Patentamt Europaisches || || 1 1| || || 1 1 1| || 1 1| || || (19) J European Patent Office

Office europeen des brevets (1 1 ) EP 0 548 157 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publicationation and mention (51) |nt. CI.6: A61 K 47/48, A61 K 49/00 of the grant of the patent: 20.05.1998 Bulletin 1998/21 (86) International application number: PCT/EP91/01780 (21) Application number: 91916129.9 (87) International publication number: (22) Date of filing : 1 3.09.1 991 WO 92/04916 (02.04.1992 Gazette 1992/08)

(54) USE OF PARTICULATE AGENTS VERWENDUNG VON SUBSTANZEN IN PARTIKELFORM UTILISATION D' AGENTS SOUS FORME DE PARTICULES

(84) Designated Contracting States: (72) Inventor: FILLER, Aaron, Gershon AT BE CH DE DK ES FR GB GR IT LI LU NL SE London SW20 ONE (GB)

(30) Priority: 14.09.1990 GB 9020075 (74) Representative: 30.10.1990 GB 9023580 Perry, Robert Edward et al 17.12.1990 GB 9027293 GILL JENNINGS & EVERY 07.01.1991 GB 9100233 Broadgate House 16.01.1991 GB 9100981 7 Eldon Street 31.01.1991 GB 9102146 London EC2M 7LH (GB) 20.05.1991 GB 9110876 30.07.1991 GB 9116373 (56) References cited: 19.08.1991 GB 9117851 WO-A-86/01112 WO-A-88/00060 30.08.1991 GB 9118676 WO-A-89/09625 WO-A-90/01295

(43) Date of publication of application: STN File Server, File Medline, accession no. 30.06.1993 Bulletin 1993/26 87239688; J.E. GALLAGHER et al.: "Sialic acid mediates the initial binding of positively charged (60) Divisional application: inorganic particles to alveolar macrophage 97119199.4 membranes" STN File Server, File Medline, accession no. (73) Proprietor: SYNGENIX LIMITED 86085167; D. MENETREY: "Retrograde tracing of Cambridge CB3 0BL (GB) neural pathways with a protein- complex. I. Light microscopic detection after intensification"

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CO Note: Within nine months from the publication of the mention of the grant of the European patent, give 10 any person may notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in o a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. Q_ 99(1) European Patent Convention). LU Printed by Xerox (UK) Business Services 2.16.3/3.4 1 EP 0 548 157 B1 2

Description anterograde labelling. The principle of improving specif- icity and uptake efficiency of a histologically identifiable This invention relates to particulate agents for use tracer was taken further by Schwab (Brain Res. in therapy. 130:190-196 (1977)) who attached nerve growth factor US-A-4849210 and US-A-4863715 demonstrate s (NGF) to HRP. It was also Schwab who showed that a the effectiveness of suspensions of ferromagnetic parti- plant lectin called wheat germ agglutinin (WGA) was an cles as intravenous MRI contrast agents. WO-A- excellent nerve adhesion molecule and again Schwab 8800060 and US-A-4827945 provide a number of addi- who introduced the use of viral fragments and toxins as tional methods of synthesis of superparamagnetic parti- labels (see Brain Res. 152:145-150 (1978) and J. Cell cles and suggest the MR intravascular use of a wide 10 Biol. 82:798-810(1979)). range of labelled particles analogous to those disclosed WGA conjugated to HRP was later suggested as a for in vitro use in US-A-4452773. tracer and this one agent has been the predominant The particles produced by these additional meth- agent of choice in many hundreds of subsequent stud- ods vary in size from 10 to 5000 nm (100 to 5,000 Ang- ies involving axonal transport. The conjugation to some stroms). They are not useful for imaging of 15 post-sacrifice visualization moiety such as HRP permit- axonal transport because most of the particles are too ted the use of a chromogen histochemical staining reac- large to be endocytosed by neurons and only a small tion. Other means of visualization of tracers included proportion will carry active targeting moiety. autoradiographic histology or immunocytochemical Brady, SMRM 10:2 (1991), verbally reported trans- techniques. port of MR detectable particles after direct injection into 20 The present invention is based on the realisation the sciatic nerve; however he exhibited only an image of that the endocytosis of nerve adhesion molecule transport after the completely severed sciatic nerve was (NAM)-labelled agents can be utilised for the remote soaked in a gel with ferrite particles. Ghosh, SMRM delivery of therapeutic agents, i.e. axoplasmic flow can 10:1042 (August 1991), similarly reported evidence of serve to transport a therapeutically-active agent com- transport of ferrite particles after direct pressure injec- 25 prising a nerve adhesion moiety from its administration tion into the brain of a frog, although no MR detection site in tissue such as muscle to a remote site where it was achieved. Neither reference teaches how pharma- exerts its pharmacological effect. This is of particular ceutical use of axonal transport could be achieved since interest where the sensitivity or accessibility of the these techniques involved irreparable destruction of remote site is such as to prevent direct administration of vital neural tissue. Intraneural injection is destructive of 30 the pharmaceutical. the nerve at the site of needle puncture and causes According to the present invention, a therapeutic forced flow of tracer in the nerve sheath which may agent comprises particles having, coupled thereto, a actually mask evidence of actual axonal transport. Mad- nerve adhesion moiety and a physiologically-active sub- ison, Brain Res. 522:90-98 (1990), also reported pres- stance, the nerve adhesion moiety serving to promote sure injection of latex nanospheres into the brain, to 35 neuronal endocytosis of said agent, the agent being deliver toxic agents for the killing of neurons after sub- capable of axonal transport following neuronal endocy- sequent photoactivation. tosis, and is used for the preparation of a medicament Non-destructive administration of toxic anthracy- for use in providing the therapeutic effect of the physio- cline antibiotics has been reported, but this was done to logically-acceptable substance on administration into a study the chemical nature of the neural uptake process 40 vascularised peripherally innervated tissue site or into and the fluorescent effect of the agents rather than to other tissue sites innervated by a spinal root. achieve any therapeutic effect, and the agents con- The pharmaceutical agent is preferably adminis- cerned were neurotoxic; see England, Brain 111:915- tered into a tissue site, such as a muscle, having a vol- 926 (1988), and Bigotte, Neurology 3J:985-992 (1987). ume of at least ten times that of the of nerve cells The existence of axonal transport (or 'axoplasmic 45 which are to transport the agent. flow') has been known for over 40 years and it has been The particulate NAM-coupled moiety may be known for twenty years that certain foreign materials coated or uncoated. If coated, the coating may be injected into muscle would be endocytosed (swallowed selected to be broken down within the neuron after up) by the axon terminus and then subsequently be endocytosis, either slowly or more rapidly, or to be main- detectable in the neuron cell body; however, until the so tained during axonal transport. developments described herein, all methods of detec- For the purposes of the present invention it should tion have required lethal interventions, generally requir- be appreciated that while natural or synthetic, essen- ing the killing of the experimental animal with tially inert, organic polymer particles (such as dextran- subsequent specialized tissue processing. coated microspheres or latex nanospheres) are capable A series of relatively non-specific substances for 55 of being endocytosed, these organic polymers unlike uptake were tried including Evans-Blue stain conju- more specific and complicated molecules such as pro- gated to albumin and also horseradish peroxidase teins, antibodies and antibody fragments are not con- (HRP) enzyme, and radio-labelled amino acids for sidered to be nerve adhesion molecules.

