Cancer-specific loss of TERT activation sensitizes glioblastoma to DNA damage.

Most glioblastomas (GBMs) achieve cellular immortality by acquiring a mutation in the telomerase reverse transcriptase (TERT) promoter. TERT promoter mutations create a binding site for a GA binding protein (GABP) transcription factor complex, whose assembly at the promoter is associated with TERT reactivation and telomere maintenance. Here, we demonstrate increased binding of a specific GABPB1L-isoform-containing complex to the mutant TERT promoter. Furthermore, we find that TERT promoter mutant GBM cells, unlike wild-type cells, exhibit a critical near-term dependence on GABPB1L for proliferation, notably also posttumor establishment in vivo. Up-regulation of the protein paralogue GABPB2, which is normally expressed at very low levels, can rescue this dependence. More importantly, when combined with frontline temozolomide (TMZ) chemotherapy, inducible GABPB1L knockdown and the associated TERT reduction led to an impaired DNA damage response that resulted in profoundly reduced growth of intracranial GBM tumors. Together, these findings provide insights into the mechanism of cancer-specific TERT regulation, uncover rapid effects of GABPB1L-mediated TERT suppression in GBM maintenance, and establish GABPB1L inhibition in combination with chemotherapy as a therapeutic strategy for TERT promoter mutant GBM.

A CRISPR toolbox for generating intersectional genetic mouse models for functional, molecular, and anatomical circuit mapping.

BACKGROUND: The functional understanding of genetic interaction networks and cellular mechanisms governing health and disease requires the dissection, and multifaceted study, of discrete cell subtypes in developing and adult animal models. Recombinase-driven expression of transgenic effector alleles represents a significant and powerful approach to delineate cell populations for functional, molecular, and anatomical studies. In addition to single recombinase systems, the expression of two recombinases in distinct, but partially overlapping, populations allows for more defined target expression. Although the application of this method is becoming increasingly popular, its experimental implementation has been broadly restricted to manipulations of a limited set of common alleles that are often commercially produced at great expense, with costs and technical challenges associated with production of intersectional mouse lines hindering customized approaches to many researchers. Here, we present a simplified CRISPR toolkit for rapid, inexpensive, and facile intersectional allele production. RESULTS: Briefly, we produced 7 intersectional mouse lines using a dual recombinase system, one mouse line with a single recombinase system, and three embryonic stem (ES) cell lines that are designed to study the way functional, molecular, and anatomical features relate to each other in building circuits that underlie physiology and behavior. As a proof-of-principle, we applied three of these lines to different neuronal populations for anatomical mapping and functional in vivo investigation of respiratory control. We also generated a mouse line with a single recombinase-responsive allele that controls the expression of the calcium sensor Twitch-2B. This mouse line was applied globally to study the effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on calcium release in the ovarian follicle. CONCLUSIONS: The lines presented here are representative examples of outcomes possible with the successful application of our genetic toolkit for the facile development of diverse, modifiable animal models. This toolkit will allow labs to create single or dual recombinase effector lines easily for any cell population or subpopulation of interest when paired with the appropriate Cre and FLP recombinase mouse lines or viral vectors. We have made our tools and derivative intersectional mouse and ES cell lines openly available for non-commercial use through publicly curated repositories for plasmid DNA, ES cells, and transgenic mouse lines.

Matrix regulators in neural stem cell functions.

BACKGROUND: Neural stem/progenitor cells (NSPCs) reside within a complex and dynamic extracellular microenvironment, or niche. This niche regulates fundamental aspects of their behavior during normal neural development and repair. Precise yet dynamic regulation of NSPC self-renewal, migration, and differentiation is critical and must persist over the life of an organism. SCOPE OF REVIEW: In this review, we summarize some of the major components of the NSPC niche and provide examples of how cues from the extracellular matrix regulate NSPC behaviors. We use proteoglycans to illustrate the many diverse roles of the niche in providing temporal and spatial regulation of cellular behavior. MAJOR CONCLUSIONS: The NSPC niche is comprised of multiple components that include; soluble ligands, such as growth factors, morphogens, chemokines, and neurotransmitters, the extracellular matrix, and cellular components. As illustrated by proteoglycans, a major component of the extracellular matrix, the NSPC, niche provides temporal and spatial regulation of NSPC behaviors. GENERAL SIGNIFICANCE: The factors that control NSPC behavior are vital to understand as we attempt to modulate normal neural development and repair. Furthermore, an improved understanding of how these factors regulate cell proliferation, migration, and differentiation, crucial for malignancy, may reveal novel anti-tumor strategies. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Confirmation of ethanol administration in racing greyhounds by LC-MS-MS.

