A phase Ib dose-escalation study of the MEK inhibitor trametinib in combination with the PI3K/mTOR inhibitor GSK2126458 in patients with advanced solid tumors.

Introduction This Phase Ib trial investigated the safety, tolerability, and recommended phase 2 dose for the pan-PI3K/mTOR inhibitor, GSK2126458 (GSK458), and trametinib combination when administered to patients with advanced solid tumors. Patients and Methods Patients with advanced solid tumors received escalating doses of GSK458 (once or twice daily, and continuous or intermittent) and trametinib following a zone-based 3 + 3 design to determine the maximum tolerated dose (MTD). Assessments included monitoring for adverse events and response, and evaluating pharmacokinetic (PK) measures. Archival tissue and circulating free DNA samples were collected to assess biomarkers of response in the PI3K and RAS pathways. Results 57 patients were enrolled onto the continuous dosing cohort and 12 patients onto an intermittent BID dosing cohort. Two MTDs were established for the continuous daily dosing: 2 mg of GSK458 with 1.0 mg of trametinib or 1.0 mg of GSK458 with 1.5 mg of trametinib; no MTD was determined in the intermittent dosing cohort. The most frequent adverse events were rash (74 %) and diarrhea (61 %). Dose interruptions due to adverse events occurred in 42 % of patients. No significant PK interaction was observed. One patient achieved partial response and 12 patients had stable disease >16 weeks. Mutations in RAS/RAF/PI3K were detected in 70 % of patients, but no pattern emerged between response and mutational status. Conclusion GSK458 plus trametinib is poorly tolerated, due to skin and GI-related toxicities. Responses were minimal, despite enrichment for PI3K/RAS pathway driven tumors, which may be due to overlapping toxicities precluding sufficient dose exposure.

Safety, pharmacokinetics, and efficacy of (+/-)-beta-2',3'-dideoxy-5-fluoro-3'-thiacytidine with efavirenz and stavudine in antiretroviral-naïve human immunodeficiency virus-infected patients.

Racivir [RCV; (+/-)-beta-2',3'-dideoxy-5-fluoro-3'-thiacytidine], a 50:50 racemic mixture of the two beta nucleoside enantiomers, is currently in development for the treatment of human immunodeficiency virus type 1 (HIV-1) infections. RCV was administered once a day orally for 14 days at doses of 200, 400, or 600 mg in combination with stavudine and efavirenz to HIV-1-infected treatment-naïve male volunteers in a phase Ib/IIa study. Six volunteers at each dose were monitored for a total of 35 days for tolerance, pharmacokinetics, and plasma HIV RNA levels. RCV in combination with stavudine and efavirenz was well tolerated at all doses tested. Pharmacokinetic parameters were dose proportional, and the maximum concentration of drug in serum at all doses exceeded the 90% effective concentration for wild-type HIV-1. Viral loads dropped as expected in all dosage groups, with mean reductions from 1.13 to 1.42 log10 by day 4 and 2.02 to 2.43 log10 by day 14. HIV RNA levels remained suppressed for more than 2 weeks in the absence of any additional therapy, with mean viral loads ranging from 2.1 to 2.6 log10 below baseline through day 28. By day 35, HIV RNA levels began to increase but still remained >1 log10 below baseline levels.

Synergistic induction of apoptosis in human myeloid leukemia cells by phorbol 12-myristate 13-acetate and flavopiridol proceeds via activation of both the intrinsic and tumor necrosis factor-mediated extrinsic cell death pathways.

