Convection-Enhanced Drug Delivery: Experimental and Analytical Studies of Infusion Behavior in an In Vitro Brain Surrogate.

Convection-enhanced drug delivery (CED) directly infuses drugs with a large molecular weight toward target cells as a therapeutic strategy for neurodegenerative diseases and brain cancers. Despite the success of many previous in vitro experiments on CED, challenges still remain. In particular, a theoretical predictive model is needed to form a basis for treatment planning, and developing such a model requires well-controlled injection tests that can rigorously capture the convective (advective) and diffusive transport of an infusate. For this purpose, we investigated the advection-diffusion transport of an infusate (bromophenol blue solution) in the brain surrogate (0.2% w/w agarose gel) at different injection rates, ranging from 0.25 to 4 µL/min, by closely monitoring changes in the color intensity, propagation distance, and injection pressures. One dimensional closed-form solution was examined with two variable sets, such as the mathematically calculated coefficient of molecular diffusion and average velocity, and the hydraulic dispersion coefficient and seepage velocity by the least squared method. As a result, the seepage velocity was greater than the average velocity to some extent, particularly for the later infusion times. The poroelastic deformation in the brain surrogate might lead to changes in porosity, and consequently, slight increases in the actual flow velocity as infusion continues. The limitation of efficiency of the single catheter was analyzed by dimensionless analysis. Lastly, this study suggests a simple but robust approach that can properly capture the convective (advective) and diffusive transport of an infusate in an in vitro brain surrogate via well-controlled injection tests.

In Vitro Evaluation of Small Molecule Delivery into Articular Cartilage: Effect of Synovial Clearance and Compressive Load.

Intra-articular injection of drug depots is considered as a therapeutic strategy for the treatment of osteoarthritis. In this study, we designed an in vitro assay in a previously described bioreactor system to evaluate the uptake of a small molecule drug mimic as a function of drug clearance by the synovium and compressive load. Bromophenol blue (BPB) loaded hydrogels were placed on top of bovine articular cartilage explants and were compressed in a dual flow bioreactor. As a control, BPB was directly injected in the bioreactor compartment mimicking the synovial fluid. Subsequently, diffusion coefficients of the dye were estimated based on Fick's law. Mimicking synovial clearance revealed that dye penetration of BPB when released from a drug delivery system placed on top of a cartilage explant was enhanced compared to direct injection of BPB into a simulated synovial fluid. Furthermore, we show the synergistic effect of the amount of load and the frequency on drug uptake by the cartilage. In the described model, we have shown that, under compressive load, drug delivery from a depot was beneficial over conventional intra-articular drug administration. The assay mimics the complexity of the knee joint in several key aspects, which results in a more close representation of the expected drug outcome. In this study, we have evaluated the penetration of a model small molecule drug into articular cartilage under compressive conditions, and future development will focus on incorporating synovial(-like) fluid, synovium, and bone to increase the predictive potential of the assay further.

State of the art in chromovitrectomy / Estado da arte da cromovitrectomia

Vitrectomy is a surgery that involves complex and delicate techniques that treat diseases such as macular hole, epiretinal membrane and diabetic macular edema. Chromovitrectomy is one of these techniques and includes the use of coloring agents such as vital dyes or crystals to enhanced visibility of transparent structures during vitrectomy. The aim of this study was to present a modern approach, based on scientific evidence, about the application and indication of vital coloring agents during vitrectomy. The use of such agents has made this surgery more predictable and has increased its post-operative prognosis. Although research on chromovitrectomy is currently expanding there is still not an established gold standard dyeing agent.

