Efficacy and safety of bevacizumab in glioblastomas.

Glioblastoma multiforme (GBM) is a common and malignant primary brain tumor arising from glial precursors the survival of which is estimated to be about 14 months after diagnosis despite current standard care with radiotherapy, surgery, and chemotherapies. Therapeutic approaches were greatly improved in the last years; however, GBM still represents the most lethal subtype of glioma. Actually, it has been estimated that only about 3.4% of patients will survive at the most five years when obtaining the best outcome from treatment; however, this depends on tumor resistance, which is generally related to repairing radiation injury, and self- improving cell growth repair and survival. All GBMs recur after initial therapy, limiting patients � survival at 20-25% within 1 year after diagnosis of recurrent disease. Moreover, for recurrent GBM response rates are less than 10% (ranging from 5% to 9%), and progression free survival at 6-month (PFS-6) rates ranges between 9% and 28% (median 15%). The development of targeted therapy based on tumor vascular blockade led to the approval of bevacizumab for recurrent or progressive glioblastoma, since it was proven that this offers a new opportunity for patients suffering from this malignancy. Bevacizumab is a recombinant antivascular monoclonal antibody binding to circulating Vascular Endothelial Growth Factor (VEGF) preventing this cytokine from reaching its receptors (VEGFR1 and VEGFR2) on endothelium, resulting in an inhibition of cells proliferation and vessels sprouting. The aim of this review is to address bevacizumab mode of action in malignant gliomas and provide a summary on currently available data on efficacy and safety.

Phase II study of ET-743 in advanced soft tissue sarcomas: a European Organisation for the Research and Treatment of Cancer (EORTC) soft tissue and bone sarcoma group trial.

PURPOSE: This nonrandomized multicenter phase II study was performed to evaluate the activity and safety of Ecteinascidin (ET-743) administered at a dose of 1.5 mg/m(2) as a 24-hour continuous infusion every 3 weeks in patients with pretreated advanced soft tissue sarcoma. PATIENTS AND METHODS: Patients with documented progressive advanced soft tissue sarcoma received ET-743 as second- or third-line chemotherapy. Antitumor activity was evaluated every 6 weeks until progression, excessive toxicity, or patient refusal. RESULTS: One hundred four patients from eight European institutions were included in the study (March 1999 to November 2000). A total of 410 cycles were administered in 99 assessable patients. Toxicity mainly involved reversible grade 3 to 4 asymptomatic elevation of transaminases in 40% of patients, and grade 3 to 4 neutropenia was observed in 52% of patients. There were eight partial responses (PR; objective regression rate, 8%), 45 no change (NC; > 6 months in 26% of patients), and 39 progressive disease. A progression arrest rate (PR + NC) of 56% was observed in leiomyosarcoma and 61% in synovialosarcoma. The median duration of the time to progression was 105 days, and the 6-month progression-free survival was 29%. The median duration of survival was 9.2 months. CONCLUSION: ET-743 seems to be a promising active agent in advanced soft tissue sarcoma, with no cumulative toxicities. The 6-months progression-free survival observed in advanced soft tissue sarcoma compares favorably with those obtained with other active drugs tested in second-line chemotherapy in previous European Organisation for the Research and Treatment of Cancer trials. The median overall survival was unusually long in these heavily pretreated patients mainly due to the high number of patients who benefit from the drug in terms of tumor control.

Imatinib mesylate (STI-571 Glivec, Gleevec) is an active agent for gastrointestinal stromal tumours, but does not yield responses in other soft-tissue sarcomas that are unselected for a molecular target. Results from an EORTC Soft Tissue and Bone Sarcoma Group phase II study.

