Publications by authors named "Daniel L Gustafson"

99 Publications

A randomized phase II study of coexpression extrapolation (COXEN) with neoadjuvant chemotherapy for bladder cancer (SWOG S1314; NCT02177695).

Clin Cancer Res 2021 Feb 10. Epub 2021 Feb 10.

Administration, CHRISTUS Santa Rosa Hospital - Medical Center.

Purpose: Dose-dense Methotrexate-Vinblastine-Adriamycin-Cisplatin (ddMVAC) and Gemcitabine-Cisplatin (GC) are accepted neoadjuvant regimens for muscle-invasive bladder cancer (BC). The aim of this study was to validate the score from a Coexpression extrapolation (COXEN) algorithm-generated gene expression model (GEM) as a biomarker in patients undergoing radical cystectomy.

Experimental Design: Eligibility included cT2-T4a N0 M0, urothelial BC, {greater than or equal to} 5 mm of viable tumor, cisplatin eligible, with plan for cystectomy; 237 patients were randomized between ddMVAC, given every 14 days for 4 cycles, and GC, given every 21 days for 4 cycles. The primary objective assessed pre-specified dichotomous treatment specific COXEN score as predictive of pT0 rate or {less than or equal to} pT1 (downstaging) at surgery.

Results: Among 167 evaluable patients, the odds ratio for pT0 with the GC GEM score in GC-treated patients was 2.63 (p=0.10; 95% CI [0.82,8.36]); for the ddMVAC COXEN GEM score with ddMVAC treatment, the OR was 1.12 (p=0.82, 95% CI [0.42, 2.95]). The GC GEM score was applied to pooled arms (GC and ddMVAC) for downstaging with an OR of 2.33 (p=0.02; 95% CI [1.11, 4.89]). In an intention to treat analysis of eligible patients (n=227), pT0 rates for ddMVAC and GC were 28% and 30% (p = 0.75); downstaging was 47% and 40% (p = 0.27), respectively.

Conclusions: Treatment-specific COXEN scores were not significantly predictive for response to individual chemotherapy treatment. The COXEN GEM GC score was significantly associated with downstaging in the pooled arms. Additional biomarker development is planned.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2409DOI Listing
February 2021

Predicting chemosensitivity using drug perturbed gene dynamics.

BMC Bioinformatics 2021 Jan 7;22(1):15. Epub 2021 Jan 7.

School of Biomedical Engineering, Colorado State University, Fort Collins, CO, USA.

Background: One of the current directions of precision medicine is the use of computational methods to aid in the diagnosis, prognosis, and treatment of disease based on data driven approaches. For instance, in oncology, there has been a particular focus on development of algorithms and biomarkers that can be used for pre-clinical and clinical applications. In particular large-scale omics-based models to predict drug sensitivity in in vitro cancer cell line panels have been used to explore the utility and aid in the development of these models as clinical tools. Additionally, a number of web-based interfaces have been constructed for researchers to explore the potential of drug perturbed gene expression as biomarkers including the NCI Transcriptional Pharmacodynamic Workbench. In this paper we explore the influence of drug perturbed gene dynamics of the NCI Transcriptional Pharmacodynamics Workbench in computational models to predict in vitro drug sensitivity for 15 drugs on the NCI60 cell line panel.

Results: This work presents three main findings. First, our models show that gene expression profiles that capture changes in gene expression after 24 h of exposure to a high concentration of drug generates the most accurate predictive models compared to the expression profiles under different dosing conditions. Second, signatures of 100 genes are developed for different gene expression profiles; furthermore, when the gene signatures are applied across gene expression profiles model performance is substantially decreased when gene signatures developed using changes in gene expression are applied to non-drugged gene expression. Lastly, we show that the gene interaction networks developed on these signatures show different network topologies and can be used to inform selection of cancer relevant genes.

Conclusion: Our models suggest that perturbed gene signatures are predictive of drug response, but cannot be applied to predict drug response using unperturbed gene expression. Furthermore, additional drug perturbed gene expression measurements in in vitro cell lines could generate more predictive models; but, more importantly be used in conjunction with computational methods to discover important drug disease relationships.
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http://dx.doi.org/10.1186/s12859-020-03947-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789515PMC
January 2021

Pharmacokinetics of a sulfadiazine and trimethoprim suspension in neonatal foals.

J Vet Pharmacol Ther 2020 Dec 1. Epub 2020 Dec 1.

Department of Clinical Sciences, James L. Voss Veterinary Teaching Hospital, Colorado State University, Fort Collins, CO, USA.

