BACKGROUND
the direct distribution of medications to specific target tissues in the central nervous system  is an attractive objective for the treatment of problems such as local pain or dermatomal viral infection.
when systemic drug delivery is used to treat a symptom affecting a small segment of the nervous system  conventional drug delivery methods can be highly inefficient because they place most of the dose in non-neuronal tissues throughout the body and in areas of the cns that do not need treatment.
dose limitation that hinders efficacy often arises from the concentration of the drug in non-target tissues.
for many compounds, this situation is aggravated because access to tissues of the central nervous system is also compromised by the relative impermeability of the blood brain barrier.
the nervous system itself provides an alternative to the vascular system or the cerebro-spinal fluid  - the intra-axonal route.
however, this has proven difficult to exploit.
methods using direct injection into nerves  <cit> , introduction of modified neurotropic viruses  <cit> , or delivery of neurotoxins such as tetanus toxin or ricin  <cit>  have been explored but each pose barriers to routine clinical use.
direct injection may damage nerves, neurotropic viruses expose the recipient to direct damage or the risk of recombination with wild type virus, and toxins for nerve destruction have limited clinical roles.
phage display has been used to generate synthetic peptides to promote axonal transport  <cit> , but it has not been clear how to exploit this.
retrograde axonal transport of exogenous molecules from the periphery to cns neuronal cell bodies is long established  <cit>  and is one of the methodological bases for mapping neuroanatomical pathways  <cit> .
the underlying physiological basis of this process and its biochemistry are increasingly well understood  <cit> .
however, although axonal transport for drug delivery has been proposed previously  <cit> , little or nothing is known about the major features of this biological system from the point of view of approaching it pharmacologically and there has been no agent demonstrated capable of accomplishing a pharmacological effect via this route.
pharmacological entrainment of axonal transport is acknowledged as a highly desirable objective since it would allow for targeted antiviral, antineuropathic or regenerative treatments to segmentally selected ganglion or cns cells.
we sought to clarify the pharmacology of intraneural drug delivery and to design agents capable of accomplishing therapeutic tasks by clinically applicable axonal transport methods employing intramuscular injection and subsequent uptake by intact nerve endings.
we designed a novel tripartite molecular complex to provide a flexible architecture capable of achieving this.
we now report on this class of pharmacological agents for selective targeted access and transport through the nervous system and demonstrate that it is capable of achieving therapeutic efficacy.
the system provides a versatile platform for many different therapies with the same targeting requirements.
the agents used are various forms of a tripartite complex composed of a first moiety acting as an "axonal transport facilitator" , an "amplifying polymer"  second moiety acting to achieve amplification of the fundamental event of synaptic endocytosis by carrying along multiple drug molecules with each saturable uptake event, and a third moiety composed of multiple copies of the therapeutic molecule, reversibly linked to the polymer .
this novel molecular architecture allowed us to independently explore and optimize general features and constraints affecting the three major aspects of the problem - a) chemical synthesis, b) optimization of uptake and transport, and c) intraneuronal drug efficacy after transport.
we used a comprehensive array of biological, physical and chemical assays in vitro and in vivo to characterize and optimize the system.
the studies are numbered  <cit>  to  <cit>  corresponding to the summarized results table .
a) we studied the synthesis and stability of the tripartite and its components addressing the following questions:  can tripartite molecules be constructed chemically to preserve efficient adsorptive endocytosis when loaded with large numbers of conjugated drug molecules?
what are the upper size limits for the tripartite complex?
can pharmacologic activity be preserved for small molecules released from the tripartite complex under intracellular conditions?
b) we analyzed interactions with axon terminals and the intra-axonal environment that would affect design of the tripartite by investigating the following questions:  - how do polymer size, molecular charge, and hydrophilicity affect the efficiency of uptake into neurons?
- what is the relative efficiency of physiologic atfs  when compared to non-physiologic atfs for uptake and transport?
can purely synthetic atf's be produced by phage display techniques that will be more effective than physiologic atfs?
will use of colchicine - an inhibitor of axonal transport serve to verify interpretations?
can a small intramuscular or intradermal injection cause significant delivery into a selected set of neurons via intact uninjured axon terminals in normal tissues?
can this clinically usable method of drug administration achieve high intra-neuronal drug concentrations when compared to the "cut neuron" methods used to introduce axonal transport tracer molecules in some anatomical studies?
during axonal transport, if the pharmacologic agents are sealed inside vesicles, will the drug or carrier be damaged or destroyed by lysosomal activity while traveling long distances in the intracellular space of the axons?
c) we investigated targeting and pharmacological efficacy:  - which classes of clinically useful sub-targets in the nervous system can be reached by clinically convenient administration techniques?
- what are the unique features of the whole body pharmacologic distribution of intraneuronal agents and how do size of the molecular complex and selection of atf affect the distribution?
can pharmacologically efficacious doses of drugs be delivered and are they functional when delivered to the interior of a cell rather than to its exterior surface?
using a tripartite construct designed according to the results of the investigations outlined above, we were able to administer a small intramuscular injection with 1/300th of the usual oral dose of gabapentin  and achieve a degree of neuropathic pain suppression that could not be obtained with tolerable amounts of oral therapy while extending the half life of the drug by more than an order of magnitude.
