U.S. patent application number 10/347672 was filed with the patent office on 2003-12-11 for particulate agents.
This patent application is currently assigned to Syngenix Limited. Invention is credited to Filler, Aaron Gershon.
Application Number | 20030228260 10/347672 |
Document ID | / |
Family ID | 10682179 |
Filed Date | 2003-12-11 |
United States Patent
Application |
20030228260 |
Kind Code |
A1 |
Filler, Aaron Gershon |
December 11, 2003 |
Particulate agents
Abstract
A novel means of pharmaceutical delivery for therapy of
prophylaxis or to assist surgical or diagnostic operations on the
living body is provided by neuronal endocytosis and axonal
transport following pharmaceutical administration into
vascularized, peripherally innervated tissue, e.g., intramuscular
injections of a nerve adhesion molecule in coupled particle
comprising a physiologically active substance or a diagnostic
marker.
Inventors: |
Filler, Aaron Gershon;
(Santa Monica, CA) |
Correspondence
Address: |
FISH & NEAVE
1251 AVENUE OF THE AMERICAS
50TH FLOOR
NEW YORK
NY
10020-1105
US
|
Assignee: |
Syngenix Limited
|
Family ID: |
10682179 |
Appl. No.: |
10/347672 |
Filed: |
January 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10347672 |
Jan 16, 2003 |
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09359884 |
Jul 26, 1999 |
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6562318 |
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09359884 |
Jul 26, 1999 |
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08473697 |
Jun 7, 1995 |
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5948384 |
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08473697 |
Jun 7, 1995 |
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07988919 |
Apr 5, 1993 |
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Current U.S.
Class: |
424/9.3 |
Current CPC
Class: |
A61L 24/0015 20130101;
A61L 2300/624 20130101; A61K 49/1863 20130101; A61K 49/1818
20130101; B82Y 5/00 20130101; C12Q 1/48 20130101; A61K 9/0019
20130101; A61L 2300/44 20130101; A61K 49/1869 20130101; A61K
49/1857 20130101; A61K 9/0024 20130101; A61L 2300/102 20130101;
A61K 2121/00 20130101; A61K 47/42 20130101; A61K 47/6923 20170801;
A61K 51/1255 20130101; A61K 51/1213 20130101 |
Class at
Publication: |
424/9.3 |
International
Class: |
A61K 049/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 14, 1990 |
GB |
9020075.9 |
Oct 30, 1990 |
GB |
9023580.5 |
Dec 17, 1990 |
GB |
9027293.1 |
Jan 7, 1991 |
GB |
9100233.7 |
Jan 16, 1991 |
GB |
9100981.1 |
Jan 31, 1991 |
GB |
9102146.9 |
May 20, 1991 |
GB |
9110876.1 |
Jul 30, 1991 |
GB |
9116373.3 |
Aug 19, 1991 |
GB |
9117851.7 |
Aug 30, 1991 |
GB |
9118676.7 |
Claims
1. A method of treatment of the living human or non-human body to
effect a desired therapeutic or prophylactic treatment or assist
diagnostic investigation or surgical treatment thereof, said method
comprising administering into a vascularized peripherally
innervated tissue site or into other tissue sites innervated by a
spinal root a particulate pharmaceutical agent comprising a nerve
adhesion moiety serving to promote neuronal endocytosis of said
agent and a physiologically active or diagnostic marker moiety
capable of axonal transport from said tissue site.
2. The use of a particulate pharmaceutical agent comprising a nerve
adhesion moiety serving to promote neuronal endocytosis of said
agent and a physiologically active or diagnostic marker moiety
capable of axonal transport following neuronal endocytosis of said
agent for the preparation of a therapeutic, prophylactic or
diagnostic composition for use on administration into a
vascularized peripherally innervated tissue site or into other
tissue sites innervated by a spinal root in a method of treatment
of the living human or non-human body to effect a desired
therapeutic or prophylactic treatment or assist diagnostic
investigation or surgical treatment thereof.
3. A pharmaceutical agent comprising a nerve adhesion molecule
coupled to an optionally-coated, particulate, physiologically
active or diagnostically marked substance, with the provisos that
for diagnostically marked substances the substance is a metal
oxide, metal sulphide or alloy.
4. A composition as claimed in claim 3 wherein said particles have
a mean particle size of 10-50 nm.
5. A composition as claimed-in either of claims 3 and 4 wherein
said particles have a spinel structure.
6. A composition as claimed in any one of claims 3 to 5 wherein
said particles are superparamagnetic.
7. A composition as claimed in many one of claims 3 to 6 wherein
said particles incorporate radionuclides.
8. A physiologically tolerable particulate metal oxide, metal
sulphide or alloy with incorporated therein a positron emitter
radionuclide.
9. A particulate metal oxide, metal sulphide or alloy as claimed in
claim 8 further containing atoms of an element having a higher
positron affinity than iron.
10. A particulate metal oxide, metal sulphide or alloy as claimed
in claim 9 containing lithium or zinc.
11. A particulate metal oxide as claimed in any one of claims 8 to
10 having a spinel or garnet structure.
12. A particulate metal oxide as claimed in claim 11 being a
ferrite.
13. A particulate metal oxide, metal sulphide or alloy as claimed
in any one of claims 8 to 12 containing Mn.sup.52, Fe.sup.52 or
Sc.sup.43.
14. A particulate metal oxide as claimed in any one of claims 8 to
13,for use in medicine.
15. A physiologically tolerable particulate garnet or spinel with
incorporated therein atoms of scandium, of a radioactive yttrium
isotope of a sixth period metal of a high MR receptivity nuclide or
of an element which on particle degradation has a desired
therapeutic or prophylactic activity.
16. A particulate spinel or garnet as claimed in claim 15
containing atoms of scandium.
17. A particulate spinel or garnet as claimed in either of claims
15 and 16 containing atoms of a lanthanide.
18. A particulate spinel or garnet as claimed in any one of claims
15 to 16 having a mean particle size of less than 100 nm and
containing on average at least 100 atoms of scandium or a sixth
period element per particle.
19. A particulate spinel or garnet as claimed in any one of claims
15 to 18 for use in medicine.
20. A process for the preparation of a particulate pharmaceutical
agent as claimed in claim 8 which process comprises conjugating a
NAM to an optionally coated particulate physiologically active or
diagnostically marked substance.
21. A process as claimed in claim 20 wherein dextran coated
particles are conjugated to a NAM.
22. A process as claimed in claim 21 wherein said dextran coated
particles are subjected to periodation prior to conjugation to the
NAM.
23. A process as claimed in any one of claims 20 to 22 wherein the
NAM is WGA.
24. A process as claimed in any one of claims 20 to 23 wherein the
NAM-conjugated particles are subjected to repeated size separations
and at least one affinity separation.
25. A process for the preparation of a physiologically tolerable
marked metal oxide, metal sulphide or alloy as claimed in claim 15
which comprises precipitating a said metal oxide or sulphide from a
solution containing a positron emitter nuclide and if desired
reducing the precipitate.
26. A process for the preparation of a modified spinel or garnet
particle as claimed in claim 15 which process comprises
precipitating di and trivalent metal ions of ionic radii such as to
permit crystals of spinel or garnet structure to form, said
precipitation being from a solution containing scandium, a
radioactive yttrium isotope, a sixth period metal, a high MR
receptivity nuclide or an element having a desired therapeutic or
prophylactic activity.
27. A process as claimed in either of claims 25 and 26 wherein the
element to be included in the precipitated particles is provided in
seed crystals which become incorporated in the precipitated
particles.
28. A radiotherapeutic composition comprising a cell adhesion
moiety-coupled radionuclide and a tissue glue.
29. A method of producing a physiologically tolerable particulate
metal oxide, said method comprising precipitating said oxide from a
biologically tolerable, physiological pH buffered solution,
optionally containing a coating agent whereby coated particles are
formed.
Description
PARTICULATE AGENTS
[0001] This invention relates to novel particulate agents for use
in diagnostics and therapy, especially in diagnostic imaging, and
more particularly diagnostic imaging or therapeutic treatment via
the neural system.
[0002] In the living body, pain, paralysis and neural dysfunction
can be inferred from electrical studies such as EMG, NCV and SSEP,
but these kinds of assessment have continued to prove awkward and
imprecise. While CT and MRI have made it possible to diagnose a
wide variety of structural problems affecting the brain and spinal
chord, and while studies on excised tissue and post mortem studies
have enabled neuronal pathways to be traced, there is currently
virtually no effective means by which diagnostic functional imaging
of the neural system, and especially the peripheral nervous system,
can be achieved in vivo.
[0003] Diagnostic imaging of nervous system function has a
multitude of potential applications which will readily be apparent
to the physician or neurosurgeon and many of these are discussed
further below. Thus for example the possibilities would exist to
visualize the impact of neurofibrillary tangles as they develop, to
locate and assess nerve compressions, to verify the effectiveness.
of surgical vagotomy and to measure the response of the injured
spinal cord to attempts at treatment.
[0004] It has now been realized that particulate agents suitable
for use as contrast agents in diagnostic imaging modalities,
especially MRI and PET, may be conjugated to nerve adhesion
molecules and that following administration into body tissue,
especially muscle, such agents are endocytosed by neurons having
axon termini in that tissue and carried along the axons by
axoplasmic flow thus allowing imaging of the axons and of the
nerves of which they form part.
[0005] The endocytosis of nerve adhesion molecule (NAM) labelled
agents can also clearly be utilized for the remote delivery of
therapeutic agents, i.e. axoplasmic flow can serve to transport a
therapeutically active agent comprising a nerve adhesion moiety
from its administration site in tissue such as muscle to a remote
site where it exerts its pharmacological effect. This is of
particular interest where the sensitivity or accessibility of the
remote site is such as to prevent direct administration of the
pharmaceutical.
[0006] Thus viewed from one aspect the invention provides a method
of treatment of the living human or non-human (preferably
mammalian) body to effect a desired therapeutic or prophylactic
treatment or assist diagnostic investigation or surgical treatment
thereof, said method comprising administering into a vascularized
peripherally innervated tissue site (preferably a muscle although
possibly also other tissue sites innervated by cranial, peripheral
or autonomic nerves) or into other tissue sites innervated by a
spinal root a particulate pharmaceutical agent comprising a nerve
adhesion moiety serving to promote neuronal endocytosis of said
agent and a physiologically active or diagnostic marker moiety
capable of axonal-transport from said tissue site, and, where said
method is to assist diagnostic investigation or surgical treatment,
detecting axonal-transport within said living body of a said agent
having a diagnostic marker moiety, preferably by generating an
image of at least part of said body.
[0007] Viewed from another aspect the invention provides the use of
a particulate pharmaceutical agent comprising a nerve adhesion
moiety serving to promote neuronal endocytosis of said agent and a
physiologically active or diagnostic marker moiety capable of
axonal transport following neuronal endocytosis of said agent for
the preparation of a therapeutic, prophylactic or diagnostic
composition for use on administration into vascularized
peripherally innervated tissue or into other tissue sites
innervated by a spinal root in a method of treatment of the living
human or non-human body to effect a desired therapeutic or
prophylactic treatment or assist diagnostic investigation or
surgical treatment thereof.
[0008] Especially in the case of therapeutic or prophylactic
treatment, the pharmaceutical agent is preferably administered into
a tissue site, such as a muscle, having a volume of at least about
ten times that of the group of nerve cells which are to transport
the agent.
