Pleiotropic stromal effects of vascular endothelial growth factor receptor 2 antibody therapy in renal cell carcinoma models.

The benefits of inhibiting vascular endothelial growth factor (VEGF) signaling in cancer patients are predominantly attributed to effects on tumor endothelial cells. Targeting non-endothelial stromal cells to further impact tumor cell growth and survival is being pursued through the inhibition of additional growth factor pathways important for the survival and/or proliferation of these cells. However, recent data suggest that VEGF receptor (VEGFR)-specific inhibitors may target lymphatic vessels and pericytes in addition to blood vessels. Here, in fact, we demonstrate that DC101 (40 mg/kg, thrice a week), an antibody specific to murine VEGFR2, significantly reduces all three of these stromal components in subcutaneous (SKRC-29) and orthotopic (786-O-LP) models of renal cell carcinoma (RCC) established in nu/nu athymic mice. Sunitinib (40 mg/kg, once daily), a receptor tyrosine kinase inhibitor of VEGFR2 and other growth factor receptors, also caused significant loss of tumor blood vessels in RCC models but had weaker effects than DC101 on pericytes and lymphatic vessels. In combination, sunitinib did not significantly add to the effects of DC101 on tumor blood vessels, lymphatic vessels, or pericytes. Nevertheless, sunitinib increased the effect of DC101 on tumor burden in the SKRC-29 model, perhaps related to its broader specificity. Our data have important implications for combination therapy design, supporting the conclusion that targeting VEGFR2 alone in RCC has the potential to have pleiotropic effects on tumor stroma.

Tumor necrosis factor-alpha and interleukin-1 antagonists alleviate inflammatory skin changes associated with epidermal growth factor receptor antibody therapy in mice.

Cancer patients receiving epidermal growth factor receptor (EGFR) antibody therapy often experience an acneiform rash of uncertain etiology in skin regions rich in pilosebaceous units. Currently, this condition is treated symptomatically with very limited, often anecdotal success. Here, we show that a monoclonal antibody targeting murine EGFR, ME1, caused a neutrophil-rich hair follicle inflammation in mice, similar to that reported in patients. This effect was preceded by the appearance of lipid-filled hair follicle distensions adjacent to enlarged sebaceous glands. The cytokine tumor necrosis factor-alpha (TNFalpha), localized immunohistochemically to this affected region of the pilosebaceous unit, was specifically up-regulated by ME1 in skin but not in other tissues examined. Moreover, skin inflammation was reduced by cotreatment with the TNFalpha signaling inhibitor, etanercept, indicating the involvement of TNFalpha in this inflammatory process. Interleukin-1, a cytokine that frequently acts in concert with TNFalpha, is also involved in this process given the efficacy of the interleukin-1 antagonist Kineret. Our results provide a mechanistic framework to develop evidence-based trials for EGFR antibody-induced skin rash in patients with cancer.

Two-year assessment of entecavir resistance in Lamivudine-refractory hepatitis B virus patients reveals different clinical outcomes depending on the resistance substitutions present.

Entecavir (ETV) is a deoxyguanosine analog approved for use for the treatment of chronic infection with wild-type and lamivudine-resistant (LVDr) hepatitis B virus (HBV). In LVD-refractory patients, 1.0 mg ETV suppressed HBV DNA levels to below the level of detection by PCR (<300 copies/ml) in 21% and 34% of patients by Weeks 48 and 96, respectively. Prior studies showed that virologic rebound due to ETV resistance (ETVr) required preexisting LVDr HBV reverse transcriptase substitutions M204V and L180M plus additional changes at T184, S202, or M250. To monitor for resistance, available isolates from 192 ETV-treated patients were sequenced, with phenotyping performed for all isolates with all emerging substitutions, in addition to isolates from all patients experiencing virologic rebounds. The T184, S202, or M250 substitution was found in LVDr HBV at baseline in 6% of patients and emerged in isolates from another 11/187 (6%) and 12/151 (8%) ETV-treated patients by Weeks 48 and 96, respectively. However, use of a more sensitive PCR assay detected many of the emerging changes at baseline, suggesting that they originated during LVD therapy. Only a subset of the changes in ETVr isolates altered their susceptibilities, and virtually all isolates were significantly replication impaired in vitro. Consequently, only 2/187 (1%) patients experienced ETVr rebounds in year 1, with an additional 14/151 (9%) patients experiencing ETVr rebounds in year 2. Isolates from all 16 patients with rebounds were LVDr and harbored the T184 and/or S202 change. Seventeen other novel substitutions emerged during ETV therapy, but none reduced the susceptibility to ETV or resulted in a rebound. In summary, ETV was effective in LVD-refractory patients, with resistant sequences arising from a subset of patients harboring preexisting LVDr/ETVr variants and with approximately half of the patients experiencing a virologic rebound.

Effect of 4-(5-chloro-2-hydroxyphenyl)-3-(2-hydroxyethyl)-6-(trifluoromethyl)-quinolin-2(1H)-one (BMS-223131), a novel opener of large conductance Ca2+-activated K+ (maxi-K) channels on normal and stress-aggravated colonic motility and visceral nociception.