RESULTS
results are summarized in table 1
chemical synthesis and stability
chemical entities
effective synthesis with loading sufficient for amplification
the synthesis strategy  achieved attachment of drug to at least 30% of dextran monomers for both gabapentin and valacyclovir.
the  <dig>  mw dextran has approximately  <dig> dextrose sub-units so each wheat germ agglutinin  molecule endocytosed in these experiments delivered around  <dig> molecules of drug.
particle based carriers can be delivered intraneuronally
edta washing of dextran coated magnetite particulates resulted in hydroxide free ferrites that were non-reactive and preserved their superparamagnetic properties during axonal transport.
after conjugation of an axonal transport facilitator to the dextran coat, they were effectively endocytosed and transported intact despite their  size .
intracellular release and drug activation
the stability of a drug is maintained through chemical linkage and biological release
in the bhk  viral plaque reduction experiments, free drug in the culture medium resulted in plaque reduction by 96% at a dose of  <dig> micrograms/well, but a dose of  <dig> micrograms/well of the drug bound to dextran was required to achieve an equivalent effect.
however we observed nearly 100% plaque reduction using a dose of  <dig> microgram/well of tripartite wga-dextran-drug conjugate.
this demonstrates that the wga efficiently promoted endocytosis of the tripartite agents while dextran-drug alone was not readily taken up by cells.
on chemical grounds, the carboxyl links binding drug molecules to dextran should be hydrolyzed after endocytosis.
for valacyclovir , the product of the hydrolysis is free and active acyclovir.
therefore, the antiviral effect of the tripartite confirms release of acyclovir from the tripartite carrier after endocytosis.
interactions with axon terminus and axonal processing
effects of polymer, linker and drug
effects of polymer size on tripartite uptake into nerves
for the two dextran sizes tested, the uptake was faster for the smaller 10k molecular weight dextran than for the larger 70k molecular weight dextran.
however, uptake of the 70k molecule was more effective from the point of view of drug delivery.
this is because each 70k dextran delivered more attached drug molecules .
with 10k dextran, there is extensive uptake within ten minutes after application and washing.
similar levels of uptake are seen with 70k dextran after  <dig> minutes.
however the differential efficacy of uptake was not large enough to outweigh the benefits of greater amplification of uptake events achieved by using the higher capacity larger molecules for drug delivery.
since molecular loading efficiency of the polymers was equivalent for various sized dextrans, the increased drug delivery with increased molecular size was linear.
however, the losses in uptake efficiency with larger molecules was not severe enough to make them less effective overall.
the consequence is that although smaller dextran molecules were endocytosed more avidly and rapidly than 70k based vehicles, more drug molecules were still delivered by the 70k based vehicles if a full hour was allowed to elapse after introduction of the test agent.
larger molecule sizes were not tested.
effects of charge on uptake and transport
the overall charge of the complex had a large impact on efficiency of uptake .
there was virtually no uptake into cultured neurons when the overall charge of the complex was negative, and effective uptake occurred when charge was neutral, or when overall charge was positive.
this finding parallels the natural movement of positively charged ions into the negatively charged internal milieu of the cell during depolarization.
similar charge findings have been reported for liposomal drug delivery systems  <cit> .
effects of side group mediated hydrophobicity on uptake and transport
acylation of fitc -labeled, wga-conjugated dextran to make the molecules progressively hydrophobic led to complete failure of uptake and transport.
we assessed various degrees of acylation and found that this effect occurred even at very low ratios of acylation.
effects of axonal transport facilitator
efficacy of physiologic, non-physiologic and synthetic atfs
the campenot chamber studies demonstrated similar efficacy for nerve growth factor  and wga for promoting uptake of the tripartite carrying fitc .
the phage display experiments demonstrated that novel purely synthetic atfs could be discovered and then produced in vitro which not only had equivalent efficiency for neuronal uptake, but which could also be more efficiently sub-targeted to different types of neuronal populations .
when colchicine - an inhibitor of axonal transport - was added to campenot chambers, it blocked intra-axonal movement of molecular complexes whose atf was derived from purely synthetic phage display techniques.
this confirms that the drug transport effect produced by the synthetic phage display products achieved delivery by axonal transport.
effects of intra-axonal processing
survival of small chemical molecules linked to the tripartite
when wga-dextran-gabapentin was used, cross staining with antibodies to gabapentin confirmed preserved antigenicity of the drug after axonal transport of the tripartite, although this method could not itself confirm that the drug was intact or active after transport and release.
dextran-gabapentin with no conjugated wga did not produce detectable gabapentin antigenicity in the histological sections.
large molecule access to spinal cord from intramuscular injections
after small im injections in muscle, wga delivered sufficient quantities of functioning hrp enzyme to label numerous motorneurons  in our primate model.
enzymatic activity of the horseradish peroxidase survived any hypothetical lysosomal degradation during transport and yielded product distributed throughout the neuron.
both epaxial and hypaxial muscles proved an effective route to reach spinal cord quickly  with no apparent difference in delivery of intact enzyme via axons despite the difference in distance of transport.
the labeled spinal cord motor neuron pool for these axial muscles had greater longitudinal extent among spinal cord segments than for appendicular muscles.
these findings are consistent with previous reports concerning wga  <cit>  and ngf  <cit>  and their intramuscular introduction  <cit> .
the rate of transport appeared to be consistent with time scales predicted by literature values of 30- <dig> mm/day  <cit> .
overall the area of spread of injectate in the spinal cord was limited to a few millimeters, although the distribution of the epaxial muscle spinal cord motorneuron pool extended over several centimeters.
this appeared to demonstrate that a group of neurons near each other in the spinal cord innervated a group of fibers in epaxial muscle that are close together despite the wider extent of the overall motor neuron pool.
intact transport of targeted nanoparticles by intact axon termini
electron microscopy showed appropriately sized ferrites within the axon, more than three centimeters from the wga-dextran-fe injection site .
the appearance of the ferrites in endosomes was similar to what has been seen in studies of similar agents in other tissues  <cit> , but in this case the particles were restricted to the axonal stream.
