First-in-Class Inhibitors of Oncogenic CHD1L with Preclinical Activity against Colorectal Cancer.

Since the discovery of CHD1L in 2008, it has emerged as an oncogene implicated in the pathology and poor prognosis of a variety of cancers, including gastrointestinal cancers. However, a mechanistic understanding of CHD1L as a driver of colorectal cancer has been limited. Until now, there have been no reported inhibitors of CHD1L, also limiting its development as a molecular target. We sought to characterize the clinicopathologic link between CHD1L and colorectal cancer, determine the mechanism(s) by which CHD1L drives malignant colorectal cancer, and discover the first inhibitors with potential for novel treatments for colorectal cancer. The clinicopathologic characteristics associated with CHD1L expression were evaluated using microarray data from 585 patients with colorectal cancer. Further analysis of microarray data indicated that CHD1L may function through the Wnt/TCF pathway. Thus, we conducted knockdown and overexpression studies with CHD1L to determine its role in Wnt/TCF-driven epithelial-to-mesenchymal transition (EMT). We performed high-throughput screening (HTS) to identify the first CHD1L inhibitors. The mechanism of action, antitumor efficacy, and drug-like properties of lead CHD1L inhibitors were determined using biochemical assays, cell models, tumor organoids, patient-derived tumor organoids, and in vivo pharmacokinetics and pharmacodynamics. Lead CHD1L inhibitors display potent in vitro antitumor activity by reversing TCF-driven EMT. The best lead CHD1L inhibitor possesses drug-like properties in pharmacokinetic/pharmacodynamic mouse models. This work validates CHD1L as a druggable target and establishes a novel therapeutic strategy for the treatment of colorectal cancer.

Pilot study of side effects and serum and urine concentrations of amoxicillin-clavulanic acid in azotemic and non-azotemic cats.

OBJECTIVES: The aims of this study were to determine the side effect frequency and serum and urine drug concentrations of amoxicillin-clavulanic acid in cats with and without azotemic chronic kidney disease (azCKD). METHODS: Owners whose cats had been prescribed amoxicillin-clavulanic acid completed a survey regarding the occurrence and type of side effects, and whether treatment was altered as a result. Cats were defined as azCKD (serum creatinine concentration >2.0 mg/dl, urine specific gravity [USG] <1.035 with a clinical diagnosis of chronic kidney disease) and without azCKD (serum creatinine concentration <2.0 mg/dl). Data were assessed with Fisher's exact test. Serum and urine samples were obtained from client-owned cats with azCKD (n = 6) and without azCKD (n = 6, serum creatinine concentration <1.8 mg/dl, USG >1.035) that were receiving amoxicillin-clavulanic acid. Amoxicillin and clavulanic acid were measured with liquid chromatography coupled to tandem mass spectrometry and compared between groups with a Mann-Whitney test. Correlation between serum creatinine and drug concentrations in urine and serum was determined using Spearman's rank test. RESULTS: Sixty-one surveys were returned (11 azCKD cats and 50 without azCKD cats). No significant difference in the presence of side effects or type of side effects was seen between groups; however, significantly more azCKD cats had more than one side effect (P = 0.02). More owners of azCKD cats reported that an alteration in treatment plan was necessitated by side effects (55% vs 12%; P = 0.008). Urine amoxicillin was significantly lower in cats with azCKD (P = 0.01) and serum amoxicillin trended toward significance (P = 0.07). Serum amoxicillin concentration was positively correlated with serum creatinine (P = 0.02; r = 0.62) and urine amoxicillin concentration was negatively correlated with serum creatinine (P = 0.01; r = -0.65). CONCLUSIONS AND RELEVANCE: The data suggest that cats with azCKD have altered pharmacokinetics of amoxicillin, which may contribute to an increased incidence of multiple side effects.

Drug Design Targeting T-Cell Factor-Driven Epithelial-Mesenchymal Transition as a Therapeutic Strategy for Colorectal Cancer.