Previous studies have shown that coexposure to marginally toxic concentrations of phorbol 12-myristate 13-acetate (PMA; 10 nM) and the cyclin-dependent kinase inhibitor flavopiridol (FP; 100-200 nM) synergistically induces apoptosis in human myeloid leukemia cells U937 and HL-60 (i.e., >50% apoptotic at 24 h). Attempts have now been made to characterize the cell death pathway(s) involved in this phenomenon. In contrast to cytochrome c release and caspase-3 activation, which occur within 2.5 h of PMA/FP coexposure, caspase-8 activation and Bid cleavage appeared as later events. Such findings implicate the mitochondria-dependent pathway in the initial induction of apoptosis by PMA/FP. However, U937 cells ectopically expressing CrmA, dominant-negative caspase-8, or dominant-negative Fas-associated death domain that were highly resistant to tumor necrosis factor (TNF)/cycloheximide-induced lethality displayed significant, albeit incomplete, resistance to PMA/FP-induced apoptosis after 24 h. Furthermore, coadministration of TNF soluble receptor significantly attenuated PMA/FP-induced apoptosis in U937 (p < 0.02) and HL-60 (p < 0.03) cells at 24 h. PMA/FP coadministration also triggered substantial increases in TNFalpha mRNA and protein secretion compared with the effects of PMA administered alone. The protein kinase C (PKC) inhibitor bisindolylmaleimide (1 microM) completely blocked PMA/FP-induced TNFalpha secretion in U937 cells and attenuated apoptosis. Taken together, these results suggest that coadministration of PMA with FP in myeloid leukemia cells initially triggers mitochondrial damage, an event followed by the PKC-dependent induction and release of TNFalpha, supporting a model in which the synergistic induction of leukemic cell apoptosis by this drug combination proceeds via both mitochondrial- and TNF receptor-related apoptotic pathways.

The cyclin-dependent kinase inhibitor (CDKI) flavopiridol disrupts phorbol 12-myristate 13-acetate-induced differentiation and CDKI expression while enhancing apoptosis in human myeloid leukemia cells.

Interactions between the cyclin-dependent kinase inhibitor (CDKI) flavopiridol (FP) and phorbol 12-myristate 13-acetate (PMA) were examined in U937 human leukemia cells in relation to differentiation and apoptosis. Simultaneous, but not sequential, exposure of U937 cells to 100 nM FP and 10 nM PMA significantly increased apoptosis manifested by characteristic morphological features, mitochondrial dysfunction, caspase activation, and poly(ADP-ribose) polymerase cleavage while markedly inhibiting cellular differentiation, as reflected by diminished plastic adherence and CD11b expression. Enhanced apoptosis in U937 cells was associated with an early caspase-independent increase in cytochrome c release and accompanied by a substantial decline in leukemic cell clonogenicity. Moreover, PMA/FP cotreatment significantly increased apoptosis in HL-60 promyelocytic leukemia cells and in U937 cells ectopically expressing the Bcl-2 protein. In U937 cells, coadministration of FP blocked PMA-induced expression and reporter activity of the CDKI p21WAF/CIP1 and triggered caspase-mediated cleavage of the CDKI p27KIP1. Coexposure to FP also resulted in a more pronounced and sustained activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase cascade after PMA treatment, although disruption of this pathway by the mitogen-activated protein kinase kinase 1 inhibitor U0126 did not prevent potentiation of apoptosis. FP accelerated PMA-mediated dephosphorylation of the retinoblastoma protein (pRb), an event followed by pRb cleavage culminating in the complete loss of underphosphorylated pRb (approximately Mr 110,000) by 24 h. Finally, gel shift analysis revealed that coadministration of FP with PMA for 8 h led to diminished E2F/pRb binding compared to the effects of PMA alone. Collectively, these findings indicate that FP modulates the expression/activity of multiple signaling and cell cycle regulatory proteins in PMA-treated leukemia cells and that such alterations are associated with mitochondrial damage and apoptosis rather than maturation. These observations also raise the possibility that combining CDKIs and differentiation-inducing agents may represent a novel antileukemic strategy.

Chronic exposure to bryostatin-1 increases the radiosensitivity of U937 leukaemia cells ectopically expressing Bcl-2 through a non-apoptotic mechanism.