The aim of this study was to assess the antitumour response and time to progression (TTP) of patients treated with imatinib mesylate (Glivec, Gleevec, formerly STI-571) who had advanced and/or metastatic gastrointestinal stroma tumours (GIST) or other soft tissue sarcomas (STS). Patients with measurable lesions and adequate organ function were entered. They were treated with imatinib mesylate at the dose of 400 mg twice daily (bid). All tumours were subject to a stringent pathological review by an expert panel. Immunohistochemical expression of KIT expression was evaluated. A total of 51 patients (27 GIST, 24 other STS), median age 53 years, median World Health Organization (WHO) performance score 1, were entered. 71% of the patients had received prior chemotherapy. The most frequent side-effects were anaemia (92%), periorbital oedema (84%), skin rash (69%), fatigue (76%), nausea (57%), granulocytopenia (47%) and diarrhoea (47%). Most of these side-effects were mild to moderate and no patient was taken off study due to side-effects. Skin rash and periorbital oedema frequently seem to be self limiting, despite continued treatment. In GIST patients, the current response rates (RRs) are 4% complete remission (CR), 67% partial remission (PR), 18% stable disease (SD) and 11% progression (PD). 73% of GIST patients are free from progression at 1 year. In the other STS group, there were no objective responses. The median time to progression in this subgroup was only 58 days. Imatinib mesylate is well tolerated at a dose of 400 mg bid. This dose is active in patients with KIT-positive GIST, but patients with other STS subtypes unselected for a molecular target are unlikely to benefit.

It is time for chronotherapy!

The EORTC Chronotherapy Group (CTG) stemmed from the International Organisation for Cancer Chronotherapy(IOCC) in 1996. The IOCC was the first to initiate large scale multicentre international chronotherapy trials, for the purpose of investigating the relevance of chronomodulated or timed administration of cancer therapy based on biological rhythms. Programmable pumps for cytotoxic chronodelivery and actigraph devices to monitor circadian rhythm alterations linked to cancer were also developed. The unique expertise of the IOCC with regard to cancer chronotherapy furthered its development within the EORTC. EORTC offers broad expertise in clinical cancer research and opportunities for scientific recognition, inter-group collaborations and translational research. Over the past 5 years, EORTC CTG has grown from 16 to 48 centres in 12 different countries. It is currently conducting seven multicentre chronotherapy trials, which test the relevance of adapting cancer treatment delivery to circadian rhythms. The group aims at developing multiple collaborations to establish a chronotherapy network involving institutions with expertise ranging from experimental chronobiology to new drug testing, disease-specific management and quality of life or survival issues.

It's time for chronotherapy!

The EORTC Chronotherapy Group (CTG) stemmed from the International Organisation for Cancer Chronotherapy (IOCC) in 1996. The IOCC was first to initiate large scale multicentre international chronotherapy trials, for the purpose of investigating the relevance of chronomodulated or timed administration of cancer therapy based on biological rhythms. Programmable pumps for cytotoxic chronodelivery and actigraph devices to monitor circadian rhythm alterations linked to cancer were also developed. The unique expertise of the IOCC with regard to cancer chronotherapy furthered its development within the EORTC. The EORTC offers broad expertise in clinical cancer research and opportunities for scientific recognition, intergroup collaborations and translational research. Over the past 5 years, the EORTC CTG has grown from 16 to 48 centres in 12 different countries. It is currently conducting seven multicentre chronotherapy trials which test the relevance of adapting cancer treatment delivery to circadian rhythms. The group aims at developing multiple collaborations to establish a chronotherapy network involving institutions with expertise ranging from experimental chronobiology to new drug testing, disease-specific management and quality of life or survival issues.

The EORTC soft tissue and bone sarcoma group. European Organisation for Research and Treatment of Cancer.

The EORTC Soft Tissue Sarcoma Group was founded 25 years ago and has since developed into one of the leading cooperative groups in the research of sarcomas and has members from 40 institutions from 13 countries. The activities of the group have primarily been within the areas of standards for local as well as systemic treatment strategies, new drug development and quality control procedures. The group has a extensive quality control programme involving a strict membership policy, central review of the responses, central review of pathology, use a systemic therapy check-list and on-site monitoring of studies. A large database with over 2500 patients included in EORTC STBSG chemotherapy trials has been developed. So far, the STBSG has conducted more than 40 clinical trials accruing more than 250 patients per year, some of which has been performed in collaboration with other prestigious groups.

Safety and efficacy of imatinib (STI571) in metastatic gastrointestinal stromal tumours: a phase I study.

