Curcumin induces apoptosis in breast cancer cell lines and delays the growth of mammary tumors in neu transgenic mice.

Breast cancer is a leading cancer in women and despite the benefits of the current therapies a significant number of patients with this tumor is at risk of relapse. Some of the alterations taking place in breast cancer cells are currently exploited by molecularly targeted drugs. Different drugs have been developed which target a single molecule but, given that the tumor originates from the dysregulation of many genes, there is the need to find new drugs that have more than one molecular target. Curcumin [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] (CUR), a polyphenolic compound found in the spice turmeric, is a pleiotropic molecule able to interact with a variety of molecular targets and has antitumor, anti-inflammatory, antioxidant, immunomodulatory and antimicrobial activities. Here we demonstrate that CUR inhibits the growth of breast cancer cell lines in a dose dependent manner, with IC50 values in the micromolar range, and induces an increase in the percentage of cells in sub-G0 phase, representing the apoptotic cell population. The activation of apoptosis was confirmed by PARP-1 cleavage and by the increased ratio between the pro-apoptotic Bax and the anti-apoptotic Bcl-2 protein. In addition, in CUR-treated cells the activity of ERK1/ERK2 MAP kinases was down-regulated. The cytotoxic effects of CUR were observed in breast cancer cells expressing either high or low levels of ErbB2/neu. The in vivo antitumor activity of CUR was tested in BALB-neuT mice transgenic for the neu oncogene, which develop atypical hyperplasia of the mammary gland at 6 weeks of age and invasive carcinoma at 16 weeks of age. CUR, administered to mice both early and in an advanced stage of mammary carcinogenesis, induced a significant prolongation of tumor-free survival and a reduction of tumor multiplicity. In addition, CUR administration was safe, since no modification of hematological and clinical chemistry parameters could be observed in BALB-neuT and BALB/c mice treated with this compound for several weeks. These findings support further studies on the therapeutic potential of CUR in combination with standard therapies in breast cancer patients.

Oxidation of methamphetamine and methylenedioxymethamphetamine by CYP2D6.

Methamphetamine (MeAmp) abuse has recently experienced a resurgence and approaches to the treatment of its addiction similar to those used with cocaine have been considered. As the treatment regimes are likely to use drugs whose metabolism is related to that of MeAmp, studies were initiated to establish the enzymology of the fate of MeAmp. This report describes investigations of the role of CYP2D6, the human isoform of the enzyme that catalyzes debrisoquine hydroxylation, in the 4-hydroxylation and N-demethylation of MeAmp. The results of studies with human liver microsomes including those from a genetically poor metabolizer with respect to CYP2D6, showing correlation between MeAmp and metoprolol hydroxylation and MDMA demethylenation, were consistent with a major involvement of CYP2D6 in the aromatic 4-hydroxylation of MeAmp. This was confirmed by studies with recombinant CYP2D6 expressed in yeast, which was also shown to effect the N-demethylation of MeAmp. The rate of the 4-hydroxylation reaction was substantially slower than the demethylenation of MDMA. In contrast to MeAmp, MDMA was not N-demethylated by CYP2D6. Since CYP2D6 participates in the major steps of MeAmp metabolism, pharmacokinetic interactions are likely with other drug substrates proposed for the treatment of MeAmp addiction. Furthermore, the genetic polymorphism associated with the enzyme could manifest itself in abnormal responses to MeAmp.

Percutaneous dilational tracheostomy after anterior cervical spine fixation.

After spinal cord injury, quadriplegic patients generally require tracheostomy for ventilatory support and airway clearance. Early tracheostomy has several advantages over translaryngeal intubation, but in patients who undergo anterior surgical fixation of the spine, it is often delayed until after recovery of the surgical wound. We report the case of a quadriplegic patient who underwent a successful percutaneous dilational tracheostomy with the Ciaglia technique after surgical fixation of the spine. The percutaneous dilational technique minimizes the injury to the adjacent structures of the neck and the risk of stomal infection. Therefore, it should be considered the technique of choice when an early tracheostomy is indicated for quadriplegic patients who have undergone anterior surgical fixation of the cervical spine.

Pharmacokinetic and pharmacodynamic evaluation of phencyclidine and its decadeutero variant.

