Transfer of rhodamine-123 into the brain and cerebrospinal fluid of fetal, neonatal and adult rats.

BACKGROUND: Adenosine triphosphate binding cassette transporters such as P-glycoprotein (PGP) play an important role in drug pharmacokinetics by actively effluxing their substrates at barrier interfaces, including the blood-brain, blood-cerebrospinal fluid (CSF) and placental barriers. For a molecule to access the brain during fetal stages it must bypass efflux transporters at both the placental barrier and brain barriers themselves. Following birth, placental protection is no longer present and brain barriers remain the major line of defense. Understanding developmental differences that exist in the transfer of PGP substrates into the brain is important for ensuring that medication regimes are safe and appropriate for all patients. METHODS: In the present study PGP substrate rhodamine-123 (R123) was injected intraperitoneally into E19 dams, postnatal (P4, P14) and adult rats. Naturally fluorescent properties of R123 were utilized to measure its concentration in blood-plasma, CSF and brain by spectrofluorimetry (Clariostar). Statistical differences in R123 transfer (concentration ratios between tissue and plasma ratios) were determined using Kruskal-Wallis tests with Dunn's corrections. RESULTS: Following maternal injection the transfer of R123 across the E19 placenta from maternal blood to fetal blood was around 20 %. Of the R123 that reached fetal circulation 43 % transferred into brain and 38 % into CSF. The transfer of R123 from blood to brain and CSF was lower in postnatal pups and decreased with age (brain: 43 % at P4, 22 % at P14 and 9 % in adults; CSF: 8 % at P4, 8 % at P14 and 1 % in adults). Transfer from maternal blood across placental and brain barriers into fetal brain was approximately 9 %, similar to the transfer across adult blood-brain barriers (also 9 %). Following birth when placental protection was no longer present, transfer of R123 from blood into the newborn brain was significantly higher than into adult brain (3 fold, p < 0.05). CONCLUSIONS: Administration of a PGP substrate to infant rats resulted in a higher transfer into the brain than equivalent doses at later stages of life or equivalent maternal doses during gestation. Toxicological testing of PGP substrate drugs should consider the possibility of these patient specific differences in safety analysis.

Influene of Pharmaceutical Excipients on the Membrane Transport of a P-glycoprotein Substrate in the Rat Small Intestine.

BACKGROUND AND OBJECTIVES: Generic drugs are generally used worldwide because of affordability compared to brand-name drugs. One of the main differences between brand-name and generic drugs is pharmaceutical excipients. We previously reported the effects of pharmaceutical excipients on the membrane permeation of drugs via the paracellular and transcellular routes, which are passive transport routes. P-glycoprotein (P-gp) is a typical ATP-binding cassette transporter and is mostly responsible for drug-drug interactions involving transporters. In the present study, rhodamine 123 (Rho123) was selected as the P-gp substrate, and the effects of pharmaceutical excipients on its membrane transport in the rat jejunum and ileum were examined. METHODS: Twenty major pharmaceutical excipients widely used in the pharmaceutical industry were selected. The in vitro diffusion chamber method using the rat jejunum and ileum was employed to investigate the effects of pharmaceutical excipients on the membrane permeation of Rho123. RESULTS: The results obtained showed that the membrane permeability of Rho123 significantly (P < 0.05) changed under certain dosage conditions of pharmaceutical excipients such as sodium carboxymethyl starch, pullulan, glyceryl monostearate and so on. Furthermore, the effects of pharmaceutical excipients were site specific in the small intestine. CONCLUSION: The present results demonstrated that some pharmaceutical excipients altered the membrane permeability of Rho123 in the rat small intestine.

Drug Clearance from Cerebrospinal Fluid Mediated by Organic Anion Transporters 1 (Slc22a6) and 3 (Slc22a8) at Arachnoid Membrane of Rats.

