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Purpose: The expression level of the transferrin receptor (TfR) on brain capillary endothelial cells (BCECs) and the endocytosis of 125I-transferrin (125I-Tf) by this receptor was investigated. Furthermore, the influence of iron, the iron scavenger deferoxamine mesylate (DFO), astrocytic factors, a GTP-ase inhibitor (tyrphostin-A8, T8), lipopolysaccharide (LPS), and the radical scavenger N-acetyl-L-cysteine (NAC) on the TfR expression was studied to gain insight in the use and optimization of the TfR for drug targeting to the brain., Methods: Experiments were performed with primary cultured bovine BCECs that were incubated with 125I-Tf at 4 degrees C (to determine binding) or at 37 degrees C (to determine endocytosis) in the absence or presence of the modulators. For full saturation curves in the absence or presence of iron or DFO, analysis was performed with a population approach using NONMEM, allowing us to estimate a single value for affinity (Kd, concentration of 50% receptor occupancy) and separate values for maximum receptor occupancy (B(max)., Results: On BCECs, the TfR is expressed extracellularly (B(max) of 0.13 fmol/microg cell protein), but also has a large intracellular pool (total B(max) of 1.37 fmol/microg cell protein), and is actively endocytosing Tf via clathrin-coated vesicles. At 4 degrees C, a Kd of 2.38 microg/ml was found, whereas the Kd at 37 degrees C was 5.03 microg/ml. Furthermore, DFO is able to increase both the extracellular as well as the total binding capacity to 0.63 and 3.67 fmol/microg cell protein, respectively, whereas it had no influence on Kd. B(max) at 37 degrees C after DFO preincubation was also increased from 0.90 to 2.31 fmol/microg cell protein. Other modulators had no significant influence on the TfR expression levels, though LPS increased cellular protein concentrations after 2-h preincubation., Conclusions: The TfR is expressed on BCECs and actively endocytoses Tf, making it a suitable target for drug delivery to the bloodbrain barrier and the CNS. DFO up-regulates the TfR expression level, which may influence targeting efficiency.

Purpose: To investigate whether P-glycoprotein (Pgp) protects the in vitro BBB against the cytotoxic effects of anti-tumour drugs., Methods: In an in vitro BBB coculture model the influence of the anti-microtubule drugs vinblastine, colchicine, paclitaxel and the non-anti-microtubule drugs doxorubicin, fluorouracil and etoposide in the absence or presence of Pgp modulators on the trans-endothelial electrical resistance (TEER), which is an indicator for the integrity, was investigated., Results: In the absence of Pgp modulators vinblastine, colchicine and paclitaxel dose dependently decreased TEER values to less than 20% of control. Non-anti-microtubule drugs did not affect TEER values. Following competitive inhibition of Pgp by various Pgp modulators and substrates, even low concentrations of vinblastine, colchicine and paclitaxel substantially decreased TEER. IC50 values of LY 335979, SDZ-PSC 833, cyclosporin A, and verapamil were 0.03, 0.25, 0.46, and 13.7 microM, respectively., Conclusions: These results indicate that Pgp normally protects the in vitro BBB against the disruptive effects of anti-microtubule drugs, but its integrity is lost when anti-microtubule drugs are used in combination with potent Pgp modulators. In addition, this procedure offers the possibility to characterize Pgp modulators and substrates with respect to their efficacy and to elucidate drug interactions at the level of Pgp.

Purpose: To investigate the influence of astrocytes on P-glycoprotein (Pgp) expression and intracellular accumulation of Pgp substrates, separate from their net transcellular transport across the blood-brain barrier (BBB)., Methods: An in vitro BBB model was used, comprising of brain capillary endothelial cells (BCEC) monolayers or BCEC co-cultured with astrocytes., Results: BCEC+astrocyte co-cultures seemed to express a higher level of Pgp compared to BCEC monolayers. Inhibition of Pgp results in an increased intracellular accumulation of Pgp substrates in both BCEC monolayers and BCEC+astrocyte co-cultures, and increased the sensitivity for vinblastine mediated disruption of the in vitro BBB (called the vinblastine exclusion assay). BCEC monolayers were more sensitive to vinblastine mediated disruption compared to BCEC+astrocyte co-cultures. In the latter, but not in BCEC monolayers, an inhibitable polar transport of Pgp substrates was only found from the brain to the blood side of the filter., Conclusions: Astrocytes increase the functional expression of Pgp in our in vitro BBB model. These results also illustrate that an important role for Pgp on the BBB is to protect the barrier against intracellular accumulation of cytotoxic BBB disrupting compounds.

