[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.

Risk of transmission of Creutzfeldt-Jakob disease via blood and blood products. The French risk-analysis over the last 15 years.

Risk of transmission of Creutzfeldt-Jakob disease (infectious agent, responsible of spongiform encephalopathy) via blood and blood components (including the plasma-derived medicinal products such as coagulation factors and immunoglobulins) have been a subject of concern for Health authorities since the early 1980s, with a regain of interest in the 1990s, with the bovine spongiform encephalopathy outbreak followed few years after with the notification of the first cases of variant Creutzfeldt-Jakob disease in humans. The risk-analysis and measures taken by the French authorities in the period 1990-2010 will be described with the various assumptions and working hypothesis used and revisited as new findings become available.

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.

[Health security--GMOs in therapeutics]. / Sécurité sanitaire des OGM utilisés en thérapeutique.

The recent progress in human therapeutics has been made possible thanks to molecular biology and its use in producing proteins having the same sequence and structure as that of human proteins. The use of GMOs allows production of proteins with high added value in therapeutics, which are of satisfactory quality. GMOs may also be directly administered to patients as gene therapy vectors. However, the use of GMOs in therapeutics must take into consideration some risks, particularly those of microbiological contamination, of neo-antigenicity as well as environmental risks with regard to the way of use of the GMO. Nevertheless, those risks are taken in due consideration in the development of these new medicinal products; solutions have been found to allow their use in therapeutics with a very positive benefit/risk ratio. Medicinal products from biotechnology have enabled considerable therapeutic progress without compromising health security.

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.

Determination of melatonin in rat pineal, plasma and retina by high-performance liquid chromatography with electrochemical detection.

A sensitive method for the routine measurement of endogenous melatonin (MEL) in pineal, retina and plasma rat tissues has been developed using reversed-phase high-performance liquid chromatography with electrochemical detection. Quantification limit for MEL was 0.2 ng/mg protein in pineal, 15 pg/ml in plasma and 2.0 pg/mg protein in retina. To improve both MEL quantification and the reproducibility of the assay, an internal standard was used when an extraction in organic solvent was required, in contrast with other available chromatographic methods. MEL values and the circadian profile obtained in this study from both rat pineal and plasma agree with those reported previously. This method allows MEL detection in mammal retina, particularly in rat, where MEL levels are very low.

Tissue distribution and metabolism in rat of an ethynesulphonamide with filaricidal activity.

1. The tissue distribution and metabolism of a new filaricidal agent P903 (N-[(2-phenylethynyl)sulfonyl]morpholine) were studied in rat. 2. After s.c. administration of 14C and 13C P903, the Tmax in the blood was observed on day 2. Elimination was slow and > 95% was bound to protein. Radioactivity was distributed in the whole organism but particularly in erythrocytes and the lymphatic channel. Four days later, > 60% of the radioactivity was excreted in urine and faeces at equal amounts and 15% remained at the injection point. 3. In all biological fluids tested no P903 was found but only its metabolites. 4. One principal metabolite, the N-[(2-phenyloxo-2-ethane) sulphonyl] morpholine or oxosulphonamide was identified in blood, urine and faeces as compared with the reference compound by GC/MS and NMR. This latter molecule was detected following hydrolysis by hydrochloric acid but not with beta glucuronidase/sulphatase. 5. Unconjugated and conjugated oxosulphonamide represented > 85% of the radioactivity at all times tested in blood but only 38 and 35% respectively of urinary and faecal radioactivity on day 1 after the administration of the labelled drug. 6. Thus, P903 is rapidly converted to a reactive metabolite, probably an oxirene, which is then conjugated with endogenous components to form conjugated oxosulphonamide and an unknown metabolite. The role of this reactive metabolite in antifilarial activity seems to be very important in understanding the mechanism of action of P903.

Dopamine inhibits melatonin synthesis in photoreceptor cells through a D2-like receptor subtype in the rat retina: biochemical and histochemical evidence.

An intrinsic oscillator, using dopamine and melatonin as antagonist signals, controls rhythmic events in the retina of nonmammals. The purpose of the present work was to localize and characterize a dopamine receptor responsible for the nocturnal inhibition of melatonin synthesis in photoreceptor cells in a mammalian retina. An antibody against the D2 receptor stained photoreceptor cell inner segments of the rat retina. alpha-Methyl-p-tyrosine, a competitive inhibitor of tyrosine hydroxylase, enhanced the nocturnal content of melatonin, suggesting the dopamine control of melatonin synthesis as in non-mammals. Clozapine, a D2c/D4 antagonist, also enhanced the nocturnal level of melatonin, whereas raclopride, a D2A antagonist, did not. Taken together, these results support the control of melatonin levels by dopamine through a D2C/D4 receptor in photoreceptor cells of the rat retina. The presence of D4 receptors in the rat retina was confirmed by reverse transcription-PCR.

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.

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.

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.

6-Fluoro-serotonin as a substrate for the neuronal serotonin transporter.

6-Fluoro-serotonin (6F-5-HT) was previously identified in the rat brain after peripheral administration of 6-fluoro-DL-tryptophan, a serotonin (5-HT) synthesis inhibitor. These present studies, performed with rat brain synaptosomes show that: i-neuronal 6F-5-HT uptake partly involved the 5-HT transporter since it was inhibited by clomipramine, a 5-HT uptake inhibitor, ii-6F-5-HT blocked the synaptosomal uptake of 3H-5-HT, with an IC50 value of 98 +/- 13 nM, and iii- 6F-5-HT induced 3H-5-HT release from preloaded synaptosomes, with an EC50 value of 95 +/- 6 nM; this release was decreased in the presence of clomipramine, suggesting the involvement of the 5-HT transporter. This release was also reduced when using synaptosomes from reserpinized rats, suggesting that the vesicular pool also participates to the 3H-5-HT release induced by 6F-5-HT. So, 6F-5-HT behaved as a substrate for the 5-HT neuronal transporter.

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.

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.

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.

Modification of cefixime bioavailability by nifedipine in humans: involvement of the dipeptide carrier system.

We studied the action of nifedipine on the bioavailability of cefixime, a molecule absorbed via the gut wall dipeptide carrier system in the rat, and on the bioavailability of D-xylose, which is absorbed via a pH (and Na(+)-)-dependent transporter. Each compound was administered alone or in combination with 20 mg of nifedipine to eight healthy male volunteers. Nifedipine significantly increased the absorption rate of cefixime (20.7 +/- 4.3 versus 16 +/- 3.5 mg/h in the absence of nifedipine). The absolute bioavailability of cefixime alone was 31% +/- 6% compared with 53% +/- 1% (P < 0.01) in the presence of nifedipine. The observed peak concentrations in serum were significantly different (2.5 +/- 0.3 mg/liter without nifedipine and 3.7 +/- 1.1 mg/liter with nifedipine; P < 0.02). In contrast, nifedipine induced no significant differences in the pharmacokinetic profile of xylose following oral administration. We conclude that (i) cefixime is absorbed in humans by an apparently active process which can be enhanced by a calcium channel blocker, in this case, nifedipine; and (ii) nifedipine does not modify the activity of the pentose transporter.