Convection-enhanced delivery to the central nervous system.

Convection-enhanced delivery (CED) is a bulk flow-driven process. Its properties permit direct, homogeneous, targeted perfusion of CNS regions with putative therapeutics while bypassing the blood-brain barrier. Development of surrogate imaging tracers that are co-infused during drug delivery now permit accurate, noninvasive real-time tracking of convective infusate flow in nervous system tissues. The potential advantages of CED in the CNS over other currently available drug delivery techniques, including systemic delivery, intrathecal and/or intraventricular distribution, and polymer implantation, have led to its application in research studies and clinical trials. The authors review the biophysical principles of convective flow and the technology, properties, and clinical applications of convective delivery in the CNS.

Real-time imaging of convection-enhanced delivery of viruses and virus-sized particles.

OBJECT: Despite recent evidence showing that convection-enhanced delivery (CED) of viruses and virus-sized particles to the central nervous system (CNS) is possible, little is known about the factors influencing distribution of these vectors with convection. To better define the delivery of viruses and virus-sized particles in the CNS, and to determine optimal parameters for infusion, the authors coinfused adeno-associated virus ([AAV], 24-nm diameter) and/or ferumoxtran-10 (24 nm) by using CED during real-time magnetic resonance (MR) imaging. METHODS: Sixteen rats underwent intrastriatal convective coinfusion with 4 microl of 35S-AAV capsids (0.5-1.0 x 10(14) viral particles/ml) and increasing concentrations (0.1, 0.5, 1, and 5 mg/ml) of a similar sized iron oxide MR imaging agent (ferumoxtran-10). Five nonhuman primates underwent either convective coinfusion of 35S-AAV capsids and 1 mg/ml ferumoxtran-10 (striatum, one animal) or infusion of 1 mg/ml ferumoxtran-10 alone (striatum in two animals; frontal white matter in two). Clinical effects, MR imaging studies, quantitative autoradiography, and histological data were analyzed. RESULTS: Real-time, T2-weighted MR imaging of ferumoxtran-10 during infusion revealed a clearly defined hypointense region of perfusion. Quantitative autoradiography confirmed that MR imaging of ferumoxtran-10 at a concentration of 1 mg/ml accurately tracked viral capsid distribution in the rat and primate brain (the mean difference in volume of distribution [Vd] was 7 and 15% in rats and primates, respectively). The Vd increased linearly with increasing volume of infusion (Vi) (R2 = 0.98). The mean Vd/Vi ratio was 4.1 +/- 0.2 (mean +/- standard error of the mean) in gray and 2.3 +/- 0.1 in white matter (p < 0.01). The distribution of infusate was homogeneous. Postinfusion MR imaging revealed leakback along the cannula track at infusion rates greater than 1.5 microl/minute in primate gray and white matter. No animal had clinical or histological evidence of toxicity. CONCLUSIONS: The CED method can be used to deliver AAV capsids and similar sized particles to the CNS safely and effectively over clinically relevant volumes. Moreover, real-time MR imaging of ferumoxtran-10 during infusion reveals that AAV capsids and similar sized particles have different convective delivery properties than smaller proteins and other compounds.

Convective delivery of glial cell line-derived neurotrophic factor in the human putamen.

OBJECT: The authors conducted an analysis of the distribution of glial cell line-derived neurotrophic factor in the human striatum following convection-enhanced delivery. METHODS: Computational examinations of the effects of differing catheters, infusion rates, infusate concentrations, and target placement on distribution were completed based on the protocols of three recent clinical trials. RESULTS: Similar drug distributions around on-target end-hole catheters were predicted in two of the trials (AmgenUT study and Bristol study), although there was slightly deeper penetration for one of the trials (Bristol) due to a higher infusate concentration. However, when positioning uncertainly located catheter tips close to gray-white matter interfaces, backflow could diminish delivery, shunting infusate across the interfaces. For delivery via a multiport catheter at a constant base infusion rate plus a periodic bolus inflow rate (Kentucky study), base inflow alone generated a somewhat smaller distribution volume relative to those in the other trials, was positioned more anteriorly in the putamen, and was somewhat elongated axially; the bolus component extended this putaminal distribution to a larger relative volume but may have been reduced by backflow loss. CONCLUSIONS: Results of these computations indicated that for catheters placed exactly on the intended target, ideal drug distributions were similar for two of the trials (AmgenUT and Bristol) and different in terms of location and extent in the third study (Kentucky); yet the pattern of trial outcomes did not reflect these same groupings. This finding suggests that other factors are at play, widely varying statistical power and the possible effects of not excluding data from patients who experienced large drug losses across gray tissue boundaries due to variation in catheter placement.

Image-guided convection-enhanced delivery of gemcitabine to the brainstem.