A cirurgia vitreorretiniana é uma cirurgia que envolve técnicas complexas e delicadas que tratam doenças como buraco macular, membrana epirretiniana e o edema macular diabético. A cromovitrectomia é uma dessas técnicas que incluem o uso de corantes compostos de pigmentos vitais ou cristais para melhorar a visibilização de estruturas transparentes durante a cirurgia de vitrectomia. O objetivo desse artigo foi apresentar uma abordagem atual, baseada em evidências, sobre a aplicação e indicação de corantes vitais durante a cirurgia vitreorretiniana. O emprego desses corantes possibilitou uma maior previsibilidade para a cirurgia, melhorando assim seu prognóstico pós-operatório. Apesar do campo da cromovitrectomia está em plena expansão de pesquisas, um corante gold standard para cromovitrectomia ainda não está estabelecido.

Pathways of infusate loss during convection-enhanced delivery into the putamen nucleus.

BACKGROUND: New strategies aiming to treat Parkinson's disease, such as delivery of trophic factors via protein infusion or gene transfer, depend upon localized intracerebral infusion, mainly into the putamen nucleus. Convection-enhanced delivery (CED) has been proposed as a method to improve intracerebral distribution of therapies. Yet analysis of controversial results during the clinical translation of these strategies suggests that intracerebral misdistribution of infusate may have affected the outcomes by limiting the amount of treatment into the target region. OBJECTIVES: This study aimed to identify possible pathways of infusate loss and their relative impact in the success of targeted CED into the postcommissural ventral putamen nucleus. METHODS: Thirteen adult macaque monkeys received intraputaminal CED infusions of 100 µl of 2.0 mM gadoteridol and bromophenol blue (0.16 mg/ml) solution at a rate of 1.0 µl/min under intraoperative magnetic resonance imaging (MRI) guidance. Quantitative maps of infusate concentration were computed at 10-min intervals throughout the procedure in a 3-Tesla MRI scanner. The fraction of tracer lost from the putamen as well as the path of loss were evaluated and quantified for each infusion. RESULTS: All injections (total 22) were successfully placed in the ventral postcommissural putamen nucleus. Four major paths of infusate loss from the putamen were observed: overflow across putamen boundaries, perivascular flow along large blood vessels, backflow along the inserted catheter and catheter tract leakage into the vacated catheter tract upon catheter removal. Overflow loss was observed within the first 30 µl of infusion in all cases. Measurable tracer loss following the path of an artery out of the putamen was observed in 15 cases, and in 8 of these cases, the loss was greater than 10% of infusate. Backflow that exited the putamen was observed in 4 cases and led to large loss of infusate (80% in 1 case) into the corona radiata. Loss into the vacated catheter tract amounted only to a few microliters. CONCLUSIONS: Our analysis demonstrates that after controlling for targeting, catheter type, infusion rate and infusate, the main issues during surgical planning are the identification of appropriate infusate volume that matches the target area, as well as mapping the regional vasculature as it may become a pathway for infusate loss. Most importantly, these results underscore the significance of presurgical planning for catheter placement and infusion, and the value of imaging guidance to ensure targeting accuracy.

Comparative effects of six intraocular vital dyes on retinal pigment epithelial cells.

PURPOSE: To evaluate and compare the effects of the following dyes on human pigmented epithelial cells: indocyanine green (ICG), infracyanine green (IfCG), trypan blue (TB), bromophenol blue (BrB), patent blue (PB), and Brilliant Blue G (BBG). METHODS: ARPE-19 cells cultured in vitro were exposed to these dyes, and acute and chronic toxicity were evaluated. Cell viability was measured by colorimetry (MTT assay), morphology was observed by phase-contrast microscopy, membrane permeability (CMP) was evaluated by flow cytometry with propidium iodide (PI), and mitochondrial membrane potential (ΔΨm) was measured with 3,3'-dihexyloxacarbocyanine (DiOC(6)(3)). RESULTS: Each of the studied dyes exhibited toxicity after acute exposure at surgical doses. The presence of light often reduced cell viability, especially when measured 3 hours after incubation in the case of ICG, TB, BrB, and BBG. Morphologic changes were induced by ICG, IfCG, and BBG. Both CMP and ΔΨm were altered after exposure to surgical doses of ICG, TB, PB, and a fourfold surgical dose of BrB. Chronic exposure to residual amounts of some dyes was associated with reduced proliferation and even cell death. CONCLUSIONS: It appears to be prudent to use the lowest possible dose of each dye, to minimize the risk of toxic effects. This precaution may be particularly important in the case of BrB, which should not be used in excess of 0.5%. In addition, abundant irrigation of the vitreous cavity after surgery to completely remove traces of dye may be of crucial importance, particularly in the case of ICG, in minimizing chronic toxicity.