[0009] The term pharmaceutical agent is used herein to designate a
substance capable of exerting a desired therapeutic or prophylactic
effect and/or acting as a tracer, label, contrast agent or other
diagnostic marker detectable in the intact living mammal. This
substance may be a single compound but more generally will comprise
a NAM coupled directly or indirectly to a physiologically active or
diagnostically marked compound. Diagnostic marking may for example
be with radiolabels, chromophores, fluorophores, by virtue of
magnetic properties, or with atoms or structures capable of higher
or lower radiation (eg. X-ray or sound) absorbance or reflectance
than surrounding body tissue. Particulate NAM-coupled moieties may
be coated or uncoated and if coated the coating may be selected to
be broken down within the neuron after endocytosis, either slowly
or more rapidly, or to be maintained during axonal transport.
[0010] For the purposes of the present invention it should be
appreciated that while natural or synthetic, essentially inert,
organic polymer particles (such as dextran coated microspheres or
latex nanospheres) are capable of being endocytosed, these organic
polymers unlike more specific and complicated molecules such as
proteins, antibodies and antibody fragments are not considered to
be nerve adhesion molecules.
[0011] The mean particle size for the particulate pharmaceutical
agents used in the invention is conveniently in the range 5 to 100
nm, especially 8-70 nm, more particularly 10 to 50 nm and
preferably about 20-30 nm.
[0012] Many of the pharmaceutical agents that may be used in the
method of invention are themselves novel and viewed from a further
aspect the invention provides a pharmaceutical agent comprising a
nerve adhesion molecule coupled (directly or indirectly) to an
optionally-coated, particulate, physiologically active or
diagnostically marked substance, with the proviso that for
diagnostically marked substances the substance is a metal oxide,
metal sulphide or alloy.
[0013] For use to assist diagnosis, the pharmaceutical agent
preferably has a diagnostic marker that can be detected
non-invasively, eg. by virtue of its radiation emission or
absorption characteristics or by virtue of its magnetic
characteristics. For use in assisting surgery, for example to
enable important nerve pathways passing through or near a wound
site or other site undergoing surgical intervention, chromophores
and fluorophores can also be used as diagnostic markers and in this
instance in particular the use of non-particulate as well as of
particulate pharmaceutical agents might be contemplated.
[0014] One especially important group of pharmaceutical agents for
use according to the invention is that of NAM-coupled particulate
inorganic compounds, for example metal oxides, sulphides or alloys,
where the inorganic material is selected for its magnetic
properties, in particular ferri- and ferromagnetism and more
particularly superparamagnetism, or includes within an otherwise
essentially inert matrix atoms or molecules which are released
gradually from the matrix to exert a therapeutic or prophylactic
effect or which function as diagnostic markers, eg. radioisotopes
or nuclides detectable upon MR spectroscopy. Many metal oxide
structures may be utilized as the inorganic particles, and spinels
and garnets have been found to be particularly useful in this
regard. It should however be stressed that other well known inert
and Preferably essentially water insoluble metal compounds may be
used, especially those having or capable of being doped to exhibit
cooperative magnetic properties and those having lattices such as
permit desired radioisotopes to be included. By alloys, mixed
metals are of course included. Organic particulate matrices may
also be used to accommodate a therapeutic compound or a diagnostic
marker.
[0015] As is clear from the above, this invention is especially
concerned with improvements in particulate pharmaceuticals which
surprisingly result in providing access to new patterns of
distribution within the body which were never previously possible.
As a result of these new patterns of distribution, a number of
previously intractable problems in medical diagnosis and treatment
have now been solved.
[0016] For the particulate pharmaceutical agents in particular, the
improvements include 1) improved control of particle size, 2)
development of an effective means of affinity purification of the
particles, 3) demonstration of a means of filter sterilization of
the concentrated product late in the synthesis, and 4) widening the
array of elements and concentration of those elements applied to
medical uses in metal oxide or sulphide crystals or in alloys.
[0017] One of the most important consequences of these advances is
the development of agents which can be delivered by, and make use
of an entirely novel intraneural pharmaceutical route (IPR). In
addition, these advances greatly simplify and reduce the cost of
production of related particulate agents with previously known
uses.
[0018] In a preferred embodiment, the agent comprises particles
with a core metal oxide crystal, eg. of spinel or garnet structure,
coated for example by dextran carbohydrate wherein the total size
of the coated particle is between 100 and 500 Angstroms (10 to 50
nanometers) and where a targeting moiety (TM) is chemically bound
to the coating in low concentration of TM per particle, preferably
1:1. The agent should preferably be virtually free of particles
lacking an active TM, and compositions thereof are preferably
sterilized by 0.2 or 0.1 micron microfiltration after final
synthesis, affinity purification and concentration.
[0019] The uses of a given version of the agent depend upon the
elements and isotopes (nuclides) used in the initial precipitation
step in which the metal oxide crystal core is precipitated and
coated and also upon the type of targeting moiety used. For each
use, the nuclide and targeting moiety may be selected to benefit
both from the general advantages of the simplicities of the
preparatory method and to take advantage of the new types of
pharmaceutical distribution which can be achieved by materials
prepared in this way.
[0020] The inorganic particles in the preferred pharmaceutical
agents used according to the invention generally fall into one of
five categories:
[0021] i) particles exhibiting cooperative magnetic properties, in
particular superparamagnetism, eg. ferrite particles such as
inverse spinel ferrites, and thus detectable by magnetic resonance
or magnetometric methods;
[0022] ii) particles incorporating a gamma or electron emitter
radionuclide, and thus detectable by gamma detectors, scintigraphy
or SPECT (single photon emission computed tomography) or thus
capable of causing radiation treatment effects;
[0023] iii) particles incorporating an element of non-zero nuclear
spin, eg. scandium, capable of being detected by magnetic resonance
spectrometry;
[0024] iv) particles incorporating a positron (.beta..sup.+)
emitter radionuclide and thus capable of detection by PET (positron
emission tomography)
[0025] v) particles incorporating a compound or element capable on
release, eg. during degradation of the particle, of effecting a
desired therapeutic or prophylactic effect.
[0026] Metal oxide particles of the first two types are clearly
known but scandium containing spinels or garnets and the particles
of the latter two types are novel and in themselves form further
aspects of the present invention.
[0027] Thus viewed from a further aspect the invention provides a
physiologically tolerable particulate metal oxide, metal sulphide
or alloy with incorporated therein a positron emitter radionuclide
and preferably an element having high positron affinity (eg. higher
than that of iron, for example lithium or zinc). Such high positron
affinity element containing particles are preferably spinels and
are referred to herein as "spinel moderated positron emitters"
(SMPE) These have several unique and surprising qualities which
enhance the image resolution of PET.
[0028] Viewed from a further aspect the invention also provides a
physiologically tolerable particulate garnet or spinel with
incorporated therein atoms of scandium, of a radioactive yttrium
isotope, of a sixth period metal (e.g. a lanthanide), of a high MR
receptivity nuclide (e.g. at least as high as .sub.71Lu.sup.175),
or of an element which on particle degradation has a desired
therapeutic or prophylactic activity.
[0029] It will be appreciated that although the metals of the metal
oxide, sulphide or alloy matrices of the particles of the invention
may have naturally occurring positron emitting isotopes the
particles according to the invention have significantly higher than
natural abundance contents of these, e.g. for positron emitters an
average of at least one, perhaps 10 or more atoms per 100 nm
crystal. The natural occurrence of many .beta..sup.+ emitters is
less than 1 in 10.sup.20 and even one emitting atom per particle
may suffice.
[0030] For other novel "doped" particles according to the
invention, the active or marker nuclei may be isotopes which occur
naturally, e.g. as impurities in naturally occurring oxides,
sulphides or alloys--in this case again the particles according to
the invention are distinguished by containing such atoms at higher
than natural values, e.g. a hundred or even more per 100 nm
particle.
[0031] The particles of the invention may be coated or uncoated and
may derive their physiological tolerability at least in part from
such a coating. They may moreover be coupled to a biotargetting
moiety, for example an antibody, an antibody fragment or another
NAM.
[0032] The particles of types i) and ii) mentioned above are also
preferably of a spinel or garnet structure--the manufacture of
particles of these types is already well known and need not be
described further here. By way of interest however it may be noted
that superparamagnetic crystals of this type have been proposed for
use as MRI contrast agents in various patent publications of
Nycomed A S, Schering A G, Advanced Magnetics Inc, etc (eg. U.S.
Pat. No. 4,863,715 (Jacobsen) and U.S. Pat. No. 4,827,945
(Groman)).
[0033] There are a wide variety of targeting moieties or NAMs which
can be used according to the invention. These include antibodies,
monoclonal antibodies, antibody fragments, receptors, peptides such
as endorphins, steroid molecules, viral fragments or coat proteins,
cell surface antigens including various carbohydrates, lectins,
immunoadhesins, neurotransmitter molecules, growth factors, and
other proteins which promote endocytosis of the pharmaceutical
agent by the axon termini. The use of lectins, such as WGA, is
particularly preferred.
[0034] The synthesis of metal oxide crystals as particulates in
stable aqueous solution has been of interest in crystalography and
in the paint pigment industry. However, many of the relevant
advances have crown out of studies of magnetism.
[0035] Many of the agents described herein involve specially
synthesized versions of magnetite (Fe.sub.3O.sub.4). The crystal
structure of magnetite is based on a mineral called spinel
MgAl.sub.2O.sub.4. However, when specific proportions of ferric and
ferrous ions are used instead of magnesium and aluminum as the
metal ions in the lattice: Fe(II)(Fe(III)).sub.2O.sub.4, a
particular set of electronic alignments and exchanges are produced
which result in spontaneous magnetization.
[0036] The basic structure of magnetite involves a close-packed,
face centred cubic crystal of oxygen atoms with metal ions placed
at interstitial spaces in the crystal (see FIG. 1). The interstices
are divided into "A" sites and "B" sites which have different
interstitial locations relative to the oxygen array and which
therefore give rise to two distinct sub-lattices within the
crystal. In the naturally occurring mineral "spinel"
(MgAl.sub.2O.sub.4) the A-sites are filled by Mg(II) and the B
sites by Al(III). The assignment of atoms to sublattices is
determined in part by size. The A-sites allow atoms of 0.3 to 0.6
angstrom radius while the B-sites allow atoms of 0.6 to 1.0
angstroms. In a normal spinel crystal, the A-sites are filled by
divalent atoms while the B-sites are filled by trivalent atoms.
[0037] Magnetite is an "inverse spinel" crystal because it has
trivalent iron in its A-sites, and a mix of divalent and trivalent
iron in its B-sites. Each crystal subunit has 32 oxygens, 8 A-site
Fe(III) atoms, 8 B-site Fe(III) atoms and 8 B-site Fe(II) atoms.
The general formula for spinel ferrites is Mt(II):
(Fe(III)).sub.2(O).sub.4, where Mt can be any divalent transition
metal or a charge balanced mix of monovalent and trivalent metals
of appropriate ionic radius.
[0038] The Fe(III) atoms in the A sublattice are positioned so as
to oppose and cancel the spin magnetization of the Fe(III) in the B
sublattice. However, after this cancellation, the 8 Fe(II)
remaining in the B sublattice have completely unopposed spin
magnetizations. For each Fe.sub.3O.sub.4 formula unit, there is a
net magnetization of 4 Bohr Magnetons due to the unopposed Fe(II)
atoms. Each crystal subunit therefore has a magnetization of 32
Bohr Magnetons packed into a cube with a face that is 837 .mu.m in
length.