OBJECT: To determine if the potent antiglioma chemotherapeutic agent gemcitabine could be delivered to the brainstem safely at therapeutic doses while monitoring its distribution using a surrogate magnetic resonance (MR) imaging tracer, the authors used convection-enhanced delivery to perfuse the primate brainstem with gemcitabine and Gd-diethylenetriamine pentaacetic acid (DTPA). METHODS: Six primates underwent convective brainstem perfusion with gemcitabine (0.4 mg/ml; two animals), Gd-DTPA (5 mM; two animals), or a coinfusion of gemcitabine (0.4 mg/ml) and Gd-DTPA (5 mM; two animals), and were killed 28 days afterward. These primates were observed over time clinically (six animals), and with MR imaging (five animals), quantitative autoradiography (one animal), and histological analysis (all animals). In an additional primate, 3H-gemcitabine and Gd-DTPA were coinfused and the animal was killed immediately afterward. In the primates there was no histological evidence of infusate-related tissue toxicity. Magnetic resonance images obtained during infusate delivery demonstrated that the anatomical region infused with Gd-DTPA was clearly distinguishable from surrounding noninfused tissue. Quantitative autoradiography confirmed that Gd-DTPA tracked the distribution of 3H-gemcitabine and closely approximated its volume of distribution (mean volume of distribution difference 13.5%). Conclusions. Gemcitabine can be delivered safely and effectively to the primate brainstem at therapeutic concentrations and at volumes that are higher than those considered clinically relevant. Moreover, MR imaging can be used to track the distribution of gemcitabine by adding Gd-DTPA to the infusate. This delivery paradigm should allow for direct therapeutic application of gemcitabine to brainstem gliomas while monitoring its distribution to ensure effective tumor coverage and to maximize safety.

Real-time, image-guided, convection-enhanced delivery of interleukin 13 bound to pseudomonas exotoxin.

PURPOSE: To determine if the tumor-targeted cytotoxin interleukin 13 bound to Pseudomonas exotoxin (IL13-PE) could be delivered to the brainstem safely at therapeutic doses while monitoring its distribution in real-time using a surrogate magnetic resonance imaging tracer, we used convection-enhanced delivery to perfuse rat and primate brainstems with IL13-PE and gadolinium-bound albumin (Gd-albumin). EXPERIMENTAL DESIGN: Thirty rats underwent convective brainstem perfusion of IL13-PE (0.25, 0.5, or 10 microg/mL) or vehicle. Twelve primates underwent convective brainstem perfusion of either IL13-PE (0.25, 0.5, or 10 microg/mL; n = 8), co-infusion of 125I-IL13-PE and Gd-albumin (n = 2), or co-infusion of IL13-PE (0.5 microg/mL) and Gd-albumin (n = 2). The animals were permitted to survive for up to 28 days before sacrifice and histologic assessment. RESULTS: Rats showed no evidence of toxicity at all doses. Primates showed no toxicity at 0.25 or 0.5 microg/mL but showed clinical and histologic toxicity at 10 microg/mL. Quantitative autoradiography confirmed that Gd-albumin precisely tracked IL13-PE anatomic distribution and accurately showed the volume of distribution. CONCLUSIONS: IL13-PE can be delivered safely and effectively to the primate brainstem at therapeutic concentrations and over clinically relevant volumes using convection-enhanced delivery. Moreover, the distribution of IL13-PE can be accurately tracked by co-infusion of Gd-albumin using real-time magnetic resonance imaging.

Surface properties, more than size, limiting convective distribution of virus-sized particles and viruses in the central nervous system.

OBJECT: Achieving distribution of gene-carrying vectors is a major barrier to the clinical application of gene therapy. Because of the blood-brain barrier, the distribution of genetic vectors to the central nervous system (CNS) is even more challenging than delivery to other tissues. Direct intraparenchymal microinfusion, a minimally invasive technique, uses bulk flow (convection) to distribute suspensions of macromolecules widely through the extracellular space (convection-enhanced delivery [CED]). Although acute injection into solid tissue is often used for delivery of oligonucleotides, viruses, and liposomes, and there is preliminary evidence that certain of these large particles can spread through the interstitial space of the brain by the use of convection, the use of CED for distribution of viruses in the brain has not been systematically examined. That is the goal of this study. METHODS: Investigators used a rodent model to examine the influence of size, osmolarity of buffering solutions, and surface coating on the volumetric distribution of virus-sized nanoparticles and viruses (adeno-associated viruses and adenoviruses) in the gray matter of the brain. The results demonstrate that channels in the extracellular space of gray matter in the brain are large enough to accommodate virus-sized particles and that the surface characteristics are critical determinants for distribution of viruses in the brain by convection. CONCLUSIONS: These results indicate that convective distribution can be used to distribute therapeutic viral vectors in the CNS.