BACKGROUND: Gastrointestinal stromal tumours (GISTs) are rare tumours of the gastrointestinal tract characterised by cell-surface expression of the tyrosine kinase KIT (CD117). No effective systemic treatment is available. Imatinib (STI571) inhibits a similar tyrosine kinase, BCR-ABL, leading to responses in chronic myeloid leukaemia, and has also been shown to inhibit KIT. We did a phase I study to identify the dose-limiting toxic effects of imatinib in patients with advanced soft tissue sarcomas including GISTs. METHODS: 40 patients (of whom 36 had GISTs) received imatinib at doses of 400 mg once daily, 300 mg twice daily, 400 mg twice daily, or 500 mg twice daily. Toxic effects and haematological, biochemical, and radiological measurements were assessed during 8 weeks of follow-up. 18Fluorodeoxy-glucose positron-emission tomography (PET) was used for response assessment in one centre. FINDINGS: Five patients on 500 mg imatinib twice daily had dose-limiting toxic effects (severe nausea, vomiting, oedema, or rash). Inhibition of tumour growth was seen in all but four patients with GISTs, resulting in 19 confirmed partial responses and six as yet unconfirmed partial responses or more than 20% regressions. 24 of 27 clinically symptomatic patients showed improvement, and 29 of 36 were still on treatment after more than 9 months. PET scan responses predicted subsequent computed tomography responses. INTERPRETATION: Imatinib at a dose of 400 mg twice daily is well tolerated during the first 8 weeks, side-effects diminish with continuing treatment, and it has significant activity in patients with advanced GISTs. Our results provide evidence of a role for KIT in GISTs, and show the potential for the development of anticancer drugs based on specific molecular abnormalities present in cancers.

Influence of retigabine on the anticonvulsant activity of some antiepileptic drugs against audiogenic seizures in DBA/2 mice.

Retigabine (D-2319, 0.5-20 mg/kg i.p.) antagonised dose dependently audiogenic seizures in DBA/2 mice. Retigabine at 0.5 mg/kg i.p., a dose that per se did not affect the occurrence of audiogenic seizures significantly, potentiated the anticonvulsant activity of carbamazepine, diazepam, felbamate, lamotrigine, phenytoin, phenobarbital and valproate against sound-induced seizures in DBA/2 mice. The degree of additivity for the effect induced by retigabine was greatest for diazepam, phenobarbital, phenytoin and valproate, less for carbamazepine and lamotrigine and least for felbamate. The increase in anticonvulsant activity was usually associated with a comparable increase in motor impairment. However, the therapeutic index of combined treatment (drugs plus retigabine), was more favourable than the same drug plus vehicle. Since retigabine had no significant influence on the total and free plasma levels of the anticonvulsant drugs, pharmacokinetic interactions, in terms of total or free plasma levels, are not probable. However, the possibility that retigabine modifies the clearance of the anticonvulsant drugs from the brain cannot be excluded. Retigabine had no significant effect on the hypothermic effects of the anticonvulsants tested. In conclusion, retigabine showed an additive effect when administered in combination with classical anticonvulsants, most notably diazepam, phenobarbital, phenytoin and valproate.

Randomised phase II trial of pegylated liposomal doxorubicin (DOXIL/CAELYX) versus doxorubicin in the treatment of advanced or metastatic soft tissue sarcoma: a study by the EORTC Soft Tissue and Bone Sarcoma Group.

CAELYX/DOXIL, pegylated liposomal doxorubicin, has shown antitumour activity and reduced toxicity compared with standard doxorubicin in other tumour types. In this prospective randomised trial, 94 eligible patients with advanced soft-tissue sarcoma (STS) were treated, 50 with CAELYX (50 mg/m(2) by a 1 h intravenous (i.v.) infusion every 4 weeks) and 44 with doxorubicin (75 mg/m(2) by an i.v. bolus every 3 weeks). Histological subtypes were evenly matched, 33% were leiomyosarcoma (CAELYX: 18; doxorubicin: 13). Primary disease sites were well matched. CAELYX was significantly less myelosuppressive, only 3 (6%) patients had grade 3 and 4 neutropenia, versus 33 (77%) on doxorubicin; febrile neutropenia occurred in 7 (16%) patients given doxorubicin, but only 1 (2%) given CAELYX. 37 (86%) patients on doxorubicin had grade 2-3 alopecia, but only 3 (6%) on CAELYX, and the major toxicity with CAELYX was to the skin. Palmar-plantar erythrodysesthesia with CAELYX was grade 1: 4 (8%) patients, grade 2: 11 (22%) patients, grade 3: 9 (18%) patients and grade 4: 1 (2%) patient. Other non-haematological grade 3 and 4 toxicities were rare. Confirmed responses were observed with both agents: CAELYX: complete response (CR) 1 (uterine), partial response (PR) 4 (response rate (RR) 10%); and doxorubicin: CR 1, PR 3 (RR of 9%); with the best response being stable disease (NC) in 16 and 18 patients, respectively. The reason for the low response rate is unknown, but it may be due partly to a high proportion of gastrointestinal stromal tumours. In conclusion, CAELYX has equivalent activity to doxorubicin in STS with an improved toxicity profile and should be considered for further investigation in combination with other agents such as ifosfamide.