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The mean particle size for the particulate pharma- thereof are preferably sterilized by 0.2 or 0.1 micron ceutical agents used in the invention is conveniently in microfiltration after final synthesis, affinity purification the range 5 to 100 nm, especially 8-70 nm, more partic- and concentration. ularly 1 0 to 50 nm and preferably about 20-30 nm. The uses of a given version of the agent depend One especially important group of pharmaceutical s upon the elements and (nuclides) used in the agents for use according to the invention is that of NAM- initial precipitation step in which the metal oxide crystal coupled particulate inorganic compounds, for example core is precipitated and coated and also upon the type metal oxides, sulphides or alloys, where the inorganic of targeting moiety used. For each use, the nuclide and material is selected for its magnetic properties, in partic- targeting moiety may be selected to benefit both from ular ferri- and ferromagnetism and more particularly w the general advantages of the simplicities of the prepar- superparamagnetism, or includes within an otherwise atory method and to take advantage of the new types of essentially inert matrix atoms or molecules which are pharmaceutical distribution which can be achieved by released gradually from the matrix to exert a therapeutic materials prepared in this way. or prophylactic effect. Many metal oxide structures may The inorganic particles in the preferred pharmaceu- be utilized as the inorganic particles, and spinels and is tical agents used according to the invention generally garnets have been found to be particularly useful in this fall into one of two categories: regard. It should however be stressed that other well known inert and preferably essentially water-insoluble i) particles incorporating a gamma or electron-emit- metal compounds may be used, especially those having ting radionuclide, and thus being capable of caus- or capable of being doped to exhibit cooperative mag- 20 ing radiation treatment effects; or netic properties and those having lattices such as per- mit desired radioisotopes to be included. By alloys, ii) particles incorporating a compound or element mixed metals are of course included. Organic particu- capable on release, e.g. during degradation of the late matrices may also be used to accommodate a ther- particle, of effecting a desired therapeutic or pro- apeutic compound. 25 phylactic effect. As is clear from the above, this invention is espe- cially concerned with improvements in particulate phar- Metal oxide particles of the first type are clearly maceuticals which surprisingly result in providing known. Particles of the latter type are novel. access to new patterns of distribution within the body The invention may utilise a physiologically-tolerable which were never previously possible. As a result of 30 particulate garnet or spinel with incorporated therein these new patterns of distribution, a number of previ- atoms of , of a radioactive , of a ously intractable problems in medical diagnosis and sixth metal (e.g. a lanthanide), of a high MR treatment have now been solved. receptivity nuclide (e.g. at least as high as 71Lu175), or For the particulate pharmaceutical agents in partic- of an element which on particle degradation has a ular, the improvements include 1) improved control of 35 desired therapeutic or prophylactic activity. particle size, 2) development of an effective means of It will be appreciated that although the metals of the affinity purification of the particles, 3) demonstration of a metal oxide, sulphide or alloy matrices of the particles of means of filter sterilization of the concentrated product the invention may have naturally-occurring positron late in the synthesis, and 4) widening the array of ele- emitting isotopes the particles according to the inven- ments and concentration of those elements applied to 40 tion have significantly higher than medical uses in metal oxide or sulphide crystals or in contents of these, e.g. for positron emitters an average alloys. of at least one, perhaps 1 0 or more atoms per 1 00 nm One of the most important consequences of these crystal. The natural occurrence of many p+ emitters is advances is the development of agents which can be less than 1 in 1020 and even one emitting atom per par- delivered by, and make use of an entirely novel intraneu- 45 ticle may suffice. ral pharmaceutical route (IPR). In addition, these For other novel "doped" particles according to the advances greatly simplify and reduce the cost of pro- invention, the active or marker nuclei may be isotopes duction of related particulate agents with previously which occur naturally, e.g. as impurities in naturally known uses. occurring oxides, sulphides or alloys - in this case again In a preferred embodiment, the agent comprises so the particles according to the invention are distin- particles with a core metal oxide crystal, eg. of spinel or guished by containing such atoms at higher than natural garnet structure, coated for example by dextran carbo- values, e.g. a hundred or even more per 100 nm parti- hydrate wherein the total size of the coated particle is cle. between 1 00 and 500 Angstroms (1 0 to 50 nanometers) The particles of the invention may be coated or and where a targeting moiety (TM) is chemically bound 55 uncoated and may derive their physiological tolerability to the coating in low concentration of TM per particle, at least in part from such a coating. They are moreover preferably 1 :1 . The agent should preferably be virtually coupled to a biotargetting moiety, for example an anti- free of particles lacking an active TM, and compositions body, an antibody fragment or another NAM.