the autoradiographic studies demonstrated sciatic nerve radioactivity and so provided similar evidence that the magnetite containing particles were transported in nerves.
the relaxivity experiments  taken together with distribution studies showed that the concentration of magnetite delivered to the axon by the tripartite was sufficient to affect the t <dig> relaxation rate of nerve.
the observation of a decrease of t <dig> relaxation time in nerves transporting superparamagnetic nanoparticles in both the micro-mri nerve channel studies  and in the high resolution mri experiments  confirmed that the carrier particles were not degraded.
any hydrolysis of the sub-domain sized particles would have eradicated their superparamagnetic effect on t <dig> relaxation time in nerve as transport progressed.
the relaxivity effect far exceeded that which would result from free iron or ferritin at the doses administered.
the wga-dextran-magnetite experiments in rabbits confirm other reports  <cit>  that particles of 5- <dig> nm are endocytosed and transported by intact nerve endings.
some studies have suggested that nerve injury is required for the transport of larger particles  <cit> , but this result suggests that intact neurons will indeed transport large  multi-molecular aggregates or "transport particles" after intramuscular injection when the particles are well solvated.
c. targeting and pharmacological efficacy
clinical target access
targeted access to clinically relevant neuronal sub-populations
intramuscular injection of the tripartite wga-dextran-fitc produced labeling of alpha motor neurons in the ventral horn and autonomic neurons in the intermediolateral cell column .
we also observed good filling of proximal sensory neuron processes  in the dorsal root entry zone and in lamina i and ii of the dorsal horn of the spinal cord .
injection of foot pad and multiple hind limb muscle each resulted in labeling of less than 50% of ganglion cells, but injection of both muscle and skin resulted in filling of nearly 90% of dorsal root ganglion  cells .
in clinical use, the objective will typically be to reach specific sub-populations rather than filling an entire ganglion per se, so these results support the expectation that a large fraction of cells in a e.g.
a subpopulation innervating a single muscle or patch of skin can readily be reached.
cross staining with an antibody to peripherin showed that many of the drg sensory neurons that were accessed were c-fiber nociceptor cells .
this helps confirm that this method of delivery does reach a selected subset of nociceptors that correlates specifically with the selected site of injection.
the atf played a major role since no detectable fluorescence was observed when dextran-fitc without atf was administered in these experiments.
distinctive pattern of distribution relative to trans-vascular
unique distribution with high fraction of drug reaching neuronal targets
in the whole body distribution studies with small calf muscle injections, the concentration of -wga detected in relevant ipsilateral peripheral nerve and dorsal root ganglia reached six times systemic concentrations .
the detected concentration in relevant nerve and ganglia  demonstrated saturability and transport time consistent with other reports  <cit> .
activity in spinal cord was less than in nerve or drg but did reach twice systemic concentrations when higher concentrations of injectate were used.
there was no significant activity detected in contralateral nerve and drgs nor in ipsilateral nerves, ganglia, or spinal cord related to cervical, thoracic, or higher lumbar segments.
the greatest activities detected in other tissues were in liver and kidney but these apparently reflected metabolized iodine lost from the compound.
ngf showed less transport to spinal cord than was seen for wga consistent with selective uptake of ngf by sensory and autonomic nerves.
detection of activity due to -wga-dextran at other systemic sites was even less than for wga or ngf alone.
considerable amounts of drug remained in the axon itself at the time points sampled.
it is known that para-nodal complexes of schwann cells at the nodes of ranvier can endocytose materials from the axoplasm  <cit> .
this may account for a peri-axonal depot effect with drug being cleared to the para-nodal complexes and then subsequently being re-released.
further studies will be needed to clarify this issue.
site of injection retains high amount of injectate
the regional view provided by the -wga gamma camera studies demonstrates that the total amount of the injectate remaining at the site of injection can be large when compared to the amount in the small volume of the nerve.
in part, this reflects the fact that wga adheres to muscle cell membranes  <cit>  as well as being taken up by adsorptive endocytosis at nerve termini.
this also explains a depot effect whereby additional amounts of the compound continue to be introduced into the axonal stream over a period of several days.
pharmacologically efficacious drug amounts were delivered
the tripartite assemblage with -labeled gabapentin produced activity levels for gabapentin in ipsilateral neurons of more than  <dig> times greater than background while counts remained at background levels in contralateral neurons.
this demonstrated the localizing effect of injection of polymer-bound drug conjugated to an axonal transport facilitator, and also allowed us to estimate the drug concentration achieved based on the specific activity of the -labeled gabapentin.
pharmacologic effects of atf mediated delivery not obtainable by trans-vascular agents
prolonged suppression of hyperalgesia
in the hyperalgesia experiments a single injection with the tripartite agent reduced the hyperalgesia  for at least four days after injection .
the total dose was  <dig>  mg/kg using the novel intraneural transport method compared to  <dig> mg/kg total dose orally for four days of the usual human oral dosing.
prolonged efficacy may have been due to a depot effect in muscle, slow redistribution from axon and schwann cells to nerve cell body, slow clearance from inside the nerve, or preemptive blocking of newly produced receptor molecules en route from ribosome to cell surface.
discussion
we have demonstrated through a comprehensive series of chemical, radiological in vitro, and in vivo biological studies that axonal transport is a valid route for drug delivery when a tripartite  complex is used.
we have used these studies to exemplify in vivo a prolonged pharmacologically effective delivery of an analgesic molecule leading to sustained significant response in a standard nociceptive model.
routine oral delivery of gabapentin results in clinical efficacy for pain relief when blood levels reach  <dig> μm.
gabapentin not only crosses the blood brain barrier but is subject to some active concentration  <cit> .