Purpose: The effect of P-glycoprotein (Pgp) on brain distribution using mdr1a (-/-) mice was investigated., Methods: Fluorescein (Flu) and FD-4 were used to check whether blood-brain barrier (BBB) integrity was maintained in mdr1a (-/-) mice. The Pgp substrate rhodamine-123 (R123) was infused and total brain, blood and brain microdialysate concentrations in mdr1a (-/-) mice and wild-type mice were compared., Results: Maintenance of BBB integrity was indicated by equal total brain/blood ratios of Flu and FD-4 in both mice types. R123 concentrations in brain after i.v. infusion were about 4-fold higher in mdr1a (-/-) than in wild-type mice (P < 0.05), without changes in blood levels. After microdialysis experiments the same results were found, excluding artifacts in the interpretation of Pgp functionality by the use of this technique. However the 4-fold ratio in brain was not reflected in corresponding microdialysates. No local differences of R123 in the brain were found. By the no-net-flux method in vivo recovery appeared to 4.6-fold lower in mdrla (-/-) mice compared with wild-type mice., Conclusions: Pgp plays an important role in R123 distribution into the brain. Using intracerebral microdialysis, changes in in vivo recovery by the absence or inhibition of Pgp (or active efflux in general) need to be considered carefully.

Purpose: The transport of the hydrophilic model compound sodium fluorescein into the cerebrospinal fluid (CSF) of rats was studied during experimental allergic encephalomyelitis (EAE), as a model for local central nervous system (CNS) inflammatory disease, and after a single injection of a pyrogenic dose of lipopolysaccharide (LPS), as a model for a general inflammation., Methods: Transport of sodium fluorescein was measured by means of serial CSF and plasma sampling. Transport of this hydrophilic model compound was studied in Lewis rats suffering from EAA and three hours after LPS administration in male Wistar rats., Results: During acute EAE, sodium fluorescein concentrations in the CSF increased twofold compared to control animals, whereas plasma kinetics were comparable within both groups. After i.v. LPS administration, however, plasma as well as CSF kinetic parameters of sodium fluorescein concentration were significantly changed from those seen in control animals. Transport of sodium fluorescein from plasma into the CSF was calculated as the ratio Area Under the Curve (AUC)CSF/AUCPLASMA. During acute EAE this ratio increased 2-fold compared to control animals, whereas after i.v. LPS administration it was not significantly different from the one obtained in control animals., Conclusions: These results suggest an opening of the blood-brain barrier (BBB) during a cerebral inflammatory response, like acute EAE, but not after LPS administration.

Purpose: The use of intracerebral microdialysis as a tool to measure the penetration of anticancer agents in brain tumor was investigated., Methods: Following intravenous (iv) administration of 75 mg/kg. concentration-time profiles of methotrexate (MTX) were determined in brain cortical dialysate and in plasma. The individual ratio of the area under the curve of MTX in brain dialysate over that in plasma (MTX penetration) was determined in normal brain, in tumor-bearing brain and in brain after sham tumor implantation. Individual brains were examined histologically on the presence of tumor, as well as for other factors that might influence local MTX penetration. Histological scores were related to the individual data on penetration of MTX., Results: MTX penetration values were higher in cortical brain at the site of the tumor, as compared to the levels measured in normal or sham implanted brain (mean increase to 250%). In the cortical brain contralateral to the tumor, MTX penetration values were found to be lower than in normal brain (mean reduction of 65%). Furthermore, it appeared that in the absence of tumor tissue, the presence of exudate around the probe was independently associated with increased penetration of MTX into the brain., Conclusions: Tumor tissue appeared to be the most important parameter in changing local MTX penetration in brain after tumor implantation. In general, it is anticipated that intracerebral microdialysis combined with histological examination can be used to investigate effects of brain tumor presence on regional (periprobe) penetration of anticancer drugs into the brain.