Ethanol is a prohibited substance in professional animal racing as its administration causes physiological effects such as depression of the central nervous system. Regulation of potential doping agents, including those that inhibit performance, is critical to ensure integrity and animal welfare in greyhound racing, but the detection of ethanol is complicated by dietary and/or environmental exposure. In response, a reliable analytical method capable of detecting recent ethanol administration in greyhound urine samples was validated and implemented. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) was used to investigate the variation in urinary ethanol metabolites; ethyl-ß-D glucuronide (EG; γ ¯ EG $$ {\overline{\gamma}}_{\mathrm{EG}} $$ = 1.0 µg/ml, s EG $$ {s}_{\mathrm{EG}} $$ = 3.3 µg/ml) and ethyl sulfate (ES; γ ¯ ES $$ {\overline{\gamma}}_{\mathrm{ES}} $$ = 0.9 µg/ml, s ES $$ {s}_{\mathrm{ES}} $$ = 1.9 µg/ml) levels from a reference population of 202 racing greyhounds. These were compared to urine samples collected following administration of ethanol to one male and one female greyhound. Results were used to establish a threshold within the national rules of greyhound racing: γ ¯ EG $$ {\overline{\gamma}}_{\mathrm{EG}} $$ and γ ¯ ES $$ {\overline{\gamma}}_{\mathrm{ES}} $$ > 20 µg/ml in urine are defensible criteria to confirm ethanol administration to greyhounds. Case studies of competition samples are provided to demonstrate the forensic translation of this work.

Cellular composition and circuit organization of the locus coeruleus of adult mice.

The locus coeruleus (LC) houses the vast majority of noradrenergic neurons in the brain and regulates many fundamental functions, including fight and flight response, attention control, and sleep/wake cycles. While efferent projections of the LC have been extensively investigated, little is known about its local circuit organization. Here, we performed large-scale multipatch recordings of noradrenergic neurons in adult mouse LC to profile their morpho-electric properties while simultaneously examining their interactions. LC noradrenergic neurons are diverse and could be classified into two major morpho-electric types. While fast excitatory synaptic transmission among LC noradrenergic neurons was not observed in our preparation, these mature LC neurons connected via gap junction at a rate similar to their early developmental stage and comparable to other brain regions. Most electrical connections form between dendrites and are restricted to narrowly spaced pairs or small clusters of neurons of the same type. In addition, more than two electrically coupled cell pairs were often identified across a cohort of neurons from individual multicell recording sets that followed a chain-like organizational pattern. The assembly of LC noradrenergic neurons thus follows a spatial and cell-type-specific wiring principle that may be imposed by a unique chain-like rule.

GABP couples oncogene signaling to telomere regulation in TERT promoter mutant cancer.

Telomerase activation counteracts senescence and telomere erosion caused by uncontrolled proliferation. Epidermal growth factor receptor (EGFR) amplification drives proliferation while telomerase reverse transcriptase promoter (TERTp) mutations underlie telomerase reactivation through recruitment of GA-binding protein (GABP). EGFR amplification and TERTp mutations typically co-occur in glioblastoma, the most common and aggressive primary brain tumor. To determine if these two frequent alterations driving proliferation and immortality are functionally connected, we combine analyses of copy number, mRNA, and protein data from tumor tissue with pharmacologic and genetic perturbations. We demonstrate that proliferation arrest decreases TERT expression in a GABP-dependent manner and elucidate a critical proliferation-to-immortality pathway from EGFR to TERT expression selectively from the mutant TERTp through activation of AMP-mediated kinase (AMPK) and GABP upregulation. EGFR-AMPK signaling promotes telomerase activity and maintains telomere length. These results define how the tumor cell immortality mechanism keeps pace with persistent oncogene signaling and cell cycling.

