Veli-Pekka Ranta

Paracellular and passive transcellular permeability in immortalized human corneal epithelial cell culture model.

A cell culture model of human corneal epithelium (HCE-model) was recently introduced [Invest. Ophthalmol. Vis. Sci. 42 (2001) 2942] as a tool for ocular drug permeation studies. In this study, passive permeability and esterase activity of the HCE-model were characterised. Immortalised human corneal epithelial cells were grown on collagen coated filters under air-lift. The sensitivity of transcellular permeability to lipophilicity was tested in studies using nine beta-blockers. The size selectivity of the paracellular route was investigated using 16 polyethylene glycol oligomers (PEG). An effusion-like approach was used to estimate porosity and pore sizes of the paracellular space in HCE membrane. Permeability and degradation of fluorescein diacetate to fluorescein in HCE-cells was used to probe the esterase activity of the HCE-model. Drug concentrations were analyzed using HPLC (beta-blockers), LC-MS (PEGs), and fluorometry (fluorescein). Permeabilities were compared to those in the excised rabbit cornea. Penetration of beta-blockers increased with lipophilicity according to a sigmoidal relationship. This was almost similar to the profile in excised cornea. No apical to basolateral directionality was seen in the permeation of beta-blockers. Paracellular permeability of the HCE-model was generally slightly higher than that of the excised rabbit cornea. The HCE-model has larger paracellular pores, but lower pore density than the excised cornea, but the overall paracellular space was fairly similar in both models. The HCE-model shows significant esterase activity (i.e. fluorescein diacetate was converted to free fluorescein). These data on permeability of 27 compounds demonstrate that the barrier of the HCE-model closely resembles that of the excised rabbit cornea. Therefore, the HCE-model is a promising alternative corneal substitute for ocular drug delivery studies.

Barrier analysis of periocular drug delivery to the posterior segment

Periocular administration is a potential way of delivering drugs to their targets in posterior eye segment (vitreous, neural retina, retinal pigment epithelium (RPE), choroid). Purpose of this study was to evaluate the role of the barriers in periocular drug delivery. Permeation of FITC-dextrans and oligonucleotides in the bovine sclera was assessed with and without Pluronic gel in the donor compartment. Computational model for subconjunctival drug delivery to the choroid and neural retina/vitreous was built based on clearance concept. Kinetic parameters for small hydrophilic and lipophilic drug molecules, and a macromolecule were obtained from published ex vivo and in vivo animal experiments. High negative charge field of oligonucleotides slows down their permeation in the sclera. Pluronic does not provide adequate rate control to modify posterior segment drug delivery. Theoretical calculations for subconjunctival drug administration indicated that local clearance by the blood flow and lymphatics removes most of the drug dose which is in accordance with experimental results. Calculations suggested that choroidal blood flow removes most of the drug that has reached the choroid, but this requires experimental verification. Calculations at steady state using the same subconconjunctival input rate showed that the choroidal and vitreal concentrations of the macromolecule is 2-3 orders of magnitude higher than that of small molecules. The evaluation of the roles of the barriers augments to design new drug delivery strategies for posterior segment of the eye.

Simultaneous determination of eight β-blockers by gradient high-performance liquid chromatography with combined ultraviolet and fluorescence detection in corneal permeability studies in vitro

A gradient HPLC method with combined ultraviolet and fluorescence detection was developed for the simultaneous determination of eight beta-blockers (alprenolol, atenolol, metoprolol, nadolol, pindolol, propranolol, sotalol and timolol) in corneal permeability studies in vitro. Fluorescence detection with excitation wavelength at 230 nm and emission at 302 nm was selective for six of the compounds, whereas UV detection at 205 nm was able to detect all the compounds. Calibration was performed with fluorescence detection for six compounds from 50 or 200 nM to 3 microM, and with UV detection for all the eight compounds from 100 or 200 nM to 30 microM. With optimized fluorescence detection, detection limits between 0.7 and 1.3 nM (0.035-0.065 pmol per 50 microl injection) were obtained for atenolol, metoprolol, nadolol and sotalol. A mixture of eight beta-blockers was used in cassette dosing permeability studies with a cultured corneal epithelium. The HPLC method revealed marked differences in the permeation between hydrophilic and lipophilic beta-blockers through the corneal epithelial cell culture model.