Randomized phase II study of docetaxel versus doxorubicin in first- and second-line chemotherapy for locally advanced or metastatic soft tissue sarcomas in adults: a study of the european organization for research and treatment of cancer soft tissue and bone sarcoma group.

PURPOSE: To assess antitumor response and time to progression (TTP) with docetaxel compared with doxorubicin in first-line treatment of advanced and/or metastatic soft tissue sarcoma. PATIENTS AND METHODS: Patients with measurable soft tissue sarcoma lesions and adequate bone marrow, liver, and renal function were entered onto the study. They were randomized to either docetaxel 100 mg/m(2) given as a 1-hour intravenous infusion every 3 weeks or doxorubicin 75 mg/m(2) given as a bolus injection every 3 weeks. A maximum of seven cycles of treatment were scheduled. The study was designed as a randomized phase III study evaluating TTP by log-rank model. There was a clause for premature closure of the trial if fewer than five responses were observed among the first 25 assessable patients in the docetaxel treatment arm. RESULTS: Eighty-six patients were entered onto the study; 85 were assessable for toxicity and 83 for response. The rate of severe granulocytopenia was not significantly different between the two arms. Nausea (P =.001), vomiting (P <.001), and stomatitis (P =.005) were more common with doxorubicin therapy, whereas neurotoxicity was more frequent with docetaxel treatment. The response rate to doxorubicin therapy was 30% (95% confidence interval, 17% to 46%), whereas no responses to docetaxel therapy were seen (P <.001). In view of this, the trial was closed prematurely and the phase III study part was not conducted. CONCLUSION: Docetaxel is inactive in soft tissue sarcomas and cannot be recommended for further use in treatment of this disease.

Effects of adenosine receptor agonists and antagonists on audiogenic seizure-sensible DBA/2 mice.

We have studied the effects of selective and non-selective adenosine receptor agonists and antagonists in audiogenic-seizure-sensitive DBA/2 mice, an animal model of generalized reflex epilepsy. With the exception of the adenosine A3 receptor agonist, N6-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine (IB-MECA), all the agonists studied prevented the development of audiogenic seizures in a dose-dependent manner. The ED50 values against the clonic phase of the audiogenic seizures were low, that is: 0.06 mg/kg, i.p., for the adenosine A1 receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), 0.02 and 0.03 mg/kg, i.p., for the adenosine A2A receptor agonists, 2-(4-(2-carboxyethyl)-phenylamino)-5'-N-ethylcarboxamidoadenosine (CGS 21680) and 2-hexynyl-5'-N-ethyl-carboxamidoadenosine (2-HE-NECA), and 0.7 mg/kg, i.p., for the adenosine A1/A3 receptor agonist, N6-2-(4-aminophenyl)ethyladenosine (APNEA). Conversely, the non-selective agonist, N-ethyl-carboxamidoadenosine (NECA), was highly potent, the ED50 being 0.0005 mg/kg, i.p. In the absence of auditory stimulation, the adenosine receptor antagonists increased the incidence of both clonic and tonic seizures in DBA/2 mice. The ED50 values were: for caffeine, 207.5 mg/kg, i.p., for the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 327.8 mg/kg i.p., for the adenosine A2A receptor antagonists, 3,7-dimethyl-1-propylxanthine (DPMX), 86.7 mg/kg i.p., for the (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3-dipropylxanthine (KF 17837), 69.1 mg/kg i.p., and 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-(4,3-c)1,2,4-triazolo(1,5 -c)-pyrimidine (SCH 58261), 321.8 mg/kg i.p. The rank order of convulsant potency in our epileptic model, following intracerebroventricular administration, was DPCPX > DMPX > 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC) > KF 17837 > Caffeine > SCH 58261 > 5-amino-9-chloro-2-(2-furyl)-1,2,4-triazolo(1,5-c)quinazoline (CGS 15943). Following a subconvulsant audiogenic stimulus of 83 dB, all adenosine receptor antagonists induced both tonic and clonic seizures. The ED50 values for such proconvulsant effects were: for caffeine 0.04 mg/kg, i.p., for the adenosine A receptor antagonist, DPCPX, 5.84 mg/kg, i.p., for the adenosine A2A receptor antagonists, DMPX, 0.02 mg/kg, i.p., CGS 15943, 0.29 mg/kg i.p., KF 17837, 0.57 mg/kg, i.p., CSC 0.12 mg/kg, i.p. and SCH 58261 0.07 mg/kg, i.p., respectively. These data suggest that stimulation of adenosine A1 and A2A receptors is involved in the suppression of seizures.