Real-time in vivo imaging of the convective distribution of a low-molecular-weight tracer.

OBJECT: Convection-enhanced delivery (CED) is increasingly used to distribute therapeutic agents to locations in the central nervous system. The optimal application of convective distribution of various agents requires the development of imaging tracers to monitor CED in vivo in real time. The authors examined the safety and utility of an iodine-based low-molecular-weight surrogate tracer for computerized tomography (CT) scanning during CED. METHODS: Various volumes (total volume range 90-150 microl) of iopamidol (MW 777 D) were delivered to the cerebral white matter of four primates (Macaca mulatta) by using CED. The distribution of this imaging tracer was determined by in vivo real-time and postinfusion CT scanning (< or = 5 days after infusion [one animal]) as well as by quantitative autoradiography (14C-sucrose [all animals] and 14C-dextran [one animal]), and compared with a mathematical model. Clinical observation (- 5 months) and histopathological analyses were used to evaluate the safety and toxicity of the tracer delivery. Real-time CT scanning of the tracer during infusion revealed a clearly definable region of perfusion. The volume of distribution (Vd) increased linearly (r2 = 0.97) with an increasing volume of infusion (V.). The overall Vd/Vi ratio was 4.1+/-0.7 (mean+/-standard deviation) and the distribution of infusate was homogeneous. Quantitative autoradiography confirmed the accuracy of the imaged distribution for a small (sucrose, MW 359 D) and a large (dextran, MW 70 kD) molecule. The distribution of the infusate was identifiable up to 72 hours after infusion. There was no clinical or histopathological evidence of toxicity in any animal. CONCLUSIONS: Real-time in vivo CT scanning of CED of iopamidol appears to be safe, feasible, and suitable for monitoring convective delivery of drugs with certain features and low infusion volumes.

Replication of a unit-copy plasmid F in the bacterial cell cycle: a replication rate function analysis.

For stability, the replication of unit-copy plasmids ought to occur by a highly controlled process. We have characterized the replication dynamics of a unit-copy plasmid F by a replication rate function defined as the probability per unit age interval of the cell cycle that a plasmid will initiate replication. Analysis of baby-machine data [J. Bacteriol. 170 (1988) 1380; J. Bacteriol. 179 (1997) 1393] by stochastics that make no detailed reference to underlying mechanism revealed that this rate function increased monotonically over the cell cycle with rapid increase near cell division. This feature is highly suggestive of a replication control mechanism that is designed to force most plasmids to replicate before cells undergo division. The replication rate function is developed anew from a mechanistic model incorporating the hypotheses that initiators are limiting and that steric hindrance of origins by handcuffing control initiation of replication. The model is based on correctly folded initiator protein monomers arising from an inactive dimer pool via chaperones in limiting amounts, their random distribution to high affinity sites (iterons) at the origin (ori) and an outside locus (incC), the statistical mechanics of bound monomer participation in pairing the two loci (cis-handcuffing), and initiation probability as proportional to the number of non-handcuffed ori-saturated plasmids. Provided cis-handcuffing is present, this model closely accounts for the shape of the replication rate function derived from experiment, and reproduces the observation that replication occurs throughout the cell cycle. Present concepts of iteron-based molecular mechanisms thus appear capable of yielding a quantitative description of unit-copy-number plasmid replication dynamics.

Direct interstitial infusion of NK1-targeted neurotoxin into the spinal cord: a computational model.

Convection-enhanced delivery of substance P (SP) nocitoxins to the spinal cord interstitium is under consideration for the treatment of chronic pain. To characterize treatment protocols, a three-dimensional finite-element model of infusion into the human dorsal column was developed to predict the distribution of SP-diphtheria toxin fusion protein (SP-DT') within normal and target tissue. The model incorporated anisotropic convective and diffusive transport through the interstitial space, hydrolysis by peptidases, and intracellular trafficking. For constant SP-DT' infusion (0.1 microl/min), the distribution of cytotoxicity in NK1 receptor-expressing neurons was predicted to reach an asymptotic limit at 6-8 h in the transverse direction at the level of the infusion cannula tip ( approximately 60% ablation of target neurons in lamina I/II). Computations revealed that SP-DT' treatment was favored by a stable SP analog (half-life approximately 60 min), high infusate concentration (385 nM), and careful catheter placement (adjacent to target lamina I/II). Sensitivity of cytotoxic regions to NK1 receptor density and white matter protease activity was also established. These data suggest that intraparenchymal infusions can be useful for treatment of localized chronic pain.

A computational model of direct interstitial infusion of macromolecules into the spinal cord.