it is reasonable to hypothesize, therefore, that clinically efficacious axonal transport for intraneural drug delivery would need to produce similar concentrations near the internal cell surface of accessed neurons to achieve similar pharmacological efficacy.
in this model, histological estimates of the ratio between the estimated intracellular volume of the accessed neurons  and the actual volume of the entire dorsal root ganglion  provide a correction factor  that allows for conversion of total drg concentration into estimated intracellular concentration in the accessed neurons.
when these considerations were employed, our counts of delivered  suggested delivery of gabapentin at intracellular doses equal to or greater than  <dig> μm.
these in vivo -gabapentin studies therefore showed that this technology can deliver drugs to target neurons at pharmacologically efficacious doses using 1/300th of the oral dose that would be required to achieve the same intracellular concentration in target cells.
in addition, this system resulted in a ten fold increase in effective half life relative to oral administration.
this appears to be because of the unique dynamics of this delivery mechanism.
this involves both a muscle injection site depot effect for the large tripartite complexes and a prolonged stream of delivery to the neuron from the axon and its associated paranodal complexes.
use of the tripartite vehicle made it possible to use a selected axonal transport facilitator to cause transport of large numbers of drug molecules and to recover the pharmaceutical effect of the drug after delivery to its target.
by varying the polymer backbone length and by use of various linker chemistries, it is possible to accommodate therapeutic molecules with a wide variety of different sizes and physico-chemical properties.
in this set of experiments, neither systemic delivery of the agent by a subcutaneous route nor administration of free drug alone to the affected limb had any significant therapeutic effect.
although dextran can itself weakly promote axonal transport  <cit> , the use of dextran/drug with no atf showed only limited effect in occasional animals and was in no case statistically significant.
this takes advantage of the greatly improved efficiency of targeted adsorptive endocytosis - the tripartite complexes specifically adhere to markers exposed on the external surface of the axon terminus at sites destined to be drawn into the interior of transport vesicles.
these markers can be used in the phage display system to develop new purely synthetic, non-viral atfs to offer selectivity for target neuron sub-types.
the system is also applicable for introducing dna to the neuronal nucleus without the risks of viral transfection systems.
it had not been clear in the past whether gabapentin acts inside nerve cells or on the external cell surface  <cit> .
further, the conjugation reaction and subsequent release of the gabapentin might have converted it to a chemically distinct metabolite or residue.
the high efficacy of this intraneurally delivered agent constructed with gabapentin demonstrates for the first time that delivery of a clinically efficacious small molecule therapeutic agent from inside the cell via an intraneural route can achieve the same pharmacological effect as delivery from outside the cell via a systemic route.
this also corroborates recent evidence that the site of action of gabapentin is indeed intracellular  <cit> .
one of the functions of retrograde axonal transport is to allow the cell body to sample conditions at the distant axon terminus in order to obtain information on requirements for replacement organelles, proteins, and small molecules.
excessive hydrolysis in lysosomal transport vesicles would interfere with this sampling process, so the preservation of therapeutic molecules during axonal transport in vesicles is consistent with this function.
chronic use of axonal transport agents raises the issue of specialized toxic effects associated with the delivery system.
in general, the amount of drug being delivered to the axonal intracellular space will be similar to normal therapeutic delivery and will be handled by the same clearance methods that the cell usually relies on.
the atf will be delivered in amounts that are physiologic and will present no special accumulation risk although there may be unintended direct intracellular signaling effects from some atfs that would need to be identified.
the dextran or other polymer linker could be subject to accumulation, however it must be kept in mind that the axon is constantly ingesting extracellular fluid as a means of sampling the environment of the axon terminus for presentation to the regulatory apparatus in the cell nucleus after transport.
the cargos delivered by the endosome/lysosome system physiologically must be degraded and removed from the cell.
we did not observe any apparent toxicity upon histological analysis of the cns or axonal structures in any of these experiments, however, this issue would be need to be carefully assessed in the course of development of any pharmaceutical for axonal delivery.
CONCLUSIONS
these studies demonstrate that intraneural delivery of pharmaceutical agents as part of a tripartite complex  results in a unique distribution in which high concentrations reach targeted cns, autonomic and peripheral nerve targets.
the resulting concentration in non-targeted cns and systemic tissues is several orders of magnitude lower than the concentration in targeted cns and pns tissues.
this effect can be achieved using a well tolerated and non-invasive clinically applicable administration route - intramuscular and intradermal injection.
whilst there are additional issues to resolve before axonal transport based medications come into regular clinical use, their eventual development now seems realistic.
previously, the relatively small number of 'uptake events' per neuron for molecules like ngf has limited their applicability for intraneural drug delivery.
this work demonstrates that a tripartite complex with a polymer linker carrying large numbers of drug molecules can amplify the pharmacological effect of each uptake event by at least two orders of magnitude.
amplification by an additional order of magnitude through alternative conjugation schemes appears to be readily achievable.
the delivery to drg neurons including nociceptors as well as to motor column neurons has positive implications for the development of medications for pain, muscle spasm, neuroprotection and anti-viral treatment.
we anticipate that intraneural pain medication will have a significant impact on the management of pain after surgery and in the treatment of patients suffering from intractable chronic pain unresponsive to existing pain medications.
some efficacious agents whose use is limited by systemic toxicity may be safely and effectively delivered by an axonal transport delivery vehicle.
it is likely that anti-viral, neuroprotectant and anti-spasmodic agents  can also be delivered to important locations in the nervous system in this manner.
the application of this new class of tripartite intraneural pharmacologic vehicles also provides a novel tool for the investigation of a number of aspects of basic neurobiology.