TERT promoter C228T mutation in neural progenitors confers growth advantage following telomere shortening in vivo.

BACKGROUND: Heterozygous TERT (telomerase reverse transcriptase) promoter mutations (TPMs) facilitate TERT expression and are the most frequent mutation in glioblastoma (GBM). A recent analysis revealed this mutation is one of the earliest events in gliomagenesis. However, no appropriate human models have been engineered to study the role of this mutation in the initiation of these tumors. METHOD: We established GBM models by introducing the heterozygous TPM in human induced pluripotent stem cells (hiPSCs) using a two-step targeting approach in the context of GBM genetic alterations, CDKN2A/B and PTEN deletion, and EGFRvIII overexpression. The impact of the mutation was evaluated through the in vivo passage and in vitro experiment and analysis. RESULTS: Orthotopic injection of neuronal precursor cells (NPCs) derived from hiPSCs with the TPM into immunodeficient mice did not enhance tumorigenesis compared to TERT promoter wild type NPCs at initial in vivo passage presumably due to relatively long telomeres. However, the mutation recruited GA-Binding Protein and engendered low-level TERT expression resulting in enhanced tumorigenesis and maintenance of short telomeres upon secondary passage as observed in human GBM. These results provide the first insights regarding increased tumorigenesis upon introducing a TPM compared to isogenic controls without TPMs. CONCLUSION: Our novel GBM models presented the growth advantage of heterozygous TPMs for the first time in the context of GBM driver mutations relative to isogenic controls, thereby allowing for the identification and validation of TERT promoter-specific vulnerabilities in a genetically accurate background.

Functional analysis of low-grade glioma genetic variants predicts key target genes and transcription factors.

BACKGROUND: Large-scale genome-wide association studies (GWAS) have implicated thousands of germline genetic variants in modulating individuals' risk to various diseases, including cancer. At least 25 risk loci have been identified for low-grade gliomas (LGGs), but their molecular functions remain largely unknown. METHODS: We hypothesized that GWAS loci contain causal single nucleotide polymorphisms (SNPs) that reside in accessible open chromatin regions and modulate the expression of target genes by perturbing the binding affinity of transcription factors (TFs). We performed an integrative analysis of genomic and epigenomic data from The Cancer Genome Atlas and other public repositories to identify candidate causal SNPs within linkage disequilibrium blocks of LGG GWAS loci. We assessed their potential regulatory role via in silico TF binding sequence perturbations, convolutional neural network trained on TF binding data, and simulated annealing-based interpretation methods. RESULTS: We built an interactive website (http://education.knoweng.org/alg3/) summarizing the functional footprinting of 280 variants in 25 LGG GWAS regions, providing rich information for further computational and experimental scrutiny. We identified as case studies PHLDB1 and SLC25A26 as candidate target genes of rs12803321 and rs11706832, respectively, and predicted the GWAS variant rs648044 to be the causal SNP modulating ZBTB16, a known tumor suppressor in multiple cancers. We showed that rs648044 likely perturbed the binding affinity of the TF MAFF, as supported by RNA interference and in vitro MAFF binding experiments. CONCLUSIONS: The identified candidate (causal SNP, target gene, TF) triplets and the accompanying resource will help accelerate our understanding of the molecular mechanisms underlying genetic risk factors for gliomas.

Patient-derived cells from recurrent tumors that model the evolution of IDH-mutant glioma.