Convection-enhanced interstitial infusion can deliver macromolecular drugs to large tissue volumes of the central nervous system. To characterize infusion into the spinal cord, an image-based three-dimensional finite element model of the rat spinal cord was developed. The model incorporated convection and diffusion through white and gray matter, including anisotropic transport due to alignment of white matter tracts. Spatial and temporal distribution of the marker substance albumin within the interstitial space was determined. Consistent with previous experiments, predicted distribution was highly anisotropic. Infusing into the dorsal column, albumin was primarily confined to, white matter with limited penetration into adjacent gray matter. Distribution was determined primarily by the ratio of fiber-parallel to fiber-perpendicular hydraulic conductivity tensor components (k(wm-z)/k(wm-x)), the ratio of transverse white and gray matter hydraulic conductivity (k(wm-x)/k(gm)), and tissue porosity. Fits to previous experimental measures of axial and transverse spread, distribution volume, and protein recovery yielded an optimum k(wm-z)/k(wm-x) of approximately 20 at 0.1 microl/min. k(wm-x)/k(gm) of 100 was sufficient to match experimental transverse distribution data. Best fits to data at 0.1 microl/min were achieved by porosities characteristic of moderate edema (e.g., 0.26). Distribution also varied with catheter placement with more medial placement resulting in greater distribution volumes.

Successful and safe perfusion of the primate brainstem: in vivo magnetic resonance imaging of macromolecular distribution during infusion.

OBJECT: Intrinsic disease processes of the brainstem (gliomas, neurodegenerative disease, and others) have remained difficult or impossible to treat effectively because of limited drug penetration across the blood-brainstem barrier with conventional delivery methods. The authors used convection-enhanced delivery (CED) of a macromolecular tracer visible on magnetic resonance (MR) imaging to examine the utility of CED for safe perfusion of the brainstem. METHODS: Three primates (Macaca mulatta) underwent CED of various volumes of infusion ([Vis]; 85, 110, and 120 microl) of Gd-bound albumin (72 kD) in the pontine region of the brainstem during serial MR imaging. Infusate volume of distribution (Vd), homogeneity, and anatomical distribution were visualized and quantified using MR imaging. Neurological function was observed and recorded up to 35 days postinfusion. Histological analysis was performed in all animals. Large regions of the pons and midbrain were successfully and safely perfused with the macromolecular protein. The Vd was linearly proportional to the Vi (R2 = 0.94), with a Vd/Vi ratio of 8.7 +/- 1.2 (mean +/- standard deviation). Furthermore, the concentration across the perfused region was homogeneous. The Vd increased slightly at 24 hours after completion of the infusion, and remained larger until the intensity of infusion faded (by Day 7). No animal exhibited a neurological deficit after infusion. Histological analysis revealed normal tissue architecture and minimal gliosis that was limited to the region immediately surrounding the cannula track. CONCLUSIONS: First, CED can be used to perfuse the brainstem safely and effectively with macromolecules. Second, a large-molecular-weight imaging tracer can be used successfully to deliver, monitor in vivo, and control the distribution of small- and large-molecular-weight putative therapeutic agents for treatment of intrinsic brainstem processes.

Effects of systemic and central nervous system localized inflammation on the contributions of metabolic precursors to the L-kynurenine and quinolinic acid pools in brain.

L-Kynurenine and quinolinic acid are neuroactive L-tryptophan-kynurenine pathway metabolites of potential importance in pathogenesis and treatment of neurologic disease. To identify precursors of these metabolites in brain, [(2)H(3) ]-L-kynurenine was infused subcutaneously by osmotic pump into three groups of gerbils: controls, CNS-localized immune-activated, and systemically immune-activated. The specific activity of L-kynurenine and quinolinate in blood, brain and systemic tissues at equilibrium was then quantified by mass spectrometry and the results applied to a model of metabolism to differentiate the relative contributions of various metabolic precursors. In control gerbils, 22% of L-kynurenine in brain was derived via local synthesis from L-tryptophan/formylkynurenine versus 78% from L-kynurenine from blood. Quinolinate in brain was derived from several sources, including: local tissue L-tryptophan/formylkynurenine (10%), blood L-kynurenine (35%), blood 3-hydroxykynurenine/3-hydroxyanthranilate (7%), and blood quinolinate (48%). After systemic immune-activation, however, L-kynurenine in brain was derived exclusively from blood, whereas quinolinate in brain was derived from three sources: blood L-kynurenine (52%), blood 3-hydroxykynurenine or 3-hydroxyanthranilate (8%), and blood quinolinate (40%). During CNS-localized immune activation, > 98% of both L-kynurenine and quinolinate were derived via local synthesis in brain. Thus, immune activation and its site determine the sources from which L-kynurenine and quinolinate are synthesized in brain. Successful therapeutic modulation of their concentrations must take into account the metabolic and compartment sources.