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The particles of type i) mentioned above are also mula unit, e.g. with the remaining 2/3 being Fe(lll) if the preferably of a spinel or garnet structure - the manufac- stoichiometry of the desired crystal structure, e.g. gar- ture of particles of these types is already well known net or spinel, is carefully considered and factors such as and need not be described further here. By way of inter- pH, temperature, and precursor metal salt and coating est however it may be noted that superparamagnetic 5 compound concentrations and the duration of heat incu- crystals of this type have been proposed for use as MRI bation after precipitation are carefully controlled, prefer- contrast agents in various patent publications of ably after optimization by routine experimentation. Thus Nycomed AS, Schering AG, Advanced Magnetics Inc, as an example, for dextran coated particles it has gen- etc (eg. US-A-4863715 (Jacobsen) and US-A-4827945 erally been found advantageous to precipitate out from (Groman)). jo a saturated dextran solution. Thus all the divalent metal There are a wide variety of targeting moieties or atoms may be replaced as opposed to the V2 or fewer NAMs which can be used according to the invention. suggested by Groman in US-A-4827945. These include antibodies, monoclonal antibodies, anti- These particles may be synthesized in such a way body fragments, receptors, peptides such as endor- that they are stably coated with dextran or other phins, steroid molecules, viral fragments or coat 15 hydrophilic molecules and the coating may then be acti- proteins, cell surface antigens including various carbo- vated and bound covalently to antibodies or any type of hydrates, lectins, immunoadhesins, neurotransmitter nerve adhesion molecule. molecules, growth factors, and other proteins which pro- Transition metal or lanthanide nuclides can be used mote endocytosis of the pharmaceutical agent by the in the synthesis of radioactive metal compounds (e.g. a axon termini. The use of lectins, such as WGA, is partic- 20 metal oxide, metal sulphide or alloy, such as a ferrite) for ularly preferred. use in monoclonal antibody-based treatment of tumours The particular agents used in the invention prefera- by irradiation. Here again, the biodistribution and clear- bly comprise therapeutically or prophylactically-loaded ance of the delivered are quite different inorganic crystals, e.g. p+ emitter-marked metal oxides. from single atoms chelated to the proteins. Intravascular There are a number of nuclides emitting positron 25 injection of Fe59-labelled particles has demonstrated a and electron p-particles, all of which can be included in biphasic plasma half-life with about 3A of the dose being metal oxides, e.g. spinels such as ferrites, either as sub- cleared to spleen, liver, marrow, and slightly to lung over stituents in the crystal lattice or as seeds, e.g. Zr02, 1-2 hours, but with a substantial fraction of the dose inside ferrite spinel shells. This provides a new and demonstrating a quite prolonged plasma half life of unique way of delivering these various nuclides to vari- 30 many hours. Each antibody molecule can be used to ous medically useful locations in the body in a wide vari- deliver several hundred or several thousand atoms of ety of new concentrations and half lives. A single ferrite the desired nuclide so achieving a high local dose. It particle can be used to attach hundreds or thousands of should also be noted that binding multiple emitter atoms p-emitting atoms to a single antibody, thus far exceeding to a single protein molecule has been known to rapidly the intensity of signal per antibody molecule available in 35 destroy the protein - this problem is substantially allevi- current preparations which generally provide one emit- ated by the SMPE particles because the emitting nuclei ting atom per antibody molecule. are up to 1 00 angstroms distant from the NAM - thus the The uses for these various beta-emitting ferrites chance of any electron, positron, or gamma-ray interact- include a number of treatment modalities. From one ing with the targeting NAM is reduced by several orders point of view, this array of possible nuclide preparations 40 of magnitude. Methods developed for antibody delivery presents a wide range of half lives and particle energies of 39Y90 can be applied with a far higher concentration which can be used for various tasks. The great simplifi- of this nuclide included in a conjugated ferrite. Another cation provided is that all of these can be manufactured treatment problem where these p-emitting ferrites could and delivered by means of essentially similar molecules be useful is in improving the current methods of intra- with effectively identical chemistry. Most of these 45 articular radiotherapy in rheumatoid conditions. nuclides decay to daughters which are also easily Defects in a crystal can cause trapping of positrons. accommodated in the crystal and therefore do not Defects in YBaCuOx perovskite crystals are particularly involve loss of integrity of the particle as decay effective at positron trapping even when these materials progresses. are not in a superconducting state, however, even In one set of embodiments, the positron-emitting so mechanical stress defects in metals are fairly effective. isotopes of (25Mn52), feeFe52), There are also effects due to the magnetic field gener- (27C055), or UsRh") are used in the synthesis ated by a moving positron and its interaction with the of spinel particles, eg. sub-domain sized, superpara- spontaneous field of a material such as magnetite, as magnetic ferrite particles. The inclusion of cobalt or well as electron interaction enhancement effects due to manganese in this type of ferrite has previously been 55 the number of unpaired, anti-spin matched electrons difficult to achieve efficiently, but it is possible to reliably from d or f orbitals in the particular spinel used for the introduce cobalt, manganese, or other metals in particulate shield. amounts up to 1/3 of the number of metal atoms per for- The consequence of these considerations is that it

4 7 EP 0 548 15757 B1 8 is possible to begin with a crystal seed of a positron- tion after intramuscular injection of the pharmaceutical emitting nuclide including several thousands atoms of agent quite unexpectedly yields dramatically high the emitter and then to precipitate a or intraneural concentration relative to other tissues. This doped, defected, magnetite shield around the positron differential in body/nerve concentration permits the use emitting core. This shield will cause a very large fraction 5 of this route with relatively small amounts of pharma- of the emitted positrons to undergo all of their ionization ceutical agent to achieve treatment effects. producing collisional losses within the particle and Non-destructive administration of toxic anthracy- therefore to annihilate without ever leaving the particle. cline antibiotics has been reported, but this was done to Those positrons that do emerge from the surface of the study the chemical nature of the neural uptake process particle without being affected by reflection or surface w and the fluorescent effect of the agents rather than to trapping effects will have a greatly reduced energy dis- achieve any therapeutic effect, and the agents con- tribution, travel far shorter distances through tissue, and cerned were neurotoxic (see England in Brain 1 1 1 :915- create far fewer ionizations in tissue per decay event 926 (1988) and Bigotte in Neurology 3_Z:985-992 than standard unshielded positron emitters. (1987)). This shielding can be used to achieve extremely is Unlike any of these previous reports, the agents limited ranges of cytotoxic ionization injury. described herein may be introduced by techniques The range of the emitted p-particles can be which do not involve the destruction of neural tissue and designed to be not much greater than the size of a sin- which then achieve a pharmacologic effect which does gle target cell, thus limiting effective irradiation to only not require any toxic injury to neural tissues. By deliver- those cells that actually ingest the particle and taking 20 ing particulate carriers it becomes possible to deliver advantage of the terminal Bragg peak effect which types of pharmaceutical agents which would be irrepa- increases the ionization rate for a low energy positron rably damaged by direct chemical conjugation to a NAM just before annihilation. A short half life emitter could be or on break up of its direct NAM-conjugate within the used to minimize the effect of increasing exposure cell. Instead the NAM is coupled to the particle and the range with digestion of the coating (which may take 25 drug is included in the particle or in the particle coating. days) and multiple treatments could then be carried out. Further, the use of particulate drug carriers permits the Larger particles can be used without magnetic aggrega- introduction of large numbers of molecules of the phar- tion by composing the shell of less magnetic nuclides. maceutical agent with each endocytotic event thus Metals other than p-emitters can be used to achieve yielding a 100 fold or up to one million fold increase of the very short range radiotherapy effect. There are a 30 uptake efficiency per NAM. This amplification effect may variety of nuclides in which decay is by K-shell capture. be crucial to achieving pharmacologically efficacious An optimal nuclide with this behaviour is 46Pd103 which doses in many situations. The methods of administra- is a pure K-capture nuclide with a 1 7 day half-life; 24Cr51 tion for these beneficial diagnostic and therapeutic uses may also advantageously be used. include topical, intravenous, intrathecal/intracisternal The particles used generally should be metal com- 35 (cerebro-spinal fluid), sub-cutaneous, intradermal, intra- pounds capable of precipitation to a stable colloid hav- nasal, eye-drop, or bladder irrigation methods, but intra- ing a particle size suitable for cell uptake and having a muscular administration is to be preferred. surface capable of being coated with or bound to bio- The agents described herein differ from all previ- chemically useful materials, e.g. carbohydrates or pro- ously used axonal tracers in that they include agents teins. 40 capable of controlled administration by safe intramuscu- Particles for use in the invention may experience lar injection with non-toxic substances and of achieving mechanical vibration and hence heating when sub- whole body distributions which permit their useful jected to resonant tuned microwave energy. Such parti- observation by various types of non-invasive imaging cles may be transported into areas of spinal cord injury modalities. The agents may be biodegradable, safe for where the development of scar prevents the regenera- 45 clinical use, and act to reveal various human disease tion of injured spinal cord tissue. In research work, this conditions which cannot be adequately demonstrated localised heating phenomenon might be used as a by existing techniques. means of inhibiting spinal cord scar formation. This Previous uses of axonal tracers have been con- effect may also be applied for selective tumour dia- cerned with optimizing the degree of post-sacrificial thermy. Intramuscular injection at various sites with dif- so staining of the neuron cell body in brain or spinal cord. It ferent resonant particle frequency types at each site will has not previously been evident that useful concentra- permit rotation of microwave heating frequencies so that tions and distributions of clinically applicable tracer only the tumour site will be stimulated by all the signals. materials could be achieved. From the above, it will be appreciated that the However, this set of agents is based on the discov- invention provides an entirely novel means of pharma- ss ery that when a nerve adhesion molecule which also ceutical distribution which involves the entrainment of a has affinity for markers on the muscle cell surface is well known physiological phenomenon called axonal used, the injected material has very minimal spread transport. A central feature is that the total body distribu- from the site of intramuscular injection. In consequence,

