Dactolisib (NVP-BEZ235) toxicity in murine brain tumour models.

BACKGROUND: Glioblastomas (GBMs) are highly malignant brain tumours with a poor prognosis, and current cytotoxic regimens provide only a limited survival benefit. The PI3K/Akt/mTOR pathway has been an attractive target for therapy due to its high activation in GBMs as well as other cancers. The dual pan-PI3K/mTOR kinase inhibitor dactolisib (NVP-BEZ235) is an anti-neoplastic compound currently under investigation. However, little is known about its efficacy in human GBMs. We aimed at evaluating the efficacy of dactolisib in human glioblastoma cells, as well as in murine models carrying human GBM xenografts. METHODS: To assess the effect of dactolisib in vitro, MTS assay, manual cell count, BrdU incorporation and Annexin V staining experiments were used to observe growth and apoptosis. Furthermore, Akt phosphorylation (S473), a downstream target of PI3K, was explored by western blotting. Animal studies utilizing orthotopic xenograft models of glioblastoma were performed in nude rats and NOD/SCID mice to monitor survival benefit or inhibition of tumor growth. RESULTS: We found that dactolisib in vitro shows excellent dose dependent anti-growth properties and increase in apoptosis. Moreover, dose dependent inhibition of Akt phosphorylation (S473), a downstream effect of PI3K, was observed by western blotting. However, in two independent animal studies utilizing nude rats and NOD/SCID mice in orthotopic xenograft models of glioblastoma, we observed no survival benefit or inhibition of tumour growth. Severe side effects were observed, such as elevated levels of blood glucose and the liver enzyme alanine transaminase (ALT), in addition to diarrhoea, hair loss (alopecia), skin rash and accumulation of saliva in the oral cavity. CONCLUSION: Taken together, our results suggest that despite the anti-neoplastic efficacy of dactolisib in glioma treatment in vitro, its utility in vivo is questionable due to toxicity.

Treatment with the PI3K inhibitor buparlisib (NVP-BKM120) suppresses the growth of established patient-derived GBM xenografts and prolongs survival in nude rats.

Glioblastomas (GBMs) are aggressive brain tumours with a dismal prognosis, despite combined surgery, radio- and chemotherapy. Close to 90 % of all GBMs harbour a deregulated PI3K pathway, which is essential in regulating central cellular functions such as proliferation, cell growth, motility and survival. Thus, PI3K represents a potential target for molecular therapy in GBM. We investigated the anti-tumour efficacy of the PI3K inhibitor buparlisib (NVP-BKM120) in GBM cell lines in vitro and in vivo, when treatment was initiated after MRI-confirmed tumour engraftment. We found that buparlisib inhibited glioma cell proliferation in a dose dependent manner, demonstrated by MTS assay, manual cell count and BrdU incorporation. A dose dependent increase in apoptosis was observed through flow cytometric analysis. Furthermore, by immunocytochemistry and western blot, we found a dose dependent inhibition of Akt phosphorylation. Moreover, buparlisib prolonged survival of nude rats harboring human GBM xenografts in three independent studies and reduced the tumours' volumetric increase, as determined by MRI. In addition, histological analyses of xenograft rat brains showed necrotic areas and change in tumour cell nuclei in buparlisib-treated animals. The rats receiving buparlisib maintained their weight, activity level and food- and water intake. In conclusion, buparlisib effectively inhibits glioma cell proliferation in vitro and growth of human GBM xenografts in nude rats. Moreover, the compound is well tolerated when administered at doses providing anti-tumour efficacy. Thus, buparlisib may have a future role in glioma therapy, and further studies are warranted to validate this compound for human use.

Drug repurposing: sulfasalazine sensitizes gliomas to gamma knife radiosurgery by blocking cystine uptake through system Xc-, leading to glutathione depletion.