Metastasis is the cause of 90% of mortality in cancer patients. For metastatic colorectal cancer (mCRC), the standard-of-care drug therapies only palliate the symptoms but are ineffective, evidenced by a low survival rate of ∼11%. T-cell factor (TCF) transcription is a major driving force in CRC, and we have characterized it to be a master regulator of epithelial-mesenchymal transition (EMT). EMT transforms relatively benign epithelial tumor cells into quasi-mesenchymal or mesenchymal cells that possess cancer stem cell properties, promoting multidrug resistance and metastasis. We have identified topoisomerase IIα (TOP2A) as a DNA-binding factor required for TCF-transcription. Herein, we describe the design, synthesis, biological evaluation, and in vitro and in vivo pharmacokinetic analysis of TOP2A ATP-competitive inhibitors that prevent TCF-transcription and modulate or reverse EMT in mCRC. Unlike TOP2A poisons, ATP-competitive inhibitors do not damage DNA, potentially limiting adverse effects. This work demonstrates a new therapeutic strategy targeting TOP2A for the treatment of mCRC and potentially other types of cancers.

The fecal microbiome and serum concentrations of indoxyl sulfate and p-cresol sulfate in cats with chronic kidney disease.

BACKGROUND: Intestinal dysbiosis has been documented in humans with chronic kidney disease (CKD) and is thought to contribute to production of the uremic toxins indoxyl sulfate (IS) and p-cresol sulfate (pCS). Characteristics of the fecal microbiome in cats with CKD and correlation to serum concentrations of uremic toxins are unknown. OBJECTIVES: To characterize the fecal microbiome and measure serum IS and pCS concentrations of cats with CKD in comparison to healthy older cats. ANIMALS: Thirty client-owned cats with CKD (International Renal Interest Society stages 2-4) and 11 older (≥8 years) healthy control cats. METHODS: Prospective, cross-sectional study. Fecal samples were analyzed by sequencing of 16S rRNA genes and Escherichia coli quantitative PCR (qPCR). Serum concentrations of IS and pCS measured using liquid chromatography tandem mass spectrometry. RESULTS: Cats with CKD had significantly decreased fecal bacterial diversity and richness. Escherichia coli qPCR showed no significant difference in bacteria count between control and CKD cats. Cats with stage 2 (P = .01) and stages 3 and 4 (P = .0006) CKD had significantly higher serum IS concentrations compared to control cats. No significant difference found between stage 2 and stages 3 and 4 CKD. The pCS concentrations were not significantly different between CKD cats and control cats. CONCLUSIONS AND CLINICAL IMPORTANCE: Decreased fecal microbiome diversity and richness is associated with CKD in cats. Indoxyl sulfate concentration is significantly increased with CKD, and cats with stage 2 CKD may suffer from a similar uremic toxin burden as do cats with later stage disease.

Drug exposure and clinical effect of transdermal mirtazapine in healthy young cats: a pilot study.

Objectives The objective of this study was to measure drug exposure and clinical effects after administration of transdermal mirtazapine (TMZ) in healthy cats. Methods Phase I: seven healthy research cats received (1) 3.75 mg and 7.5 mg TMZ once aurally with 48 h serum sampling (serum samples were obtained via the jugular catheter at 0, 0.5, 1, 2, 5, 9, 12, 24, 36 and 48 h); (2) 7.5 mg TMZ and placebo daily aurally for 6 days then 48 h serum sampling; (3) 1.88 mg mirtazapine orally once with serum sampling at 1, 4 and 8 h. Phase II: 20 client-owned cats were enrolled in a randomized, double-blind, placebo-controlled, three-way crossover clinical effect study. Treatments consisted of 6 days of aural 7.5 mg TMZ or placebo gel at home, and 1.88 mg mirtazapine orally once in the clinic. Owners documented appetite, rate of food ingestion, begging activity and vocalization daily at home. On day 6, food consumed, activity and vocalization were documented in hospital, and trough and peak serum mirtazapine levels were obtained. Serum mirtazapine and gel concentrations were measured using liquid chromatography/tandem mass spectrometry. Results Phase I: administration of TMZ achieved measureable serum mirtazapine concentrations. Area under the curve0-48 of multidose 7.5 mg TMZ was significantly higher than single-dose 1.88 mg oral mirtazapine (OMZ) ( P = 0.02). Phase II: client-owned cats administered TMZ had a significant increase in appetite ( P = 0.003), rate of food ingestion ( P = 0.002), activity ( P = 0.002), begging ( P = 0.002) and vocalization ( P = 0.002) at home. In hospital there was a significant increase in food ingested with both TMZ and OMZ compared with placebo ( P <0.05). Gel concentrations ranged from 87%-119% of target dose. Conclusions and relevance TMZ 7.5 mg daily achieves measureable serum concentrations and produces significant appetite stimulation despite variance in compounded gel concentrations, but side effects denote a lower dose is indicated.