Although arachnoid mater epithelial cells form the blood-arachnoid barrier (BAB), acting as a blood-CSF interface, it has been generally considered that the BAB is impermeable to water-soluble substances and plays a largely passive role. Here, we aimed to clarify the function of transporters at the BAB in regulating CSF clearance of water-soluble organic anion drugs based on quantitative targeted absolute proteomics (QTAP) and in vivo analyses. Protein expression levels of 61 molecules, including 19 ATP-binding-cassette (ABC) transporters and 32 solute-carrier (SLC) transporters, were measured in plasma membrane fraction of rat leptomeninges using QTAP. Thirty-three proteins were detected; others were under the quantification limits. Expression levels of multidrug resistance protein 1 (Mdr1a/P-gp/Abcb1a) and breast cancer resistance protein (Bcrp/Abcg2) were 16.6 and 3.27 fmol/µg protein (51.9- and 9.82-fold greater than in choroid plexus, respectively). Among those organic anion transporters detected only at leptomeninges, not choroid plexus, organic anion transporter 1 (oat1/Slc22a6) showed the greatest expression (2.73 fmol/µg protein). On the other hand, the protein expression level of oat3 at leptomeninges was 6.65 fmol/µg protein, and the difference from choroid plexus was within two-fold. To investigate oat1's role, we injected para-aminohippuric acid (PAH) with or without oat1 inhibitors into cisterna magna (to minimize the contribution of choroid plexus function) of rats. A bulk flow marker, FITC-inulin, was not taken up from CSF up to 15 min, whereas uptake clearance of PAH was 26.5 µL/min. PAH uptake was completely blocked by 3 mM cephalothin (inhibits both oat1 and oat3), while 17% of PAH uptake was inhibited by 0.2 mM cephalothin (selectively inhibits oat3). These results indicate that oat1 and oat3 at the BAB provide a distinct clearance pathway of organic anion drugs from CSF independently of choroid plexus.

Pharmacokinetic interaction of green tea beverage containing cyclodextrins and high concentration catechins with P-glycoprotein substrates in LLC-GA5-COL150 cells in vitro and in the small intestine of rats in vivo.

OBJECTIVES: Possible interaction of green tea beverage (GT) containing cyclodextrins and high concentration catechins, a drinking water, with P-glycoprotein (P-gp) substrates was examined in vitro and in vivo. METHODS: Effects of GT on the uptake of rhodamine 123 by LLC-GA5-COL150 cells and intestinal efflux of rhodamine 123 from blood, intestinal absorption of quinidine from ileum loop and oral absorption of digoxin were examined in rats. Effects of GT and GT components on digoxin solubility were also examined. KEY FINDINGS: Green tea increased the uptake of rhodamine 123 by LLC-GA5-COL150 cells, suppressed the intestinal efflux of rhodamine 123 from blood and increased the absorption of quinidine in the ileum of rats. Also, GT increased the solubility of digoxin, and ingestion of GT significantly increased the oral absorption of digoxin given at a high dose in rats. CONCLUSIONS: Green tea suppressed the P-gp-mediated efflux transport of hydrophilic compounds and increased the solubility of lipophilic compounds. Thus, GT may cause interaction with various P-gp substrates, due to the combined effects of catechins and cyclodextrins. Especially, cyclodextrin alone can cause interaction with various low-solubility compounds in vivo. In taking low-solubility drugs including low-solubility P-gp substrates, cyclodextrin-containing foods and beverages such as GT should be avoided.

Non-invasive topical drug delivery to spinal cord with carboxyl-modified trifunctional copolymer of ethylene oxide and propylene oxide.

In this study the effect of oxidative modification on micellar and drug delivery properties of copolymers of ethylene oxide (EO) and propylene oxide (PO) was investigated. Carboxylated trifunctional copolymers were synthesized in the reaction with chromium(VI) oxide. We found that carboxylation significantly improved the uniformity and stability of polymeric micelles by inhibiting the microphase transition. The cytotoxicity of copolymers was studied in relation to their aggregative state on two cell types (cancer line vs. primary fibroblasts). The accumulation of rhodamine 123 in neuroblastoma SH-SY5Y cells was dramatically increased in the presence of the oxidized block copolymer with the number of PO and EO units of 83.5 and 24.2, respectively. The copolymer was also tested as an enhancer for topical drug delivery to the spinal cord when applied subdurally. The oxidized copolymer facilitated the penetration of rhodamine 123 across spinal cord tissues and increased its intraspinal accumulation. These results show the potential of using oxidized EO/PO based polymers for non-invasive delivery of protective drugs after spinal cord injury.

