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.

P-glycoprotein Restricts Ocular Penetration of Loperamide across the Blood-Ocular Barriers: a Comparative Study in Mdr1a Knock-out and Wild Type Sprague Dawley Rats.

The current research was undertaken to determine the existence and magnitude of P-glycoprotein (P-gp) expression on the blood-ocular barriers by studying the ocular penetration of loperamide, a specific P-gp substrate, in P-gp (Mdr1a) knock-out (KO) and wild type (WT) Sprague Dawley rats. A clear, stable, sterile solution of loperamide (1 mg/mL), for intravenous administration, was formulated and evaluated. Ocular distribution was studied in P-gp KO and WT rats following intravenous administration of loperamide (at two doses). The drug levels in plasma, aqueous humor (AH), and vitreous humor (VH) samples were determined with the aid of UHPLC-Q-TOF-MS/MS, and the AH/plasma (D AH ) and VH/plasma (D VH ) distribution ratios were estimated. Electroretinography (ERG), ultrastructural analyses, and histology studies were carried out, in both KO and WT rats, to detect any drug-induced functional and/or structural alterations in the retina. Dose-related loperamide levels were observed in the plasma of both WT and KO rats. The loperamide concentrations in the AH and VH of KO rats were significantly higher compared to that observed in the WT rats, at the lower dose. However, a marked increase in the D AH and D VH was noted in the KO rats. ERG, ultrastructure, and histology studies did not indicate any drug-induced toxic effects in the retina under the test conditions. The results from these studies demonstrate that P-gp blocks the penetration of loperamide into the ocular tissues from the systemic circulation and that the effect is more pronounced at lower plasma loperamide concentrations.

Nano-engineered mesenchymal stem cells as targeted therapeutic carriers.

Poor availability in deep-seated solid tumors is a significant challenge that limits the effectiveness of currently used anticancer drugs. Approaches that can specifically enhance drug delivery to the tumor tissue can potentially improve therapeutic efficacy. In our current studies, we used nano-engineered mesenchymal stem cells (nano-engineered MSCs) as tumor-targeted therapeutic carriers. In addition to their exquisite tumor homing capabilities, MSCs overexpress efflux transporters such as P-glycoprotein and are highly drug resistant. The inherent tumor-tropic and drug-resistant properties make MSCs ideal carriers for toxic payload. Nano-engineered MSCs were prepared by treating human MSCs with drug-loaded polymeric nanoparticles. Incorporating nanoparticles in MSCs did not affect their viability, differentiation or migration potential. Nano-engineered MSCs induced dose-dependent cytotoxicity in A549 lung adenocarcinoma cells and MA148 ovarian cancer cells in vitro. An orthotopic A549 lung tumor model was used to monitor the in vivo distribution of nanoengineered MSCs. Intravenous injection of nanoparticles resulted in non-specific biodistribution, with significant accumulation in the liver and spleen while nano-engineered MSCs demonstrated selective accumulation and retention in lung tumors. These studies demonstrate the feasibility of developing nano-engineered MSCs loaded with high concentration of anticancer agents without affecting their tumor-targeting or drug resistance properties.

Mixed molecular weight copolymer nanoparticles for the treatment of drug-resistant tumors: formulation development and cytotoxicity.

Nanoparticles composed of both high- and low-molecular-weight methoxy poly(ethylene glycol)-block-poly(caprolactone) (MePEG-b-PCL) diblock copolymers (termed "mixed molecular weight nanoparticles") were investigated for the encapsulation and delivery of the taxane drugs paclitaxel (PTX) and docetaxel (DTX). These nanoparticles were prepared using nanoprecipitation and emulsion methods. These 80 nm nanoparticles were prepared with high yields, efficiently solubilized PTX and DTX up to 500 and 1300 µg/mL, respectively, and demonstrated controlled release of these drugs over 14 days. The taxane-sensitive (MDCKII) and taxane-resistant [P-glycoprotein (P-gp) overexpressing] MDCKII-MDR cell lines were used to establish the cytotoxic profiles of these nanoparticles. Because of the coencapsulation of the previously demonstrated P-gp inhibitor, a low-molecular-weight MePEG-b-PCL copolymer (MePEG17 -b-PCL5 ), these drug-loaded mixed molecular weight nanoparticles dramatically reduced the viability of P-gp overexpressing MDCKII-MDR cells and restored sensitivity to taxane drugs in these cells.

Sex differences in cyclosporine pharmacokinetics and ABCB1 gene expression in mononuclear blood cells in African American and Caucasian renal transplant recipients.