 the direct distribution of medications to specific target tissues in the central nervous system is an attractive objective for the treatment of problems such as local pain or dermatomal viral infection. when systemic drug delivery is used to treat a symptom affecting a small segment of the nervous system conventional drug delivery methods can be highly inefficient because they place most of the dose in non-neuronal tissues throughout the body and in areas of the cns that do not need treatment. dose limitation that hinders efficacy often arises from the concentration of the drug in non-target tissues. for many compounds, this situation is aggravated because access to tissues of the central nervous system is also compromised by the relative impermeability of the blood brain barrier. the nervous system itself provides an alternative to the vascular system or the cerebro-spinal fluid - the intra-axonal route. however, this has proven difficult to exploit. methods using direct injection into nerves, introduction of modified neurotropic viruses, or delivery of neurotoxins such as tetanus toxin or ricin have been explored but each pose barriers to routine clinical use. direct injection may damage nerves, neurotropic viruses expose the recipient to direct damage or the risk of recombination with wild type virus, and toxins for nerve destruction have limited clinical roles. phage display has been used to generate synthetic peptides to promote axonal transport, but it has not been clear how to exploit this. retrograde axonal transport of exogenous molecules from the periphery to cns neuronal cell bodies is long established and is one of the methodological bases for mapping neuroanatomical pathways. the underlying physiological basis of this process and its biochemistry are increasingly well understood. however, although axonal transport for drug delivery has been proposed previously, little or nothing is known about the major features of this biological system from the point of view of approaching it pharmacologically and there has been no agent demonstrated capable of accomplishing a pharmacological effect via this route. pharmacological entrainment of axonal transport is acknowledged as a highly desirable objective since it would allow for targeted antiviral, antineuropathic or regenerative treatments to segmentally selected ganglion or cns cells. we sought to clarify the pharmacology of intraneural drug delivery and to design agents capable of accomplishing therapeutic tasks by clinically applicable axonal transport methods employing intramuscular injection and subsequent uptake by intact nerve endings. we designed a novel tripartite molecular complex to provide a flexible architecture capable of achieving this. we now report on this class of pharmacological agents for selective targeted access and transport through the nervous system and demonstrate that it is capable of achieving therapeutic efficacy. the system provides a versatile platform for many different therapies with the same targeting requirements. the agents used are various forms of a tripartite complex composed of a first moiety acting as an "axonal transport facilitator", an "amplifying polymer" second moiety acting to achieve amplification of the fundamental event of synaptic endocytosis by carrying along multiple drug molecules with each saturable uptake event, and a third moiety composed of multiple copies of the therapeutic molecule, reversibly linked to the polymer. this novel molecular architecture allowed us to independently explore and optimize general features and constraints affecting the three major aspects of the problem - a) chemical synthesis, b) optimization of uptake and transport, and c) intraneuronal drug efficacy after transport. we used a comprehensive array of biological, physical and chemical assays in vitro and in vivo to characterize and optimize the system. the studies are numbered to corresponding to the summarized results table. a) we studied the synthesis and stability of the tripartite and its components addressing the following questions: can tripartite molecules be constructed chemically to preserve efficient adsorptive endocytosis when loaded with large numbers of conjugated drug molecules? what are the upper size limits for the tripartite complex? can pharmacologic activity be preserved for small molecules released from the tripartite complex under intracellular conditions? b) we analyzed interactions with axon terminals and the intra-axonal environment that would affect design of the tripartite by investigating the following questions: - how do polymer size, molecular charge, and hydrophilicity affect the efficiency of uptake into neurons? - what is the relative efficiency of physiologic atfs when compared to non-physiologic atfs for uptake and transport? can purely synthetic atf's be produced by phage display techniques that will be more effective than physiologic atfs? will use of colchicine - an inhibitor of axonal transport serve to verify interpretations? can a small intramuscular or intradermal injection cause significant delivery into a selected set of neurons via intact uninjured axon terminals in normal tissues? can this clinically usable method of drug administration achieve high intra-neuronal drug concentrations when compared to the "cut neuron" methods used to introduce axonal transport tracer molecules in some anatomical studies? during axonal transport, if the pharmacologic agents are sealed inside vesicles, will the drug or carrier be damaged or destroyed by lysosomal activity while traveling long distances in the intracellular space of the axons? c) we investigated targeting and pharmacological efficacy: - which classes of clinically useful sub-targets in the nervous system can be reached by clinically convenient administration techniques? - what are the unique features of the whole body pharmacologic distribution of intraneuronal agents and how do size of the molecular complex and selection of atf affect the distribution? can pharmacologically efficacious doses of drugs be delivered and are they functional when delivered to the interior of a cell rather than to its exterior surface? using a tripartite construct designed according to the results of the investigations outlined above, we were able to administer a small intramuscular injection with 1/300th of the usual oral dose of gabapentin and achieve a degree of neuropathic pain suppression that could not be obtained with tolerable amounts of oral therapy while extending the half life of the drug by more than an order of magnitude.  