Intraneural Drug Delivery

Molecular Synthetics intraneural drug delivery technology is based on the natural phenomenon of axonal transport. This is the mechanism that makes it possible for a neuron in the spinal cord to determine the chemical contents of its distant nerve ending in the hand or foot. In tens of thousands of academic studies, axonal transport has been used to deliver anatomical tracer compounds. Molecular Synthetics is harnessing this potential for drug delivery.

Molecular Synthetics technology includes developments related to a wide variety of compounds. In many cases, the Molecular Synthetics vehicle is able to carry dozens of molecules of a useful medication into a nerve and deliver it at high concentration in the particular location in the nervous system where it will be most effective.

Axonal Transport

Introduction

Currently, medications are generally delivered through the bloodstream. This is the case whether they are administered by mouth, by intramuscular injection, or by an intravenous route. By nature ‘blood-delivered’ medications are distributed evenly through the entire body except where there are special exclusions such as the blood brain barrier.

Molecular Synthetics is developing a revolutionary class of drug delivery vehicles and medications that nerve enter the blood stream on their path to their target. Instead, these medications travel up the inside of nerves – the entirely novel ‘intraneural route.’ This is a feat that has been dreamed of by neuroscientists and pharmacologists for decades. Now, by applying its expertise and intellectual property position in nanoscale, supramolecular design, Molecular Synthetics has developed the technology that makes this dream a reality.

The resulting intraneural medications can accomplish tasks that no blood-delivered medication ever will. A single injected treatment at 1/10,000th of the oral dose can produce six days of near complete pain relief. Injection of medications of this class are expected to transform the way surgical patients are managed and also is expected to help solve many of the most intractable problems in pain management.

So, how is this remarkable technological feat accomplished? This overview is intended to provide a detailed scientific overview of the biological phenomenon of axonal transport on which these medications are based.

In a healthy nerve cell, a large fraction of all metabolic effort is expended in managing the intracellular movement of molecules and organelles. The nucleus of a motor neuron in the high lumbar terminus of the spinal cord, must manage events taking place in its synapses in e.g. a muscle in the foot nearly three feet away. The neuromuscular junction is dependent on the distant nucleus for much of its regulatory control and the nucleus needs information on chemical events taking place at the axon terminus. This remarkable intracellular informational and metabolic challenge is managed through a phenomenon called axonal transport. During the 1990’s it began to be apparent that magnetic resonance imaging could be capable of evaluating the physiology and pathology of axonal transport by the means of a novel class of intraneural contrast agents. That work led to fundamental discoveries about pharmacological aspects of axonal transport and its potential for therapeutic use.

There is strong evidence that interference with axoplasmic flow precedes interference with the transmission of electrical impulses or actual vascular compromise of the nerve.(1,2) Axonal transport continues in crushed nerves with accumulation of transported substances both proximal and distal to the site of injury(3) and so could be used to mark the location of the compression. Axonal transport is disordered in diabetic neuropathy(4,5) and amyotrophic lateral sclerosis,(6) there is at least reason to hope that clinical imaging of transport could aid in diagnosis and management. More importantly, however, it is now increasingly clear that axonal transport can be used to do the work of drug delivery for purely pharmaceutical effect. Treatable pathologies affecting the nerves, spinal cord, and the brain have typically presented severe technical challenges for the delivery of useful medications to the target sites of action. Axonal transport provides a powerful highway for transporting useful pharmaceuticals, nucleic acids, and other physiologically active compounds from easily accessible sites in the periphery to important targets behind the blood/nerve and blood/brain barrier.

There is an impressive array of compounds which have been developed for the detection of axonal transport and its sequellae in laboratory settings using agents intended only for animal experiments with histologic detection. In the 1990’s years, a number of groups(7,8,9) began to explore the development of clinically applicable analogs for the histologic tracer compounds. This effort remains at a fairly preliminary stage, but a number of promising early results have been reported.(10,11) This review is intended to lay out the cellular and biochemical background which underlies this body of work as well as to assess progress and prospects.

