Comparative safety, pharmacokinetics, and off-target assessment of 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane in mouse and dog: implications for therapeutic development.

The modified phytochemical derivative, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane (C-DIM12), has been identified as a potential therapeutic platform based on its capacity to improve disease outcomes in models of neurodegeneration and cancer. However, comprehensive safety studies investigating pathology and off-target binding have not been conducted. To address this, we administered C-DIM12 orogastrically to outbred male CD-1 mice for 7 days (50 mg/kg/day, 200 mg/kg/day, and 300 mg/kg/day) and investigated changes in hematology, clinical chemistry, and whole-body tissue pathology. We also delivered a single dose of C-DIM12 (1 mg/kg, 5 mg/kg, 25 mg/kg, 100 mg/kg, 300 mg/kg, 1,000 mg/kg) orogastrically to male and female beagle dogs and investigated hematology and clinical chemistry, as well as plasma pharmacokinetics over 48-h. Consecutive in-vitro off-target binding through inhibition was performed with 10 µM C-DIM12 against 68 targets in tandem with predictive off-target structural binding capacity. These data show that the highest dose C-DIM12 administered in each species caused modest liver pathology in mouse and dog, whereas lower doses were unremarkable. Off-target screening and predictive modeling of C-DIM12 show inhibition of serine/threonine kinases, calcium signaling, G-protein coupled receptors, extracellular matrix degradation, and vascular and transcriptional regulation pathways. Collectively, these data demonstrate that low doses of C-DIM12 do not induce pathology and are capable of modulating targets relevant to neurodegeneration and cancer.

Plasma Concentrations of Oral Ondansetron in Hospitalized Dogs Exhibiting Clinical Signs of Nausea.

The purpose of this study was to evaluate plasma ondansetron (OND) concentrations in a population of dogs with naturally occurring nausea after oral OND administration. Twenty-four dogs were randomly assigned to receive one of the following doses of oral OND: 0.5 mg/kg q8h, 0.5 mg/kg q12h, 1 mg/kg q8h, or 1 mg/kg q12h. Blood samples for plasma OND measurements were collected at baseline and 2, 4, and 8 h after administration of the first dose of OND. OND concentrations averaged over an 8 h time period were not significantly different between dose groups (0.5 mg/kg group: median 8.5 ng/mL [range 1-96.8 ng/mL], 1 mg/kg group: median 7.4 ng/mL [range 1-278.7 ng/mL]). The mean maximum concentrations in the 0.5 mg/kg and 1 mg/kg groups were 35.8 ± 49.0 ng/mL and 63.3 ± 121.1 ng/mL, respectively. OND concentrations were below the lower limit of quantification (LLOQ) in 50% (18/36) of samples in the 0.5 mg/kg groups and 39% (14/36) of samples in the 1 mg/kg groups. Six dogs (6/24, 25%) did not have OND detected at any time. The mean nausea scores at baseline were similar amongst all groups and decreased over time. The bioavailability of oral OND appears to be poor. Despite low plasma OND concentrations, nausea scores improved over time.

PHARMACOKINETICS AND EFFICACY OF A SINGLE TOPICAL DOSE OF EPRINOMECTIN IN GIRAFFE (GIRAFFA SPP.).

Growing resistance to current antiparasitic medications, both in livestock and in zoological species under human care, makes it imperative to evaluate available drugs on the market, such as eprinomectin. In this prospective study, five males and one female of reticulated (Giraffa reticulata; n = 2), Masai (Giraffa tippelskirchii; n = 1), Nubian (Giraffa camelopardalis; n = 2), and hybrid subspecies (n = 1) of giraffe, received 1.5 mg/kg eprinomectin topically along the dorsum. Using high-performance liquid chromatography, concentrations of eprinomectin in plasma samples collected at 0, 4, 24, and 48 h, and 7, 14, 21, and 28 d were evaluated following drug administration. Complete blood cell counts and biochemistry panels were performed before (n = 6) and after (n = 3) eprinomectin administration. Samples for modified double centrifugal fecal flotation (n = 6) were evaluated prior to eprinomectin administration to evaluate for endoparasites and were repeated after the study (n = 5). Noncompartmental pharmacokinetic analysis was applied to the data. The observed maximum plasma concentration was 11.45 ng/ml and the time of observed maximum concentration was 2.67 d. The mean terminal half-life was 5.16 d. No adverse effects were observed related to eprinomectin administration and no blood work changes were observed. Parasite loads decreased (n = 3) or did not change (n = 2) after eprinomectin administration. The mean peak plasma concentration of eprinomectin in giraffe was similar to that achieved in cattle, despite using three times the dose.