PURPOSE: Ionizing radiation (IR) produced a dose-dependent increase in apoptosis in U937/pCEP4 cells which was attenuated by the stable over expression of Bcl-2 (U937/Bcl-2). A dose of 2 Gy IR was selected for further analyses to determine if subsequent exposure to 10nM bryostatin- would overcome the resistance to IR-induced apoptosis conferred by Bcl-2 over expression. METHODS AND RESULTS: Although bryostatin- did not increase IR-induced apoptosis in U937/pCEP4 or U937/Bcl-2 cells, it impaired mitochondrial function and increased the antiproliferative effects of IR in both cell lines. The effects were more pronounced in U937/Bcl-2 cells. Bryostatin-1 also exerted differential effects on cell-cycle distributions of U937 transfectant cells, producing a significant G0/G1 arrest in U937/Bcl-2 cells, while decreasing IR-induced G2/M arrest in U937/pCEP4 cells. Although Bcl-2 over expression attenuated IR-induced apoptosis, clonogenic survival was similar in U937/pCEP4 and U937/Bcl-2 cells following 2 Gy IR treatment. Treatment with 10nM bryostatin-1 after 2 Gy IR further reduced clonogenic survival in both cell lines. Moreover, U937/Bcl-2 cells were more susceptible to the growth-inhibitory effects of IR/bryostatin-1 than U937/pCEP4 cells. CONCLUSIONS: Bryostatin-1 increased the radiosensitivity of U937 transfectant cell lines without enhancing apoptosis; furthermore, U937/Bcl-2 cells were more susceptible to IR/bryostatin-1-mediated antiproliferative effects than their empty-vector counterparts.

Evaluation of a model of the cochlear neural membrane. I. Physiological measurement of membrane characteristics in response to intrameatal electrical stimulation.

To understand the auditory neural response to electrical stimuli similar to those used in a cochlear implant, it will be necessary to understand the neural refraction and summation response kinetics. Evidence exists indicating that the cell soma may alter the auditory neural response kinetics and could be the site of conduction failure for excitation initiated on the peripheral process. There is, however, reason to believe that the excitation site in some healthy, type I neurons and in pathological, type III neurons is the central process of the cell. To characterize the neural response to activation at a controlled central process site, cat auditory neurons were stimulated with an intrameatal electrode, and the summation and refraction response kinetics were measured. This approach was used to: (1) characterize the behavior of the neural response to central process excitation; (2) make comparisons between intrameatal excitation at a known central site and scala tympani excitation at an unknown site; and (3) provide membrane characterization free from the possible alteration of membrane kinetics produced by the cell soma. The membrane kinetics measured using intrameatal stimulation differ from those recorded with scala tympani stimulation indicating that the mechanisms for scala tympani and intrameatal stimulation differ.

Evaluation of a model of the cochlear neural membrane. II: comparison of model and physiological measures of membrane properties measured in response to intrameatal electrical stimulation.

This study examines existing equation sets describing neural membrane ionic currents, such as the Hodgkin-Huxley (1952) equations, used to define the membrane currents in a numerical model of the auditory neuron and determines their adequacy for modeling the summation and refraction properties of auditory neurons in response to electrical stimulation. Specifically, the summation and refraction time constants of each equation set are compared to physiological measures of these time constants. Since previous studies have shown the cell body and peripheral process of the auditory neuron may influence the measurement of neural time constants, the physiological time constants used for comparison are those which were recorded using intrameatal electrical stimulation. The intrameatal electrode should stimulate the neuron in an area where the axon has a uniform geometry. Accordingly, the neural model used to duplicate this experiment was also of uniform geometry. Of the membrane equation sets evaluated, none was clearly superior for modeling both the refraction and summation properties of the auditory neuron, though some equation sets were capable of accurately modeling either the refraction or the summation properties, provided operating temperatures were adjusted to provide appropriate kinetics.

Inhibition of the mitogen activated protein kinase pathway potentiates radiation-induced cell killing via cell cycle arrest at the G2/M transition and independently of increased signaling by the JNK/c-Jun pathway.

