Immunohistohemical expression of the chemokine fractalkine and its receptor in the human brain cortex after severe traumatic brain injury and brain hemorrhage.

AIM: Recent experimental studies have suggested that chemokines, a subclass of chemoattractant cytokines which play an important role in regulating leukocyte migration and intercellular communication, participate in brain responses of traumatic injury. Fractalkine (CX3CL1) is a peculiar chemokine, the only one with a CX3C motif, existing both as a soluble and a membrane-anchored molecule. In the brain, Fractalkine has been suggested to have a role in neuroprotection under experimental conditions of brain injury. METHODS: Eighteen human brain samples were obtained during surgery of decompressive craniotomy for severe traumatic brain injury (TBI) or after spontaneous intracranial haemorrhage (ICH). Five normal brain samples were obtained during surgery for unruptured intracranial aneurysms (standard gyrectomy). Immunohistochemistry of formalin fixed and paraffin embedded tissues was performed in order to verify the expression of fractalkine and its receptor (CX3CR1). The values of chemokine and receptor expression were correlated with the clinical parameters of the patients. RESULTS: The chemokine fractalkine was significantly upregulated in the neural compartment after brain injury, compared to normal brain samples. Intensity scores were significantly higher when the interval between injury and surgery was >5 h, (P=0.015). In the glial compartment, Fractalkine expression was significantly associated with less severe clinical conditions and lower intracranial pressure at surgery (P=0.014). Expression of the receptor CX3CR1 was detected, at low intensity, on both glial and neurons. Higher expression in neurons was associated with better clinical conditions (Glasgow score) of patients at admission (P=0.037). CONCLUSION: The results of this study highlights for the first time that fractalkine and its receptor CX3CR1 are expressed in the human brain after TBI and ICH and may be involved in the limitation of tissue damage.

[Cell therapy for stroke: from myth to reality]. / La thérapie cellulaire de l'accident vasculaire cérébral ischémique : du mythe à la réalité

INTRODUCTION: Stroke is one of the leading causes of death and disability worldwide. Intravenous recombinant tissue plasminogen activator is the only available therapy for acute ischemic stroke, but its use is limited by a narrow therapeutic window and cannot stimulate endogenous repair and regeneration of damaged brain tissue. Stem cell-based approaches hold much promise as potential novel treatments to restore neurological function after stroke. STATE OF THE ART: In this review, we summarize data from preclinical and clinical studies to investigate the potential application of stem cell therapies for treatment of stroke. Stem cells have been proposed as a potential source of new cells to replace those lost due to central nervous system injury, as well as a source of trophic molecules to minimize damage and promote recovery. Various stem cells from multiple sources can generate neural cells that survive and form synaptic connections after transplantation in the stroke-injured brain. Stem cells also exhibit neurorevitalizing properties that may ameliorate neurological deficits through stimulation of neurogenesis, angiogenesis and inhibition of inflammation. PERSPECTIVES/CONCLUSION: Performed in stroke, cell therapy would decrease brain damage and reduce functional deficits. After the damage has been done, it would still improve neurological functions by activating endogenous repair. Nevertheless, many questions raised by experimental studies particularly related to long-term safety and technical details of cell preparation and administration must be resolved before wider clinical use.

The microvascular network of the pituitary gland: a model for the application of fractal geometry to the analysis of angioarchitecture and angiogenesis of brain tumors.

In geometrical terms, tumor vascularity is an exemplary anatomical system that irregularly fills a three-dimensional Euclidean space. This physical characteristic, together with the highly variable vessel shapes and surfaces, leads to considerable spatial and temporal heterogeneity in the delivery of oxygen, nutrients and drugs, and the removal of metabolites. Although these biological features have now been well established, quantitative analyses of neovascularity in two-dimensional histological sections still fail to view tumor architecture in non-Euclidean terms, and this leads to errors in visually interpreting the same tumor, and discordant results from different laboratories. A review of the literature concerning the application of microvessel density (MVD) estimates, an Euclidean-based approach used to quantify vascularity in normal and neoplastic pituitary tissues, revealed some disagreements in the results and led us to discuss the limitations of the Euclidean quantification of vascularity. Consequently, we introduced fractal geometry as a better means of quantifying the microvasculature of normal pituitary glands and pituitary adenomas, and found that the use of the surface fractal dimension is more appropriate than MVD for analysing the vascular network of both. We propose extending the application of this model to the analysis of the angiogenesis and angioarchitecture of brain tumors.