Transport of stearic acid-based solid lipid nanoparticles (SLNs) into human epithelial cells.

Development of drug delivery systems, as much as the drug molecule itself, is an important consideration for improving drug absorption and bioavailability. The mechanisms by which drug carriers enter target cells can differ depending on their size, surface properties and components. Solid lipid nanoparticles (SLNs) have gained an increased attention in recent years and are the drug carriers of interest in this paper. They are known to breach the cell-membrane barrier and have been actively sought to transport biomolecules. Previous studies by our group, and also other groups, provided an extensive characterization of SLNs. However, few studies have investigated the uptake of SLNs and these have had limited mechanistic focus. The aim of this work was to investigate the pathway of uptake of SLNs by human epithelial cells i.e., lung A549 and cervical HeLa cells. To the best of our knowledge, this is first study that investigates the cellular uptake of SLNs by human epithelial cells. The mechanism of cellular uptake was deciphered using pharmacologic inhibitors (sucrose, potassium-free buffer, filipin and cytochalasin B). Imaging techniques and flow assisted cell sorting (FACS) were used to assess the cellular uptake of SLNs loaded with rhodamine 123 as a fluorescent probe. This study provided evidence that the cellular uptake of SLNs was energy-dependent, and the endocytosis of SLNs was mainly dependent on clathrin-mediated mechanisms. The establishment of entry mechanism of SLNs is of fundamental importance for future facilitation of SLNs as biological or drug carriers.

Permeation of Therapeutic Drugs in Different Formulations across the Airway Epithelium In Vitro.

BACKGROUND: Pulmonary drug delivery is characterized by short onset times of the effects and an increased therapeutic ratio compared to oral drug delivery. This delivery route can be used for local as well as for systemic absorption applying drugs as single substance or as a fixed dose combination. Drugs can be delivered as nebulized aerosols or as dry powders. A screening system able to mimic delivery by the different devices might help to assess the drug effect in the different formulations and to identify potential interference between drugs in fixed dose combinations. The present study evaluates manual devices used in animal studies for their suitability for cellular studies. METHODS: Calu-3 cells were cultured submersed and in air-liquid interface culture and characterized regarding mucus production and transepithelial electrical resistance. The influence of pore size and material of the transwell membranes and of the duration of air-liquid interface culture was assessed. Compounds were applied in solution and as aerosols generated by MicroSprayer IA-1C Aerosolizer or by DP-4 Dry Powder Insufflator using fluorescein and rhodamine 123 as model compounds. Budesonide and formoterol, singly and in combination, served as examples for drugs relevant in pulmonary delivery. RESULTS AND CONCLUSIONS: Membrane material and duration of air-liquid interface culture had no marked effect on mucus production and tightness of the cell monolayer. Co-application of budesonide and formoterol, applied in solution or as aerosol, increased permeation of formoterol across cells in air-liquid interface culture. Problems with the DP-4 Dry Powder Insufflator included compound-specific delivery rates and influence on the tightness of the cell monolayer. These problems were not encountered with the MicroSprayer IA-1C Aerosolizer. The combination of Calu-3 cells and manual aerosol generation devices appears suitable to identify interactions of drugs in fixed drug combination products on permeation.

Reversal of ABCB1-related Multidrug Resistance of Colonic Adenocarcinoma Cells by Phenothiazines.

BACKGROUND: The most common mechanism that reduces the efficacy of anticancer agents is overexpression of ATP-binding cassette (ABC) drug transporters. Phenothiazines and structurally-related compounds can sensitize multidrug-resistant (MDR) cells to chemotherapeutics. MATERIALS AND METHODS: Phenothiazine derivatives were investigated regarding their anticancer and MDR-reversing effect on colonic adenocarcinoma cells. The anti-proliferative and cytotoxic effects of the derivatives were assessed by the thiazolyl blue tetrazolium bromide (MTT) method, the modulation of the ABCB1 activity was measured by rhodamine 123 accumulation assay using flow cytometry. RESULTS: All phenothiazines exhibited potent cytotoxic effect on the sensitive and MDR colon adenocarcinoma cell lines. The inhibition of the ABCB1 transporter was greater in the presence of the phenothiazine derivatives than for the known ABCB1 inhibitor verapamil. CONCLUSION: It can be concluded that these derivatives show synergism in the presence of doxorubicin and could have potential as ABCB1 inhibitors.