results are summarized in table 1 chemical synthesis and stability chemical entities effective synthesis with loading sufficient for amplification the synthesis strategy achieved attachment of drug to at least 30% of dextran monomers for both gabapentin and valacyclovir. the mw dextran has approximately dextrose sub-units so each wheat germ agglutinin molecule endocytosed in these experiments delivered around molecules of drug. particle based carriers can be delivered intraneuronally edta washing of dextran coated magnetite particulates resulted in hydroxide free ferrites that were non-reactive and preserved their superparamagnetic properties during axonal transport. after conjugation of an axonal transport facilitator to the dextran coat, they were effectively endocytosed and transported intact despite their size. intracellular release and drug activation the stability of a drug is maintained through chemical linkage and biological release in the bhk viral plaque reduction experiments, free drug in the culture medium resulted in plaque reduction by 96% at a dose of micrograms/well, but a dose of micrograms/well of the drug bound to dextran was required to achieve an equivalent effect. however we observed nearly 100% plaque reduction using a dose of microgram/well of tripartite wga-dextran-drug conjugate. this demonstrates that the wga efficiently promoted endocytosis of the tripartite agents while dextran-drug alone was not readily taken up by cells. on chemical grounds, the carboxyl links binding drug molecules to dextran should be hydrolyzed after endocytosis. for valacyclovir, the product of the hydrolysis is free and active acyclovir. therefore, the antiviral effect of the tripartite confirms release of acyclovir from the tripartite carrier after endocytosis. interactions with axon terminus and axonal processing effects of polymer, linker and drug effects of polymer size on tripartite uptake into nerves for the two dextran sizes tested, the uptake was faster for the smaller 10k molecular weight dextran than for the larger 70k molecular weight dextran. however, uptake of the 70k molecule was more effective from the point of view of drug delivery. this is because each 70k dextran delivered more attached drug molecules. with 10k dextran, there is extensive uptake within ten minutes after application and washing. similar levels of uptake are seen with 70k dextran after minutes. however the differential efficacy of uptake was not large enough to outweigh the benefits of greater amplification of uptake events achieved by using the higher capacity larger molecules for drug delivery. since molecular loading efficiency of the polymers was equivalent for various sized dextrans, the increased drug delivery with increased molecular size was linear. however, the losses in uptake efficiency with larger molecules was not severe enough to make them less effective overall. the consequence is that although smaller dextran molecules were endocytosed more avidly and rapidly than 70k based vehicles, more drug molecules were still delivered by the 70k based vehicles if a full hour was allowed to elapse after introduction of the test agent. larger molecule sizes were not tested. effects of charge on uptake and transport the overall charge of the complex had a large impact on efficiency of uptake. there was virtually no uptake into cultured neurons when the overall charge of the complex was negative, and effective uptake occurred when charge was neutral, or when overall charge was positive. this finding parallels the natural movement of positively charged ions into the negatively charged internal milieu of the cell during depolarization. similar charge findings have been reported for liposomal drug delivery systems. effects of side group mediated hydrophobicity on uptake and transport acylation of fitc -labeled, wga-conjugated dextran to make the molecules progressively hydrophobic led to complete failure of uptake and transport. we assessed various degrees of acylation and found that this effect occurred even at very low ratios of acylation. effects of axonal transport facilitator efficacy of physiologic, non-physiologic and synthetic atfs the campenot chamber studies demonstrated similar efficacy for nerve growth factor and wga for promoting uptake of the tripartite carrying fitc. the phage display experiments demonstrated that novel purely synthetic atfs could be discovered and then produced in vitro which not only had equivalent efficiency for neuronal uptake, but which could also be more efficiently sub-targeted to different types of neuronal populations. when colchicine - an inhibitor of axonal transport - was added to campenot chambers, it blocked intra-axonal movement of molecular complexes whose atf was derived from purely synthetic phage display techniques. this confirms that the drug transport effect produced by the synthetic phage display products achieved delivery by axonal transport. effects of intra-axonal processing survival of small chemical molecules linked to the tripartite when wga-dextran-gabapentin was used, cross staining with antibodies to gabapentin confirmed preserved antigenicity of the drug after axonal transport of the tripartite, although this method could not itself confirm that the drug was intact or active after transport and release. dextran-gabapentin with no conjugated wga did not produce detectable gabapentin antigenicity in the histological sections. large molecule access to spinal cord from intramuscular injections after small im injections in muscle, wga delivered sufficient quantities of functioning hrp enzyme to label numerous motorneurons in our primate model. enzymatic activity of the horseradish peroxidase survived any hypothetical lysosomal degradation during transport and yielded product distributed throughout the neuron. both epaxial and hypaxial muscles proved an effective route to reach spinal cord quickly with no apparent difference in delivery of intact enzyme via axons despite the difference in distance of transport. the labeled spinal cord motor neuron pool for these axial muscles had greater longitudinal extent among spinal cord segments than for appendicular muscles. these findings are consistent with previous reports concerning wga and ngf and their intramuscular introduction. the rate of transport appeared to be consistent with time scales predicted by literature values of 30- mm/day. overall the area of spread of injectate in the spinal cord was limited to a few millimeters, although the distribution of the epaxial muscle spinal cord motorneuron pool extended over several centimeters. this appeared to demonstrate that a group of neurons near each other in the spinal cord innervated a group of fibers in epaxial muscle that are close together despite the wider extent of the overall motor neuron pool. intact transport of targeted nanoparticles by intact axon termini electron microscopy showed appropriately sized ferrites within the axon, more than three centimeters from the wga-dextran-fe injection site. the appearance of the ferrites in endosomes was similar to what has been seen in studies of similar agents in other tissues, but in this case the particles were restricted to the axonal stream. the autoradiographic studies demonstrated sciatic nerve radioactivity and so provided similar evidence that the magnetite containing particles were transported in nerves. the relaxivity experiments taken together with distribution studies showed that the concentration of magnetite delivered to the axon by the tripartite was sufficient to affect the t relaxation rate of nerve. the observation of a decrease of t relaxation time in nerves transporting superparamagnetic nanoparticles in both the micro-mri nerve channel studies and in the high resolution mri experiments confirmed that the carrier particles were not degraded. any hydrolysis of the sub-domain sized particles would have eradicated their superparamagnetic effect on t relaxation time in nerve as transport progressed. the relaxivity effect far exceeded that which would result from free iron or ferritin at the doses administered. the wga-dextran-magnetite experiments in rabbits confirm other reports that particles of 5- nm are endocytosed