[0039] The magnetization of a ferrite can be altered by
substituting different metals into the various interstices. For
instance, Mn(II) has a magnetization of 5 Bohr Magnetons, so
creation of an inverse spinel with the formula
Mn(II)(Fe(III)).sub.2O.sub.4 should yield crystals with 5 Bohr
Magnetons per unit. The use of Zn(II) has a quite different effect.
It has no unfilled d-orbitals and so has zero magnetic moment.
However, zinc tends to enter A sites causing a normal spinel
organization for the crystal. Therefore, at each formula unit, a
zero moment zinc opposes an Fe(III) with a moment of 5 Bohr
Magnetons resulting in a net moment of 5 for the pair, the
remaining Fe(III) are also unopposed, so the net moment is 10 Bohr
Magnetons per formula unit (80 Bohr Magnetons per crystal
subunit).
[0040] In actuality, this situation can prevail only for a low
percentage of the total number of sites in a larger crystal. Zn(II)
is actually too large for the A sites (0.77 angstrom radius) so
that as the concentration of zinc exceeds 50%, there is a
transformation into inverse spinel structure. In this arrangement,
Fe(III) opposes Fe(III) cancelling each other out, and the
unopposed Zn(II) have no moment, so the ferrite has a net
magnetization of zero. This is sometimes useful in applications
such as the heteronuclear tracers described below in which
magnetization is not necessarily desirable.
[0041] In 1955 the term superparamagnetism was proposed to describe
the behaviour of extremely small magnetic particles. The
fundamental idea is that there is sufficient thermal agitation in a
small particle that the tendency for the magnetic dipole axis to
flip into various orientations is greater than the tendency to
align as a coherent domain with a single fixed axis.
[0042] As the particle size increases above a critical size in the
range of 10.sup.6 atoms, it becomes stable and coherently aligned
as a spontaneously magnetized single domain. Below this critical
size, the magnetic susceptibility is temperature and size
dependent. Smaller particles at higher temperatures require
stronger external fields to become detectably magnetized. Once
magnetization is achieved, however, the total magnetization is
related directly to the size of the particle.
[0043] The behaviour of a superparamagnetic particle is described
by a relaxation rate which reflects the rate at which local
magnetic moments within the particle will flip spontaneously. In
order to flip, an energy barrier which is proportional to the
volume of the particle and to the anisotropy of the material must
be overcome. In a domain sized particle, the magnetization settles
along one single axis because the energy barrier is too great to
permit flipping at the temperature of the experiment. At sub-domain
size, the energy barrier is low enough that the flip rate becomes
exceedingly rapid. The size at which this transition occurs is
temperature dependent and also dependent on the composition of the
particle. (For present purposes the relevant temperature for
determining whether or not a substance is superparamagnetic is body
temperature).
[0044] By the substitution of some metals such as cobalt in place
of some of the Fe(II) in the lattice, the crystals become more
anisotropic and this tends to slow the rate of flipping and so
lower the critical size for a stable domain.
[0045] When larger ions are included in the crystal matrix, the
spinel structure cannot accommodate them. This is particularly
important for the use of elements from the lanthanide series.
However, lanthanides may be accommodated by the garnet crystal
structure. The natural form of this crystal is
Ca.sub.3Al.sub.2(SiO.sub.4).sub.3 or 3CaO.Al.sub.2O.sub.3.3SiO-
.sub.2. An analogous structure is achieved with the composition
Ln.sub.3Fe.sub.5O.sub.12, wherein Ln is a lanthanide element. (A
common example made using Yttrium is called YIG or
Yttrium-Iron-Garnet and is used for instance in lasers) Although
small amounts of the lanthanides are accommodated within spinel
crystals, stoichiometries which favour garnet formation are more
important as larger percentages of lanthanides are included.
[0046] A novel type of spinel crystal developed and synthesized
according to the invention uses scandium in place of aluminum in
the preparation of coated, colloidal spinel crystals. The most
stable of these are Mg(II)(Sc(III)).sub.2O.sub.4 or magnesium
scandites. These are helpful vehicles in several of the
applications described below. These crystals are not magnetic.
Scandium has stable trivalent chemistry but, unlike yttrium and
lanthanides, is similar in ionic size to the remaining transition
metals.
[0047] Methods for precipitating ferrites from metal salts date
back into the 1800's and several investigators have modified these
methods in attempts to develop improved ferrofluids. Elmore in
Phys. Rev. 54: 309-310 (1938) explored ammonia precipitation of
ultrafine ferrite particles in aqueous solutions and first
demonstrated that their aggregation increased when they approached
an applied magnetic field.
[0048] A further step towards developing stable colloidal
ferrofluids came in 1965 with the development of a method for
grinding magnetic materials into fine powders and then suspending
them in oleic acid by sonication (see U.S. Pat. No. 3,215,572).
Takada and Kiyama in Proc. Int. Conf. (ICF-1), U. Tokyo Press (Ed.
Hoshino et al), p.69-71 (1970) reexplored a variety of methods for
precipitating ultrafine crystals of magnetite and developed a new
oxidation method although this body of work did not address the
problem of keeping the particles in suspension.
[0049] Reimers and Khalafalla in Bu Mines TPR 59:13 (1972) used an
ammonia peptization method to create aqueous suspensions of ground
particles. In their initial method, an acid treatment followed by
sonication is used to induce interaction with solvent molecules to
prevent clumping of the particles and maintain suspension.
Subsequently, they developed a modification of Elmore's ammonia
precipitation method to create more stable, dilutable suspensions
in which molecules of dodecanoic acid are chemically adsorbed onto
the surface of the magnetite particle (see Khalafalla and Reimers
in IEEE Trans Mag 16: 178-183 (1980)). This yielded dilution-stable
solutions of superparamagnetic particles.
[0050] Biologists became interested in small magnetic particles as
potential means of carrying out biochemical separations and
developed various means of incorporating domain sized particles
into beads. These did not need to be soluble in the form initially
used. However, building on methods used to create dense
immunospecific labels for electron microscopy, an aqueous technique
developed by Molday (see U.S. Pat. No. 4,452,773 and J. Immunol.
Meth 52: 353-367 (1982)) opened the way to a variety of biological
applications.
[0051] The Molday method involves an ammonia precipitation
synthesis in which dextrans are used to coat the magnetite. This
results in an aqueous suspension of superparamagnetic particles
which can be conjugated to a wide variety of types of molecules
including antibodies and so used to carry out various types of
separations. The advantage of the superparamagnetism of the Molday
particles is that they do not tend to aggregate magnetically unless
they are in an applied magnetic field. This simplifies the
preparation of more elaborate compounds while permitting recovering
of the magnetic properties when they are wanted after the synthesis
is completed.
[0052] Whitehead et al (U.S. Pat. No. 4,554,088) developed a silane
binding technique in which clusters of superparamagnetic magnetite
particles each about 30 nm in size are bound in groups into larger
particles about 500 nm in diameter (now marketed as "AMI-25"). In
the silane matrix, the small particles are held apart from each
other and so retain their superparamagnetism. They therefore do not
aggregate and remain relatively soluble. However, the total
magnetic moment of the entire larger particle is quite large so
that biological separations can be carried out.
[0053] Sub-micron coated iron oxide particles have been proposed
for use as intravascular X-ray contrast agents and a number of
other medical uses have been described for other superparamagnetic
particles including magnetic confinement for blockage of fistulas
and thrombosis of aneurysms, use in producing focal diathermy for
treatment of infection, selective removal of tumour cells from bone
marrow, and use as MRI contrast agents.
[0054] There have now been a variety of clinical studies in which
MRI contrast is achieved by intravenous injection of ferrites for
evaluation of liver and spleen tumours and also after oral intake
as a gastrointestinal contrast agent. In these cases, it is the
particulate nature of the material that is used to achieve useful
distributions in the body, either by their uptake by
reticuloendothelial cells in liver and spleen, or by their
confinement to the GI tract because of their indigestible
nature.
[0055] Paramagnetic contrast agents such as gadolinium-DTPA act
primarily by altering T.sub.1 relaxation rates. superparamagnetic
agents cause their MRI contrast enhancing effect in a rather
different fashion. When the external main MR field is applied, the
particles are organized into acting as powerful microscopic magnets
scattered through the tissue being imaged. These particles
therefore result in large numbers of local inhomogeneities in the
larger field to which the protons are exposed. In the vicinity of
an activated magnetite particle, therefore, the Larmor frequency of
the protons is shifted away from resonance with the RF pulse (away
from 200 MHz in a 4.7 Tesla field) and so generate a less intense
signal. At larger distances from a magnetite particle, the contrast
agent's field will cause smaller changes in Larmor frequency, so
that although the RF pulse is still fully absorbed, the slight
differences will accelerate dephasing of the protons (i.e. shorten
T.sub.2). This is similar to the effect caused by the local field
inhomogeneities in the main magnet. However, because the magnetite
particles are themselves tumbling and moving over relevant
time-scales in the signal collection sequence, the rephasing pulses
are ineffective. Therefore, particularly in T.sub.2 weighted
images, a single magnetite particle can have tremendous impact.
[0056] Indeed, experiments conducted in our laboratory show that a
single 40 nm particle of magnetite can drive T.sub.2 to less than
10 milliseconds in an area more than 10 microns in diameter. This
is why exceedingly low concentrations of magnetite particles in the
range of 50 picomoles/liter can be effective. Even greater
sensitivity may be achieved by using specially designed pulse
sequences based on current gradient echo techniques which are
particularly sensitive to local variations in magnetic field
homogeneity.
[0057] Widder (U.S. Pat. No. 4,849,210) and Jacobsen (U.S. Pat. No.
4,863,715) demonstrated the effectiveness of suspensions of
ferromagnetic particles as intravenous MRI contrast agents with
various methods of synthesis. Groman (U.S. Pat. No. 4,827,945)
provided a number of additional methods of synthesis of
superparamagnetic particles and suggested the MR intravascular use
of a wide range of labelled particles analogous to those disclosed
for in vitro use by Molday. Although the compounds they describe
are physically very similar to those disclosed by Molday (U.S. Pat.
No. 4,452,773) they discuss sterilization techniques and methods of
use involving diagnostic MRI.
[0058] However, the particles produced by the methods of Groman
vary in size from 100 to 5,000 Angstroms, cannot be filter
sterilized in concentrated final form, and cannot be effectively
purified by affinity chromatography since, like the compounds of
Molday, they contain many constituents which will not pass readily
through agarose based affinity media late in the preparation.
Because of the need for autoclaving of the Groman products, the use
of delicate protein ligands is severely limited because they cannot
withstand autoclaving. It is possible to carry out the synthesis of
Groman using ultraclean facilities so that final sterilization of
the product is less important but this adds considerably to the
expense of manufacture.
[0059] The products of the Groman synthesis are not useful for
radionuclide imaging of axonal transport because most of the
particles are too large to be endocytosed by neurons and only a
small proportion will carry active targeting moiety thus leading to
unnecessarily large doses of radiation to achieve the required
intraneural dose and leading to unnecessarily high tissue radiation
background levels. Similarly, for MRI applications, the product of
the Groman synthesis requires unnecessarily large injections into
muscle to achieve the needed dose of small, specifically labelled
sterilized particles desired.
