Comparative single-dose pharmacokinetics of clonazepam following intravenous, intramuscular and oral administration to healthy volunteers.

The objective was to assess the single-dose pharmacokinetics of clonazepam following i.m., p.o. and i.v. administration. In an open-label, three-way crossover study, 12 healthy volunteers were randomized to receive a single dose of 2 mg clonazepam either by the i.m., p.o. or i.v. route. Serial blood samples were collected up to 120 h after drug administration. Plasma concentrations of clonazepam were determined by electron-capture gas-liquid chromatography. The absorption rates of clonazepam after i.m. and p.o. administration of clonazepam were significantly different from each other, as reflected by the respective mean values of maximum plasma concentration (C(max) 11.0 vs. 14.9 ng.ml(-1)) and time to reach maximum concentration (t(max) 3.1 vs. 1.7 h). Secondary plasma peaks of clonazepam were observed in 9 volunteers after i.m. injection (C(max) 9.9 ng.ml(-1); t(max) 10.4 h). A comparison of the area under the plasma concentration-time curves (AUC) shows that the i.m. route is equivalent to the oral route (AUC(0- infinity ) 620 vs. 561 ng.h.ml(-1)). Clonazepam was almost completely absorbed after i.m. and p.o. administration, as shown by the mean absolute bioavailability of 93 and 90%, respectively. No significant differences existed between the elimination half-lives (i.v. 38.0 h; i.m. 43.6 h; p.o. 39.0 h). The average clearance and volume of distribution (V(Z)) were 55 ml.min(-1) and 180 liters, respectively. In conclusion, the observed differences in C(max) and t(max) after i.m. and p.o. administration were consistent with a slower absorption rate of clonazepam after i.m. injection. The systemic exposure to clonazepam was not affected by the route of extravascular administration. Statistical evaluation of these kinetic data showed differences in the absorption rate, so that clonazepam given by the i.m. route is not bioequivalent to the oral route. On the basis of the results of this study, we would recommend the same i.m. and p.o. dose in epileptic patients, but therapeutic response would be expected to be less predictable and to occur later in the case of i.m. administration.

Phosphate deprivation induces overexpression of two proteins related to the rat renal phosphate cotransporter NaPi-2.

Polyclonal antibodies were raised in rabbits against the C-terminal portion of the rat renal brush-border membrane sodium/phosphate cotransporter NaPi-2. Antibody specificity and molecular sizes of proteins related to NaPi-2 were assayed by Western blot analysis. Proteins of 40 and 70-75 kDa (p40 and p70) were immunodetected in rat and mouse brush-border membranes and proteins of 72 and 82 kDa were detected in rabbit. The absence or presence of beta-EtSH in the samples before electrophoresis greatly influenced the immunodetection profile of the rat proteins. Since the 40 kDa protein (p40) can only be detected under reducing conditions, it probably originates from reduction of disulfide bonds in p70. Tryptic cleavage of p40 and p70 revealed identical protein fragments showing the close structural identity of those proteins. Both proteins were more abundant in the outer cortex portion of the rat kidney than in the juxtamedullary portion. Furthermore, rats fed a low-phosphate diet for 24 h showed a 20- and 14-fold increase in the amount of p40 and p70, respectively, compared to control rats, showing that the adaptation to P(i) deprivation by increasing renal phosphate reabsorption is not only the result of overproduction of p70, as previously shown, but is also due to the novel p40 which most probably derives from p70.

Apolipoprotein E4 allele as a predictor of cholinergic deficits and treatment outcome in Alzheimer disease.