and transported by intact nerve endings. some studies have suggested that nerve injury is required for the transport of larger particles, but this result suggests that intact neurons will indeed transport large multi-molecular aggregates or "transport particles" after intramuscular injection when the particles are well solvated. c. targeting and pharmacological efficacy clinical target access targeted access to clinically relevant neuronal sub-populations intramuscular injection of the tripartite wga-dextran-fitc produced labeling of alpha motor neurons in the ventral horn and autonomic neurons in the intermediolateral cell column. we also observed good filling of proximal sensory neuron processes in the dorsal root entry zone and in lamina i and ii of the dorsal horn of the spinal cord. injection of foot pad and multiple hind limb muscle each resulted in labeling of less than 50% of ganglion cells, but injection of both muscle and skin resulted in filling of nearly 90% of dorsal root ganglion cells. in clinical use, the objective will typically be to reach specific sub-populations rather than filling an entire ganglion per se, so these results support the expectation that a large fraction of cells in a e.g. a subpopulation innervating a single muscle or patch of skin can readily be reached. cross staining with an antibody to peripherin showed that many of the drg sensory neurons that were accessed were c-fiber nociceptor cells. this helps confirm that this method of delivery does reach a selected subset of nociceptors that correlates specifically with the selected site of injection. the atf played a major role since no detectable fluorescence was observed when dextran-fitc without atf was administered in these experiments. distinctive pattern of distribution relative to trans-vascular unique distribution with high fraction of drug reaching neuronal targets in the whole body distribution studies with small calf muscle injections, the concentration of -wga detected in relevant ipsilateral peripheral nerve and dorsal root ganglia reached six times systemic concentrations. the detected concentration in relevant nerve and ganglia demonstrated saturability and transport time consistent with other reports. activity in spinal cord was less than in nerve or drg but did reach twice systemic concentrations when higher concentrations of injectate were used. there was no significant activity detected in contralateral nerve and drgs nor in ipsilateral nerves, ganglia, or spinal cord related to cervical, thoracic, or higher lumbar segments. the greatest activities detected in other tissues were in liver and kidney but these apparently reflected metabolized iodine lost from the compound. ngf showed less transport to spinal cord than was seen for wga consistent with selective uptake of ngf by sensory and autonomic nerves. detection of activity due to -wga-dextran at other systemic sites was even less than for wga or ngf alone. considerable amounts of drug remained in the axon itself at the time points sampled. it is known that para-nodal complexes of schwann cells at the nodes of ranvier can endocytose materials from the axoplasm. this may account for a peri-axonal depot effect with drug being cleared to the para-nodal complexes and then subsequently being re-released. further studies will be needed to clarify this issue. site of injection retains high amount of injectate the regional view provided by the -wga gamma camera studies demonstrates that the total amount of the injectate remaining at the site of injection can be large when compared to the amount in the small volume of the nerve. in part, this reflects the fact that wga adheres to muscle cell membranes as well as being taken up by adsorptive endocytosis at nerve termini. this also explains a depot effect whereby additional amounts of the compound continue to be introduced into the axonal stream over a period of several days. pharmacologically efficacious drug amounts were delivered the tripartite assemblage with -labeled gabapentin produced activity levels for gabapentin in ipsilateral neurons of more than times greater than background while counts remained at background levels in contralateral neurons. this demonstrated the localizing effect of injection of polymer-bound drug conjugated to an axonal transport facilitator, and also allowed us to estimate the drug concentration achieved based on the specific activity of the -labeled gabapentin. pharmacologic effects of atf mediated delivery not obtainable by trans-vascular agents prolonged suppression of hyperalgesia in the hyperalgesia experiments a single injection with the tripartite agent reduced the hyperalgesia for at least four days after injection. the total dose was mg/kg using the novel intraneural transport method compared to mg/kg total dose orally for four days of the usual human oral dosing. prolonged efficacy may have been due to a depot effect in muscle, slow redistribution from axon and schwann cells to nerve cell body, slow clearance from inside the nerve, or preemptive blocking of newly produced receptor molecules en route from ribosome to cell surface. discussion we have demonstrated through a comprehensive series of chemical, radiological in vitro, and in vivo biological studies that axonal transport is a valid route for drug delivery when a tripartite complex is used. we have used these studies to exemplify in vivo a prolonged pharmacologically effective delivery of an analgesic molecule leading to sustained significant response in a standard nociceptive model. routine oral delivery of gabapentin results in clinical efficacy for pain relief when blood levels reach μm. gabapentin not only crosses the blood brain barrier but is subject to some active concentration. it is reasonable to hypothesize, therefore, that clinically efficacious axonal transport for intraneural drug delivery would need to produce similar concentrations near the internal cell surface of accessed neurons to achieve similar pharmacological efficacy. in this model, histological estimates of the ratio between the estimated intracellular volume of the accessed neurons and the actual volume of the entire dorsal root ganglion provide a correction factor that allows for conversion of total drg concentration into estimated intracellular concentration in the accessed neurons. when these considerations were employed, our counts of delivered suggested delivery of gabapentin at intracellular doses equal to or greater than μm. these in vivo -gabapentin studies therefore showed that this technology can deliver drugs to target neurons at pharmacologically efficacious doses using 1/300th of the oral dose that would be required to achieve the same intracellular concentration in target cells. in addition, this system resulted in a ten fold increase in effective half life relative to oral administration. this appears to be because of the unique dynamics of this delivery mechanism. this involves both a muscle injection site depot effect for the large tripartite complexes and a prolonged stream of delivery to the neuron from the axon and its associated paranodal complexes. use of the tripartite vehicle made it possible to use a selected axonal transport facilitator to cause transport of large numbers of drug molecules and to recover the pharmaceutical effect of the drug after delivery to its target. by varying the polymer backbone length and