Effects of hepatic ischemia-reperfusion injury on the P-glycoprotein activity at the liver canalicular membrane and blood-brain barrier determined by in vivo administration of rhodamine 123 in rats.

PURPOSE: To investigate the effects of normothermic hepatic ischemia-reperfusion (IR) injury on the activity of P-glycoprotein (P-gp) in the liver and at the blood-brain barrier (BBB) of rats using rhodamine 123 (RH-123) as an in vivo marker. METHODS: Rats were subjected to 90 min of partial ischemia or sham surgery, followed by 12 or 24 h of reperfusion. Following intravenous injection, the concentrations of RH-123 in blood, bile, brain, and liver were used for pharmacokinetic calculations. The protein levels of P-gp and some other transporters in the liver and brain were also determined by Western blot analysis. RESULTS: P-gp protein levels at the liver canalicular membrane were increased by twofold after 24 h of reperfusion. However, the biliary excretion of RH-123 was reduced in these rats by 26%, presumably due to IR-induced reductions in the liver uptake of the marker and hepatic ATP concentrations. At the BBB, a 24% overexpression of P-gp in the 24-h IR animals was associated with a 30% decrease in the apparent brain uptake clearance of RH-123. The pharmacokinetics or brain distribution of RH-123 was not affected by the 12-h IR injury. CONCLUSIONS: Hepatic IR injury may alter the peripheral pharmacokinetics and brain distribution of drugs that are transported by P-gp and possibly other transporters.

Functional characterization of hepatic transporters using intravital microscopy.

A better understanding of the role of hepatic transporters in drug elimination is of crucial importance for drug development and therapy. This study examined the usefulness of intravital microscopy to quantitatively evaluate the function of hepatic transporters in the exposed liver of anesthetized rats. In one experiment the function of the organic anion transporting polypeptide (Oatp) in sinusoidal uptake was investigated by administering an Oatp inhibitor, rifampicin, prior to the probe substrate Na-fluorescein. In another experiment, rhodamine 123 was used to quantify the biliary canalicular transporter P-glycoprotein (P-gp, Abcb1a/b) with cyclosporin A as an inhibitor of P-gp activity. Calibrated fluorescence intensity time curves measured in sinusoids and hepatocytes together with cumulative biliary excretion data from control and inhibitor treated animals were analyzed with a three-compartment model. A robust parameter estimation was achieved using nonlinear mixed effects modeling. Rifampicin reduced the hepatic uptake clearance of Na-fluorescein to 25% of the control (p<0.05) without affecting other parameters. In the presence of cyclosporin A, biliary excretion of rhodamine 123 decreased to 7% of the control (p<0.01). The novelty of this approach is that it allows a quantitative evaluation of transporter function in the in vivo rat liver.

Contribution of radixin to P-glycoprotein expression and transport activity in mouse small intestine in vivo.

The ERM proteins, ezrin, radixin, and moesin, are membrane-cytoskeleton cross-linkers with multiple physiological functions. We previously showed that radixin is involved in posttranslational regulation of P-glycoprotein (P-gp) in human hepatoblastoma HepG2 cells. Here, we investigated the physiological role of radixin in regulating P-gp expression and activity in the small intestine by comparing wild-type- and radixin knockout (Rdx) mice. In intestinal tissue homogenates, P-gp protein levels increased markedly from the upper part to the lower part of the small intestine in both wild-type- and Rdx(-/-) mice. In the membrane fractions, a similar pattern was seen in wild-type mice. However, the membrane expression of P-gp protein remained at the same level from the upper to the lower part of the small intestine in Rdx(-/-) mice. When rhodamine123 (Rho123), a substrate of P-gp, was orally administered to Rdx(-/-) and wild-type mice, the absorption phase of Rho123 was greater in Rdx(-/-) than in wild-type mice, whereas the elimination phase in Rdx(-/-) mice was not different from that of wild-type mice. Our results indicate that radixin plays an important role in regulating P-gp localization and P-gp functional activity at the intestinal membrane.

