The subcutaneous administration of biopharmaceuticals is advantageous over intravenous administration, particularly with regard to improved patient compliance. However, in highly concentrated protein formulations lower viscosity of the formulation and stability of the protein is difficult to achieve. One approach involves using the viscosity-reducing excipients to diminish the interactions between protein molecules. In this context, the main objective of the study was to develop an optimal formulation for a model monoclonal antibody (mAb) and to evaluate new test compounds as viscosity-reducing agents. The test compounds were investigated both individually at increasing concentrations up to 200 mM and in combinations for their viscosity-reducing effect. Our results showed that all individual test compounds reduced the viscosity of the mAb formulation by more than 30 %, with reduction achieved by the six test compounds exceeding that achieved by proline (Pro). A reduction in the viscosity of the formulation below the 20 mPas threshold was achieved either by combining two test compounds or by increasing the concentration of a single compound above 25 mM. An accelerated stability study showed similar stabilization effects regardless of whether the test compounds were used alone or in combination. The percentage of aggregates was below 5 % in most formulations. These viscosity-reducing and stabilization effects corresponded to the dynamic light scattering results, which indicated that the test compounds reduced the attractive forces between the mAb molecules.

Polysorbates (PS) are widely used surfactants in biopharmaceutical formulations playing a crucial role in protecting proteins against mechanical stress and interface-induced damage. However, their susceptibility to degradation can compromise their function and lead to particle formation. Recent studies suggest that monoclonal antibodies (mAbs) may mitigate PS degradation catalyzed by histidine chloride buffer, indicating the presence of protein-PS interactions. In this study, we investigated these interactions using NMR, starting with 1H T2 CPMG filter experiments and methyl fingerprinting, which failed to detect interactions. To enhance sensitivity, we synthesized spin-labeled PS (SLPS), enabling paramagnetic relaxation enhancement (PRE) NMR experiments, specifically amide fingerprinting, which successfully revealed interactions. Complementary electron paramagnetic resonance (EPR) measurements of SLPS and mAb also detected interactions, but only in the presence of sucrose, underscoring their weak and transient nature. Additionally, molecular dynamics simulations identified potential interaction hotspots on the antibody structure, providing mechanistic insights into these interactions.

The excipient selection process plays a crucial role in biopharmaceutical formulation development to ensure the long-term stability of the drug product. Though there are numerous options approved by regulatory authorities, only a subset is commonly utilized. Previous research has proposed various stabilization mechanisms, including protein-excipient interactions. However, identifying these interactions remains challenging due to their weak and transient nature. In this study, we present a comprehensive approach to identify such interactions. Using the 1H T2 CPMG (Carr-Purcel-Meiboom-Gill) filter experiment we identified interactions of rituximab with certain buffers and amino acids, shedding light on its Fc fragment instability that manifested during the enzymatic cleavage of the antibody. Moreover, chemometric analyses of 2D NMR fingerprints revealed interactions of selected excipients with antibody fragments. Furthermore, molecular dynamics simulations revealed potential interacting hotspots without NMR spectra assignment. Our results highlight the importance of an orthogonal methods approach to uncovering these critical interactions, advancing our understanding of excipient stabilization mechanisms and rational formulation design in biopharmaceutics.

Administration of monoclonal antibodies (mAbs) is currently focused on subcutaneous injection associated with increased patient adherence and reduced treatment cost, leading to sustainable healthcare. The main bottleneck is low volume that can be injected, requiring highly concentrated mAb solutions. The latter results in increased solution viscosity with pronounced mAb aggregation propensity because of intensive protein-protein interactions. Small molecule excipients have been proposed to restrict the protein-protein interactions, contributing to reduced viscosity. The aim of the study was to discover novel compounds that reduce the viscosity of highly concentrated mAb solution. First, the chemical space of proline analogs was explored and 35 compounds were determined. Viscosity measurements revealed that 18 proline analogs reduced the mAb solution viscosity similar to or more than proline. The compounds forming both electrostatic and hydrophobic interactions with mAb reduced the viscosity of the formulation more efficiently without detrimentally effecting mAb physical stability. A correlation between the level of interaction and viscosity-reducing effect was confirmed with molecular dynamic simulations. Structure rigidity of the compounds and aromaticity contributed to their viscosity-reducing effect, dependent on molecule size. The study results highlight the novel proline analogs as an effective approach in viscosity reduction in development of biopharmaceuticals for subcutaneous administration.