Glioblastomas (GBMs) are aggressive brain tumors that always recur after radiotherapy. Cystine, mainly provided by the system X(c)(-) antiporter, is a requirement for glioma cell synthesis of glutathione (GSH) which has a critical role in scavenging free radicals, for example, after radiotherapy. Thus, we hypothesized that the X(c)(-)-inhibitor sulfasalazine (SAS) could potentiate the efficacy of radiotherapy against gliomas. Here, we show that the catalytic subunit of system X(c)(-), xCT, was uniformly expressed in a panel of 30 human GBM biopsies. SAS treatment significantly reduced cystine uptake and GSH levels, whereas it significantly increased the levels of reactive oxygen species (ROS) in glioma cells in vitro. Furthermore, SAS and radiation synergistically increased DNA double-strand breaks and increased glioma cell death, whereas adding the antioxidant N-acetyl-L-cysteine (NAC) reversed cell death. Moreover, SAS and gamma knife radiosurgery (GKRS) synergistically prolonged survival in nude rats harboring human GBM xenografts, compared with controls or either treatment alone. In conclusion, SAS effectively blocks cystine uptake in glioma cells in vitro, leading to GSH depletion and increased ROS levels, DNA damage and cell death. Moreover, it potentiates the anti-tumor efficacy of GKRS in rats with human GBM xenografts, providing a survival benefit. Thus, SAS may have a role as a radiosensitizer to enhance the efficacy of current radiotherapies for glioma patients.

A comparison of actual registered costs and costs derived from diagnosis-related groups (DRGs) for patients undergoing heart transplantation, lung transplantation, and thoracotomy for other lung diseases.

The Norwegian health care system, like other health care systems in the world, is in the midst of a changing financial environment for hospital reimbursement for patient care. Since 1997 the Norwegian government has introduced a new financing model of block grant and activity-based financing. In this model, diagnosis-related groups (DRGs) play an important role in hospital financing. The initial motive for developing the DRGs was to improve hospital productivity and efficiency and to develop a tool to control increasing hospital costs better. We raised the question as to whether the DRG system in fact covers actual costs in patient groups undergoing heart transplantation (n = 12), lung transplantation (n = 4), and thoracotomy for other diseases (n = 10). A new prospective cost model was developed to measure actual costs related to individual patients. The patients were closely observed and the related data collected during the hospital stay. Each patient's hospital stay was divided into four different categories of resource requirements, defined as heavy intensive care, light intensive care, intermediate care, and ordinary care. In addition, the number of staff involved and the duration of surgery and procedures were recorded, as were medicine costs and material costs. Based on these data, the actual costs for each patient were calculated. These were then compared with the respective DRG reimbursement (100 % coverage) for the corresponding group. We found that the median cost for heart transplantation was US$ 50,590 (1 US$ = 7.5 NOK based on the exchange rate at the time of the study), while the respective DRG reimbursement was US$ 65,662. For lung transplantation, the respective figures were US$ 46,668 vs US$ 65,662, and for thoracotomy, US$ 24,307 vs. US$ 11,004. We found that our method was applicable to a hospital setting. DRG coverage for heart and lung transplantation seems to overestimate the actual costs. For the thoracotomy procedure, the DRG coverage did not cover the actual costs.

Effects of valine, leucine, isoleucine, and a balanced amino acid solution on the seizure threshold to picrotoxin in rats.

During infusion of branched-chain amino acids (BCAAs) in humans, changes in ventilatory drive, sleeping pattern, and appetite have been reported. The mechanism by which BCAA exerts their effects on CNS remains unclear. An infusion of a BCAA solution (300 mg/kg) has previously been found to increase the seizure threshold in rats to the proconvulsant drug picrotoxin, an antagonist on the GABA-benzodiazepine receptor complex. In this study, each of the BCAAs given separately (valine, leucine, isoleucine; 300 mg/kg) (n = 10) increased the mean latency time to onset of seizures vs. placebo as an indication of an increased seizure threshold. A balanced amino acid solution (Vamin-Glucose) had no effect on the seizure threshold. Thus, these CNS effects are specific for BCAAs and occur with all three.

Branched-chain amino acids increase the seizure threshold to picrotoxin in rats.

During infusion of branched-chain amino acids (BCAAs) in humans, changes in ventilatory drive, appetite, and sleep have been reported. The mechanism by which BCAAs exert their effects on CNS remains unclear. Picrotoxin is a proconvulsant drug, acting as an antagonist on the GABA-benzodiazepine receptor complex. Twenty rats were randomized to receive either an IP injection with 4% BCAAs (300 mg/kg; 8 ml/kg) (n = 10) or placebo (saline 8 ml/kg) (n = 10). The mean latency time from injection to onset of seizures was recorded as an indication of the seizure threshold. Latency time was significantly longer for BCAAs than for placebo, 11.2 (+/- 1.9) vs. 8.3 (+/- 1.8) min. Thus, a BCAA injection increased the seizure threshold to picrotoxin (p < 0.03). This suggests that BCAA infusion may exert effects on the GABA-benzodiazepine receptor complex.