The work of SynGenix LTD in applying axonal transport to drug delivery to treat pain, relieve muscle spasm, and possibly to provide neuroprotection in a variety of conditions is an exciting and very recent development.

The History of Axonal Transport Studies

The slow movement of molecules along axons was first described nearly 40 years ago,(12) but with the proof of retrograde transport of proteins from axon termini in muscle back to the CNS,(13) an extensive research program began to emerge. The initial studies in adult animals were done using Evans-Blue labeled Albumin (EBA) and horseradish peroxidase (HRP).(14) At about the same time, Cowan et al(15) reported the use of radiolabelled leucine as an anterograde tracer for study of connections between nuclei in the CNS and it was subsequently demonstrated that HRP also underwent anterograde transport within the CNS.(16,17)

Neuroanatomists have since carried out thousands of studies involving the application of a tracer at one site followed 24 to 72 hours later by sacrifice of the animal, sectioning of tissue, and histologic identification of the axon termini and neuron cell bodies to which the tracers have been transported. The principal techniques used have been fluorescent studies of small dye molecules,(18,19) autoradiography of tritiated amino acids and proteins, and a variety of techniques involving HRP.

The biology of transport of HRP has been particularly well studied in the course of attempts to improve the technique. Native HRP enters damaged axon termini at injection sites, but is also taken up by “fluid phase endocytosis.” Once inside the cell, it becomes associated with vesicles that are transported towards the neuron (somatopetal transport) at rates up to 300mm/day.(20) It remains in the cell body for up to two weeks. Histologic studies use HRP as a catalyst for a peroxidase reaction to precipitate a chromogen, usually tetramethyl benzidine (TMB) as per a protocol introduced by Mesulam.(21,22)

Uptake of HRP at the cell body with subsequent anterograde transport was not demonstrated conclusively until Mesulam and Mufson(23) showed with ocular intravitreal injections that no injury to the neuron was required for uptake by retinal ganglion cells.

An improvement in sensitivity of 40 to 50 times is achieved by the binding of some plant lectins to the HRP molecule(24) with some variation in activity associated with the method used for conjugating the lectin to the HRP.(25) Selection of particular lectins can provide selective staining of different types of neurons,(26) however, the most effective and most widely studied of these is wheat germ agglutinin (WGA) derived from Triticum vulgaris. Unconjugated wheat germ agglutinin labeled with [125I] was shown to be a highly avid tracer,(27) and is actually up to 10 times more sensitive than WGA-HRP conjugates.(28,29)

Content continues below the following illustration section

Tripartite Drug Vehicles In Axonal Transport Stream: Tripartite drug vehicles include a targeting component (blue spheres) called an axonal transport facilitator (ATF), a long chain polymer such as dextran (red molecular chain), and numerous drug molecules attached to the polymer (yellow molecules). The drawing represents carriers wich actually can carry more than a hundred drug molecules in each tripartite complex. Complexes adhere to molecule in the synaptic membrane and are then carried in the axonal transport stream, along the microtubules, to reach the cell body.

Design and Assembly of Tripartite Drug Delivery Vehicles: Tripartite drug vehicles include a targeting component (blue spheres) called an axonal transport facilitator (ATF), a long chain polymer such as dextran (red molecular chain), and numerous drug molecules attached to the polymer (yellow molecules). The drawing represents carriers wich actually can carry more than a hundred drug molecules in each tripartite complex. Design of the targeting compounds is based on Molecular Synthetics breakthrough discovery technology

Delivery of Functioning Enzyme to Axoplasm Volume of Neurons: Results of early experiments on MSI technology conducted at Harvard University. These spinal cord motor neurons have been filled with an orange colored reaction product generated from an enzyme – horseradish peroxidase – that was delivered by an intraneural route. The enzyme was conjugated to wheat germ agglutinin (WGA) – a natural but non-physiological ATF (axonal transport facilitator). The complex is injected in to muscle, taken up into the nerve endings, and transported in transport vesicles. These vesicles do not destroy or digest the enzyme and it remains functional when this histological staining procedure was carried out days later. Note that the interiors of the cell bodies and their processes make up only a small fraction of the spinal cord interior.

