One of the main difficulties with primary rat brain endothelial cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood-brain barrier. As P-glycoprotein expression is known to be much higher in RBECs than in any contaminating cells, we have tested the effect of five P-glycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating cells would not. Treatment with either 4 lg/mL puromycin for the first 2 days of culture or 3 lg/mL puromycin for the first 3 days showed the best results without causing toxicity to the cells. Transendothelial electrical resistance was significantly increased in cell monolayers treated with puromycin compared with untreated cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycin-treated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75 · 10 )6 cm/s) and a high electrical resistance (up to 500 W · cm 2 ). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood-brain barrier for cellular and molecular biology studies as well as pharmacological investigations. Keywords: blood-brain barrier, in vitro model, P-glycoprotein, puromycin, rat brain microvessel endothelium. In the last decade, many efforts have been made to produce reliable in vitro models in order to study the blood-brain barrier (BBB). It is indeed important to better understand the complex cellular and molecular interactions at the interface between blood and brain. The BBB regulates the passage of physiological substances into and out of the CNS and protects it against potentially harmful substances present in the blood. It also prevents the passage of pharmacological substances into the CNS. In order to optimize drug delivery to the CNS, it is important to gain knowledge about the passage of drug candidates through the BBB, especially their effects on the CNS and their toxicity to this barrier (Begley 1996;Tsuji and Tamai 1997). The better we understand BBB regulation, the better we will be able to conceive treatments for CNS pathologies, including neurodegenerative diseases and brain tumours

Although it has been well established that the drug efflux pump P-glycoprotein (P-gp) protects the brain against the entry of cytotoxic drugs, its real in situ localization, i.e., at brain capillary endothelial cells or on astrocyte foot processes, is still controversial. The aim of this study was to compare the expression of P-gp and of multidrug resistance-associated protein (Mrp1), another drug efflux pump, in cultured neonatal rat brain astrocytes and in cultured brain capillary endothelial cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the mdr1b gene was preferentially expressed in astrocytes, whereas both mdr1a and mdr1b mRNA were detected in endothelial cells. Moreover, the mrp1 gene encoding Mrp1 was expressed in both cell types. Western blotting analysis revealed higher expression of P-gp in endothelial cells as compared with astrocytes, but higher expression of Mrp1 in astrocytes. Moreover, P-gp and Mrp1 expression was not modified in more differentiated astrocytes obtained when cultured with db-cAMP for 48 hr. Our functional analysis of P-gp showed a modest effect of P-gp modulators (CsA, verapamil, PSC 833) on the uptake of colchicine (a substrate of P-gp) by astrocytes, whereas they increased by about 50% the uptake of vincristine (a common substrate of P-gp and MRP) by astrocytes. MRP modulators (genistein, probenecid, and sulfinpyrazone) did not modify the uptake of colchicine but increased that of vincristine with a major effect found for sulfinpyrazone. Moreover, indomethacin, probenecid, and sulfinpyrazone increased the uptake of fluorescein (a substrate of MRP but not of P-gp). Taken together, our results provide the first biochemical and functional evidence supporting the expression of P-gp and Mrp1 in rat cultured astrocytes.

1 The toxicokinetics of paraquat were studied in 18 cases of acute human poisoning using a specific radioimmunoassay. Plasma paraquat concentration exhibited a mean distribution half-life ( t½ α) of 5 h and a mean elimination half-life ( t½ β) of 84 h. Cardiovascular collapse supervened early during the course of the intoxication and was associated with the distribution phase. Death related to pulmonary fibrosis occurred late and was associated with the elimination phase. 2 Pharmacokinetic analysis of urine paraquat excretion confirmed the biphasic decline of paraquat. Moreover, renal paraquat and creatinine clearances were not correlated but renal paraquat clearance was never higher than the renal creatinine clearance. 3 Tissue paraquat distribution was ubiquitous with an apparent volume of distribution ranging from 1.2 to 1.6 l/kg. Muscle could represent an important reservoir explaining the long persistence of paraquat in plasma and urine for several weeks or months after poisoning.

Because few pharmacokinetic studies of antibodies and their fragments have compared the influence of species origin and antibody size, the plasma pharmacokinetics of a single intravenous dose (0.7 mg kg-1) of 125I-labelled mouse, rat and human immunoglobulin G (IgG), and mouse F(ab')2 and Fab were investigated in the rat. IgG reached equilibrium after six distribution half-lives, i.e. only 36-50 h post-dosing, and the distribution volume was about four times the rat plasma volume. IgG elimination half-lives ranged from 5.33 to 8.10 days. Fragmentation of IgG into smaller fragments, F(ab')2 and Fab, resulted in pharmacokinetics that were molecular-weight-dependent with volume of distribution and systemic clearance values inversely related to antibody size. We conclude that antibody variability in terms of species origin and size influences antibody pharmacokinetics and should be carefully studied before selection of the best antibody for a clinical application.