Branched-chain amino acids and respiration.

A series of investigations suggest a specific role for BCAA in the regulation of respiration. In vitro incubation studies have shown that BCAAs improve the recovery of muscle force after fatigue. Further investigations revealed that leucine plays a key role in this action and acts in a manner not dependent on its use as an energy substrate. In humans, solutions enriched with BCAA have decreased PCO2 and stimulated the ventilatory response to hypercapnia, thereby corresponding to an enhanced ventilatory sensitivity with the administration of BCAA. The mechanisms for these actions are unknown. The most viable hypothesis is based on the ability of BCAA to decrease the synthesis of serotonin due to altered transport of AAs, including tryptophan, to the brain. Clinical studies have suggested a potency of BCAA in the treatment of respiratory dysfunction of preterm infants, as well as of patients with sleep apnea related to various disease states. The clinical applications of BCAA-enriched mixtures in respiratory diseases are still experimental, and many controversies exist concerning the validity of BCAA in clinical practice. Most TPN regimens contain BCAA approximating the average intake of BCAA in the Western diet. The question therefore remains whether additional BCAA supplementation is useful to achieve the suggested metabolic and pharmacological effects. Meticulous future studies are needed to establish the therapeutic value of BCAA in the treatment of various respiratory functions.

Long-term use of gamma-linolenic acid-enriched fat emulsion (PFE 4501): case report.

A new gamma-linolenic acid-enriched fat emulsion (PFE 4501) was given for 13 mo as part of a home parenteral nutrition regimen to a 28-yr-old patient with cystic fibrosis. Blood biochemistry, hematological data, and fatty acid profile were followed. No side effects were reported, and pulmonary function tests remained stable. gamma-Linolenic acid appears to be safe for clinical use and may benefit patients with derangements in essential fatty acid metabolism. The potential role of a gamma-linolenic acid-enriched intravenous fat emulsion is discussed.

Branched-chain amino acid in chronic renal failure patients: respiratory and sleep effects.

Sleep disorders, including a high incidence of sleep apnea, have been recognized as a significant problem in chronic renal failure (CRF) patients. In a preliminary study, we examined CRF patients on maintenance hemodialysis for three nights; one control night, and thereafter randomized to infusion of saline (placebo) for one night and 4% branch-chain amino acid (BCAA) solution for one night. Polysomnographic and respiratory data [respiratory rate, oxygen saturation and end-tidal CO2 (ETCO2)] was recorded continuously throughout the nights and data from each hour compared with baseline (awake) values. The patients studied were characterized by reduced sleep quality and decreased amount of rapid eye movement (REM) sleep. The BCAA infusion was associated with a return of REM sleep to normal and a significant decrease in ETCO2 during both REM and non-REM sleep (P less than 0.05). Our findings demonstrate respiratory stimulation during sleep with infusion of BCAA; this stimulatory effect on respiration (in contrast to many respiratory stimulants) is associated with an increased amount of REM sleep.

Parenteral nutrition and oral intake: effect of glucose and fat infusions.

The effect of intravenous nutrition on voluntary oral intake was studied in healthy male volunteers. Subjects were confined to the Surgical Metabolic Unit for the 17 to 19 day study and were restricted to commercial liquid diet. Each study consisted of three consecutive phases: (1) Ringer's lactate (RL), (2) peripheral parenteral nutrition (PPN) administered for 5 or 6 days as a combination of glucose (caloric load equal to 34% resting energy expenditure, REE), fat (34% REE), and amino acids (17% REE) or a single nutrient infusion of glucose (68% REE), fat (68% REE), glucose (34% REE), or fat (34% REE), and (3) RL for the third period. When all three nutrients or glucose alone (68% REE) were given, subjects decreased daily voluntary food intake within 24 to 48 hr by an amount that closely compensated for the infused calories. Intake was reduced by only 20% to 40% of the infused calories when fat alone (68% REE) was given. There were no significant effects when the lower levels of glucose and fat were given. These data suggest the presence of a postabsorptive control of food intake in humans that is sensitive to the circulating supply of fuels.

Parenteral nutrition and oral intake: effect of branched-chain amino acids.

