Synergistic Interferon-Alpha-Based Combinations for Treatment of SARS-CoV-2 and Other Viral Infections

Background: There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects. Methods: Here, we tested the antiviral properties of interferons (IFNs), alone and with other drugs in vitro. Results: While IFNs alone were insufficient to completely abolish replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), IFNα, in combination with remdesivir, EIDD-2801, camostat, cycloheximide, or convalescent serum, proved to be more effective. Transcriptome and metabolomic analyses revealed that the IFNα–remdesivir combination suppressed SARS-CoV-2-mediated changes in Calu-3 cells and lung organoids, although it altered the homeostasis of uninfected cells and organoids. We also demonstrated that IFNα combinations with sofosbuvir, telaprevir, NITD008, ribavirin, pimodivir, or lamivudine were effective against HCV, HEV, FLuAV, or HIV at lower concentrations, compared to monotherapies. Conclusions: Altogether, our results indicated that IFNα can be combined with drugs that affect viral RNA transcription, protein synthesis, and processing to make synergistic combinations that can be attractive targets for further pre-clinical and clinical development against emerging and re-emerging viral infections.

Highly synergistic combinations of nanobodies that target SARS-CoV-2 and are resistant to escape

The emergence of SARS-CoV-2 variants threatens current vaccines and therapeutic antibodies and urgently demands powerful new therapeutics that can resist viral escape. We therefore generated a large nanobody repertoire to saturate the distinct and highly conserved available epitope space of SARS-CoV-2 spike, including the S1 receptor binding domain, N-terminal domain, and the S2 subunit, to identify new nanobody binding sites that may reflect novel mechanisms of viral neutralization. Structural mapping and functional assays show that indeed these highly stable monovalent nanobodies potently inhibit SARS-CoV-2 infection, display numerous neutralization mechanisms, are effective against emerging variants of concern, and are resistant to mutational escape. Rational combinations of these nanobodies that bind to distinct sites within and between spike subunits exhibit extraordinary synergy and suggest multiple tailored therapeutic and prophylactic strategies.

Systematic identification of synergistic combinations of targeted agents and immunotherapies in breast cancer using intratumor multiplex implantable microdevice assay

Systematically identifying synergistic combinations between targeted agents and immunotherapies in cancer based on genomic or other static biomarkers remains elusive. Here we integrate two novel high-content and high-throughput techniques, an implantable microdevice to administer multiple drugs into different sites in tumors at nanodoses; and spatial systems analysis of tumor microenvironmental states to describe tumor cell and immunological response signatures and rapidly, within days, identify effective combinations from among numerous agents. We demonstrate in systemic follow-up studies across three mammary carcinoma models that combinations identified by this approach lead to highly synergistic effects. Biomarkers associated with resistance to each agent allowed us to prioritize at least five novel treatment strategies of which the panobinostat/venetoclax/anti-CD40 was the most effective inducing complete tumor control across models. We show that spatial association of cancer stem cells with dendritic cells during immunogenic cell death is a potential mechanism of action underlying long-term breast cancer control.

Exploration of the antimicrobial synergy between selected natural substances on Streptococcus mutans to identify candidates for the control of dental caries

Dental caries is caused by dental plaque, a community of micro-organisms embedded in an extracellular polymer matrix as a biofilm on the tooth surface. Natural products that are widely available could be used as an alternative or adjunctive anti-caries therapy. Sometimes, when two products are used together, they yield a more powerful antimicrobial effect than the anticipated additive effect. These synergistic combinations are often better treatment options because individual agents may not have sufficient antimicrobial action to be effective when used alone. Cranberries contain phenolic compounds like proanthocyanidins (PAC) that disrupt biofilm formation. Manuka honey has high concentrations of the agent methylglyoxal, which is cariostatic. Because these agents have varied modes of antimicrobial action, they show potential for possible synergistic effects when paired. Various cranberry extracts were tested pairwise with manuka honey or methylglyoxal by well-diffusion assays and 96-well checkerboard assays in the presence of Streptococcus mutans to test for synergy. Synergy was demonstrated in two of the cranberry extracts paired with manuka honey. The synergistic combinations found in this research thus can be considered as candidates for the formulation of a dentifrice that could be used to inhibit the formation of dental plaque and thereby avoid the development of caries.

