Marios Marselos

Dose-Ranging Study of Metronomic Oral Vinorelbine in Patients with Advanced Refractory Cancer

Aim: To determine the safe dose range and pharmacokinetics of metronomic oral vinorelbine and obtain preliminary data on biomarkers and efficacy in patients with advanced cancer. Methods: Successive cohorts of patients received escalated doses of oral vinorelbine given thrice a week until disease progression, unacceptable toxicity (UT), or consent withdrawal. UT was any grade 4 toxicity, or grade 2 or 3 toxicity that would result to longer than 2-week break during the first 2 months of treatment. Blood samples were collected for pharmacokinetics and quantification of angiogenesis regulatory proteins. Results: Sixty-two patients (median age, 60 years) enrolled at six dose levels from 20 to 70 mg and received treatment for median 12.25 weeks (range, 2-216+). Unacceptable toxicity occurred in two of six patients treated at 60 mg (leucopenia grade 4 and epistaxis grade 2) and in one at 70 mg (leucopenia grade 2). The upper metronomic dose was 50 mg. Objective antitumor response documented in eight cases and 32% of patients experienced disease stability for minimum 6 months. Three responders (renal cancer, medullary thyroid carcinoma, and Kaposi sarcoma) received nonstop treatment for over 3 years without overt toxicity. Low pretreatment levels of circulating interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor were found predictors of efficacy. Steady-state concentrations of vinorelbine and its active metabolite ranged from 0.5 to 1.5 ng/mL. Conclusions: Metronomic administration of oral vinorelbine is feasible at doses up to 50 mg thrice a week and can yield sustainable antitumor activity without overt toxicity, probably through antiangiogenic mechanism. Further clinical investigation is warranted. (Clin Cancer Res 2009;15(20):6454–61)

The effect of sclareol on growth and cell cycle progression of human leukemic cell lines.

Sclareol, a labdane-type diterpene, was tested for cytotoxic effect against a panel of established human leukemic cell lines. The compound showed an IC50 lower than 20 microg/ml in most cell lines tested, while it was higher for resting peripheral blood mononuclear leukocytes (PBML). Furthermore, the compound was tested for cytostatic activity against four of the leukemic cell lines used. At a concentration of 20 microg/ml the compound showed a significant cytostatic effect as soon as 4 h after continuous incubation against two from B and two from T lineage cell lines. The morphology and the kind of death induced from sclareol in three cell lines, was also investigated. The effect of sclareol on the cell cycle progression of two cell lines, using flow cytometry, was examined. The results show that sclareol kills cell lines, through the process of apoptosis. The appearance of the apoptotic signs is time and dose dependent. From the flow cytometry experiments, a delay of the cell population on G0/1 seems to take place. This is the first report, that a labdane type diterpene kills tumor cells via a phase specific mechanism which induces apoptosis.

Starvation and phenobarbital treatment effects on drug hydroxylation and glucuronidation in the rat liver and small intestinal mucosa

Abstract Drug hydroxylation and glucuronidation enzyme levels were measured in the liver and small intestinal mucosa of male rats after starvation for 3 days and after starvation combined with phenobarbital treatment (80 mg/kg, 3 days). After simple starvation liver microsomal cytochrome P-450 content and NADPH cytochrome c reductase activity were unaffected, while p -nitroanisole demethylase activity was increased. Specific activities of the UDPglucose dehydrogenase, total β-glucuronidase and 3-hydroxy-acid dehydrogenase were increased, UDPglucuronosyltransferase was unaffected and glucuronolactone dehydrogenase was decreased. When activities were calculated per whole liver, all enzymes tested were decreased during starvation due to the reduction of the liver weight. In the small intestinal mucosa specific enzyme activities were lower in the starved animals, with the exception of UDPglucuronosyl-transferase which was not changed. The excretion into the urine of d -glucaric and l -ascorbic acid, two final products of the glucuronic acid pathway in the rat, was decreased by fasting. Phenobarbital treatment proved more effective in inducing several enzymes in the starved animals than in those fed ad lib . This compensated for the reduction of total activities due to the loss of liver weight. Despite fasting, the excretion into the urine of d -glucaric and l -ascorbic acid was enhanced after treatment with phenobarbital, and d -glucaric acid reached the levels found in normally fed rats. These findings suggest that starvation impairs drug-metabolism in the rat liver and small intestinal mucosa. Inducibility of the drug-metabolizing enzymes, however, is not depressed by this condition, but on the contrary it is markedly enhanced.