Cross Sectional Anatomy of Nerve and Axon: A nerve is made up of several fascicles. Inside each fascicle is a large number of axons. The inside of the fascicle is protected from tbe blood stream by a blood/nerve barrier located in the perineurium. The axons them selves are wrapped inside the lipid laden Schwann cells which act as electrical insulation. The microtubules of the axonskeleton are inside the axon itself.

Nerve Components – The axoplasm inside the axon is only a small fraction of total nerve volume: The total volume of the cytoplasm of a nerve makes up only a small fraction of the total volume of a nerve. For this reason, the concentration of a drug in a nerve reflects as much two orders of magnitude greater concentration in the axoplasm itself.

Anatomy of Junction Between Nerve and Spinal Cord: A sensory neuron has its cell body in the dorsal root ganglion. One end of the neuron travels out to the periphery throught the peripheral nerve (dorsal and ventral ramus), while the other end travels into the spinal cord through the dorsal root. Inside the spinal cord, sensory neurons carrying pain signals (nociceptors) typically have a “synapse” just past the dorsal root entry zone where they pass their electrical signal on to another neuron. Other sensory neurons may continue up to the base of the brain before arriving at their first synapse. Motor neuron cell bodies are in the ventral horn of the spinal cord. The axons of these neurons travel outward through the ventral root.

Anatomy and size of spinal motor neurons: The nucleus inside the cell body of neuron must direct production of the various proteins needed by the cell. However, neurons have long axonal processes, so neurons rely on axonal transport to rapidly carry the molecules produced in the nucleus to where they are needed to do their work in the axon termins. Reverse or retrrograde transport takes place as well. A neuron can be an enormous single cell with an axon three feet in length.

Terminal expansion of an axon: An axon comes to an end at a terminal bouton. This is an enlargement where the neuron can respond to arriving electrical impulses by releasing neurotransmitter chemicals in to the synaptic space. Axonally delivered medications travel this route in reverse by entering the terminal bouton then traveling up the axon towards the cell body.

Molecular Mechanics of Axonal Transport: The axon is surrounded by the lipid laden insulation of the Schwann cells. Inside the axon there is a skeleton of microtubules. Dynein and Kinesin are energy driven motile protiens which pull molecules and vesicles along the axoskeleton and so make axonal transport take place.

Overview of Relevant Axonal Physiology

The physiology of axonal transport per se has been a very active area of research, with a literature that parallels and in some matters, overlaps the work on tracer methods carried out by neuroanatomists and histochemists and has been reviewed extensively.(30,31,32) Interest in transport includes studies on the mechanisms by which various substances enter the neuron, the rates at which various substances travel, and in the various skeletal and motile proteins involved.

Endocytosis

The ability of neurons to engulf large intact proteins was a surprise finding that grew out of early electron microscope work, and it was immediately apparent that this process involved the formation of membrane bound vesicles.(33)

In general, cellular endocytosis of receptor-ligand complexes involves a first stage event of creation of “endosomes.” The endosomes have a highly acidic pH which tends to dissociate the ligand.(34,35) In this sequence, the receptor is then recycled back to the cell membrane for reuse while the ligand is passed along to a lysosome for degradation. It is known that this sequence can be altered for e.g. transferrin when carrying colloidal gold. In this situation, the receptor and ligand remain complexed and are passed onto the lysosome as a unit.(36) This sort of unit, packaged in the lysosome, would then undergo fast retrograde transport in the neuron such as is known for WGA-HRP.

Retrograde transport of exogenous molecules apparently involves three distinct mechanisms of intake into the cell. Fluid phase endocytosis and adsorptive endocytosis as observed with HRP and WGA-HRP respectively both result in these substances being introduced into lysosomes.(37) The third mechanism, transcytosis(38,39) is slower than normal retrograde transport, takes place in a limited compartment immediately beneath the axolemma, and differs from other mechanisms in that the receptor and ligand are not subject to lysosomal digestion during transport. [125I] WGA has been used to study this route, and it also seems to be the route taken by NGF with NGF receptor and by lofentanil bound to the opiate receptor.