Purpose: The evaluation of the inhibitory action of two mucoadhesive poly(acrylates), polycarbophil and carbomer, registered by the Food and Drug Administration (FDA), on the intestinal proteolytic enzyme trypsin., Methods: The effect of the polymers on trypsin activity by measuring the degradation of a trypsin specific substrate. Binding of Ca2+ ions and proteins (125I-BSA) to the poly(acrylates). The influence of the polymers on the secondary trypsin structure by circular dichroism., Results: Trypsin inhibition was found to be time-dependent upon addition of Ca2+ in the degradation experiment. Only when Ca2+ was added within 10 min after trypsin incubation, recovery of the enzyme could be observed. Both polymers showed a strong Ca2+ binding ability. Carbomer, which had a higher inhibitory effect on trypsin activity, also revealed a higher Ca2+ binding affinity than polycarbophil. The amount of Ca2+ depleted out of the trypsin structure and the reduction of enzyme activity were comparable. Immobilization of trypsin by binding to the polymers could not be observed at pH 6.7. Circular dichroism studies suggested that, under depletion of Ca2+ from trypsin, the secondary structure changed its conformation, followed by an increased autodegradation of the enzyme., Conclusions: The poly(acrylates) investigated may have potential to protect peptides from tryptic degradation and may be used to master the peroral delivery of peptide drugs.

The aim of this study was to determine whether changes in the transport of drugs into the brain could be determined by in vivo intracerebral microdialysis. Atenolol was used as a model drug to determine blood-brain barrier (BBB) transport characteristics. In rats, unilateral opening of the blood-brain barrier was achieved by infusion of hyperosmolar mannitol (25%, w/v) into the left internal carotid artery. BBB transport, expressed as the ratio of the area under the curve (AUC) of atenolol in brain extracellular fluid over plasma, was three times higher for the mannitol treated hemisphere as compared with the contralateral brain or after infusion of saline, being (mean +/- SEM) 0.094 +/- 0.024 (n = 16), 0.029 +/- 0.007 (n = 12) and 0.030 +/- 0.009 (n = 12) respectively. Further evaluation of the data indicated that for experiments performed in the morning the mannitol infusion had little effect on the extent of transport of atenolol into the brain, while in the afternoon BBB transport was about 10-fold higher than in the contralateral and saline group. The mean "afternoon" ratios +/- SEM were 0.155 +/- 0.038 (n = 8), 0.012 +/- 0.003 (n = 6) and 0.018 +/- 0.006 (n = 6) respectively. It is concluded that intracerebral microdialysis is capable of revealing changes in BBB transport and regional and time-dependent differences in drug levels can be demonstrated with the use of this technique.

Confocal laser scanning microscopy (CLSM) was used to quantify and visualize the transport of the octapeptide and somatostatin analogue, octreotide (SMS 201-995, Sandostatin), across monolayers of bovine cerebrovascular endothelial cells, an in vitro model of the blood-brain barrier. The concentrations of octreotide and its conjugates in the cell culture medium were determined by radioimmunoassay (RIA). Two fluorescent conjugates of octreotide (FITC- and NBD-octreotide) were used to obtain CLSM images. The peptides did not undergo significant degradation in the presence of brain endothelial cell monolayers. The transport rate of octreotide expressed as clearance (Cl) and endothelial permeability (Pe) did not depend on either the initial concentration (between 10 nM and 1 microM) or the site of administration (luminal or abluminal side of the monolayer), indicating the absence of saturable and/or asymmetrical transport mechanisms. The Pe of octreotide and that of the paracellular permeability marker fluorescein correlated well. Although the conjugates are more lipophilic than octreotide itself, they exhibited lower Cl and Pe, values probably because of their larger molecular size. On the CLSM images, FITC-octreotide was present only in the intercellular space, while the cells did not exhibit detectable fluorescence. Transport studies and CLSM images suggest that octreotide passes the endothelial monolayer primarily via the paracellular route without significant contribution of carrier-mediated transport.

The transport of the antiviral nucleoside analogue zidovudine (3'-azido-3'-deoxythymidine; AZT) into the central nervous system (CNS) was characterized in vitro and in vivo. The in vitro model consisted of primary cultures of isolated bovine capillary endothelial cells. The transport rate of AZT across the monolayer, expressed as endothelial permeability P, was determined following luminal and abluminal administration. P did not differ between the two administration sites (luminal, 1.65 +/- 0.44 cm/min/10(3); abluminal, 1.63 +/- 0.28 cm/min/10(3)). The transport of AZT across the endothelial cell monolayer was found to be concentration independent in the range between 0.4 and 50 micrograms/mL. AZT transport was not affected by pretreatment of the cells with either metabolic inhibitors (DODG and DODG/NaN3) or probenecid. This suggests that AZT passes the monolayer mainly by passive diffusion. The in vivo transport of AZT across the blood-brain barrier and the blood-CSF barrier was studied in male Wistar rats after coadministration of potential inhibitors of active transport of AZT: probenecid (organic anion transport) and thymidine (nucleoside transport). Intracerebroventricular and intravenous coadministration of probenecid caused a significant (P < 0.001) increase in the CSF/plasma concentration ratio compared to the control phase, indicating that the organic anion carrier is involved in AZT transport from CSF to blood. Since there was no effect of probenecid on the transport of AZT in vitro, it is suggested that this carrier is located at the choroid plexus. Coadministration of thymidine did not affect the CSF/plasma concentration ratio, suggesting that a nucleoside carrier system is not involved in AZT transport into or out of the CNS.