Accuracy of ultrasound-guided injections of thoracolumbar articular process joints in horses: a cadaveric study.

REASONS FOR PERFORMING STUDY: Arthrosis of the articular process joints (APJs) in the caudal thoracolumbar region of horses may cause back pain and subsequent reduced performance or lameness. Ultrasound-guided injections of the APJs of the equine back have been described only briefly in the literature. OBJECTIVES: To evaluate factors affecting the accuracy of intra-articular injections of the APJs in the caudal thoracolumbar region. METHODS: One-hundred-and-fifty-four injections with blue dye were performed on APJs including the T14-L6 region in 12 horses subjected to euthanasia for reasons unrelated to back problems. The backs were subsequently dissected to verify the location of the injectate in relation to the APJs. RESULTS: Twenty-seven percent of the injections were found to be intra-articular and a total of 77% found to be within 2 mm of the joint capsule including the intra-articular deposits. Application of a medial approach and 18 gauge needle were significantly associated with an intra-articular injection or deposition close to the joint capsule. Operator, APJ (location) and back number (chronological) did not significantly affect the accuracy of injection. CONCLUSIONS AND POTENTIAL RELEVANCE: Injection of the vertebral APJ in the thoracolumbar region using ultrasound guidance is a reliable method, as most of the injections were either in or within 2 mm of the joint. Based on the findings of this cadaver study, the medial approach is expected to be the most accurate in live horses. Further investigations are required to evaluate the diagnostic and therapeutic potential of this method in clinical practice.

Formulation and evaluation of albumin microspheres and its enteric coating using a spray-dryer.

This study optimized and evaluated the conditions for surface coating of microspheres using a spray-dryer. Four formulations of Bromophenol blue (BPB)-loaded albumin microspheres were prepared using a spray-dryer, cross-linked at different concentrations and time periods. One of the optimized formulations with the desired characteristics was selected for enteric coating with Eudragit L100-55. The procedure involved suspending BPB microspheres in polymer solution and spray-drying it. Four enteric coated formulations were prepared with different concentrations of microspheres in suspension (0.25 and 0.5%w/v) and polymer concentrations (0.25 and 0.5%w/v). Change in the mean particle size after coating was determined using a Laser Particle Counter. The surface coating technique employed did not significantly increase the particle size. Enteric coating efficiency was determined in simulated gastric fluid. Compared to the uncoated microspheres the cumulative amount of drug released from coated microspheres was significantly lower for 3 h, implying efficient surface coating.

Bromophenol blue staining of tumors in a rat glioma model.

OBJECTIVE: For patients with gliomas, decreasing the tumor burden with macroscopic surgical resection may affect quality of life, time to tumor progression, and survival. Injection of bromophenol blue (BPB) may enhance intraoperative visualization of an infiltrating tumor and its margins and improve the extent of resection. In this study, we investigated the uptake of BPB in experimental rat brain tumors. METHODS: We first conducted a toxicity study with bolus intravenous injections of 5, 60, and 360 mg/kg doses of BPB in nontumor-bearing Fischer 344 rats. No adverse effects were observed in any of the animals during the 60 day observation period. We then injected 9L tumor cells intracerebrally into Fischer 344 rats and approximately 2 weeks later, administered a bolus intravenous injection of 5 to 360 mg/kg BPB. Fifteen minutes after BPB injection, we sacrificed the animals and removed their brains. In a subsequent study, we injected 180 mg/kg BPB and sacrificed animals at several time points to monitor tumor staining over time. RESULTS: The stain was clearly visible and localized to the tumor for all BPB concentrations 60 mg/kg or greater, and in an additional experiment, we found that tumor staining persisted for at least 8 hours after BPB injection. CONCLUSION: We conclude that BPB helped visualize experimental tumors at time points from a few minutes to several hours after injection. Because BPB also proved to be nontoxic to the animals at effective concentrations, we believe the compound may be potentially useful in helping neurosurgeons visualize brain tumors in humans.