Apiaceae Essential Oils: Boosters of Terbinafine Activity against Dermatophytes and Potent Anti-Inflammatory Effectors

Dermatophyte infections represent an important public health concern, affecting up to 25% of the world’s population. Trichophyton rubrum and T. mentagrophytes are the predominant dermatophytes in cutaneous infections, with a prevalence accounting for 70% of dermatophytoses. Although terbinafine represents the preferred treatment, its clinical use is hampered by side effects, drug–drug interactions, and the emergence of resistant clinical isolates. Combination therapy, associating terbinafine and essential oils (EOs), represents a promising strategy in the treatment of dermatophytosis. In this study, we screened the potential of selected Apiaceae EOs (ajowan, coriander, caraway, and anise) to improve the antifungal activity of terbinafine against T. rubrum ATCC 28188 and T. mentagrophytes ATCC 9533. The chemical profile of EOs was analyzed by gas chromatography. The minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of EOs/main compounds were determined according to EUCAST-AFST guidelines, with minor modifications. The checkerboard microtiter method was used to identify putative synergistic combinations of EOs/main constituents with terbinafine. The influence of EOs on the viability and pro-inflammatory cytokine production (IL-1β, IL-8 and TNF-α) was determined using an ex vivo human neutrophils model. The binary associations of tested EOs with terbinafine were found to be synergistic against T. rubrum, with FICI values of 0.26–0.31. At the tested concentrations (6.25–25 mg/L), EOs did not exert cytotoxic effects towards human neutrophils. Anise EO was the most potent inhibitor of IL-1β release (46.49% inhibition at 25 mg/L), while coriander EO displayed the highest inhibition towards IL-8 and TNF-α production (54.15% and 54.91%, respectively). In conclusion, the synergistic combinations of terbinafine and investigated Apiaceae EOs could be a starting point in the development of novel topical therapies against T. rubrum-related dermatophytosis.

Systematic Review of Antimicrobial Combination Options for Pandrug-Resistant Acinetobacter baumannii

Antimicrobial combinations are at the moment the only potential treatment option for pandrug-resistant A. baumannii. A systematic review was conducted in PubMed and Scopus for studies reporting the activity of antimicrobial combinations against A. baumannii resistant to all components of the combination. The clinical relevance of synergistic combinations was assessed based on concentrations achieving synergy and PK/PD models. Eighty-four studies were retrieved including 818 eligible isolates. A variety of combinations (n = 141 double, n = 9 triple) were tested, with a variety of methods. Polymyxin-based combinations were the most studied, either as double or triple combinations with cell-wall acting agents (including sulbactam, carbapenems, glycopeptides), rifamycins and fosfomycin. Non-polymyxin combinations were predominantly based on rifampicin, fosfomycin, sulbactam and avibactam. Several combinations were synergistic at clinically relevant concentrations, while triple combinations appeared more active than the double ones. However, no combination was consistently synergistic against all strains tested. Notably, several studies reported synergy but at concentrations unlikely to be clinically relevant, or the concentration that synergy was observed was unclear. Selecting the most appropriate combinations is likely strain-specific and should be guided by in vitro synergy evaluation. Furthermore, there is an urgent need for clinical studies on the efficacy and safety of such combinations.

555 A high-throughput in situ screen to identify synergistic combinations of immune-oncology drugs with targeted and cytotoxic agents in a patient-derived humanized mouse model of renal cancer

BackgroundIdentifying how to optimally combine immunotherapies with other available anti-cancer therapies is a major challenge in oncology. A systematic method to screen many potential combination therapies ideally in vivo has remained elusive. We have utilized an implantable microdevice (IMD) performing cassette microdosing that measures intratumor drug responses and anti-tumor immunity for 20 agents in parallel. For each of the agents, local tumor response is measured by cyclical immunofluorescence for deep cellular response phenotyping. This approach is combined with systemic administration of checkpoint inhibitors to examine whether local immunogenic cell death (ICD) induced by a given drug microdose potentiates the immunotherapy’s anti-tumor effect.MethodsThe measurements were performed in a humanized mouse model of renal cancer, patient derived xenograft (PDX) RXF488. The PDX is derived from a 68 year old male patient suffering from clear cell renal carcinoma. RXF488 was implanted subcutaneously in 30 NSG mice. Animals were stratified into 6 groups with n= 4–6. Humanization was performed by the intravenous injection of 5x10e6 human peripheral blood mononuclear cells (PBMC) prior to the first treatment. Systemic anti-PD1 treatment was applied in the presence and absence of the microdevice loaded with eleven different drugs. Control groups received the microdevice in the presence or absence of PBMC. Beside the histological examination of the tumor tissue, flow cytometry (FC) was performed on bone marrow, spleen and tumor tissue to determine infiltration of human immune cells.ResultsFC analyses revealed no influence of the treatment on the human immune cells in bone marrow and spleen. The anti-PD1 treatment induced an increase in huCD45+ cells specifically in the tumor tissue and a decrease of the CD4/CD8 ratio in these cells only 48h after treatment. Our combination screen identified LXH254, Sorafenib and Doxorubicin exhibiting the highest increase in apoptosis induction when combined with checkpoint inhibitors. The increased efficacy from immunotherapy administration coincided with increased induction of ICD. We were able to verify the results of the screening experiment in a conventional setting with systemic combination treatment in the same PDX model.ConclusionsOur results demonstrate that local tumor response signatures of ICD can be used to systemically identify synergistic combinations of a range of drugs with immunotherapy on a tumor specific basis. The approach may represent a new paradigm for efficient in vivo screening of novel combinations, particularly with combinations involving immunotherapies.

