Fitzpatrick LR, O’Connell R, Talbott G et al. A novel ROR-gamma T inhibitor (VPR-254) attenuates key parameters of innate immune colitis in mice. Digestive Disease Week annual meeting, Chicago IL, May 2017.

Abstract: Injection of an agonistic CD40 monoclonal antibody to T and B cell-deficient mice induces an innate inflammatory response with colonic inflammation. This colitis is dependent on ROR-γT and various cytokines (IL-23, IL-17, IL-22 and GM-CSF [Pearson et al., eLIFE, 2015]. Therefore, we hypothesized that this colitis would be attenuated by oral treatment with a novel ROR-gamma T inverse agonist (VPR-254). Methods: Female scid mice were injected by the ip route with 12 mg/kg of anti-CD40 monoclonal antibody to induce innate acute colitis. On the same day (study day 0), mice (n = 10 per group) were treated (days 0–7) with vehicle, VPR-254 (50 mg/kg, po, bid), or ip with anti-IL-12 p40 antibody (0.5mg/mouse, on study days 0 and 3). A naïve group of four mice served as the non-colitis control group. Mice were euthanized on study day 7. Efficacy evaluations included: animal body weight, colon weight and length measurements, colonic histology (12 point severity scale, on coded slides) and the level of colonic GM-CSF (determined by ELISA). Colonic tissue samples (n = 4 per group) were also processed for immunohistochemistry, utilizing specific antibodies for IL-17A and GM-CSF. The percent areas of cytokine immunostaining in the mouse colon were determined with an online grid software program. Results: Vehicle control mice lost body weight (≈ 15%) during the study. VPR-254 modestly protected mice from weight loss; while anti-IL-12 antibody treated mice maintained their body weight throughout the study. Colonic weights (mg) were: 189±12 (naïve), 298±21 (vehicle), 248±12 (VPR-254) and 212±6 (anti-IL-12 antibody). Both drug treatments significantly reduced (p< 0.05) this morphometric parameter of colitis compared to vehicle treatment. Interestingly, VPR-254 treated mice had the longest colon length (p < 0.05 vs. vehicle) in this study. Colonic histology scores were: 2.6±0.2 (naïve), 9.0±0.5 (vehicle), 6.2±0.4 (VPR-254) and 3.7±0.3 (anti-IL-12 antibody). Both drug treatments significantly reduced (p< 0.05) this histological parameter of colitis compared to vehicle treatment. ELISA results showed that colonic GM-CSF antibody levels were significantly reduced in mice treated with VPR-254 (24% reduction), or anti-IL-12 (p40) (42% reduction), as compared to vehicle treatment. The mean percent areas of GM-CSF immunostaining were: 36±4 (naïve), 65±5 (vehicle), 56±7 (VPR-254) and 41±5 (anti-IL-12 ab, p<0.05 vs. vehicle). A somewhat similar pattern of immunostaining was found for IL-17A. There was a significant association (r = 0.704, p = 0.002) between colonic GM-CSF staining and histopathology scores. Summary: A novel ROR-γT Inhibitor (VPR-254), as well as an anti-IL-12 p40 antibody, reduced key parameters of innate immune colitis in mice. Conclusion: These results further support ROR-gamma T inhibition as a possible pharmacological approach for colonic inflammation. 



Kreys ED, Frei CR , Villarreal SM, Bollinger MJ et al.  Medication adherence and persistence of Dasatinib and Nilotinib in a national cohort of veterans. Managed Care Pharmacy Annual Meeting, Denver CO,   March 2017.

Abstract:  The introduction of tyrosine kinase inhibitor, imatinib, was a breakthrough in the treatment of CML that drastically improved outcomes. Medication adherence is essential to achieve these outcomes. Second-generation TKIs, dasatinib and nilotinib, are commonly used as second-line treatment after imatinib failure, yet studies comparing adherence between the two drugs have revealed conflicting results. Objective  To compare treatment persistence and medication adherence between dasatinib and nilotinib as second-line treatment. Methods This observational study utilized internal Veterans Health Administration (VHA) databases for the time period of 10/1/2000-9/20/2012. The study included VHA beneficiaries, age 18-89 years, with ≥1 encounter at any of the VHA institution with a diagnosis code for CML (ICD-9 205.1x). Patients had to have filled ≥1 prescription of nilotinib or dasatinib as second-line treatment, defined as TKI treatment subsequent of previous treatment of one other TKI. Primary study endpoints included medication adherence, as measured by Proportion of Days (PDC), and treatment persistence in the first year of treatment. Appropriate level of adherence was defined as achieving PDC ≥80%. Persistence was evaluated using Kaplan-Meier analysis in conjunction with a log-rank test. Multivariable analyses were performed to control for pertinent confounding variables. Results  As second line treatment 457 patients received dasatinib while 124 received nilotinib. The average PDC was 51% (±35%) for dasatinib-treated patients as compared to 47% (±34%) of nilotinib-treated patients, resulting in a mean difference of 4% (95% CI: -3.5 - 11.0%, p= 0.309). Thirty-three percent of the dasatinib-treated patients were deemed adherent during first-year of treatment relative to 28% of nilotinib-treated patients resulting in an adjusted odds ratio (OR) of 1.24 (95% CI: 0.78-1.95, p= 0.361). A Kaplan-Meier analysis did not demonstrate a statistically significant difference in time to discontinuation between dasatinib and nilotinib (p= 0.740). Forty-five percent of patients receiving dasatinib continued therapy beyond one year relative to 44% of patients receiving nilotinib resulting in an adjusted OR of 1.10 (95% CI 0.71-1.68, p= 0.680). Conclusion In this national cohort of VHA patients receiving dasatinib or nilotinib as second-line treatment, no significant differences in the persistence or adherence were identified.