Pharmacokinetics of orally administered low-dose rapamycin in healthy dogs.

OBJECTIVE: To determine the pharmacokinetics of orally administered rapamycin in healthy dogs. ANIMALS: 5 healthy purpose-bred hounds. PROCEDURES: The study consisted of 2 experiments. In experiment 1, each dog received rapamycin (0.1 mg/kg, PO) once; blood samples were obtained immediately before and at 0.5, 1, 2, 4, 6, 12, 24, 48, and 72 hours after administration. In experiment 2, each dog received rapamycin (0.1 mg/kg, PO) once daily for 5 days; blood samples were obtained immediately before and at 3, 6, 24, 27, 30, 48, 51, 54, 72, 75, 78, 96, 96.5, 97, 98, 100, 102, 108, 120, 144, and 168 hours after the first dose. Blood rapamycin concentration was determined by a validated liquid chromatography-tandem mass spectrometry assay. Pharmacokinetic parameters were determined by compartmental and noncompartmental analyses. RESULTS: Mean ± SD blood rapamycin terminal half-life, area under the concentration-time curve from 0 to 48 hours after dosing, and maximum concentration were 38.7 ± 12.7 h, 140 ± 23.9 ng•h/mL, and 8.39 ± 1.73 ng/mL, respectively, for experiment 1, and 99.5 ± 89.5 h, 126 ± 27.1 ng•h/mL, and 5.49 ± 1.99 ng/mL, respectively, for experiment 2. Pharmacokinetic parameters for rapamycin after administration of 5 daily doses differed significantly from those after administration of 1 dose. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that oral administration of low-dose (0.1 mg/kg) rapamycin to healthy dogs achieved blood concentrations measured in nanograms per milliliter. The optimal dose and administration frequency of rapamcyin required to achieve therapeutic effects in tumor-bearing dogs, as well as toxicity after chronic dosing, need to be determined.

Comparison of the stability and pharmacokinetics in dogs of modified ciclosporin capsules stored at -20°C and room temperature.

BACKGROUND: Placement of ciclosporin (Atopica(®); Novartis Animal Health, Greensboro, NC, USA) capsules in a freezer prior to administration may reduce the incidence of vomiting in dogs. However, its impact on ciclosporin stability and pharmacokinetics is unknown. HYPOTHESIS/OBJECTIVES: The purpose of this study was to evaluate the stability of Atopica(®) capsules and pharmacokinetics of ciclosporin in dogs after storage at -20°C in comparison with storage of capsules at 15-25°C. We hypothesized that there would be no difference in stability or pharmacokinetic parameters between freezer-stored and room-temperature Atopica(®) capsules. ANIMALS: Eight healthy research beagle dogs received one 5.0 mg/kg oral dose each of freezer-stored and room-temperature Atopica(®) capsules with a 1 week washout period between. METHODS: Ciclosporin concentrations of all available Atopica(®) capsule strengths were assessed for stability after -20°C storage at five time points over 30 days and at room temperature (15-25°C). A blinded, randomized cross-over study was also performed to compare blood concentrations of ciclosporin after capsule storage for 28 days at -20 versus 15-25°C. Blood samples were obtained over a 24 h period after administration. Capsule and whole-blood ciclosporin concentrations were assessed via high-performance liquid chromatography-tandem mass spectrometry. RESULTS: There was no significant difference in stability between freezer-stored and room-temperature Atopica(®) capsules at any time point. In the cross-over study, there were no significant differences in pharmacokinetic parameters assessed. CONCLUSIONS AND CLINICAL IMPORTANCE: Placing Atopica(®) capsules in a -20°C freezer for 28 days does not affect stability or absorption in the dog.

Comparative pharmacokinetic properties and antitumor activity of the marine HDACi Largazole and Largazole peptide isostere.

