Osteopontin Inhibits Interleukin-1β-Stimulated Increases in Matrix Metalloproteinase Activity in Adult Rat Cardiac Fibroblasts: Role of Protein Kinase C-ζ

We have shown that osteopontin (OPN), an extracellular matrix protein, plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Interleukin-1β (IL-1β), increased in the heart following MI, increases matrix metalloproteinase (MMP) activity in cardiac fibroblasts in vitro. Here, we show that OPN alone has no effect on MMP activity or expression. However, it reduces IL-1β-stimulated increases in MMP activity and expression in adult rat cardiac fibroblasts. Pretreatment with bovine serum albumin had no effect on MMP activity or protein content, whereas GRGDS (glycine-arginine-glycine-aspartic acid-serine)- pentapeptide (which interrupts binding of RGD-containing proteins to cell surface integrins) and monoclonal antibody m7E3 (a rat β3 integrins antagonist) inhibited the effects of OPN. Inhibition of PKC using chelerythrine inhibited the activities of both MMP-2 and MMP-9. Stimulation of cells using IL-1β increased phosphorylation and translocation of PKC to membrane fractions, which was inhibited by OPN. OPN inhibited IL-1β-stimulated increases in translocation of PKC-ζ from cytosolic to membrane fractions. Furthermore, the levels of phospho-PKC-ζ were lower in the cytosolic fractions of OPN knock-out mice hearts as compared with wild type 6 days post-MI. Inhibition of PKC-ζ using PKC-ζ pseudosubstrate inhibited IL-1β-stimulated increases in MMP-2 and MMP-9 activities. These observations suggest that OPN, acting via 3 integrins, inhibits IL-1β-stimulated increases in MMP-2 and MMP-9 activity, at least in part, via the involvement of PKC-ζ. Thus, OPN may play a key role in collagen deposition during myocardial remodeling following MI by modulating cytokine-stimulated MMP activity

In vivo microdialysis to determine subcutaneous interstitial fluid penetration and pharmacokinetics of fluconazole in intensive care unit patients with sepsis

The objective of the study was to describe the subcutaneous interstitial fluid (ISF) pharmacokinetics of fluconazole in critically ill patients with sepsis. This prospective observational study was conducted at two tertiary intensive care units in Australia. Serial fluconazole concentrations were measured over 24 h in plasma and subcutaneous ISF using microdialysis. The concentrations in plasma and microdialysate were measured using a validated high-performance liquid chromatography system with electrospray mass spectrometer detector method. Noncompartmental pharmacokinetic analysis was performed. Twelve critically ill patients with sepsis were enrolled. The mean in vivo fluconazole recovery rates +/- standard deviation (SD) for microdialysis were 51.4% +/- 16.1% with a mean (+/- SD) fluconazole ISF penetration ratio of 0.52 +/- 0.30 (coefficient of variation, 58%). The median free plasma area under the concentration-time curve from 0 to 24 h (AUC(0-24)) was significantly higher than the median ISF AUC(0-24) (340.4 versus 141.1 mg . h/liter; P = 0.004). There was no statistical difference in median fluconazole ISF penetration between patients receiving and not receiving vasopressors (median, 0.28 versus 0.78; P = 0.106). Both minimum and the maximum concentrations of drug in serum (C-max and C-min) showed a significant correlation with the fluconazole plasma exposure (Cmax, R-2 = 0.86, P < 0.0001; Cmin, R-2 = 0.75, P < 0.001). Our data suggest that fluconazole was distributed variably, but incompletely, from plasma into subcutaneous interstitial fluid in this cohort of critically ill patients with sepsis. Given the variability of fluconazole interstitial fluid exposures and lack of clinically identifiable factors by which to recognize patients with reduced distribution/exposure, we suggest higher than standard doses to ensure that drug exposure is adequate at the site of infection

SWOG 1815: A phase III randomized trial of gemcitabine, cisplatin, and nab-paclitaxel versus gemcitabine and cisplatin in newly diagnosed, advanced biliary tract cancers