Changes in absorption and excretion of rhodamine 123 by sodium nitroprusside.

Nitric oxide (NO) donors increase the permeability of water-soluble compounds with neither loss of cell viability nor lactate dehydrogenase release, but the involved mechanism is not fully understood. In this study, we focused on permeation via the transcellular route and P-glycoprotein, which is a typical ABC transporter. We examined the effect of sodium nitroprusside (SNP), which is an NO donor, on the membrane permeation of rhodamine 123 (Rho123), a representative P-gp substrate, and the change in expression level of ileal P-gp. We used an in situ closed loop method in rat ileum to study changes in the permeation of Rho123. The effects of SNP (1 and 10mg/kg) on the mdr-1a mRNA and P-gp protein expression levels were examined by real-time RT-PCR and Western blotting, respectively. The absorption and excretion of Rho123 were significantly increased in an SNP dose-dependent manner when compared with those with no addition, but no changes in protein expression level of P-gp in ileal BBM were observed by SNP administration. The relative activity of P-gp was not changed by SNP administration. On the other hand, the expression level of mdr-1a mRNA was induced by SNP administration. We indicated that SNP could increase the mucosal permeation of Rho123 via the transcellular route without an influence on P-gp, and we showed that this effect is temporary. SNP has no influence on P-gp function and protein expression level in the short term, but they may change in the long term.

Time-dependent interaction of ritonavir in chronic use: the power balance between inhibition and induction of P-glycoprotein and cytochrome P450 3A.

Ritonavir (RTV) is not only an inhibitor but also an immunoreactive inducer of both P-glycoprotein (Pgp) and cytochrome P450 (CYP) 3A in terms of its chronic use. The aim of present study was to test the hypothesis that the power balance between inhibition effects of RTV and induced activities of Pgp and CYP3A depends on the time after last RTV treatment (TimeR) in the chronic use of RTV; rhodamine 123 (Rho) and midazolam (MDZ) were administered at predetermined TimeR to rats pretreated with RTV for 7 days. After oral administration of Rho and MDZ to rats pretreated with RTV for 7 days, the areas under the plasma concentration-time curve of Rho and MDZ were significantly altered depending on TimeR: 1.27-, 0.79-, 0.95-, and 0.11-fold increases over that of the control for Rho at TimeR = 0, 3, 9, and 24 h and 3.12-, 1.50-, 1.27-, and 0.17-fold increases over that of the control for MDZ at TimeR = 0, 3, 9, and 24 h, respectively. These results revealed the presence of the time-dependent interaction of RTV with concomitant drugs in chronic use and should be taken into account in therapeutic strategies for HIV infection.

Design and in vitro evaluation of a novel polymeric excipient for buccal applications.

BACKGROUND: The objective of this study was to develop and evaluate a more effective mucoadhesive thiomer for buccal drug-delivery systems. METHODS: 2-iminothiolane was covalently attached to a chitosan backbone. A preactivation step followed, mediated by 6,6´dithionicotinamide, thiol groups were modified by disulfide bonds formation. Mucoadhesion studies were performed on buccal mucosa. In addition, water-uptake capacity, disintegration, release and cytotoxicity studies were completed. RESULTS: The obtained chitosan-thiobutylamidine conjugate displayed 647.4 ± 85.23 µmol/g immobilized thiol groups. Due to the preactivation, approximately 60% of thiol groups were modified by formation of disulfide bonds. Chitosan-thiobutylamidine-mercaptonicotinamide displayed 1.8-fold higher stability and 1.6-fold higher mucoadhesive properties, respectively. The release study demonstrated a 1.4-fold more prolonged drug release compared with corresponding thiomers. CONCLUSION: According to these results, preactivated thiomers demonstrate an improved stability and increased mucoadhesive properties. Therefore, they seem to be advantageous for buccal administration over corresponding thiomers and chitosan.