There is limited investigation of neonatal foal pharmacokinetic parameters for the antimicrobial combination of sulfadiazine (SDZ) and trimethoprim (TMP). Neonatal pharmacokinetic investigation of the sulfadiazine-trimethoprim combination is required to ensure safe and effective utilization in this population. The purpose of this study was to determine the pharmacokinetics of sulfadiazine-trimethoprim in five healthy neonatal foals with oral administration at 24 mg/kg every 12 hr (hrs) for 10 days. Blood samples were collected at serial time points at approximately 72 hr of age (steady-state) and at days 5 and 10 to monitor the influence of age within the neonatal period. Pharmacokinetic parameters were determined using a one-compartment model analysis, and mean ± SD was calculated. C was 37.8 ± 13.4 μg/ml (SDZ) and 1.92 ± 0.25 μg/ml (TMP). T was 1.4 ± 0.6 hr (SDZ) and 1.4 ± 0.4 hr (TMP). C for SDZ and TMP was 16.84 ± 8.46 μg/ml and 0.46 ± 0.24 μg/ml, respectively. Elimination half-life was 10.8 ± 6.1 hr (SDZ) and 6.5 ± 2 hr (TMP). AUC was 667 ± 424 μg × hr/ml (SDZ) and 21.1 ± 5.3 μg × hr/ml (TMP). Foals remained healthy, and the plasma concentration of sulfadiazine-trimethoprim reached levels above MIC for Streptococcus equi ssp. (SDZ/TMP): 9.5/0.5 μg/ml).
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http://dx.doi.org/10.1111/jvp.12930DOI Listing
December 2020

Lysosomal Biogenesis and Implications for Hydroxychloroquine Disposition.

J Pharmacol Exp Ther 2021 Feb 10;376(2):294-305. Epub 2020 Nov 10.

Colorado State University, School of Biomedical Engineering (K.P.C., S.W., D.L.G.) and Department of Clinical Sciences (D.L.G., J.W.C.), Colorado State University, Fort Collins, Colorado; University of Colorado Cancer Center, Anschutz Medical Campus, Aurora, Colorado (D.L.G.); and University of Akron, Department of Chemistry, Akron, Ohio (Y.P.)

Lysosomes act as a cellular drug sink for weakly basic, lipophilic (lysosomotropic) xenobiotics, with many instances of lysosomal trapping associated with multiple drug resistance. Lysosomotropic agents have also been shown to activate master lysosomal biogenesis transcription factor EB (TFEB) and ultimately lysosomal biogenesis. We investigated the role of lysosomal biogenesis in the disposition of hydroxychloroquine (HCQ), a hallmark lysosomotropic agent, and observed that modulating the lysosomal volume of human breast cancer cell lines can account for differences in disposition of HCQ. Through use of an in vitro pharmacokinetic (PK) model, we characterized total cellular uptake of HCQ within the duration of static equilibrium (1 hour), as well as extended exposure to HCQ that is subject to dynamic equilibrium (>1 hour), wherein HCQ increases the size of the lysosomal compartment through swelling and TFEB-induced lysosomal biogenesis. In addition, we observe that pretreatment of cell lines with TFEB-activating agent Torin1 contributed to an increase of whole-cell HCQ concentrations by 1.4- to 1.6-fold, which were also characterized by the in vitro PK model. This investigation into the role of lysosomal volume dynamics in lysosomotropic drug disposition, including the ability of HCQ to modify its own disposition, advances our understanding of how chemically similar agents may distribute on the cellular level and examines a key area of lysosomal-mediated multiple drug resistance and drug-drug interaction. SIGNIFICANCE STATEMENT: Hydroxychloroquine is able to modulate its own cellular pharmacokinetic uptake by increasing the cellular lysosomal volume fraction through activation of lysosomal biogenesis master transcription factor EB and through lysosomal swelling. This concept can be applied to many other lysosomotropic drugs that activate transcription factor EB, such as doxorubicin and other tyrosine kinase inhibitor drugs, as these drugs may actively increase their own sequestration within the lysosome to further exacerbate multiple drug resistance and lead to potential acquired resistance.
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http://dx.doi.org/10.1124/jpet.120.000309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7841421PMC
February 2021

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

Preclinical and Dose-Finding Phase I Trial Results of Combined Treatment with a TORC1/2 Inhibitor (TAK-228) and Aurora A Kinase Inhibitor (Alisertib) in Solid Tumors.

Clin Cancer Res 2020 Sep 15;26(17):4633-4642. Epub 2020 May 15.

University of Colorado Cancer Center, Aurora, Colorado.

Purpose: The purpose of this study was to evaluate the rational combination of TORC1/2 inhibitor TAK-228 and Aurora A kinase inhibitor alisertib in preclinical models of triple-negative breast cancer (TNBC) and to conduct a phase I dose escalation trial in patients with advanced solid tumors.