Background: Biliary tract cancers (BTCs) are a heterogeneous group of malignancies with a dismal prognosis. Gemcitabine-based regimens are the standard of care in advanced disease, but median overall survival (OS) is roughly 12 months. The addition of albumin-bound paclitaxel to gemcitabine and cisplatin (GAP) demonstrated promising efficacy in a 60 patient, single-arm phase II study (Shroff et al, JAMA Oncol 2019), with observed median OS of 19.2 months. Methods: SWOG 1815 is a randomized, open-label, phase III trial comparing GAP to gemcitabine/cisplatin (GC). The study included newly diagnosed advanced BTC patients (pts), randomized 2:1 to GAP vs. GC. GAP included gemcitabine at 800 mg/m2, cisplatin at 25 mg/m2 and albumin-bound paclitaxel at 100 mg/m2 on days 1 and 8 of a 21-day cycle. GC included standard dosing of gemcitabine at 1000 mg/m2 and cisplatin at 25 mg/m2 on days 1 and 8 of a 21-day cycle. Pts were treated until progression. The primary endpoint was overall survival (OS) with a target hazard ratio of 0.7 with 90% power and a 1-sided alpha of 0.025; randomization was stratified by disease site (intrahepatic cholangiocarcinoma [CCA] vs gallbladder adenocarcinoma [GBC] vs extrahepatic CCA), disease stage (locally advanced vs metastatic), and Zubrod PS 0 vs 1. Results: Of 441 eligible pts randomized, 55% were female. 67% of patients had intrahepatic CCA, 16% had GBC and 17% had extrahepatic CCA. Most pts had metastases (73%). Median OS with GAP vs. GC was 14 vs. 12.7 mo respectively (HR 0.93, 95% CI 0.74-1.19, p=0.58), ORR (confirmed and unconfirmed) 34% vs25% (p=0.11) and median PFS 8.2 vs 6.4 mo (HR 0.92, 95% CI 0.72-1.16, p=0.47), respectively. Grade 3 and 4 treatment related adverse events (TRAEs) in ≥10% of pts for GAP and GC were anemia, neutropenia, and thrombocytopenia. GAP had more ≥ grade 3 hematologic AEs compared to the GC arm (60% vs. 45%, p=0.003). Discontinuation due to toxicity was at 24% vs 19% (p=0.26) with GAP vs GC. In exploratory subset analyses, GAP vs GC improved OS in pts with locally advanced disease (medians 19.2 vs 13.7 mo; HR 0.67, 95% CI 0.42- 1.06, p=0.09) and in GBC pts (medians 17.0 vs 9.3 mo; HR 0.74, 95% CI 0.41-1.35, p=0.33). ORR for GAP vs GC in GBC was 50% vs 24% (p=0.09) and for locally advanced disease 28 vs 21% p=0.74. Conclusions: SWOG 1815 did not result in a statistically significant improvement in median OS with GAP vs. GC. The GAP regimen had higher rates of TRAEs without a statistically significant difference in discontinuation rates. Pts with locally advanced disease and GBC may benefit from the use of GAP. Further analyses are ongoing to understand potential benefit of GAP in subsets of BTC pts. Funding: NIH/National Cancer Institute grants CA180888, CA180819, CA180820, CA180821, and CA180868; and in part by Celgene Corporation, Summit, NJ (subsidiary of Bristol Myer Squibb)

combined pik3ca and fgfr inhibition with alpelisib and infigratinib in patients with pik3ca mutant solid tumors with or without fgfr alterations