BACKGROUND: IDH-mutant lower-grade gliomas (LGGs) evolve under the selective pressure of therapy, but well-characterized patient-derived cells (PDCs) modeling evolutionary stages are lacking. IDH-mutant LGGs may develop therapeutic resistance associated with chemotherapy-driven hypermutation and malignant progression. The aim of this study was to establish and characterize PDCs, single-cell-derived PDCs (scPDCs), and xenografts (PDX) of IDH1-mutant recurrences representing distinct stages of tumor evolution. METHODS: We derived and validated cell cultures from IDH1-mutant recurrences of astrocytoma and oligodendroglioma. We used exome sequencing and phylogenetic reconstruction to examine the evolutionary stage represented by PDCs, scPDCs, and PDX relative to corresponding spatiotemporal tumor tissue and germline DNA. PDCs were also characterized for growth and tumor immortality phenotypes, and PDX were examined histologically. RESULTS: The integrated astrocytoma phylogeny revealed 2 independent founder clonal expansions of hypermutated (HM) cells in tumor tissue that are faithfully represented by independent PDCs. The oligodendroglioma phylogeny showed more than 4000 temozolomide-associated mutations shared among tumor samples, PDCs, scPDCs, and PDX, suggesting a shared monoclonal origin. The PDCs from both subtypes exhibited hallmarks of tumorigenesis, retention of subtype-defining genomic features, production of 2-hydroxyglutarate, and subtype-specific telomere maintenance mechanisms that confer tumor cell immortality. The oligodendroglioma PDCs formed infiltrative intracranial tumors with characteristic histology. CONCLUSIONS: These PDCs, scPDCs, and PDX are unique and versatile community resources that model the heterogeneous clonal origins and functions of recurrent IDH1-mutant LGGs. The integrated phylogenies advance our knowledge of the complex evolution and immense mutational load of IDH1-mutant HM glioma.

Mechanisms of Resistance to EGFR Inhibition Reveal Metabolic Vulnerabilities in Human GBM.

Amplification of the epidermal growth factor receptor gene (EGFR) represents one of the most commonly observed genetic lesions in glioblastoma (GBM); however, therapies targeting this signaling pathway have failed clinically. Here, using human tumors, primary patient-derived xenografts (PDX), and a murine model for GBM, we demonstrate that EGFR inhibition leads to increased invasion of tumor cells. Further, EGFR inhibitor-treated GBM demonstrates altered oxidative stress, with increased lipid peroxidation, and generation of toxic lipid peroxidation products. A tumor cell subpopulation with elevated aldehyde dehydrogenase (ALDH) levels was determined to comprise a significant proportion of the invasive cells observed in EGFR inhibitor-treated GBM. Our analysis of the ALDH1A1 protein in newly diagnosed GBM revealed detectable ALDH1A1 expression in 69% (35/51) of the cases, but in relatively low percentages of tumor cells. Analysis of paired human GBM before and after EGFR inhibitor therapy showed an increase in ALDH1A1 expression in EGFR-amplified tumors (P < 0.05, n = 13 tumor pairs), and in murine GBM ALDH1A1-high clones were more resistant to EGFR inhibition than ALDH1A1-low clones. Our data identify ALDH levels as a biomarker of GBM cells with high invasive potential, altered oxidative stress, and resistance to EGFR inhibition, and reveal a therapeutic target whose inhibition should limit GBM invasion.

Coordinated cerebellar climbing fiber activity signals learned sensorimotor predictions.

The prevailing model of cerebellar learning states that climbing fibers (CFs) are both driven by, and serve to correct, erroneous motor output. However, this model is grounded largely in studies of behaviors that utilize hardwired neural pathways to link sensory input to motor output. To test whether this model applies to more flexible learning regimes that require arbitrary sensorimotor associations, we developed a cerebellar-dependent motor learning task that is compatible with both mesoscale and single-dendrite-resolution calcium imaging in mice. We found that CFs were preferentially driven by and more time-locked to correctly executed movements and other task parameters that predict reward outcome, exhibiting widespread correlated activity in parasagittal processing zones that was governed by these predictions. Together, our data suggest that such CF activity patterns are well-suited to drive learning by providing predictive instructional input that is consistent with an unsigned reinforcement learning signal but does not rely exclusively on motor errors.

Disruption of the ß1L Isoform of GABP Reverses Glioblastoma Replicative Immortality in a TERT Promoter Mutation-Dependent Manner.

