[The radiolabeled blood cell: Finding a legal qualification]. / La cellule sanguine marquée par un radionucléide : recherche d'une qualification juridique.

Radiolabelling of blood cells is a technique commonly used as a diagnostic tool in nuclear medicine; it has never been legally defined. This lack of legal status is a factor of uncertainty for both patients and health care professionals. The aim of this work was to identify what could be the legal nature of the radiolabelled blood cells by comparing their constitutive elements to the various existing legal categories applicable, according to the law applicable to living organisms as well as the regulations for other health products. The study concludes that, as it stands, the radiolabelled blood cells undoubtedly belong to the category of a drug by function for diagnostic purpose. More precisely, it is compared to a radiopharmaceutical medicinal product resulting from a well characterised manufacturing process. In order to increase visibility and thus the actors' awareness of the constraints arising from this specific status, it is proposed to create a specific legal regime for the radiolabelled blood cells by including in Article L. 5121-1 of the French Public Health Code a new category of health product which could be called: radiopharmaceutical preparation of cellular blood component for diagnostic purposes. The consequence of this proposal will mechanically place the radiolabelled blood cell preparation under the exclusive competences of radiopharmacists practising in an hospital pharmacy. Another important consequence will be that the radiolabelling process of blood cells will have to fulfil the rules of the French good hospital pharmacy practices and good preparation practices, for the benefit of patient protection and safety.

Taking immunogenicity assessment of therapeutic proteins to the next level.

Therapeutic proteins provide innovative and effective therapies for numerous diseases. However, some of these products are associated with unwanted immunogenicity that may lead to clinical consequences such as reduced or loss of efficacy, altered pharmacokinetics (PK), general immune and hypersensitivity reactions, and neutralisation of the natural counterpart (e.g. the physiological hormone). Regulatory guidance on immunogenicity assessment needs to take into consideration a great diversity of products, indications and patient populations as well as constantly advancing manufacturing technologies. Such guidance needs to be sufficiently specific while, at the same time, allowing interactive discussion and adjusted benefit-risk weighing of each product on a case-by-case basis, e.g. for a unique treatment of a life threatening disease acceptable treatment risks may differ considerably from the ones in case of less serious disease. This theme was the focus of the international conference "Taking immunogenicity assessment of therapeutic proteins to the next level", held at the Paul-Ehrlich-Institut in Langen, Germany, on the 10-11. June 2010. The objectives of the conference were to highlight how the field could move from that of a mere description of risk factors to a system of risk assessment and mitigation, as well as an understanding of the impact of unwanted immunogenicity on the overall benefit/risk consideration for a medicinal product. More than 150 experts from industry, academia and regulatory authorities worldwide discussed the phenomenon of undesired immunogenicity from different perspectives. The conference focussed on issues relevant to three areas: (1) new European guidelines that are currently the subject of discussion; (2) testing strategies for immunogenicity assessment; and (3) scientific progress on the product-related factors that may contribute to the development of pathogenesis of immunogenicity, in particular in the field of protein aggregation and post-translational modifications. This report provides an overview of issues, insights, and conclusions that were discussed and achieved during the meeting.

[Transfusion risk analysis with regard to vCJD in France]. / Evaluation du risque transfusionnel vis-à-vis de la variante de la maladie de Creutzfeldt-Jakob en France.

The latest updates (February 2004 and February 2005) of the analysis of the risk of transmission of the agent of Creutzfeldt-Jakob disease (CJD) by blood and blood products in France firstly reported in 2000, were triggered by the two cases of probable transmission of variant CJD (vCJD) by transfusion reported in the UK, and the notification of two French cases of vCJD who had been blood donors on several occasions before clinical onset. Even though some figures of the quantitative assumption used in the risk analysis have been modified since 2000, the conclusion as regards the risk for blood cellular component is considered unchanged: it can be assumed that one unit of labile blood products will contain more than one infectious unit if the donor is incubating the disease. Therefore, the residual risk of receiving by transfusion one infectious blood unit is depending on the prevalence of subjects incubating the disease in the blood donor population. For this particular aspect, the expected number of clinical vCJD cases to occur in France has been lowered since 2000. However, the worst-case scenario of 300 cases in the next 60 years has been maintained in the risk analysis, leading to the hypothesis that one blood donor per 120,000 could be infectious. In conclusion, the risk of getting one infectious blood unit is considered probable to a level of 1/120,000, but the benefit outweighs the risk if the use of transfusion is restricted to well justified indications and if patients are informed a priori and a posteriori.

Changes in retinal dopaminergic cells and dopamine rhythmic metabolism during the development of a glaucoma-like disorder in quails.

