[The Ethics Committee meeting in Belgium May 7, 2004, on human experimentation]. / Les comités d'éthique face a la loi belge du 7 mai 2004 sur l'expérimentation humaine.

The European directive 2001/20/EC concerning the performance or clinical trials has been transposed in the Belgian law of May 7, 2004, entitled "Loi relative aux expérimentations sur la personne humaine" (Moniteur belge, May 18, 2004)". The range of the law is larger than those of the directive. Several new elements must be underlined: 1. Any experimentation on a human being may only begin if the promoter received a positive advice from an ethical committee according to the provision of the law. 2. For multicentric assays, a single opinion, identical for all the participating centres must be obtained. This implies a close dialogue between the ethical committees. A central committee is proposed by the industrial promoter. Each clinical trial of a drug must have a unique identification number obtained at the European agency for the evaluation of medicinal products (EMEA). 3. Taking into account the fear of the pharmaceutical industry that some clinical studies could be "delocalized", the authorities accepted to reduce to twenty- eight days (instead of 60) the delay granted to the ethical committee in order to produce their single opinion. 4. The law of May 7, 2004, clearly defines the dispositions related to the protection of the participants. Particular dispositions are foreseen for the protection of minors, incapable adults and patients in emergency. 5. The promoter endorses, even without fault, the responsibility of the damage caused to the participant or his descendants. An insurance covering this risk must be contracted.

Electrophysiological effects of neurotensin on dopaminergic neurones of the ventral tegmental area of the rat in vitro.

The effects of neurotensin on the spontaneous firing rate of presumed dopaminergic neurones of the ventral tegmental area of the rat, were studied in a slice preparation of brain by extracellular single-cell recordings. Bath-applied neurotensin excited all cells which were studied (N = 25). This effect was concentration-dependent; the threshold was 10(-10) M and maximal activation (about 30 spikes/10 sec) was obtained with 10(-6) M. The EC50 (half-maximal effective concentration) was roughly estimated at 35 nM. The action of neurotensin was mimicked by neurotensin 8-13 (N = 6), but not neurotensin 1-8 (N = 6). It persisted in low-calcium, high-magnesium solutions (N = 5) and therefore probably resulted from a direct activation of neurotensin receptors. The responses to neurotensin were long-lasting (30-60 min after a 10 min 10(-7) M infusion) and exhibited little tachyphylaxis. Dose-response curves to the dopaminergic agonist BHT920 showed that, during the infusion of 10(-7) M neurotensin, dopaminergic autoreceptors of some neurones were less sensitive than in control conditions. This was not a non-specific effect produced by the excitation, since it was not observed during the infusion of another excitant, N-methyl-D-aspartate (NMDA). These results show that neurotensin potently activates presumed dopaminergic neurones in the ventral tegmental area in vitro; it may also decrease the effectiveness of the autoreceptors of some neurones.

Evidence for a non-GABAergic action of quaternary salts of bicuculline on dopaminergic neurones.

Intracellular recordings were made from neurones, presumed to be dopaminergic, in the rat midbrain slice preparation. Bicuculline methiodide (BMI) and methochloride (BMC) reversibly blocked the slow, apamin-sensitive component of the afterhyperpolarization in these cells. The IC50 for this effect was about 26 microM. In comparison, BMC antagonized the input resistance decrease evoked by muscimol (3 microM) with an IC50 of 7 microM. The base of bicuculline was less potent in blocking the slow afterhyperpolarization. SR95531 (2-[carboxy-3'-propyl]-3-amino-6-paramethoxy-phenyl-pyridaziniu m bromide), another competitive GABA(A) antagonist, and picrotoxin, a non-competitive GABA(A) antagonist, also blocked the action of muscimol (IC50s: 2 and 12 microM respectively), but had no effect on the afterhyperpolarization at a concentration of up to 100 microM. Moreover, 100 microM SR95531 did not affect the blockade of the afterhyperpolarization by BMC. This blockade persisted in the presence of tetrodotoxin and was apparently not due to a reduction of calcium entry, suggesting that it involved a direct action on the channels which mediate this afterhyperpolarization. These results strongly suggest that quaternary salts of bicuculline act on more than one target in the central nervous system. Thus, the involvement of GABA(A) receptors in a given effect cannot be proven solely on the basis of its blockade by these agents.

Action of rilmenidine on locus ceruleus and dorsal raphe cells in vivo and in vitro.