Alterations in central monoaminergic neurotrasmission induced by polycyclic aromatic hydrocarbons in rats

SummaryBenzo[α]pyrene (B[α]P) is a product derived from incomplete combustion of organic material and is considered responsible for chemically-induced cancer in humans. In the present study, the levels of noradrenaline (NA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) were measured in the brains of female Wistar rats 6, 12, 24 and 96 h after a single dose of B[α]P (50 mg kg−1 b.w., i.p.) and also after repeated administration of B[α]P (50 mg kg−1 b.w., i.p., 2×wk, 1 mo). The brain regions studied were the striatum, hypothalamus, midbrain and cortex. Catecholamines were measured using high performance liquid chromatography (HPLC) and electrochemical detection. Significant changes were observed in the striatum where NA, DA, DOPAC were decreased after 24 h and HVA was decreased after 6h. In contrast, no major alterations occurred in 5-HT and 5-HIAA. In the hypothalamus, a significant decrease in NA was observed after 96 h. In the midbrain, the most important change observed was the decrease in NA after 24h. A trend toward an increase in 5-HIAA was observed in the cortex after 6 h. The results demonstrate that B[α]P induces alterations in the dopaminergic and serotoninergic systems throughout the brain. These alterations may lead to behavioural and hormonal disturbances.

Evaluation of the cytotoxicity of selected systemic and intravitreally dosed drugs in the cultures of human retinal pigment epithelial cell line and of pig primary retinal pigment epithelial cells

The cytotoxicity of the selected systemic and intravitreally dosed drugs tamoxifen, toremifene, chloroquine, 5-fluorouracil, gentamicin and ganciclovir was studied in retinal pigment epithelium (RPE) in vitro. The cytotoxicity was assayed in the human RPE cell line D407 and the pig RPE cell culture using the WST-1 test, which is an assay of cell proliferation and viability. The effects of experimental conditions on the WST-1 test (cell density, serum content in the culture medium, the exposure time) were evaluated. The EC50 values in tamoxifen-treated D407 cells ranged between 6.7 and 8.9 micromol/l, and in pig RPE cells between 10.1 and 12.2 micromol/l, depending on the cell density used. The corresponding values for toremifene were 7.4 to 11.1 micromol/l in D407 cells and 10.0 to 11.6 micromol/l in pig RPE cells. In chloroquine-treated cells, the EC50 values were 110.0 micromol/l for D407 cells and 58.4 micromol/l for pig RPE cells. Gentamicin and ganciclovir did not show any toxicity in micromolar concentrations. The exposure time was a significant factor, especially when the drug did not induce cell death, but was antiproliferative (5-fluorouracil). Serum protected the cells from the toxic effects of the drugs. Both cell cultures were most sensitive to tamoxifen and toremifene, and next to chloroquine. The drug toxicities obtained in the present study were quite similar in both cell types; that is, the pig RPE cells and the human D 407 cell line, despite the differences in, for example, the growth rate and melanin contents of the cell types. Owing to the homeostatic functions important for the whole neuroretina, RPE is an interesting in vitro model for the evaluation of retinal toxicity, but, in addition to the WST-1 test, more specific tests and markers based on the homeostatic functions of the RPE are needed.

Early maternal deprivation-induced modifications in the neurobiological, neurochemical and behavioral profile of adult rats.

Early maternal deprivation (MD) is an animal model of neurodevelopmental stress associated with a variety of abnormalities during adulthood. The present study investigated specific behavioral, neurochemical and neurobiological parameters related to dopaminergic and serotonergic function in adult rats subjected to early life MD. Behavioral responses, including the reaction to novelty, the response to d-amphetamine (d-AMP) and the susceptibility to apomorphine (APO) were evaluated in adulthood. Dopamine (DA) and serotonin (5-HT) levels, their metabolites along with their turnover ratios were assessed in distinct rat brain regions. The impact of MD on DARPP-32 protein, D2 and 5-HT2A receptor expression was also estimated in the same brain regions during adulthood. Our results indicated that MD rats were more reactive to novelty behavior and more sensitive to dopaminergic agonists compared to controls. MD rats displayed elevated dopaminergic and serotonergic function in the amygdala and prefrontal cortex, whereas in the striatum only the dopaminergic activity was also increased. Interestingly, MD induced a region-dependent modulation of D2, 5-HT2A receptor and DARPP-32 protein expression. Our findings clearly indicated that early MD stress produces long term behavioral impairments and region-dependent modifications in various neurochemical and neurobiological indices of dopaminergic and serotonergic function in brain regions holding critical roles in the pathophysiology of central nervous system disorders.

Individual differences in the effects of cannabinoids on motor activity, dopaminergic activity and DARPP-32 phosphorylation in distinct regions of the brain

This study explored the behavioural, neurochemical and molecular effects of Delta9-tetrahydrocannabinol (Delta9-THC) and WIN55,212-2, in two rat phenotypes, distinguished on the basis of their vertical activity upon exposure to a novel environment, as high responders (HR) and low responders (LR). Motor effects were assessed under habituated vs. non-habituated conditions. Dopaminergic activity and DARPP-32 phosphorylation were measured in the dorsal striatum, nucleus accumbens, prefrontal cortex and amygdala. These cannabinoids influenced motor activity in a biphasic manner, i.e. low doses stimulated, whereas high doses suppressed motor activity. Dopamine (DA) biosynthesis was increased in most brain regions studied following Delta9-THC administration mainly in HR rats, and low-dose WIN55,212-2 increased DA biosynthesis in HR rats only. Both high and low doses of Delta9-THC increased DARPP-32 phosphorylation in most brain regions studied in both phenotypes, an effect that was also observed following high-dose WIN55,212-2 administration only in the striatum. The present results provide further support for a key role of cannabinoids in the regulation of motoric responses and elements of dopaminergic neurotransmission and reveal their complex differential effects in distinct rat phenotypes, as seen with other drugs of abuse.