The ability of low dose ionizing radiation (2 Gy) to modulate the activities of the mitogen activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK1) cascades in human monocytic leukemia (U937/pREP4) cells and in cells over-expressing dominant negative c-Jun (TAM67) (U937/TAM67) was investigated. Radiation exposure caused prolonged ( approximately 1 h) MAPK activations in U937 cells. In contrast, low dose irradiation weakly modulated JNK1 activity in these cells. Inhibition of the MAPK pathway by use of the specific MEK1/2 inhibitor (10 microM PD98059) in both U937/pREP4 and U937/TAM67 cells prior to radiation exposure permitted strong prolonged radiation-induced activations of JNK1. Expression of TAM67 decreased the ability of radiation to cause apoptosis compared to control transfected cells. However, combined MEK1/2 inhibition and radiation exposure in both cell types caused a large decrease in suspension culture growth and a large increase in apoptosis, when compared to either treatment alone. Reduced proliferation after combined irradiation and PD98059 treatment in both cell types correlated with prolonged cell cycle arrest in G2/M phase. Prolonged growth arrest was abolished when MEK1/2 inhibitor was removed 6 h following irradiation, which was associated with a reduction in apoptosis. The ability of MEK1/2 inhibition to cause prolonged G2/M growth arrest was reduced in U937 cells stably transfected with a p21Cip-1/WAF1 antisense construct (U937/p21AS). This data correlated with an enhancement of radiation-induced apoptosis and a reduced ability of MEK1/2 inhibition to potentiate apoptosis. Collectively our data demonstrate that inhibition of MEK1/2 function increases the radiation sensitivity of U937 cells, independently of c-Jun function, and decreases the ability of these cells to recover from the radiation-induced G2/M cell cycle checkpoint arrest. In addition, our data also demonstrate that the ability of MEK1/2 inhibition to potentiate radiation-induced cell death in U937 cells in part requires an ability of cells to express low levels of p21Cip-1/WAF1.

Gemcitabine induces programmed cell death and activates protein kinase C in BG-1 human ovarian cancer cells.

PURPOSE: Cytosine arabinoside induces apoptosis and this cell death process is influenced by protein kinase C signaling events in leukemic cells. We present findings that extend these observations to include another deoxycytidine analog, gemcitabine, which is more potent in solid tumors. METHODS AND RESULTS: Gemcitabine induced programmed cell death in BG-1 human ovarian cancer cells based on biochemical and morphologic analyses. The DNA was fragmented in BG-1 cells exposed to gemcitabine (0.5 microM, 1.0 microM and 10 microM) for 8 h, but gemcitabine treatment did not induce internucleosomal DNA degradation. Scanning and transmission electron microscopy of BG-1 cells showed morphologic changes associated with apoptosis in response to gemcitabine: membrane blebbing, the formation of apoptotic bodies and chromatin condensation. Thus, BG-1 cells undergo programmed cell death in response to gemcitabine treatment without internucleosomal DNA fragmentation. Furthermore, gemcitabine (10 microM) activated protein kinase C in BG-1 cells and the phosphorylation of the endogenous protein kinase C substrate, myristoylated alanine-rich C kinase substrate, was increased following exposure of BG-1 cells to gemcitabine for up to 6 h. Clonogenicity studies with gemcitabine in combination with various protein kinase C-modulating agents demonstrated that gemcitabine cytotoxicity was influenced by protein kinase C signaling events in BG-1 cells. Short-term (1 h) exposure to TPA (1 or 10 nM) followed by gemcitabine (0.5 microM for 4 h) did not alter the response to gemcitabine. However, a 24-h exposure to TPA followed by gemcitabine resulted in synergistic cytotoxicity, while coincubation of TPA with a PKC inhibitor (e.g. bisindolylmaleimide or calphostin-C) in this regimen abrogated the synergistic response. CONCLUSIONS: Based on our findings, it is plausible that gemcitabine therapy could be improved by modulating PKC signaling events linked to drug-induced apoptosis/cytotoxicity.

The effects of gemcitabine and TPA on PKC signaling in BG-1 human ovarian cancer cells.

