Paul Reddell

Intratumoural drug delivery can achieve high local therapeutic exposure while limiting systemic effects, but heterogeneous distribution may compromise treatment efficacy and precision. This proof-of-concept study investigated open flow microperfusion (OFM) as a novel platform for local delivery of tigilanol tiglate (TT), an intratumoural antineoplastic agent with immunomodulatory and vascular-disrupting effects. Four horses with histologically confirmed cutaneous neoplasia (three sarcoids and one squamous cell carcinoma) underwent OFM using TT concentrations of 0.01–0.04 mg/mL delivered at 9 µL/min for 7–24 h over one to three consecutive days. OFM systems were well tolerated and remained functional for up to three days. Localized vascular disruption, polymorphonuclear cell recruitment, oncosis and tissue sloughing developed in association with perfused regions, consistent with recognized TT-induced tissue responses. Two sarcoids exhibited biologic responses that did not result in complete local tumour ablation. Tumour ablation was achieved initially in one squamous cell carcinoma, although deep-seated disease subsequently progressed, while one sarcoid was ablated and remained tumour-free 497 days after treatment. These preliminary findings demonstrate that OFM allows precise, sustained intratumoural TT challenge, resulting in characteristic tissue responses supporting further investigation of this local delivery platform for tumour-targeted therapies.

The unrestricted use of colistin in animal husbandry has led to the emergence of mobile plasmid-borne colistin resistance ( mcr ). The antimicrobial epoxytigliane, EBC-1013, has been shown to be effective in the topical treatment of biofilm-mediated infections in vivo . Hypothesizing that EBC-1013 interacts with the modified bacterial outer membrane of colistin-resistant Escherichia coli, we investigated its ability to potentiate the activity of colistin using fractional inhibitory concentration (FIC) index determinations against environmental mcr Enterobacteriaceae. Molecular dynamics (MD) simulations were used to visualize EBC-1013 interactions with the outer membrane in silico . Modification of outer membrane surface chemistry and adhesion properties was characterized using X-ray photoelectron spectroscopy (XPS) and hydrophobicity and bacterial membrane permeability assays. Anti-biofilm activity was quantified using confocal laser scanning microscopy (CLSM). Checkerboard assays revealed synergistic effects of EBC-1013 with colistin (FIC ≤ 0.5) in 6/9 strains. MD simulations demonstrated weak EBC-1013 interactions with colistin and the outer membrane. XPS suggested modification of the outer membrane in both colistin-sensitive and colistin-resistant E. coli, as well as the interaction of colistin and EBC-1013 with the phosphoethanolamine (pEtN)-substituted mcr lipid moiety. EBC-1013 binding significantly decreased bacterial hydrophilicity and potentiated membrane permeability induced by colistin ( P < 0.05). In the biofilm model, potentiation was also evident, with a marked decrease in bacterial biomass in EBC-1013/colistin combined treatments ( P < 0.0001) for colistin-resistant E. coli . The results highlight the ability of EBC-1013 to modify the bacterial membrane of colistin-resistant E. coli and potentiate the activity of colistin in the treatment of multidrug-resistant wound bacteria. IMPORTANCE: Resistance to colistin, an antibiotic of last resort for hard-to-treat infections, is on the increase. Therefore, the need to develop new antimicrobials to tackle antimicrobial resistance is of paramount importance. The epoxytiglianes represent an exciting range of molecules with a diverse range of biological effects in human and veterinary applications, including antimicrobial properties. In this study, we show how EBC-1013 interacts with the outer surface of colistin-resistant Escherichia coli cells, inducing chemical and structural changes to the cell wall, making it susceptible again to colistin treatment. This ability of EBC-1013 to enhance the activity of colistin against a range of colistin-resistant E. coli suggests that EBC-1013, alone (or as a combination therapy), has potential as a new treatment strategy to treat antibiotic-resistant bacterial wound infections and reduce antibiotic usage. This study is registered with ClinicalTrials.gov as (Australian New Zealand Clinical Trials Register: ACTRN12624000544572 ).

Seven new arylalkenyl α,β-unsaturated-δ-lactones, triplinones I-O (1-7), were isolated from the leaves of the Australian rainforest plant Cryptocarya triplinervis B. Hyland (Lauraceae). The chemical structures of these compounds were established by NMR spectroscopic data analysis, while their relative and absolute configurations were established using a combination of Mosher ester analysis utilizing both Riguera's and Kishi's methods and CD experiments. Compounds 2 and 4 were identified as epimers, where the relative stereochemistry of the 1,3-diol moiety in both structures was first confirmed by Kishi's method, followed by their absolute configuration determination using Riguera's method. Compounds 1-7 exhibited good inhibitory activities toward nitric oxide (NO) production in lipopolysaccharide (LPS) and interferon (IFN)-γ-induced RAW 264.7 macrophages, in particular compounds 6, 2, and 4, with IC₅₀ values of 2.1 ± 1.4, 8.5 ± 0.6, and 8.5 ± 0.7 μM, respectively.