A realistic brain tissue phantom for intraparenchymal infusion studies.

OBJECT: The goal of this study was to validate a simple, inexpensive, and robust model system to be used as an in vitro surrogate for in vivo brain tissues in preclinical and exploratory studies of infusion-based intraparenchymal drug and cell delivery. METHODS: Agarose gels of varying concentrations and porcine brain were tested to determine the infusion characteristics of several different catheters at flow rates of 0.5 and 1 microl per minute by using bromophenol blue (BPB) dye (molecular weight [MW] approximately 690) and gadodiamide (MW approximately 573). Magnetic resonance (MR) imaging and videomicroscopy were used to measure the distribution of these infusates, with a simultaneous measurement of infusion pressures. In addition, the forces of catheter penetration and movement through gel and brain were measured. Agarose gel at a 0.6% concentration closely resembles in vivo brain with respect to several critical physical characteristics. The ratio of distribution volume to infusion volume of agarose was 10 compared with 7.1 for brain. The infusion pressure of the gel demonstrated profiles similar in configuration and magnitude to those of the brain (plateau pressures 10-20 mm Hg). Gadodiamide infusion in agarose closely resembled that in the brain, as documented using T1-weighted MR imaging. Gadodiamide distribution in agarose gel was virtually identical to that of BPB dye, as documented by MR imaging and videomicroscopy. The force profile for insertion of a silastic catheter into agarose gel was similar in magnitude and configuration to the force profile for insertion into the brain. Careful insertion of the cannula using a stereotactic guide is critical to minimize irregularity and backflow of infusate distribution. CONCLUSIONS: Agarose gel (0.6%) is a useful surrogate for in vivo brain in exploratory studies of convection-enhanced delivery.

Accuracy of ultrasound-guided intra-articular injection of cervical facet joints in horses: a cadaveric study.

REASONS FOR PERFORMING STUDY: Intra-articular facet joint injection is an established diagnostic procedure in human medicine but there are no reports on its reliability in equine practice. OBJECTIVES: To investigate the accuracy of ultrasound-guided intra-articular injections of the cervical facet joints and to estimate factors influencing the accuracy. METHODS: Sixty injections with blue dye were performed on the facet joints between 2nd and 7th cervical vertebra (C2-C7) on horses subjected to euthanasia for nonorthopaedic reasons. The facet joints were subsequently dissected to verify accuracy of deposition. RESULTS: Seventy-two percent of the injections were found to be intra-articular, 17% were intracapsular and a total of 98% were within 1 mm of the joint capsule. There was a marked effect of gained experience (P < 0.01), but not of other factors tested. CONCLUSIONS AND POTENTIAL RELEVANCE: The results of the present study do not translate directly to injections performed in live horses, but they indicate that the method can be applied as a diagnostic as well as therapeutic procedure in C2 to C7 and that is advisable to practise injections on cadaver specimens before applying the technique.

Absorption of phenol red and bromphenol blue as model drugs from the peritoneal cavity around the liver surface in rats.

The importance of the injection site on the pharmacokinetics of phenol red and bromphenol blue as model drugs after intraperitoneal administration into rat was examined. Their absorption rate from the peritoneal cavity was faster after intraperitoneal administration to the liver surface than that after intraperitoneal administration to the distal small intestine, as shown by the increase in maximum concentration and decrease in mean residence time in plasma. A similar tendency was observed in the biliary excretion pattern. The enhanced absorption rate was supported by the significantly smaller amount of both drugs remaining in the peritoneal cavity at 15 min after liver surface administration than that after small intestine administration. The liver concentration of the model drugs at 15 min after liver surface administration was 1.5-2.0 times that after small intestine administration. Accordingly, liver surface administration was shown to be effective with good absorption and efficient drug delivery to the liver.