PURPOSE: To examine the possible correlation between a dysfunction of the daily rhythm of retinal dopamine (DA) and the development of a glaucoma-like disorder in an animal model, the al mutant quail (Coturnix coturnix japonica). METHODS: The morphology and density of DA-containing cells labeled immunohistochemically with an anti-tyrosine hydroxylase (TH) antibody were correlated with the diurnal and nocturnal contents of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) determined by high-pressure liquid chromatography with electrochemical detection. RESULTS: The number of TH-immunoreactive cells was lower than normal in mutant quails suffering from the disorder. There were considerably fewer cells in the central retina, and the DA metabolism was reduced in parallel. The nocturnal DA content was lower than the diurnal level in normal quails, but there was no such circadian fluctuation in mutant quails. CONCLUSIONS: This glaucoma-like disorder in quails is correlated with the degeneration of DA-containing amacrine cells and a dysfunction of the circadian rhythmicity of DA synthesis.

Albumin binding and brain uptake of 6-fluoro-DL-tryptophan: competition with L-tryptophan.

We investigated potential competition between L-tryptophan (TRP) and 6-fluoro-DL-tryptophan (6-F-TRP) for binding to albumin and for passage through the blood-brain barrier (BBB). In experiments based on equilibrium dialysis, albumin (600 microM) bound about 80% of TRP and 50% of 6-F-TRP with affinity constants (Ka) of 3.7 +/- 0.04 x 10(4) and 0.62 +/- 0.01 x 10(4) M-1, respectively. Competitive inhibition was assessed as the decrease in the apparent Ka (K' a) of TRP in the presence of 6-F-TRP, with no modification of the N value. Competition between TRP, 6-F-TRP and L-valine (VAL) for passage across the BBB was demonstrated using two approaches. When administered concomitantly with TRP or 6-F-TRP to rats, VAL decreased brain uptake of TRP and 6-F-TRP and reversed their action on serotonin. In Oldendorf's model, 6-F-TRP and VAL decreased the brain uptake of TRP.

Metabolism of 6-fluoro-DL-tryptophan and its specific effects on the rat brain serotoninergic pathway.

We administered 6-fluoro-DL-tryptophan (6F-Trp) to rats (50-200 mg/kg i.p.) and evaluated its neurochemical effects on central catechole and indole compounds; we also determined the time course of its action, together with its metabolism and kinetics in four rat brain areas. Neither norepinephrine nor dopamine and its major metabolites were affected by 6F-Trp. With regard to serotonin (5-HT), 6F-Trp induced a transient depletion in all the brain areas studied, with a maximum of about 60-65% obtained between 1 and 3 hr depending on the dose administered. After 6 hr, 5-HT levels generally returned to control values. 5-Hydroxyindolacetic acid (5-HIAA) levels were also reduced 3 hr after administration (-40 to -60%). A large dose-dependent increase in tryptophan (Trp) was observed in the four brain areas, possibly because of an inhibition of Trp incorporation into protein, as suggested by experiments with mouse neuroblastoma cells. The brain elimination half-life of 6F-Trp was estimated at 0.5-1 hr. Regarding 6F-Trp metabolism, three new compounds were detected in all four brain areas after 6F-Trp administration. They were identified by means of liquid chromatography with electrochemical detection and/or radioenzymology, in comparison with fluorinated standards, or after NSD 1015 or pargyline coadministration with 6F-Trp. The first two 6F-Trp metabolites detected were probably 6-fluoro-5-hydroxytryptophan and 6-fluoro-5-HIAA. The third, identified and quantified by means of the two analytical methods, was 6-fluoro-5-HT (6F-5-HT). These findings suggest that 6F-Trp could be used as the in vivo precursor of 6F-5-HT with a view to tracing neuronal serotoninergic pools, as has already been done with platelets.

Day and night dysfunction in intraretinal melatonin and related indoleamines metabolism, correlated with the development of glaucoma-like disorder in an avian model.

As previous studies have suggested that melatonin and serotonin may be involved in the regulation of intraocular pressure, retinal concentrations of melatonin, 5-HT, and related indoleamines measured at day and at night were studied during the development of a glaucoma-like disorder with increased intraocular pressure in the al mutant quail. Indoleamine levels were determined by HPLC with electrochemical detection in 1-month-, 3-month-, and 7-month-old al mutant and control quails. Morphology and numbers of melatonin-synthesizing and 5-HT-containing cells, labelled immunohistochemically with an anti-hydroxyindol-0-methyltransferase (HIOMT) antibody and an anti-5-HT antibody, respectively, were studied. Major findings were that: (1) no significant changes in morphology of melatonin-synthesizing cells or in the morphology and density of 5-HT-containing amacrine cells were observed during the development of glaucoma: (2) 5-HT metabolism was modified during the night at 1 month of age and during the day after 3 months; and (3) melatonin metabolism was modified during the night at 7 months and during the day after 3 months. These results demonstrate a relationship between the temporal evolution of this avian glaucoma and a dysfunction in indoleamine retinal metabolism.

