Publications by authors named "Dominique Ramirez"

8 Publications

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Use of in vitro assays to identify antibiotics that are cytotoxic to normal equine chondrocytes and synovial cells.

Equine Vet J 2020 Jun 16. Epub 2020 Jun 16.

Translational Medicine Institute, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.

Background: Intra-articular (IA) antibiotic usage is prevalent in equine practice. However, recent emergence of antimicrobial resistance prompts re-evaluation of antibiotic selection, particularly when used prophylactically. Furthermore, many commonly used antibiotics exert direct cytotoxicity to equine cells, and appropriate IA doses have not been defined.

Objectives: To screen antibiotics in vitro as an initial assessment of cytotoxicity against normal equine joint cells in monolayer culture and explant tissues.

Study Design: In vitro experimental study.

Methods: Chondrocytes and synovial cells were harvested from three horses and plated on 24-well plates (100 000 cells/wells in triplicate) for 48 hours prior to addition of antibiotics. Joint cells were exposed to antibiotics (n = 15) at various doses (25-0.39 mg/mL in complete DMEM media) for 24 hours and viability was assessed by trypan blue dye exclusion. The half maximal inhibitory concentration (IC50) was determined for each antibiotic. Cartilage explants were obtained from 3 horses, minced and exposed to antibiotics (n = 5) for 72 hours. Live/dead staining was performed, and fluorescence was visualised using Olympus IX83 spinning disk confocal microscope. Percentage of live vs dead cells was quantified.

Results: Antibiotics from different antimicrobial classes expressed dose-dependent but variable cytotoxicity to equine joint cells in vitro. Aminoglycosides and doxycycline had the lowest IC50 (most toxic). Ampicillin sulbactam, imipenem, tobramycin, ceftiofur sodium and amoxicillin had IC50 > 25 mg/mL for at least one cell line, representing potentially less cytotoxic alternatives.

Main Limitations: Further studies are necessary to extrapolate these in vitro data results to the in vivo joint environment.

Conclusions: Targeted IA antibiotic therapy would involve selection of the safest antibiotics (highest IC50) with efficacy based on bacterial culture/sensitivity. Antimicrobial selection and evidence-based dosing may minimise damage to native articular cartilage and synovial cells and development of antimicrobial resistance when IA antibiotics are used in equine practice.
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http://dx.doi.org/10.1111/evj.13314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738387PMC
June 2020

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

Mol Cancer Ther 2020 08 4;19(8):1598-1612. Epub 2020 Jun 4.

The Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, The University of Colorado Anschutz Medical Campus, Aurora, Colorado.

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 pharmacokinetics and pharmacodynamics. Lead CHD1L inhibitors display potent 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.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665848PMC
August 2020

Identification of a Small-Molecule Inhibitor That Disrupts the SIX1/EYA2 Complex, EMT, and Metastasis.

Cancer Res 2020 06 27;80(12):2689-2702. Epub 2020 Apr 27.

Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFβ signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFβ signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis. SIGNIFICANCE: These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510951PMC
June 2020

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

J Med Chem 2019 11 18;62(22):10182-10203. Epub 2019 Nov 18.

Clinical Sciences, School of Biomedical Engineering , Colorado State University , Fort Collins , Colorado 80523 , United States.

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.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723234PMC
November 2019

Doxorubicin area under the curve is an important predictor of neutropenia in dogs with naturally occurring cancers.

Vet Comp Oncol 2019 Jun 4;17(2):147-154. Epub 2019 Feb 4.

Flint Animal Cancer Center, College of Veterinary Medicine and Biological Sciences, Colorado State University, Fort Collins, Colorado.

Doxorubicin (DOX) area-under-the-curve (AUC) was calculated for 40 dogs with spontaneously occurring cancers using a previously validated limited-sampling approach. All dogs were administered a dose of 30 mg/m by intravenous infusion and serum samples were collected at 5, 45 and 60 minutes post-infusion. DOX and its major metabolite, doxorubicinol (doxol), were quantified in serum samples using high-performance liquid chromatography tandem-mass spectrometry. Wide interpatient variability was observed in the predicted DOX AUC with a coefficient of variation of 34%. A significant relationship was found between DOX AUC and absolute white blood cell count (P = 0.003), absolute neutrophil count (ANC; P = 0.002) and surviving fraction of neutrophils (P = 0.03) approximately 1 week after dosing (nadir). No changes in other hematologic parameters (red blood cells, platelets, lymphocytes, haemoglobin) were found to correlate with DOX AUC. The absolute dose (mg) and the dose per unit body weight (mg/kg) were not significantly correlated with nadir ANC. No relationships were found between maximum serum doxol concentration and myelosuppression. Baseline ANC was also significantly correlated to nadir ANC and a model was constructed using baseline ANC and DOX AUC that significantly described the nadir ANC. These findings demonstrate the important relationship between systemic DOX exposure and degree of neutropenia in dogs, and suggest a potential for individualized, pharmacokinetically-guided DOX dosing in dogs.
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http://dx.doi.org/10.1111/vco.12455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478528PMC
June 2019

Kinetics of Cyclophosphamide Metabolism in Humans, Dogs, Cats, and Mice and Relationship to Cytotoxic Activity and Pharmacokinetics.

Drug Metab Dispos 2019 03 19;47(3):257-268. Epub 2018 Dec 19.

Department of Clinical Sciences (D.A.R., A.E.A., D.L.G.) and School of Biomedical Engineering (K.P.C., K.A.C., D.L.G.), Colorado State University, Fort Collins, and University of Colorado Cancer Center, Aurora (D.L.G.), Colorado.