Association of Molecular Subtypes with Pathologic Response, PFS, and OS in a Phase II Study of COXEN with Neoadjuvant Chemotherapy for Muscle-invasive Bladder Cancer.

PURPOSE: The Coexpression Extrapolation (COXEN) gene expression model with chemotherapy-specific scores [for methotrexate, vinblastine, adriamycin, cisplatin (ddMVAC) and gemcitabine/cisplatin (GC)] was developed to identify responders to neoadjuvant chemotherapy (NAC). We investigated RNA-based molecular subtypes as additional predictive biomarkers for NAC response, progression-free survival (PFS), and overall survival (OS) in patients treated in S1314. EXPERIMENTAL DESIGN: A total of 237 patients were randomized between four cycles of ddMVAC (51%) and GC (49%). On the basis of Affymetrix transcriptomic data, we determined subtypes using three classifiers: TCGA (k = 5), Consensus (k = 6), and MD Anderson (MDA; k = 3) and assessed subtype association with path response to NAC and determined associations with COXEN. We also tested whether each classifier contributed additional predictive power when added to a model based on predefined stratification (strat) factors (PS 0 vs. 1; T2 vs. T3, T4a). RESULTS: A total of 155 patients had gene expression results, received at least three of four cycles of NAC, and had pT-N response based on radical cystectomy. TCGA three-group classifier basal-squamous (BS)/neuronal, luminal (Lum), Lum infiltrated, and GC COXEN score yielded the largest AUCs for pT0 (0.59, P = 0.28; 0.60, P = 0.18, respectively). For downstaging (

Individualized chemotherapy drug dose escalation in dogs with multicentric lymphoma.

BACKGROUND: This study was performed to determine the ability to escalate drug doses in a 15-week CHOP protocol in dogs with multicentric lymphoma. HYPOTHESIS: We hypothesized that at least 50% of dogs could successfully be escalated in at least 1 drug. Secondary aims were to establish objective response rate (ORR), progression-free interval (PFI), and overall survival time (OST). ANIMALS: Thirty dogs with newly diagnosed multicentric lymphoma were prospectively treated with a 15-week CHOP protocol. METHODS: This was a prospective cohort study. Drug doses that did not cause dose-limiting adverse effects (AEs) were increased using a standardized escalation protocol. AEs and response were assessed using VCOG criteria. Serial blood samples were collected after the first dose of each drug for pharmacokinetic analysis. RESULTS: Of the 23 dogs with the opportunity to dose escalate, at least 1 drug was successfully escalated in 18 (78%). Vincristine was successfully escalated to 0.8 mg/m2 or higher in 11 dogs, cyclophosphamide to 300 mg/m2 or higher in 16 dogs, and doxorubicin to 35 mg/m2 or 1.4 mg/kg or higher in 9 dogs. Three of the 23 dogs (13%) were hospitalized at least once because of drug-induced AEs. Neutropenia was the most common dose-limiting toxicosis for all drugs. Peak doxorubicin concentrations were significantly lower in dogs where doxorubicin was successfully escalated. The objective response rate was 100%. The median progression free interval was 171 days. The median overall survival time was 254 days. CONCLUSIONS: Drugs in the CHOP protocol can often be escalated safely with manageable AEs.

The efficacy and safety of cannabidiol as adjunct treatment for drug-resistant idiopathic epilepsy in 51 dogs: A double-blinded crossover study.