Focal extra-axial hemorrahagic mass with subdural hemorrhage secondare to extramedullary hematopoiesis in idiopathic myelodysplastic sindrome.

Idiopathic myelodysplastic syndrome is a disease characterized by a clonal stem cell disorder in which megacaryocitic and granulocytic lineages are mainly involved; extramedullary myeloid metaplasia is due to abnormal location of myeloid tissue in other organs than bone marrow. Rarely the central nervous system is involved. When it happens, it is typical to find masses around the brain and pachymeningeal thickening, but it is very rare to find it associated with subdural haemorrhage, as in the case we describe in the present article. Considering our case and the literature we can suggest that radiological images associated with the clinical history of the patient suggestive for extramedullary hematopoiesis can be sufficient for a correct diagnosis and for a radiotherapy treatment, demanding surgery in the case of diagnostic doubts, massive hemorrahages or neurological decifits caused by the focal lesions.

Lack of toxicity of hydroethanolic extract from Mitragyna inermis (Willd.) O. Kuntze by gavage in the rat.

In traditional medicine in Mali, extracts derived from Mitragyna inermis (Willd.) O. Kuntze (Family: Rubiaceae) are commonly used to treat malaria. The antimalarial activity and the lack of genotoxicity in vitro and in vivo have been demonstrated in previous studies. Acute and chronic evaluation of the toxicity of the hydroethanolic extract of Mitragyna inermis leaves was performed in this study, according to the recommendations (cahier de l'Agence no. 3) of the French Drug Office. Two dosages (300 mg/kg and 3 g/kg) were given in one single administration by gavage to male and female rats. No animal died and no behavioral signs of acute toxicity were observed. Chronic toxicity studies over 28 days showed no changes in body weight and no macroscopic abnormality in the 14 organs examined after the animals were sacrificed. With the 3 g/kg/d drug dosage (100-fold higher than those proposed in man), only slight histological abnormalities were observed. Statistically significant differences, compared to control animals, in the weight of some organs and the values of some haematological or biochemical parameters were observed. However, these values always remained in the range given by the breeder for naive animals of the same strain. These investigations thus seemed to indicate the safety of repeated oral administration (up to 3 g/kg/d) of the hydroethanolic extract of Mitragyna inermis leaves, which can therefore be continuously used with safety by the African population in traditional treatment of malaria.

Differential regulation by protein kinases of activity and cell surface expression of glutamate transporters in neuron-enriched cultures.

This study described the involvement of short-term PKA, PKC or PI3K phosphorylation-mediated processes in the regulation of activity and trafficking of the excitatory amino acid transporters EAAC1, GLAST and GLT-1 endogenously expressed in neuron-enriched cultures. Glutamate uptake was dose-dependently decreased by inhibitors of protein kinase A (PKA), [N-[2-(p-bromocinnamylamino)-ethyl]-5-(isoquinolinesulfonamide)] (H89) or phosphatidylinositol 3-kinase (PI3K) (wortmannin), but not altered after protein kinase C (PKC) inhibition (staurosporine) or activation phorbol-12-myristate-13-acetate (PMA). Biotinylation and immunoblotting results (% of controls) showed that EAAC1 membrane expression was significantly decreased by H89 (71.9+/-4.7%) and wortmannin (63.3+/-20.0%) and increased by PMA (137.7+/-15.5%). H89 and PMA induced a significant decrease of the cell surface fraction of GLAST (54.0+/-34.1% and 73.3+/-14.3%, respectively) whereas wortmannin significantly increased this fraction (119.8+/-9.3%). After treatment with H89, the GLT-1 membrane level showed a two-fold increase (179.4+/-19.7%). Conversely, PMA and wortmannin induced a significant decrease of the cell surface expression of GLT-1 (49.0+/-15.4% and 40.7+/-33.7%, respectively). Confocal microscopy revealed a wortmannin-induced clustering of EAAC1 in the intracellular compartment. These data suggest that trafficking of glutamate transporters can be differentially regulated by PKA-, PKC- and PI3K-dependent signaling pathways and could therefore control total glutamate uptake activity. These processes may represent rapid adaptive responses to changes in the cellular environment, which significantly contribute to regulation of EAA transmission and further prevent possible excitotoxic events.