[0060] The current invention achieves particular improved
characteristics through the discovery that the use of repeated
purification steps during the synthesis greatly improves the
performance of the particles as biochemical reagents. These
purifications remove dissolved metal ions as they appear during the
synthesis since they can precipitate as hydrous oxides which impair
the gel flow characteristics of the preparation during the
synthesis. In addition, by using serial filtration steps after the
initial precipitation, particles may be selected which are less
than 500 angstroms in size (including the dextran coat). This helps
assure the flow characteristics of the particles through the
remainder of the synthesis and results in the production of only
100-500 angstrom particles which have a number of physiological
advantages but are all large enough to retain superparamagnetic
function.
[0061] Finally, and most importantly, when all these measures are
taken, it is possible to take advantage of the versatility and
convenience of re-usable agarose based affinity chromatography
media to remove all particles which are not bound to a targeting
moiety as well as permitting the discard of all particles whose
bound targeting moiety has been inactivated or otherwise lost its
specificity during the synthetic process. The potential to use
these media is quite important since this permits the preparation
of affinity media with a wide variety of ligands which can be used
to purify a correspondingly wide variety of targeted particles.
Although there are many applications of large (10 to 40 micron)
magnetic beads as supports for affinity separations of other kinds
of (non-magnetic) molecules and cells no previous preparation has
achieved the affinity purification of the small superparamagnetic
particles themselves upon a standard affinity chromatography
matrix.
[0062] The final result is an agent with very nearly one active
targeting moiety per particle with all particles selectively active
and small enough for effective use. This can then be concentrated
or formulated as desired and filter sterilized in small volume if
necessary. The final sterilization can be with conventional 0.2
micron filters for bacterial clearance or with 0.1 micron filters
to assure removal of small mycobacterial contaminants.
[0063] An alternative method of obtaining high specific activity is
to actually coat all of the particles in the preparation with a
large number of molecules of the targeting moiety. This has the
undesirable effects of greatly increasing the expense of the
product when the targeting moiety is expensive to produce,
increasing the antigenicity of the particle, and in many cases,
altering the distribution of the particle in undesirable ways. It
is well known from work in-affinity chromatography on solid
supports that spacing and density of affinity ligands are crucial
determinants of efficacy.
[0064] There has been considerable interest in the medical uses of
various types of microspheres and nanospheres. The composition of
such particles include latex polymers from various methacrylates,
polylactic acid, protein/albumin, lipids and various other
materials (see for example Proc. Soc. Exp. Biol. Med 58: 141-146
(1978), AJR 149: 839-843 (19.+-.7), J. Cell Biol. 64: 75-88 (1975),
J. Microencaps 5: 147-157 (1988), and Radiol. 163: 255-258 (1987)).
These particles have been used as drug delivery systems, imaging
agents, and for histological studies of axonal transport. They
offer unique patterns of metabolism and biodistribution and
continue to be the subject of intense investigation by many groups.
The uses of such particles for in vivo diagnostic imaging of axonal
transport or for the delivery of large numbers of atoms for
heteronuclear imaging are among the new uses for microspheres
described herein. The use of such particles as part of a drug
delivery system that employs an intraneural route and axonal
transport is also described here for the first time.
[0065] As mentioned above, the particles and the particulate agents
of the invention preferably comprise therapeutically or
prophylactically loaded or diagnostically marked inorganic
crystals, e.g. .beta..sup.+ emitter marked metal oxides.
[0066] Currently, the principal uses of positron emission
tomography are in situations in which relatively short half-life
emitters such as .sub.6C.sup.11 (t.sup.1/2=20.3 minutes, 0.960
MeV), .sub.7N.sup.13 (t.sup.1/2=9.96 minutes, 1.190 MeV),
.sub.8O.sup.15 (t.sup.1/2=2.03 minutes, 1.723 MeV), and
.sub.9F.sup.18 (t{fraction (1/2)}=109.7 minutes, 0.635 MeV) are
effective. However, for diagnostic or treatment situations such as
the use of monoclonal anti-tumour antibodies or for imaging of
axonal transport, it is sometimes necessary to allow several days
for adequate tissue distributions to be achieved. There are a
variety of relatively long half-life positron emitting nuclides,
however, all of these have previously proven to be difficult to
keep firmly bound to proteins over the necessary two to three
days.
[0067] As demonstrated in FIG. 2, there are a number of nuclides
emitting positron and electron particles all of which can be
included in metal oxides, eg. spinels such as ferrites, either as
substituents in the crystal lattice or as seeds, eg. ZrO.sub.2,
inside ferrite spinel shells. This provides a new and unique way of
delivering these various nuclides to various medically useful
locations in the body in a wide variety of new concentrations and
half lives. A single ferrite particle can be used to attach
hundreds or thousands of .beta.-emitting atoms to a single
antibody, thus far exceeding the intensity of signal per antibody
molecule available in current preparations which generally provide
one emitting atom per antibody molecule.
[0068] The uses for these various beta emitting ferrites are
protean and include imaging tasks as well as a number of treatment
modalities. From one point of view, this array of possible nuclide
preparations presents a wide range of half lives and particle
energies which can be used for various tasks. The great
simplification provided is that all of these can be manufactured
and delivered by means of essentially similar molecules with
effectively identical chemistry. Most of these nuclides decay to
daughters which are also easily accommodated in the crystal and
therefore do not involve loss of integrity of the particle as decay
progresses.
[0069] In one set of embodiments, the positron emitting isotopes of
manganese (.sub.25Mn.sup.52), iron (.sub.26Fe.sup.52), cobalt
(.sub.27Co.sup.55), or rhodium (.sub.45Rh.sup.99) are used in the
synthesis of spinel particles, eg. sub-domain sized,
superparamagnetic ferrite particles. The inclusion of cobalt or
manganese in this type of ferrite has previously been difficult to
achieve efficiently, but it is possible to reliably introduce
cobalt, manganese, or other metals in amounts-up to 1/3 of the
number of metal atoms per formula unit, e.g. with the remaining 2/3
being Fe(III) if the stoichiometry of the desired crystal
structure, e.g. garnet or spinel, is carefully considered and
factors such as pH, temperature, and precursor metal salt and
coating compound concentrations and the duration of heat incubation
after precipitation are carefully controlled, preferably after
optimization by routine experimentation. Thus as an example, for
dextran coated particles it has generally been found advantageous
to precipitate out from a saturated dextran solution. Thus all the
divalent metal atoms may be replaced as opposed to the 1/2 or fewer
suggested by Groman in U.S. Pat. No. 4,827,945.
[0070] These particles may be synthesized in such a way that they
are stably coated with dextran or other hydrophlic molecules and
the coating may then be activated and bound covalently to
antibodies or any type of nerve adhesion molecule. Particles so
fashioned will be detectable upon Positron Emission Tomography
(PET) as positron sources, and also upon Magnetic Resonance Imaging
(MRI) as superparamagnetic particles. Some of these will also be
detectable upon Magnetic Resonance Spectroscopy (MRS) as high
receptivity nuclei at selected frequencies or on X-ray CT scanning
where the Z-number and particle concentration is sufficient.
[0071] In positron ferrites made with .sub.25Mn.sup.52 the emission
detection is based on the 0.511 MeV annihilation photons due to
positron decay (.beta.+27.9%, 0.575 MeV, E.C. 72.1%) with a half
life of 5.59 days and associated gamma emissions of (100%, 1.434
MeV; 94.5%, 0.935 MeV; 90%, 0.744 MeV; 5%, 1.33 MeV; 4%, 1.25 MeV;
3%, 0.85 MeV) to .sub.24Cr.sup.52 which is stable. This is a decay
half life which is quite well suited to long nerve transports and
to full monoclonal antibody distribution for tumour studies.
Further, with a relatively low positron energy of just 0.575 MeV,
the spatial resolution is substantially better than any positron
emitter in active clinical use including .sub.9F.sup.18. The high
gamma emission may make .sub.25Mn.sup.52 less attractive for
clinical use in some situations, but as indicated by FIG. 2, there
are many alternatives.
[0072] Positron ferrites can also be made with .sub.26Fe.sup.52
which undergoes positron decay (.beta.+56%, 0.804 MeV; EC 43.5%)
with a half life of 8.275 hours and associated gamma emissions
(99.2%, 0.169 MeV) to .sub.25,Mn.sup.52m which is metastable and
decays with a half life of 21.1 minutes by positron decay
(.beta.+96.27%, 2.631 MeV; EC 1.53%) and associated gamma emission
(97.8%, 1.434 MeV) to stable .sub.24Cr.sup.52 as well as by
isomeric internal conversion (2.2%, 0.378 MeV) to
.sub.25Mn.sup.52.
[0073] This type of positron ferrite has the advantage of a strong
positron emission signal during the day of injection with a fairly
rapid decline towards the continuing positron emission of the
.sub.25Mn.sup.52 with a 5.7 day half life. This is particularly
useful in neuropathy studies where an initial assessment of rate of
transport is desired with a follow-up study done at several days to
assess the amount of transport. At the time of the initial study,
only a small fraction of the intramuscular dose will have entered
the nerve, so a relatively high activity injection is needed.
However, after several days, the amount in the nerve will be much
larger, and it is then helpful to minimize the continuing absorbed
dose to the patient by delivering it as the 2.2% of the
.sub.25Mn.sup.52 converted to .sub.25Mn.sup.52.
[0074] An intermediate half life can be provided by positron
ferrites made with .sub.27Co.sup.55 which undergoes positron decay
(.beta.+77%, 1.54 MeV; EC 23%) with a half life of 17.5 hours and
associated gamma emissions (75%, 0.93 MeV; 16.5%, 1.41 MeV; 20.3%,
0.477 MeV; 7%, 1.32 MeV; 3%, 1.37 MeV) to .sub.26Fe.sup.55. This
nuclide of iron then decays slowly by K-shell electron capture
(0.006 MeV) with a half life of 2.7 years to .sub.25,Mn.sup.55
which is stable.
[0075] Although the half life of this cobalt positron emitter may
be useful for some studies, its use is inhibited by the decay
pattern of .sub.26Fe.sup.55; the energy of the photon is quite low,
but the irradiation continues for a long time and virtually all the
energy is deposited within tissue as non-penetrating radiation.
This type of ferrite, however, does have the advantage of yielding
a ferrous ferrite which is a chemically quite stable metal oxide
that is cleared from the body differently than ionic iron. Further,
unlike positron ferrites decaying towards an increasing composition
of chromium or titanium, these compositions result chemically
stable ferrite particles with good magnetic properties and so
remain effective superparamagnetic MR contrast agents as decay
progresses.
[0076] A fourth type of positron ferrite can be synthesized with
.sub.45Rh.sup.99 which undergoes positron decay (1.03 MeV) with a
half-life of 16.0 days and no associated gamma emission to
.sub.44Ru.sup.99 which is stable. This is a longer half life than
will generally be needed but may be helpful in trans neuronal
transport studies intended to cross a synapse for transport in a
second nerve in a chain. In particular, this could be helpful in
studies of spinal cord injury. Also this sort of positron ferrite
could be used in studies intended to assess the acute effect of
surgery, where a diagnostic study is done and then a second study
is required several days after the surgery to assess whether an
accumulation of transported molecules at a compression site had
commenced to clear.