PURPOSE: Largazole is a potent class I-selective HDACi natural product isolated from the marine cyanobacteria Symploca sp. The purpose of this study was to test synthetic analogs of Largazole to identify potential scaffold structural modifications that would improve the drug-like properties of this clinically relevant natural product. METHODS: The impact of Largazole scaffold replacements on in vitro growth inhibition, cell cycle arrest, induction of apoptosis, pharmacokinetic properties, and in vivo activity using a xenograft model was investigated. RESULTS: In vitro studies in colon, lung, and pancreatic cancer cell lines showed that pyridyl-substituted Largazole analogs had low-nanomolar/high-picomolar antiproliferative activity, and induced apoptosis and cell cycle arrest at concentrations equivalent to or lower than the parent compound Largazole. Using IV bolus delivery at 5 mg/kg, two compartmental pharmacokinetic modeling on the peptide isostere analog of Largazole indicated improved pharmacokinetic parameters. In an A549 non-small cell lung carcinoma xenograft model using a dosage of 5 mg/kg administered intraperitoneally every other day, Largazole, Largazole thiol, and Largazole peptide isostere demonstrated tumor growth inhibition (TGI%) of 32, 44, and 66%, respectively. Largazole peptide isostere treatment was statistically superior to control (p = 0.002) and to Largazole (p = 0.006). Surprisingly, tumor growth inhibition was not observed with the potent pyridyl-based analogs. CONCLUSIONS: These results establish that replacing the depsipeptide linkage in Largazole with an amide may impart pharmacokinetic and therapeutic advantage and that alternative prodrug forms of Largazole are feasible.

Pharmacokinetics and pharmacodynamics of propofol with or without 2% benzyl alcohol following a single induction dose administered intravenously in cats.

OBJECTIVE: To compare the pharmacokinetics and pharmacodynamics of propofol with or without 2% benzyl alcohol administered intravenously (IV) as a single induction dose in cats. STUDY DESIGN: Prospective experimental study. ANIMALS: Six healthy adult cats, three female intact, three male castrated, weighing 4.8 ± 1.8 kg. METHODS: Cats received 8 mg kg(-1) IV of propofol (P) or propofol with 2% benzyl alcohol (P28) using a randomized crossover design. Venous blood samples were collected at predetermined time points to 24 hours after drug administration to determine drug plasma concentrations. Physiologic and behavioral variables were also recorded. Propofol and benzyl alcohol concentrations were determined using high pressure liquid chromatography with fluorescence detection. Pharmacokinetic parameters were described using a 2-compartment model. Pharmacokinetic and pharmacodynamic parameters were analyzed using repeated measures anova (p < 0.05). RESULTS: Plasma concentrations of benzyl alcohol were below the lower limits of quantification (LLOQ) at all time points for two of the six cats (33%), and by 30 minutes for the remaining four cats. Propofol pharmacokinetics, with or without 2% benzyl alcohol, were characterized by rapid distribution, a long elimination phase, and a large volume of distribution. No differences were noted between treatments with the exception of clearance from the second compartment (CLD2), which was 23.6 and 38.8 mL kg(-1)  minute(-1) in the P and P28 treatments, respectively. Physiologic and behavioral variables were not different between treatments with the exception of heart rate at 4 hours post administration. CONCLUSIONS AND CLINICAL RELEVANCE: The addition of 2% benzyl alcohol as a preservative minimally altered the pharmacokinetics and pharmacodynamics of propofol 1% emulsion when administered as a single IV bolus in this group of cats. These data support the cautious use of propofol with 2% benzyl alcohol for induction of anesthesia in healthy cats.

Pharmacokinetics of sustained-release analgesics in mice.