Rates of Transport

It has been clear for many years that the rate of transport of a given substance is independent of electrical activity within an axon.(40) It was also appreciated early on that different classes of substances moved at different rates.(41,42)

Anterograde axonal transport has been shown to have a major fast and a slow component(43,44). The slow component is divided into “slow component a” and “slow component b” at rates of approximately 1 and 3 mm/day respectively. These slow components apparently reflect gradual structural repair and replacement of the subunits of the cytoskeleton and are not involved in the fast components important for tracer studies.(45,46)

The fast component of transport demonstrates distinct maximal rates for anterograde (300-400mm/day) and retrograde (150-300mm/day) transport. The maximal rates of transport apply to small membrane vesicles. Further, there are a variety of “waves” or distinct sets of slower transport rates exhibited in characteristic fashion by various molecules.(47,48) Rates of progression and spreading of wave fronts have been assessed using pulse labeled [35S]-protein in rat sciatic nerve subsequently excised, and “desheathed” for measurement with a linear array of proportional ß-counters.(49,50)

Cytoskeletal mechanisms

There are three major cytoskeletal elements in the axon: 1) microtubules made up of alpha and ß-tubulin monomers, 2) microfilaments composed of actin monomers, and 3) neurofilaments which are a type of intermediate filament composed of three distinct subunits.(51) A variety of studies involving intact and skinned axons with specific antibodies and various specific poisons and depolymerizing agents have proven that it is the microtubules which are the most directly involved in axonal transport.

The various cytoskeletal elements are effectively stationary in the axon.(52) Anterograde transport involves the packaging of various proteins into the membranes of transport vesicles. In anterograde transport, these vesicles and similarly sized mitochondria are pulled along the microtubules by movable bridges of “kinesin” at a characteristic rate.(53,54,55) The various slower rates of transport in anterograde motion appear to reflect molecules that either bind poorly with kinesin or that pass in and out of the vesicles during transport. Retrograde transport moves along the same microtubules but the driving protein is dynein which operates in a direction opposite to kinesin(56,57) (see figure 1).

All of this movement is ATP and calcium dependent. The metabolism involved is local, i.e., mitochondria bound to the axolemma as well as mitochondria being transported on the microtubules use glucose and oxygen absorbed through the cell membrane along the axon to generate ATP locally.

When a nerve is ligated, transport continues for many hours both proximal and distal to the ligation. Transported vesicles, proteins, and mitochondria pile up at the ligation site. Some signal which turns around and heads back to the cell body then causes anterograde fast transport to halt.(58,59) This situation continues until a critical concentration of cytoskeletal subunits accumulates via slow transport and at that time, axonal growth begins.

Physiologic Axonal Transport Facilitators

Axons will transport a tremendous variety of substances, several of which are relatively well studied. The earliest view into these was provided by studies of amino acids which are incorporated into proteins in the cell body and then transported anterograde. However, there are a variety of cell surface receptors that first bind ligands and then are endocytosed and transported anterograde or retrograde. When these endogenous ligands are conjugated to exogenous substances, the ligands act to facilitate transport of the exogenous substance via an unique and specific intraneural pathway.

Trophic Factors

The uptake and retrograde transport of Nerve Growth Factor (NGF) by adrenergic sympathetic neurons was first demonstrated by Hendry et al(60) by injection of [I125]-NGF into the anterior chamber of the eye with subsequent transport to the superior cervical ganglion. The same group also showed that NGF is transported by sensory neurons after injection into the forepaw,(61) but is not taken up or transported by motor neurons.(62) Similar studies with an antibody to dopamine ß-hydroxylase showed uptake and transport by adrenergic neurons only.(63)

