Tri-partite complex for axonal transport drug delivery achieves pharmacological effect

Abstract

Background: Targeted delivery of pharmaceutical agents into selected populations of CNS (Central Nervous System) neurons is an extremely compelling goal. Currently, systemic methods are generally used for delivery of pain medications, anti-virals for treatment of dermatomal infections, anti-spasmodics, and neuroprotectants. Systemic side effects or undesirable effects on parts of the CNS that are not involved in the pathology limit efficacy and limit clinical utility for many classes of pharmaceuticals. Axonal transport from the periphery offers a possible selective route, but there has been little progress towards design of agents that can accomplish targeted delivery via this intraneural route. To achieve this goal, we developed a tripartite molecular construction concept involving an axonal transport facilitator molecule, a polymer linker, and a large number of drug molecules conjugated to the linker, then sought to evaluate its neurobiology and pharmacological behavior.

Results: We developed chemical synthesis methodologies for assembling these tripartite complexes using a variety of axonal transport facilitators including nerve growth factor, wheat germ agglutinin, and synthetic facilitators derived from phage display work. Loading of up to 100 drug molecules per complex was achieved. Conjugation methods were used that allowed the drugs to be released in active form inside the cell body after transport. Intramuscular and intradermal injection proved effective for introducing pharmacologically effective doses into selected populations of CNS neurons. Pharmacological efficacy with gabapentin in a paw withdrawal latency model revealed a ten fold increase in half life and a 300 fold decrease in necessary dose relative to systemic administration for gabapentin when the drug was delivered by axonal transport using the tripartite vehicle.

Conclusion: Specific targeting of selected subpopulations of CNS neurons for drug delivery by axonal transport holds great promise. The data shown here provide a basic framework for the intraneural pharmacology of this tripartite complex. The pharmacologically efficacious drug delivery demonstrated here verify the fundamental feasibility of using axonal transport for targeted drug delivery.

Figure 1

Tripartite delivery vehicle. (a) The drug delivery vehicles include a targeting element - the axonal transport facilitator or ATF (blue sphere), and a polymer such as dextran (red repeating units) that carry multiple drug molecules (purple). (b) They are designed to be injected in muscle or skin and then travel via an "intraneural" route to deliver the drug molecules to the cell body. (c) Late during transport and on arrival in the cell body, lytic processes release the active drug molecule by breakdown of the linker components.

Figure 2

Outline of conjugation and synthesis. A polymer such as dextran is first activated with cyanogen bromide to generate a carboxy modified dextran that can be conjugated to linker molecules attached at each monomer. The linkers are then conjugated with drug molecules. A second cyanogen bromide activation step is then carried out and followed by conjugation with an axonal transport facilitator molecule (ATF). An affinity purification step is then used to obtain drug loaded polymer complexes with ATF attached.

Figure 3

Effects of molecular charge on neuronal uptake and transport. (a) lack of uptake of WGA-dextran-FITC after carboxyl derivatization resulting in negative charge.(b) good uptake with neutral charge from 50:50 carboxyl and amine derivatization. (c) good uptake with positive charge from amine derivatization. Scale bar is 120 μm.

Figure 4

Axonal Transport of NGF and WGA in Campenot Chambers. Top panel shows a transmission image of the central and side compartments of a Campenot chamber. WGA-FITC or NGF-Texas Red were added to the side compartment and left overnight. At higher magnification, the "red series" of panels are fluorescent images obtained from the arrowed areas of the chamber following NGF-TR administration. Similarly, the "green" series of panels shows fluorescent images taken of the chambers following WGA-FITC administration. The left most panels in both series show uptake of fluorescence in cell bodies derived from the axonal transport of these labeled ATFs.

Figure 5

Axonal transport to spinal cord neurons (longitudinal). (a-c) Magnified, back illuminated view of an individual motor neuron in an oblique longitudinal section through a portion of the ventral horn of the spinal cord (Macaca fascicularis), and seen at lower magnification in figures (b) and (c). The dark orange material seen inside the cell and filling the cell body and dendritic processes is the product of a chemical reaction carried out by an administered enzyme, horseradish peroxidase. To introduce this exogenous enzyme into the cell, it was conjugated to WGA, an ATF (axonal transport facilitator), then injected into a muscle innervated by the axons which arise from these neuron cell bodies. Scale bars (a) = 50 μm, (b) = 200 μm, (c) = 400 μm.