Parvovirus infection in children with AIDS: high prevalence of B19-specific immunoglobulin M and G antibodies.

OBJECTIVE: Investigation of the prevalence and pathogenic role of parvovirus B19 infection in Italian and Rumanian children with AIDS, compared with age-matched HIV-negative children (controls) with various recurrent infections of unknown aetiology. DESIGN: Detection of B19-specific immunoglobulin (Ig) M and IgG antibodies as the most indicative markers of past or current B19 infection. METHODS: B19 antibodies were detected by two enzyme immunoassays using synthetic peptide or recombinant protein, which corresponded to different B19 epitopes, as coating antigens. RESULTS: B19 IgM and IgG were seen in 10 out of 20 (50%) Italian and in 20 out of 51 (39.2%) Rumanian children with AIDS, in contrast to none out of 17 Italian and one out of 22 Rumanian controls (P less than 0.001). In addition, two Italian controls (11.8%), two Rumanian children with AIDS (3.9%), and two Rumanian controls (9.1%) had B19 IgM alone. Specific IgG alone was detected in eight (40%) Italian and 14 (27.5%) Rumanian children with AIDS, and in seven (41.2%) Italian and four (10.2%) Rumanian controls. CONCLUSIONS: While it is possible to attribute some B19 infections in Rumanian children to blood transfusion, the source was unknown for Italian children. However, in three of the Italian children who had B19 IgM and IgG persistently for 15-22 months, and in a 2-month-old Italian infant with B19 IgM and IgG, HIV might have activated a congenital or perinatally-acquired B19 infection.

Pronounced effect of caprylocaproyl macrogolglycerides on nasal absorption of IS-159, a peptide serotonin 1B/1D-receptor agonist.

OBJECTIVES: This double-blind, randomized, two-way crossover study in 12 healthy male subjects investigated the influence of caprylocaproyl macrogolglycerides on the pharmacokinetics of IS-159 (serotonin-carboxylmethyleneoxy-L-tyrosylglycinamide), a peptide serotonin 1B/1D-receptor agonist, after intranasal administration. METHODS: A dose of 4 mg IS-159 was administered in a volume of 200 microL, once in the presence and once in the absence of 2% caprylocaproyl macrogolglycerides. Plasma concentrations of IS-159 were measured over a period of 12 hours for determination of pharmacokinetic parameters. Systemic and local tolerability were assessed at regular time points, the latter by rhinoscopy and visual analog scales. RESULTS: Caprylocaproyl macrogolglycerides significantly increased the maximum plasma concentration (from 4.7 +/- 1.7 to 48 +/- 17 ng/mL) and the area under the plasma concentration-time curve (from 12 +/- 4.7 to 56 +/- 22 ng x h/mL) of IS-159. The time to maximum concentration (15 to 20 minutes) and the elimination half-life (2.0 to 2.3 hours) were not different between the two treatments. Rhinoscopic examination revealed no differences between treatments, but in the presence of caprylocaproyl macrogolglycerides subjects reported more local and systemic adverse events and on the visual analog scales greater nasal obstruction and rhinorrhea. CONCLUSION: 2% caprylocaproyl macrogolglyceride markedly increased the absorption of IS-159 through the nasal mucosa and elicited only mild irritant effects.

Activation of the adenylate cyclase-dependent protein kinase pathway increases high affinity glutamate uptake into rat striatal synaptosomes.