Apolipoprotein E (apoE) is critical in the modulation of cholesterol and phospholipid transport between cells of different types. Human apoE is a polymorphic protein with three common alleles, APO epsilon 2, APO epsilon 3, and APO epsilon 4. ApoE4 is associated with sporadic and late-onset familial Alzheimer disease (AD). Gene dose was shown to have an effect on risk of developing AD, age of onset, accumulation of senile plaques in the brain, and reduction of choline acetyltransferase (ChAT) activity in the hippocampus of AD subjects. To characterize the possible impact of the apoE4 allele on cholinergic markers in AD, we examined the effect of apoE4 allele copy number on pre- and postsynaptic markers of cholinergic activity. ApoE4 allele copy number showed an inverse relationship with residual brain ChAT activity and nicotinic receptor binding sites in both the hippocampal formation and the temporal cortex of AD subjects. AD cases lacking the apoE4 allele showed ChAT activities close or within age-matched normal control values. The effect of the apoE4 allele on cholinomimetic drug responsiveness was assessed next in a group (n = 40) of AD patients who completed a double-blind, 30-week clinical trial of the cholinesterase inhibitor tacrine. Results showed that > 80% of apoE4-negative AD patients showed marked improvement after 30 weeks as measured by the AD assessment scale (ADAS), whereas 60% of apoE4 carriers had ADAS scores that were worse compared to baseline. These results strongly support the concept that apoE4 plays a crucial role in the cholinergic dysfunction associated with AD and may be a prognostic indicator of poor response to therapy with acetylcholinesterase inhibitors in AD patients.

Molecular study of P-glycoprotein in multidrug resistance using surface plasmon resonance.

P-Glycoprotein is an integral membrane protein which mediates the energy-dependent efflux of various antitumor agents from multidrug-resistant cancer cells. Surface plasmon resonance was used for the detection of P-glycoprotein after solubilization from drug-resistant and drug-sensitive Chinese hamster ovary cells and for the analysis of its interaction with cyclosporin A, a competitive inhibitor of drug efflux. Detection of P-glycoprotein relied on its binding to the monoclonal antibody C219 which was immobilized on a sensor chip. Binding of Zwittergent 3-14-solubilized P-glycoprotein to the antibody was concentration-dependent and reflected the relative abundance of P-glycoprotein in both cell lines. It was abolished when C219 was omitted or replaced by a rabbit anti-mouse IgG antibody and considerably reduced after precipitation of P-glycoprotein with wheat germ agglutinin. Preincubation of solubilized proteins with cyclosporin A increased the amount of protein bound to the antibody by approximately 30%. These results indicate that surface plasmon resonance is well suited to the detection of P-glycoprotein from biological samples and shows promise as a tool for the study of its interaction with different drugs.

Reduction of plasma alanine aminotransferase during vigabatrin treatment.

In 9 drug-resistant patients with partial seizures treated with vigabatrin, gamma-vinyl GABA (VGB), alanine aminotransaminase (ALAT) activity in plasma was significantly reduced. Comparison of in vitro with in vivo measurements led us to conclude that this reduction is mainly an in vivo phenomenon, perhaps due to cross-enzyme inhibition. The assessment of two biological variables linked with ALAT, glucose and alanine levels under fasting conditions, failed to show any significant metabolic alterations. VGB is an effective drug for partial epilepsy. Our observations do not suggest that reduced ALAT activity is of clinical concern.

Factors affecting the stability of the renal sodium/phosphate symporter during its solubilization and reconstitution.

Phosphate is reabsorbed across the brush-border membrane of the proximal tubule by a specific sodium-dependent symporter. Like the other brush-border membrane transport proteins of the kidney, the phosphate carrier remains to be isolated in a functional state. To establish a set of parameters that allow to preserve its biological activity, the phosphate carrier was solubilized under systematically varied conditions and reconstituted into proteoliposomes. Successful reconstitution was achieved only when the extraction buffer contained lipids extracted from the renal brush-border membrane. Glycerol, an osmolyte which reduces the water activity of the solution, was also required. It could however be replaced by 150 mM sodium or potassium phosphate. Below this concentration and in the presence of glycerol, the ionic strength of the solution had little effect on the stability of the transporter, but sodium phosphate could not be replaced by sodium chloride. Phosphate transport in reconstituted vesicles depended on the concentration of detergent and pH of the extraction buffer. Finally, transport activity was increased when solubilization was carried out in the presence of a reducing agent, dithiothreitol. These results should be helpful during the purification and further characterization of the renal phosphate symporter.