Experimental Design: TNBC cell lines and patient-derived xenograft (PDX) models were treated with alisertib, TAK-228, or the combination and evaluated for changes in proliferation, cell cycle, mTOR pathway modulation, and terminal cellular fate, including apoptosis and senescence. A phase I clinical trial was conducted in patients with advanced solid tumors treated with escalating doses of alisertib and TAK-228 using a 3+3 design to determine the maximum tolerated dose (MTD).

Results: The combination of TAK-228 and alisertib resulted in decreased proliferation and cell-cycle arrest in TNBC cell lines. Treatment of TNBC PDX models resulted in significant tumor growth inhibition and increased apoptosis with the combination. In the phase I dose escalation study, 18 patients with refractory solid tumors were enrolled. The MTD was alisertib 30 mg b.i.d. days 1 to 7 of a 21-day cycle and TAK-228 2 mg daily, continuous dosing. The most common treatment-related adverse events were neutropenia, fatigue, nausea, rash, mucositis, and alopecia.

Conclusions: The addition of TAK-228 to alisertib potentiates the antitumor activity of alisertib , resulting in increased cell death and apoptosis. The combination is tolerable in patients with advanced solid tumors and should be evaluated further in expansion cohorts with additional pharmacodynamic assessment.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864382PMC
September 2020

Dose-Escalation and Pharmacokinetic Study Following a Single Dose of Oxaliplatin in Cancer-Bearing Dogs.

J Am Anim Hosp Assoc 2020 Jul/Aug;56(4):206-214. Epub 2020 May 15.

From the Department of Small Animal Clinical Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia (S.K., N.D., J.A.); and Clinical Sciences Department, Pharmacology and Biomedical Engineering, Colorado State University, Fort Collins, Colorado (D.L.G.).

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http://dx.doi.org/10.5326/JAAHA-MS-7007DOI Listing
May 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

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

J Feline Med Surg 2020 08 29;22(8):729-735. Epub 2019 Oct 29.

Department of Clinical Sciences, Colorado State University, Fort Collins, CO, USA.

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 ( = 0.02). More owners of azCKD cats reported that an alteration in treatment plan was necessitated by side effects (55% vs 12%;  = 0.008). Urine amoxicillin was significantly lower in cats with azCKD ( = 0.01) and serum amoxicillin trended toward significance ( = 0.07). Serum amoxicillin concentration was positively correlated with serum creatinine ( = 0.02;  = 0.62) and urine amoxicillin concentration was negatively correlated with serum creatinine ( = 0.01;  = -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.
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http://dx.doi.org/10.1177/1098612X19881537DOI Listing
August 2020

The pharmacokinetics of cytarabine administered at three distinct subcutaneous dosing protocols in dogs with meningoencephalomyelitis of unknown origin.

J Vet Pharmacol Ther 2019 Nov 6;42(6):588-592. Epub 2019 Sep 6.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO.

The objective of this study was to evaluate the pharmacokinetics of the standard cytarabine (Ara-C) protocol (50 mg/m subcutaneously every 12 hr for 2 days) used for dogs with neuroinflammatory disease and compare it to two more practical protocols (a single 200 mg/m subcutaneous dose and two 100 mg/m subcutaneous doses every 12 hr). Four client-owned dogs previously diagnosed with meningoencephalomyelitis of unknown origin were administered three distinct Ara-C protocols with a 21-day washout between each protocol. A complete blood count was performed seven days after each dosing protocol to assess for clinically relevant myelosuppression. No adverse events were observed. Plasma Ara-C concentrations were measured using a validated liquid chromatography coupled to tandem mass spectrometry assay. The mean maximal concentrations in this study were 4,230, 9,293, and 16,675 ng/ml for a single dose of 50, 100, and 200 mg/m , respectively. There was a linear relationship between dose and drug exposure. Drug exposure was similar regardless of the dosing protocol when the total dose was analyzed, with an area under the concentration versus time curve of 37,026, 38,465, and 32,510 ng × hr/ml for 50, 100, and 200 mg/m , respectively.
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http://dx.doi.org/10.1111/jvp.12809DOI Listing
November 2019

Drug dose and drug choice: Optimizing medical therapy for veterinary cancer.

Vet Comp Oncol 2020 Jun 12;18(2):143-151. Epub 2019 Sep 12.

Flint Animal Cancer Center, Colorado State University, Fort Collins, Colorado.

Although novel agents hold great promise for the treatment of animal neoplasia, there may be room for significant improvement in the use of currently available agents. These improvements include altered dosing schemes, novel combinations, and patient-specific dosing or selection of agents. Previous studies have identified surrogates for "individualized dose intensity,", for example, patient size, development of adverse effects, and pharmacokinetic parameters, as potential indicators of treatment efficacy in canine lymphoma, and strategies for patient-specific dose escalation are discussed. Strategies for treatment selection in individual patients include conventional histopathology, protein-based target assessment (eg, flow cytometry, immunohistochemistry, and mass spectrometry), and gene-based target assessment (gene expression profiling and targeted or global sequencing strategies). Currently available data in animal cancer evaluating these strategies are reviewed, as well as ongoing studies and suggestions for future directions.
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http://dx.doi.org/10.1111/vco.12537DOI Listing
June 2020

Cancer Cells Upregulate NRF2 Signaling to Adapt to Autophagy Inhibition.