Effects of some AMPA receptor antagonists on the development of tolerance in epilepsy-prone rats and in pentylenetetrazole kindled rats.

The non-selective alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor antagonists, 2,3-benzodiazepine derivatives CFM-1 (3,5-dihydro-7,8-dimethoxy-1-phenyl-4H-2,3-benzodiazepin-4-one) and CFM-2 (1-(4'-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin -4-one), following intraperitoneal (i.p.) administration, were studied against audiogenic seizures in genetically epilepsy-prone rats (GEPRs) or pentylenetetrazole induced kindling in rats. After acute i.p. administration the ED50 values of CFM-1 against the clonic and tonic phases of the audiogenic seizures 30 min after pretreatment were 40 (16-100) and 13 (8-25) micromol kg(-1), respectively. The animals used for chronic study were treated i.p. daily (at 10 h) for 4 weeks with CFM-1 (20 or 50 micromol kg(-1)). Chronic treatment for 2 weeks with CFM-1 gave ED50 values against clonic and tonic seizures of 39 (22-69) and 16 (8-25) micromol kg(-1), respectively, whereas chronic treatment for 4 weeks gave ED50 values against clonic and tonic seizures of 42 (18-98) and 17 (7-41.3) micromol kg(-1), respectively. The duration of anticonvulsant activity observed between 0.5 and 4 h following administration of CFM-1 was similar for acute and chronic treatment. Two groups of Sprague-Dawley rats received CMF (20 or 50 micromol kg(-1)) 30 min before a subconvulsant dose of pentylentetrazole (25 mg kg(-1) i.p.) which is able to increase seizure severity in control animals (i.e., chemical kindling). Pretreatment with CFM-2 delayed the progression of seizure rank during repeated administration of pentylentetrazole. At the end of the period of repeated pentylentetrazole treatment (6 weeks) the mean seizure score was 0 in vehicle treated controls, 4.3 in animals treated with vehicle + pentylentetrazole, 2.2 in rats treated chronically with CFM-2 (20 micromol kg(-1) i.p.) + pentylentetrazole and 1.0 in rats treated repeatedly with CFM-2 (50 micromol kg(-1) i.p.) + pentylenetetrazole. CFM-2 was also able to antagonize the long-term increase in sensitivity of the convulsant effects of GABA function inhibitors in pentylentetrazole-kindled animals. Thus, the administration of a challenge dose of pentylentetrazole (15 mg kg(-1) i.p.) or picrotoxin (1.5 mg kg(-1) i.p.) 15 or 30 days after the end of the repeated treatment showed that animals treated with CFM-2 were significantly protected against seizures induced by pentylentetrazole or picrotoxin. The data suggest that, following repeated treatment, tolerance to the novel AMPA receptor antagonists does not develop (CFM-1 in genetically epilepsy-prone rats and CFM-2 in the pentylentetrazole kindling model of epilepsy). Thirteen minutes after drug injection on days 1, 14 and 28 of chronic treatment the motor impairment induced by these compounds was studied with a rotarod apparatus. The TD50 values for CFM-1 or CFM-2-induced impairment of locomotor performance were similar following acute and repeated treatment. The data also suggest that some novel 2,3-benzodiazepines may have clinical potential for some types of epilepsy.

Is the mitochondrial benzodiazepine receptor involved in the control of airway smooth muscle tone?