Protein kinase C (PKC) signaling pathways play an important role in cell survival and anticancer drug-induced apoptosis. We observed in clonogenicity assays of BG-1 human ovarian cancer cells that gemcitabine cytotoxicity was increased synergistically when drug treatment was followed or preceded by a 24-h exposure to 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA). Coincubation of 10 nM TPA with pharmacological inhibitors of PKC abrogated the synergism of TPA and gemcitabine. These observations prompted further investigation of PKC signaling events linked to TPA and gemcitabine cytotoxicity in BG-1 cells. Because PKC isoforms are differentially expressed in various cell types, we determined that BG-1 cells express the alpha, beta, delta, epsilon, and zeta isoforms of PKC. In addition, 1-h exposures to 10 microM gemcitabine triggered cytosol to membrane translocation of PKC isoforms alpha, delta, and epsilon, indicating these isoforms were activated by gemcitabine. We also explored the PKC mechanism(s) responsible for the synergism of TPA and gemcitabine, and determined that treatment with 10 nM TPA for 24 h in BG-1 cells: 1) downregulated PKCdelta and PKCalpha, without affecting PKCepsilon, 2) did not affect cell cycle distribution into S phase. 3) increased extracellular signal-regulated kinase signaling, and 4) increased intracellular alkaline phosphatase activity, a biochemical marker of cellular differentiation. Chronic exposure (24 h) to TPA enhanced gemcitabine cytotoxicity, perhaps by inducing cellular differentiation pathways in BG-1 cells. Therefore, the use of differentiating agents in combination with gemcitabine may improve its clinical efficacy.

Induction of apoptosis by gemcitabine in BG-1 human ovarian cancer cells compared with induction by staurosporine, paclitaxel and cisplatin.

A variety of chemotherapeutic agents induce cell death via apoptosis. We had shown previously that gemcitabine (2',2'-difluorodeoxycytidine) induced an atypical apoptosis in BG-1 human ovarian cancer cells; therefore, further studies were conducted to characterize more precisely gemcitabine-induced apoptosis in BG-1 cells compared to a general inducer of apoptosis, staurosporine. BG-1 cells exposed to 0.5, 1.0 and 10 microM gemcitabine for 8 h, or staurosporine (1.0 microM) for 6 h, exhibited high molecular weight DNA fragmentation (50 kbp); however, only staurosporine treatment produced internucleosomal DNA fragments (200 bp) in a laddered pattern on the agarose gel. Staurosporine (1.0 microM) rapidly induced phosphatidylserine plasma membrane translocation that increased linearly with time as measured by annexin V-FITC binding, and similar kinetics were observed for caspase activation by staurosporine in BG-1 cells. In contrast, 10 microM gemcitabine increased phosphatidylserine expression in a small fraction of cells (5-10%) vs. untreated controls over the course of 48 h and significant caspase activity was detected within 12 h of drug exposure. Time-lapse video microscopy of BG-1 cells exposed to 1.0 microM staurosporine or 10 microM gemcitabine for up to 72 h showed that the morphologic changes and kinetics of cell death induced by these agents differed significantly. We also evaluated the apoptosis induced by paclitaxel (a mitotic poison) and cisplatin (an agent not dependent on cell cycle functions) in BG-1 cells by these methods because these drugs are used clinically to treat ovarian cancer. Our findings demonstrate that the kinetics of apoptotic cell death induced by gemcitabine and other chemotherapeutic agents should be taken into account when designing treatment strategies for ovarian cancer.

Cochlear implant studies at Research Triangle Institute and Duke University Medical Center.

Examples from several areas of cochlear implant research are presented, with emphasis on the continuous interleaved sampling (CIS) approach to speech processor design. Within-subject comparisons of such processors with the compressed analog (CA) approach of the clinical Ineraid device are reviewed, and ongoing similar comparisons with the clinical Nucleus spectral peak (SPEAK) strategy are outlined. Correlations between chronic performance levels with clinical CA processors and initial performance levels with CIS, data on further improvements in performance with chronic use of CIS, and instances of substantial benefit from custom fitting of CIS parameters are presented as examples of findings with immediate clinical implications. New studies are described, involving the measurement of intracochlear evoked potentials in response to cochlear implant stimulation, and the integration of such work with computer modeling studies.

Evaluation of GM-CSF mouthwash for prevention of chemotherapy-induced mucositis: a randomized, double-blind, dose-ranging study.