Studies of the kinetics and transport capacity of NGF(64,65,66) have demonstrated that various NGF receptor molecules are responsible for the transport specificity. A monoclonal antibody (“192-IgG”) raised against an NGF receptor on pheochromocytoma cells has made it possible to show that the NGF molecule binds to the receptor protein and that the entire complex is then transported up the axon to the cell body.(67) Studies intended to demonstrate NGF localization during transport and within the cell body have shown that i.e. NGF-HRP conjugates are also endocytosed without loss of the specificity and high transport capacity of NGF.(68) Other neurotrophic factors such as CNTF (ciliary neurotrophic factor),(69) BDNF (brain derived neurotrophic factor), and NT-3 (neurotrophin-3) are transported in similar fashions but provide access to different subsets of axons.(70)

Receptors and neurotransmitters

Another interesting ligand/receptor complex involves [3H]-lofentanil and the opiate receptor which are endocytosed and transported by sensory neurons.(71,72) PET studies with [11C]-carfentanil(73,74) have been used to assess the general distribution of opiate receptors, but this approach has never been tried as a means of tracing selected tracts via axonal transport. Similar studies with GABA, D-aspartate, dopamine, norepinephrine, and serotonin have shown that uptake and transport of some neurotransmitters is a widespread phenomenon in the CNS(75,76) as is the transport of receptors.(77)

Acetylcholinesterase uptake and transport has been studied for many years because of its ease of use as a histochemical marker.(40,78) Other studies have demonstrated transport of a wide variety of substances including Vasoactive Intestinal Polypeptide (VIP),(79) cholecystokinin,(80) substance P and somatostatin,(81) and neuropeptide-Y.(82)

Non-Physiologic Transport Facilitators

The various plant lectins bind to complex carbohydrates on the cell surface of axon termini and cell bodies and, facilitate uptake by adsorptive endocytosis.(83) Most lectins have some degree of local cytotoxicity, indeed, some studies make use of the severe toxicity of lectins such as ricin and abrin to produce chromatolytic degeneration of neurons after labeling(84,85) in studies of what is called “suicide transport.” The uptake and subsequent retrograde transport of WGA in all types of peripheral neurons was first demonstrated by Stoeckel et al.(86) A variety of fluorescent small molecules such as adriamycin(87) and fluoro-gold have also been identified as subject to efficient transport.(88,89)

Binding fragments of some bacterial toxins have proven to be even more effective than WGA for increasing adsorptive specificity of conjugated HRP labeling. Both the B-chain of cholera toxin,(90,91) and the C fragment of tetanus toxin(92,93) have been used extensively.

Yet another set of studies worth mentioning involves the use of neurotrophic viruses such as Herpes Simplex,(94,95) and rabies virus(96,97) as tracers, in some cases with cDNA probes used to locate the endpoint of transport.(98)

Monoclonal Antibodies as Transport Facilitators

Using a WGA affinity chromatography column, Fabian et al(99) isolated axonal fragments that bind this lectin and used these fractions to generate polyclonal antibodies against WGA-binding glycoproteins. These antibodies, and other anti-synaptosomal Ab’s(100,101,102) were transported retrograde only. Subsequent work with monoclonal antibodies(103) identified MoAb’s subject to both anterograde and retrograde transport, with different MoAb’s following different routes of intracellular processing and transport. Various MoAb’s may also be directed to specific sets of neurons to further increase specificity of uptake.(104) Ritchie et al(106) found much greater staining of motorneurons after uptake and transport of intramuscular injections of anti-synaptosomal (polyclonal) antibodies than after WGA injections. Exogenous Ab’s administered IV or intraperitoneally are taken up by peripheral axons throughout the CNS.(105)

Transneuronal transport

Another important phenomenon is transneuronal transport wherein a transport facilitator permits a tracer to be extruded back onto the cell surface after transport thus acting to produce a sort of second injection at the next synapse in the chain.(106,107) Tetanus toxin appears to move in a specifically transsynaptic fashion,(93) but WGA and WGA-HRP are found in glia after anterograde transport of WGA-HRP,(108) and synaptic structures therefore need not be involved.