The effect of rilmenidine (S 3341) and clonidine on the firing rate of locus ceruleus (LC) and dorsal raphe (DR) cells was studied in vivo after systemic and microiontophoretic administration. The effect of the 2 drugs was also studied in vitro on brain slices containing LC cells. Rilmenidine and clonidine inhibited the firing of LC and DR neurons. Complementary experiments performed with yohimbine and prazosin demonstrated that the effect of LC is related to an agonistic action on alpha 2 somatodendritic receptors, whereas the effect observed on DR cells is indirect. On LC cells, clonidine is 30 to 60 times as potent as rilmenidine. These observations may be related to less sedative effects of rilmenidine than of clonidine.

Central dopaminergic and noradrenergic receptor blockade in a patient with neuroleptic malignant syndrome.

During treatment with clomipramine and haloperidol, a 54-year-old depressed woman exhibited a typical neuroleptic malignant syndrome (NMS). Among results of biologic tests performed at least 2 weeks after discontinuation of all psychotropic treatment, the absence of normal growth hormone response after both apomorphine and clonidine challenge tests and increased levels of cerebrospinal fluid homovanillic acid and urinary 3-methoxy-4-hydroxyphenylglycol suggest that NMS may be related to central dopaminergic and possible alpha-noradrenergic receptor blockade.

Pimobendane (UD-CG 115 BS) in chronic congestive heart failure. Short-term and one-month effects of a new inotropic vasodilating agent.

We studied the effects of oral administration of pimobendane on hemodynamics, blood gas levels, the renin-angiotensin system, and plasma catecholamines in 11 patients who were affected by severe chronic congestive heart failure. Following the administration of 5 mg, the cardiac index increased from 2.0 +/- 0.2 to 2.5 +/- 0.2 L/min/m2 (p less than 0.01), and the pulmonary wedge pressure decreased from 28 +/- 3 to 17 +/- 4 mm Hg (p less than 0.01). The maximal changes were noted five hours after intake of the drug. In spite of a significant decrease in arterial oxygen pressure (PaO2) (from 81 +/- 4 to 67 +/- 5 mm Hg; p less than 0.05), a significant increase in the oxygen delivery index was seen (from 322 +/- 32 to 436 +/- 38 ml/min/m2; p less than 0.01). The patients who were submitted to long-term treatment (5 mg twice daily) and who were reassessed after at least one month exhibited an improved cardiac index from 1.9 +/- 0.2 to 2.5 +/- 0.1 L/min/m2 (p less than 0.01), as well as a decreased pulmonary wedge pressure from 26 +/- 2 to 14 +/- 4 mm Hg (p less than 0.01). The norepinephrine levels were significantly reduced after one month (from 1,496 +/- 185 to 678 +/- 95 pg/ml; p less than 0.01), whereas the plasma renin activity was not. One patient died suddenly during the one-month follow-up period. With the exception of one case, which was also treated with heparin, a transient cutaneous rash and a drop in the level of blood platelets were observed, pimobendane was well tolerated. This new inotropic and vasodilating drug thus seems to have promise for the treatment of chronic congestive heart failure.

Hemodynamic and clinical response to three-day infusion of sulmazol (AR-L 115 BS) in severe congestive heart failure.

Sulmazol (AR-L 115 BS) is a new positively inotropic drug with arterial and venous vasodilating properties. We studied the effects of sulmazol (three-day infusion) on clinical tolerance, hemodynamics, and blood gas levels in ten patients with severe chronic heart failure. The hemodynamic monitoring included a Swan-Ganz catheter in the pulmonary artery and a radial catheter. Blood gas levels were determined on samples of arterial and mixed venous blood. After 24 hours of infusion, there was a significant increase in cardiac index (2 to 2.5 L/min/sq m; p less than 0.005) and a significant decrease in pulmonary wedge pressure (28 to 19 mm Hg; p less than 0.001) and in right atrial pressure (7 to 4 mm Hg; p less than 0.001) without significant changes in heart rate and systolic blood pressure. These beneficial effects lasted during the three days of infusion. Oxygen delivery was significantly increased (350 to 443 ml/min/sq m; p less than 0.005) without significant change in arterial oxygen tension. The side effects included nausea, vomiting, anorexia, and mild thrombocytopenia. We conclude that sulmazol is a potent drug which may improve severely deteriorated left and right ventricular function in patients with chronic refractory heart failure without affecting the heart rate and the systolic blood pressure.

The inclusion of fluoxetine into gamma-cyclodextrin increases its bioavailability: behavioral, electrophysiological and pharmacokinetic studies.