This study examined the effects of various agents known to alter protein phosphorylation through protein kinase A or C on high affinity glutamate uptake measured in vitro on rat striatal homogenates. Incubation of synaptosomes with the phosphatase inhibitor, okadaic acid, dramatically increased glutamate uptake indicating that underlying phosphorylation mechanisms may be involved in the regulation of this transport process. The protein kinase C activator, phorbol-12,13-dibutyrate, or inhibitor, staurosporine, did not significantly modify glutamate uptake. In contrast, forskolin, which activates adenylate cyclase, induced a dose-dependent increase in glutamate uptake. Saturation kinetic analysis indicated that forskolin increased the Vmax without modifying the Km of the transport process as compared to control values. The effect of forskolin was mimicked by dibutyryl adenosine monophosphate, an analog of cAMP which activates protein kinase A, and counteracted by high doses of N-[2-(methylamino) ethy1]-5-isoquinoline sulfonamide, a protein kinase A inhibitor. These results suggest that an adenylate cyclase-dependent protein kinase may be involved in the post-translational regulation of glutamate transporters.

Effects of PKA and PKC modulators on high affinity glutamate uptake in primary neuronal cell cultures from rat cerebral cortex.

In this study, the effects of various agents known to alter protein phosphorylation, via protein kinase C or A, on high affinity glutamate uptake were investigated in primary neuronal cell cultures of rat cerebral cortex. Incubating the culture dishes with chelerythrine or H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), which inhibit PKC and PKA, respectively, dramatically decreased the glutamate uptake in a dose-dependent manner. Saturation kinetic analysis showed that chelerythrine and H89 decreased the Vmax (chelerythrine: -61%, P < 0.06; -59%, P < 0.05) without affecting the Km of the transport process as compared to the control values. These inhibitory effects were counteracted by the corresponding protein kinase activators, i.e. PMA (phorbol-12-myristate 13-acetate) in the case of PKC and forskolin in the case of PKA, although these protein kinase activators alone did not significantly affect the glutamate uptake. These results provide evidence that, in primary cultures of neuronal cells, the high affinity glutamate uptake may be regulated by both PKA and PKC-mediated phosphorylation processes.

Involvement of the macrolide antibiotic inducible cytochrome P-450 LM3c in the metabolism of midazolam by microsomal fractions prepared from rabbit liver.

This report characterizes the cytochrome P-450 isozyme involved in midazolam metabolism. This study was undertaken into liver microsomal fractions prepared from untreated rabbits or animals treated with drugs known to specifically induce various cytochrome P-450 isozymes such as form LM2 by phenobarbital, LM4 and LM6 by 3-methylcholanthrene and beta-naphthoflavone, LM3a by ethyl alcohol and acetone, and LM3c by macrolide antibiotics (rifampicin, erythromycin and triacetyloleandomycin). Among this library of characterized microsomal preparations, only those obtained from macrolide antibiotic-treated rabbits exhibited a Type I binding spectrum upon addition of midazolam (Ks = 3.2-5.3 micrograms/ml; 10.6-17.5 microM) and significantly metabolized midazolam to its various hydroxylated metabolites (Km = 2.52 +/- 0.22 micrograms/ml; 8.32 +/- 0.73 microM and Vmax = 20 micrograms metabolites formed/min/mg proteins; 66 nmoles metabolites formed/min/mg proteins). The following observations further confirmed the specific involvement of the cytochrome P-450 LM3c isozyme: (i) only anti-cytochrome P-450 LM3c isozyme antibodies intensively inhibited midazolam metabolism, (ii) incubation of microsomes, prepared from TAO-treated rabbits, with midazolam in the presence of potassium ferricyanide which restored the functional cytochrome P-450 LM3c isozyme, increased midazolam metabolism to a similar extent, and (iii) in the presence of Cyclosporin A, a specific substrate of the rabbit cytochrome P-450 LM3c isozyme, midazolam metabolism was inhibited in a concentration-dependent manner. These data demonstrated that the rabbit cytochrome P-450 LM3c isozyme was predominantly involved in midazolam metabolism.

Developmental expression and activity of high affinity glutamate transporters in rat cortical primary cultures.