Figure 6

Intra-axonal location of transported agents. (a) Electron micrograph of rabbit tibial nerve four days after injection of the gastrocnemius muscle with ferrite-WGA tracer. (b) magnified view of a vesicle seen in (a) to 195,000×. (ly) lysosomal vesicle, (fp) small particles transporting on microtubules (mi), (ep) larger particles in vesicles.

Figure 7

T2 relaxivity of hydroxide free magnetite preparations. T2 relaxivity curves for polyacrylamide "tissue" gels polymerized with uniform distributions of various dextran coated magnetite particle preparations. Relaxivity is compared with the concentration of particles in the each gel preparation as assessed by ferrozine assay of iron content after the imaging. 1/T2 was measured in a 4.7Tesla Sisco MR spectrometer. At concentrations comparable to what was achieved in nerve by axonal transport, a T2 below 30 milliseconds would be expected for any of these particle preparations. D10 = 10,000 MW dextran coating, D40 - 40,000 MW, D70 - 70,000 MW, Mgn = magnetite, WGA is wheat germ agglutinin, Seph II - sepharose separated to reduce contamination of magnetite by non-superparamagnetic ferrites.

Figure 8

Microscopic MRI evaluation of sciatic nerve magnetite contrast effect. Microscopic MRI image of sciatic nerve in calibrating gel chamber in anaesthetized rabbit. The experimental set up is detailed in Figure 19. The wall of the silastic cuff was opened and placed surgically around the sciatic nerve. Serial images with a 2 cm surface coil allows for measurement of the relative T2 intensity of the sciatic nerve by comparison with the T2 of the calibration gels in the three surrounding chambers with the elapse of 8 hours between the pre-injection image and the post-transport image. The injection was carried out immediately following the pre-injection image. The T2 of the sciatic nerve decreases relative to the calibration chambers as axonal transport of the WGA-dextran-magnetite agent progresses. Scale bar is 4 millimeters.

Figure 9

Median nerve contrast study by solenoid coil high resolution, high field MRI. All images are from a single image slice of rabbit upper arm. Image (a) is collected with a STIR (short tau inversion recovery) sequence which suppresses signal from fat - it reduces the marrow signal (6), and also identifies structure (1) as the median nerve[44], structure (7) as the ulnar nerve, structure (8) as the flow void of he brachial artery and structure 9 as the flow void of the basilic vein. Images (b) & (c)are colorized spin echo studies obtained at 90 minutes and 360 minutes after injection, respectively. Note that the marrow (3) appears shifted out the humerus (partially overlapped dark circle) by chemical shift effects. The shift at 4.7 Tesla is 1.85 millimeters. Similar shifts are seen at (4), and serve at (5) to leave two bright structures in a gap between triceps and biceps. (a-m), (b-m), (c-m) are magnified views of the space between the biceps and triceps on the medial aspect of the upper arm. Structure (2) is a small amount of fatty tissue that is actually located on the inferior left surface of the brachial artery, but chemical shift has placed its fat image into the midst of the basilic vein. Structure (2) disappears in the STIR image due to fat suppression. Based on this identification, the median nerve (1/1a) is compared to the non-neural structure (2/2a) and is seen to lose intensity in the four and half hour interval between images (b) and (c) reflecting transport of the WGA-magnetite contrast agent injected in the forearm flexor muscles [24]. The image conspicuity of this structure was measured by multiplying its volume times the intensity in grayscale and this reveals a decrease of 52% in the 270 minute interval. Scale bars are 6 mm for (a), (b), and (c) and 3 mm for (am), (bm) and (cm).

Figure 10

Axonal transport to spinal motor neurons and autonomic neurons. (a) Section of rat spinal cord showing retrogradely transported WGA-FITC in the motor neuron cell bodies (v) and in cells in the autonomic intermediolateral cell column (i). (b) magnified view of motor neurons seen in (a). Scale bars (a)= 120 μm, (b) = 30 μm.

Figure 11

Dorsal root entry zone access by transported agents. Section of spinal cord showing retrogradely transported NGF-TR (Nerve Growth Factor - Texas Red) in the dorsal horn. (a+b) The ipsilateral and contralateral dorsal horn viewed using darkfield microscopy. (c+d) The same fields as in a+b viewed using fluorescence microscopy. The arrow heads delineate the area of DREZ lamina I and II where the proximal axons of the dorsal root ganglia cells terminate on nociceptor neurons. Scale bars = 120 μm.