BACKGROUND: Approximately 30% of dogs with idiopathic epilepsy (IE) are drug-resistant. Recent studies have suggested cannabidiol (CBD) may be an effective anticonvulsant in dogs with IE. OBJECTIVE: To evaluate the addition of CBD to antiseizure drugs (ASDs) on seizure frequency and to report adverse events in dogs with drug-resistant IE. ANIMALS: Fifty-one dogs. Dogs having at least 2 seizures per month while receiving at least 1 ASD were included in the trial. METHODS: Double-blinded placebo-controlled crossover study. The 5 mg/kg/day dosage met futility requirements after 12 dogs, and a dosage of 9 mg/kg/day was used in the next 39 dogs. Dogs were randomly assigned to receive CBD or placebo for 3 months, with a 1-month washout period between oils. Total numbers of seizures and seizure days were recorded. Diagnostic testing was performed periodically throughout the trial. RESULTS: At the 9 mg/kg/day dose, the decrease in total seizure frequency was significant compared with placebo. A 24.1% decrease in seizure days occurred in dogs receiving CBD and a 5.8% increase occurred in dogs receiving placebo (P ≤ .05). No significant difference was found in the number of responders (≥50% decrease in total seizures or seizure days). Liver enzyme activities increased at both dosages. Decreased appetite and vomiting were more common in the CBD phase (P ≤ .05). CONCLUSIONS AND CLINICAL IMPORTANCE: Cannabidiol decreased total seizures and seizure days compared to placebo when administered to dogs PO at 9 mg/kg/day. Liver enzymes should be monitored with administration of CBD in dogs.

Sensitivity of osteosarcoma cell lines to autophagy inhibition as determined by pharmacologic and genetic manipulation.

Pharmacologic inhibition of autophagy can be achieved using lysosomotropic agents such as hydroxychloroquine (HCQ) that interfere with fusion of the autophagosome to the lysosome thus preventing completion of the recycling process. The goal of the present study is to determine the sensitivity of eight canine (cOSA) and four human (hOSA) osteosarcoma tumour cell lines to antiproliferative and cytotoxic effects of lysosomal autophagy inhibitors, and to compare these results to the autophagy-dependence measured using a CRISPR/Cas9 live-cell imaging assay in OSA and other tumour cell lines. Antiproliferative and cytotoxic response to HCQ and Lys05 was determined using live cell imaging and YOYO-1 staining. CRISPR/Cas9 live cell imaging screen was done using species specific guide RNA's and transfection of reagents into cells. Response to autophagy core genes was compared to response to an essential (PCNA) and non-essential (FOXO3A) gene. cOSA and hOSA cell lines showed similar antiproliferative and cytotoxic responses to HCQ and Lys05 with median lethal dose (Dm ) values ranging from 4.6-15.8 µM and 2.1-5.1 µM for measures of anti-proliferative response, respectively. A relationship was observed between antiproliferative responses to HCQ and Lys05 and VPS34 CRISPR score with Dm values correlating with VPS34 response (r = 0.968 and 0.887) in a species independent manner. The results show that a subset of cOSA and hOSA cell lines are autophagy-dependent and sensitive to HCQ at pharmacologically-relevant exposures.

Long-term Outcomes from a Phase 2 Study of Neoadjuvant Chemotherapy for Muscle-invasive Bladder Cancer (SWOG S1314; NCT02177695).