Efflux Protein Expression in Human Retinal Pigment Epithelium Cell Lines

PurposeThe objective of this study was to characterize efflux proteins (P-glycoprotein (P-gp), multidrug resistance proteins (MRP1–6) and breast cancer resistance protein (BCRP)) of retinal pigment epithelium (RPE) cell lines.MethodsExpression of efflux proteins in two secondary (ARPE-19, D407) and two primary (HRPEpiC and bovine) RPE cell lines was measured by quantitative RT-PCR and western blotting. Furthermore, activity of MRP1 and MRP5 of ARPE-19 cell line was assessed with calcein-AM and carboxydichlorofluorescein (CDCF) probes.ResultsSimilar efflux protein profile was shared between ARPE-19 and primary RPE cells, whereas D407 cell line was notably different. D407 cells expressed MRP2 and BCRP, which were absent in other cell lines and furthermore higher MRP3 transcript expression was found. MRP1, MRP4 and MRP5 were identified from all human RPE cell lines and MRP6 was not expressed in any cell lines. The pattern of efflux protein expression did not change when ARPE-19 cells were differentiated on filters. The calcein-AM and CDCF efflux tests provided evidence supporting MRP1 and MRP5 activity in ARPE-19 cells.ConclusionsMRP1, MRP4 and MRP5 are the main efflux transporters in RPE cell lines. There are differences in efflux protein expression between RPE cell lines.

Filter-cultured ARPE-19 cells as outer blood-retinal barrier model

Retinal pigment epithelium (RPE) regulates drug transfer between posterior eye segment and blood circulation, but there is no established RPE cell model for drug delivery studies. We evaluated ARPE-19 filter culture model for this purpose. Passive permeability of 6-carboxyfluorescein, betaxolol and FITC-dextran (40kDa) and active transport of 6-carboxyfluorescein, sodium fluorescein, rhodamine 123, cyclosporine A and digoxin in ARPE-19 model were investigated and compared with isolated bovine RPE-choroid tissue. In addition, barrier properties, and mRNA expression of RPE-specific and melanogenesis-related genes (RPE65, VMD2, CRALBP, OTX-2, MITF-A, TRP-1, tyrosinase) were measured in various culture conditions. The filter grown ARPE-19 cell model showed reasonable barrier properties (TER close to 100Omegacm(2)), but its permeability was slightly higher than that of isolated bovine RPE/choroid specimens. In active transport studies the ARPE-19 model mimics qualitatively the permeability profile of bovine RPE-choroid, but ARPE-19 model underestimates the importance of active transport relative to passive diffusion. Long-term filter-cultured ARPE-19 cells expressed various RPE-specific and melanogenesis-related genes at higher levels than the ARPE-19 cells cultured short-term in flasks. ARPE-19 model can be used to study drug permeation processes in the RPE.

Intracellular DNA release and elimination correlate poorly with transgene expression after non-viral transfection

The intracellular limiting steps in non-viral gene delivery are still unclear. The purpose of this study was to quantize intracellular DNA release and elimination kinetics after transfection with various non-viral carriers (calcium phosphate precipitates, branched poly(ethyleneimine), poly-L-lysine, DOTAP, DOTAP/DOPE) and to correlate these factors with transgene expression. Intracellular kinetics of DNA was determined by novel quantitative method based on qRT-PCR and DNase treatment. Intracellular elimination of DNA after calcium phosphate transfection was rapid (half-life of 0.37 h) whereas the amount of DNA in the cells was stable for at least 136 h after poly(ethyleneimine) mediated transfection. Intracellular elimination half-lives for DNA delivered by other carrier systems ranged from 9 to 27 h. Calcium phosphate precipitates are not able to protect DNA, which explains the short elimination half-life. In the case of other carriers DNA is after complex removal mostly carrier bound but after 24 h the major fraction of DNA is in the released or loosened state. Overall, neither total nor released amount of intracellular DNA correlates with the transgene expression.

Ocular pharmacokinetic modeling using corneal absorption and desorption rates from in vitro permeation experiments with cultured corneal epithelial cells.

AbstractPurpose. To determine corneal absorption and desorption rate constants in a corneal epithelial cell culture model and to apply them to predict ocular pharmacokinetics after topical ocular drug application. Method. In vitro permeation experiments were performed with a mixture of six β-blockers using an immortalized human corneal epithelial cell culture model. Disappearance of the compounds from the apical donor solution and their appearance in the basolateral receiver solution were determined and used to calculate the corneal absorption and desorption rate constants. An ocular pharmacokinetic simulation model was constructed for timolol with the Stella® program using the absorption and desorption rate constants and previously published in vivo pharmacokinetic parameters. Results. The corneal absorption rates of β-blockers increased significantly with the lipophilicity of the compounds. The pharmacokinetic simulation model gave a realistic mean residence time for timolol in the cornea (57 min) and the aqueous humor (90 min). The simulated timolol concentration in the aqueous humor was about 1.8 times higher than the previously published experimental values. Conclusions. The simulation model gave a reasonable estimate of the aqueous humor concentration profile of timolol. This was the first attempt to combine cell culture methods and pharmacokinetic modeling for prediction of ocular pharmacokinetics. The wider applicability of this approach remains to be seen.