The expression and activity of glutamate transporters (EAAC1, GLAST and GLT1) were examined during the development of cortical neuron-enriched cultures. Protein content and mitochondrial respiration both increased during the first 7 days, later stabilized and decreased from DIV14. Glutamate transport and extracellular concentration were relatively constant from DIV3 to 18. The kinetic parameters of glutamate transport were at DIV7: K(m)=19+/-3 microM and V(max)=1068+/-83 pmol/mg protein/min and at DIV14: K(m)=40.8+/-9.3 microM and V(max)=1060+/-235 pmol/mg protein/min. The shift in K(m) towards higher values suggest a more important participation of GLAST after DIV14. At DIV7 and 14, glutamate transport was poorly sensitive to dihydrokaïnate (DHK) suggesting a weak participation of GLT1 in glutamate transport. Western blot experiments and immunocytochemistry showed that EAAC1 was expressed by neurons whatever the stage of the culture. GLAST was found in astrocytes as soon as DIV3 and labeling increased during the development of the culture. There was little neuronal GLT1 immunoreactivity at DIV7, only detected by immunocytochemistry. From DIV10 to 18, an increasing astrocytic expression of GLT1 was observed, also detected by Western blotting. These results show that: (1) glutamate uptake remains stable all along the development of the cultures although the pattern of expression of the different transporters is changing, suggesting that glutamate transport is highly regulated; (2) neuronal EAAC1 may play a critical role during the early stages of the culture when it is expressed alone; and (3) the developmental expression pattern of glutamate transporters in cortical neuron-enriched cultures is quite similar to that observed in vivo during early postnatal development.

Hepatitis in children with thalassemia major.

Since thalassemia major patients are transfusion dependent, they are at a particularly high risk of contracting post-transfusion hepatitis. In this study, 36 transfusion-dependent children were followed up for evidence of viral hepatitis. Of 23 with increased ALT levels, 17 were anti-CMV and 12 were anti-HCV positive, 9 were positive for both CMV and HCV. Of 13 children with normal transaminase levels, 5 were CMV positive and 3 were HCV positive. These results show that CMV may be a very common cause of non-A, non-B hepatitis in transfusion dependent thalassemic children.

Involvement of the glutamatergic metabotropic receptors in the regulation of glutamate uptake and extracellular excitatory amino acid levels in the striatum of chloral hydrate-anesthetized rats.

The microdialysis technique was used to assess in vivo the putative functional role of metabotropic excitatory amino acid receptors in regulating extracellular levels of the excitatory amino acids glutamate and aspartate in the striatum of chloral hydrate-anesthetized rats. Addition of the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) (10(-3) or 4 x 10(-3) M) in the dialysis probe did not modify the basal extracellular levels of glutamate and aspartate but induced a significant dose-dependent decrease in the KCl-elicited elevation of glutamate and aspartate extracellular levels. The effect of MCPG on glutamate extracellular concentration under K+ stimulation was reduced by the simultaneous superfusion of the metabotropic glutamate receptor agonist (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine) (L-CCG) (10(-3) M) which had no significant effect when tested alone. In contrast, L-CCG alone significantly potentiated the KCl-elicited elevation of aspartate extracellular concentrations but failed to modify the MCPG effect on this amino acid concentration. In a parallel series of experiments, high-affinity glutamate uptake was measured ex vivo 20 min after an in vivo injection of 10 pmol of MCPG in the striatum. MCPG was found unable to modify the glutamate uptake rate. In vitro, MCPG (1-1000 microM) again had no effect on glutamate transport rate. These data suggest that metabotropic excitatory amino acid receptors (1) may act to increase the extracellular levels of glutamate and aspartate under depolarizing conditions, and (2) may not have a major role in the regulation of high affinity glutamate uptake under basal conditions. In addition, it can be assumed that the control of glutamate and aspartate extracellular levels may involve different metabotropic receptors.

Ketamine effects on bupivacaine local anaesthetic activity and pharmacokinetics of bupivacaine in mice.