Figure 12

Axonal transport to dorsal root ganglion neurons. (a) Delivery to rat dorsal root ganglion cells from different peripheral sources. Section of L4 dorsal root ganglia showing retrogradely transported FITC (green) injected intra-muscular and TRITC (red) injected intra-plantar, in the sensory neuron cell bodies (fp - footpad injection, ga - gastrocnemius injection) (b) higher resolution view. Scale bars (a) = 170 μm, (b) = 45 μm.

Figure 13

WGA-dex-FITC transport to dorsal root ganglion cells. (a) Section of L4 dorsal root ganglia showing retrogradely transported FITC in the sensory neuron cell bodies. (b) The FITC can be seen in sensory dendrites arriving at the cell. Ax - axon, DRG - dorsal root ganglion, Ne - neuron cell body. Scale bars (a) = 150 μm, (b) = 80 μm.

Figure 14

Demonstration of transport to C-type nociceptor cells in dorsal root ganglion. Section of rat L5 dorsal root ganglia showing retrogradely transported FITC combined with immunohistochemistry for the specific C-fiber nociceptor marker, Peripherin. (a) - Retrogradely transported FITC, (b) - the same field as in (a) showing cells that are immuno-positive for peripherin, (c) - overlay of (a) and (b) showing FITC is present in the nociceptors (v - same neuron in a, b, &c). There are 26 cells seen with FITC in (a) versus 59 cells with peripherin in (b) & (c). (d) - Two sensory neurons containing retrogradely transported FITC, a third neuron is unlabeled, (e) - the same field as in (d) showing that all three neurons are positive for Peripherin, (f) - overlay of (d) and (e) (^ - same neuron in d, e, &f). Scale bars (a-c) = 150 μm, (d-f) = 40 μm.

Figure 15

Tissue distribution data for intramuscular [¹²⁵I]-WGA. Results are standardized by dividing all results for a given animal by the cpm/mg in blood for that animal. This results in a dimensionless figure proportional to concentration in tissue relative to concentration in blood for each tissue in each animal. Data are arrayed in chronological experimental sequence order as follows: (a) and (b) - incision with suture closure and 3 day survival; (c) - similar to (a) & (b) but longer survival time; (d) - superglue seal on the incision, animals kept in a metabolic cage; (e), (f), and (g) - the [¹²⁵I]-WGA was concentrated into one tenth the volume; (h) - large dose with 20 injection locations of 0.5 microliters each and longer survival; (i) and (j) - ten small injections; (k) - 10 gm-cm spinal cord injury; (l)- 25 gm-cm spinal cord injury; (m) - very long survival of 10 days (multiplied by 10 to emphasize very low retention).

Figure 16

Effect of gabapentin delivered by intraneural vehicle. The effect of Gabapentin tripartite administration, to mono-neuropathic animals, on thermal nociceptive threshold (con, contralateral; ips, ipsilateral; GPN, Gabapentin). Results are the mean ± SEM (n = 6). The difference between the ipsilateral and contralateral side is significant from 4 days post surgery to the end of the experiment in all treatment groups. Treatment with Gabapentin tripartite to the injured limb caused a significant elevation in paw withdrawal latency which was evident within 2 days and lasted for a further 4 days, after which the latency returned to that of the other groups (e.g. day 12, P = 0.0049 between Dx-GPN and GPN tripartite ipsilaterally). This effect was not observed in any other group.

Figure 17

Campenot chamber principles for compartmented cultures. The culture dish is coated with collagen, and then the substratum is scarified with a pin rake. A shaped Teflon gasket with silicon grease on its bottom edges is placed in the chamber over the scarified tracks. Dissociated mouse superior cervical ganglion neurons are then plated in the central chamber. Neurite outgrowths are then confirmed as they project into the adjacent chambers.

Figure 18

Magnified detail of cultured sympathetic ganglion cells. Ganglion cells growing in a compartmented chamber with an axon growing across the divider into the axon terminus chamber.

Figure 19

Microscopic MRI silastic cuff imaging set up. (a) The rabbit is positioned on gantry outside MRI scanner with surface coil over thigh. (b) Blow up shows the surface coil over the incision line. (c) The silastic cuff is shown in place on the tibial nerve in a cross section of the thigh. (d) The 9161 silastic cuff is shown with three outer channels for contrast gel standards and a center channel with access slit for the nerve to be imaged.