Synergistic Antibacterial and Antibiofilm Activity of the MreB Inhibitor A22 Hydrochloride in Combination with Conventional Antibiotics against Pseudomonas aeruginosa and Escherichia coli Clinical Isolates

In the era of antibiotic resistance, the bacterial cytoskeletal protein MreB is presented as a potential target for the development of novel antimicrobials. Combined treatments of clinical antibiotics with anti-MreB compounds may be promising candidates in combating the resistance crisis, but also in preserving the potency of many conventional drugs. This study aimed to evaluate the synergistic antibacterial and antibiofilm activities of the MreB inhibitor A22 hydrochloride in combination with various antibiotics. The minimum inhibitory concentration (MIC) values of the individual compounds were determined by the broth microdilution method against 66 clinical isolates of Gram-negative bacteria. Synergy was assessed by the checkerboard assay. The fractional inhibitory concentration index was calculated for each of the A22-antibiotic combination. Bactericidal activity of the combinations was evaluated by time-kill curve assays. The antibiofilm activity of the most synergistic combinations was determined by crystal violet stain, methyl thiazol tetrazolium assay, and confocal laser scanning microscopy analysis. The combined cytotoxic and hemolytic activity was also evaluated toward human cells. According to our results, Pseudomonas aeruginosa and Escherichia coli isolates were resistant to conventional antibiotics to varying degrees. A22 inhibited the bacterial growth in a dose-dependent manner with MIC values ranging between 2 and 64 μg/mL. In combination studies, synergism occurred most frequently with A22-ceftazidime and A22-meropemen against Pseudomonas aeruginosa and A22-cefoxitin and A22-azithromycin against Escherichia coli. No antagonism was observed. In time-kill studies, synergism was observed with all expected combinations. Synergistic combinations even at the lowest tested concentrations were able to inhibit biofilm formation and eradicate mature biofilms in both strains. Cytotoxic and hemolytic effects of the same combinations toward human cells were not observed. The findings of the present study support previous research regarding the use of MreB as a novel antibiotic target. The obtained data expand the existing knowledge about the antimicrobial and antibiofilm activity of the A22 inhibitor, and they indicate that A22 can serve as a leading compound for studying potential synergism between MreB inhibitors and antibiotics in the future.

The Synergistic Activity of Bortezomib and TIC10 against A2058 Melanoma Cells

Combination antitumor treatments are essential parts of modern tumor therapy as—compared to monotherapies—(i) they are more effective; (ii) the dose of the compounds can be reduced; and (iii) therefore the side effects are improved. Our research group previously demonstrated the antitumor character of bortezomib (BOZ) in A2058 melanoma cells. Unfortunately, dose-related side effects are common during BOZ therapy, which could be prevented by reducing the dose of BOZ. This study aimed to characterize synergistic combinations of BOZ with a TRAIL (TNF-related apoptosis-inducing ligand) -inducing compound (TIC10), where the doses can be cut down but the efficacy is preserved. Endpoint cell viability assays were performed on A2058 cells, and synergism of BOZ and TIC10 was observed after 72 h. Synergism was further validated in a real-time impedimetric assay, and our results showed that BOZ-treated melanoma cells survived the treatment, an effect not registered in the co-treatments. Treatment with the combinations resulted in increased apoptosis, which was not accompanied by enhanced LDH release. Nevertheless, the expression of death receptor 5 (DR5) was increased on the cell surface without transcriptional regulation. In summary, our findings support the theory that the application of BOZ and TIC10 in combination could provide higher efficacy in vitro.