BACKGROUND: The COXEN gene expression model was evaluated for prediction of response to neoadjuvant chemotherapy for muscle-invasive bladder cancer (MIBC). OBJECTIVE: To conduct a secondary analysis of the association of each COXEN score with event-free survival (EFS) and overall survival (OS) and by treatment arm. DESIGN, SETTING, AND PARTICIPANTS: This was a randomized phase 2 trial of neoadjuvant gemcitabine-cisplatin (GC) or dose-dense methotrexate-vinblastine-adriamycin-cisplatin (ddMVAC) in MIBC. INTERVENTION: Patients were randomized to ddMVAC (every 14 d) or GC (every 21 d), both for four cycles. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: EFS events were defined as progression or death before scheduled surgery, a decision to not undergo surgery, recurrence, or death due to any cause after surgery. Cox regression was used to evaluate the COXEN score or treatment arm association with EFS and OS. RESULTS AND LIMITATIONS: A total of 167 evaluable patients were included in the COXEN analysis. The COXEN scores were not significantly prognostic for OS or EFS in the respective arms, but the GC COXEN score had a hazard ratio (HR) of 0.45 (95% confidence interval [CI] 0.20-0.99; p = 0.047) when the arms were pooled. In the intent-to-treat analysis (n = 227), there was no significant difference between ddMVAC and GC for OS (HR 0.87, 95% CI 0.54-1.40; p = 0.57) or EFS (HR 0.86, 95% CI 0.59-1.26; p = 0.45). Among the 192 patients who underwent surgery, pathologic response (pT0 vs downstaging vs no response) was strongly correlated with superior postsurgical survival (5-yr OS 90%, 89% and 52%, respectively). CONCLUSIONS: The COXEN GC score has prognostic value for patients receiving cisplatin-based neoadjuvant treatment. The randomized, prospective design provides estimates of OS and EFS for GC and ddMVAC in this population. Pathologic response (

EYA2 tyrosine phosphatase inhibition reduces MYC and prevents medulloblastoma progression.

BACKGROUND: Medulloblastoma is the most common pediatric brain malignancy. Patients with the Group 3 subtype of medulloblastoma (MB) often exhibit MYC amplification and/or overexpression and have the poorest prognosis. While Group 3 MB is known to be highly dependent on MYC, direct targeting of MYC remains elusive. METHODS: Patient gene expression data were used to identify highly expressed EYA2 in Group 3 MB samples, assess the correlation between EYA2 and MYC, and examine patient survival. Genetic and pharmacological studies were performed on EYA2 in Group 3 derived MB cell models to assess MYC regulation and viability in vitro and in vivo. RESULTS: EYA2 is more highly expressed in Group 3 MB than other MB subgroups and is essential for Group 3 MB growth in vitro and in vivo. EYA2 regulates MYC expression and protein stability in Group 3 MB, resulting in global alterations of MYC transcription. Inhibition of EYA2 tyrosine phosphatase activity, using a novel small molecule inhibitor (NCGC00249987, or 9987), significantly decreases Group 3 MB MYC expression in both flank and intracranial growth in vivo. Human MB RNA-seq data show that EYA2 and MYC are significantly positively correlated, high EYA2 expression is significantly associated with a MYC transcriptional signature, and patients with high EYA2 and MYC expression have worse prognoses than those that do not express both genes at high levels. CONCLUSIONS: Our data demonstrate that EYA2 is a critical regulator of MYC in Group 3 MB and suggest a novel therapeutic avenue to target this highly lethal disease.

Vinblastine pharmacokinetics in mouse, dog, and human in the context of a physiologically based model incorporating tissue-specific drug binding, transport, and metabolism.

Vinblastine (VBL) is a vinca alkaloid-class cytotoxic chemotherapeutic that causes microtubule disruption and is typically used to treat hematologic malignancies. VBL is characterized by a narrow therapeutic index, with key dose-limiting toxicities being myelosuppression and neurotoxicity. Pharmacokinetics (PK) of VBL is primarily driven by ABCB1-mediated efflux and CYP3A4 metabolism, creating potential for drug-drug interaction. To characterize sources of variability in VBL PK, we developed a physiologically based pharmacokinetic (PBPK) model in Mdr1a/b(-/-) knockout and wild-type mice by incorporating key drivers of PK, including ABCB1 efflux, CYP3A4 metabolism, and tissue-specific tubulin binding, and scaled this model to accurately simulate VBL PK in humans and pet dogs. To investigate the capability of the model to capture interindividual variability in clinical data, virtual populations of humans and pet dogs were generated through Monte Carlo simulation of physiologic and biochemical parameters and compared to the clinical PK data. This model provides a foundation for predictive modeling of VBL PK. The base PBPK model can be further improved with supplemental experimental data identifying drug-drug interactions, ABCB1 polymorphisms and expression, and other sources of physiologic or metabolic variability.