Pharmaceutical agents known to produce disulfiram-like reaction: effects on hepatic ethanol metabolism and brain monoamines.

Several pharmaceutical agents produce ethanol intolerance, which is often depicted as disulfiram-like reaction. As in the case with disulfiram, the underlying mechanism is believed to be the accumulation of acetaldehyde in the blood, due to inhibition of the hepatic aldehyde dehydrogenases. In the present study, chloramphenicol, furazolidone, metronidazole, and quinacrine, which are reported to produce a disulfiram-like reaction, as well as disulfiram, were administered to Wistar rats and the hepatic activities of alcohol and aldehyde dehydrogenases (1A1 and 2) were determined. The expression of aldehyde dehydrogenase 2 was further assessed by Western blot analysis, while the levels of brain monoamines were also analyzed. Finally, blood acetaldehyde was evaluated after ethanol administration in rats pretreated with disulfiram, chloramphenicol, or quinacrine. The activity of aldehyde dehydrogenase 2 was inhibited by disulfiram, chloramphenicol, and furazolidone, but not by metronidazole or quinacrine. In addition, although well known for metronidazole, quinacrine also did not increase blood acetaldehyde after ethanol administration. The protein expression of aldehyde dehydrogenase 2 was not affected at all. Interestingly, all substances used, except disulfiram, increased the levels of brain serotonin. According to our findings, metronidazole and quinacrine do not produce a typical disulfiram-like reaction, because they do not inhibit hepatic aldehyde dehydrogenase nor increase blood acetaldehyde. Moreover, all tested agents share the common property to enhance brain serotonin, whereas a respective effect of ethanol is well established. Therefore, the ethanol intolerance produced by these agents, either aldehyde dehydrogenase is inhibited or not, could be the result of a “toxic serotonin syndrome,” as in the case of the concomitant use of serotonin-active medications.

Comparison of phenobarbital- and carcinogen-induced aldehyde dehydrogenases in the rat.

Abstract There is a genetically determined variation in the inducibility of a high- K m cytoplasmic aldehyde dehydrogenase activity in the rat liver by treatment with phenobarbital. In the present experiments this activity increased after phenobarbital administration in the phenobarbital-responsive rats also in the intestinal postmitochondrial supernatant fraction. Phenobarbital-nonresponsive rats did not exhibit such an increase after drug treatment. Intraperitoneal administration of 2,3,7,8,-tetrachlorodibenzo- p dioxin, strongly enchanced the cytoplasmic enzyme activity in the liver of both responsive and nonresponsive rats. This effect was also seen in the serum but not in the intestinal or hte kidney. Intragastric administration of 3-methylcholanthrene, 3,4,-benzpyrene or chrysene induced the activity in liver and intestine but not in serum or kidney. The activity in liver was also induced by long-term feeding with 2-acetamido-fluorene. The activities induced by tetrachlorodibenzodioxin or the carcinogens had similar behaviour in isoelectric focusing in gel slabs and in gel chromatography, suggesting a possible common identity of these induced enzymes. The activity induced by these agents could be clearly differentiated both from the activity induced by phenobarbital and from the normal cytoplasmic activities.

Modifications of drug metabolism by disulfiram and diethyldithiocarbamate I. Mixed-function oxygenase

Disulfiram and diethyldithiocarbamate were administered to rats for 4 days alone (300 mg/kg, daily, per os) or in combination with phenobarbital (80 mg/kg, daily, i.p.), in order to observe the effects of these compounds on the microsomal membrane components and on the mixed-function oxygenase system. Both disulfiram and diethyldithiocarbamate increased the liver to body weight ratio, and the total hepatic protein content. Disulfiram significantly increased also the microsomal protein and phospholipid contents. Diethyldithiocarbamate and disulfiram partially prevented the increase of microsomal protein and phospholipid contents caused by phenobarbital. Disulfiram and diethyldithiocarbamate decreased the amount of cytochrome P-450 and P-420, and the activity of p-nitroanisole O-demethylase. These changes were more pronounced after diethyldithiocarbamate than after disulfiram treatment. On the contrary, the activity of NADPH-cytochrome c reductase was enhanced only by disulfiram. The induction by phenobarbital of cytochrome P-450 and p-nitrosanisole O-demethylase was partially prevented on concomitant treatment with disulfiram and diethyldithiocarbamate. These compounds. however, had an additive effect with phenobarbital in enhancing the microsomal NADPH-cytochrome c reductase activity.