Molecular size of the functional complex and protein subunits of the renal phosphate symporter.

The oligomeric structure of the rabbit renal brush-border membrane sodium/phosphate cotransporter was examined with the radiation inactivation and fragmentation technique. The size of its functional complex (its "radiation inactivation size") was estimated from the rate of decay of its sodium-dependent transport activity as a function of the radiation dose. A radiation inactivation size of 223 +/- 42 kDa was obtained. The polypeptide constituting the monomeric unit of the Na1+/Pi symporter was detected by immunoblotting with polyclonal anti-peptide antibodies directed against the 14 amino acid C-terminal portion of the symporter molecule. Its apparent molecular size estimated by comparison with standards following SDS-polyacrylamide gel electrophoresis was 64,000. This value is in good agreement with its known molecular mass of 51,797 Da calculated from the amino acid sequence deducted from the nucleotide sequence of its gene since this protein is probably glycosylated. The loss of labeling intensity of the polypeptide of M(r) = 64,000 was also measured as a function of radiation dose. The molecular size calculated from these data (its "target size") was 165 +/- 20 kDa. The target size estimated for the rat phosphate cotransporter was 184 +/- 46 kDa, and its previously reported radiation inactivation size was 234 +/- 14 kDa. These results strongly suggest that the renal Na1+/Pi cotransporter exists as an oligomeric protein, probably a homotetramer. The fact that the values obtained for the target size are about 3/4 those obtained for the radiation inactivation size of these cotransport proteins indicates that their subunits are closely associated since most of their subunits appear to be fragmented by a single ionizing radiation hit.

Immunodetection and characterization of proteins implicated in renal sodium/phosphate cotransport.

Polyclonal antibodies raised against the 14-amino acid C-terminal portion of the rabbit renal brush-border membrane Na+/Pi cotransporter, as deduced from the nucleotide sequence of the cloned NaPi-1 gene, were used for Western blot analysis of renal brush-border membrane proteins from rat, rabbit and beef. Proteins of 65 kDa from the rat, 64 kDa from the rabbit, and 38, 66, 77, 92, 110, 176 and 222 kDa from the beef were specifically labelled. The affinity of the antibodies was much greater, however, for the proteins of the rat and rabbit than for those of the beef. The rat 65-kDa antigen was readily detected in brush-border membranes isolated from kidney cortex, but was absent from the basolateral membrane and the cytosolic and microsomal fractions of this tissue, in agreement with the subcellular localization of the Na+/Pi cotransporter. This antigen was however several-fold more abundant in the juxtamedullary portion of the cortex than in the outer portion. Despite a strong stimulation in phosphate transport, a low-phosphate diet had little influence on the amount of antigen detected. An additional peptide-displaceable band corresponding to a protein of 250 kDa appeared when beta-mercaptoethanol was omitted during electrophoresis, in agreement with the possibility that disulfide bonds may be involved in the regulation of renal phosphate transport activity.

Molecular size of the renal sodium/phosphate symporter in native and reconstituted systems.

The size of the renal sodium/phosphate symporter was estimated with the radiation inactivation technique in isolated bovine brush border membrane vesicles and after reconstitution in proteoliposomes. The functional unit of the native phosphate carrier had a radiation inactivation size of 172 +/- 17 kDa. Identical values were obtained for the reconstituted carrier whether it was irradiated before or after the formation of the proteoliposomes (161 +/- 9 and 159 +/- 11 kDa, respectively). The sodium-independent uptake of phosphate was not affected significantly by radiation doses up to 10 Mrad. This activity is therefore not due to the reconstitution of a large phosphate-binding protein such as alkaline phosphatase. Furthermore, bromotetramisole, a specific inhibitor of phosphate binding to this enzyme, had no significant effect on the uptake of phosphate by the proteoliposomes.