This study was designed to document possible changes in bupivacaine (B) local anaesthetic activity and pharmacokinetics in mice after a ketamine (K) injection. In the experiments, bupivacaine (8.25 mg.kg(-1)), was injected into the popliteal space of the right posterior limb: the local anaesthetic activity was assessed according to a sciatic nerve blockade method with three different doses (2, 10 and 40 mg/kg) of ketamine and the kinetics were studied after a 10 mg/kg dose. When ketamine was associated, the local anesthetic activity of bupivacaine was significantly enhanced as well as its elimination half-life. Significantly lower levels of the main metabolite, PPX, were observed, when ketamine was associated, suggesting a metabolic inhibition phenomenon. The ketamine-induced increase in the total anaesthetic effect of bupivacaine may thus be explained by kinetic modifications i.e. a possible inhibiting effect of ketamine on the metabolism of bupivacaine.

Identification of the human and animal hepatic cytochromes P450 involved in clonazepam metabolism.

This report characterizes the cytochrome P450 isozyme involved in clonazepam metabolism. This study was undertaken using a library of liver microsomal fractions prepared from untreated rabbits or those treated with drugs known to specifically induce various cytochrome P450 isozymes (ie P450 2B4 by phenobarbital, P450 1A1 and P450 1A2 by 3-methylcholanthrene and beta-naphthoflavone, P450 2E1 by acetone and ethyl alcohol, and P450 3A6 by erythromycin). Only microsomes obtained from phenobarbital-treated rabbits exhibited a type II binding spectrum upon addition of clonazepam (Ks(app) = 31.4 +/- 3.8 microM) and significantly metabolized clonazepam to 7-aminoclonazepam. Benzphetamine, which is a known substrate for P450 2B1 was also extensively metabolized by microsomes prepared from phenobarbital treated rabbits. This indicates that the same isozyme (P450 2B subfamily) was involved in the biotransformation of both substrates. Experiments performed on 14 human liver microsomal preparations showed a wide interindividual variability (from 1-4) and a good correlation (r = 0.70) between benzphetamine and clonazepam metabolism. Since P450 3A4 (nf25) was involved in benzphetamine metabolism, clonazepam was probably nitroreduced by the same isozyme. An oligonucleotide specific for the P450 3A4 gene subfamily was synthetized and used for hybridization on total RNA from human liver samples. Two transcripts of 2.2 and 3.0 kb were detected and the level of the 2.2 kb mRNA expression was significantly correlated (r = 0.61) with the intensity of clonazepam nitroreduction by the corresponding microsome batches.

1H nuclear magnetic resonance and clinical studies of interaction of calcium antagonists and hypoglycemic sulfonylureas.

The hypoglycemic effect of gliclazide is mainly due to its action on ATP stimulated K+ channels, but the calcium ionophoretic effect of this drug may also be involved in its physiological properties. Using 1H NMR we demonstrated the antiionophoretic effect of nifedipine and diltiazem. We attempted to verify whether this in vitro interaction also occurs in vivo. A clinical trial, was performed on patients treated concomitantly with gliclazide and nifedipine or diltiazem. Results showed that no in vivo interaction occurred. The discrepancy between in vivo and in vitro results may be explained by a too weak plasma concentration in the case of nifedipine and by a large plasma protein binding in the case of diltiazem.

Pharmacokinetic and pharmacodynamic analysis of sedative and amnesic effects of lorazepam in healthy volunteers.