1. In addition to binding to GABAA receptors in the central nervous system, benzodiazepines have also been reported to recognize high affinity binding sites in several different peripheral tissues. 2. These peripheral benzodiazepine receptors likely consist of distinct integral membrane proteins, which are predominantly localized in the outer mitochondrial membrane and may be associated to form a heteropolymeric receptor complex. One such protein, identified for its ability to bind a class of benzodiazepines and isoquinolines, has been purified and the corresponding complementary DNA (cDNA) has been cloned and characterized. Furthermore, the structure of the rat gene encoding this protein has been clarified, thus potentially opening new insights into the molecular mechanisms responsible for receptor regulation. 3. Although the exact physiologic and/or pharmacologic role of peripheral benzodiazepine receptors is still unknown, their wide tissue distribution suggests an involvement in many cellular phenomena. 4. In particular, several lines of investigation indicate that these receptors, densely expressed on airway smooth muscle of various species, may contribute to the modulation of bronchomotor tone and perhaps to the pathogenesis of asthma and airway hyperresponsiveness.

Repeated treatment with adenosine A1 receptor agonist and antagonist modifies the anticonvulsant properties of CPPene.

The effects of repeated administration of the selective adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the selective adenosine A2 receptor agonist 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA), the non-selective adenosine A1/A2 receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3 dipropylxanthine (DPCPX) and the selective adenosine A2 receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-(4,3-e)1,2,4-triazolo(1,5 -c)pyrimidine (SCH 58261) on the anticonvulsant activity of 3-(2-carboxypiperazine-4y)propenyl-1-phosphonic acid (CPPene), a selective N-methyl-D-aspartate receptor antagonist, were evaluated in audiogenic sensible dilute brown agouti mice DBA/2J (DBA/2). Mice were treated intraperitoneally twice daily for 7 days with CCPA 0.11 mg/kg, 2HE-NECA 0.056 mg/kg, NECA 0.11 mg/kg, DPCPX 0.5 mg/kg and SCH 58261 0.5 mg/kg followed by 2 vehicle injections (the wash-out period of 1 day) and subsequently CPPene was administered intracerebroventricularly. Audiogenic seizures were delivered 30 min after CPPene administration. Repeated treatment with CCPA significantly reduced the anticonvulsant properties of CPPene against audiogenic seizures. A weak and not significant reduction of anticonvulsant effects of CPPene was observed following repeated administration of NECA, whilst the repeated administration of 2HE-NECA did not decrease the antiseizure activity of CPPene. Conversely, repeated administration of DPCPX markedly potentiated the anticonvulsant properties of CPPene, whilst the repeated treatment with SCH 58261 did not increase the anticonvulsant activity of CPPene. The present results indicate that repeated treatment with CPPA, a selective adenosine A1 receptor agonist, decreases the anticonvulsant properties of CPPene, whilst the repeated administration of DPCPX, a selective adenosine A1 receptor antagonist, potentiates the anticonvulsant effects of CPPene. The compounds acting as selective agonists or antagonists of adenosine A2 receptors do not affect the antiseizure activity of CPPene. In conclusion, the repeated interaction of agonists or antagonists with adenosine A1 receptors seems to induce changes on anticonvulsant activity of CPPene, whereas drugs acting at adenosine A2 receptors do not.

Tolerance to anticonvulsant effects of some benzodiazepines in genetically epilepsy prone rats.