The formulation of biopharmaceutical drugs is designed to eliminate chemical instabilities, increase conformational and colloidal stability of proteins, and optimize interfacial stability. Among the various excipients involved, buffer composition plays a pivotal role. However, conventional buffers like histidine and phosphate buffers may not always be the optimal choice for all monoclonal antibodies (mAbs). In this study, we investigated the effects of several alternative buffer systems on seven different mAbs, exploring various combinations of ionic strengths, concentrations of the main buffer component, mAb concentrations, and stress conditions. Protein stability was assessed by analyzing soluble aggregate formation through size exclusion chromatography. At low protein concentrations, protein instability after temperature stress was exclusively observed in the bis-TRIS/ glucuronate buffer. Conversely, freeze-thaw stress led to a significant increase in aggregate formation in tested formulations, highlighting the efficacy of several alternative buffers, particularly arginine/ citrate, in preserving protein stability. Under temperature stress, the introduction of arginine to histidine buffer systems provided additional stabilization, while the addition of lysine resulted in protein destabilization. Similarly, the incorporation of arginine into histi-dine/HCl buffer further enhanced protein stability during freeze--thaw cycles. At high protein concentrations, the histidine/citrate buffer emerged as one of the most optimal choices for addressing temperature and light-induced stress. The efficacy of histidine buffers in combating light stress might be attributed to the light-absorbing properties of histidine molecules. Our findings demonstrate that the development of biopharmaceutical formulations should not be confined to conventional buffer systems, as numerous alternative options exhibit comparable or even superior performance.

For the development of concentrated monoclonal antibody formulations for subcutaneous administration, the main challenge is the high viscosity of the solutions. To compensate for this, viscosity reducing agents are commonly used as excipients. Here, we applied two computational chemistry approaches to discover new viscosity-reducing agents: fingerprint similarity searching, and physicochemical property filtering. In total, 94 compounds were selected and experimentally evaluated on two model monoclonal antibodies, which led to the discovery of 44 new viscosity-reducing agents. Analysis of the results showed that using a simple filter that selects only compounds with three or more charge groups is a good ‘rule of thumb’ for selecting potential viscosity-reducing agents for two model monoclonal antibody formulations.

Spumigins are marine natural products derived from cyanobacteria Nodularia spumigena, which mimics the structure of the d-Phe-Pro-Arg sequence and is crucial for binding to the active site of serine proteases thrombin and factor Xa. Biological evaluation of spumigins showed that spumigins with a (2S,4S)-4-methylproline central core represent potential lead compounds for the development of a new structural type of direct thrombin inhibitors. Herein, we represent synthesis and thrombin inhibitory activity of a focused library of spumigins analogues with indoline ring or l-proline as a central core. Novel compounds show additional insight into the structure and biological effects of spumigins. The most active analogue was found to be a derivative containing l-proline central core with low micromolar thrombin inhibitory activity.

We have prepared three alkaloids from the Agelas sponges, clathrodin, hymenidin and oroidin, and a series of their synthetic analogues, and evaluated their inhibitory effect against six isoforms of the Kv1 subfamily of voltage-gated potassium channels, Kv1.1-Kv1.6, expressed in Chinese Hamster ovary (CHO) cells using automated patch clamp electrophysiology assay. The most potent inhibitor was the (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-4,5-dichloro-1H-pyrrole-2-carboxamide (6g) with IC50 values between 1.4 and 6.1 μM against Kv1.3, Kv1.4, Kv1.5 and Kv1.6 channels. All compounds tested displayed selectivity against Kv1.1 and Kv1.2 channels. For confirmation of their activity and selectivity, compounds were additionally evaluated in the second independent system against Kv1.1-Kv1.6 and Kv10.1 channels expressed in Xenopus laevis oocytes under voltage clamp conditions where IC50 values against Kv1.3-Kv1.6 channels for the most active analogues (e.g. 6g) were lower than 1 μM. Because of the observed low sub-micromolar IC50 values and fairly low molecular weights, the prepared compounds represent good starting points for further optimisation towards more potent and selective voltage-gated potassium channel inhibitors.