This study describes for the first time the pharmacokinetic and pharmacodynamic modeling of the psychomotor and amnesic effects of a single 2-mg oral dose of lorazepam in healthy volunteers. Twelve healthy volunteers were included in this randomized, double-blinded, placebo-controlled two-way crossover study. The effect of lorazepam was examined for a battery of tests that explored mood, vigilance, psychomotor performance, and memory. The pharmacokinetic and pharmacodynamic modeling of these tests was performed using the indirect response model. Vigilance and psychomotor performance were significantly impaired. Short-term memory was not affected, but a paradoxical tendency to improvement of the score was observed 0.4 hours after drug intake. Significant impairment was observed for immediate and delayed cued verbal recall, for immediate and delayed free recall, and for picture recognition as well as for visual-verbal recall, but not for cued visual-spatial recall or priming. Globally, the different effects were greatest between 0.4 to 3 hours after dosing. However, the time course profile of the recovery period suggests a possible dissociation between the kinetics of the effects of lorazepam on vigilance, psychomotor performance, and visual episodic memory, on the one hand, and on verbal episodic memory, on the other. The pharmacokinetic and pharmacodynamic model used two compartments with first-order absorption to describe the lorazepam concentrations and an indirect response model with inhibition or stimulation of Kin to describe the effects. The mean values for calculated median effective concentration (EC50) derived from the pharmacokinetic and pharmacodynamic modeling of the different tests ranged from 11.3 to 39.8 ng/mL. According to these EC50 values, lorazepam seemed to be more potent on the delayed-recall trials than on the immediate-recall trials; similar observations were made concerning the free-recall versus cued-recall trials. The previously stated results suggest that the tests performed in this study represent sensitive measurements of the effects of lorazepam on the central nervous system. Moreover, the parameter values derived from pharmacokinetic and pharmacodynamic modeling, especially, the EC50 values, may provide sensitive indices that can be used to compare the central nervous system effects of benzodiazepines.

Fundamental aspects of radioreceptor assays.

Significant advances in understanding the actions of hormones, neurotransmitters and drugs resulted from the development of new analytical procedures. Among these advances the discovery that, for a given drug (or hormone or neurotransmitter), there exists a receptor site on membranes to which the substance must attach in order to evoke a biological response, led to the setting up of the radioreceptor assays. Thus ligand-binding techniques can be used as assays for measuring drug levels in biological tissues and fluids. Radioreceptor assays are based on the principle of competitive protein-binding methods; when a radioligand and an unlabelled ligand are present together with a specific receptor preparation, the amount of radioactive ligand bound to the receptor is a quantitative function of the amount of unlabelled ligand present in the incubation medium. The technology of the radioreceptor assays is simple and rapid to perform when optimal conditions for binding are determined. Radioreceptor assays are highly sensitive, reliable, precise and accurate. Their chief limitation relates to specificity as any substance having an appreciable affinity for the receptor displaces the specifically bound radioligand. Paradoxically this lack of specificity may be in some cases advantageous in that it allows for the detection of the parent compound and active metabolites in proportion to their affinity for the specific receptor. During recent years, radioreceptor assays have been applied to quantitative determination of hormones, neurotransmitters and drugs. Among these, the radioreceptor assay of benzodiazepines is a typical example. In addition to drug analysis and drug monitoring, radioreceptor assays have been found to be versatile tools for basic studies on the receptor itself, on the endogenous ligand(s) for the receptor and on the discovery and the identification of biologically active chemical entities (pharmacological screening). The future of radioreceptor assays in the pharmacological sciences appears to be promising.

Plasma concentrations and pharmacokinetics of midazolam during anaesthesia.

Midazolam and 1-hydroxymidazolam plasma concentrations have been monitored and pharmacokinetic parameters of midazolam estimated during anaesthesia induced and maintained by its repeated injection according to two protocols (3 X 0.3 mg kg-1 at 45 min intervals or an induction dose of 0.3 mg kg-1 with maintenance doses of 0.15 mg kg-1 at 30 min intervals). Minimum plasma concentrations of midazolam measured just before each injection were 258.8 +/- 108.4 ng ml-1 for the first protocol and 353.1 +/- 55.2 ng ml-1 for the second protocol; maximum midazolam concentrations, measured 5 min after the last administration, were 1103.1 +/- 237.9 ng ml-1 and 743.0 +/- 103.2 ng ml-1, respectively, suggesting that a continuous infusion of midazolam after a loading dose should be better than repeated injections at keeping the concentration close to the sedative level of 400 ng ml-1. The estimated pharmacokinetic parameters were similar to those already published, except for the beta elimination half-life of midazolam (3.24 +/- 0.90 h for protocol 1 and 3.34 +/- 1.47 h for protocol 2) which was slightly longer than that reported for single dose studies. The comparison of plasma determinations, obtained either by gas-liquid chromatography or by a radioreceptor assay technique, clearly showed that 1-hydroxymidazolam, even after repeated midazolam administration, was not present at a concentration sufficient to affect the overall pharmacological activity of the parent drug.