Pharmacokinetics of escalating single-dose administration of cannabidiol to cats.

This study aimed to assess the single-dose pharmacokinetics and tolerability of a cannabidiol (CBD) isolate in sunflower oil with escalating oral doses in eight healthy, purpose-bred cats. Eight cats were randomized into six dosing groups of four cats each. Cats were administered a single 2.5, 5, 10, 20, 40, or 80 mg/kg dose orally with at least a two-week washout in between doses. Behavior scoring, complete blood count, serum biochemistry analysis, physical examination, and CBD plasma levels were evaluated before and after dosing. All cats successfully completed the study. CBD was measured in the plasma of all cats dosed with CBD oil. The Cmax and AUC increased in a dose-proportional fashion across all dosing groups. There were no major bloodwork or behavioral changes although the BUN and creatinine values decreased after treatment across all doses. No adverse effects were observed, and behavioral changes were limited to head shaking, lip smacking, and hypersalivation immediately following dose administration. Single orally administered CBD doses up to 80 mg/kg were safe and well tolerated in this cohort of cats and display dose-proportional pharmacokinetics across a broad concentration.

Pharmacokinetics and anti-nausea effects of intravenous ondansetron in hospitalized dogs exhibiting clinical signs of nausea.

The purpose of this study was to evaluate the pharmacokinetics of intravenous (IV) ondansetron in a population of hospitalized dogs exhibiting clinical signs of nausea. The causes of nausea included pancreatitis, gastroenteritis, endocarditis, chemotherapy-induced nausea, diabetes mellitus and ketoacidosis, acute kidney injury with aspiration pneumonia, pyometra, uroabdomen, neoplasia, and hepatopathy. Twenty-four dogs were randomly assigned to one of the following IV ondansetron protocols: 1 mg/kg q12h, 0.5 mg/kg q12h, 1 mg/kg q8h, 0.5 mg/kg q8h. Serum was collected at 0, 0.25, 0.5, 1, 2, 4, 8, 16, and 24 h after the first dose, and nausea scores were recorded at multiple time points. Ondansetron and arginine vasopressin (AVP) concentrations were measured via high-performance liquid chromatography coupled to tandem mass spectrometry and ELISA, respectively. Noncompartmental pharmacokinetic modeling and dose interval modeling were performed. Ondansetron displayed linear pharmacokinetics. In the 0.5 mg/kg group, mean Cmax  = 214 ng/ml, AUC0-8h  = 463 ng/ml*h, and calculated half-life was 1.9 h. In the 1 mg/kg group, mean Cmax  = 541 ng/ml, AUC0-8h  = 1057 ng/ml*h and calculated half-life was 1.6 h. Serum ondansetron concentrations were not significantly different between dogs that required rescue anti-nausea medication (non-responders) and dogs that did not require rescue therapy (responders). In total, 83.3% of patients in the 0.5 mg/kg q8h, 0.5 mg/kg q12h, and 1 mg/kg q8h groups had improvement in nausea scores. In total, 66.7% of patients in the 1 mg/kg q12h group had improvement in nausea scores. In total, 33% of patients had resolution of nausea in the 0.5 mg/kg q8h, 1 mg/kg q8h, and 1 mg/kg q12h groups, and 16% of patients had resolution of nausea in the 0.5 mg/kg q12h group. AVP concentrations were highly variable and did not correlate with nausea scores. Nausea scores significantly decreased regardless of dosage protocol. AVP was not a reliable biomarker of nausea in this group of dogs.

A small molecule that disrupts S. Typhimurium membrane voltage without cell lysis reduces bacterial colonization of mice.