5 9 EP 0 548 157 B1 10 a relatively large amount of the substance is transported rally incomplete at this site so that tracers reversibly into the nerve while relatively little spreads throughout adherent to muscle or emerging from small blood ves- the body. The initial distribution assay results with ani- sels passing near neuromuscular synapses can present mal studies using 125l labelled WGA are shown in Fig. a variety of molecules and particles directly to the neu- 1 , the accompanying drawing. This graph depicts the 5 ronal cell surface for uptake after specific adhesion to results of the 125I-WGA distribution study, in which the the neuronal cell surface at the axon or dendrite termi- vertical axis gives counts per minute per gm of tissue nus. normalised by dividing by the cpm/gm of blood for the The site of injection or administration will preferably individual animal in the series, various tissues are dis- be determined only by knowledge of the nerves which played along the long Y axis, and lines 1 through 8 w project to that site. For example, a pain in the large toe reflect the results from the different animals with varying will be known to the neurologist to involve the dorsal treatment/survival times and doses. DRG signifies spi- root ganglion of the fifth lumbar nerve root. He will then nal roots and dorsal root ganglia and demonstrates con- choose an injection site somewhere in the dermatome centrations 5-10 times higher than any other tissue or myotome served by that nerve root in order to label except the muscle and lymph nodes near the injection 15 the part of the nerve he believes to be impaired or to site which are not shown. deliver, for instance, a pain medication to the ganglion The delivery of particulate pharmaceuticals by the or spinal cord dorsal root entry zone connected to the intraneural route is an entirely novel means of drug fifth lumbar spinal nerve root. For drugs where systemic administration. The largest number of drugs in current spread is to be minimised, it is particularly important to use depend in some way upon the bloodstream to 20 be able to achieve high uptake by neurons per unit achieve their distribution. This vascular dependence amount injected and to minimise spread away from the includes not only drugs given by intravenous or intra- injection site. arterial routes, but also most orally administered drugs The nerve adhesion molecules used to initiate the which must be absorbed into the bloodstream to reach uptake and transport of the pharmaceutical agents used target tissues, most intramuscularly administered drugs 25 according to the invention all have in common with each which are absorbed by the muscles blood vessels, other some tendency to promote uptake by neurons. many inhaled agents, most intranasally applied drugs, Some molecules with no particular affinity may be taken some rectally administered drugs such as paraldehyde, up and transported inefficiently by neurons. However, and many topically administered agents such as molecules which interact with and bind to specific cell transdermal nitroglycerine. There are, however some 30 surface markers or receptors on the nerve ending of the drugs which are delivered into and distributed by the selected nerve type are far more efficient and are pre- cerebrospinal fluid (intrathecal route), some oral drugs ferred. An additional degree of efficiency can be which are not absorbed (kaolin, oral vancomycin), a obtained when the compound also has some affinity for variety of topical and intravaginal agents, and some the cell surface of muscle cells, since this will promote administered percutaneously for local effect or intraar- 35 the depot effect at the injection site and decrease the ticular effect such as local anaesthetics, and locally tendency for the agent to diffuse away or be carried administered steroids. away by the bloodstream prior to uptake by the neuron. Various new drug forms and methods of use For investigative work involving animals, wheat described below involve delivery via an intraneural germ agglutinin (WGA) can be used to provide specifi- route. Access to this route may be obtained by oral 40 city for active uptake and transport. However, there are ingestion, topical, intra-articular, intrathecal, intrave- a wide range of nerve adhesion molecules which can be nous and, preferably, intramuscular administration. used to cause selective and active adsorptive endocyto- However, common to all these new methods, is that the sis into nerves. This class of nerve adhesion molecules dosing and active site of the agent is determined by a includes: route which involves endocytosis by nerve endings with 45 subsequent transport to a different and distant part of 1) Anti-synaptosomal monoclonal (and non-mono- the neuron. clonal) antibodies which are purified or generated In some experimental studies, various agents for based upon their affinity for nerve membranes. transport have been introduced by intraneural injection These can be made by using crude nerve homoge- or by application to the cut end of a severed nerve. In so nates and then testing for endocytotic efficacy in these methods, the 'blood brain barrier' due to the cultured neuroblasoma cells or by direct measure- perineurium is traversed by mechanical injury, and ment of uptake of radiolabeled forms after intramus- much of the uptake of tracer is due to direct presenta- cular injection in laboratory animals. These agents tion at cut nerve endings where specific nerve adhesion may involve entire antibodies of, preferably, the molecules may be irrelevant. The intramuscular tech- ss fragment of the antibody responsible for recognition nique (and also the intravenous application) depend without the Fc region. Similar considerations apply upon the natural defect in the perineurium which occurs for antibodies to dopamine-beta-hydroxylase. at axon terminus. Thus the blood brain barrier is natu-