Phytochemical investigation of the Australian rainforest plant Cryptocarya rhodosperma B. Hyland (Lauraceae) led to the isolation and structural elucidation of seven new unique spiroketals fused with an α,β-unsaturated δ-lactone, namely, rhodospermals A-G (1 - 7), and a new alkylbenzodioxole α,β-unsaturated-δ-lactone rhodopyrone A (8) along with the known compounds EBC-23 (9) and EBC-73 (10). The chemical structures of these compounds were established by NMR spectroscopic data analysis, relative stereochemistry confirmed by distinct J coupling, NOESY correlations, and a close comparison of the NMR data and optical rotation of the previously reported compounds (9, 10). X-ray crystallography analysis and Mosher ester analysis of 1 confirmed the absolute stereochemistry of the fused spiroketal with an α,β-unsaturated δ-lactone. A plausible identical stereochemistry of the common fused spiroketal with an α,β-unsaturated δ-lactone observed in the remaining analogues (2-7) was proposed. The absolute stereochemistry of 8 was determined by performing a Mosher ester analysis and distinct J coupling. Compounds 1-10 exhibited cytotoxic activity in several cancer cell lines, highlighting their potential use in the treatment of human tumors.

Background: Intralesional chemotherapeutic administration represents an important treatment option for equine cutaneous neoplasia. Tigilanol‐tiglate (TT), a novel molecule extracted from Fontainea picrosperma , an Australian rainforest plant, is registered for intratumoural treatment of canine MCT, leading to rapid oncosis and tumour slough. Evidence from horses is limited but suggests that efficacy may be similar. Objectives: To evaluate the response to intratumoural TT treatment in horses with sarcoids (fibroblastic/nodular) and cutaneous melanomas. Study Design: Two noncontrolled prospective multicentre clinical trials, one for each of sarcoids and melanomas. Methods: Cases were enrolled across multiple sites and treated by the same site‐specific clinician with intralesional TT (sarcoids: 0.35 mg/cm ³ ; melanomas: 0.2 mg/cm ³ of tumour volume − T _vol ; max dose 2 mg). Quantitative ( T _vol regression) and qualitative outcomes (likely tumour free (LTF) per expert opinion) were recorded, and potential determinants of efficacy were assessed using random effects logistic models. A full clinical response was complete T _vol regression and a LTF treatment site. Results: Forty‐one sarcoids and 97 melanomas were enrolled and treated. 73/74% of treated sarcoids/melanomas showed complete T _vol regression. 64/61% (sarcoids/melanomas) showed a full clinical response at medians of 546/247 days post final treatment. For both tumour types, this response was dependent on initial tumour volume ( P _sarcoids = 0.006; P _melanomas T _vol = 1 cm ³ ) than for the maximum study volume ( T _vol = 6 cm ³ ). For melanomas in the perineal region, this was 11 times greater for T _vol ≤0.3 cm ³ than for tumours ≥2.0 cm ³ . For melanomas, tumour location further affected treatment efficacy = 0.005). In total, 5 adverse events were reported. Main Limitations: Lack of treatment control and histologic/biomolecular follow‐up data. Conclusions: The observed therapeutic efficacy of TT supports clinical use as well as early interventions in horses. Successful use necessitates knowledge of the drug's mode of action and management of associated local site responses.

Our ongoing exploration of Australian rainforest plants for the biodiscovery of anti-inflammatory agents led to the isolation and structural elucidation of eight new arylalkenyl α,β-unsaturated-δ-lactones, triplinones A-H (1-8), from the leaves of the Australian rainforest plant Cryptocarya triplinervis B. Hyland (Lauraceae). The chemical structures of these compounds were established by NMR spectroscopic data analysis, while their relative and absolute configurations were established using a combination of Mosher ester analysis utilizing both Riguera's and Kishi's methods, ECD experiments, and X-ray crystallography analysis. Compounds 1-8 exhibited good inhibitory activities toward nitric oxide (NO) production in lipopolysaccharide (LPS) and interferon (IFN)-γ induced RAW 264.7 macrophages, in particular compounds 1-3 and 5, with IC₅₀ values of 7.3 ± 0.5, 6.0 ± 0.3, 5.6 ± 0.3, and 5.4 ± 2.5 μM, respectively.