The development of tolerance to the anticonvulsant effects of clonazepam, clobazam, and diazepam were studied in genetically epilepsy-prone rats following intraperitoneal (IP) or oral administration. The anticonvulsant effects were evaluated on seizures evoked by means of auditory stimulation (109 dB, 12-16 kHz). All compounds showed 60 min after IP injection antiseizure activity with ED50 against clonus of 0.24 mumol kg-1 for clonazepam, 0.72 mumol kg-1 for diazepam, and 3.9 mumol kg-1 for clobazam. After 120 min of oral administration the ED50 against clonus of 2.37 mumol kg-1 for clonazepam, 15.8 mumol kg-1 for diazepam, and 30 mumol kg-1 for clobazam. The dose chosen for the chronic treatment were 2.5 mumol kg-1 for clonazepam, 15 mumol kg-1 for diazepam, and 30 mumol kg-1 for clobazam. The animals were treated three times daily for 4 or 6 weeks. Auditory stimulation was administered 60 min after drug IP injection on various days. During treatment, tolerance was observed as a loss of drug anticonvulsant effects. No changes of occurrence of audiogenic seizures was observed in rats treated with vehicle. Tolerance to the anticonvulsant activity developed most rapidly during clobazam treatment, less rapidly following diazepam treatment, and most slowly during clonazepam treatment. Sixty minutes after IP injection on various days of chronic treatment the motor impairment induced by these benzodiazepines was also studied by means of a rotarod apparatus. The tolerance to the motor impairment developed more rapidly than the anticonvulsant effects. The response to auditory stimulation to benzodiazepines was stopped 24 and 48 h after chronic treatment with these compounds, showing no residual drug effects and that rats were still tolerant. The genetically epilepsy-prone rats is a reliable and sensitive model for studying long-term effects of anticonvulsant drugs.

Effect of propionyl-l-carnitine and enalapril on cardiac function of pressure-overloaded rats during increase in load.

Chronic administration of propionyl-l-carnitine has been recently shown to correct hypertrophy related abnormalities in muscle mechanics. Accordingly, this study investigated whether the drug would similarly improve cardiac dynamics in rats with pressure overload. Enalapril was used for comparison. Drugs were administered in the drinking water for 3 weeks to Wistar Kyoto rats with a 2 week abdominal aortic constriction. Cardiac function was studied under urethane anaesthesia in basal conditions, during increase in preload, and during increase in afterload. Basal cardiac function was comparable in pressure-overloaded and sham-operated animals. Neither propionyl-l-carnitine nor enalapril affected the basal performance. Compared to sham-operated animals, untreated pressure-overloaded rats showed an impaired cardiac response (cardiac output, stroke volume) to preload increase induced by saline i.v. infusion. Propionyl-l-carnitine dose dependently improved cardiac function in the range 30-180 mg/kg, without affecting cardiac hypertrophic growth. Enalapril (3 mg/kg) reduced cardiac hypertrophy and improved cardiac function. The two effects were unrelated. The afterload increase by total aortic occlusion evidenced a reduction in the left ventricle pressure generating capacity of hypertrophied hearts. Propionyl-l-carnitine did not modify this parameter, while enalapril afforded a significant improvement. Results show that propionyl-l-carnitine significantly improves in vivo cardiac dynamics under conditions of increased energy demand. The effect is not due to inotropic efficacy, but presumably to increased cardiac efficiency.

Propionyl-L-carnitine limits chronic ventricular dilation after myocardial infarction in rats.

To determine whether propionyl-L-carnitine (PLC) administration ameliorates ventricular remodeling after myocardial infarction, we performed coronary occlusion in rats and examined the long-term effects of the drug 19-24 wk after surgery. In view of the well-established role of angiotensin-converting enzyme (ACE) inhibitors in the reduction of ventricular dilation after infarction, the therapeutic impact of oral PLC (60 mg/kg) was compared with that of enalapril (1 mg/kg). Infarct size measured planimetrically was found to be comparable in untreated, PLC-treated, and enalapril-treated rats, averaging 40-46% of the left ventricular free wall. Heart weight was increased 14, 16, and 11% with no treatment, with PLC, and with enalapril, respectively. The relationship between left ventricular filling pressure and chamber volume demonstrated that PLC and enalapril significantly prevented the expansion in cavitary size after infarction. These protective influences were observed throughout the range of filling pressures measured, from 0 to 30 mmHg. At a uniform reference point of filling pressure of 4 mmHg, untreated infarcted hearts showed an expansion in ventricular volume of 2.17-fold (P < 0.0001). Corresponding increases in this parameter after PLC and enalapril were 36 and 43%, respectively, both not statistically significant. Moreover, PLC was capable of reducing the alterations in myocardial compliance associated with myocardial infarction. In conclusion, PLC reduces the magnitude of decompensated eccentric hypertrophy produced by myocardial infarction in a manner similar to that found with ACE inhibition.

Desensitization and down-regulation of neurotensin receptors in murine neuroblastoma clone N1E-115 by [D-Lys8] neurotensin(8-13).