Reconstitution of the renal brush-border membrane sodium/phosphate co-transporter.

A simple and rapid procedure was developed for the reconstitution of Na(+)-dependent phosphate-transport activity from bovine kidney brush-border membranes. The phosphate transporter appears to be particularly sensitive to extraction conditions. To prevent its inactivation, the phosphate carrier was solubilized in a buffer containing its substrates, Na+ and phosphate, CHAPS, dithiothreitol, brush-border membrane lipids and glycerol. The uptake of phosphate by reconstituted vesicles was strongly stimulated by the presence of a transmembrane Na+ gradient. This stimulation was abolished when the Na+ gradient was dissipated by monensin. The affinity of the carrier for phosphate was similar in proteoliposomes and in brush-border membrane vesicles (apparent Kt = 40 microM). The transporter was also stimulated by the presence of a high concentration of phosphate on the trans side of the membrane. The reconstituted transport activity was inhibited by arsenate, a known inhibitor of phosphate transport. However, the bovine phosphate carrier, intact or reconstituted, was much less sensitive to inhibition by phosphonoformic and phosphonoacetic acids than were those of other species studied so far. SDS/PAGE revealed that only a small number of brush-border membrane proteins were incorporated into the proteoliposomes. This reconstitution procedure should be useful for the purification and identification of the carrier protein.

[Antiserums against carcino-embryonic antigen (CEA) can induce a specific lysis of colon carcinoma cells by normal lymphocytes]. / Des antisérums dirigés contre l'antigène carcino-embryonnaire (CEA) peuvent induire une lyse spécifique des cellules de carcinome du côlon par des lymphocytes normaux.

Antibody-dependent cell-mediated cytotoxicity (ADCC) against human colon carcinoma cells grown in vitro was demonstrated with two specific rabbit anti-carcinoembryonic antigen (cea) antisera. The same antisera did not lyse the colon carcinoma cells in the presence of complement but without lymphocytes. The normal human lymphocytes in the absence of anti-CEA antiserum had a very low cytotoxic activity during the three hours 51Cr release assay used in this study. Two colon carcinoma cell lines, HT-29 and Co-115, expressing CEA on their surface as demonstrated by immunofluorescence, were significantly lysed in the ADCC test, whereas control tumor cell lines, not expressing CEA, were not affected by the anti-CEA sera and the lymphocytes. The specificity of the reaction was further demonstrated by the inhibition of antibody-dependent cell-mediated cytotoxicity after the addition of increasing amounts of purified CEA to the antiserum. The absorption of the anti-CEA antisera was controlled by radioimmunoassay. Absorption of the antisera by normal lung extracts and red cells of different blood groups did not decrease the cytotoxicity.

Antibody-dependent cell-mediated cytolysis of human colon carcinoma cells induced by specific antisera against carcinoembryonic antigen.

Antibody-dependent lymphocyte cytotoxicity against human colon carcinoma cells grown in vitro was demonstrated with rabbit anti-carcinoembryonic antigen (CEA) antisera and normal human lymphocytes. The same antisera produced no tumor cell lysis in a complement-dependent cytotoxicity test. The specificity of the reaction was demonstrated by the inhibition of antibody-dependent lymphocyte cytotoxicity after the addition of increasing amounts of purified CEA to the antiserum and by the fact that only tumor cell lines expressing CEA on their surface were lysed. Antibody-dependent lymphocyte cytotoxicity was also observed against two colon carcinoma cell lines that expressed Blood Group A antigen, using a human serum containing anti-Blood Group A antibodies of the immunoglobulin G class. This reaction was specifically inhibited by absorption with Blood Group A red cells, whereas the anti-CEA-dependent cytotoxicity was not inhibited by absorption with red cells of different blood groups.