The role of metabolism in the in vivo actions of phencyclidine (PCP) was examined by comparing deuterium-substituted drug with drug of normal isotopic abundance. PCP elicits two responses that differ in their time course, ataxia, which is observable immediately after dosage, and hypothermia, which peaks approximately 90 to 120 min after drug administration. The role of metabolism in these responses was determined by comparing bioavailabilities of deuterium enriched (d10) and normal (d0) PCP with the two responses. Plasma concentration was determined after the i.v. and i.p. administration of d10 and d0 drug and the bioavailability of the d10 was found to be 1.3 to 1.5 times the d0. The clearance of the d10 was also smaller than the d0. The d10, which is pharmacologically equivalent in vitro, is metabolized more slowly than the d0 in vitro. The pharmacokinetic and pharmacodynamic bioavailabilities exhibited comparable isotope effects, indicating that both responses are due to the actions of the parent drug.

Mechanism-based inhibition of cytochrome P-450 by heterocyclic analogues of phencyclidine.

Three homologues of 1-(1-phenylcyclohexyl)piperidine (PCP) containing the five-, six-, and seven-membered heterocyclic ring (1-(1-phenylcyclohexyl)pyrrolidine (PCPY), PCP, 1-(1-phenylcyclohexyl)hexamethyleneimine (PCHMI) were preincubated with microsomes from phenobarbital-induced rabbit liver. The microsomes were then diluted, an additional charge of NADPH was added, and N-demethylation of benzphetamine was determined. Preincubation of the microsomes with the analogues lowered P-450-dependent N-demethylation by a process that was NADPH-dependent, reduced CO binding to microsomes, and followed pseudo-first order kinetics. The relative rates of inactivation, PCP greater than or equal to PCPY greater than PCHMI, agreed with the order of inhibition of CO binding to reduced microsomes. This mechanism-based inhibition was not observed with phenylcyclohexylamine, indicating that the substituted nitrogen is necessary. The substituted nitrogen must also be part of a heterocyclic ring since the diethylamino analogue of PCP did not exhibit the same type of inhibition a heterocyclic ring is involved. These trends correlated with the expected relative stabilities of the cyclic form of the carbinolamine suggesting that the inhibitory species was formed from the closed ring isomer.

Spectral and inhibitory interactions of (+/-)-3,4-methylenedioxyamphetamine (MDA) and (+/-)-3,4-methylenedioxymethamphetamine (MDMA) with rat hepatic microsomes.

Incubation of racemic methylenedioxyamphetamine (MDA) or methylenedioxymethamphetamine (MDMA) with rat hepatic microsomes, in the presence of NADPH, generated a spectrally observed inhibitory complex with cytochrome P-450. The complex inhibited product formation from MDA and MDMA as well as other P-450 dependent reactions such as benzphetamine demethylation and CO binding. In the absence of NADPH, MDMA and MDA generated type I and type IIa difference spectra, respectively, suggesting differences in their binding to the enzyme active site. The N-demethylation of MDMA was partially inhibited by methimazole suggesting involvement of the hepatic flavin-containing monooxygenase.

Mechanism of oxidation of N-hydroxyphentermine by superoxide.

Cytochrome P-450 oxidizes N-hydroxyphentermine (MPPNHOH) by an indirect pathway involving superoxide. The chemical details of this oxidation, in which N-hydroxyphentermine is converted to 2-methyl-2-nitro-1-phenylpropane (MPPNO2), have been elucidated by examining the interaction of MPPNHOH with superoxide in aqueous and organic solvents. The role of peroxide, hydroperoxy radicals, and oxygen in the reaction was also examined. The results indicate that superoxide itself is oxidizing MPPNHOH to a nitroxide that disproportionates to MPPNHOH and 2-methyl-2-nitroso-1-phenylpropane (MPPNO). MPPNO is then oxidized to MPPNO2 by O2 or hydroperoxide. Two possible mechanisms for the superoxide oxidation were considered, a proton abstraction and a hydrogen atom abstraction. Stoichiometric and oxygen evolution studies favor the hydrogen abstraction pathway.

Role of superoxide in the N-oxidation of N-(2-methyl-1-phenyl-2-propyl)hydroxylamine by the rat liver cytochrome P-450 system.