As pathogenic bacteria become increasingly resistant to antibiotics, antimicrobials with mechanisms of action distinct from current clinical antibiotics are needed. Gram-negative bacteria pose a particular problem because they defend themselves against chemicals with a minimally permeable outer membrane and with efflux pumps. During infection, innate immune defense molecules increase bacterial vulnerability to chemicals by permeabilizing the outer membrane and occupying efflux pumps. Therefore, screens for compounds that reduce bacterial colonization of mammalian cells have the potential to reveal unexplored therapeutic avenues. Here we describe a new small molecule, D66, that prevents the survival of a human Gram-negative pathogen in macrophages. D66 inhibits bacterial growth under conditions wherein the bacterial outer membrane or efflux pumps are compromised, but not in standard microbiological media. The compound disrupts voltage across the bacterial inner membrane at concentrations that do not permeabilize the inner membrane or lyse cells. Selection for bacterial clones resistant to D66 activity suggested that outer membrane integrity and efflux are the two major bacterial defense mechanisms against this compound. Treatment of mammalian cells with D66 does not permeabilize the mammalian cell membrane but does cause stress, as revealed by hyperpolarization of mitochondrial membranes. Nevertheless, the compound is tolerated in mice and reduces bacterial tissue load. These data suggest that the inner membrane could be a viable target for anti-Gram-negative antimicrobials, and that disruption of bacterial membrane voltage without lysis is sufficient to enable clearance from the host.

Pharmacokinetics, Safety, and Synovial Fluid Concentrations of Single- and Multiple-Dose Oral Administration of 1 and 3 mg/kg Cannabidiol in Horses.

Cannabidiol (CBD) products are widely marketed to horse owners, trainers, and veterinarians and are readily available to the consumer despite minimal pharmacokinetic or safety data being available. The objectives of this study were to determine the plasma pharmacokinetics, short-term safety, and synovial fluid levels of CBD following oral administration in horses. A prospective, randomized, controlled study design was used. Twelve horses were administered sunflower lecithin oil-based CBD at either 1 mg/kg (Group 1) or 3 mg/kg (Group 2) for a 24-hour pharmacokinetic study. Horses then received 0.5 mg/kg (Group 1) or 1.5 mg/kg (Group 2) CBD q12 PO for 6 weeks, with steady state and elimination sampling performed up to 96 hours post-final dose. Synovial fluid CBD concentrations were evaluated at 12 and 24 hours, and 5 weeks. Horses were monitored daily and clinicopathologic parameters were evaluated. Mean ± SD Cmax and tmax were 4.3 ± 2.1 ng/ml and 4.1 ± 4.1 hours, and 19.9 ± 15.6 ng/ml and 5.0 ± 3.7 hours for Groups 1 and 2, respectively. CBD was detectable in synovial fluid in 8/12 horses during steady state. Mild hypocalcemia was seen in all horses and elevated liver enzymes were observed in 8/12 horses, but these changes improved or normalized within 10 days after the final CBD dose. CBD had dose-dependent, but variable, oral bioavailability at 1 mg/kg and 3 mg/kg daily dosing and was consistently detectable at steady state in synovial fluid at the higher dose. Further investigation is needed to establish clinically effective doses.

Design, Synthesis, and Biological Evaluation of the First Inhibitors of Oncogenic CHD1L.

Chromodomain helicase DNA-binding protein 1 like (CHD1L) is an oncogene implicated in tumor progression, multidrug resistance, and metastasis in many types of cancer. In this article, we described the optimization of the first lead CHD1L inhibitors (CHD1Li) through drug design and medicinal chemistry. More than 30 CHD1Li were synthesized and evaluated using a variety of colorectal cancer (CRC) tumor organoid models and functional assays. The results led to the prioritization of six lead CHD1Li analogues with improved potency, antitumor activity, and drug-like properties including metabolic stability and in vivo pharmacokinetics. Furthermore, lead CHD1Li 6.11 proved to be an orally bioavailable antitumor agent, significantly reducing the tumor volume of CRC xenografts generated from isolated quasi mesenchymal cells (M-phenotype), which possess enhanced tumorigenic properties. In conclusion, we reported the optimization of first-in-class inhibitors of oncogenic CHD1L as a novel therapeutic strategy with potential for the treatment of cancer.