Cyclosporine exhibits pharmacokinetic and pharmacodynamic variability in renal transplant recipients (RTR) attributed to P-glycoprotein (P-gp), an ABCB1 efflux transporter that influences bioavailability and intracellular distribution. Data on race and sex influences on P-gp in RTR are lacking. We investigated sex and race influences on cyclosporine pharmacokinetics and ABCB1 gene expression in peripheral blood mononuclear cells (PBMC). Fifty-four female and male African American and Caucasian stable RTR receiving cyclosporine and mycophenolic acid completed a 12-hour study. ABCB1 gene expression was assessed in PBMCs pre-dose and 4 hours after cyclosporine. Statistical analysis used mixed effects models on transformed, normalized ABCB1 expression and cyclosporine pharmacokinetics. Sex and race differences were observed for the dose-normalized area under the concentration curve (AUC0-12 /Dose) [P = .0004], apparent clearance [P = .0004] and clearance/body mass index (CL/BMI) [P = .027] with slowest clearance and greatest drug exposure in females. Sex and race differences were found pre-dose and 4 hours for ABCB1 [P < .0001] with females having less expression than males. ABCB1 differences were observed between pre-dose and 4 hours [P = .0009]. Female RTR had slower cyclosporine clearance and lower ABCB1 gene expression in PBMC suggesting reduced efflux activity and greater intracellular drug exposure.

Reversal of multidrug resistance by magnetic chitosan-Fe3O4 nanoparticle-encapsulated MDR1 siRNA in glioblastoma cell line.

OBJECTIVE: To investigate the reversal effects of MDR1 gene on multidrug resistance in the glioblastoma cell line BT325 by magnetic chitosan-Fe3O4 nanoparticle-encapsulated MDR1 siRNA. METHODS: The shRNA expression vector was constructed and the recombinant plasmids were cloned. Magnetic chitosan-Fe3O4 nanoparticles were prepared and the encapsulation rate was determined. After transfection, the BT325 cells were cultured to assay the transfection efficiency. The changing of MDR1 mRNA level and P-gp protein was evaluated. And the sensitivity to different chemotherapeutic drugs was assessed in BT325-siRNA transfected cell and untransfected cell by IC50 values. RESULTS: The MDR1 RNAi plasmid was successfully designed and preparation. The encapsulation efficiency of the magnetic chitosan-Fe3O4 nanoparticle was 98-99%. The transfection efficiency of the siRNA-nanoparticles in BT325 cells was 70-80%. And the MDR1 mRNA levels were downregulated by reverse transcription (RT)-PCR assay. Furthermore, the results of P-gp protein expression decreased on immunocytochemical assay, Western blot and flow cytometry compared with control group. The IC50 values of DOX and VCR were decreased between the transfected cell and normal BT325 cell. CONCLUSION: After targeted transfection of the glioblastoma cell line with magnetic chitosan-Fe3O4 nanoparticle-encapsulated MDR1 siRNA, the expression of MDR1 at both the mRNA and protein level decreased, which increased sensitivity to chemotherapy in vitro. It might provide a basis for investigation of the mechanism involved in multidrug resistance in glioma.

Permeability dominates in vivo intestinal absorption of P-gp substrate with high solubility and high permeability.

Three purposes are presented in this study: (1) to study the in vivo regional dependent intestinal absorption of a P-gp substrate with high solubility and high permeability, (2) to study the gene expression difference in the various regions of the intestine, and (3) to study the contributions of P-gp or any other transporters for the absorption of a P-gp substrate. The in vivo permeability of verapamil and propranolol were determined by single-pass in situ intestinal perfusion in rat. The gene expression profiles were measured using Affymetrix GeneChip. Correlation analysis between drug in vivo permeability and expression of 3500 genes was performed with nonparametric bootstrap and ANOVA analysis. The permeability of verapamil and propranolol did not demonstrate regional dependency even though significant differences in gene expression were observed in various regions of the intestine. Verapamil permeability significantly correlates with propranolol permeability in both jejunum and ileum, but did not correlate with the permeability of other hydrophilic compounds (valacyclovir, acyclovir, and phenylalanine). Four different regions (duodenum, jejunum, ileum, and colon) showed distinct gene expression patterns with more than 70-499 genes showing at least 5-fold expression differences. Interestingly, P-gp expression is gradually increased by 6-fold from the duodenum to colon. Despite the distinct gene expression patterns in the various regions of the intestine, verapamil permeability did not correlate with any gene expression from 3500 expressed genes in the intestine. A 2-6-fold P-gp expression difference did not seem to associate verapamil permeability in the various intestinal regions in vivo. These data suggest that P-gp plays a minimal role in the in vivo intestinal absorption process of verapamil with high water solubility and high membrane permeability. The intestinal absorption of verapamil in vivo is primarily dominated by its high permeability. However, it is important to note that the findings in this paper do not undermine the importance of P-gp in oral drug bioavailability, drug disposition from the liver, drug efflux from the blood-brain barrier, and drug-drug interaction.

Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis.

Previous studies have demonstrated leukemic complications in mice after high-copy retroviral gene transfer of the multidrug resistance 1 (MDR1) cDNA, encoding a membrane-located efflux pump expressed in hematopoietic stem cells. In contrast, no such complications or MDR1-associated alterations of hematopoiesis were observed in numerous other studies exploring MDR1 gene transfer into cell lines, mice, dogs, nonhuman primates, and human subjects. Here, we show that leukemias associated with retroviral expression of MDR1 depend on high vector dose, and involve the selection of clones with combinatorial insertional mutagenesis of proto-oncogenes or other signaling genes. Compared with insertion patterns in normal long-term repopulating hematopoietic cells, such hits were overrepresented in leukemic clones, pointing to a causal role. A similar constellation of insertion sites was also observed in a leukemia arising after high-copy retroviral gene transfer of a fluorescent protein. Spectral karyotyping demonstrated additional chromosomal translocations in a subset of cases, indicative of secondary genetic instability. We also show that insertional mutants can be amplified in vitro prior to transplantation. On the basis of these findings, we suggest the use of preclinical dose-escalation studies to define a therapeutic index for retroviral transgene delivery.

Cerebral uptake of mefloquine enantiomers with and without the P-gp inhibitor elacridar (GF1210918) in mice.

1. Mefloquine is a chiral neurotoxic antimalarial agent showing stereoselective brain uptake in humans and rats. It is a substrate and an inhibitor of the efflux protein P-glycoprotein. 2. We investigated the stereoselective uptake and efflux of mefloquine in mice, and the consequences of the combination with an efflux protein inhibitor, elacridar (GF120918) on its brain transport. 3. Racemic mefloquine (25 mg kg(-1)) was administered intraperitoneally with or without elacridar (10 mg kg(-1)). Six to seven mice were killed at each of 11 time-points between 30 min and 168 h after administration. Blood and brain concentrations of mefloquine enantiomers were determined using liquid chromatography. 4. A three-compartment model with zero-order absorption from the injection site was found to best represent the pharmacokinetics of both enantiomers in blood and brain. (-)Mefloquine had a lower blood and brain apparent volume of distribution and a lower efflux clearance from the brain, resulting in a larger brain/blood ratio compared to (+)mefloquine. Elacridar did not modify blood concentrations or the elimination rate from blood for either enantiomers. However, cerebral AUC(inf) of both enantiomers were increased, with a stronger effect on (+)mefloquine. The efflux clearance from the brain decreased for both enantiomers, with a larger decrease for (+)mefloquine. 5. After administration of racemic mefloquine in mice, blood and brain pharmacokinetics are stereoselective, (+)mefloquine being excreted from brain more rapidly than its antipode, showing that mefloquine is a substrate of efflux proteins and that mefloquine enantiomers undergo efflux in a stereoselective manner. Moreover, pretreatment with elacridar reduced the brain efflux clearances with a more pronounced effect on (+)mefloquine.

Ciclosporina A e seus análogos como reversores da resistência a múltiplas drogas em células tumorais / Cyclosporin A and analogues as multidrug resistance modulating agents in tumour cell

A resistência a múltiplas drogas (MDR) é um dos principais obstáculos no tratamento quimioterápico de pacientes com câncer. O processo de resistência é multifatorial, mas o mecanismo mais bem caracterizado é a superexpressão da glicoproteína P (Pgp), uma proteína de membrana plasmática que funciona como uma bomba de efluxo levando a uma diminuição da concentração intracelular do quimioterápico. A circunvenção da resistência pode ser obtida utilizando-se agentes reversores capazes de inibir a atividade funcional da Pgp e de outras bombas de efluxorelacionadas. Nesta revisão, discutimos o uso do imunossupressor Ciclosporina A (CSA) e de seus análogos como agentes reversores da MDR. Aspectos como sua combinação com ciclofilinas, sua capacidade de inibir a atividade funcional da Pgp e seu uso clínico, especialmente em leucemias, são discutidos baseados tanto na literatura quanto em resultados dos próprios autores.

