Consumption of psychotropic drugs is a major economic and public health challenge in Australia. Wastewater-based epidemiology (WBE) and drug checking have emerged as complementary tools to monitor this market. WBE estimates drug use within communities for target compounds and their metabolites, while drug checking is a free service available in some Australian jurisdictions, which allows individuals to verify drug purity and composition. In this work, wastewater samples were collected from five sites across Queensland (n = 3), New South Wales (n = 1) and Victoria (n = 1) over the 2024-25 New Year holidays, while drug checking was carried out at a site in central Canberra over December-January. Wastewater samples were analysed for alcohol, illicit drugs and new psychoactive substances (NPS) using liquid chromatography-tandem mass spectrometry. Methamphetamine was the most consumed illicit substance across all sites, while cocaine and MDMA showed distinct increases over the New Year period. Among NPS, bromazolam and 2'-fluoro-2-oxo-PCE were most prevalent, while ketamine was consistently detected at regional sites with consumption 2.8-3-fold higher on New Year's days. Drug checking revealed prevalence of MDMA, cocaine and ketamine. NPS were detected in 15%-20% of total drug samples submitted, with N,N-dimethylpentylone and 2'-fluoro-2-oxo-PCE the most frequently found. This study is the first to demonstrate the potential synergy between WBE and drug checking, highlighting how their complementary roles can enhance drug surveillance, early warnings for emerging psychoactive substances and monitoring of changes in drug consumption during high-risk periods.

Higenamine is prohibited in sports as a β2 -agonist by the World Anti-Doping Agency. As a key component of a great variety of plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion of Annona fruit could lead to higenamine adverse analytical findings. Single-dose administration studies including three Annona species (i.e., Annona muricata, Annona cherimola, and Annona squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10 ng/mL in urine. In consideration of cmax values (7.8 ng/mL) observed for higenamine up to 24 h, a multidose administration study was also conducted, indicating cumulative effects, which can increase the risk of exceeding the applicable MRL doping after Annona fruit ingestion. In this study, however, the MRL was not exceeded at any time point. Further, the major urinary excretion of higenamine in its sulfo-conjugated form was corroborated, its stability in urine was assessed, and in the absence of reference material, higenamine sulfo-conjugates were synthesized and comprehensively characterized, suggesting the predominant presence of higenamine 7-sulfate. In addition, the option to include complementary biomarkers of diet-related higenamine intake into routine doping controls was investigated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and a yet not fully characterized bismethylated higenamine glucuronide was observed after Annona ingestion but not after supplement use, providing a promising dataset of urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs.

SNAT2 (SLC38A2) is a sodium-dependent neutral amino acid transporter, which is important for the accumulation of amino acids as nutrients, the maintenance of cellular osmolarity, and the activation of mTORC1. It also provides net glutamine for glutaminolysis and consequently presents as a potential target to treat cancer. A high-throughput screening assay was developed to identify new inhibitors of SNAT2 making use of the inducible nature of SNAT2 and its electrogenic mechanism. Using an optimized FLIPR membrane potential (FMP) assay, a curated scaffold library of 33934 compounds was screened to identify 3-(N-methyl (4-methylphenyl)sulfonamido)-N-(2-trifluoromethylbenzyl)thiophene-2-carboxamide as a potent inhibitor of SNAT2. In two different assays an IC50 of 0.8–3 µM was determined. The compound discriminated against the close transporter homologue SNAT1. MDA-MB-231 breast cancer and HPAFII pancreatic cancer cell lines tolerated the SNAT2 inhibitor up to a concentration of 100 µM but in combination with tolerable doses of the glucose transport inhibitor Bay-876, proliferative growth of both cell lines was halted. This points to synergy between inhibition of glycolysis and glutaminolysis in cancer cells.

16 Alkaloids including galanthamine (1) and codeine (2) are reported to be positive allosteric 17 modulators of nicotinic acetylcholine receptors (nAChRs) but the binding sites responsible 18 for this activity are not known with certainty. Analogues of galanthamine (1), codeine (2) and 19 morphine (3) with reactivity towards cysteine thiols were synthesised including conjugated 20 enone derivatives of the three alkaloids 4-6 and two chloro-alkane derivatives of codeine 7 21 and 8. The stability of the enones was deemed sufficient for use in buffered aqueous solutions 22 and their reactivity towards thiols was assessed by determining the kinetics of reaction with a 23 cysteine derivative. All three enone derivatives were of sufficient reactivity and stability to be 24 used in covalent trapping, an extension of the substituted cysteine accessibility method 25 (SCAM), to elucidate the allosteric binding sites of galanthamine and codeine at nAChRs. 26