In vivo evidence for carrier-mediated brain uptake of a new 2-amino-2-oxazoline (COR3224) via the purine transport system in rat.

We studied the brain uptake of a new 2-amino-2-oxazolamines derivative (COR3224) in the rat by means of the rapid intracarotid injection technique described by Oldendorf. The brain uptake index (BUI) of labelled COR3224 decreased progressively from 10% to 5% when concentrations of unlabelled compound were increased. The effect of various compounds indicated that COR3224 is transported into the brain by the purine carrier. The affinity of COR3224 for this carrier (Km = 5.68 microM) was higher than that of adenine.

Microdialysis monitoring of variations in extracellular levels of serotonin, GABA and excitatory amino acids in the frontal cortex of awake rats in response to a single peripheral or central administration of dexfenfluramine.

The effects of a single dexfenfluramine (D-fen) administration on the release of endogenous serotonin (5-hydroxytryptamine, 5-HT), excitatory (glutamate, Glu, aspartate, Asp) and inhibitory (gamma-aminobutyric acid, GABA) amino acids from the frontal cortex were studied by using in vivo microdialysis in freely-moving rats. Extracellular levels of these neurotransmitters were measured with HPLC coupled to electrochemical detection or with capillary electrophoresis coupled to laser-induced fluoresence detection (CE-LIFD). In a first study, single intraperitoneal administration of D-fen (0.5, 1.3, 5 and 10 mg/kg) increased extracellular 5-HT levels in a dose-dependent manner (maximal increase by 982% over baseline for the highest dose) while changes in Glu, Asp or GABA never reached statistical significance. In a second study, 73 nM of D-fen applied locally through the frontocortical dialysis probe, at a flow rate of 1.5 microliters/min in 30 microliters of perfusion fluid for 20 min, increased extracellular 5-HT and Asp levels [the maximal increases were to 1804% and 280% of the respective basal values (100%)] without altering extracellular levels of Glu and GABA. Thus, the order of magnitude of the changes induced by systemic administration or local infusion of D-fen on frontocortical extracellular levels of several neurotransmitters (5-HT > > Asp > GABA = Glu) demonstrate that D-fen, an indirect serotoninergic agonist, mainly increases 5-HT release while producing slight (Asp) or no (Glu, GABA) short-term in vivo variations in amino acid extracellular levels in the rat frontal cortex.

Benzodiazepine receptors are involved in tabernanthine-induced tremor: in vitro and in vivo evidence.

Tabernanthine, an indol alkaloid, is structurally related to carbolines (harmane, harmaline) which, in vitro, displace specific flunitrazepam binding to brain benzodiazepine receptors. In vivo, both tabernanthine and carbolines cause a fine general tremor, suggesting that a possible interaction with benzodiazepine receptors could be involved in the activity of tabernanthine. This hypothesis was validated by the in vitro and in vivo antagonism of benzodiazepine by tabernanthine. In vitro, tabernanthine inhibited specific flunitrazepam binding in a competitive manner with an affinity (IC50 150 microM) in the same range as harmane. Tabernanthine appeared as a benzodiazepine receptor inverse agonist in a discriminant in vitro binding assay. In vivo, the time course of tremorigenic activity was related to the tabernanthine concentration in brain (half-life = 2 h). Moreover, tabernanthine-induced tremor was inhibited reversibly by flunitrazepam or by Ro-15 1788 (an antagonist of benzodiazepine-receptors). These results suggest that part of the action of tabernanthine may be mediated by an interaction at the benzodiazepine receptor level.

Structure-activity relationships for substrates and inhibitors of pineal 5-hydroxytryptamine-N-acetyltransferase: preliminary studies.