Dev Cell 2019 09 1;50(6):690-703.e6. Epub 2019 Aug 1.

Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA. Electronic address:

While autophagy is thought to be an essential process in some cancer cells, it is unknown if or how such cancer cells can circumvent autophagy inhibition. To address this, we developed a CRISPR/Cas9 assay with dynamic live-cell imaging to measure acute effects of knockout (KO) of autophagy genes compared to known essential and non-essential genes. In some cancer cells, autophagy is as essential for cancer cell growth as mRNA transcription or translation or DNA replication. However, even these highly autophagy-dependent cancer cells evolve to circumvent loss of autophagy by upregulating NRF2, which is necessary and sufficient for autophagy-dependent cells to circumvent ATG7 KO and maintain protein homeostasis. Importantly, however, this adaptation increases susceptibly to proteasome inhibitors. These studies identify a common mechanism of acquired resistance to autophagy inhibition and show that selection to avoid tumor cell dependency on autophagy creates new, potentially actionable cancer cell susceptibilities.
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http://dx.doi.org/10.1016/j.devcel.2019.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7233142PMC
September 2019

A systematic analysis of genomics-based modeling approaches for prediction of drug response to cytotoxic chemotherapies.

BMC Med Genomics 2019 06 17;12(1):87. Epub 2019 Jun 17.

School of Biomedical Engineering, Colorado State University, Fort Collins, 80523, CO, USA.

Background: The availability and generation of large amounts of genomic data has led to the development of a new paradigm in cancer treatment emphasizing a precision approach at the molecular and genomic level. Statistical modeling techniques aimed at leveraging broad scale in vitro, in vivo, and clinical data for precision drug treatment has become an active area of research. As a rapidly developing discipline at the crossroads of medicine, computer science, and mathematics, techniques ranging from accepted to those on the cutting edge of artificial intelligence have been utilized. Given the diversity and complexity of these techniques a systematic understanding of fundamental modeling principles is essential to contextualize influential factors to better understand results and develop new approaches.

Methods: Using data available from the Genomics of Drug Sensitivity in Cancer (GDSC) and the NCI60 we explore principle components regression, linear and non-linear support vector regression, and artificial neural networks in combination with different implementations of correlation based feature selection (CBF) on the prediction of drug response for several cytotoxic chemotherapeutic agents.

Results: Our results indicate that the regression method and features used have marginal effects on Spearman correlation between the predicted and measured values as well as prediction error. Detailed analysis of these results reveal that the bulk relationship between tissue of origin and drug response is a major driving factor in model performance.

Conclusion: These results display one of the challenges in building predictive models for drug response in pan-cancer models. Mainly, that bulk genotypic traits where the signal to noise ratio is high is the dominant behavior captured in these models. This suggests that improved techniques of feature selection that can discriminate individual cell response from histotype response will yield more successful pan-cancer models.
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http://dx.doi.org/10.1186/s12920-019-0519-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580596PMC
June 2019

Identifying Candidate Druggable Targets in Canine Cancer Cell Lines Using Whole-Exome Sequencing.

Mol Cancer Ther 2019 08 7;18(8):1460-1471. Epub 2019 Jun 7.

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

Cancer cell culture has been a backbone in cancer research, in which analysis of human cell line mutational profiles often correlates with oncogene addiction and drug sensitivity. We have conducted whole-exome sequence analyses on 33 canine cancer cell lines from 10 cancer types to identify somatic variants that contribute to pathogenesis and therapeutic sensitivity. A total of 66,344 somatic variants were identified. Mutational load ranged from 15.79 to 129.37 per Mb, and 13.2% of variants were located in protein-coding regions (PCR) of 5,085 genes. PCR somatic variants were identified in 232 genes listed in the Cancer Gene Census (COSMIC). Cross-referencing variants with human driving mutations on cBioPortal identified 61 variants as candidate cancer drivers in 30 cell lines. The most frequently mutated cancer driver was TP53 (15 mutations in 12 cell lines). No drivers were identified in three cell lines. We identified 501 non-COSMIC genes with PCR variants that functionally annotate with COSMIC genes. These genes frequently mapped to the KEGG MAPK and PI3K-AKT pathways. We evaluated the cell lines for ERK1/2 and AKT(S473) phosphorylation and sensitivity to the MEK1/2 inhibitor, trametinib. Twelve of the 33 cell lines were trametinib-sensitive (IC < 32 nmol/L), all 12 exhibited constitutive or serum-activated ERK1/2 phosphorylation, and 8 carried MAPK pathway cancer driver variants: NF1(2), BRAF(3), N/KRAS(3). This functionally annotated database of canine cell line variants will inform hypothesis-driven preclinical research to support the use of companion animals in clinical trials to test novel combination therapies.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-1346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679748PMC
August 2019

Assessment of compounded transdermal mirtazapine as an appetite stimulant in cats with chronic kidney disease.