The inclusion of a drug into cyclodextrin generally results in the modification of its physical and chemical properties and sometimes can increase its oral bioavailability. The aim of this study was to compare the effects of the fluoxetine HCl/gamma-cyclodextrin complex to that of traditional fluoxetine HCl. In the forced swimming test in mice, fluoxetine HCl/gamma-cyclodextrin was more effective than fluoxetine HCl, the ED30s being, respectively, 9.5 and 16.9 mg/kg PO. Both compounds (10 mg/kg PO) were able to reduce the firing rate of dorsal raphe neurons in the rat. However, between-groups comparisons showed no significant differences between fluoxetine HCl treated animals and the vehicle group, while fluoxetine HCl/gamma-cyclodextrin appeared significantly more effective than vehicle from minute 25 of the measurement period. In healthy volunteers, the relative oral bioavailability, calculated as the ratio AUC 0-infinity fluoxetine HCl/gamma-cyclodextrin on AUC 0-infinity fluoxetine HCl (20 mg PO), was equal to 249.9%. The three experiments taken together suggest that the complexation of fluoxetine HCl into gamma-cyclodextrin increases its pharmacological efficacy in animals, this effect being related to an enhancement of its oral bioavailability as demonstrated in human healthy subjects.

Evidence for a modulatory role of Ih on the firing of a subgroup of midbrain dopamine neurons.

A previous investigation has suggested that the hyperpolarization-activated cation current Ih does not contribute to the spontaneous firing of midbrain dopaminergic neurons. This conclusion was reached using Cs(-1). We have re-examined this question with extracellular recordings in slices using the more specific blocker ZD7288. In two-thirds of the cells, low concentrations of ZD7288 induced a decrease of the spontaneous firing. The maximal inhibition was about 40% and the mean IC50 was 1.6 microM. This effect was probably direct because it persisted in the presence of antagonists of various receptors. These concentrations of ZD7288 had no effect in the remaining one third of the examined cells. However, the highest concentration of ZD7288 (300 microM) abolished the firing of all dopaminergic neurons, probably by a mechanism unrelated to the blockade of Ih. We conclude that Ih controls to a certain extent the firing of a majority of midbrain dopaminergic neurons.

Transmammary passage of cefoxitin: additional results.

It has been shown previously that cefoxitin was not detectable in the milk following an intramuscular injection of 1 Gm to mothers. In the present study, using a more sensitive HPLC method, we have shown in five women that low concentrations of cefoxitin (0.25 to 0.65 micrograms/ml) are measured in the milk after the intramuscular injection of 2 Gm.

A study of the transplacental transfer and the mammary excretion of cefoxitin in humans.

Cefoxitin is a new semisynthetic cephamycin derivative with broad bactericidal activities. In order to determine the extent of the transplacental transfer of cefoxitin, 35 pregnant women received 1 Gm cefoxitin intramuscularly 15 to 180 minutes before normal or Caesarean delivery. Cefoxitin was measured microbiologically in maternal blood (multiple-time samples), umbilical blood (one-time sample), and amniotic fluid in the cases of Caesarean sections. The mammary excretion of cefoxitin injected at the same dose was investigated by measuring cefoxitin in the milk of 16 nursing mothers. In the maternal blood, a peak plasma level of approximately 25 microgram/ml was reached 30 minutes after the 1-Gm intramuscular injection. A clear-cut passage of cefoxitin in the umbilical cord blood was demonstrated. In the fetal blood, a peak level of 15 microgram/ml was obtained 45 minutes after the injection. No cefoxitin was detectable in any of the milk samples from 30 minutes to 24 hours after the 1-Gm intramuscular injection.

Influence of food on tianeptine and its main metabolite kinetics.

The influence of a test meal on the absorption and disposition of tianeptine (Stablon), a new antidepressant, was investigated in 12 healthy subjects in a two-way, randomized, open cross-over study. Single 12.5-mg oral doses of tianeptine were administered following a night of fasting or immediately after a standardized breakfast. When subjects received tianeptine under fasting conditions the lag time before absorption onset, and the time of the maximum plasma concentration were 0.55 +/- 0.26 hours and 1.29 +/- 0.29 hours, respectively. The maximum plasma concentration was 322 +/- 44 ng/mL, and the total area under the curve 994 +/- 248 ng/hr/mL. When tianeptine was given at the end of the meal, several significant changes were found for tianeptine kinetic parameters; the lag time increased by 0.3 hour and the maximum plasma concentration was lowered (decreased by 25%) and occurred later (tmax increased by 0.5 hour). However, no significant change was found in the area under the plasma concentration-time curve. The trend and extent of changes in the MC5 metabolite parameters were similar to those observed for the parent drug. Absorption of tianeptine is slightly delayed and slowed down without modification of its extent when tianeptine is given at the end of a meal. These slight changes are not clinically relevant for an antidepressant administered three times a day. Despite the changes observed, tianeptine may be given at meal times to improve compliance with treatment.