The effect of peripheral parenteral nutrition (PPN) on voluntary food intake was examined in healthy male subjects. Each study (lasting 17 to 19 days) was divided into three phases: Ringer's lactate (RL); PPN administered as a combination of glucose, fat, and amino acids; and finally RL. During the middle phase, some of the subjects received a parenteral formula in which half of the amino acids had been replaced with the branched-chain amino acids (BCPPN). When PPN was infused, subjects reduced their food intake within 48 hours by approximately 80% of the infused calories (p less than 0.001) within 48 hours, whereas intake was reduced by less than 40% of the infused calories when BCPPN was infused. Use of branched-chain amino acid-enriched parenteral nutrition may minimize the reduction in food intake seen during intravenous nutrition, possibly hastening a return to normal eating.

Antagonism of flunitrazepam-induced sedative effects by flumazenil in patients after surgery under general anaesthesia. A double-blind placebo-controlled investigation of efficacy and safety.

This study aimed to assess the efficacy and safety of flumazenil and a placebo in reversing the residual effects of flunitrazepam used to induce anaesthesia for elective laparoscopy in 49 female patients aged 16 to 52 years. In contrast to the placebo, flumazenil gave reductions in amnesia, sedation score, mood rating for mental sedation and physical sedation, and time taken to complete a psychomotor performance test which lasted throughout the study. There were no significant changes in pulse rate, respiration rate or blood pressure, and no unwanted effects were attributed to flumazenil.

Intravenous fat emulsions and lung function: a review.

Numerous studies have reported varying degrees of apparent pulmonary dysfunction when iv fat emulsions (IVFE) are given. These changes have generally not been of sufficient magnitude to carry clinical significance. The lung dysfunction observed has been attributed to an associated hyperlipemia. Recent studies, however, suggest that the associated impairment in lung function is due to alterations in pulmonary vascular tone (which results in ventilation/perfusion inequalities) caused by an IVFE-related increase in prostaglandin (PG) production. The polyunsaturated fatty acids in the IVFE serve as precursors to the PGs. Due to the varied effects of PGs on inflammation and pulmonary vasomotor tone, infusion of IVFE could have profound physiologic and pharmacologic actions aside from the provision of lipid calories. In some circumstances, IVFE may, in fact, be beneficial to the lung via alterations in

Long-term bupivacaine infusion does not alter the acute seizure threshold to bupivacaine in rats.

Rats were given a continuous subcutaneous infusion of either bupivacaine (n = 18) or placebo (n = 17) for 4 days. On the 5th day the acute seizure threshold to intravenous bupivacaine was determined in both groups. Blood and brain concentrations of bupivacaine at the onset of seizures showed no difference between the two groups, whereas the seizure dose was decreased in the bupivacaine group. The tachyphylaxis observed when bupivacaine is used for a regional nerve blockade does not seem to evolve for the central nervous system effects.

Improved exercise tolerance with long-term parenteral nutrition in cystic fibrosis.

Two cystic fibrosis (CF) patients with severe pulmonary disease and malnutrition were followed during a course of long-term home total parenteral nutrition (TPN), which included iv fat emulsion. They gained 7 and 12 kg in body weight, respectively, and their ability to participate in daily activities increased. Progressive exercise testing before and during TPN showed a marked increase in maximal work load: 100% and 30%, respectively. At any given work load, oxygen uptake was increased while the respiratory quotient remained lower after the course of TPN. We believe that the lower respiratory quotient and greater oxygen consumption during exercise were due to a reduction in anaerobic metabolism after TPN.

Antagonism of diazepam-induced sedative effects by Ro15-1788 in patients after surgery under lumbar epidural block. A double-blind placebo-controlled investigation of efficacy and safety.

The aim of this study was to assess the efficacy of Ro15-1788 and a placebo in reversing diazepam-induced effects after surgery under epidural block, and to evaluate the local tolerance and general safety of Ro15-1788. Fifty-seven patients were sedated with diazepam for surgery under epidural anaesthesia. Antagonism of diazepam-induced effects by Ro15-1788 was investigated postoperatively in a double-blind placebo-controlled trial. The patient's subjective assessment of mood rating, an objective test of performance, a test for amnesia, and vital signs were recorded for up to 300 min after administration of the trial drug. No significant differences between the two groups were observed for mood rating, amnesia, or vital signs. The Ro15-1788 group showed a significant improvement in the performance test up to 120 min after administration of the drug. There was no evidence of reaction at the injection site.