J Feline Med Surg 2020 04 4;22(4):376-383. Epub 2019 Jun 4.

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

Objectives: The aim of this study was to assess the appetite stimulation properties of compounded transdermal mirtazapine (CTM) in cats with chronic kidney disease (CKD).

Methods: Two sequential double-blind placebo-controlled crossover prospective studies were performed in client-owned cats with stable stage 2 or 3 CKD and a history of decreased appetite. In the first study nine CKD cats were randomized to receive 3.75 mg/0.1 ml CTM gel or placebo on the inner pinna every other day for 3 weeks, then, after a 4 day washout period, the cats were crossed over to the alternate 3 week treatment. In a second study, 10 CKD cats were randomized to receive 1.88 mg/0.1 ml CTM or placebo on the same schedule. Physical examination and serum biochemistry were performed before and after each treatment period, and owners kept daily logs of appetite, activity and eating behaviors. Mirtazapine concentrations in CTM gels and steady-state mirtazapine serum concentrations were measured using liquid chromatography/tandem mass spectrometry.

Results: Administration of both 3.75 mg and 1.88 mg CTM resulted in a statistically significant increase in weight ( = 0.002 for both), increase in appetite ( = 0.01 and = 0.005, respectively), and increase in rate of food consumption ( = 0.03 and = 0.008, respectively). No significant difference in activity or vocalization was seen at either dose; however, individual cats experienced excessive meowing. Median weight increase for the 3.75 mg arm was 0.22 kg (range 0.04-0.44 kg), while median weight increase for the 1.88 mg arm was 0.26 kg (range -0.25 to 0.5 kg). Improvement in body condition score was seen in 5/9 cats in the 3.75 mg arm (P = 0.04) and 6/10 cats in the 1.88 mg arm (P = 0.004).

Conclusions And Relevance: CTM increased appetite and resulted in weight gain in CKD cats despite significant inconsistencies in compounding, and may benefit cats in countries where an approved product is not available.
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http://dx.doi.org/10.1177/1098612X19851303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099811PMC
April 2020

Randomized blinded controlled clinical trial to assess the effect of oral cannabidiol administration in addition to conventional antiepileptic treatment on seizure frequency in dogs with intractable idiopathic epilepsy.

J Am Vet Med Assoc 2019 Jun;254(11):1301-1308

Objective: To assess the effect of oral cannabidiol (CBD) administration in addition to conventional antiepileptic treatment on seizure frequency in dogs with idiopathic epilepsy.

Design: Randomized blinded controlled clinical trial.

Animals: 26 client-owned dogs with intractable idiopathic epilepsy.

Procedures: Dogs were randomly assigned to a CBD (n = 12) or placebo (14) group. The CBD group received CBD-infused oil (2.5 mg/kg [1.1 mg/lb], PO) twice daily for 12 weeks in addition to existing antiepileptic treatments, and the placebo group received noninfused oil under the same conditions. Seizure activity, adverse effects, and plasma CBD concentrations were compared between groups.

Results: 2 dogs in the CBD group developed ataxia and were withdrawn from the study. After other exclusions, 9 dogs in the CBD group and 7 in the placebo group were included in the analysis. Dogs in the CBD group had a significant (median change, 33%) reduction in seizure frequency, compared with the placebo group. However, the proportion of dogs considered responders to treatment (≥ 50% decrease in seizure activity) was similar between groups. Plasma CBD concentrations were correlated with reduction in seizure frequency. Dogs in the CBD group had a significant increase in serum alkaline phosphatase activity. No adverse behavioral effects were reported by owners.

Conclusions And Clinical Relevance: Although a significant reduction in seizure frequency was achieved for dogs in the CBD group, the proportion of responders was similar between groups. Given the correlation between plasma CBD concentration and seizure frequency, additional research is warranted to determine whether a higher dosage of CBD would be effective in reducing seizure activity by ≥ 50%.
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http://dx.doi.org/10.2460/javma.254.11.1301DOI Listing
June 2019

Identifying the ErbB/MAPK Signaling Cascade as a Therapeutic Target in Canine Bladder Cancer.

Mol Pharmacol 2019 07 2;96(1):36-46. Epub 2019 May 2.