The N-oxidation of N-(2-methyl-1-phenyl-2-propyl)hydroxylamine (N-hydroxyphentermine, MPPNHOH) and the N-hydroxylation of 2-methyl-1-phenyl-2-propylamine (phentermine) by reconstituted systems that contained cytochromes P-450 purified from rat liver microsomes were demonstrated. The oxidation of MPPNHOH, but not of phentermine, could also be mediated by a superoxide and hydrogen peroxide generating system that contained xanthine and xanthine oxidase. Superoxide dismutase completely inhibited the oxidation of MPPNHOH by the xanthine/xanthine oxidase system and inhibited by 70% the oxidation mediated by a reconstituted cytochrome P-450 oxidase system. The majority of the microsomal oxidation was inhibited by an antibody raised against the major isozyme of cytochrome P-450 purified from livers of phenobarbital-pretreated rats. 2-Methyl-2-nitroso-1-phenylpropane (MPPNO) was found to be an intermediate in the overall oxidation of MPPNHOH to 2-methyl-2-nitro-1-phenylpropane (MPPNO2). Superoxide dismutase appeared to inhibit the first step, the conversion of MPPNHOH to MPPNO. These observations are accounted for by a sequence of two mechanistically distinct P-450-mediated oxidations. In the first reaction, N-hydroxylation of phentermine occurs by a normal cytochrome P-450 pathway. The formed hydroxylamine then uncouples the cytochrome P-450 system to generate superoxide and hydrogen peroxide. The superoxide oxidizes MPPNHOH to MPPNO which is then oxidized to MPPNO2, the ultimate product. This superoxide-mediated oxidation represents another pathway for N-oxidation by cytochrome P-450.

Nerve impulse propagation along central and peripheral fast conducting motor and sensory pathways in man.

Forty-four limbs from 11 healthy volunteers were examined. Spinal and scalp somatosensory evoked potentials to median and peroneal nerve stimulation were recorded and the peripheral (wrist-Erb, Erb-cervical, knee-thoracic spine) and central (cervical-scalp, thoracic-cervical spine, spine-scalp) conduction times and velocities (CTs, CVs) were calculated. Sensory and mixed trunks of median and peroneal nerves were also stimulated and their motor and sensory CVs in mid-distal districts were measured. Motor responses to scalp (motor areas for hand and leg muscles) and spinal cord stimulation (cervical and lower thoracic levels) were carried out through skin rectangular plate electrodes delivering high voltage (880-1870 V) brief anodal pulses. The intracranial (scalp-cervical) and intraspinal (cervical-thoracic spine) CTs and CVs of motor pathways were measured. The elbow-cervical and knee-thoracic spines CTs of motor fibres were also calculated through the F wave method, which gave values almost superimposable on those obtained through direct spine stimulation. Nerve propagation was faster in sensory than in motor fibres in peripheral nerve mid-distal districts, while this difference was reduced or reversed in more proximal segments, including nerve roots. The scalp-cervical CT was slightly shorter in motor than in sensory fibres after subtraction of synaptic delays (6.12 vs. 6.18 msec). The scalp-lower thoracic spine, as well as the intraspinal, CVs were 7-12% faster in sensory than in motor pathways (45.3 vs. 38.7 m/sec for the former; 62.65 vs. 55.4 m/sec for the latter). The reported method allows the evaluation of fast conducting motor and sensory pathways along 'central' and 'peripheral' nerve structures of the entire body. Preliminary findings on scalp stimulation of brain motor areas with low voltage pulses are also included.

Short-latency scalp somatosensory evoked potentials and central spine to scalp propagation characteristics during peroneal and median nerve stimulation in multiple sclerosis.

Peripheral (cauda-lumbar, wrist-Erb, Erb-cervical) and central (cauda-vertex, cervical-scalp) nervous impulse propagation velocities and times to peroneal and median nerve stimulation were investigated in 34 patients suffering from definite (17 cases), probable (6 cases) and possible (11 cases) forms of multiple sclerosis (MS). In 6 cases short- and intermediate-latency scalp somatosensory evoked potentials to peroneal nerve stimulation were recorded with 'open' (1-5000 Hz, -6 dB) bandpass filters and subsequently digitally filtered through a 'narrow' bandpass (200-5000 Hz, -6 dB). The lumbar response was abnormal in 2.95% of legs, while the Erb response was always within normal limits. The cauda-vertex conduction was altered in 75% of the examined limbs (86.2% definite, 58.3% probable, 63.6% possible MS). Absent scalp responses to peroneal stimulation were often encountered during narrow bandpass recording (54.9%), while a slowed central conduction was less frequent (33.3%). Scalp responses when recorded with open bandpass were always identifiable, being delayed in 3 out of 6 cases. In 5 of these the short-latency wavelets were either absent or showed a prolonged interpeak time even when open filter records were normal. Median nerve SEPs were altered in 60.3% of cases, more frequently because of a delayed scalp response or of a prolonged cervical-scalp conduction time than because of an absent cervical or scalp response. When peroneal and median nerve data were considered together, the rate of abnormality rose to 88.2% of patients. Due to their length, afferent pathways from the lower limb might suffer from a loss of high frequency impulse coding as an early sign of defective impulse propagation.