Acute amphetamine-induced subsensitivity of A10 dopamine autoreceptors in vitro.

Extracellular recordings were obtained from spontaneously active, presumed dopamine (DA) neurons of the ventral tegmental area (VTA) of the rat in a slice preparation. Bath-applied (+)-amphetamine (AMPH) (1-30 microM) induced a concentration-dependent decrease in the firing rate of these neurons, which tended to saturate with the highest concentrations used (n = 11). This inhibitory effect was dependent on the activation of D2 receptors since it was reversed by the D2 antagonist sulpiride (n = 8). However, the most striking effect of AMPH was the induction of a prominent subsensitivity of DA autoreceptors: whereas in 18 out of 20 control neurons, the D2 agonist BHT 920 (100 nM) produced a rapid and complete inhibition of the firing, this was observed in none out of 11 neurons 10 min after the end of the application of AMPH (1-30 microM) (P less than 0.001). In these cells, the mean percent inhibition produced by BHT 920 was only 47 +/- 8%. This subsensitivity remained unchanged after 20 min and declined after one hour. This effect was specific, since the sensitivity of GABAB receptors to baclofen (500 nM-1 microM) was not modified by the application of AMPH (n = 12). These results suggest that AMPH-induced DA autoreceptor subsensitivity can be produced acutely and may be the first step in a cascade of events leading to behavioral sensitization to this compound.

Evidence for the presence of N-methyl-D-aspartate receptors in the ventral tegmental area of the rat: an electrophysiological in vitro study.

Extracellular recordings were obtained from spontaneously active, presumed dopaminergic neurons of the ventral tegmental area (VTA) of the rat in a slice preparation. Bath-applied N-methyl-D-aspartate (NMDA) (1-20 microM) activated all neurons tested (n = 36). This effect was clearly concentration-dependent (n = 14), quickly reversible and reproducible. No bursting type of discharge was observed during NMDA infusion. The NMDA receptor blocker DL-2-amino-5-phosphonovaleric acid (50 microM) reversibly antagonized the increase in cell firing produced with 10 microM NMDA by 83.5 +/- 3% (mean +/- S.E.M.) (n = 8, P less than 0.05). Lowering the Mg2+ concentration of the perfusion fluid to one-third of its normal value significantly enhanced the excitatory effect of 5 microM NMDA (n = 7, P less than 0.05), but not of 500 nM carbachol (n = 6). Finally, NMDA did not modify the sensitivity of dopaminergic autoreceptors of VTA neurons (n = 8), when compared to controls (n = 10). These observations strongly support the presence of specific NMDA receptors in the VTA.

Hydrogen peroxide hyperpolarizes rat CA1 pyramidal neurons by inducing an increase in potassium conductance.

It has been suggested that hydrogen peroxide is involved in cascades of pathological events affecting neural cells. The aim of this study was therefore to examine whether this molecule is able by itself to modify membrane properties of pyramidal neurons in the CA1 region of the rat hippocampus. Intracellular recordings in the slice preparation showed that 3.3 mM hydrogen peroxide hyperpolarized all neurons tested (n = 41) by 11 +/- 3 mV. This effect persisted in the presence of tetrodotoxin. It developed slowly, was reversible and reproducible. In the presence of tetrodotoxin, the extrapolated reversal potential of this effect was -95 +/- 5 mV in 2.5 mM external potassium. This value was not significantly different from the one obtained with the GABAB agonist baclofen (10 microM) (-98 +/- 5 mV). It shifted when the concentration of external potassium was increased to 10.5 mM (from -96 +/- 5 to -62 +/- 4 mV), in close agreement with the Nernst equation potassium ions. The hyperpolarization was significantly reduced (by 65 +/- 22%) by the potassium channel blocker barium (100 microM). We suggest that hydrogen peroxide is able to induce an increase in potassium conductance in rat CA1 pyramidal neurons. The exact mechanism by which it produces this effect (direct action on channels or indirect effect) remains to be determined.

Influence of fenfluramine and norfenfluramine stereoisomers on the firing rate of central monoaminergic neurons in the rat.