[0077] The decay for .sub.21SC.sup.43 (.beta.+78%, 1.22 MeV; EC
22%) and associated gamma emission (, 0.373 MeV) with half life of
3.9 hours to stable .sub.20Ca.sup.43 make this very promising for
clinical-work. Particularly for rate of transport studies in the
lower extremity which are carried out after a few hours, this may
be a completely adequate half life to allow observation of the
advancing front of the transport pulse. The substantial increase in
ionic radius and the tendency to change from trivalence to
divalence upon transition from Sc to Ca will be disruptive to the
spinel crystal, but this may aid in the more rapid metabolism of
the particles.
[0078] Except for calcium, all of these nuclides are accommodated
in the spinel ferrite crystal, although the chromium decay products
from .sub.25Mn.sup.52 and .sub.26Fe.sup.52 will generate some
regions of spinel chromite (FeCr.sub.2O.sub.4) within the inverse
spinel ferrite (Mt[II]O:Fe[III].sub.2O.sub.3) crystal. Similarly,
some regions of ilmenite, perovskite, and titanium spinel will form
in consequence of eg. .sub.23V.sup.48 decay. In any case, the
transitions due to nuclear decay will not affect the
biodistribution of the tracers on the time scale of the imaging
studies. The particles degrade slowly after initial concentration
in the reticuloendothelial system of liver, lungs, and spleen.
[0079] The optimal method for producing .sub.26Fe.sup.52 with
minimal .sub.26Fe.sup.55 contamination is by the irradiation of
.sub.24Cr.sup.50 enriched chromium with cyclotron generated 38 MeV
.sub.2He.sup.4 beams (.sub.24Cr.sup.50(.alpha.,2n).sub.26Fe.sup.52)
with subsequent acid extraction, oxidation, evaporative drying,
ether phase separation, redrying and filtration for sterilization
(see Zweit Int. J. Radiat. Appl. Instrum. Part A, Appl. Radiat,
Isol 39: 1197-1201 (1988)). other reactions available for the
production of .sub.26Fe.sup.52 include .sub.25Mn.sup.55(p,4
n).sub.26Fe.sup.52, .sub.24Cr.sup.nat (.alpha.,
xn).sub.26Fe.sup.52,
.sub.24Cr.sup.nat(.sub.2He.sup.3,xn).sub.26Fe.sup.52- ,
.sub.28Ni.sup.nat(p,spall).sub.26Fe.sup.52 with subsequent acid
extraction and purification by anion exchange chromatography,
wherein .sub.24Cr.sup.nat includes .sub.24Cr.sup.50 (4.35%),
.sub.24Cr.sup.52 (83.79%), .sub.24Cr.sup.53 (9.50%), and
.sub.24Cr.sup.54 (2.36%).
[0080] .sub.25Mn.sup.52 may also be synthesized by standard
techniques including .sub.2He.sup.3 activation of Vanadium
.sub.23V.sup.51(.sub.2He.- sup.3, 2 n) .sub.25Mn.sup.52 (see Sastri
Int. J. Appl. Rad. Isol. 32: 246-247 (1981)) or other cylcotron
reactions including .sub.24Cr.sup.52(p,n).sub.25Mn.sup.52,
.sub.24Cr.sup.52(d,2 n) .sub.25Mn.sup.52. Methods for
.sub.27Co.sup.55 include .sub.26Fe.sup.54(d,n).sub.27Co.sup.55,
.sub.26Fe.sup.56(p, 2 n).sub.27Co.sup.55,
.sub.26Fe.sup.nat(.sub.2He.sup.3,xnp).sub.27Co.sup.55- ,
.sub.25Mn.sup.55(.sub.2He.sup.3, 3 n).sub.27Co.sup.55,
.sub.25Mn.sup.55(.alpha.,4 n).sub.27Co.sup.55, wherein
.sub.26Fe.sup.nat is composed of .sub.26Fe.sup.54(5.82%),
.sub.26Fe.sup.56(91.8%), .sub.26Fe.sup.57(2.1%), and
.sub.26Fe.sup.58(0.28%).
[0081] Generator techniques in which a longer half-life parent
nuclide is synthesized and transported to the clinical site with
subsequent extraction of the clinically useful daughter nuclide
just prior to use can be arranged for several useful metals. These
include .sub.26Pd.sup.100 (4.0 d K,.gamma.).sub.45Rh.sup.100 (20 h
.beta.+), .sub.74W.sup.188 (69 d .beta.-: 188 m,18 m
.gamma.).sub.75Re (16.7 h .beta.-), and
.sub.76Os.sup.154(6.0.gamma. .beta.-).sub.77Ir.sup.194 (17.4 h
.beta.-)
[0082] A proposed cyclotron .sub.21Sc.sup.43 synthesis involves the
following scheme which would apply for alpha particle bombardment
of .sub.20Ca.sup.40 (thermal neutron cross section=0.43 barns):
1
[0083] The calcium and scandium are readily separated either by
phase separation (see Hara in Int. J. Appl. Rad. 24: 373-376
(1973)) or by chromatography (see Kuroda in J. Chrom 22: 143-148
(1966)) which also permits separation of any titanium.
[0084] These and other transition metal or lanthanide nuclides can
be used in the synthesis of radioactive metal compounds (e.g. a
metal oxide, metal sulphide or alloy, such as a ferrite) for use in
monoclonal antibody based treatment of tumours by irradiation. Here
again, the biodistribution and clearance of the delivered
radionuclides is quite different from single atoms chelated to the
proteins. Intravascular injection of Fe.sup.59 labelled particles
of the type described demonstrated a biphasic plasma half-life with
about 3/4 of the dose being cleared to spleen, liver, marrow, and
slightly to lung over 1-2 hours, but with a substantial fraction of
the dose demonstrating a quite prolonged plasma half life of many
hours. Each antibody molecule can be used to deliver several
hundred or several thousand atoms of the desired nuclide so
achieving a high local dose. It should also be noted that binding
multiple emitter atoms to a single protein molecule has been known
to rapidly destroy the protein--this problem is substantially
alleviated by the SMPE particles because the emitting nuclei are up
to 100 angstroms distant from the NAM--thus the chance of any
electron, positron, or gamma-ray interacting with the targeting NAM
is reduced by several orders of magnitude. Methods developed for
antibody delivery of .sub.39Y.sup.90 can be applied with a far
higher concentration of this nuclide included in a conjugated
ferrite. Another treatment problem where these .beta.-emitting
ferrites could be useful is in improving the current methods of
intra-articular radiotherapy in rheumatoid conditions.
[0085] Another means of delivery for .beta.-emitting ferrites is by
preparing suspensions of particles in the pre-mix of various tissue
glues. After a surgical resection of a tumour, particularly when
near an eloquent area of brain, it is often necessary to leave a
thin shell of tumour behind on the brain surface. Common practice
currently involves the use of various tissue glues to attach a
number of radioactive seeds to the residual tumour surface. This
method is tedious, leaves no means for removal of the metal without
repeat surgery, and causes artefact on future CT and MR scans which
makes it difficult to assess the results of therapy. If a colloidal
solution of .beta.-emitting ferrite is prepared in a tissue glue
component, this can be applied rapidly to the tumour surface,
minimizing exposure to the surgeon and operating staff. The
particles are biodegradable, so will be resorbed over weeks. This
method also permits the use of a variety of different nuclides
depending on the energy, penetration, or half-life desired.
[0086] Thus viewed from a still further aspect the invention
provides a composition comprising a cell adhesion moiety-coupled
radionuclide and a tissue glue.
[0087] The tissue glue may for example be based on a clottable
protein such as fibrinogen; thus for example a glue such as Tisseel
(available from Imuno Danmark A/S of Copenhagen) may be used. In
two part systems such as this the NAM-conjugated particles are
preferably in the protein containing component.
[0088] Magnetic properties of the .beta.-emitting vehicle can also
be used to help control delivery. This method can be used with or
without conjugation with antibodies, and employs the selective
catheterization techniques of interventional radiology. An arterial
catheter can be introduced near the tumour, or ideally, at a tumour
feeding vessel. A magnetic field can be applied by means of
multiple external current rings so as to be strongest in the
vicinity of the tumour. This can be achieved with the magnetic
stereotaxy device described in U.S. Pat. No. 4,869,247. Finally a
venous catheter system which is itself strongly magnetized and
furnished with a magnetized intravascular filter is introduced
downstream of the tumour in one or several draining veins or
centrally in the atrium. A highly energetic .beta.-emitting ferrite
is slowly injected via the arterial catheter. The progress of the
particles through the tumour is slowed by the external field and by
any antibodies which have affinity for tumour antigens detectable
in the vasculature. After passage through the tumour, the particles
are collected on the venous magnetic catheter/filter and so can be
removed without exposing the remainder of the body to the
radiation. If a positron emitter is used, it is possible to verify
the effectiveness of the control of the speed. of the ferrite, and
if the filtration is effective, then a second stage treatment can
be done with ferrite particles including highly toxic alpha
emitting nuclides such as .sub.78Pt.sup.186 (K, .alpha. 4.23 MeV, 3
h). Particles can also be heated with tuned microwave irradiation
during their transit for a synergistic diathermy effect.
[0089] Turning now to PET image resolution, one of the limitations
on scanning resolution is a result of the distance travelled by the
positron after the decay event but before electron-positron
annihilation. This distance is dependent upon the energy of the
characteristic .beta. emission for a given nuclide. The maximum
range for an .sub.9F.sup.18 positron emitted at 0.64 MeV is 2.6 mm
while the particles from .sub.37Rb.sup.82 decay emitted at 3.35 MeV
travel up to 16.5 mm before annihilation. Along this path (see FIG.
3), the positron loses energy by interacting with the electrons of
atoms it passes, causing a variety of ionizations and excitations.
only when most of the kinetic energy is expended does the positron
interact with an electron in a matter-antimatter annihilation
reaction generating two 0.511 MeV photons travelling approximately
1800 away from each other. The residual momentum of the positron at
the time of the annihilation imparts some translational momentum to
the emitted photons resulting in an angle between the two which
differs from 1800. Measurements of this angle reflect the nuclide
and the medium in which the energy losses and subsequent
annihilation take place.
[0090] It has been known for some time that the distance of travel
of the positron prior to annihilation is proportional to the
density of the medium. The density of magnetite is 5,180
kg/m.sup.3, just over five times greater than most animal tissues
and, according to classical calculations based on electron range
measurements, this potentially results in an 80% decrease in the
maximum distance travelled by a positron travelling in magnetite as
opposed to travelling in tissue. There is an increase in
Brehmsstrahlung braking radiation proportional to the effective Z
number of magnetite (which=52), but this only accounts for 1% of
energy loss for a population of positrons.
[0091] The numbers stated above for travel of the positron before
annihilation reflect maxima. In fact during positron emission, the
decay energy is divided between the positron and a neutrino and the
division is variable, thus resulting in a population of energies.
The mean energy of a positron from a given nuclide is about 1/3 of
the maximum usually given as the particle energy. The means
positron energy from .sub.25Mn.sup.52 is 0.19 MeV and in magnetite
this classically would result in a range of about 20 microns if the
travel were entirely in magnetite.
[0092] However various elements have characteristic positron
affinities and these have profound impact on positron lifetimes.
Therefore, the classical view of positron range in relation to a
general density measurement proves to be a substantial
oversimplification.
[0093] The positron affinities of a variety of nuclides are
included in FIG. 2. It can be seen that by using high affinity
nuclides such as lithium in the .beta.-emitter loaded particles,
the positron range can be further decreased.