Tryptamine, (1-naphthyl)ethylamine and phenethylamine derivatives were tested as substrates of ovine pineal serotonin-N-acetyl transferase (5-HT-NAT), a key enzyme involved in the synthesis of melatonin. Almost all of the indole derivatives possessed affinity similar to that of tryptamine (Km = 0.05 mM), while the substituted naphthalene and phenyl derivatives were less potent. However, the Km values seem be influenced by the steric hindrance and polar properties of the substituent. Vmax values for the naphthyl and phenyl derivatives were generally 10-20-fold higher than those of the indole derivatives and no clear structure-activity relationship was observed. Melatonin and several bioisosteric derivatives were shown to be inhibitors of 5-HT-N-acetyltransferase. Preliminary data suggested that over the 5-50-microM concentration range, melatonin was a competitive inhibitor (IC50 = 10 microM) with a concentration-dependent inhibitory effect on its own synthesis in the pineal gland. However, the bioisosteric naphthalene derivatives were characterized instead as mixed inhibitors. (1-Napthyl)ethylacetamido, a putative melatoninergic antagonist, was also shown to be an inhibitor of 5-HT-N-acetyltransferase (IC50 = 8 microM) and is a promising tool for the regulation of melatonin synthesis and the understanding of its role.

Effects of food intake and body weight on a serotonergic turnover index in rat hypothalamus.

Absolute levels and ratio of serotonergic compounds in food-related hypothalamic nuclei were determined with a chromatographic method comparing for control (C) versus lean (L) versus obese (O) rats. In all three groups, a second parameter, i.e., fasting (F) versus satiation (S) was superimposed on the body weight factor. Indoleamine levels failed to show statistical differences depending on the body weight and/or the nutritional status. A significant negative correlation between an index of the serotonin turnover, the 5-HIAA/5-HT ratio, and body weight was found for the paraventricular (PVN) nucleus in the C and L groups as well as for the lateral (LH) nucleus for the C and O groups. A positive correlation was found for the ventromedian (VMH) nucleus in the O group. These data suggest that the body weight factor is implicated in the central serotonergic regulation more than the feeding parameter.

Putative neurotransmitters in three experimental filariasis models.

Some putative neurotransmitters in three experimental filariasis models were investigated by a new relevant chromatographic method, sensitive and specific. No catecholaminergic compounds have been detected, but serotonin was found in Dipetalonema vitae. However, further investigations revealed very high levels of gamma amino butyric acid (GABA) in the macro-filariae. These data allow us to foresee new fields in filariasis therapeutics.

Time course of brain serotonin metabolism after cessation of long-term fluoxetine treatment in the rat.

The effects of repeated fluoxetine (Flx) administration (5, 10, or 15 mg/kg i.p., twice daily for 21 days) on serotonin and 5-HIAA metabolism were examined in the hypothalamus, hippocampus, pons medulla and cerebral cortex of rats killed 1-28 days after the last dose. Dose-dependent weight loss was observed during treatment, followed by gradual and complete recovery of body weight over the following two weeks. Chronic Flx treatment caused a dose-dependent decrease in brain 5-HT levels (by between 10 and 50% depending on the region examined), lasting for 3-7 days after cessation of treatment with the lowest and intermediate doses, and for 7-14 days after cessation of the highest dose. 5-HIAA levels decreased more markedly (-20; -60% depending on the region examined) than those of 5-HT, and tended to overshoot during the recovery period. The prolonged reduction in brain 5-HT levels after chronic Flx treatment was similar to that seen in rats given very high doses of dexfenfluramine (d-fen), a drug which both blocks 5-HT uptake and increases its release. These data suggest that brain 5-HT and 5-HIAA depletion may reflect similar dose-related expressions of the drug's mechanisms of action.

Changes in dopamine metabolism in rat forebrain regions after cessation of long-term fluoxetine treatment: relationship with brain concentrations of fluoxetine and norfluoxetine.

We examined the effects of repeated administration of the selective serotonin uptake inhibitor (SSRI) fluoxetine (Flx) (5, 10, or 15 mg/kg i.p., twice daily for 21 days) on brain and plasma concentrations of the parent drug and its active desmethyl metabolite, norfluoxetine (NFlx), in rats during the 21-day regimen as well as after cessation of drug treatment. We also measured dopamine (DA) levels in 2 midbrain regions (the striatum, St and nucleus accumbens, NAc) in rats killed 1-14 days after the last dose. NFlx concentrations in plasma and brain were ten times higher than those of Flx during the period of drug treatment. Although Flx accumulated more markedly in the rat brain than NFlx, it disappeared completely from plasma and brain after treatment stopped, while NFlx persisted up to Day P7. Chronic Flx treatment caused a persistent decrease in brain DA levels of -60% to -70% in St and NAc; this lasted for 7-14 days after cessation of treatment, depending on the dose used. The levels of DA metabolites decreased by 20-40%, and, except for 3-MT, tended to overshoot during the recovery period. Our data suggest that the long-term inhibition of DA neurons after cessation of Flx treatment parallels the inhibition previously observed for 5-HT neurons. Thus, besides blocking 5-HT uptake, Flx is likely to also inhibit in vivo DA uptake in forebrain regions, following prolonged administration.