The possibility of developing bioadhesive drug delivery systems on the basis of molecules which selectively bind to the small intestinal epithelium by specific, receptor-mediated mechanisms was investigated using a lectin isolated from tomato fruits (Lycopersicum esculentum). The tomato lectin (TL) was found to bind specifically onto both isolated, fixed pig enterocytes and monolayers of human Caco-2 cell cultures with a similar affinity. TL-coated polystyrene microspheres (0.98 micron) also showed specific binding to enterocytes in vitro. Lectin binding was found to be favored at neutral pH and to be reduced in an acidic environment. Crude pig gastric mucin, however showed a marked cross-reactivity in vitro, indicating that lectin binding to the cell surface in vivo might be inhibited by mucus.

The blood-brain barrier transport characteristics of racemic baclofen and the separate R- and S-enantiomers have been determined in vivo in rats by using the unit impulse response methodology. Transport rate was determined as blood-brain barrier clearance, the volume of plasma per unit time cleared of baclofen by transport across the blood-brain barrier. Plasma elimination kinetics and CSF elimination kinetics did not differ among racemic baclofen and the R- and S-enantiomers. Transport of each compound could be described by a linear V(t) curve, suggesting the absence of saturable transport processes in the concentration range studied. However, for R-baclofen the blood-brain barrier clearance (4.7 +/- 1.0 microliters/min, mean +/- SE; n = 6) and cumulative transported amount (0.085 +/- 0.007%; n = 6) were significantly higher than these values for the S-enantiomer (1.1 +/- 0.3 microliters/min, 0.031 +/- 0.005%; n = 6) and racemic baclofen (1.0 +/- 0.1 microliters/min, 0.036 +/- 0.003%; n = 6). These findings indicate that there is stereoselective transport of baclofen across the blood-brain barrier.

The pharmacokinetic characteristics of desglycinamide-arginine vasopressin (DGAVP) with respect to its transport across the blood-brain barrier (BBB) were studied with the use of serial CSF sampling in an individual animal and the unit impulse response methodology. Transport rate is determined as BBB clearance, the volume of plasma per unit time cleared of the peptide by BBB transport, and the extent of transport as the percentage of the administered dose transported into the central nervous system. Plasma kinetics of DGAVP were shown to be linear within the dose range studied (50-150 micrograms), plasma mean residence time (MRT) being 18 +/- 4 min (mean +/- SE; n = 9). Elimination of DGAVP from CSF after icv administration was linear, with an MRT of 10 +/- 1 min (n = 9). After iv administration of 100 micrograms DGAVP, CSF concentrations were detectable for 90 min. Transport from plasma to the central nervous system was linear. The BBB transport clearance value was 1.0 +/- 0.3 microliters/min, and 0.026 +/- 0.007% of the administered dose was transported into the central nervous system. Results demonstrate that, within the concentration range studied, DGAVP is transported across the BBB by passive diffusion, although to a very low extent.

The bile salt derivative sodium tauro-24,25-dihydrofusidate (STDHF) has been reported to promote nasal absorption of insulin. In the present study the effect of STDHF on rectal insulin absorption was investigated in rats. At concentrations of 1 and 4% (w/v) it enhanced insulin bioavailability from 0.2 +/- 0.2 (control) to 4.2 +/- 3.2 and 6.7 +/- 2.1%, respectively, as assessed by radioimmunoassay. Insulin preparations with STDHF reduced blood glucose concentrations considerably in a concentration-dependent way. Coadministration of STDHF with Na2EDTA (0.25%, w/v) tended to increase further insulin bioavailability and hypoglycemic response. Varying the site of rectal administration did not influence these parameters.