[0094] In addition, it has been learned that defects in a crystal
can cause trapping of positrons. Defects in YBaCuO.sub.x perovskite
crystals are particularly effective at positron trapping even when
these materials are not in a superconducting state, however, even
mechanical stress defects in metals are fairly effective. There are
also effects due to the magnetic field generated by a moving
positron and its interaction with the spontaneous field of a
material such as magnetite, as well as electron interaction
enhancement effects due to the number of unpaired, anti-spin
matched electrons from d or f orbitals in the particular spinel
used for the particulate shield.
[0095] The consequence of these considerations is that it is
possible to begin with a crystal seed of a positron emitting
nuclide including several thousands atoms of the emitter and then
to precipitate a lithium or zinc doped, defected, magnetite shield
around the positron emitting core. This shield will cause a very
large fraction of the emitted positrons to undergo all of their
ionization producing collisional losses within the particle and
therefore to annihilate without ever leaving the particle. Those
positrons that do emerge from the surface of the particle without
being affected by reflection or surface trapping effects will have
a greatly reduced energy distribution, travel far shorter distances
through tissue, and create far fewer ionizations in tissue per
decay event than standard unshielded positron emitters.
[0096] The annihilation photons themselves are relatively
unaffected by the presence of ferrite as opposed to tissue in their
surroundings. Therefore, there will be a very large decrease in
tissue ionizations with only a trivial decrease in photon
emissions. Further, the photon emissions will all take place far
closer to the location of the actual tracer atom, typically within
several microns rather than within millimetres and this will result
in an improvement in the spatial resolution of the PET scan.
Further, the annihilations, as a population, will have lower
momentum and this will shift the population annihilation angle
closer to 180.degree., further improving the resolution of the
scan.
[0097] Where .beta. particles are used for treatment rather than
primarily for imaging, this shielding can be used to achieve
extremely limited ranges of cytotoxic ionization injury.
[0098] The range of the emitted .beta.-particles can be designed to
be not much greater than the size of a single target cell, thus
limiting effective irradiation to only those cells that actually
ingest the particle and taking advantage of the terminal Bragg peak
effect which increases the ionization rate for a low energy
positron just before annihilation. A short half life emitter could
be used to minimize the effect of increasing exposure range with
digestion of the coating (which may take days) and multiple
treatments could then be carried out. Larger particles can be used
without magnetic aggregation by composing the shell of less
magnetic nuclides.
[0099] A quite different set of particles, e.g. mixed spinels, may
be used for spectroscopic tracing and heteronuclear imaging
methods. When large percentages of .sub.3Li, .sub.21Sc, .sub.27Co,
.sub.25Mn, .sub.29Cu, .sub.59Pr, .sub.71Lu, or .sub.75Re are
introduced into ferrite crystals these become vehicles for
delivering large groups of those atoms to a desired site. These
elements and their various isotopes have high nuclear resonant
receptivity when in the appropriate oxidation state and
electron/chemical environment and so the MR machine can be used as
a spectrometer to detect the presence of these crystals. Table I
lists a series of nuclei with relatively high receptivity. Any high
receptivity metal in an oxidation state where electrons do not
produce confounding relaxation (e.g. Mn.sup.7+, Co.sup.3+) or in
which d-electron orbitals are entirely empty (Sc.sup.3+) or full
(Zn.sup.2+) are particularly amenable. The chemical environment is
also important to minimize the effects of quadrupolar relaxation
for nuclei with I>1/2.
[0100] Nuclei such as F.sup.19 and In.sup.115 can be included in
compounds which can then be included or embedded in microspheres of
latex, protein, polylactic acid or other polymers and these can
then be introduced into the axon in sufficient quantity to achieve
F.sup.19 or In.sup.115 imaging. F.sup.19 is also quite suitable for
labelling a variety of small molecules which are susceptible to
effective axonal transport. Compounds incorporating such nuclei may
also be included in the coating of metal compound particles with a
targeting moiety also present in the coating.
[0101] One optimal method in this regard is to use individual
chelated scandium atoms where the chelate is conjugated to a small
nerve adhesion molecule. By using a very small carrier, it can be
assured that the tumbling rate of the scandium atoms is high enough
to permit standard MR detection. Where particles are used, the
breakdown of the particle inside the neuron will slowly release
scandium ions which will become imageable as they are freed from
the particle and so begin to tumble rapidly.
[0102] Because of the very great abundance of Na.sup.23, imaging
with this nucleus to create a generally useful anatomical image of
the patient is readily achieved. Superparamagnetic particles such
as ferrous ferrites are effective relaxation agents for sodium and
so can be used as axonally transported contrast agent to study
nerves upon sodium imaging.
1TABLE I Nuclides with usefully large MR receptivity Frequency in
MHz Nuclide MR receptivity at 4.7 T .sub.1H.sup.1 1.000 200.0
.sub.3Li.sup.7 .270 77.6 .sub.9F.sup.19 .830 188.2 .sub.11Na.sup.23
.093 53.0 .sub.15P.sup.31 .066 81.0 .sub.21Sc.sup.45 .301 48.6
.sub.23V.sup.51 .381 52.6 .sub.25Mn.sup.55 .175 49.4
.sub.27Co.sup.59 .277 47.2 .sub.29Cu.sup.63 .064 53.0
.sub.41Nb.sup.93 .482 48.8 .sub.49In.sup.115 .332 43.8
.sub.53I.sup.127 .093 40.0 .sub.59Pr.sup.141 .260 54.0
.sub.71Lu.sup.175 .048 22.6 .sub.75Re.sup.187 .086 45.6
.sub.81Tl.sup.205 .140 115.4 .sub.83Bi.sup.209 .137 32.2
[0103] and their corresponding frequency for 4.7 T MRS. Nuclei in
italics (H, F, Na, P) are commonly used in MR spectroscopy but are
not readily included in metal oxide particles.
[0104] Using a double tuned coil or multiple coil MR system, a high
gradient proton image may be made and a selected voxel may then be
evaluated at the appropriate MR observation frequency. The presence
of the given nucleus with the appropriate spectral appearance
confirms the presence of the tracer and also makes quantitation
possible.
[0105] The sensitivity of these various nuclei for NMR is
sufficiently great that actual scandium, or other tracer, imaging
can be carried out when delivered quantities are sufficient. This
produces a positive image roughly similar in appearance to those
resulting from some current nuclear medicine imaging studies. These
uses of these nuclides are also applicable to several of their
isotopes, both stable and radioactive with some variation in
gyromagnetic ratio for the various nuclides. These variations also
can provide multiple additional frequency selectable tracers for
spectroscopy or heteronuclear imaging.
[0106] Using particle types and delivery targeting systems as
described above, a different group of metals can be used instead of
the .beta.-emitters to achieve-the very short range radiotherapy
effect. These are a variety of nuclides in which decay is by
K-shell capture. Although decay in these nuclides involves collapse
of an electron into the nucleus, the resulting vacancy causes
effects among the remaining electrons which result in Auger and
Coster-Kronig electron emissions. These have extremely low energies
and resulting ranges of micron and sub-micron distances, although
several such electrons may be emitted for each single decay event.
An optimal nuclide with this behaviour is .sub.46Pd.sup.103 which
is a pure K-capture nuclide with a 17 day half life;
.sub.24Cr.sup.51 may also advantageously be used.
[0107] By analogy with the multiple tracer methods described above
for MR spectroscopic nuclides, it is also possible to use various
transition or lanthanide metal radionuclides to prepare multiple
metal conjugated antibody tracers with the intent of providing them
with characteristic gamma emission signatures. Here, the positron
emission or MR contrast effect could be used for localization and
then the gamma emissions could be evaluated for energy
level/frequency. In this fashion multiple different gamma labels
could be distinguished as a means for image based tumour diagnosis
by multiple antibody labels.
[0108] The particles used generally should be metal compounds
capable of precipitation to a stable colloid having a particle size
suitable for cell uptake and having a surface capable of being
coated with or bound to biochemically useful materials, e.g.
carbohydrates or proteins.
[0109] As a dense material, ferrite particles are effective X-ray
contrast agents. By substituting high Z metals (e.g. elements of
atomic number 50 and above, especially sixth period elements) into
the lattice, their effectiveness can be further enhanced and the
necessary dose thus decreased. This is illustrated by FIG. 4 hereto
which shows a CT image of a phantom with wells containing similar
concentrations of Mg/Tb and Fe/Fe particles showing the greater
X-ray opacity of the former. Wells 11 to 17 contained the following
X-ray contrast media:
2 Concentration Well No. Material (mg/ml) Field Units 11 Mg/Tb
(III) 30* 411 12 Mg/Tb (III) 10* 150 13 Fe/Fe (III) 30* 101 14
Fe/Fe (III) 10* 0 15 Metrizamide 33 250 16 Air -- -1017 17
Metrizamide 100 447 *Concentration of the trivalent metal
[0110] This sort of technique is particularly useful for axon
transport imaging techniques and the evaluation of spinal root
compression by herniated disks by CT scanning. Since higher
particle concentrations are needed for CT than for MRI, the best
uses of this phenomenon include CT scanning of the injection site
for confirmation of optimal localization or actual CT guided
placement of the injection where necessary with immediate
confirmation of location and dose amount delivered. The superior
spatial linearity of CT compared to MR, makes CT preferable for
stereotactic placement tests. CT is also effective for these agents
at the concentrations achieved in lymphatics after subcutaneous or
intramuscular injection.
[0111] When an alternating magnetic field is applied to a magnet, a
number of resonant interactions can come into play which can
completely destroy the net magnetization. The main resonance is to
do with the precession frequency of the dipoles around the main
axis. There are also resonance effects in bulk magnet to do with
movements of the Bloch walls between domains as well as with the
size of domains. In a sub-domain sized superparamagnetic particle,
the principal determinant of resonant behaviour is generally the
intrinsic flipping frequency due to the temperature, particle size,
and compositional anisotropy. Exploration of the impact of
radiofrequency signals on the resonant behaviour of
superparamagnetic particles of well defined sizes is suggestive of
numerous useful effects.
[0112] For more specific separation of the particles according to
resonant behaviour, a chromatography column or a very long coil of
narrow bore tubing can be placed inside a high field magnet such as
a 2.0 Tesla MRI magnet, and the column then surrounded by an
elongated solenoid coil with various switchable capacitors,
resistors, and inductors attached. This apparatus can be used to
subject the chromatography column to a series of selected
radiofrequency fields. During the irradiation of the column with a
particulate field frequency, those particles that are relatively
demagnetized at that selected frequency will commence moving down
the column while the remainder of the assortment of particles will
remain fixed in the external magnet's field. That fraction of
resonant selected particles is collected, and then the frequency of
the applied field is changed to permit elution of a second resonant
selected fraction, and so on in this fashion until a series of
different resonant selected fractions are collected. The
demagnetization can be achieved either by pulsed RF irradiation
which flips the coherent particle axis into a transverse
orientation, or, more efficiently, by introducing sufficient energy
to induce non-coherent flipping of sub-unit dipoles.
[0113] By the use of these alterations in size, composition and
intrinsic resonant magnetic behaviour, a series of particles is
produced with differing resonant behaviour which can optimize them
for use in an MRI device of a given field strength and proton
Larmor frequency. Also, by producing highly purified resonant
engineered particles in this fashion, it becomes possible to
produce the phenomenon of Selective Radiofrequency Flipping
Alteration (SRFA). The resonant engineered, purified particles are
subjected to a selected radiofrequency signal (by means of
additional coils around the imaging subject) and sufficient energy
is introduced to overcome the coherent alignment of the crystalline
sub-units with the applied external field. This results in an
effective demagnetization of the particles and a sudden reduction
of their contrast effect in an MR image.