Yadao MA, Kreys ED,  Phung O.  Trends in hospitalizations as a result of chemotherapy-induced adverse effects. Annual Conference of the Hematology/Oncology Pharmacy Association,  Anaheim CA, March 2017.

Abstract: Chemotherapy often causes severe complications that may lead to hospitalizations and negatively affect clinical outcomes. Over time new treatments have been developed and guidelines have been updated to limit the negative impact of these complications. Objective: To evaluate the incidence and trends of hospitalizations as a result of certain chemotherapy-induced adverse effects among cancer patients. Methods: This was a retrospective cohort study that utilized the National Inpatient Sample database, within the Healthcare Cost and Utilization Project, to evaluate hospitalizations of cancer patients from 1997 to 2010 Hospitalizations were selected using ICD-9 diagnosis codes for aplastic anemia (284), neutropenia (288.00), nausea and vomiting (787.01 – 787.03), and pneumocystis (136.3). Cancer prevalence data, from the Surveillance, Epidemiology, and End Results program, were used to derive the incidence of hospitalizations due to chemotherapy-induced adverse effects per 100,000 cancer patients. Linear regression was performed to assess trends in incidence while logistic regression assessed trends in mortality. Multivariable analyses were used to adjust for age, gender, race, and primary source of payment. Results: Among the observed chemotherapy-induced adverse effects, the adjusted changes in incidence per year per 100,000 persons with cancer were observed for patients hospitalized due to anemia (4.9 [-24.6 – 34.5]), neutropenia (-23.7 [-45.4 – -2.1]), nausea and vomiting (-4.9 [-9.6 – -0.1]), and pneumocystis (-0.8 [-1.4 – -0.3]). Additionally, the adjusted decreases in the odds of mortality per year were observed for hospitalizations due to anemia (0.947 [0.946 – 0.948]), neutropenia (0.946 [0.944 – 0.948]), nausea and vomiting (0.949 [0.946 – 0.953]), and pneumocystis (0.964 [0.959 – 0.968]). Conclusion: Among this national cohort of cancer patients hospitalized due to chemotherapy-induced adverse effects, small but statistically significant decreases in hospitalization incidence and mortality may suggest that newer treatment strategies and practices may reduce adverse effects and their impact.



Vinall R, Chen Q, Talbott G, Hubbard N et al. Use of a genetically engineered mouse model and RNA-Seq to identify genes that can be regulated by mutant p53 in prostate cells following irradiation. AACR annual meeting, Washington DC, April 2017.

Abstract: Our group has previously demonstrated that the Trp53 R270H mutation can drive prostate cancer (CaP) initiation in a genetically engineered mouse model, and that the human equivalent, TP53 R273H, can promote development of castration resistant growth of LNCaP cells as well as resistance to commonly-used therapeutic agents. The primary objective of the current study was to identify genes that may contribute to the development of these gain-of-function phenotypes.

Wildtype mice and mice that were heterozygous or homozygous for the Trp53 R270H mutation (referred to as Trp53 +/+, Trp53 +/ R270H, or Trp53 R270H/R270H, respectively) that were ~3 months old were exposed to 5 Gy radiation to activate and stabilize p53, consequently increasing its expression. Mouse prostates were harvested 6 hours post-irradiation and either processed for subsequent histological/immunohistochemistry (IHC) analysis or snap-frozen for subsequent RNA extraction and transcriptome profiling with RNA-Sequencing (RNA-Seq) analysis. P53 expression was determined by IHC. RNA-Seq data were processed to quantify transcript levels and to assess differential gene expression between the 3 groups.  