The role of inter-segmental differences in P-glycoprotein expression and activity along the rat small intestine in causing the double-peak phenomenon of substrate plasma concentration.

  Conflicting results have been reported on segmental differences in expression of P-glycoprotein (P-gp) along the small intestine of animals and humans. In this study, we investigated P-gp mRNA and protein levels within each of nine segments of rat small intestine. In addition, P-gp activity in each segment was evaluated in terms of permeability of rhodamine123 (Rho123), a typical P-gp substrate, using the serial intestinal non-everted sac method. The P-gp mRNA levels tended to increase from the duodenum to the ileum, with peaks in the upper and lower ileum, while P-gp protein level reached its maximum in the middle ileum. The activity of P-gp was also the highest in the middle ileum, and was highly correlated with P-gp protein level. The double-peaked plasma concentration profile that was observed following oral administration of Rho123 to rats could be well reproduced by an intestinal compartmental kinetic model incorporating inter-segmental differences of absorption and excretion rate constants. Our results suggest that the heterogeneous distribution of P-gp along the small intestine plays a key role in causing the double-peak of plasma concentration of P-gp substrates following oral administration to rats.

Mechanism of inhibition of P-glycoprotein mediated efflux by Pluronic P123/F127 block copolymers: relationship between copolymer concentration and inhibitory activity.

The aim of this study was to clarify the relationship between the concentration of Pluronic P123/F127 block copolymers and P-glycoprotein (P-gp) inhibitory potency. Modulation of multidrug resistance (MDR) by Pluronic P123/F127 was evaluated in P-gp over-expressing human breast cancer cell line MCF-7/ADR and its non-P-gp over-expressing counterpart MCF-7 cells. Four different probes (known as P-gp substrates) including rhodamine 123 (R-123), rhodamine 6G (R-6G), doxorubicin (DOX), and paclitaxel (PTX) were applied to investigate the impact of Pluronic P123/F127 copolymers with different concentrations on the intracellular accumulation of these probes. Additionally, the intracellular ATP and mitochondrial transmembrane potential in MCF-7/ADR cells were determined over a wide concentration range of Pluronic P123/F127. Furthermore, the endocytic mechanisms of Pluronic micelles were performed. It was suggested that P-gp substrate hydrophobicity and the concentration of P123/F127 copolymers had little impact on P-gp inhibitory activity of Pluronic P123/F127 itself. Intracellular ATP depletion was the main mechanism of Pluronic P123/F127 for P-gp inhibition. In vitro cytotoxicity study was also conducted in order to compare cytotoxic effect among different PTX formulations. It indicated that the IC50 of PTX-loaded Pluronic P123/F127 mixed micelles was 6.3-fold lower than free PTX and 2.3-fold lower than Taxol, respectively. Therefore, Pluronic P123/F127 polymeric micelles could be considered a promising drug delivery system to overcome MDR in cancer therapy.

Biodegradable hyperbranched polyglycerol with ester linkages for drug delivery.

Biodegradable hyperbranched polyglycerols (dHPGs) were synthesized through oxyanionic initiating hybrid polymerization of glycerol and glycidyl methacrylate. Due to the introduction of ester linkages into the hyperbranched polyglycerol backbone, dHPGs showed good biodegradability and low cytotoxicity. Benefiting from the existence of terminal hydroxyls and methacryloyl groups, both the anticancer drug methotrexate (MTX) and fluorescent probe Rhodamine-123 could be conjugated onto the surface of dHPGs easily. The resultant MTX-conjugated polymers (dHPG-MTXs) exhibited an amphiphilic character, resulting in the formation of micelles in an aqueous solution. The release of MTX from micelles was significantly faster at mildly acidic pH of 5.0 compared to physiological pH of 7.4. dHPG-MTX micelles could be efficiently internalized by cancer cells. MTT assay against cancer cells showed dHPG-MTXs micelles had high anticancer efficacy. On the basis of their good biodegradability and low cytotoxicity, dHPGs provide an opportunity to design excellent drug delivery systems.

Amphiphilic HPMA-LMA copolymers increase the transport of Rhodamine 123 across a BBB model without harming its barrier integrity.