Cambios en el contenido de glicoproteinas en las cataratas diabéticas / Glycoproteins level variations in diabetic cataracts

Las cataratas que son más comunes en las personas diabéticas que en la población general, por debajo de los 40 años, son morfológicamente similares a las cataratas seniles. Los cambios en el contenido de las glicoproteinas en las cataratas diabéticas son poco conocidos. En la presente investigación se determino el contenido de diversas fracciones glicoprotéicas en 40 cataratas de pacientes diabéticos (glicemia en ayunas, 180 ± 10 mg/ dl), y se comparo con lo que sucede en cataratas seniles extraídas de 40 pacientes no diabéticos (glicemia, 80±7 mg/ dL). Los resultados obtenidos demuestran un incremento significativo (p<0.05) en la concentración de la hexosa y del ácido siálico unidos a las proteínas en las cataratas diabéticas al comparar con la seniles. El análisis de regresión lineal demostró que el incremento en la concentración de estas fracciones en las cataratas diabéticas es directamente proporcional a la glicemia en ayunas y a la edad de los pacientes, mientras que el sexo no tiene influencia significativa. Estos hallazgos se explican muy fácilmenten ya que en la diabetes, una entidad clínica que cursa con deficiencia de insulina, la glucosa se utiliza en el cristalino, y en otras estructuras oculares, principalmente en la síntesis de glicoproteínas. La significación fisiológica de estos hallazgos será investigada en futuras experiencias a realizar por nuestro equipo de investigación

Enhanced cellular accumulation of a P-glycoprotein substrate, rhodamine-123, by Caco-2 cells using low molecular weight methoxypolyethylene glycol-block-polycaprolactone diblock copolymers.

A series of diblock copolymers based on methoxypolyethylene glycol-block-poly(caprolactone) (MePEG-b-PCL) was synthesized and evaluated for enhancing the cellular accumulation of a P-glycoprotein (P-gp) substrate, rhodamine-123 (R-123), into caco-2 cells. Altering MePEG:caprolactone feed weight ratio allowed diblocks with varying PCL lengths to be synthesized onto MePEG of molecular weight 750 or 2000. The critical micelle concentration (CMC) and the hydrophilic-lipophilic balance all decreased with increasing degree of polymerization of PCL. R-123 accumulation by caco-2 cells increased to a maximum in the presence of increasing concentrations of MePEG-b-PCL diblock copolymers (compared to R-123 alone) beginning at concentrations at or above the CMC, with little or no R-123 accumulation enhancement observed below the CMC. Further increases in MePEG-b-PCL concentration resulted in a decrease in R-123 uptake back to baseline levels. It is suggested that the higher concentrations of diblock above the CMC were required to serve as a 'depot' for free unimer partitioning into the cell membrane in order to obtain a critical concentration of diblock in the membrane for P-gp modulation. Alternatively, MePEG-b-PCL micelles may increase R-123 accumulation via endocytosis of micellized R-123.

The effects of polyethyleneglycol (PEG)-derived lipid on the activity of target-sensitive immunoliposome.

In this study, serum stability and target-sensitivity of phosphatidylethanolamine (PE) immunoliposomes prepared with dioleoylphosphatidylethanolamine (DOPE), HYB-241 monoclonal antibody that targets p-glycoproteins, and various levels of polyethyleneglycol 2000 dioleoylphosphatidylethanolamine (PEG(2000)-DOPE) were determined. Incubation of calcein-laden pegylated immunoliposomes prepared with different levels of PEG(2000)-DOPE (0.3, 0.5 and 1.0 mol%) with p-glycoprotein rich bovine brain microvessel endothelial cells in 10% serum cell culture medium, all resulted in time-dependent release of calcein from the liposomes. The release of calcein was greatest for immunoliposomes prepared with 0.3 mol% PEG(2000)-DOPE (66% in 1 h). Contrarily, the release of calcein from the other two immunoliposomes reached only approximately 10-3% after same period of incubation. When serum-induced leakage of calcein was investigated for the above liposome preparations, liposomes prepared with 0.3 and 0.5 mol% PEG(2000)-DOPE had the highest leakage level (10% in 1 h). Contrarily, the release of calcein from liposomes prepared with 1.0 mol% PEG(2000)-DOPE reached only 3% after same period of incubation. Together, it would appear that release of calcein from the immunoliposomes prepared with 0.3 mol% PEG(2000)-DOPE is a result of both serum-induced and target-induced destabilization of liposomes. The net release of calcein due to target-induced destabilization of liposomes is calculated to be at approximately 56%. In contrast, there is no target-induced leakage of calcein from immunoliposomes prepared with either 0.5 or 1.0 mol% PEG(2000)-DOPE.