[0114] Two MR images may then be collected a few hundred
milliseconds apart, with the first being contrasted and the second
being non-contrasted. These two images are then subtracted from one
another by the computer and a substraction image results. This
yields a "contrast neurography" by which only the nerves and any
other tissues with high concentration of the particles are seen.
The process can be repeated at a different appropriate frequency
for each type of resonant tuned particle injected. In this fashion,
several different nerve roots could be visualized, each in a
different image if their respective muscles of innervation had been
injected with different resonant tracers. The lymphatics will
collect all the tracers and so will be subtracted from all the
images.
[0115] This SRFA subtraction technique may also be applied to other
ferrite MRI contrast methods such as antibody based labelling of
tumours or infection sites wherein several different antibodies
could each be attached to a different resonant particle group. Then
by using an apparatus that can generate multiply tuned frequencies
within the MR magnet during imaging to serially change the
frequency at which the subtraction image is obtained, the external
images can be used to determine which antibody is adhering to the
area of interest.
[0116] Another distinct use of these resonant modifications is to
prepare particles with frequencies in the microwave range. Such
particles experience mechanical vibration and hence hearing when
subjected to resonant tuned microwave energy. In this method, the
particles would be transported into areas of spinal cord injury
where the development of scar prevents the regeneration of injured
spinal cord tissue. In research work, this localized heating
phenomenon might be used as a means of inhibiting spinal cord scar
formation. This effect may also be applied for selective tumour
diathermy. Intramuscular injection at various sites with different
resonant particle frequency types at each site will permit rotation
of microwave heating frequencies so that only the tumour site will
be stimulated by all the signals.
[0117] From the above, it will be appreciated that the method of
the invention provides an entirely novel means of pharmaceutical
distribution which involves the entrainment of a well known
physiological phenomenon called axonal transport. A central feature
is that the total body distribution after intramuscular injection
of the pharmaceutical agent quite unexpectedly yields dramatically
high intraneural concentration relative to other tissues. This
differential in body/nerve concentration permits the use of this
route with relatively small amounts of pharmaceutical agent to
achieve nerve based imaging and treatment effects.
[0118] Insofar as the method of the invention is concerned, it may
be helpful to review the background to the present understanding of
axonal transport processes.
[0119] A neuron which innervates a muscle in. the human foot is an
enormous single cell (see FIG. 5) nearly three feet in length whose
nucleus in the spinal cord must manage chemical metabolic events
taking place far away in the axon terminus. The supply of newly
synthesized proteins, membrane vesicles, and organelles such as
mitochondria is accomplished by first producing these items in the
cell body, then transferring them along the axon at rates of up to
a meter per day. This `anterograde` flow could result in a
tremendous accumulation of material in the axon terminus unless
compensated by a return or `retrograde` flow at similar rates and
by a similar mechanism.
[0120] Although there are various rates and mechanisms of axonal
transport, the fast anterograde and retrograde flows (see FIG. 11)
are carried out by motile proteins (kinesin and dynein
respectively) which drag molecules and vesicles along the
microtubules of the axoskeleton. The materials transported include
not only structural and metabolic molecules, but also molecules
sampled from the external environment of the axon terminus which
are passed back up to the neuron cell body to inform it of the
environment. Such signals include various trophic or growth factors
originating in cells near the axon terminus which are endocytosed
by the axon, encapsulated in lipid vesicles, and various trophic or
growth factors originating in cells near the axon terminus which
are endocytosed by the axon, encapsulated in lipid vesicles, and
then passed up to the cell body for processing or analysis via the
axonal transport system (see FIG. 12).
[0121] The rate of transport of a given substance is independent of
electrical activity within a neuron but does vary with the type of
molecule being transported. Anterograde axonal transport has a
major fast and a slow component. The slow component is divided into
"slow component a" and "slow component b" at rates of approximately
1 and 3 mm/day respectively. These slow components apparently
reflect gradual structural repair and replacement of the subunits
of the cytoskeleton and are not involved in the fast components
important for tracer studies.
[0122] The fast component of transport demonstrates distinct
maximal rates for anterograde (300-400 mm/day) and retrograde
(150-300 mm/day) transport and some rates up to a meter/day have
been reported. The maximal rates of transport apply to small
membrane vesicles. Further, there are a variety of "waves" or
distinct sets of slower transport rates exhibited in characteristic
fashion by various molecules.
[0123] All of this movement is ATP and calcium dependent. The
metabolism involved is local, i.e. mitochondria bound to the
axolemma as well as mitochondria being transported on the
microtubules use glucose and oxygen absorbed through the cell
membrane along the axon to generate ATP locally.
[0124] The existence of axonal transport (or `axoplasmic flow`) has
been known for over 40 years and it has been known for twenty years
that certain foreign materials injected into muscle would be
endocytosed (swallowed up) by the axon terminus and then
subsequently be detectable in the neuron cell body; however, until
the developments described herein, all methods of detection have
required lethal interventions, generally requiring the killing of
the experimental animal with subsequent specialized tissue
processing.
[0125] A series of relatively non-specific substances for uptake
were tried including Evans-Blue stain conjugated to albumin and
also horseradish peroxidase (HRP) enzyme, and radio-labelled amino
acids for anterograde labelling. The principal of improving
specificity and uptake efficiency of a histologically identifiable
tracer was taken further by Schwab (Brain Res. 130:190-196 (1977))
who attached nerve growth factor (NGF) to HRP. It was also Schwab
who showed that a plant lectin called wheat germ agglutinin (WGA)
was an excellent nerve adhesion molecule and again Schwab who
introduced the use of viral fragments and toxins as labels (see
Brain Res. 152:145-150 (1978) and J. Cell Biol. 82:798-810
(1979)).
[0126] WGA conjugated to HRP was later suggested as a tracer and
this one agent has been the predominant agent of choice in many
hundreds of subsequent studies involving axonal transport. The
conjugation to some post-sacrifice visualization moiety such as HRP
permitted the use of a chromogen histochemical staining reaction.
Other means of visualization of tracers included autoradiographic
histology or immunocytochemical techniques.
[0127] Once endocytosed, WGA-HRP conjugates are found in
Golgi/Endoplasmic Reticulum/and Lysosomes (GERL) and are
transported at a slower rate than HRP alone. Many of the agents
which employ plant lectins, viral toxins and surface fragments, and
some anti-synaptosomal antibodies as targeting moieties are taken
into the cell by "adsorptive endocytosis".
[0128] There is also a route called "transcytosis" taken by
unconjugated lectins. These molecules also bind to receptors before
endocytosis but are then transported within the cell without being
first introduced into lysosomes. This mechanism has also been shown
with a monoclonal antibody ("192-IgG") raised against an NGF
receptor on pheochromocytoma cells and has made it possible to show
that the NGF molecule binds to the receptor protein and that the
entire complex is then transported up the axon to the cell
body.
[0129] Another interesting ligand/receptor complex involves
[H.sup.3]-Lofentanil and the opiate receptor which are endocytosed
and transported by sensory neurons. PET studies with
[C.sup.11]-carfentanil have been used to assess the general
distribution of opiate receptors, but this approach has never been
tried as a means of tracing selected tracts via axonal transport in
humans. Similar studies with GABA, D-aspartate, dopamine,
norepinephrine, and serotonin have shown that uptake and transport
of neurotransmitters is a widespread phenomenon in the CNS as is
the transport of receptors.
[0130] Acetylcholinesterase uptake and transport has been studied
for many years because of its ease of use as a histochemical
marker. Other studies have demonstrated transport of a wide variety
of substances including Vasoactive Intestinal Polypeptide (VIP),
cholecystokinin, substance P and somatostatin, neuropeptide-Y, and
adriamycin. These types of tracers have sometimes been introduced
by intravenous injection with subsequent uptake by neurons as well
as by actual tissue injection in or near the neurons of
interest.
[0131] Yet another set of studies has involved neurotrophic viruses
such as Herpes Simplex, poliovirus and bacterial neurotoxins, e.g.
tetanus toxin. Of the various tracers, tetanus toxin is the most
effective for "transsynaptic" labelling in which the next neuron in
a synapsing series is also labelled. It is possible that killed
vaccines, or toxoid versions of these could be useful. As with
physiologic molecules, they offer high avidity for the neuron and
their transport kinetics have been previously studied.
[0132] Another important phenomenon is transneuronal transport
wherein tracer is apparently extruded back onto the cell surface
after transport thus acting to produce a sort of second injection
at the next synapse in the chain (see Gerfen in Exp. Brain Res.
48:443-448 (1982)). Tetanus toxin appears to move in a specifically
transsynaptic fashion, but WGA and WGA-HRP are found in glia after
anterograde transport of WGA-HRP, and synaptic structures need not
therefore be involved.
[0133] Another area of advance has been in the use of particulate
tracers. Olsson in Neurosci Lett., 8:265 (1978) suggested-the use
of a non-specific very small particulate iron-dextran complex in
which the iron was in gamma iron oxide form and in which
post-sacrificial detection involved microscopic study after
chemical staining for iron. Other important particulate tracers
used for histological light and electron microscopy have included a
large protein with a ferritin core, 1-10 nm non bio-degradable
colloidal gold particles and colloidal fluorescent particles some
15-20 nanometers in diameter. Latex microspheres with fluorescent
labels and ranging from 50 to 200 nanometers in size have also been
used. However, there has been a continuing belief that larger
particles can only be transported after neuronal injury and most of
the particle studies have involved transport between locations in
the central nervous system after traumatic needle injection into
the brain substance (see Colin, Brain Res. 486:334-339 (1989)).
[0134] Detection of transport in living neurons has been
accomplished in several ways. Thus for example, the neuron may be
rapidly removed intact from the killed animal and placed over a
series of proportional .beta.-particle counters to detect the
passage of a radioactive tracer pulse along the axon. It is also
possible to directly observe the movement of organelles along such
excised neurons via microscopic video interference contrast
techniques.
[0135] There is however no prior art for in vivo imaging use of
nerve adhesion molecules coupled to clinically imageable tracer
molecules which does not involve direct inspection of neural
tissue. Further there is no prior art for any entrainment of axonal
transport to achieve desired distributions of any actual
pharmaceuticals for human or veterinary therapeutic use. Axonal
transport has been much studied as a physiological process
(analogous to the study of DNA prior to the advent of industrial
biotechnology) and it has been used extensively for studies in
which the delivered agent is effective only after the death of the
organism (as in histology) or achieves its effectiveness only
through the killing of nerve cells which transport various toxins.
However, there are no prior clinical uses, or uses in which the
effect is achieved in a living animal or human with intended
diagnostic or therapeutic rather than neurotoxic effects.
[0136] Very recently (after the priority date hereof), Brady SMRM
10:2 (1991) verbally reported transport of MR detectable particles
after direct injection into the sciatic nerve; however he exhibited
only an image of transport after the completely severed sciatic
nerve was soaked in a gel with ferrite particles. Ghosh in SMRM
10:1042 (August 1991) similarly reported evidence of transport of
ferrite particles after direct pressure injection into the brain of
a frog, although no MR detection was achieved. Neither taught how
pharmaceutical use of axonal transport could be achieved since
these techniques involved irreparable destruction of vital neural
tissue. Intraneural injection is destructive of the nerve at the
site of needle puncture and causes forced flow of tracer in the
nerve sheath which may actually mask evidence of actual axonal
transport. Madison in Brain Res. 522:90-98 (1990) also reported
pressure injection of latex nanospheres into the brain wherein the
spheres were used to deliver toxic agents for the killing of
neurons after subsequent photoactivation. These reports can,
indeed, be taken as evidence of the non-obviousness of the
non-destructive techniques described herein.