Due to the widespread emergence of resistant bacterial strains, an urgent need for the development of new antibacterial agents with novel modes of action has emerged. The discovery of naturally occurring monocyclic β‐lactams in the late 1970s, mainly active against aerobic Gram‐negative bacteria, has introduced a new approach in the design and development of novel antibacterial β‐lactam agents. The main goal was the derivatization of the azetidin‐2‐one core in order to improve their antibacterial potency, broaden their spectrum of activity, and enhance their β‐lactamase stability. In that respect, our review covers the updates in the field of monocyclic β‐lactam antibiotics during the last three decades, taking into account an extensive collection of references. An overview of the relationships between the structural features of these monocyclic β‐lactams, classified according to their N ‐substituent, and the associated antibacterial or β‐lactamase inhibitory activities is provided. The different paragraphs disclose a number of well‐established classes of compounds, such as monobactams, monosulfactams, monocarbams, monophosphams, nocardicins, as well as other known representative classes. Moreover, this review draws attention to some less common but, nevertheless, possibly important types of monocyclic β‐lactams and concludes by highlighting the recent developments on siderophore‐conjugated classes of monocyclic β‐lactams.

Structural modification of the marine alkaloid oroidin resulted in improved apoptosis inducing activity in HepG2 and THP-1 cell lines.

Clathrodin is a marine alkaloid and believed to be a modulator of voltage-gated sodium (Na(V)) channels. Since there is an urgent need for small molecule Na(V) channel ligands as novel therapeutics, clathrodin could represent an interesting lead compound. Therefore, clathrodin was reinvestigated for its potency and Na(V) channel subtype selectivity. Clathrodin and its synthetic analogues were subjected to screening on a broad range of Na(V) channel isoforms, both in voltage clamp and patch clamp conditions. Even though clathrodin was not found to exert any activity, some analogues were capable of modulating the Na(V) channels, hereby validating the pyrrole-2-aminoimidazole alkaloid structure as a core structure for future small molecule-based Na(V) channel modulators.

Marine organisms produce secondary metabolites that may be valuable for the development of novel drug leads as such and can also provide structural scaffolds for the design and synthesis of novel bioactive compounds. The marine alkaloids, clathrodin and oroidin, which were originally isolated from sponges of the genus, Agelas, were prepared and evaluated for their antimicrobial activity against three bacterial strains (Enterococcus faecalis, Staphylococcus aureus and Escherichia coli) and one fungal strain (Candida albicans), and oroidin was found to possess promising Gram-positive antibacterial activity. Using oroidin as a scaffold, 34 new analogues were designed, prepared and screened for their antimicrobial properties. Of these compounds, 12 exhibited >80% inhibition of the growth of at least one microorganism at a concentration of 50 µM. The most active derivative was found to be 4-phenyl-2-aminoimidazole 6h, which exhibited MIC90 (minimum inhibitory concentration) values of 12.5 µM against the Gram-positive bacteria and 50 µM against E. coli. The selectivity index between S. aureus and mammalian cells, which is important to consider in the evaluation of a compound’s potential as an antimicrobial lead, was found to be 2.9 for compound 6h.

A convenient strategy for the scalable synthesis of the 2-aminoimidazole alkaloids, clathrodin, oroidin, and hymenidin derived from marine Agelas species and their analogs possessing a saturated or unsaturated linker moiety is described. The key intermediates, 4-(3-aminopropyl)-1H-imidazol-2-amine and (E)-4-(3-aminoprop-1-en-1-yl)-1H-imidazol-2-amine were obtained through two different synthetic pathways starting from l-ornithine and benzyl 1,2-dihydropyridine-1-carboxylate respectively, using (i) an innovative combination of Weinreb amide strategy with di-Boc protection, and (ii) a modified pyridine-1(2H)-carboxylate based strategy. Convenient access to these 2-aminoimidazole amines is crucial for the synthesis of libraries of clathrodin, oroidin, and hymenidin analogs.

Herein, we describe indole-based analogues of oroidin as a novel class of 2-aminoimidazole-based inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of Streptococcus mutans biofilm formation. This study highlighted the indole moiety as a dibromopyrrole mimetic for obtaining inhibitors of S. aureus and S. mutans biofilm formation. The most potent compound in the series, 5-(trifluoromethoxy)indole-based analogue 4b (MBIC50 = 20 μM), emerged as a promising hit for further optimisation of novel inhibitors of S. aureus and S. mutans biofilms.

2-Aminoimidazole skeleton represents a unique building block which is often used in the design of modulators of different targets and small molecule drugs. Alkaloids isolated from marine sponges are known to be one of the most common sources of the 2-aminoimidazole compounds and from a medicinal chemistry perspective, the marine alkaloids' fascinating structures as well as their broad spectrum of pharmacological activity make them promising lead compounds for various druggable targets. This review will focus on the presentation of biologically evaluated 2-aminoimidazole compounds showing a variety of pharmacological properties and their structure-activity relationship. The importance of 2-aminoimidazoles as bioisosteres of guanidine, acylguanidine, benzamidine and triazole groups will be highlighted.