Parkinsonism, basal ganglia calcification and epilepsy as late complications of postoperative hypoparathyroidism.

A patient with post-thyroidectomy hypoparathyroidism, basal ganglia calcification, parkinsonism and seizures is reported. The parkinsonism was resistant to levodopa therapy but was not significantly improved by the correction of hypoparathyroidism. Previously reported cases are discussed, as well as the relationship between hypoparathyroidism, calcification of basal ganglia, parkinsonism and epilepsy.

Effects of intravenously administered L-acetylcarnitine on somatosensory-evoked potentials. Studies of healthy and diseased volunteers with focal cerebral lesions.

The acute effects of intravenously administered L-acetylcarnitine (L-AC) were evaluated in 5 healthy and 20 diseased volunteers (17 vascular, 3 tumoral cerebral lesions). Short-latency scalp somatosensory-evoked potentials (SEPs) to simultaneous median, and separate unilateral peroneal nerve stimulation were carried out before and after L-AC administration (at 10-, 30- and 60-min intervals). L-AC did not influence peak and interpeak latencies; however, in a percentage of healthy and diseased volunteers a clear-cut amplitude increase was evident affecting all those peaks generated between the thalamus and the cortex. While in normal and tumoral volunteers the voltage increase was bilaterally balanced, the amplitude increments were more evident on the 'affected' hemisphere in vascular patients, partially reversing the abnormal amplitude ratios between homologous peaks on 'healthy' and 'affected' hemispheres. In no case were transient clinical changes, either of an objective or subjective nature, associated with SEP amplitude changes; these were still present at the 60th minute, having reached their nadir at the 30th minute in 'responders'.

Brain-stem auditory evoked responses (BAERs) in patients with chronic renal failure.

Brain-stem auditory evoked responses have been investigated in 28 patients suffering from chronic renal failure (17 on diet, 11 on chronic dialysis) during monaural stimulation with 70 dB HL clicks. These responses were abnormal in 32.15% of cases. In 13 out of 15 years with altered BAERs a normal response morphology was present, with progressively more significant latency delays involving all peaks following wave I, while in the remaining two ears some of the components always found in healthy controls were not identifiable. The most prominent abnormality in patients with symptoms referable to peripheral neuropathy was the presence of a prolonged I-II interpeak latency, while in those without symptomatology the I-V and III-V interpeak times and the peak V latency were the most strikingly altered parameters. The incidence of altered BAERs was higher in the group of patients treated with diet than in the dialysed group. When BAERs were followed-up in the hours preceding and following the first dialysis in two patients not included in the previous group, the I-V interpeak time significantly decreased after 26 h from the end of dialysis.

Nervous impulse propagation along peripheral and central fibres in patients with chronic renal failure.

In thirty-one patients with chronic renal failure we measured the conduction velocities in the proximal regions of median and peroneal nerves and concurrently evaluated the propagation properties of an afferent input along the related central (spinal and supraspinal) sensory pathways. In 26 of these patients the sensorimotor velocities of median, peroneal and sural nerves were also calculated. Lower limb peripheral conduction was more frequently abnormal than that of upper limb, the distal segment being involved to a greater degree. Since central propagation was more impaired during lower (11 out of 27 cases) than during upper limb (1 out of 27 patients) stimulation and the intracranial propagation time was rarely prolonged, a predominantly spinal defective impulse conduction was hypothesized. Neurophysiological data seem to confirm that early stages of defective neural conduction in CRF are in agreement with the model of central-peripheral distal axonopathy. However, an abnormal impulse propagation was also rarely found in proximal districts of peripheral fibres as well as along the intracranial sensory pathways. Correlations were found between some of the impulse propagation properties and the duration of CRF or dialysis. Serum parathyroid hormone, creatinine and BUN levels were correlated with some peripheral and central conduction indexes.