Flint Animal Cancer Center, Department of Clinical Sciences (K.E.C., B.G.H., D.L.G., D.L.D.), and Cell and Molecular Biology Graduate Program (K.E.C., D.L.G., D.L.D.), Colorado State University, Fort Collins, Colorado; and University of Colorado Cancer Center, Aurora, Colorado (D.L.G., D.L.D.)

Transitional cell carcinoma (TCC) of the bladder comprises 2% of diagnosed canine cancers. TCC tumors are generally inoperable and unresponsive to traditional chemotherapy, indicating a need for more effective therapies. BRAF, a kinase in the mitogen-activated protein kinase (MAPK) pathway, is mutated in 70% of canine TCCs. In this study, we use BRAF mutant and wild-type TCC cell lines to characterize the role of BRAF mutations in TCC pathogenesis and assess the efficacy of inhibition of the MAPK pathway alone and in combination with other gene targets as a treatment for canine TCC. Analysis of MAPK target gene expression and assessment of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation following serum starvation indicated constitutive MAPK activity in all TCC cell lines. BRAF mutant TCC cell lines were insensitive to the BRAF inhibitor vemurafenib, with IC values greater than 5 M, but exhibited greater sensitivity to a paradox-breaking BRAF inhibitor (IC: 0.2-1 M). All TCC cell lines had IC values less than 7 nM to the mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor trametinib independent of their BRAF mutation status. ERK1/2 phosphorylation decreased after 6-hour treatments with MAPK inhibitors, but rebounded by 24 hours, suggesting the presence of resistance mechanisms. Microarray analysis identified elevated expression of the ErbB family of receptors and ligands in TCC cell lines. The pan-ErbB inhibitor sapitinib synergized with BRAF inhibition in BRAF mutant Bliley TCC cells and synergized with MEK1/2 inhibition in Bliley and BRAF wild-type Kinsey cells. These findings suggest the potential for combined MAPK and ErbB receptor inhibition as a therapy for canine TCC. SIGNIFICANCE STATEMENT: The results of this study (1) identify a novel combination strategy for canine bladder cancer treatment: targeting the ErbB/MAPK signaling cascade and (2) establish the utility of canine bladder cancer as a naturally-occurring model for human MAPK-driven cancers.
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http://dx.doi.org/10.1124/mol.119.115808DOI Listing
July 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

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

J Vet Intern Med 2019 Mar 18;33(2):662-669. Epub 2018 Dec 18.

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

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.
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http://dx.doi.org/10.1111/jvim.15389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430892PMC
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

A phase II clinical trial of the Aurora and angiogenic kinase inhibitor ENMD-2076 for previously treated, advanced, or metastatic triple-negative breast cancer.

Breast Cancer Res 2018 08 2;20(1):82. Epub 2018 Aug 2.

Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, USA.

Background: Triple-negative breast cancer (TNBC) remains an aggressive breast cancer subtype with limited treatment options. ENMD-2076 is a small-molecule inhibitor of Aurora and angiogenic kinases with proapoptotic and antiproliferative activity in preclinical models of TNBC.

Methods: This dual-institution, single-arm, two-stage, phase II clinical trial enrolled patients with locally advanced or metastatic TNBC previously treated with one to three prior lines of chemotherapy in the advanced setting. Patients were treated with ENMD-2076 250 mg orally once daily with continuous dosing in 4-week cycles until disease progression or unacceptable toxicity occurred. The primary endpoint was 6-month clinical benefit rate (CBR), and secondary endpoints included progression-free survival, pharmacokinetic profile, safety, and biologic correlates in archival and fresh serial tumor biopsies in a subset of patients.

Results: Forty-one patients were enrolled. The 6-month CBR was 16.7% (95% CI, 6-32.8%) and included two partial responses. The 4-month CBR was 27.8% (95% CI, 14-45.2%), and the average duration of benefit was 6.5 cycles. Common adverse events included hypertension, fatigue, diarrhea, and nausea. Treatment with ENMD-2076 resulted in a decrease in cellular proliferation and microvessel density and an increase in p53 and p73 expression, consistent with preclinical observations.

Conclusions: Single-agent ENMD-2076 treatment resulted in partial response or clinical benefit lasting more than 6 months in 16.7% of patients with pretreated, advanced, or metastatic TNBC. These results support the development of predictive biomarkers using archival and fresh tumor tissue, as well as consideration of mechanism-based combination strategies.

Trial Registration: ClinicalTrials.gov, NCT01639248 . Registered on July 12, 2012.
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http://dx.doi.org/10.1186/s13058-018-1014-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090978PMC
August 2018

Phase Ib Results of the Rational Combination of Selumetinib and Cyclosporin A in Advanced Solid Tumors with an Expansion Cohort in Metastatic Colorectal Cancer.