PURPOSE Concurrent PIK3CA mutations and fibroblast growth factor receptor (FGFR) alterations occur in multiple cancer types, including estrogen receptor–positive breast cancer, bladder cancer, and endometrial cancer. In this first-in-human combination trial, we explored safety and preliminary efficacy of combining the PI3Kα selective inhibitor alpelisib with the FGFR1-4 selective inhibitor infigratinib. PATIENTS AND METHODS Patients with PIK3CA-mutant advanced solid tumors, with or without FGFR1-3 alterations, were enrolled in the dose escalation or one of three molecular-defined dose-expansion cohorts. The primary end point was the maximum tolerated dose. Secondary end points included safety, pharmacokinetics, and response. Archival tumor samples were sequenced to explore genomic correlates of response. RESULTS In combination, both agents were escalated to full, single-agent recommended doses (alpelisib, 300 mg per day continuously; infigratinib, 125 mg per day 3 weeks on followed by 1 week off). The toxicity profile of the combination was consistent with the established safety profile of each agent, although 71% of all patients required at least one treatment interruption or dose reduction. Molecularly selected dose expansions in breast cancer and other solid tumors harboring PIK3CA mutations, alone or in combination with FGFR alterations, identified sporadic responses, predominately in tumor types and genotypes previously defined to have sensitivity to these agents. CONCLUSION The combination of alpelisib and infigratinib can be administered at full single-agent doses, although the high rate of dose interruption or reduction suggests long-term tolerability may be challenging. In exploratory signal-seeking cohorts of patients harboring dual PIK3CA and FGFR1-3 alterations, no clear evidence of synergistic activity was observed

Phase I Trial of the Human Double Minute 2 Inhibitor MK-8242 in Patients With Advanced Solid Tumors.

Purpose To evaluate MK-8242 in patients with wild-type TP53 advanced solid tumors. Patients and Methods MK-8242 was administered orally twice a day on days 1 to 7 in 21-day cycles. The recommended phase II dose (RP2D) was determined on the basis of safety, tolerability, pharmacokinetics (PK), and by mRNA expression of the p53 target gene pleckstrin homology-like domain, family A, member 3 ( PHLDA3). Other objectives were to characterize the PK/pharmacodynamic (PD) relationship, correlate biomarkers with response, and assess tumor response. Results Forty-seven patients received MK-8242 across eight doses that ranged from 60 to 500 mg. Initially, six patients developed dose-limiting toxicities (DLTs): grade (G) 2 nausea at 120 mg; G3 fatigue at 250 mg; G2 nausea and G4 thrombocytopenia at 350 mg; and G3 vomiting and G3 diarrhea at 500 mg. DLT criteria were revised to permit management of GI toxicities. Dosing was resumed at 400 mg, and four additional DLTs were observed: G4 neutropenia and G4 thrombocytopenia at 400 mg and G4 thrombocytopenia (two patients) at 500 mg. Other drug-related G3 and G4 events included anemia, leukopenia, pancytopenia, nausea, hyperbilirubinemia, hypophosphatemia, and anorexia. On the basis of safety, tolerability, PK, and PD, the RP2D was established at 400 mg (15 evaluable patients experienced two DLTs). PK for 400 mg (day 7) showed Cmax 3.07 μM, Tmax 3.0 hours, t1/2 (half-life) 6.6 hours, CL/F (apparent clearance) 28.9 L/h, and Vd/F (apparent volume) 274 L. Blood PHLDA3 mRNA expression correlated with drug exposure ( R(2) = 0.68; P < .001). In 41 patients with postbaseline scans, three patients with liposarcoma achieved a partial response (at 250, 400, and 500 mg), 31 showed stable disease, and eight had progressive disease. In total, 27 patients with liposarcoma had a median progression-free survival of 237 days. Conclusion At the RP2D of 400 mg twice a day, MK-8242 activated the p53 pathway with an acceptable safety and tolerability profile. The observed clinical activity (partial response and prolonged progression-free survival) provides an impetus for further study of HDM2 inhibitors in liposarcoma

Neurophysiological modeling of bladder afferent activity in the rat overactive bladder model

The overactive bladder (OAB) is a syndrome-based urinary dysfunction characterized by “urgency, with or without urge incontinence, usually with frequency and nocturia”. Earlier we developed a mathematical model of bladder nerve activity during voiding in anesthetized rats and found that the nerve activity in the relaxation phase of voiding contractions was all afferent. In the present study, we applied this mathematical model to an acetic acid (AA) rat model of bladder overactivity to study the sensitivity of afferent fibers in intact nerves to bladder pressure and volume changes. The afferent activity in the filling phase and the slope, i.e., the sensitivity of the afferent fibers to pressure changes in the post-void relaxation phase, were found to be significantly higher in AA than in saline measurements, while the offset (nerve activity at pressure ~0) and maximum pressure were comparable. We have thus shown, for the first time, that the sensitivity of afferent fibers in the OAB can be studied without cutting nerves or preparation of single fibers. We conclude that bladder overactivity induced by AA in rats is neurogenic in origin and is caused by increased sensitivity of afferent sensors in the bladder wall

Tumor-derived interleukin-10 as a prognostic factor in stage III patients undergoing adjuvant treatment with an autologous melanoma cell vaccine.