Buprenorphine and carprofen, 2 of the most commonly used analgesics in mice, must be administered every 8 to 12 h to provide sustained analgesia. Sustained-release (SR) formulations of analgesics maintain plasma levels that should be sufficient to provide sustained analgesia yet require less frequent dosing and thus less handling of and stress to the animals. The pharmacokinetics of SR formulations of buprenorphine (Bup-SR), butorphanol (Butp-SR), fentanyl (Fent-SR), carprofen (Carp-SR), and meloxicam (Melox-SR) were evaluated in mice over 72 h and compared with those of traditional, nonSR formulations. Bup-SR provided plasma drug levels greater than the therapeutic level for the first 24 to 48 h after administration, but plasma levels of Bup-HCl fell below the therapeutic level by 4 h. Fent-SR maintained plasma levels greater than reported therapeutic levels for 12 h. Therapeutic levels of the remaining drugs are unknown, but Carp-SR provided plasma drug levels similar to those of Carp for the first 24 h after administration, whereas Melox-SR had greater plasma levels than did Melox for the first 8 h. Butp-SR provided detectable plasma drug levels for the first 24 h, with a dramatic decrease over the first 4 h. These results indicate that Bup-SR provides a stable plasma drug level adequate for analgesia for 24 to 48 h after administration, whereas Carp-SR, Melox-SR, Fent-SR, and Butp-SR would require additional doses to provide analgesic plasma levels beyond 24 h in mice.

A phase I dose-finding study of silybin phosphatidylcholine (milk thistle) in patients with advanced hepatocellular carcinoma.

PURPOSE: To determine the maximum tolerated dose per day of silybin phosphatidylcholine (Siliphos) in patients with advanced hepatocellular carcinoma (HCC) and hepatic dysfunction. EXPERIMENTAL DESIGN: Patients with advanced HCC not eligible for other therapies based on poor hepatic function were enrolled in a phase I study of silybin phosphatidylcholine. A standard phase I design was used with 4 planned cohorts, dose escalating from 2, 4, 8, to 12 g per day in divided doses for 12 weeks. RESULTS: Three participants enrolled in this single institution trial. All enrolled subjects consumed 2 g per day of study agent in divided doses. Serum concentrations of silibinin and silibinin glucuronide increased within 1 to 3 weeks. In all 3 patients, liver function abnormalities and tumor marker α-fetoprotein progressed, but after day 56 the third patient showed some improvement in liver function abnormalities and inflammatory biomarkers. All 3 participants died within 23 to 69 days of enrolling into the trial, likely from hepatic failure, but it could not be ruled out that deaths were possibly due to the study drug. CONCLUSION: Short-term administration of silybin phosphatidylcholine in patients with advanced HCC resulted in detectable increases in silibinin and its metabolite, silibinin glucuronide. The maximum tolerated dose could not be established. Since patients died soon after enrollment, this patient population may have been too ill to benefit from an intervention designed to improve liver function tests.

Neuroprotective efficacy and pharmacokinetic behavior of novel anti-inflammatory para-phenyl substituted diindolylmethanes in a mouse model of Parkinson's disease.

There are currently no registered drugs that slow the progression of neurodegenerative diseases, in part because translation from animal models to the clinic has been hampered by poor distribution to the brain. The present studies examined a selected series of para-phenyl-substituted diindolylmethane (C-DIM) compounds that display anti-inflammatory and neuroprotective efficacy in vitro. We postulated that the pharmacokinetic behavior of C-DIM compounds after oral administration would correlate with neuroprotective efficacy in vivo in a mouse model of Parkinson's disease. Pharmacokinetics and metabolism of 1,1-bis(3'-indolyl)-1-(p-methoxyphenyl)methane (C-DIM5), 1,1-bis(3'-indolyl)-1-(phenyl)methane, 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (C-DIM8), and 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (C-DIM12) were determined in plasma and brain of C57Bl/6 mice after oral and intravenous administration at 10 and 1 mg/Kg, respectively. Putative metabolites were measured in plasma, liver, and urine. C-DIM compounds given orally displayed the highest area under the curve, Cmax, and Tmax levels, and C-DIM12 exhibited the most favorable pharmacokinetics of the compounds tested. Oral bioavailability of each compound ranged from 6% (C-DIM8) to 42% (C-DIM12). After pharmacokinetic studies, the neuroprotective efficacy of C-DIM5, C-DIM8, and C-DIM12 (50 mg/Kg per oral) was examined in mice exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid for 14 days, a model of progressive neurodegeneration with a strong neuroinflammatory component. C-DIM5 and C-DIM12 given orally once daily after one week of exposure to MPTP and probenecid prevented further loss of dopaminergic neurons in the substantia nigra pars compacta and striatal dopamine terminals, indicating that these compounds could be effective therapeutic agents to prevent neurodegeneration.