TERT promoter mutations reactivate telomerase, allowing for indefinite telomere maintenance and enabling cellular immortalization. These mutations specifically recruit the multimeric ETS factor GABP, which can form two functionally independent transcription factor species: a dimer or a tetramer. We show that genetic disruption of GABPß1L (ß1L), a tetramer-forming isoform of GABP that is dispensable for normal development, results in TERT silencing in a TERT promoter mutation-dependent manner. Reducing TERT expression by disrupting ß1L culminates in telomere loss and cell death exclusively in TERT promoter mutant cells. Orthotopic xenografting of ß1L-reduced, TERT promoter mutant glioblastoma cells rendered lower tumor burden and longer overall survival in mice. These results highlight the critical role of GABPß1L in enabling immortality in TERT promoter mutant glioblastoma.

Heparan Sulfate Glycosaminoglycans in Glioblastoma Promote Tumor Invasion.

Glioblastoma (GBM) is the most common primary malignant brain tumor of adults and confers a poor prognosis due, in part, to diffuse invasion of tumor cells. Heparan sulfate (HS) glycosaminoglycans, present on the cell surface and in the extracellular matrix, regulate cell signaling pathways and cell-microenvironment interactions. In GBM, the expression of HS glycosaminoglycans and the enzymes that regulate their function are altered, but the actual HS content and structure are unknown. However, inhibition of HS glycosaminoglycan function is emerging as a promising therapeutic strategy for some cancers. In this study, we use liquid chromatography-mass spectrometry analysis to demonstrate differences in HS disaccharide content and structure across four patient-derived tumorsphere lines (GBM1, 5, 6, 43) and between two murine tumorsphere lines derived from murine GBM with enrichment of mesenchymal and proneural gene expression (mMES and mPN, respectively) markers. In GBM, the heterogeneous HS content and structure across patient-derived tumorsphere lines suggested diverse functions in the GBM tumor microenvironment. In GBM5 and mPN, elevated expression of sulfatase 2 (SULF2), an extracellular enzyme that alters ligand binding to HS, was associated with low trisulfated HS disaccharides, a substrate of SULF2. In contrast, other primary tumorsphere lines had elevated expression of the HS-modifying enzyme heparanase (HPSE). Using gene editing strategies to inhibit HPSE, a role for HPSE in promoting tumor cell adhesion and invasion was identified. These studies characterize the heterogeneity in HS glycosaminoglycan content and structure across GBM and reveal their role in tumor cell invasion.Implications: HS-interacting factors promote GBM invasion and are potential therapeutic targets. Mol Cancer Res; 15(11); 1623-33. ©2017 AACR.

GBM heterogeneity as a function of variable epidermal growth factor receptor variant III activity.

Abnormal activation of the epidermal growth factor receptor (EGFR) due to a deletion of exons 2-7 of EGFR (EGFRvIII) is a common alteration in glioblastoma (GBM). While this alteration can drive gliomagenesis, tumors harboring EGFRvIII are heterogeneous. To investigate the role for EGFRvIII activation in tumor phenotype we used a neural progenitor cell-based murine model of GBM driven by EGFR signaling and generated tumor progenitor cells with high and low EGFRvIII activation, pEGFRHi and pEGFRLo. In vivo, ex vivo, and in vitro studies suggested a direct association between EGFRvIII activity and increased tumor cell proliferation, decreased tumor cell adhesion to the extracellular matrix, and altered progenitor cell phenotype. Time-lapse confocal imaging of tumor cells in brain slice cultures demonstrated blood vessel co-option by tumor cells and highlighted differences in invasive pattern. Inhibition of EGFR signaling in pEGFRHi promoted cell differentiation and increased cell-matrix adhesion. Conversely, increased EGFRvIII activation in pEGFRLo reduced cell-matrix adhesion. Our study using a murine model for GBM driven by a single genetic driver, suggests differences in EGFR activation contribute to tumor heterogeneity and aggressiveness.

Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17alpha-alkyl anabolic steroid metabolites.

Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17beta-hydroxy-17alpha-methyl-5alpha-androstan-3-one) or 16beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16beta,17beta-dihydroxy-17alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17alpha-methyl-pyrazolo[4',3':2,3]-5alpha-androstan-17beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17alpha-methylandrost-5-ene-3beta,17beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.