Characterizing the molecular and immune landscape of canine bladder cancer.

Transitional cell carcinoma (TCC), also known as urothelial carcinoma, is the most common bladder cancer in humans and dogs. Approximately one-quarter of human TCCs are muscle-invasive and associated with a high risk of death from metastasis. Canine TCC (cTCC) tumours are typically high-grade and muscle-invasive. Shared similarities in risk factors, histopathology, and clinical presentation suggest that cTCC may serve as a model for the assessment of novel therapeutics that may inform therapies for human muscle-invasive TCC. The goal of this study was to characterize cTCC at the molecular level to identify drivers of oncogenesis and druggable targets. We performed whole exome sequencing (WES) of 11 cTCC tumours and three matched normal samples, identifying 583 variants in protein-coding genes. The most common variant was a V-to-E missense mutation in BRAF, identified in 4 out of 11 samples (36%) via WES. Sanger sequencing identified BRAF variants in 8 out of the same 11 cTCC samples, as well as in 22 out of 32 formalin-fixed paraffin embedded (FFPE) cTCC samples, suggesting an overall prevalence of 70%. RNA-Seq was performed to compare the gene expression profiles of cTCC tumours to normal bladder tissue. cTCC tumours exhibited up-regulation of genes involved in the cell cycle, DNA repair, and antiviral immunity. We also analysed the immune landscape of cTCC using immune gene signatures and immunohistochemical analysis. A subset of tumours had characteristics of a hot tumour microenvironment and exhibited high expression of signatures associated with complete response to PD-1/PD-L1 blockade in human bladder cancer.

Losartan Blocks Osteosarcoma-Elicited Monocyte Recruitment, and Combined With the Kinase Inhibitor Toceranib, Exerts Significant Clinical Benefit in Canine Metastatic Osteosarcoma.

PURPOSE: There is increasing recognition that progress in immuno-oncology could be accelerated by evaluating immune-based therapies in dogs with spontaneous cancers. Osteosarcoma (OS) is one tumor for which limited clinical benefit has been observed with the use of immune checkpoint inhibitors. We previously reported the angiotensin receptor blocker losartan suppressed metastasis in preclinical mouse models through blockade of CCL2-CCR2 monocyte recruitment. Here we leverage dogs with spontaneous OS to determine losartan's safety and pharmacokinetics associated with monocyte pharmacodynamic endpoints, and assess its antitumor activity, in combination with the kinase inhibitor toceranib. PATIENTS AND METHODS: CCL2 expression, monocyte infiltration, and monocyte recruitment by human and canine OS tumors and cell lines were assessed by gene expression, ELISA, and transwell migration assays. Safety and efficacy of losartan-toceranib therapy were evaluated in 28 dogs with lung metastatic OS. Losartan PK and monocyte PD responses were assessed in three dose cohorts of dogs by chemotaxis, plasma CCL2, and multiplex cytokine assays, and RNA-seq of losartan-treated human peripheral blood mononuclear cells. RESULTS: Human and canine OS cells secrete CCL2 and elicit monocyte migration, which is inhibited by losartan. Losartan PK/PD studies in dogs revealed that a 10-fold-higher dose than typical antihypertensive dosing was required for blockade of monocyte migration. Treatment with high-dose losartan and toceranib was well-tolerated and induced a clinical benefit rate of 50% in dogs with lung metastases. CONCLUSIONS: Losartan inhibits the CCL2-CCR2 axis, and in combination with toceranib, exerts significant biological activity in dogs with metastatic osteosarcoma, supporting evaluation of this drug combination in patients with pediatric osteosarcoma. See related commentary by Weiss et al., p. 571.

Drug-drug interaction between cannabidiol and phenobarbital in healthy dogs.