Cyclophosphamide (CP), a prodrug that is enzymatically converted to the cytotoxic 4-hydroxycyclophosphamide (4OHCP) by hepatic enzymes, is commonly used in both human and veterinary medicine to treat cancers and modulate the immune system. We investigated the metabolism of CP in humans, dogs, cats, and mice using liver microsomes; apparent , , and intrinsic clearance (/) parameters were estimated. The interspecies and intraspecies variations in kinetics were vast. Dog microsomes were, on average, 55-fold more efficient than human microsomes, 2.8-fold more efficient than cat microsomes, and 1.2-fold more efficient than mouse microsomes at catalyzing CP bioactivation. These differences translated to cell-based systems. Breast cancer cells exposed to 4OHCP via CP bioactivation by microsomes resulted in a stratification of cytotoxicity that was dependent on the species of microsomes measured by IC: dog (31.65 M), mouse (44.95 M), cat (272.6 M), and human (1857 M). The contributions of cytochrome P450s, specifically, CYP2B, CYP2C, and CYP3A, to CP bioactivation were examined: CYP3A inhibition resulted in no change in 4OHCP formation; CYP2B inhibition slightly reduced 4OHCP in humans, cats, and mice; and CYP2C inhibition drastically reduced 4OHCP formation in each species. Semiphysiologic modeling of CP metabolism using scaled metabolic parameters resulted in simulated data that closely matched published pharmacokinetic profiles, determined by noncompartmental analysis. The results highlight differential CP metabolism delineated by species and demonstrate the importance of metabolism on CP clearance.
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http://dx.doi.org/10.1124/dmd.118.083766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939680PMC
March 2019

Simultaneous absolute quantitation of ATP-binding cassette transporters in normal dog tissues by signature peptide analysis using a LC/MS/MS method.

Res Vet Sci 2019 Feb 12;122:93-101. Epub 2018 Nov 12.

Department of Clinical Sciences, Flint Animal Cancer Center, Colorado State University, 300 West Drake Road, Fort Collins, Colorado 80525, United States.

Membrane transport proteins are fundamental components of blood-tissue barriers and affect the absorption, distribution and elimination, and interactions of many of the drugs commonly used in veterinary medicine. A quantitative, simultaneous measurement of these proteins across dog tissues is not currently available, nor is it possible with current immune-based assays such as western blot. In the present study, we aimed to develop a sensitive and specific liquid chromatography tandem-mass spectrometry (LC/MS/MS) based quantitation method that can simultaneously quantitate 14 ATP-binding cassette transporters. We applied this method to a panel of normal canine tissues and compared the LC/MS/MS results with relative messenger RNA (mRNA) abundance using quantitative real-time polymerase chain reaction (qRT-PCR). Our LC/MS/MS method is sensitive, with lower limits of quantitation ranging from 5 to 10 fmol/μg of protein. We were able to detect and/or quantitate each of the 14 transporters in at least one normal dog tissue. Relative protein and mRNA abundance within tissues did not demonstrate a significant correlation in all cases. The results presented here will provide for more accurate predictions of drug movement in dogs through incorporation into physiologically based pharmacokinetic (PBPK) models; the method described here has wide applicability to the quantitation of virtually any proteins of interest in biologic samples where validated canine antibodies do not exist.
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http://dx.doi.org/10.1016/j.rvsc.2018.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522139PMC
February 2019

In vivo and in vitro assessment of mirtazapine pharmacokinetics in cats with liver disease.

J Vet Intern Med 2018 Nov 11;32(6):1951-1957. Epub 2018 Oct 11.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, 300 West Drake Road, Fort Collins, Colorado.

Background: Liver disease (LD) prolongs mirtazapine half-life in humans, but it is unknown if this occurs in cats with LD and healthy cats.

Hypothesis/objectives: To determine pharmacokinetics of administered orally mirtazapine in vivo and in vitro (liver microsomes) in cats with LD and healthy cats.

Animals: Eleven LD and 11 age-matched control cats.

Methods: Case-control study. Serum was obtained 1 and 4 hours (22 cats) and 24 hours (14 cats) after oral administration of 1.88 mg mirtazapine. Mirtazapine concentrations were measured by liquid chromatography with tandem mass spectrometry. Drug exposure and half-life were predicted using limited sampling modeling and estimated using noncompartmental methods. in vitro mirtazapine pharmacokinetics were assessed using liver microsomes from 3 LD cats and 4 cats without LD.

Results: There was a significant difference in time to maximum serum concentration between LD cats and control cats (median [range]: 4 [1-4] hours versus 1 [1-4] hours; P = .03). The calculated half-life of LD cats was significantly prolonged compared to controls (median [range]: 13.8 [7.9-61.4] hours versus 7.4 [6.7-9.1] hours; P < .002). Mirtazapine half-life was correlated with ALT (P = .002; r = .76), ALP (P < .0001; r = .89), and total bilirubin (P = .0008; r = .81). The rate of loss of mirtazapine was significantly different between microsomes of LD cats (-0.0022 min , CI: -0.0050 to 0.00054 min ) and cats without LD (0.01849 min , CI: -0.025 to -0.012 min ; P = .002).

Conclusions And Clinical Importance: Cats with LD might require less frequent administration of mirtazapine than normal cats.
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http://dx.doi.org/10.1111/jvim.15237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272035PMC
November 2018