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2) Various growth factors such as nerve growth fac- depending on whether primarily mixed or primarily sen- tors, epidermal growth factors, insulin-related sory nerves are to be imaged; however, various other growth factors and other proteins and peptides in molecules may be optimal for particular sorts of phar- this functional category which are known to have or maceutical tasks. discovered to have efficacy at causing the neuronal 5 These proteins and other nerve adhesion mole- uptake of themselves and of other agents with cules can be attached to the therapeutic or prophylactic which they are conjugated. moiety or to the coating (e.g. dextran coating) thereof by various methods including a periodate oxidation reac- 3) Lectins of various sorts which are proteins hav- tion carried out under mild conditions which does not ing a high degree of affinity for particular carbohy- 10 affect the oxidation state of ferrite crystals or the binding drate and other types of cell surface markers. This affinity of the targeting molecule. Other binding reac- is meant to include both plant lectins as well as var- tions include carbodiimide binding, glutaraldehyde bind- ious endogenous vertebrate, mammal, or human ing, biotin/avidin linking, or noncovalent coating with the lectins as are known or may be discovered. molecule of interest. Metal oxide particles provide a 15 core of useful size which can be securely coated by a 4) Fragments of neurotrophic viruses such as Her- variety of types of agents under mild, non-denaturing pes simplex, pseudorabies virus or poliovirus or conditions - they are therefore useful as a particle seed proteins or other markers from the viral coat even when their special metallic or magnetic properties responsible for their highly efficacious uptake and are not relevant to the task at hand. transport. These fragments or inactivated whole 20 All of the above conditions may be optimized so as viral particles, or cloned and produced copies of the to use this new drug administration method to efficiently crucial proteins are of particular interest for trans- deliver a specially designed pharmaceutical to a spe- neuronal transport. cific and preselected location within the nervous sys- tem. The sites will include peripheral nerves along their 5) Fragments of bacterial toxins such as the B- 25 continuous lengths, sensory ganglia, autonomic gan- chain of cholera toxin and non-toxic fragments of glia, spinal cord, and brainstem or the olefactory tract tetanus toxin such as the C fragment as well as system upon intranasal administration. When the route modified versions or cloned portions of other safely of administration is intrathecal, or topically upon the administered proteins with high neural affinity. brain during surgery or in the brain during open surgery 30 or during stereotactic surgical procedures, then a wide 6) A wide variety of peptides and small proteins range of sites within the central nervous system can be such as endorphins, vasoactive intestinal polypep- reached. In these cases of direct application in the tide, calcitonin gene-related peptide, cholecystoki- brain, it may be the purpose of the agent to be trans- nin, substance P, somatostatin, and neuropeptide Y ported to a surgically inaccessible site by axonal trans- or the relevant portions of such peptides for the 35 port from a surgically accessible site, or in any case to a encouragement of neuronal uptake and transport. site different from the site of application. This may also be used to demonstrate a successful incorporation of 7) Enzymes which are selectively endocytosed for grafted neural tissue which will become capable of synaptic recycling purposes such as acetylcho- transporting included agents from the graft to distant linesterase and dopamine beta hydroxylase or por- 40 sites. It will occasionally be helpful to achieve transport tions of these enzymes which are effective at with a small molecule such as an amino acid or protein inducing neuronal uptake. labelled with e.g. a positron emitting nuclide; however, in many of the applications, the preferred type of agent 8) Various cell adhesion molecules including pep- will include a particle which is up to 50 nanometers in tides, proteins and various simple and complex car- 45 diameter and may contain many atoms, crystal subu- bohydrates which are effective at promoting nits, or molecules of the active ingredient for diagnosis neuronal uptake and transport of conjugated phar- or therapy. maceutical agents. The exact relation between particle core size and resulting T2 relaxivity per microgram or iron is depend- 9) Neurotransmitters and neurotransmitter analogs 50so ent on a variety of factors in the particle synthesis and such as GABA, D-aspartate, dopamine, norepine- upon the biochemical environment in which the particle phrine, serotonin, and benzodiazepine drugs which must exist prior to imaging. Shen in SMRM 10:871 can be so constructed as to maintain their efficacy (1991) reports a relation between relaxivity and crystal in promoting transport after the conjugation to phar- size which greatly favours the use of the largest possible maceutical^ useful agents. 55 particle. Relaxivity data on particles produced by the inventor showed values of up to 4.0 x 105 sec"1M"1 Two optimal nerve adhesion molecules for most which are greater than those reported by Shen and are applications are transferrin and p-nerve growth factor consistent with this trend. Thus, an optimal relaxation