The influence of acute administration of stereoisomers of fenfluramine and norfenfluramine on the firing rate of central monoaminergic neurons was investigated in rats anaesthetized with chloral hydrate. The firing rate of dorsal raphe (DR) and locus coeruleus (LC) neurons was inhibited. The parent drugs were more active on DR neurons than on LC neurons, and the converse was true for the demethylated metabolites. In both cases the d isomers were more active than the l isomers. No effect was observed on the electrical activity of A10 dopaminergic neurons. These differences in potency and selectivity could have therapeutic implications.

Effect of various antidepressant drugs on the spontaneous firing rate of locus coeruleus and dorsal raphe neurons of the rat.

The spontaneous firing rate of the noradrenergic neurons of the locus coeruleus and of the serotonergic neurons of the dorsal raphe was recorded with extracellular microelectrodes in chloral hydrate-anesthetized rats. A quantitative comparison of the effect of five tricyclic antidepressants, of tranylcypromine and of mianserin on the spontaneous activity of these two types of cells was performed. All drugs tested, except mianserin reduced the frequency of discharge of the noradrenergic neurons. Intravenous perfusion of the drugs allowed the doses required for inhibition of firing to 50% of the baseline rate (ID50) to be determined. Secondary aminated antidepressants (desipramine and nortriptyline) were more potent inhibitors than their tertiary aminated analogues (imipramine, chlorimipramine and amitriptyline). All drugs tested, except desipramine decreased the rate of firing of the serotonergic cells. In this case, the tertiary aminated antidepressants were much more potent than their secondary analogues. Mianserin was only active at very high doses. These results are in good agreement with the relative potencies of the tricyclic antidepressants for blocking the uptake of noradrenaline and serotonin into central and peripheral neurons.

Galanin decreases the activity of locus coeruleus neurons in vitro.

A brain slice preparation was used to examine the effects of galanin on the spontaneous firing rate of locus coeruleus noradrenergic neurons. Galanin (10(-9)-10(-7) M), added to the bath, inhibited the firing of 14 out of 19 neurons in a concentration-dependent manner. The observed effect was quite variable, ranging from 20 to 100% at 10(-7) M. Experiments performed in low-Ca2+, high-Mg2+ medium also showed a significant inhibition by galanin (10(-7) M) in three out of five neurons, which suggests that the peptide acts directly.

Differential effects of picrotoxin and RO 15-1788 on high and low ethanol concentrations on rat locus coeruleus in vitro.

In an in vitro electrophysiological single-cell recording model, ethanol had an inhibitory effect on locus coeruleus (LC) neurons at both low (0.1 mmol/l) and high (500 mmol/l) concentrations. In order to test if the benzodiazepine-GABA (gamma-aminobutyric acid) receptor complex could be implicated in this effect, we tested the interaction of these ethanol concentrations with picrotoxin (100 mmol/l) and RO 15-1788 (10 nmol/l). RO 15-1788 reversed the inhibitory effect induced by ethanol 500 mmol/l, but not by ethanol 0.1 mmol/l; picrotoxin reversed the effects of both concentrations. This indicates that the mechanisms of action of ethanol on LC neurons are not the same for high and low concentrations. Furthermore, the effect of concentrations related to a behavioral effect (greater than 10 mmol/l) was reversed by a low-calcium medium that abolishes transmitter release. Therefore, the inhibition induced by ethanol 500 mmol/l seems to be due to the release of an endogenous benzodiazepine-like compound.

Propofol protects cultured brain cells from iron ion-induced death: comparison with trolox.

The anesthetic propofol (PPF) has been shown to be an antioxidant in acellular experiments. This study was designed to assess the ability of PPF to protect primary-cultured brain cells against iron-mediated toxicity. A comparison with trolox (TX), a hydrosoluble vitamin E analogue, was performed. Rat cortical cells were exposed to 10 microM FeSO(4), PPF and/or TX. After a 4-h incubation, PPF and TX improved cell survival (lactate dehydrogenase measurements) in a concentration-dependent manner. The respective EC(50s) of each substance were 4 and 4.6 microM. The maximal effect was obtained at a 25-microM concentration which is similar to concentrations of PPF used clinically. The combination of both drugs at certain concentrations showed a complete protection of the cells, a significant decrease in intracellular peroxide production (dichloro-fluorescein diacetate (DCF-DA) fluorescence, 4-h incubation), in lipoperoxidation (thiobarbituric acid reactive substances fluorescence, PPF 6.25 microM+TX 12.5 microM) and an additive protective effect. This was true after 4- and 16-h incubation. These data suggest that PPF is neuroprotective. Moreover, the combination with a vitamin E analogue confers long duration protection against oxidative stress.