The detailed mechanisms of transneuronal transport of WGA-HRP after retrograde transport are still not completely clear, but Harrison et al(109) demonstrated that such transport did take place, and that it seemed to be enhanced by neuronal activity. Subsequent studies(110,111) have taken advantage of this phenomenon in attempts to identify interneurons involved in central pattern generators for various stereotypic movements.

Of the various transport facilitators, tetanus toxin is the most effective for “transsynaptic” labeling in which the next neuron in a synapsing series is also labeled(112) and HSV-1 has been useful for multi synapse transneuronal studies.(113,114)

Quantitative Aspects of Uptake and Transport

Margolis and LaVail(115) sought to demonstrate quantitatively the nature of anterograde transport of the WGA molecule. They performed intravitreal injections of [125I] -WGA and assessed delivery of tracer to the optic tectum. It was possible to reduce transport by 75% with excess native WGA at 1.1mM. This made it possible to calculate that the transport capacity was for 550 fmoles of WGA per 24 hours. Estimating 2.6 x 106 ganglion cells in the system, this represented 5,000 molecules of WGA transported per cell per hour. Their previous study(116) demonstrated that approximately 85% of injected [125I]-WGA arrives in the optic tectum as the same 18,000 dalton molecule injected 25 hours earlier.

Access to Transport Stream from Intra-Muscular Injection

Many studies of motorneuron organization have been carried out by the application of native HRP directly to isolated muscular branches of peripheral nerves.(117,118,119) Brushart and Mesulam(120) demonstrated the effectiveness of intramuscular and intradermal injections of WGA-HRP, attributing the effect to improved uptake and higher axon transport capacity than for native HRP. However, in an attempt to achieve distinct labeling of motorneurons in small, closely juxtaposed muscle fiber groups, it has been noted that WGA-HRP remain tightly bound at the site of injection providing impressive control of injection site specificity.(121,122,123,124) It was also found in these studies that extremely small quantities of WGA-HRP could be used without diminishing central labeling.

Injections into muscle or near sensory nerve endings work because the axon terminus presents a breach in the blood brain barrier. The perineurium, which generally is the site of the barrier in peripheral nerves, does not quite extend onto the bouton of the neuromuscular junction. Further, many axons branch extensively as they enter muscle, thus providing a relatively very large surface area which, because of the organization of motor units, may be distributed quite extensively throughout a given muscle.

Intravenous administration of tracers/transport facilitators leads to uptake at neuromuscular junctions throughout the body (125) and via brain regions which have an incomplete blood brain barrier.(126,127)

There are no quantitative studies of intramuscular uptake comparable to the anterior chamber and intravitreal studies noted above, but several aspects of the phenomenon are explained by muscle studies. In the same year that Stoeckel et al(86) discovered neuron uptake of WGA, Barchi et al(128) found that WGA had a great specificity for muscle cell membranes (sarcolemma). In addition, DeSantis and Paul(129) have shown high affinity for various parts of the muscle spindle. Local non-neuronal binding may also serve as a continuing fixed pool which continues to provide e.g. WGA for neuronal uptake for hours or days after the initial injection, thus helping to avoid limitations due to saturation of uptake receptor sites on the axon termini.

There is substantial evidence that intramuscular injections can yield high intraneural concentrations of tracer with minimal spread away from the injection site.(127) Only very recently has it become clear that the efficiency of uptake of axonal tracers after intramuscular injection is sufficient to permit consideration of clinical uses. Preliminary reports appearing during 1991 reveal that labeled proteins amenable to nuclear medicine imaging can achieve useful concentrations in nerve after intramuscular injection.(130)

Design Considerations for Intraneural Agents

Information derived from the effort to develop intraneural contrast agents for magnetic resonance provides several key findings relevant to design factors for intraneural agents in general. The bulk of MRI enhancement research in neuroimaging has involved small molecules, mostly gadolinium-DTPA, and has been based on interruptions in the blood brain barrier which permit selective enhancement of tissues involved in pathologic processes.(131) Attempts to explore the use of magnetite as a superparamagnetic enhancement agent for MRI(132,133) had been directed towards developing an intracellular agents for hepato-biliary work,(134,135) oral contrast for GI studies(136,137) and selective antibody based labels for tumors.(138,139)