Tigilanol tiglate (TT, also known as EBC-46) is a novel, plant-derived diterpene ester possessing anticancer and wound-healing properties. Here, we show that TT-evoked PKC-dependent S⁹⁸⁵ phosphorylation of the tyrosine kinase MET leads to subsequent degradation of tyrosine phosphorylated p-Y¹⁰⁰³ and p-Y^1234/5 MET species. PKC inhibition with BIM-1 blocked S⁹⁸⁵ phosphorylation of MET and led to MET cell surface accumulation. Treatment with metalloproteinase inhibitors prevented MET-ECD release into cell culture media, which was also blocked by PKC inhibitors. Furthermore, unbiased secretome analysis, performed using TMT-technology, identified additional targets of TT-dependent release of cell surface proteins from H357 head and neck cancer cells. We confirm that the MET co-signalling receptor syndecan-1 was cleaved from the cell surface in response to TT treatment. This was accompanied by rapid cleavage of the cellular junction adhesion protein Nectin-1 and the nerve growth factor receptor NGFR^p75/TNFR16. These findings, that TT is a novel negative regulator of protumorigenic c-MET and NGFRp⁷⁵/TNFR16 signalling, as well as regulating Nectin-1-mediated cell adhesion, further contribute to our understanding of the mode of action and efficacy of TT in the treatment of solid tumours.

To investigate the role of the secondary 5-hydroxy group in the activity of the anticancer drug tigilanol tiglate (2b) (Stelfonta), oxidation of this epoxytigliane diterpenoid from the Australian rainforest plant Fontainea picrosperma was attempted. Eventually, 5-dehydrotigilanol tiglate (3a) proved too unstable to be characterized in terms of biological activity and, therefore, was not a suitable tool compound for bioactivity studies. On the other hand, a series of remarkable skeletal rearrangements associated with the presence of a 5-keto group were discovered during its synthesis, including a dismutative ring expansion of ring A and a mechanistically unprecedented dyotropic substituent swap around the C-4/C-10 bond. Taken together, these observations highlight the propensity of the alpha-hydroxy-beta-diketone system to trigger complex skeletal rearrangements and pave the way to new areas of the natural products chemical space.

Peri-implantitis has become an inexorable clinical challenge in implantology. Topical immunomodulatory epoxy-tiglianes (EBCs), derived from the Queensland blushwood tree, which induce remodeling and resolve dermal infection via induction of the inflammasome and biofilm disruption, may offer a novel therapeutic approach. In vitro antimicrobial activity of EBC structures (EBC-46, EBC-1013 and EBC-147) against Streptococcus mutans, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in minimum inhibitory concentration, growth curve and permeabilization assays were determined. Antibiofilm activity was assessed using minimum biofilm eradication concentration (MBEC) experiments. Biofilm formation and disruption assays were analyzed using confocal laser scanning microscopy, scanning electron microscopy and direct plate counting. The observed antimicrobial efficacy of the tested compounds (EBC-1013 > EBC-46 > EBC-147) was directly related to significant membrane permeabilization and growth inhibition (p < 0.05) against planktonic S. mutans and P. gingivalis. Antibiofilm activity was evident in MBEC assays, with S. mutans biofilm formation assays revealing significantly lower biomass volume and increased DEAD:LIVE cell ratio observed for EBC-1013 (p < 0.05). Furthermore, biofilm disruption assays on titanium discs induced significant biofilm disruption in S. mutans and P. gingivalis (p < 0.05). EBC-1013 is a safe, semi-synthetic, compound, demonstrating clear antimicrobial biofilm disruption potential in peri-implantitis.

The management of antibiotic-resistant, bacterial biofilm infections in chronic skin wounds is an increasing clinical challenge. Despite advances in diagnosis, many patients do not derive benefit from current anti-infective/antibiotic therapies. Here, we report a novel class of naturally occurring and semisynthetic epoxy-tiglianes, derived from the Queensland blushwood tree ( Fontainea picrosperma) , and demonstrate their antimicrobial activity (modifying bacterial growth and inducing biofilm disruption), with structure/activity relationships established against important human pathogens. In vitro, the lead candidate EBC-1013 stimulated protein kinase C (PKC)–dependent neutrophil reactive oxygen species (ROS) induction and NETosis and increased expression of wound healing–associated cytokines, chemokines, and antimicrobial peptides in keratinocytes and fibroblasts. In vivo, topical EBC-1013 induced rapid resolution of infection with increased matrix remodeling in acute thermal injuries in calves. In chronically infected diabetic mouse wounds, treatment induced cytokine/chemokine production, inflammatory cell recruitment, and complete healing (in six of seven wounds) with ordered keratinocyte differentiation. These results highlight a nonantibiotic approach involving contrasting, orthogonal mechanisms of action combining targeted biofilm disruption and innate immune induction in the treatment of chronic wounds.