The successful non-invasive treatment of diseases associated with the central nervous system (CNS) is generally limited by poor brain permeability of various developed drugs. The blood-brain barrier (BBB) prevents the passage of therapeutics to their site of action. Polymeric drug delivery systems are promising solutions to effectively transport drugs into the brain. We recently showed that amphiphilic random copolymers based on the hydrophilic p(N-(2-hydroxypropyl)-methacrylamide), pHPMA, possessing randomly distributed hydrophobic p(laurylmethacrylate), pLMA, are able to mediate delivery of domperidone into the brain of mice in vivo. To gain further insight into structure-property relations, a library of carefully designed polymers based on p(HPMA) and p(LMA) was synthesized and tested applying an in vitro BBB model which consisted of human brain microvascular endothelial cells (HBMEC). Our model drug Rhodamine 123 (Rh123) exhibits, like domperidone, a low brain permeability since both substances are recognized by efflux transporters at the BBB. Transport studies investigating the impact of the polymer architecture in relation to the content of hydrophobic LMA revealed that random p(HPMA)-co-p(LMA) having 10mol% LMA is the most auspicious system. The copolymer significantly increased the permeability of Rh123 across the HBMEC monolayer whereas transcytosis of the polymer was very low. Further investigations on the mechanism of transport showed that integrity and barrier function of the BBB model were not harmed by the polymer. According to our results, p(HPMA)-co-p(LMA) copolymers are a promising delivery system for neurological therapeutics and their application might open alternative treatment strategies.

Enhanced in vitro cellular uptake of P-gp substrate by poloxamer-modified liposomes (PMLs) in MDR cancer cells.

Poloxamer-modified liposomes (PMLs) were prepared using poloxamers (P85 and F68) by the thin-film hydration method for overcoming the multidrug resistance and thereby enhancing the intracellular uptake of specific substrates of P-gp, rhodamine 123 (R123). The prepared liposomes, plain liposomes (PLs) and PMLs, were characterized by particle size, zeta potential and drug entrapment efficiency, and assessed by in vitro cellular uptake using KB and KBV20C (P-gp over-expression cell line) cells. The transmission electron microscopy study revealed the spherical shape of the prepared liposomes. No significant difference was observed between the PMLs and liposome without poloxamer (PLs) in the particle size (∼160 nm) and zeta potential (∼-5 mV). The in vitro cellular uptake study showed that P85-modified liposomes (PML-P85) significantly increased the internalization of R123 in MDR tumour cells. Our results showed that PML-P85 could be an effective carrier for anticancer drugs in MDR cancer therapy.

Thiolated chitosans: influence of various sulfhydryl ligands on permeation-enhancing and P-gp inhibitory properties.

PURPOSE: The influence of various sulfhydryl ligands on permeation-enhancing and P-glycoprotein (P-gp) inhibitory properties of the six established thiolated chitosan conjugates was investigated using Rhodamine-123 (Rho-123) and fluorescein isothiocyanate-dextran 4 (FD4) as model compounds. METHODS: Permeation of these compounds was tested on freshly excised rat intestine in Ussing-type chambers. Apparent permeability coefficients (Papp) were calculated and compared to values obtained from the buffer only control. RESULTS: The lyophilized polymers had a thiol group content in the range of 230-520 µmol/g. Results of this study led to the following rank order in permeation enhancement: chitosan-6-mercaptonicotinic acid (chitosan-6MNA) > chitosan-cysteine (chitosan-Cys) > chitosan-glutathione (chitosan-GSH) > chitosan-4-thiobutylamidine (chitosan-TBA) > chitosan-thioglycolic acid (chitosan-TGA) > chitosan-N-acetyl cysteine (chitosan-NAC). In P-gp inhibition studies, 0.5% (m/v) chitosan-NAC showed the highest inhibitory effect on P-gp, where the Papp was determined to be 3.78-fold increased compared with the buffer control. Among these thiolated chitosans, chitosan-NAC and chitosan-6MNA are the most effective polymers being responsible for P-gp inhibition and permeation enhancement, respectively. CONCLUSION: These thiolated chitosans would therefore be advantageous tools for enhancing the noninvasive bioavailability of active pharmaceutical ingredients.