by use of various linker chemistries, it is possible to accommodate therapeutic molecules with a wide variety of different sizes and physico-chemical properties. in this set of experiments, neither systemic delivery of the agent by a subcutaneous route nor administration of free drug alone to the affected limb had any significant therapeutic effect. although dextran can itself weakly promote axonal transport, the use of dextran/drug with no atf showed only limited effect in occasional animals and was in no case statistically significant. this takes advantage of the greatly improved efficiency of targeted adsorptive endocytosis - the tripartite complexes specifically adhere to markers exposed on the external surface of the axon terminus at sites destined to be drawn into the interior of transport vesicles. these markers can be used in the phage display system to develop new purely synthetic, non-viral atfs to offer selectivity for target neuron sub-types. the system is also applicable for introducing dna to the neuronal nucleus without the risks of viral transfection systems. it had not been clear in the past whether gabapentin acts inside nerve cells or on the external cell surface. further, the conjugation reaction and subsequent release of the gabapentin might have converted it to a chemically distinct metabolite or residue. the high efficacy of this intraneurally delivered agent constructed with gabapentin demonstrates for the first time that delivery of a clinically efficacious small molecule therapeutic agent from inside the cell via an intraneural route can achieve the same pharmacological effect as delivery from outside the cell via a systemic route. this also corroborates recent evidence that the site of action of gabapentin is indeed intracellular. one of the functions of retrograde axonal transport is to allow the cell body to sample conditions at the distant axon terminus in order to obtain information on requirements for replacement organelles, proteins, and small molecules. excessive hydrolysis in lysosomal transport vesicles would interfere with this sampling process, so the preservation of therapeutic molecules during axonal transport in vesicles is consistent with this function. chronic use of axonal transport agents raises the issue of specialized toxic effects associated with the delivery system. in general, the amount of drug being delivered to the axonal intracellular space will be similar to normal therapeutic delivery and will be handled by the same clearance methods that the cell usually relies on. the atf will be delivered in amounts that are physiologic and will present no special accumulation risk although there may be unintended direct intracellular signaling effects from some atfs that would need to be identified. the dextran or other polymer linker could be subject to accumulation, however it must be kept in mind that the axon is constantly ingesting extracellular fluid as a means of sampling the environment of the axon terminus for presentation to the regulatory apparatus in the cell nucleus after transport. the cargos delivered by the endosome/lysosome system physiologically must be degraded and removed from the cell. we did not observe any apparent toxicity upon histological analysis of the cns or axonal structures in any of these experiments, however, this issue would be need to be carefully assessed in the course of development of any pharmaceutical for axonal delivery.  these studies demonstrate that intraneural delivery of pharmaceutical agents as part of a tripartite complex results in a unique distribution in which high concentrations reach targeted cns, autonomic and peripheral nerve targets. the resulting concentration in non-targeted cns and systemic tissues is several orders of magnitude lower than the concentration in targeted cns and pns tissues. this effect can be achieved using a well tolerated and non-invasive clinically applicable administration route - intramuscular and intradermal injection. whilst there are additional issues to resolve before axonal transport based medications come into regular clinical use, their eventual development now seems realistic. previously, the relatively small number of'uptake events' per neuron for molecules like ngf has limited their applicability for intraneural drug delivery. this work demonstrates that a tripartite complex with a polymer linker carrying large numbers of drug molecules can amplify the pharmacological effect of each uptake event by at least two orders of magnitude. amplification by an additional order of magnitude through alternative conjugation schemes appears to be readily achievable. the delivery to drg neurons including nociceptors as well as to motor column neurons has positive implications for the development of medications for pain, muscle spasm, neuroprotection and anti-viral treatment. we anticipate that intraneural pain medication will have a significant impact on the management of pain after surgery and in the treatment of patients suffering from intractable chronic pain unresponsive to existing pain medications. some efficacious agents whose use is limited by systemic toxicity may be safely and effectively delivered by an axonal transport delivery vehicle. it is likely that anti-viral, neuroprotectant and anti-spasmodic agents can also be delivered to important locations in the nervous system in this manner. the application of this new class of tripartite intraneural pharmacologic vehicles also provides a novel tool for the investigation of a number of aspects of basic neurobiology. 
pharmacological efficacy with gabapentin in a paw withdrawal latency model revealed a ten fold increase in half life and a fold decrease in necessary dose relative to systemic administration for gabapentin when the drug was delivered by axonal transport using the tripartite vehicle. intramuscular and intradermal injection proved effective for introducing pharmacologically effective doses into selected populations of cns neurons. 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. 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. specific targeting of selected subpopulations of cns neurons for drug delivery by axonal transport holds great promise. the pharmacologically efficacious drug delivery demonstrated here verify the fundamental feasibility of using axonal transport for targeted drug delivery. currently, systemic methods are generally used for delivery of pain medications, anti-virals for treatment of dermatomal infections, anti-spasmodics, and neuroprotectants. conjugation methods were used that allowed the drugs to be released in active form inside the cell body after transport. loading of up to drug molecules per complex was achieved. targeted delivery of pharmaceutical agents into selected populations of cns neurons is an extremely compelling goal. the data shown here provide a basic framework for the intraneural pharmacology of this tripartite complex.
 targeted delivery of pharmaceutical agents into selected populations of cns 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.  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 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 fold decrease in necessary dose relative to systemic administration for gabapentin when the drug was delivered by axonal transport using the tripartite vehicle.  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. 