We evaluated the effects of 4-(5-chloro-2-hydroxyphenyl)-3-(2-hydroxyethyl)-6-(trifluoromethyl)-quinolin-2(1H)-one (BMS-223131), an opener of large conductance Ca(2+)-activated potassium (maxi-K) channels, on normal and stress-exacerbated colonic motility and visceral nociception in the rat. Fecal output was employed as an index of motility. Visceral nociception, in response to intracolonic balloon distension (10-90 mm Hg; 30 s duration), was evaluated using one of three indices: change in blood pressure, abdominal withdrawal, or myoelectrical activity. BMS-223131 (2, 6, or 20 mg/kg i.p.) produced a small but dose-dependent and significant reduction in cumulative 24-h fecal output. Fecal output in response to stress (1-h restraint plus bursts of air to the face) was markedly inhibited by BMS-223131, and moisture content was significantly reduced. With regard to visceral pain, the transient and distention-dependent reduction in arterial pressure in anesthetized animals was inhibited by BMS-223131 in a dose-dependent manner. Distension-induced abdominal withdrawal in conscious rats was also dose-dependently attenuated by BMS-223131. BMS-223131 at a dose of 20 mg/kg markedly attenuated the increase in myoelectrical activity evoked by balloon distention in conscious animals. BMS-223131 was also evaluated in viscerally hypersensitive rats (sensitized as neonates by intracolonic mustard oil) where it produced a robust dose-dependent attenuation of the abdominal withdrawal response. Compared with naive animals, BMS-223131 was more potent in the sensitized animals. Thus, BMS-223131 effectively reduced stress-induced colonic motility and visceral nociception supporting the potential utility of maxi-K channel openers for the treatment of bowel disorders involving dysfunctional motility and visceral sensitivity.

BL-1249 [(5,6,7,8-tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine]: a putative potassium channel opener with bladder-relaxant properties.

BL-1249 [(5,6,7,8-tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine] produced a concentration-dependent membrane hyperpolarization of cultured human bladder myocytes, assessed as either a reduction in fluorescence of the voltage-sensitive dye bis-(1,2-dibutylbarbituric acid)trimethine oxonol (EC50 = 1.26 +/- 0.6 microM) or by direct electrophysiological measurement (EC50 = 1.49 +/- 0.08 microM). BL-1249 also produced a membrane hyperpolarization of acutely dissociated rat bladder myocytes. Voltage-clamp studies in human bladder cells revealed that BL-1249 activated an instantaneous, noninactivating current that reversed near E(K). The BL-1249-evoked outward K+ current was insensitive to blockade by glyburide, tetraethylammonium, iberiotoxin, 4-aminopyridine, apamin, or Mg2+. However, the current was inhibited by extracellular Ba2+ (10 mM). In in vitro organ bath experiments, BL-1249 produced a concentration-dependent relaxation of 30 mM KCl-induced contractions in rat bladder strips (EC50 = 1.12 +/- 0.37 microM), yet had no effect on aortic strips up to the highest concentration tested (10 microM). The bladder relaxation produced by BL-1249 was partially blocked by Ba2+ (1 and 10 mM) but not by apamin, iberiotoxin, 4-aminopyridine, glyburide, or tetraethylammonium. In an anesthetized rat model, BL-1249 (1 mg/kg i.v.) decreased the number of isovolumic contractions, without significantly affecting blood pressure. Thus, BL-1249 behaves as a potassium channel activator that exhibits bladder versus vascular selectivity both in vitro and in vivo. A survey of potassium channels exhibiting sensitivity to extracellular Ba2+ at millimolar concentration revealed that the expression of the K2P2.1 (TREK-1) channel was relatively high in human bladder cells versus human aortic cells, suggesting this channel as a possible candidate target for BL-1249.

1-(2-pyrimidinyl)-piperazine, a buspirone metabolite, modulates bladder function in the anesthetized rat.

AIMS: To examine the effects of 1-(2-pyrimidinyl)-piperazine (1-PP), a buspirone metabolite, on bladder function in vivo. METHODS: Micturition reflexes in the rat were evaluated in two models of bladder function; a constant infusion model employing 0.5% acetic acid and an isovolumic model. RESULTS: In the constant infusion model, 1-PP (0.14-1.32 mg/kg) dose-dependently and significantly decreased the number of bladder contractions measured during a 30 min recording period, with little effect on the pressure developed during each contraction. 1-PP is an alpha2-adrenergic receptor antagonist. The alpha2 antagonists BRL44408 (alpha2A vs. alpha2B selective; 0.3 and 1 mg/kg), imiloxan (alpha(2B) vs. alpha2A selective; 1 mg/kg), and yohimbine (non-subtype selective; 1 mg/kg; but not 0.3 mg/kg) also significantly reduced the number of contractions. Vehicle was without effect. In the isovolumic model, 1-PP (0.03-1.0 mg/kg) produced a dose-dependent and significant reduction in the number of bladder contractions recorded during a 15 min assessment period, with the maximum effect observed at 0.3 mg/kg. 1-PP had little effect on blood pressure; the only effect was observed at the highest dose (1 mg/kg) where it produced a transient 17% decrease in pressure. Cromakalim and tolterodine served as comparitors in all studies. CONCLUSIONS: 1-PP decreased the number of bladder contractions evoked by the micturition reflex at doses that had little effect on either the pressure developed during each bladd er contraction or on blood pressure. The effects of 1-PP are likely mediated primarily by alpha2 receptor antagonism.