Cancer Res 2018 09 24;78(18):5398-5407. Epub 2018 Jul 24.

University of Colorado, Denver, Colorado.

MEK inhibition is of interest in cancer drug development, but clinical activity in metastatic colorectal cancer (mCRC) has been limited. Preclinical studies demonstrated Wnt pathway overexpression in -mutant cell lines resistant to the MEK inhibitor, selumetinib. The combination of selumetinib and cyclosporin A, a noncanonical Wnt pathway modulator, demonstrated antitumor activity in mCRC patient-derived xenografts. To translate these results, we conducted a NCI Cancer Therapy Evaluation Program-approved multicenter phase I/IB trial (NCT02188264) of the combination of selumetinib and cyclosporin A. Patients with advanced solid malignancies were treated with the combination of oral selumetinib and cyclosporin A in the dose escalation phase, followed by an expansion cohort of irinotecan and oxaliplatin-refractory mCRC. The expansion cohort utilized a single-agent selumetinib "run-in" to evaluate FZD2 biomarker upregulation and -WT and -MT stratification to identify any potential predictors of efficacy. Twenty and 19 patients were enrolled in dose escalation and expansion phases, respectively. The most common adverse events and grade 3/4 toxicities were rash, hypertension, and edema. Three dose-limiting toxicities (grade 3 hypertension, rash, and increased creatinine) were reported. The MTD was selumetinib 75 mg twice daily and cyclosporin A 2 mg/kg twice daily on a 28-day cycle. stratification did not identify any differences in response between -WT and -MT cancers. Two partial responses, 18 stable disease, and 10 progressive disease responses were observed. Combination selumetinib and cyclosporin A is well tolerated, with evidence of activity in mCRC. Future strategies for concept development include identifying better predictors of efficacy and improved Wnt pathway modulation. These findings translate preclinical studies combining selumetinib and cyclosporin into a phase I first-in-human clinical trial of such a combination in patients with advanced solid malignancies. .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139073PMC
September 2018

Hydroxychloroquine: A Physiologically-Based Pharmacokinetic Model in the Context of Cancer-Related Autophagy Modulation.

J Pharmacol Exp Ther 2018 06 8;365(3):447-459. Epub 2018 Feb 8.

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

Hydroxychloroquine (HCQ) is a lysosomotropic autophagy inhibitor being used in over 50 clinical trials either alone or in combination with chemotherapy. Pharmacokinetic (PK) and pharmacodynamic (PD) studies with HCQ have shown that drug exposure in the blood does not correlate with autophagy inhibition in either peripheral blood mononuclear cells or tumor tissue. To better explain this PK/PD disconnect, a PBPK was developed for HCQ describing the tissue-specific absorption, distribution, metabolism, and excretion as well as lysosome-specific sequestration. Using physiologic and biochemical parameters derived from literature or obtained experimentally, the model was first developed and validated in mice, and then adapted to simulate human HCQ exposure in whole blood and urine through allometric scaling and species-specific parameter modification. The human model accurately simulated average steady-state concentrations (Css) of those observed in five different HCQ combination clinical trials across seven different doses, which was then expanded by comparison of the Css distribution in a virtual human population at this range of doses. Value of this model lies in its ability to simulate HCQ PK in patients while accounting for PK modification by combination treatment modalities, drug concentrations at the active site in the lysosome under varying pH conditions, and exposure in tissues where toxicity is observed.
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http://dx.doi.org/10.1124/jpet.117.245639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931434PMC
June 2018

Canine sarcomas as a surrogate for the human disease.

Pharmacol Ther 2018 08 9;188:80-96. Epub 2018 Mar 9.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA; Flint Animal Cancer Center, Colorado State University, Fort Collins, CO 80523, USA; University of Colorado Cancer Center, Anschutz Medical Campus, Aurora, CO 80045, USA.

Pet dogs are becoming increasingly recognized as a population with the potential to inform medical research through their treatment for a variety of maladies by veterinary health professionals. This is the basis of the One Health initiative, supporting the idea of collaboration between human and animal health researchers and clinicians to study spontaneous disease processes and treatment in animals to inform human health. Cancer is a major health burden in pet dogs, accounting for approximately 30% of deaths across breeds. As such, pet dogs with cancer are becoming increasingly recognized as a resource for studying the pharmacology and therapeutic potential of anticancer drugs and therapies under development. This was recently highlighted by a National Academy of Medicine Workshop on Comparative Oncology that took place in mid-2015 (http://www.nap.edu/21830). One component of cancer burden in dogs is their significantly higher incidence of sarcomas as compared to humans. This increased incidence led to canine osteosarcoma being an important component in the development of surgical approaches for osteosarcoma in children. Included in this review of sarcomas in dogs is a description of the incidence, pathology, molecular characteristics and previous translational therapeutic studies associated with these tumors. An understanding of the patho-physiological and molecular characteristics of these naturally occurring canine sarcomas holds great promise for effective incorporation into drug development schemas, for evaluation of target modulation or other pharmacodynamic measures associated with therapeutic response. These data could serve to supplement other preclinical data and bolster clinical investigations in tumor types for which there is a paucity of human patients for clinical trials.
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http://dx.doi.org/10.1016/j.pharmthera.2018.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6432917PMC
August 2018

Activation of the unfolded protein response in sarcoma cells treated with rapamycin or temsirolimus.