An MRI tracer must have a powerful effect on proton relaxivity to be detectable in tracer quantities and gadolinium would require concentrations much greater than can be achieved intraneurally by IM injection. This is because uptake events are essentially saturable. However, a single magnetite particle can have an impact on relaxivity that is orders of magnitude greater than a single molecule of gadolinium DTPA.(140,141,142) Since the number of uptake events at the neuromuscular junction limits the amount of tracer which can be introduced into the transport stream, several groups have sought to develop magnetite based labels for axonal transport imaging, including studies with the highly effective axonal transport facilitator WGA(143) conjugated to the superparamagnetic particle. In this case, use of a ferrite contrast material with 100,000 times greater imaging effect per unit than a gadolinium agent effectively amplifies the effects of the limited number of uptake events.

Most histologic studies of axonal transport used small molecules or proteins, however, it has proven possible to use several types of particulate tracers such as colloidal gold,(144) iron dextran,(145) and fluorescent labeled latex microspheres with diameters ranging up to 200 nm.(146) Natural particulate substances in the 80-120nm size range(147) as well as larger organelles are transported(148) physiologically.

A study by Colin et al(149) with particles found that nerve injury is required to permit entrance and transport of the particles. However , the injections undertaken in that study were not intramuscular and did not involve attachment of an axonal transport facilitator. Hollander et al(150) have demonstrated uptake of latex microspheres up to 50 nm in diameter in intact cultured sympathetic neurons.

Brady(8) reported transport of MR detectable particles after direct injection into the sciatic nerve and after the completely severed sciatic nerve was soaked in a gel with ferrite particles. Ghosh et al(7) reported evidence of transport of ferrite particles after direct pressure injection into the brain of a frog although no MR detection was achieved. Enochs et al(10) report slow transport of ferrite particles after DMSO assisted intraneural injection, but could not achieve MR detectable quantities of transport with intramuscular injection.

Particles such as colloidal gold histology labels can be endocytosed and transported by nerves after intramuscular injection,(151) and it has been known for many years that dextran coated iron oxide particles are transported after intramuscular injection.(152) It has now been reported that biodegradable superparamagnetic particles, made by a modification of the Molday and MacKenzie(153) method, can also be endocytosed and transported by nerves after intramuscular injection with intraneural concentrations sufficient for detection in magnetic resonance images.(9) However, the relative concentration in spinal cord relative to nerve was much lower than was achieved with unconjugated WGA alone.(130)

Substantial amounts of some transported axonal tracers are actually deposited at nodes of Ranvier and not transported the entire distance up the nerve to the cell body(154) and there is evidence that iron dextran particles are diverted into the myelin sheath in the CNS.(155) These phenomena may account for the predominantly axonal rather than neuronal distribution and may be critical in setting a limit due to toxicity of degraded iron oxide. However, this predominantly neural rather than central distribution is preferable for the requirements of nerve imaging.

This work is relevant to broader issues affecting intraneural pharmaceuticals. It demonstrated that even large molecular complexes ranging up to 50 nm can be endocytosed and transported. Further, it demonstrates that amplification of the uptake events by delivering particles with thousands of iron atoms instead of just one gadolinium atom is an effective and desirable strategy.

Prospects for Clinical Use

Development of the imaging agents continues to be at a very preliminary stage, however, very recent advances in therapeutic drug delivery at Molecular Synthetics have now virtually assured that axonal transport will play a major role in pharmacological therapy in the new millennium.

It is now clear that small molecule drugs can be delivered in highly efficacious quantity into the interior of selected nerve groups. When pain medicines are delivered in this fashion, the result is highly effective analgesia lasting several days after a single injection using amounts of drug that are orders of magnitude less that what is required for systemic application. Intraneural drug delivery promises to usher in a new era in pharmacological therapy in which selected targets in the nervous system can be treated effectively with a wide variety of types of drugs with out the systemic and nervous system side effects which have plagued neurological pharmacology in the past.

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