Clone N1E-115 cells have specific, high-affinity binding sites for neurotensin (NT). These receptors mediate formation of cyclic GMP and other second messengers. We studied the effects of extended exposure of cells to [D-Lys8]NT(8-13) (NT2), a peptidase-resistant NT analog, on NT's ability to stimulate cyclic GMP synthesis and to bind [3H]NT to its sites on these cells. When intact N1E-115 cells were preincubated with NT2 (1 microM) for various times (15 min to 12 hr) at 37 degrees C, maximal desensitization and binding site (Bmax) loss occurred after only 15 min. Receptor affinity for [3H]NT did not change. At 0 degrees C NT2 for 15 min caused no down-regulation. After 15 min of preincubation with NT2 at 37 degrees C, the recovery of receptor binding and function after a 10 to 20 min lag-time was rapid (T1/2 = 15 min and 19 min, for binding and cyclic GMP response, respectively). After 12 hr of NT2 preincubation, it was slow (T1/2 = 212 min). Incubation of cells with cycloheximide (70 microM) for 5 hr after their exposure to NT2 for 15 min did not change the rate or extent of recovery of NT Bmax. However, cycloheximide did decrease the recovery of NT Bmax after exposure of cells to NT2 for 12 hr. Cycloheximide alone did not change NT Bmax. These data suggest that this decrease of NT receptor binding after short (15 min) and long (12 hr) exposure times to agonist involves two consecutive steps: intracellular sequestration of recyclable NT receptors, followed by receptor degradation causing true down-regulation.

L-arginine potentiates excitatory amino acid-induced seizures elicited in the deep prepiriform cortex.

Microinjection of N-methyl-D-aspartate (NMDA; 1 and 2.5 nmol) or kainate (KA; 50 pmol) into the deep prepiriform cortex elicited behavioral signs of seizure activity. No epileptiform activity was observed after deep prepiriform cortex microinjection of either L-arginine (L-Arg, 5 and 10 nmol) or its D-enantiomer, D-arginine (D-Arg, 2.5-10 nmol). However, both the seizure score and the incidence of electroencephalographic (EEG) epileptic discharges elicited by NMDA (1 and 2.5 nmol) and KA (50 pmol) were significantly increased by L- but not D-Arg. The facilitatory effects of L-Arg on seizure activity elicited by both NMDA and KA were dose-dependent and could be prevented by co-administration of L-Arg (10 nmol) and the nitric oxide (NO) synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 20 nmol). Motor and electrocortical seizures were observed after microinjection of the NO donor sodium nitroprusside (SNP; 5 to 20 nmol) into the deep prepiriform cortex. Infusion of methylene blue (20 nmol), a soluble guanylate cyclase inhibitor, protected against SNP-induced seizures. Furthermore, prior infusion of a subconvulsant dose of SNP into the deep prepiriform cortex significantly potentiated the seizure activity elicited by either NMDA (1 and 2.5 nmol) or KA (50 pmol). These results support the proposal that NO is formed from L-Arg upon excitatory amino acid receptor activation within the deep prepiriform cortex, thereby contributing to the genesis of seizure activity.

Effects of antiepileptic drugs, calcium channel blockers and other compounds on seizures induced by activation of voltage-dependent L calcium channel in DBA/2 mice.

1. The convulsant activity of the calcium voltage L-channel agonist Bay k 8644 was studied in genetically epilepsy prone DBA/2 mice. 2. Seizures were induced by intracerebroventricular injection of Bay k 8644. 3. These seizures were reversed by some calcium channel blockers such as dihydropyridines, some excitatory amino acid antagonists such as 2-amino-7-phosphonoeptanoate and CPPene, 2-chloro-adenosine, some anticonvulsant drugs such as magnesium valproate, diazepam and clonazepam and two kappa opioid agonists (U-50488H and U-54494A). 4. The remaining antiepileptic drugs (carbamazepine, phenytoin, phenobarbital and trimethadione) were ineffective in this respect. Other anticonvulsant compounds such as dizocilpine (MK 801), ketamine and drugs enhancing GABAergic transmission did not significantly affect the clonic phase of the seizures induced by Bay k 8644. 5. These results show that Bay k 8644 seizures are relatively resistant to some anticonvulsant compounds. The role of some neurotransmitters on seizures induced by Bay k 8644 is discussed.