7 13 EP 0 548 157 B1 14

effect per amount of iron to be cleared by the nerve may depended upon vascular uptake from the injection site be achieved with the largest particles endocytosed and in order to achieve the best available distribution. Alter- transported efficiently. natively, cumbersome procedures in which special cath- When more diffuse effects are desired, an injection eters are threaded into place near the spinal cord have into the bloodstream or into the cerebro-spinal fluid can s been undertaken with attendant dangers of spinal cord be used to cause uptake by a specific type of neuron injury. There has never been any route except by whole wherever it contacts the fluid. However, for specific body vascular distribution to deliver any drugs to gan- delivery to an individual peripheral nerve, the intramus- glia. cular technique will be preferred. However, by taking advantage of the drug distribu- The usefulness of the intramuscular administration 10 tions achievable by axonal transport where particulates derives from the fact that individual axons which enter a are used, a dramatic change in pharmaceutical practice muscle divide multiply and fan out to innervate numer- can be achieved. When the desired drug is trapped in a ous individual muscle cells making up a 'motor unit'. polymer, liposome, or protein nanosphere with an Such a motor unit is a group of muscle cells which fire attached nerve adhesion molecule, it becomes possible simultaneously whenever their shared axon delivers a is to carry out an intramuscular injection of an extremely depolarization signal. Due to the dictates of muscle small amount at a location to which the desired part of energetics and control mechanisms, the cells of a unit the nervous system is connected by a peripheral nerve. are generally scattered through a muscle rather than Most of the injectate will stay in the muscle at the site of concentrated in a single area. Thus an injection at any injection while the drug particles are ingested by nerve one site will usually be near the axon termini of a large 20 endings and transported to the ganglionic or spinal cord number of different motor units. This presents the injec- sites toward which treatment is to be directed. tate with a relatively large axon terminal surface area Liposomes with phosphatidlycholine and phos- and provides access to a mix of axons distributed phatidylethanolamine (e.g. as described by Grant in evenly through the incoming nerve. Additionally, Mag. Res. Med. 11:236-243 (1989)), derivatized for because all muscles have a rich sensory supply to mus- 25 attachment of a nerve adhesion molecule can be pre- cle spindle tension, length, and rate receptors, distrib- pared in the necessary size range for efficient uptake uted through the muscle, there is simultaneous access and neuronal transport. In this fashion, a wide variety of to sensory and to motor fibers. hydrophilic and hydrophobic drugs can be packaged for When l125-NGF was injected in muscle, high delivery direct to their intended neural sites of action intraneural concentrations were achieved because of 30 while minimizing systemic dose. uptake by the various types of sensory endings in the The particles yield a large amplification of the spindle organs of the muscle. uptake process since many drug molecules are Intramuscular injections with intraneuronal pharma- ingested by the cell with each event and helps minimize ceuticals present no greater risk of nerve injury than any spread of the drug away from the injection site. other sort of routine intramuscular injection. However, it 35 While there are a number of well known methods is well documented that any fine axonal branch which is for producing particulate drug carriers, many of these injured causes no lasting muscle impairment. This is are not suitable for intraneural drug delivery. Many of the because of the short distance of regrowth and the ten- known techniques are not applicable because they dency for axon terminal branches to recolonize any result in the production of particles which are far too denervated muscle cell. It should be noted that although 40 large to permit access to the axon terminus in general injury to some axonal branches may occur, the distribu- and synaptic cleft in particular as needed to promote tion of tracer in nerve and in cell bodies upon histologic neural uptake. For instance, many polylactic acid parti- inspection of the spinal cord is consistent with uptake by cles can only be produced in sizes near 100 microns intact cells rather than by any important contribution which is three orders of magnitude too large. Liposomes from uptake by injured cells. 45 similarly tend to be too large unless they are produced It is often necessary to administer drugs whose as small unilamellar vesicles (SUV) which require harsh intended site of action is in the spinal cord or dorsal root physical and chemical conditions for synthesis. ganglia (DRG). However, in the past there has been no Polycyanoacrylate/vinyl particulates (many of easy way of safely administering such drugs near their which are termed "latex" microspheres or nanoparti- site of action. While injections are commonly used to so cles) can be readily produced in the appropriate size achieve local drug effects in muscle or in joints, it is range, but require the use of organic solvents and other exceedingly hazardous to introduce a needle percuta- treatments which are destructive to many biological neously and blindly into the vicinity of the spinal cord or molecules of therapeutic interest. Albumin or other pro- sensory ganglia. Therefore, in order to achieve pharma- tein microspheres of useful size can be produced from cologically efficacious doses of various drugs at these 55 sonicated emulsions, but these require denaturing by locations it has been necessary to give very high sys- heating over 100°C or chemical crosslinking to achieve temic doses by intravenous and oral routes, or by intra- stability and this similarly limits the range of potential muscular routes wherein the actual delivery of the drug pharmaceuticals.

8 15 EP 0 548 157 B1 16

Metal oxide particles are particularly convenient as the crystals at physiological pH in biochemically tolera- drug carriers. They readily form stable colloids of appro- ble buffer solutions. priate size and can be coated by a wide variety of mole- Thus a suitable method of producing a physiologi- cules. When the particles are prepared by adding a cally tolerable particulate metal oxide comprises precip- strong base to the metal chlorides in saturated dextran, 5 itating said oxide from a biologically tolerable, a strong bond between the particle and the dextran is physiological pH buffered solution, optionally containing formed. Subsequently, some proteins can be covalently a coating agent whereby coated particles are formed. bound to the dextran molecules. However, this type of For instance, a strong buffer such as 1 molar synthesis precludes the use of any drugs which cannot HEPES pH 7.4 is prepared with the desired coating mol- tolerate the strong acidity of the metal salts or the rapid w ecules in the buffer solution. The mixed metal salt solu- shift to a strong alkali typically used in the precipitation tion, either with or without dextran, is then added to the reaction. buffer solution in dropwise fashion. This method has The predominant solution to this problem has been resulted in the production of stable coated colloids pro- to precipitate the particles with no coating, and then duced at physiological pH. This new method of produc- apply the coating at a later step under more mild condi- 15 tion of these crystals greatly broadens the range of tions. However, whether NaOH or NH4OH is used, and pharmaceuticals which can be included in the particle whether the metal salts are added to the alkali or vice coat of these 10-100 nm particles for delivery by versa, uncoated particles always aggregate and despite intraneural or other routes. high power sonication of the resuspended pellets, it is Treatment of HTLV-I Associated Myelopathy: A focal difficult to produce a stable colloid. 20 infection of the spinal cord associated with HTLV-1 Precipitation by addition to ammonia is more effec- (Human T-Cell Leukaemia Virus, type 1) in which there tive than using NaOH because of a clear peptizing effect is involvement of the phagocytic microglia and oli- reconfirmed in experiments conducted by the inventor. godendrocytes as well as of neurons has presented a Such uncoated ammonia precipitates become stable for very forbidding picture for any possible treatment centrifugal ultrafiltration even without sonification. This 25 options. What few anti-viral agents have been available preparation is sufficiently reactive as to permit reliably have poor penetration of the blood brain barrier and so stable but non-covalent binding of a wide range of mol- must be administered intravenously in very high con- ecules. In one series of preparations made by the inven- centration to achieve potentially therapeutic dose levels tor, coatings were made with alpha-cyclodextrin which in the spinal cord. Similarly, intrathecal administration is capable of binding relatively non-polar drugs and is 30 into the CSF allows uncontrolled spread of the anti-viral well known as a drug vehicle. Further, particles were agent throughout the CNS, affecting various sites of prepared which were first used to adsorb tritiated dex- reduced blood brain barrier (e.g. median eminence of amethasone and then to adsorb WGA. These particles the hypothalamus, pineal gland, and area prostrema) were finally coated with dextran or bovine serum albu- more than the infected site. Further, intrathecal^ admin- min to cover unused reactive sites on the particle sur- 35 istered drugs tend to be swept out of the CSF into the face. Preparations of this sort were subjected to blood stream over a relatively rapid time course com- repeated washing, ultrafiltration, and N-acetylglu- pared to what is required for anti-viral therapy. cosamine affinity purification. These steps demon- However, because of the focal nature of the lesion, strated a very slow release of the labelled it is quite easy to identify the dermatome supplied by the dexamethasone, but also showed that the affinity puri- 40 involved location of the spinal cord. Therefore, by under- fied particles carried with them a high concentration of taking intramuscular injections in neck/back muscles as bound dexamethasone. well as in muscles placed at greater distances from the Particles of this sort can be used to introduce this spinal cord, it is possible to set up a continuous inflow of helpful steroid drug into neurons for delivery to areas of specific medication into the CNS neurons, and by trans- spinal cord injury. As has also been shown by the inven- 45 cellular transport, into the surrounding oligodendrocytes tor, such particles are extruded from the neuron into the and microglia. The active agents used can include small endoneurial space and so are then placed in a position molecules conjugated to a nerve adhesion molecule, to properly interact with and activate cell surface gluco- small liposomes or other types of nanoparticles carrying corticoid receptors at otherwise inaccessible locations antiviral drugs. The administration of steroids by the inside the blood brain barrier. so same route can also help to reduce injury due to inflam- An entirely novel means of producing metal oxide matory components of the disease. particles under mild conditions suitable for delicate pro- Where transport in the affected dermatome is teins and peptides has also been developed by the severely impaired by extensive cell death, injections on inventor. This method is based on the tendency of the contralateral side of the body and at dermatomes mixed iron salts to commence precipitation when the pH ss below and above those affected will still bring the drugs is raised above 4.0. Thus, rather than raising the pH of into a relatively high concentration at close proximity to the solution to pH 9, 10 or 1 1 as is done in all previously the lesion and usefully across the blood brain barrier. known methods, the new method involves precipitating Pain: Among the most common of all tasks faced by the