Effect of L-dopa loading on 5-HTP decarboxylation in rat brain areas.

The time course of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) concentrations in four rat brain areas (hypothalamus, hippocampus, striatum and olfactory bulbs) were investigated after treatment with L-dopa (125 mg/kg, ip) + benserazide (50 mg/kg, ip). 5-HTP levels increased as early as 0.5 h, showed maximum accumulation at 1.5 h and returned to control levels within 4 h, while 5-HT was markedly decreased in all four structures, with a maximum effect at 1.5 h (approximately -70%) in the four areas. The decrease in 5-HT was not accompanied by changes in 5-HIAA levels. In agreement with previous studies, these data demonstrate that L-dopa loading interferes with serotonin metabolism in the rat brain. However, in addition to the releasing action of newly-synthesized dopamine, the accumulation of 5-HTP and the parallel decrease in 5-HT indicate a reduction in 5-HT synthesis. This inhibition could be explained by a competitive effect of L-dopa for aromatic aminoacid decarboxylase activity.

Ontogeny of brain monoamines in lean and obese female Zucker rats.

Differences in monoamine and metabolite levels were previously observed between lean and obese 16 week-old female Zucker rats. In order to test whether these variations exist initially in young animals, catecholamines (CA), serotonin (5-HT) and some of their metabolites were assayed in brain areas of Zucker rats between 5 and 16 weeks of age. The levels of most compounds in all areas studied increased with age in both groups. At 5 weeks of age, there was no difference between lean and obese rats. At 8 weeks, a reduction of dopamine (DA) metabolites appeared in the striatum and cortex of obese rats and persisted up to 16 weeks. A reduction of the 5-HT metabolite, 5-hydroxyindolacetic acid (5-HIAA), occurred at 8 weeks only. At 16 weeks, increases of DA and 5-HT in the hypothalamus and decreases of norepinephrine (NE), 5-HT and 5-HIAA in the hippocampus appeared. These data suggest that the development of obesity occurs before any monoamine alterations.

Adaptive computer program for determination of absorption profiles by numerical deconvolution: application to amoxicillin absorption.

We have developed a specific numerical deconvolution program for the Apple Macintosh microcomputer. After comparison with other methods, we used the program to evaluate the influence of nifedipine on the absorption and bioavailability of amoxicillin. The technique provided a model-independent approach. This study shows that the simultaneous administration of nifedipine with amoxicillin leads to a significant increase in both the total quantity of amoxicillin absorbed (+22%) and the rate of absorption. Parameters of clearance, volume of distribution, and elimination were unaffected. Numerical deconvolution analysis showed that nifedipine did not modify the absorption kinetics of amoxicillin, which are characterized by a lag time followed by a constant rate of absorption, suggesting zero-order kinetics with first-order kinetics at the end of the process. The results suggest the existence of a specialized, saturable transport molecule for this antibiotic.

High-performance liquid chromatography of a new 2-amino-2-oxazoline: application to pharmacokinetic studies in dogs.

5-(1'-Phenyl-4'-piperazinomethyl)-2-amino-2-oxazoline (1; COR3224), a derivative of 2-amino-2-oxazolines with antidepressant properties in rats, was assayed in plasma by high-performance liquid chromatography. After back extraction, 1 and a ortho-O-methyl derivative of 1 as the internal standard were separated by reversed-phase liquid chromatography and measured by UV detection (235 nm). The method is rapid and specific: the detection was linear in the range 125-1000 micrograms.L-1, with a detection limit of 50 micrograms.L-1. This method allowed the determination of pharmacokinetic parameters in six beagle dogs after intravenous and oral administration of 14C-labeled 1 ([14C]1) in a crossover study. The comparison of the results obtained from total radioactivity counting and unchanged product evaluated by HPLC-UV suggest the presence of metabolites.

Pharmacokinetic studies of N-butyric acid mono- and polyesters derived from monosaccharides.

The pharmacokinetics of seven butyric esters derived from monosaccharides were studied after iv administration of a bolus dose to rabbits. Results obtained showed that a constant plasma level of butyric acid is maintained due to the slow disappearance of butyric acid esters from the plasma in contrast to the case of salts, such as arginine butyrate, which are rapidly cleared. The maintenance of these covalent compounds in the body can increase concentrations of n-butyric acid in the tumor area for more efficient chemotherapy. These results seem to be directly related to the in vitro anticellular activity of butyric esters and the prolonged therapeutic protection in tumor-bearing animals.