OBJECTIVES: Interleukin-10 (IL-10) downregulates T-cell-mediated immune responses. We studied the association between IL-10 production by freshly isolated melanoma cell suspensions in vitro and overall survival in patients undergoing adjuvant treatment with a vaccine prepared from the same autologous melanoma cells modified with a hapten, dinitrophenyl (DNP). METHODS: Forty-four patients with cutaneous melanoma (29 stage III and 15 stage IV) were prospectively evaluated. Tumor cells were extracted from metastatic deposits for production of DNP-modified autologous melanoma cell vaccine. Small aliquots of the melanoma cell suspensions were separated prior to vaccine processing and cultured overnight for IL-10 production. Based on a blind assessment of the distribution of IL-10 levels in the culture supernatants, a cutoff of 200 pg/ml was used to define high versus low IL-10 producers. Cox regression model was used for multivariate analysis. Overall survival was calculated using the Kaplan-Meier method, and survival curves were compared with the log-rank test. RESULTS: Out of 44 patients, 29 were low and 15 were high IL-10 producers. The median OS was significantly worse for high compared with low IL-10 producers (10.5 months vs. 42 months; P = 0.022). In stage III patients, the multivariate hazard ratio for high versus low IL-10 producers was 2.92 (95% CI, 1.04-8.20; P = 0.041). The corresponding hazard ratio in stage IV patients was 0.92 (95% CI, 1.04-8.20; P = 0.888). CONCLUSIONS: High IL-10 production in the tumor microenvironment could be a determinant of clinical outcomes in stage III melanoma patients receiving autologous melanoma cell vaccine

Effect of obesity on the population pharmacokinetics of fluconazole in critically Ill patients

Our objective was to describe the population pharmacokinetics of fluconazole in a cohort of critically ill nonobese, obese, and morbidly obese patients. Critically ill patients prescribed fluconazole were recruited into three body mass index (BMI) cohorts, nonobese (18.5 to 29.9 kg/m(2)), obese (30.0 to 39.9 kg/m(2)), and morbidly obese (>= 40 kg/m(2)). Serial fluconazole concentrations were determined using a validated chromatographic method. Population pharmacokinetic analysis and Monte Carlo dosing simulations were undertaken with Pmetrics. Twenty-one critically ill patients (11 male) were enrolled, including obese (n = 6) and morbidly obese (n = 4) patients. The patients mean +/- standard deviation (SD) age, weight, and BMI were 54 +/- 15 years, 90 +/- 24 kg, and 31 +/- 9 kg/m(2), respectively. A two-compartment linear model described the data adequately. The mean +/- SD population pharmacokinetic parameter estimates were clearance (CL) of 0.95 +/- 0.48 liter/h, volume of distribution of the central compartment (V-c) of 15.10 +/- 11.78 liter, intercompartmental clearance from the central to peripheral compartment of 5.41 +/- 2.28 liter/h, and intercompartmental clearance from the peripheral to central compartment of 2.92 +/- 4.95 liter/h. A fluconazole dose of 200 mg daily was insufficient to achieve an area under the concentration-time curve for the free, unbound drug fraction/MIC ratio of 100 for pathogens with MICs of >= 2 mg/liter in patients with BMI of >30 kg/m(2). A fluconazole loading dose of 12 mg/kg and maintenance dose of 6 mg/kg/day achieved pharmacodynamic targets for higher MICs. A weight-based loading dose of 12 mg/kg followed by a daily maintenance dose of 6 mg/kg, according to renal function, is required in critically ill patients for pathogens with a MIC of 2 mg/liter