PLoS One 2017 19;12(9):e0185089. Epub 2017 Sep 19.

Tumor Metastasis Biology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

Activation of the unfolded protein response (UPR) in eukaryotic cells represents an evolutionarily conserved response to physiological stress. Here, we report that the mTOR inhibitors rapamycin (sirolimus) and structurally related temsirolimus are capable of inducing UPR in sarcoma cells. However, this effect appears to be distinct from the classical role for these drugs as mTOR inhibitors. Instead, we detected these compounds to be associated with ribosomes isolated from treated cells. Specifically, temsirolimus treatment resulted in protection from chemical modification of several rRNA residues previously shown to bind rapamycin in prokaryotic cells. As an application for these findings, we demonstrate maximum tumor cell growth inhibition occurring only at doses which induce UPR and which have been shown to be safely achieved in human patients. These results are significant because they challenge the paradigm for the use of these drugs as anticancer agents and reveal a connection to UPR, a conserved biological response that has been implicated in tumor growth and response to therapy. As a result, eIF2 alpha phosphorylation and Xbp-1 splicing may serve as useful biomarkers of treatment response in future clinical trials using rapamycin and rapalogs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185089PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605117PMC
October 2017

Pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in cats after oral, intravenous, and intraperitoneal administration of cyclophosphamide.

Am J Vet Res 2017 Jul;78(7):862-866

OBJECTIVE To characterize pharmacokinetics of cyclophosphamide and 4-hydoxycyclophosphamide (4-OHCP) in the plasma of healthy cats after oral, IV, and IP administration of cyclophosphamide. ANIMALS 6 healthy adult cats. PROCEDURES Cats were randomly assigned to receive cyclophosphamide (200 mg/m) via each of 3 routes of administration (oral, IV, and IP); there was a 30-day washout period between successive treatments. Plasma samples were obtained at various time points for up to 8 hours after administration. Samples were treated with semicarbazide hydrochloride to trap the 4-OHCP in stable form, which allowed for cyclophosphamide and trapped 4-OHCP to be simultaneously measured by use of tandem mass spectrometry. Pharmacokinetic parameters were determined from drug concentration-versus-time data for both cyclophosphamide and 4-OHCP. RESULTS Cyclophosphamide was tolerated well regardless of route of administration. Pharmacokinetic parameters for 4-OHCP were similar after oral, IV, and IP administration. Area under the concentration-time curve for cyclophosphamide was lower after oral administration than after IV or IP administration. CONCLUSIONS AND CLINICAL RELEVANCE Cyclophosphamide can be administered interchangeably to cats as oral, IV, and IP formulations, which should provide benefits with regard to cost and ease of administration to certain feline patients.
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http://dx.doi.org/10.2460/ajvr.78.7.862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6143145PMC
July 2017

Assessment of absorption of transdermal ondansetron in normal research cats.

J Feline Med Surg 2017 Dec 23;19(12):1245-1248. Epub 2017 Jan 23.

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

Objectives The objective of this study was to assess the absorption of transdermal ondansetron in healthy cats. Methods Five research cats with unremarkable complete blood count, biochemistry and urinalysis were used for both single- and multiple-dose application studies. For single-dose application, 4 mg ondansetron in 0.1 ml Lipoderm gel was applied once to the internal ear pinna. Blood samples were collected via jugular catheter over a 48 h period following administration (0, 15 mins, 30 mins, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h). For multiple-dose application, 4 mg ondansetron in 0.1 ml Lipoderm gel was applied for five consecutive days before blood samples were obtained in the same manner. Serum was separated and frozen prior to analysis. Ondansetron was measured via liquid chromatography coupled to tandem mass spectrometry. Results Analysis revealed no clinically relevant drug levels in serum after either single- or multiple-dose administration of 4 mg transdermal ondansetron. Conclusions and relevance Transdermal application of 4 mg ondansetron does not result in clinically relevant serum concentrations of drug. Despite characteristics of the drug that imply suitability for transdermal application, this does not appear to be an acceptable method of drug delivery for this medication at this dose. This study highlights the importance of assessing the suitability of each medication for transdermal administration.
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http://dx.doi.org/10.1177/1098612X16688807DOI Listing
December 2017