PIN lesions were observed in 3-month-old Trp53 R270H/R270H mice prostates, but not in Trp53 +/+ or Trp53 +/ R270H mice prostates. IHC analysis demonstrated that p53 was stabilized in the majority of prostate cells from Trp53 +/+, Trp53 +/ R270H, or Trp53 R270H/R270H mice 6 hours post-irradiation. RNA-Seq analysis of RNA isolated from irradiated mice prostates identified 1,444 genes that were differentially expressed in Trp53 +/+ versus Trp53 R270H/R270H mice prostate cells, and 796 genes that were differentially expressed in Trp53 +/+ versus Trp53 +/ R270H mice. Statistically significant differences in gene expression between the 3 groups were observed for 1,378 genes, including a number of p53 target genes, such as Cdkn1a, Bax, Bcl2, Kras, Mdm2, and Id4.

Our data identify multiple genes that may contribute to prostate cancer initiation and/or progression through p53 gain-of-function and loss-of-function mechanisms. It is possible that further analysis of these genes may lead to the development of new therapies and/or biomarkers for prostate cancer patients as well as guide the usage of currently available therapies in men at risk of developing CaP and CaP patients who harbor TP53 mutations.



Fitzpatrick LR, Talbott G, Mokrushin E, Woldemariam T. Ex Vivo Effects of Silymarin Fractions on Pro-inflammatory Cytokine Secretion from Colonic Strips of Mice with DSS-Induced Colitis. Digestive Disease Week annual meeting, Chicago IL, May 2017.

Abstract: Silymarin is an extract of milk thistle seeds. The silymarin complex consists of four major classes of flavonoligans: silibinin, silychristin, silydianin and isosilibinin. These compounds have both antioxidant and anti-inflammatory activities. Previously, we found that certain silymarin-derived fractions attenuated pro-inflammatory cytokine (TNF-α and IL-8) secretion from macrophage and colonic epithelial cell lines [Gastroenterology 150: S376-S377, 2016]. Methods: Silymarin fraction compositions were confirmed by (LC/MS) analyses. Silibinin and isosilibinin were obtained from a commercial source. Dextran Sulfate Sodium (DSS) was given to male C57BL/6 mice (n = 16) in the drinking water (2% concentration), for a six day period, in order to induce colitis. Control mice (n = 8) received water. The ex vivo effects of crude silymarin extract, two distinct silymarin fractions, as well as silibinin and isosilibinin (20 to 200 μg/ml concentration range) were tested in our 24 hour colonic culture system [Fitzpatrick et al., Inflammopharmacology, 2014]. We utilized ≈ 4 mm colonic strips (n = 4 to 7 per flavonoligan treatment group) from mice with DSS-induced colitis. The secretion of TNF-α and MIP-2 were determined in the cell culture media by ELISA. Some colonic strips were stimulated with IL-1β (10 ng/ml) plus IL-23 (10 ng/ml) to induce IL-17 secretion. This cytokine was measured with an ELISA kit. Results: Mice that were administered DSS showed clear evidence of colitis (enhanced diseases activity indices and reduced colon lengths). Ex vivo treatment with fraction 2 (containing mainly isosilibinin and silibinin), silibinin, or isosilibinin were most effective for inhibiting dual cytokine stimulated IL-17 secretion. Specifically, IL-17 values (pg/ml) were: 55±7 (Vehicle), 26±5 (fraction 5), 20±7 (crude extract), 13±5 (fraction 2), 11±5 (silibinin) and 9±2 (isosilibinin). All treatments significantly reduced (p < 0.05 vs. vehicle) IL-17 secretion. Fraction 2 and isosilibinin (at 200 μg/ml) also significantly (p< 0.05 vs. vehicle treatment) attenuated TNF-α secretion to the level found in colonic strips from non-DSS treated mice. Other silymarin-derived fractions and compounds also reduced TNF-α secretion, but not to the same degree. For attenuating MIP-2 secretion: the mean percent inhibition (compared to vehicle treatment) was: Fraction 2 (96%) = Isosilibinin (96%) > Silibinin (92%) > crude extract (75%) > fraction 5 (69%). Summary: A silibinin/isosilibinin containing fraction of silymarin, as well as these two compounds, prominently inhibited basal and stimulated pro-inflammatory cytokine secretion from colonic strips of mice with DSS-induced colitis. Conclusion: These data further contribute to the identification of optimal silymarin-derived anti-inflammatory flavonoligans, which can be used for follow-up in vivo testing in murine models of colitis.


California Northstate University College of Pharmacy ♦ 9700 West Taron Drive ♦ Elk Grove, CA 95757 ♦ Phone: (916) 686-7400