Comparison of Oxycodone Pharmacokinetics after Buccal and Sublingual Administration in Children

ObjectiveWe evaluated and compared the pharmacokinetics of two oral administration routes of oxycodone parenteral liquid (10 mg/mL) - single buccal and sublingual administration - in 30 generally healthy awake children, aged 6–91 months.MethodsTwo groups of children undergoing inpatient surgery were enrolled. In a randomised fashion, children received a single dose of oxycodone 0.2 mg/kg buccally (n = 15) or sublingually (n = 15). Regular blood samples were collected for up to 12 hours, and plasma was analysed for oxycodone, oxymorphone and noroxycodone using gas chromatography-mass spectrometry.ResultsBioavailability was similar after administration at the two instillation sites. The area under the plasma concentration-time curve from time zero extrapolated to infinity (AUC∞) was 2400–8000 ng · min/mL (median 4200 ng · min/mL) in the buccal group and 2700-7900 ng · min/mL (median 5500 ng · min/mL) in the sublingual group. After buccal administration, maximum plasma concentration (Cmax) was 5.4–39 ng/mL (16 ng/mL) after buccal and 5.5–42 ng/mL (22 ng/mL) after sublingual administration. Twelve of the 15 children in both groups reached the oxycodone analgesic concentration of 12 ng/mL, which was sustained for 43–209 minutes (median 160 minutes) in the children with buccal oxycodone and for 32–262 minutes (median 175 minutes) in the children with sublingual oxycodone. The terminal elimination half-lives were closely similar in the two groups: 104–251 minutes (median 140 minutes) in the buccal group and 110–190 minutes (150 minutes) in the sublingual group.ConclusionThe results of this study show that in young children the absorption of oxycodone is similar after buccal and sublingual instillation.

Passive oral drug absorption can be predicted more reliably by experimental than computational models--fact or myth.

This study assessed the prediction power of experimental and computational models that are widely used to predict human passive intestinal absorption. The models evaluated included two cell lines, three artificial membrane models, in vivo rat experiments, and seven previously described computational quantitative structure property relationship models based on human absorption values. The data sets used in the assessment of the models were carefully chosen from the literature, and different models were compared using the same compounds to ensure objective results. Three of the computational models were found to be significantly more reliable in predicting human passive intestinal absorption than the artificial membrane models that have been developed for the prediction of passive intestinal absorption. Two of these computational models were found to be as reliable as the Caco-2 and the 2/4/A1 cell lines and, furthermore, one of the models was able to predict the absorption of a set of 65 drugs nearly as well as absorption studies in rats. The unexpectedly good prediction power of the simple computational models with high throughput makes them ideal tools in the early screening of drug candidates, whereas laborious cell culture models and animal studies can be useful in the later phases when detailed information about the transport mechanisms is needed.

Central nervous system penetration of oxycodone after intravenous and epidural administration

BACKGROUND Despite being increasingly used for pain management, only two studies, with controversial results, have evaluated the epidural use of oxycodone. METHODS Twenty-four women, aged 26-64 yr, undergoing elective gynaecological surgery were enrolled in this randomized, double-blinded, parallel group study. The subjects were administered either i.v. oxycodone and epidural placebo (IV group; n=12) or epidural oxycodone and i.v. placebo (EPI group; n=12) after operation. Oxycodone was administered as a single dose of 0.1 mg kg(-1). An epidural catheter for drug administration was placed at T12/L1 and a spinal catheter for cerebrospinal fluid (CSF) sampling at L3/4. Plasma and CSF were frequently collected for the analysis of oxycodone and its major metabolites. The primary outcomes were the peak concentration (C(max)), time to peak concentration (T(max)), and the exposure (AUC(last)) of oxycodone in CSF and plasma. The secondary outcome was the analgesic efficacy, measured as the total dose of rescue fentanyl during the first four postoperative hours. RESULTS In the EPI group, the median oxycodone Cmax and AUC(last) in the CSF were 320- and 120-fold higher, respectively, compared with the IV group. The total dose of rescue fentanyl was significantly lower in the EPI group (seven subjects needed 16 doses) than in the IV group [12 subjects needed 71 doses (P=0.001)]. No serious or unexpected adverse events were reported. CONCLUSIONS Epidural oxycodone provides much higher CSF concentrations and possibly better analgesic efficacy than does i.v. oxycodone. CLINICAL TRIAL REGISTRATION EudraCT reference number: 2011-000125-76.