Phencyclidine metabolism in vitro. The formation of a carbinolamine and its metabolites by rabbit liver preparations.

Four products of the in vitro oxidative metabolism of the piperidine ring of phencyclidine, 5-(1-phenylcyclohexylamino)valeraldehyde, V; N-(1-phenylcyclohexyl)-1,2,3,4-tetrahydropyridine, VIII; 5-(1-phenylcyclohexylamino)valeric acid, VII; and 1-phenylcyclohexylamine, IX, have been identified following derivatization, by GC/MS with stable isotope labeling and/or synthesis. The enamine, VIII, may be a work-up elimination product of a carbinolamine, alpha-hydroxy-N-(1-phenylcyclohexyl)piperidine, IV. The formation of all metabolites requires microsomal enzymes, but VII and the previously described N-(5-hydroxypentyl)-1-phenylcyclohexylamine, VI, also require soluble enzymes. Quantitative or semiquantitative data show that VI and VIII appear and disappear with time, whereas VII seems to be a terminal metabolite.

The metabolism of (R)-(-)-amphetamine by rabbit liver microsomes. Initial products.

The metabolism of 1-amphetamine in rabbit liver is complex in that several routes may give rise to the same intermediate. In this study, the subsequent metabolism of the primary products of N- and C-oxidation were blocked by selecting appropriate incubation conditions. The resulting simplified system was used to investigate the enzymes involved in the two pathways. Phenobarbital treatment increased N- and C-hydroxylation, whereas 3-methylcholanthrene treatment had an inhibitory effect on both pathways. Inhibitors of cytochrome P-450 were either nonselective or were partially selective in inhibiting the two routes of amphetamine metabolism. Induction modulated the sensitivity toward the inhibitors of N- and C-oxidation.

VM-26 monochemotherapy trial in the treatment of recurrent supratentorial gliomas: preliminary report.

Twenty-five adults with recurrent supratentorial malignant gliomas were treated with VM-26 in a monochemotherapy schedule, the antiblastic region covering a period of 24 weeks. Five cases were excluded from the final evaluation, 4 because of protocol violations and 1 due to intolerance to the drug. In the group of 20 patients who met the criteria for evaluation, 7 (35%) showed a partial or complete response, with clinical or radiological tumor aggression, and the conditions of 7 (35%) were unchanged at the end of the chemotherapy courses. The progression-free intervals turned out to be 10.4 months and 8.5 months, respectively. Six patients (30%) complained of tumor progression despite the treatment. Most of the patients are still alive, and the actuarial survival rate is encouraging. Toxicity was mainly hematologic, but usually moderate and easily reversible.

Multidisciplinary treatment of medulloblastoma: a 5-year experience with the SIOP trial.

A 5-year experience of the multidisciplinary treatment of pediatric medulloblastoma with the SIOP (International Society of Pediatric Oncology) trial is presented. 33 eligible patients finally reduced to a total of 30 evaluative cases were treated with major surgical resection, extensive irradiation, and combined chemotherapy (vincristine + CCNU). The overall survival rate without recurrence was encouraging, and the actuarial survival rate is satisfactory. At follow-up controls, most of the patients showed a good performance status and a promising neurological stage. However, the problem concerning quality of life remains unsolved: morbidity and sequelae following high-dose radiotherapy and concomitant antiblastic treatment were noticeable.

A gas-liquid chromatography assay for phencyclidine and its metabolites.

A GLC assay for phencyclidine (PCP) is described, which also simultaneously measures three primary hydroxylated metabolites formed from incubating PCP in tissue homogenates. Using the FID detector, the limits of reliable detection of PCP and both monohydroxy metabolites, 4-phenyl-4-piperidino-cyclohexanol, 2, and 1-(1-phenylcyclohexyl)-4-hydroxypiperidine, 3, are 0.02 mumol per injection and 0.05 mumol for the dihydroxy metabolite, 4-(4'-hydroxypiperidino)-4-phenylcyclohexanol, 2A. Baseline separation of an compounds was achieved and coefficients of variation (between-run) was 3-6% for PCP, and both monohydroxy metabolites, and 12% for the dihydroxy metabolite. A GCMS assay is also reported herein for the analysis of PCP at low levels, and can detect 5 pmol per injection of PCP, with a linear standard curve from 50 to 2000 pmol.