The pharmacokinetics of colchicine were studied in six healthy male and four elderly female volunteers after i.v. and oral administration. Plasma samples were collected over 72 h and assayed for colchicine by a specific and sensitive radioimmunoassay. Plasma concentration-time curves were fitted using a three-compartmental model after i.v. administration of 0.5 mg (healthy volunteers) and 1 mg (elderly group) colchicine. The first distribution half-life (t1/2 lambda 1) was short: 9.2 min in healthy volunteers and 3.0 min in the elderly group; the second distribution half-life (t1/2 lambda 2) was of the same order for both groups, 1.2 h. Plasma elimination half-lives were also in the same range: 30 h for healthy volunteers versus 34 h for the elderly subjects. Mean residence time was also in the same range in the two groups: 27 h in healthy volunteers and 21 h for elderly subjects. The volume of distribution (Vz) was 6.7 l.kg-1 for the healthy group and 6.3 l.kg-1 for the elderly group, while Vss was smaller: 4.2 l.kg-1 for healthy volunteers and 2.9 l.kg-1 for elderly subjects. Total body clearance was 10.5 l.h-1 for healthy and 5.5 l.h-1 for elderly subjects. After oral administration of 1 mg, lag-time was 14 min in healthy volunteers and 11 min in elderly subjects. Maximal plasma concentration was 5.5 ng.ml-1 at 62 min in the healthy group, while in the elderly group Cmax was 12 ng.ml-1 at 87 min. Mean absolute bioavailability of the tablet was the same in both groups, 44% for healthy volunteers and 45% for elderly subjects.

1 A specific and sensitive radioimmunoassay was used to study the toxicokinetics of colchicine in seven cases of acute human poisoning. Post-mortem tissue concentrations of colchicine were measured in three further cases. Depending on the time of patient admission, two disposition processes could be observed. The first, in three patients, admitted early, showed a bi-exponential plasma colchicine decrease, with distribution half-lives of 30, 45 and 90 min. The second, in four patients, admitted late, showed a mono-exponential decrease. Plasma terminal half-lives ranged from 10.6 to 31.7 h for both groups. 2 Pharmacokinetic analysis of urine colchicine data was performed for two patients. The fraction of unchanged colchicine excreted in urine was about 30%, renal clearance was about 131 h-1 and three-fold less than total body clearance (391 h-1). The apparent volume of distribution was 211 kg-1. 3 Post-mortem tissue analysis showed an ubiquitous colchicine distribution. Colchicine accumulated at high concentrations in the bone marrow (more than 600 ng g-1), testicle (400 ng g-1), spleen (250 ng g-1), kidney (200 ng g-1), lung (200 ng g-1) and heart (95 ng g -1); it was also found in the brain (125 ng g-1). 4 This toxicokinetic study shows that after massive ingestion, the disposition parameters and kinetics of colchicine are not markedly modified from those occuring in healthy volunteers. The absorption process was not delayed and the distribution and elimination half-lives were in the range known to occur with therapeutic doses.

The 190 kDa multidrug resistance protein MRP1 is likely to be involved in the multidrug resistance phenotype of human gliomas. MRP1 expression was evaluated in surgical tumor samples from 17 patients with gliomas. In addition, the impact of the MRP's inhibitor, indomethacin, on the chemosensitivity to etoposide (VP16) and vincristine (VCR) of two glioblastoma cell lines expressing MRP1 (GL15 and 8MG) was investigated. When evaluated in tumor samples, MRP1 expression was observed in all of them with more than 90% of stained tumor cells in 14/15 high-grade gliomas. MRP1 was also strongly expressed at the membrane of the vascular endothelial cells in the same 14 tumor samples, suggesting that the permeability to anticancer drugs could be also limited across brain tumor vessels. At concentrations comprised between 5 and 50 microM, indomethacin significantly increased the cytotoxic effect of etoposide in both cell lines but it was more efficient in increasing the cytotoxicity of VCR on GL15 cells, as compared with 8MG cells. These results suggest that the association of indomethacin to VCR or etoposide could be of interest in the clinical management of gliomas.

1 Colchicine poisoning, which is relatively rare, is associated with significant morbidity and mortality. Whilst a new treatment modality, in the form of colchicine-specific Fab fragments is on the horizon, currently available therapy is largely supportive. 2 The elimination of colchicine occurs primarily by hepatic metabolism, following a first-order process, with significant enterohepatic circulation. Renal extraction is responsible for approximately 20% of colchicine elimination. 3 We report a case of colchicine intoxication, compli cated by the presence of co-ingestants, in which serum colchicine concentrations remained quasi-constant over the 3 days of the patient's survival, consistent with marked alterations both in metabolism and excretion. The initial presentation was relatively benign but the subsequent course was one of severe colchicine poisoning, resulting in death. 4 Severe colchicine toxicity appears to have resulted in a vicious cycle of progressive organ dysfunction and impaired elimination. 5 Josamycin, one of the co-ingestants and an inhibitor of P-glycoprotein, the membrane pump responsible for multidrug resistance, may have played a significant role in impeding the cellular and biliary elimination of colchicine. Co-ingested opioid and anticholinergic compounds may have altered colchicine absorption and gastrointestinal transit. 6 This case serves as a reminder of the need for attention to co-ingested drugs, to early aggressive therapy, and if available, to consideration of immunotherapy.