Absorption of organic anions as model drugs following application to rat liver surface in-vivo.

Absorption of organic anions (phenol red, bromphenol blue and bromosulphonphthalein) has been studied after their application to rat liver surface in-vivo, employing a cylindrical glass cell (i.d. 9 mm, area 0.64 cm2). Each drug appeared gradually in the blood with the peak level at about 1 h, after which its concentration declined slowly. Absorbed model drug was efficiently excreted into the bile. These observations appear to indicate the possibility of drug absorption from liver surface membrane. Absorption of model drugs was estimated to be more than 59% in 6 h. The biliary recovery and metabolism of phenol red did not change as compared with that after intravenous administration.

Amphiphilic networks: II. Biocompatibility and controlled drug release of poly[isobutylene-co-2-(dimethylamino)ethyl methacrylate].

Biocompatibility and diffusional characteristics of recently synthesized unique amphiphilic networks, i.e., copolymers of methacryloyl-capped polyisobutylene (MA-PIB-MA) with 2-(dimethylamino)-ethyl methacrylate (DMAEMA) have been examined. Excellent biocompatibility and biostability were obtained after 7 weeks with films containing 53-58% MA-PIB-MA implanted dorsally or abdominally in rats. In contrast, networks with higher or lower MA-PIB-MA contents showed decreased biocompatibility. These amphiphilic networks were also studied as potential implantable drug release systems. Bromophenol blue and folic acid were used as model compounds for drug release. Diffusion of these chemicals from loaded networks into water showed a marked pH dependence. Under specific well-defined conditions (MA-PIB-MA/DMAEMA content, pH, time range) release was observed to be independent of time (zero-order release).

Effect of nonuniform bile flow rate on the rate of biliary excretion of bromophenol blue in the beagle.

Following the intravenous administration of bromophenol blue to beagle dogs, graphs of the biliary excretion rate versus time displayed drastic fluctuations which render them of little value for standard pharmacokinetic modeling purposes. It was shown that these fluctuations in excretion rate are highly correlated with corresponding fluctuations in the bile flow rate. An expression was derived which accounts for the primary effect of nonuniform bile flow rate on the biliary excretion rate. This treatment would enable the use of such biliary excretion rate data for pharmacokinetic modeling. Secondary effects of nonuniform bile flow on the biliary excretion rate are also discussed. It is suggested that the modeling of other flow rate-dependent elimination processes could benefit from such a treatment.

Dose-dependent pharmacokinetics and biliary excretion of bromophenol blue in the rat.

Concentrations of bromophenol blue (I) in plasma, urine, and bile were determined spectrophotometrically after intravenous bolus injections and infusions in rats. The plasma concentrations were found to decrease monoexponentially after all doses except the highest, where the decrease was biexponential. Although the disposition kinetics of I were apparently first-order at all doses, the half-life increased with increasing dose. The area under the plasma concentration-time curve (AUC0-infinity) increased disproportionately with increasing dose. The binding of I to rat plasma proteins, as determined by equilibrium dialysis, showed that the fraction bound (96%) remained constant in the concentration range of 10-300 micrograms/ml. Plasma concentrations were determined at time zero after intravenous administration and after a second dose administered 20 min later when plasma concentrations from the first dose were minimal. The apparent first-order elimination rate constant for the plasma concentration decline following the second dose was significantly less than after the first dose, indicating that the residual dye in the liver altered the elimination of I after the second dose. The fraction of the dose in the liver decreased with increasing dose, indicating a saturable uptake process. The biliary excretion profile reflected the uptake saturation that occurred in the liver and demonstrated that the biliary excretion of I depended on the amount present in the liver. When liver damage was induced by exposure to carbon tetrachloride, dye concentrations in the plasma, liver, and kidney increased markedly.