates Addressing H1N1 Influenza A Virus Infection.

Influenza A viruses (IAV), including the pandemic 2009 (pdm09) H1N1 or avian influenza H5N1 virus, may advance into more pathogenic, potentially antiviral drug-resistant strains (including loss of susceptibility against oseltamivir). Such IAV strains fuel the risk of future global outbreaks, to which this study responds by re-engineering Interferon-α2a (IFN-α2a) bioconjugates into influenza therapeutics. Type-I interferons such as IFN-α2a play an essential role in influenza infection and may prevent serious disease courses. We site-specifically conjugated a genetically engineered IFN-α2a mutant to poly(2-ethyl-2-oxazoline)s (PEtOx) of different molecular weights by strain-promoted azide-alkyne cyclo-addition. The promising pharmacokinetic profile of the 25 kDa PEtOx bioconjugate in mice echoed an efficacy in IAV-infected ferrets. One intraperitoneal administration of this bioconjugate, but not the marketed IFN-α2a bioconjugate, changed the disease course similar to oseltamivir, given orally twice every study day. PEtOxylated IFN-α2a bioconjugates may expand our therapeutic arsenal against future influenza pandemics, particularly in light of rising first-line antiviral drug resistance to IAV.

Polymer selection impacts the pharmaceutical profile of site-specifically conjugated Interferon-α2a.

Conjugation of poly(ethylene glycol) (PEG) to biologics is a successful strategy to favorably impact the pharmacokinetics and efficacy of the resulting bioconjugate. We compare bioconjugates synthesized by strain-promoted azide-alkyne cycloaddition (SPAAC) using PEG and linear polyglycerol (LPG) of about 20 kDa or 40 kDa, respectively, with an azido functionalized human Interferon-α2a (IFN-α2a) mutant. Site-specific PEGylation and LPGylation resulted in IFN-α2a bioconjugates with improved in vitro potency compared to commercial Pegasys. LPGylated bioconjugates had faster disposition kinetics despite comparable hydrodynamic radii to their PEGylated analogues. Overall exposure of the PEGylated IFN-α2a with a 40 kDa polymer exceeded Pegasys, which, in return, was similar to the 40 kDa LPGylated conjugates. The study points to an expanded polymer design space through which the selected polymer class may result in a different distribution of the studied bioconjugates.

Molecular Insights into Site-Specific Interferon-α2a Bioconjugates Originated from PEG, LPG, and PEtOx.

Conjugation of biologics with polymers modulates their pharmacokinetics, with polyethylene glycol (PEG) as the gold standard. We compared alternative polymers and two types of cyclooctyne linkers (BCN/DBCO) for bioconjugation of interferon-α2a (IFN-α2a) using 10 kDa polymers including linear mPEG, poly(2-ethyl-2-oxazoline) (PEtOx), and linear polyglycerol (LPG). IFN-α2a was azide functionalized via amber codon expansion and bioorthogonally conjugated to all cyclooctyne linked polymers. Polymer conjugation did not impact IFN-α2a's secondary structure and only marginally reduced IFN-α2a's bioactivity. In comparison to PEtOx, the LPG polymer attached via the less rigid cyclooctyne linker BCN was found to stabilize IFN-α2a against thermal stress. These findings were further detailed by molecular modeling studies which showed a modulation of protein flexibility upon PEtOx conjugation and a reduced amount of protein native contacts as compared to PEG and LPG originated bioconjugates. Polymer interactions with IFN-α2a were further assessed via a limited proteolysis (LIP) assay, which resulted in comparable proteolytic cleavage patterns suggesting weak interactions with the protein's surface. In conclusion, both PEtOx and LPG bioconjugates resulted in a similar biological outcome and may become promising PEG alternatives for bioconjugation.

Targeting interleukin-4 to the arthritic joint.

Anti-inflammatory cytokines are a promising class of therapeutics for treatment of rheumatoid arthritis (RA), but their use is currently limited by a rapid clearance and systemic toxicity. Interleukin-4 is a small cytokine with potential for RA therapy. To increase its pharmacokinetic features, we engineered a murine IL4 conjugate by incorporating an unnatural amino acid through genetic code expansion to which PEG-folate, as a targeting moiety and PEG alone as control, were site-specifically bound. Both IL4 conjugates retained bioactivity and induced primary murine macrophage polarization into an alternatively activated (M2) related phenotype. The PEGylated conjugates had a terminal half-life of about four hours in healthy mice compared to unPEGylated IL4 (0.76 h). We showed that both conjugates successfully accumulated into arthritic joints in an antigen-induced arthritis (AIA) mouse model, as assessed by non-invasive fluorescence imaging. The modular nature of the IL4 conjugate chemistry presented herein facilitates easy adaption of PEG chain length and targeting moieties for further improvement of half-life and targeting function for future efficacy studies.

Biodistribution of Site-Specific PEGylated Fibroblast Growth Factor-2.

Fibroblast growth factor 2 (FGF-2) is a small 18 kDa protein with clinical potential for ischemic heart disease, wound healing, and spinal cord injury. However, the therapeutic potential of systemic FGF-2 administration is challenged by its fast elimination. Therefore, we deployed genetic codon expansion to integrate an azide functionality to the FGF-2 N-terminus, which was site-directly decorated with poly(ethylene glycol) (PEG) through bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC). PEGylated FGF-2 was as bioactive as wild-type FGF-2 as demonstrated by cell proliferation and Erk phosphorylation of fibroblasts. The PEGylated FGF-2 conjugate was radiolabeled with [111In] Indium cation ([111In]In3+) to study its biodistribution through noninvasive imaging by single-photon emission computed tomography (SPECT) and by quantitative activity analysis of the respective organs in healthy mice. This study details the biodistribution pattern of site-specific PEGylated FGF-2 in tissues after intravenous (iv) administration compared to the unconjugated protein. Low accumulation of the PEGylated FGF-2 variant in the kidney and the liver was demonstrated, whereas specific uptake of PEGylated FGF-2 into the retina was significantly diminished. In conclusion, site-specific PEGylation of FGF-2 by SPAAC resulted in a superior outcome for the synthesis yield and in conjugates with excellent biological performances with a gain of half-life but reduced tissue access in vivo.

Fluorine walk: The impact of fluorine in quinolone amides on their activity against African sleeping sickness.

Human African Trypanosomiasis, also known as African sleeping sickness, is caused by the parasitic protozoa of the genus Trypanosoma. If there is no pharmacological intervention, the parasites can cross the blood-brain barrier (BBB), inevitably leading to death of the patients. Previous investigation identified the quinolone amide GHQ168 as a promising lead compound having a nanomolar activity against T. b. brucei. Here, the role of a fluorine substitution at different positions was investigated in regard to toxicity, pharmacokinetics, and antitrypanosomal activity. This 'fluorine walk' led to new compounds with improved metabolic stability and consistent activity against T. b. brucei. The ability of the new quinolone amides to cross the BBB was confirmed using an 18F-labelled quinolone amide derivative by means of ex vivo autoradiography of a murine brain.