OBJECTIVE: To assess drug-drug interactions between cannabidiol (CBD) and phenobarbital (PB) when simultaneously administered to healthy dogs. ANIMALS: 9 healthy, purpose bred Beagles. PROCEDURES: A 3-phase prospective, randomized pharmacokinetic (PK) interaction study of CBD and PB was performed as follows: phase 1, CBD PK determination and evaluation of CBD tolerability by 3 single-dose CBD (5 mg/kg, 10 mg/kg, and 20 mg/kg) protocols followed by 2-week CBD dosing; phase 2, a single-dose, 3-way, crossover PK study of CBD (10 mg/kg), PB (4 mg/kg), or CBD (10 mg/kg) administration plus PB (4 mg/kg); and phase 3, evaluation of chronic PB (4 mg/kg, q 30 d) administration followed by single-dose CBD (10 mg/kg) PK study. RESULTS: Although there were variations in CBD PK variables in dogs receiving CBD alone or in conjunction with PB, significance differences in CBD PK variables were not found. No significant difference was observed in PB PK variables of dogs receiving PB alone or with CBD. During chronic CBD administration, mild gastrointestinal signs were observed in 5 dogs. At daily CBD doses of 10 to 20 mg/kg/d, hypoxia was observed in 5 dogs and increased serum alkaline phosphatase (ALP) activities (range, 301 to 978 U/L) was observed in 4 dogs. A significant increase in ALP activity was observed with chronic administration of CBD during phase 1 between day 0 and day 14. CONCLUSIONS AND CLINICAL RELEVANCE: No significant PK interactions were found between CBD and PB. Dose escalation of CBD or adjustment of PB in dogs is not recommended on the basis of findings of this study.

Immune pathways and TP53 missense mutations are associated with longer survival in canine osteosarcoma.

Osteosarcoma affects about 2.8% of dogs with cancer, with a one-year survival rate of approximately 45%. The purpose of this study was to characterize mutation and expression profiles of osteosarcoma and its association with outcome in dogs. The number of somatic variants identified across 26 samples ranged from 145 to 2,697 with top recurrent mutations observed in TP53 and SETD2. Additionally, 47 cancer genes were identified with copy number variations. Missense TP53 mutation status and low pre-treatment blood monocyte counts were associated with a longer disease-free interval (DFI). Patients with longer DFI also showed increased transcript levels of anti-tumor immune response genes. Although, T-cell and myeloid cell quantifications were not significantly associated with outcome; immune related genes, PDL-1 and CD160, were correlated with T-cell abundance. Overall, the association of gene expression and mutation profiles to outcome provides insights into pathogenesis and therapeutic interventions in osteosarcoma patients.

Plasma and joint tissue pharmacokinetics of two doses of oral cannabidiol oil in guinea pigs (Cavia porcellus).

Cannabidiol (CBD) has gained widespread popularity as a treatment for osteoarthritis (OA) in pets; however, there is minimal scientific evidence regarding safe and effective dosing. This study determined plasma and tissue pharmacokinetics after oral CBD oil suspension administration in Hartley guinea pigs (Cavia porcellus), which spontaneously develop OA at 3 months of age. Ten, 5-month-old, male guinea pigs were randomly assigned to receive 25 (n = 5) or 50 mg/kg (n = 5) CBD oil once orally. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h timepoints. Open-field enclosure monitoring revealed no adverse effects. After euthanasia, stifle cartilage and infrapatellar fat pads were collected to quantitate CBD. CBD concentrations were determined using a validated liquid chromatography-mass spectrometry method, and pharmacokinetic parameters were calculated using noncompartmental analysis. The area under the plasma concentration-versus-time curve was 379.5 and 873.7 h*ng/mL, maximum plasma concentration was 42 and 96.8 ng/mL, time to maximum plasma concentration was 1.6 and 4.8 h, and terminal phase half-life was 8.1 and 10.8 h for the 25 and 50 mg/kg doses, respectively. CBD was detected in joint tissues of all animals. Further studies, including work in female guinea pigs, are needed to determine the efficacy of CBD for OA.