Glycosylation alterations in lung and brain cancer.

Alterations in glycosylation are common in cancer and are thought to contribute to disease. Lung cancer and primary malignant brain cancer, most commonly glioblastoma, are genetically heterogeneous diseases with extremely poor prognoses. In this review, we summarize the data demonstrating that glycosylation is altered in lung and brain cancer. We then use specific examples to highlight the diverse roles of glycosylation in these two deadly diseases and illustrate shared mechanisms of oncogenesis. In addition to alterations in glycoconjugate biosynthesis, we also discuss mechanisms of postsynthetic glycan modification in cancer. We suggest that alterations in glycosylation in lung and brain cancer provide novel tumor biomarkers and therapeutic targets.

Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography-mass spectrometry.

A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS(2) were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabolites, respectively, in horse urine samples collected following the administration of boldenone undecylenate by intramuscular injection.

Detection of stanozolol and its metabolites in equine urine by liquid chromatography-electrospray ionization ion trap mass spectrometry.

The equine phase I and phase II metabolism of the synthetic anabolic steroid stanozolol was investigated following its administration by intramuscular injection to a thoroughbred gelding. The major phase I biotransformations were hydroxylation at C16 and one other site, while phase II metabolism in the form of sulfate and beta-glucuronide conjugation was extensive. An analytical procedure was developed for the detection of stanozolol and its metabolites in equine urine using solid phase extraction, acid solvolysis of phase II conjugates and analysis by positive ion electrospray ionization ion trap LC-MS.

The metabolism of anabolic-androgenic steroids in the greyhound.

BACKGROUND: Effective control of the use of anabolic-androgenic steroids (AASs) in animal sports is essential in order to ensure both animal welfare and integrity. In order to better police their use in Australian and New Zealand greyhound racing, thorough metabolic studies have been carried out on a range of registered human and veterinary AASs available in the region. RESULTS: Canine metabolic data are presented for the AASs boldenone, danazol, ethylestrenol, mesterolone, methandriol, nandrolone and norethandrolone. The principal Phase I metabolic processes observed were the reduction of A-ring unsaturations and/or 3-ketones with either 3α,5ß- or 3ß,5α-stereochemistry, the oxidation of secondary 17ß-hydroxyl groups and 16α-hydroxylation. The Phase II ß-glucuronylation of sterol metabolites was extensive. CONCLUSION: The presented data have enabled the effective analysis of AASs and their metabolites in competition greyhound urine samples.

Acepromazine pharmacokinetics: a forensic perspective.

Acepromazine (ACP) is a useful therapeutic drug, but is a prohibited substance in competition horses. The illicit use of ACP is difficult to detect due to its rapid metabolism, so this study investigated the ACP metabolite 2-(1-hydroxyethyl)promazine sulphoxide (HEPS) as a potential forensic marker. Acepromazine maleate, equivalent to 30mg of ACP, was given IV to 12 racing-bred geldings. Blood and urine were collected for 7days post-administration and analysed for ACP and HEPS by liquid chromatography-mass spectrometry (LC-MS). Acepromazine was quantifiable in plasma for up to 3h with little reaching the urine unmodified. Similar to previous studies, there was wide variation in the distribution and metabolism of ACP. The metabolite HEPS was quantifiable for up to 24h in plasma and 144h in urine. The metabolism of ACP to HEPS was fast and erratic, so the early phase of the HEPS emergence could not be modelled directly, but was assumed to be similar to the rate of disappearance of ACP. However, the relationship between peak plasma HEPS and the y-intercept of the kinetic model was strong (P=0.001, r(2)=0.72), allowing accurate determination of the formation pharmacokinetics of HEPS. Due to its rapid metabolism, testing of forensic samples for the parent drug is redundant with IV administration. The relatively long half-life of HEPS and its stable behaviour beyond the initial phase make it a valuable indicator of ACP use, and by determining the urine-to-plasma concentration ratios for HEPS, the approximate dose of ACP administration may be estimated.