9 17 EP 0 548 157 B1 18 physician or surgeon is the treatment of a severe local- been appreciated that extremely high doses of methyl- ized pain whose duration will probably be only a few prednisolone given intravenously over the first 24 to 48 days but which will cause considerable distress and dis- hours post-injury can be significantly beneficial to even- comfort to the patient. This might be from a bone frac- tual neurological outcome. Of course, the actual site of ture, ankle sprain, a surgical incision, abscess, severe 5 action is at the injury location in the spinal cord, but the back muscle spasm, exacerbation of arthritis, dental dose is limited by the extremely large amounts of steroid extraction, burn, or tumour among other problems. The which must be distributed to the rest of the body for no available pharmaceutical options at present fall into four useful purpose. main categories, an oral drug such as aspirin or aceta- These steroids can be incorporated into liposomes minophen which acts at the site of injury by altering the 10 or other particles of appropriate size, conjugated to a effects of prostacyclin related pain mediators, a range of nerve adhesion molecule and injected intramuscularly oral or locally injected anti-inflammatory drugs of both in back or neck muscles at the level of sensory or motor steroidal and non-steroidal composition, locally injected loss. When this is done, they are transported directly anaesthetics such as marcaine or lignocaine which into the nervous system at a continuing flow and arrive reduce neural activity, and the systemic administration is precisely at the site of injury. Wherever actual nerve of opiates or opiate analogs by oral, intramuscular or compression or injury has occurred, the transported intravenous routes. There are a few extraordinary treat- agent will accumulate and automatically achieve partic- ments most commonly undertaken in cancer patients ularly high concentrations. Drug leaking out of torn or such as the placement of continuous infusion morphine inflamed cells, and extruded into the nervous tissue cannulas near the spinal cord but these are little used 20 around the affected neurons will act on other injured tis- because of difficulty in preventing infection and the com- sues in the vicinity. This will also assure good delivery plexity of placing them. Further, such treatments cannot into the spinal cord grey matter even when the fracture be used for pain in the arm or neck because free opiates has caused venous congestion which is slowing vascu- in spinal fluid of the upper spinal cord may reach the lar delivery to the most affected areas. medulla and cause respiratory arrest. 25 Certain pharmaceutical agents useful in the If opiate or anti-inflammatory drugs are included in method according to the invention are already known - particles, bound to nerve adhesion molecules, and then others may be produced by methods analogous to injected in the dermatome/myotome which is involved in those used for producing the known agents, e.g. for par- the pain, then an extremely efficient distribution can be ticulate agents: obtaining particles of a matrix material achieved. This permits the extended administration of 30 comprising a physiologically active agent or diagnostic an opiate at an extremely high concentration in the dor- marker; optionally coating said particles with a physio- sal root ganglion and dorsal root entry zone of the spinal logically tolerable optionally biodegradable coating cord at the involved level, while resulting in negligible material, e.g. a natural or synthetic polymer or derivative systemic levels of the drug. For opiates, this avoids tol- thereof such as latex, polylactic acid, proteins, albumin, erance, sedation, respiratory depression and addiction 35 polysaccharides, starches, dextrans, polymerized sugar and provides for steady long term administration over alcohols, etc (see for example EP-A-1 84899 (Jacob- days after a single injection. For steroids and non-ster- sen)); and conjugating said particle (optionally via cou- oidal anti-inflammatory drugs, this will help reduce the pling to a said coating, optionally after appropriate risk of gastric irritation and internal bleeding as well as derivatization thereof e.g. to provide a binding site or to the other side effects of steroids. 40 block excess binding sites) to a nerve adhesion mole- Some types of chronic pain syndromes are remark- cule, preferably with a NAM: particle ratio of up to 10, ably resistant to all standard pain medications including especially up to 5 more especially up to 2 and most pref- the various opiates and opiate derivatives. The proto- erably about 1 ; optionally separating NAM-conjugated type for this sort of pain is trigeminal neuralgia and particles so formed from unconjugated particles, preter- indeed the sine qua non of this syndrome is that the ms ably by size separation, especially preferably by pain can be relieved only by anticonvulsant medications repeated size separation followed by at least one affinity such as carbamazepine. This sort of chronic pain is separation; optionally sterilizing the NAM-conjugated often viewed as a kind of focal sensory epilepsy. The particles, if desired after formulation thereof with a phar- problem in treating such pain is that there is often great maceutical carrier and optionally with further conven- difficulty in achieving adequate doses at the dorsal root 50 tional pharmaceutical excipients, e.g. viscosity entry zone of the spinal cord or the trigeminal nuclei in enhancing agents, pH regulators, osmolality adjusting the brainstem without causing unacceptable systemic agents, etc. side effects. By use of pharmaceutical agents according The matrix material used may be an inorganic to the invention, agents such as carbamazepine can be matrix, e.g. a metal oxide, or an organic matrix, e.g. a delivered via an intraneural route after intramuscular 55 polymer such as a cross-linked starch or dextran, and it injection or intradermal injection. may serve as a carrier for the physiologically active Spinal Cord Injury: Although acute spinal cord injury agent or diagnostic marker or it may itself serve as the has long proven very difficult to treat, it has recently active agent or marker, as would for example be the