Uncontrolled clinical trials have shown that parenteral administration of GM-CSF reduces the frequency of chemotherapy-induced mucositis. The mechanism of this effect could be related to acceleration of haematopoiesis and/or increase in functional activation of WBC. We conducted a double-blind, placebo-controlled, dose ranging study of GM-CSF (mol-gramostim) mouthwash in patients with breast cancer during the first treatment cycle of a combination chemotherapy regimen which has historically produced dose-limiting (grade > or = 3) mucositis in approximately 39% of patients. Subjects were randomized to receive either placebo mouthwash (0.1 percent albumin) or one of four concentrations of GM-CSF mouthwash (0.01, 0.1, 1.0 or 10 mcg/ml). The primary endpoint was to evaluate the relationship between dose of GM-CSF mouthwash received and probability of grade > or = 3 mucositis using a logistic model. Solutions were administered four times daily starting within 24 hours of chemotherapy initiation and continuing until the end of the cycle (day 21). Mucositis was assessed on days 1-6, 10, 15 and 21. Day 6 plasma samples were assayed for GM-CSF. Forty-five patients were evaluable for response (nine per dosing group). A 42% risk (15/36) of mucositis grade > or = 3 was evident on day 15 in patients receiving GM-CSF compared to 2 of 9 patients on the placebo arm. No evidence of dose response was found by logistic regression. Five patients had a detectable plasma concentration of GM-CSF (56-209 pg/ml). A positive correlation between GM-CSF dose and leukocyte recovery was noted (P = 0.04).

Active response of a one-dimensional cardiac model with gap junctions to extracellular stimulation.

To study the response of cardiac tissue to electrical stimulation, a one-dimensional model of cardiac tissue has been developed using linear core-conductor theory and the DiFrancesco-Noble model of Purkinje tissue. The cable lies in a restricted extracellular medium and includes a representation of the junctional resistances known to interconnect cardiac cells. Two electrode geometries are considered: (a) a semi-infinite cable with a monopolar electrode at the end of the cable and (b) a terminated cable with one electrode at each end of the cable. In a series of simulations, stimuli of varying magnitude and polarity are applied at three different times during the plateau of the action potential. The results at the stimulus site show that the action potential duration may either decrease or increase in response to the stimulus, depending on the polarity and application time of the stimulus. The spatial behaviour of the cable in response to the stimulus indicates that sites greater than 200 cells from the stimulating electrode are not affected by the stimulus.

The effect of cellular discontinuities on the transient subthreshold response of a one-dimensional cardiac model.

Previous studies have examined the influence of the gap-junction discontinuity on the steady-state response of a cardiac cable to electrical defibrillation. It is important to understand when steady-state conditions may be assumed. For this reason, the transient, subthreshold behavior of a discontinuous cardiac cable is examined in this study. The behavior of the cable reflects two characteristics: 1) the continuous nature of the entire cable and 2) the isolated behavior of individual cells imposed by the junction discontinuity. The results show two effective time constants of activation: a large time constant corresponding to the time constant of a continuous cable of equivalent length, and a small time constant reflecting the rapid activation of an isolated cell. The rapid activation establishes a voltage gradient across each cell of the cable with one end of the cell hyperpolarized and the opposite end depolarized. This pattern of hyperpolarization and depolarization reaches a maximum value in approximately 3 microseconds and may play an important role in the mechanism of defibrillation.

The transient subthreshold response of spherical and cylindrical cell models to extracellular stimulation.

The effect of extracellular stimulation on excitable tissue is evaluated using analytical models. Primary emphasis is placed on the determination of the rate of rise of the membrane potential in response to subthreshold stimulation. Three models are studied: 1) a spherical cell in a uniform electric field, 2) an infinite cylindrical fiber with a point source stimulus, and 3) a finite length cable with sealed ends and a stimulus electrode at each end. Results show that the rate of rise of the transmembrane potential was more rapid than the step response of a space-clamped membrane for all geometries considered. The response of the cylindrical fiber to extracellular stimulation is compared to previously reported studies of the cylindrical fiber response to intracellular stimulation. It is found that the location of the stimulus has little effect on the infinite fiber response. For terminated cables, however, an accurate model of stimulus response must discriminate between intracellular and extracellular stimulation.