Lidocaine was administered to healthy volunteers at different sites in the rectum. Unchanged drug and monoethylglycinexylidide (MEGX) concentrations were measured in plasma with a newly developed gas chromatographic method. Lidocaine was given rectally by means of an osmotic system (Osmet(®)) which delivered 25 mg/h at zero-order rate. In a pilot experiment in two subjects it was shown that lidocaine administration close to the anus for 5 h resulted in higher lidocaine plasma levels as compared to administration at 15 cm from the anus. Six other subjects participated in three separate experiments, in which lidocaine was administered rectally close to the anus and at 7.5 and 15 cm from the anus. A zero-order infusion plasma level profile was found for both the parent compound and its metabolite. The MEGX/lidocaine plasma concentration ratio was calculated for all experiments. After administration most proximal to the anus the mean metabolite/parent drug concentration ratio was significantly less than that obtained after administration at 15 cm from the anus, whereas at approximately 7.5 cm from the anus the values were in-between. Comparison of the AUC lidocaine/AUC MEGX ratios gave similar results; the highest value, 3.2 ± 1.3 (mean ± S. D.), was found after administration close to the anus, while at 15 cm from the anus the ratio was 1.6 ± 0.3 (p < 0.01). The terminal elimination half-lives of lidocaine and MEGX did not differ for the three sites of administration, and the mean values were 110 and 180 min respectively. The results of this study demonstrate that the site of drug administration in the human rectum determines the degree of hepatic "first-pass" elimination of high-clearance drugs. Maximal avoidance of presystemic elimination is achieved when administration takes place close to the anus.

The extent of "first-pass" elimination of racemic propranolol and dextropropranolol in doses of 0.25 or 0.50 mg was investigated in relation to the site of drug administration in the rectum of rats. The compounds were given orally, i.v., and rectally at distances of 2 and 1 cm from and directly at the anus by low volume zero-order 30 min infusion. Unchanged propranolol was determined in blood, and propranolol and three metabolites were measured in urine. The systemic availability of propranolol after oral administration was approximately 6 %. Rectal administration at 2 cm, at 1 cm and directly at the anus (0.2 cm) gave two, three and six times higher values, respectively. The more distal application site produced urinary metabolite profiles that were comparable to those observed after oral administration, while application directly at the anus was similar to i.v. dosing. In all experiments log-linear elimination phases with comparable elimination half-lives (range 12-18 min) were found, except with the 0.50 mg dose after i.v. and rectal administration close to the anus which showed a non-linear profile. The mean systemic availability after rectal administration of 0.25 mg dextro-propranolol close to the anus was 50 and 64 % as compared to a 0.25 and 0.125 mg i.v. dose, respectively. The rectal route may be used for propranolol to partially prevent hepatic first-pass metabolism. However, avoidance of presys-temic elimination is maximal only in the immediate vicinity of the anus as the venous blood supply of the upper part of the rectum of rats appears to be connected to the portal system and the lower part to the general circulation.

The stability of the neuroleptic peptide des-enkephalin-gamma-endorphin (DE gamma E; Org 5878) in the rectal lumen and the rectal bioavailability of DE gamma E were investigated in conscious rats. Furthermore, the influence of peptidase inhibition, peptidase saturation, and absorption enhancement on DE gamma E bioavailability were evaluated. Na2EDTA (0.25%, w/v) prolonged the degradation half-life of DE gamma E in the ligated colon from 33 +/- 7 to 93 +/- 45 min. Without adjuvant, tritium-labeled DE gamma E was absorbed from the rat rectum to a very low extent (0-4%). After administration of an excess of unlabeled DE gamma E or with Na2EDTA, comparable results were obtained. The medium-chain glyceride preparation MGK markedly enhanced the rectal DE gamma E bioavailability, up to 8-20%, which was further increased to 10-44% by coadministration of Na2EDTA. No substantial influence of varying the rectal delivery rate was observed. The results suggest that absorption enhancement and enzyme inhibition both are essential for effective increase of rectal peptide bioavailability.

The objective of this study was to assess the relative absorption promoting potency in terms of concentration-effect relationships of the medium-chain fatty acids hexanoic acid, octanoic acid, decanoic acid, and dodecanoic acid in conscious rats, using cefoxitin sodium as the rectally delivered model compound. Rectal uptake of cefoxitin, which was absorbed to a limited extent without enhancer (30 +/- 25%), proved to be significantly enhanced by 2.0 M sodium hexanoate, 0.69 M sodium octanoate, and 0.22 M sodium decanoate, resulting in mean bioavailabilities of 102 +/- 24, 68 +/- 25, and 68 +/- 10%, respectively. Thus, increasing fatty acid chain length results in increased enhancing potency from hexanoic acid to decanoic acid. However, using dodecanoate a statistically significant effect could not be reached, because of its limited aqueous solubility. Optimal chain length for absorption enhancement by medium-chain fatty acids is probably determined by interplay of intrinsic effects on mucosal permeability and solubility of the medium-chain fatty acid.