10 19 EP 0 548 157 B1 20 case with superparamagnetic ferrite crystals. Claims Incorporation of the agent or marker within a carrier matrix can be achieved by conventional techniques, for 1. Use of a therapeutic agent comprising particles example by co-precipitation, by steeping a porous having, coupled thereto, a nerve adhesion moiety matrix material to impregnate it with the desired agent s and a physiologically-active substance, the nerve or marker, by exposing the agent to ultrasonically sus- adhesion moiety serving to promote neuronal pended, uncoated metal oxide particles, or by means of endocytosis of said agent, the agent being capable the buffered precipitation technique described herein. of axonal transport following neuronal endocytosis, The matrix particles should desirably be relatively for the preparation of a medicament for providing uniformly dimensioned, e.g. within the ranges dis- to the therapeutic effect of the physiologically-accept- cussed above, and this may be achieved for example by able substance on administration into a vascular- conventional screening or particle precipitation tech- ised peripherally innervated tissue site or into other niques. Monodisperse particles will be preferred. tissue sites innervated by a spinal root. Where the agent used according to the invention is non-particulate it may again be produced by conven- is 2. Use as claimed in claim 1 , wherein the agent has a tional techniques, e.g. by binding a desired nuclide mean particle size of 5-100 nm. directly or via a chelant molecule to a NAM or by binding a chromophore or f luorophore or a physiologically active 3. Use as claimed in claim 2, wherein the particle size molecule to a NAM, optionally and indeed preferably so is 10-50 nm. as to provide a biodegradable bonding which will permit 20 liberation of the physiologically active agent after endo- 4. Use as claimed in any preceding claim, wherein the cytosis. particles have a spinel structure. For axonal delivery of therapeutic or prophylactic substances, in certain cases it may be desirable in the 5. Use as claimed in any preceding claim, wherein the method of the invention to select physiologically active 25 particles are superparamagnetic. substances which occur naturally in neurons or which are analogues of such naturally-occurring substances. 6. Use as claimed in any preceding claim, wherein the A suitable process for the preparation of the modi- particles incorporate radionuclides. fied spinel and garnet particles used in the invention comprises precipitating di and trivalent metal ions of 30 7. Use as claimed in any preceding claim, wherein the ionic radii such as to permit crystals of spinel or garnet particles are coated with dextran. structure to form, said precipitation being from a solu- tion containing scandium, a radioactive yttrium isotope, Patentanspruche a sixth period metal, a high MR receptivity nuclide or an element having a desired therapeutic or prophylactic 35 1. Verwendung eines therapeutischen Mittels, wel- activity. ches Teilchen umfaBt, an die eine Nervadhasions- In these particle precipitation processes the physio- Einheit und eine physiologisch aktive Substanz logically active, marker, or high positron affinity ele- gekoppelt sind, wobei die Nervadhasions-Einheit ments to be incorporated into the particles may dazu dient, eine neuronale Endozytose des Mittels themselves be in solution or alternatively they may be in 40 zu fordern, wobei das Mittel im AnschluB an die fine "seed" crystals which become included in the pre- neuronale Endozytose zu einem Neurit-Transport cipitating particles. in der Lage ist, zur Herstellung eines Medikaments For administration in vivo, the dosages used will fur die Bereitstellung der therapeutischen Wirkung clearly depend upon a wide range of factors such as the der physiologisch akzeptablen Substanz bei Verab- patient's weight, the specificity of the NAM (for NAM- 45 reichung in eine vaskularisierte, peripher inner- conjugated agents), the nature of the physiologically vierte Gewebestelle oder in andere durch eine active or diagnostic marker component of the pharma- Spinalwurzel innervierte Gewebestellen. ceutical agent, the extent or severity of the injury or ail- ment that is being investigated or treated, the distance 2. Verwendung nach Anspruch 1, worin das Mittel over which axonal transport is required, etc. The appro- so eine mittlere TeilchengroBe von 5 - 100 nm auf- priate dosage however can readily be determined tak- weist. ing these factors into account. However the intramuscular administration accord- 3. Verwendung nach Anspruch 2, worin die Teilchen- ing to the invention of NAM-targetted agents offers the groBe 10 - 50 nm betragt. possibility of very efficient and very specific delivery. ss 4. Verwendung nach irgendeinem vorhergehenden Anspruch, worin die Teilchen eine Spinellstruktur aufweisen.

11 21 EP 0 548 157 B1

5. Verwendung nach irgendeinem vorhergehenden Anspruch, worin die Teilchen superparamagnetisch sind.

6. Verwendung nach irgendeinem vorhergehenden s Anspruch, worin die Teilchen Radionuklide enthal- ten.

7. Verwendung nach irgendeinem vorhergehenden Anspruch, worin die Teilchen mit Dextran beschich- 10 tet sind.

Revendications

1. Utilisation d'un agent therapeutique comprenant is des particules auxquelles sont couples un groupe- ment d'adhesion neurale et une substance physio- logiquement active, le groupement d'adhesion neurale servant a activer I'endocytose neuronale de cet agent, agent qui est apte au transport axonal 20 apres endocytose neuronale, pour la preparation d'un medicament destine a engendrer I'effet thera- peutique de la substance physiologiquement acceptable par administration dans un site tissu- laire vascularise a innervation peripherique ou 25 dans d'autres sites tissulaires innerves par une racine spinale.

2. Utilisation suivant la revendication 1 , dans laquelle I'agent a un diametre moyen de particules de 5 a 30 100 nm.

3. Utilisation suivant la revendication 2, dans laquelle le diametre de particules est compris dans I'inter- valle de 10 a 50 nm. 35

4. Utilisation suivant I'une quelconque des revendica- tions precedentes, dans laquelle les particules ont une structure de spinelle. 40 5. Utilisation suivant I'une quelconque des revendica- tions precedentes, dans laquelle les particules sont des particules supraparamagnetiques.

6. Utilisation suivant I'une quelconque des revendica- 45 tions precedentes, dans laquelle les particules ren- ferment des radionuclides.

7. Utilisation suivant I'une quelconque des revendica- tions precedentes, dans laquelle les particules sont so enrobees avec du dextrane.

55