[0137] Non-destructive administration of toxic anthracycline
antibiotics has been reported, but this was done to study the
chemical nature of the neural uptake process and the fluorescent
effect of the agents rather than to achieve any therapeutic effect,
and the agents concerned were neurotoxic (see England in Brain
111:915-926 (1988) and Bigotte in Neurology 37:985-992 (1987)).
[0138] Unlike any of these previous reports, the agents described
herein may be introduced by techniques which do not involve the
destruction of neural tissue and which then achieve a pharmacologic
effect which does not require any toxic injury to neural tissues.
By delivering particulate carriers it becomes-possible to deliver
types of-pharmaceutical agents which would be irreparably damaged
by direct chemical conjugation to a NAM or on break up of its
direct NAM-conjugate within the cell. Instead the NAM is coupled to
the particle and the drug is included in the particle or in the
particle coating. Further, the use of particulate drug carriers
permits the introduction of large numbers of molecules of the
pharmaceutical agent with each endocytotic event thus yielding a
100 fold or up to one million fold increase of uptake efficiency
per NAM. This amplification effect may be crucial to achieving
pharmacologically efficacious doses in many situations. The methods
of administration for these beneficial diagnostic and therapeutic
uses include topical, intravenous, intrathecal/intracisternal
(cerebro-spinal fluid), sub-cutaneous, intradermal, intra-nasal,
eye-drop, or bladder irrigation methods, but intramuscular
administration is to be preferred.
[0139] The agents described herein differ from all previously used
axonal tracers in that they include agents capable of controlled
administration by safe intramuscular injection with non-toxic
substances and of achieving whole body distributions which permit
their useful observation by various types of non-invasive imaging
modalities. The agents may be biodegradable, safe for clinical use,
and act to reveal various human disease conditions which cannot be
adequately demonstrated by existing techniques.
[0140] Previous uses of axonal tracers have been concerned with
optimizing the degree of post-sacrificial staining of the neuron
cell body in brain or spinal cord. It has not previously been
evident that useful concentrations and distributions of clinically
applicable tracer materials could be achieved.
[0141] However, this set of agents is based on the discovery that
when a nerve adhesion molecule which also has affinity for markers
on the muscle cell surface is used; the injected material has very
minimal spread from the site of intramuscular injection. In
consequence, a relatively large amount of the substance is
transported into the nerve while relatively little spreads
throughout the body. The initial distribution assay results with
animal studies using .sup.125I labelled WGA are shown in FIG. 13.
This showed that the concentration in peripheral nerve was up to
ten times higher than in any other tissue excluding the site of
injection. The injection site could be masked out of an image so
this suggested that the distribution after intramuscular injection
might be consistent with imaging.
[0142] However, although the concentration in the nerve was 10 to
50 times higher than for example in surrounding muscle, the total
volume of the nerve relative to the volume of surrounding tissue
was quite small. Thus only an imaging technique which could collect
signals from a very small `voxel` size could successfully recover
the signal. At this relative concentration, simple labelling of a
small molecule or protein with a gamma emitter for SPECT detection
would be inadequate. Substitution of a relatively long half life
positron emitter (.sup.124Iodine) for the .sup.125Iodine would
provide nearly adequate voxel size but would involve substantial
spread of radioactive iodine through the body. Other relatively
long half life positron emitters presented similar problems.
[0143] The use of a magnetic resonance small molecule contrast
agent such as gadolinium-DTPA (diethylene-triaminepentaacetic acid)
required the introduction of a very high concentration into the
nerve and this amount was beyond what could be achieved. However,
by synthesizing a particulate magnetic resonance contrast agent
based on a ferrous ferrite core, coated with dextran and conjugated
to WGA, a series of useful solutions to the problem were revealed.
very surprisingly, the distribution results with even a crude
preparation of this type of agent which was not affinity purified
were up to an order of magnitude better then the previous results
with iodinated WGA (I.sup.125-WGA). The concentrations in nerve
were 50 to 100 times higher than in any other tissue excluding the
injection site and local lymph nodes (see FIG. 14). However, unlike
the I.sup.125-WGA result, the concentration in the nerve was
actually considerably higher than in the neuronal cell bodies of
the spinal cord. This distribution will often be advantageous since
most of the metabolism of the particle carrier will take place in
the nerve and surrounding Schwann cells while passing mostly only
smaller molecules on to the cell body in the central nervous
system. Using highly purified, affinity specific product,
exceedingly desirable distributions result, with effectively nil
detectable agent in any tissue, but for traces in liver despite
very good intraneural concentrations. Non-specific particles eluted
from the affinity column without using the affinity eluant, but
injected in identical concentration and amount yielded no evidence
of axonal transport. Only particulate tracer conjugated to affinity
purified NAM entered the nerve in high concentration.
[0144] Using ferrite doped polyacrylamide gel phantoms, it was
observed that this preparation could reduce the T.sub.2 relaxation
time of nerve below 30 milliseconds if the intraneural
concentration of iron were greater than 5 micrograms/ml. The
injections with the crude preparations actually achieved
concentrations in nerve of over 50 micrograms/ml (see FIG. 15). An
experimental imaging magnet was modified to carry out confirmatory
tests which permitted an image resolution with voxel size of only
{fraction (1/10)} of a millimeter and using this system it was
possible to measure and callibrate nerve contrast distinguishing
the tibial nerve of the injected from the uninjected leg (see FIGS.
16 to 20).
[0145] Nerves which were subsequently excised and measured for
exact T.sub.2 in the magnet confirmed the desired 50% reduction of
T.sub.2. Electron microscopy confirmed uptake and transport of the
intact particles (see FIGS. 21 and 22) and the T.sub.2 results
showed that their rate of metabolism in the nerve was slow enough
for their superparamagnetic properties to be maintained until the
time of imaging. The electron microscopy also revealed that most of
the particles were being passed out of the neuron into the
endoneurial fluid surrounding the nerve. This export of the tracer
was accomplished by. the paranodal complex at the nodes of Ranvier
(see FIG. 10). From the endoneurial fluid, the particles were being
attached to the outer surfaces of the Schwann cells which surround
the axon due to affinity of the WGA label for the Schwann cell
surface.
[0146] In parallel with these studies, a positron emitter,
.sup.52Manganese, was used to make spinel moderated positron
emitters and these were prepared in gels to duplicate the
concentrations achieved with ferrous ferrites. This study confirmed
the physical prediction that with as low as 25:1 contrast ratio, a
1 nm object could be readily detected and distinguished from a
larger object simulating a lymph node one centimeter away (see FIG.
23). Thus, the SMPE version of the agent was shown to be adequate
for PET observation of the transported agent. These distributions
also permit the use of SPECT labelled crystals for nerve imaging
studies in humans.
[0147] The delivery of particulate pharmaceuticals by the
intraneural route is an entirely novel means of drug
administration. The largest number of drugs in current use depend
in some way upon the bloodstream to achieve their distribution.
This vascular dependence includes not only drugs given by
intravenous or intra-arterial routes, but also most orally
administered drugs which must be absorbed into the bloodstream to
reach target tissues, most intramuscularly administered drugs which
are absorbed by the muscles blood vessels, many inhaled agents,
most intranasally applied drugs, some rectally administered drugs
such as paraldehyde, and many topically administered agents such as
transdermal nitroglycerine. There are, however some drugs which are
delivered into and distributed by the cerebrospinal fluid
(intrathecal route), some oral drugs which are not absorbed
(kaolin, oral vancomycin), a variety of topical and intravaginal
agents, and some administered percutaneously for local effect or
intraarticular effect such as local anaesthetics, and locally
administered steroids.
[0148] Various new drug forms and methods of use described below
involve delivery via an intraneural route. Access to this route may
be obtained by oral ingestion, topical, intra-articular,
intrathecal, intravenous and, preferably, intramuscular
administration. However, common to all these new methods, is that
the dosing and active site of the agent is determined by a route
which involves endocytosis by nerve endings with subsequent
transport to a different and distant part of the neuron.
[0149] In some experimental studies, various agents for transport
have been introduced by intraneural injection or by application to
the cut end of a severed nerve. In these methods, the `blood brain
barrier` due to the perineurium is traversed by mechanical injury,
and much of the uptake of tracer is due to direct presentation at
cut nerve endings where specific nerve adhesion molecules may be
irrelevant. The intramuscular technique (and also the intravenous
application) depend upon the natural defect in the perineurium
which occurs at axon terminus. Thus the blood brain barrier is
naturally incomplete at this site so that tracers reversibly
adherent to muscle or emerging from small blood vessels passing
near neuromuscular synapses can present a variety of molecules and
particles directly to the neuronal cell surface for uptake after
specific adhesion to the neuronal cell surface at the axon or
dendrite terminus.
[0150] The site of injection or administration will preferably be
determined only by knowledge of the nerves which project to that
site. For example, a pain in the large toe will be known to the
neurologist to involve the dorsal root ganglion of the fifth lumbar
nerve root. He will then choose an injection site somewhere in the
dermatome or myotome served by that nerve root in order to label
the part of the nerve he believes to be impaired or to deliver, for
instance, a pain medication to the ganglion or spinal cord dorsal
root entry zone connected to the fifth lumbar spinal nerve root.
For radionuclide imaging as well as for drugs where sytemic spread
is to be minimised, it is particularly important to be able to
achieve high uptake by neurons per unit amount injected and to
minimise spread away from the injection-site.
[0151] When imaging is done, the image will preferably be collected
at high resolution of a site which is different from the site of
injection, but which is connected to the injection site by a nerve.
The imaging will also be done at a time which allows the agent to
be transported the necessary distance from the injection site to
the active or imaging site at a natural rate related to the size
and type of the injected intraneural drug.
[0152] Where the intraneurally administered agent is a negative
(T.sub.2-reducing) MRI contrast agent, contrast may be further
enhanced by administration of a positive MRI contrast agent (e.g.
Salutar's SO41, Squibb's Pro-Hance or of course Schering's
Magnevist) so as to distribute into the tissues surrounding the
neuronal pathway under investigation. It has been shown that STIR
and CSI (Chemical Shift Imaging) sequences help sharply distinguish
nerves from any surrounding fat and so emphasise the impact of the
contrast agent. For example a STIR (Short Tau Inversion Recorvery)
with tau=160 ms, t.sub.c/2=30 ms and a long d.sub.1=2 seconds to
decrease saturation will accomplish this desirable effect to best
demonstrate the axonal imaging effect of ferrite tracers.
[0153] The nerve adhesion molecules used to initiate the uptake and
transport of the pharmaceutical agents used according to the
invention all have in common with each other some tendency to
promote uptake by neurons. Some molecules with no particular
affinity may be taken up and transported inefficiently by neurons.
However, molecules which interact with and bind to specific cell
surface markers or receptors on the nerve ending of the selected
nerve type are far more efficient and are preferred. An additional
degree of efficiency can be obtained when the compound also has
some affinity for the cell surface of muscle cells, since this will
promote the depot effect at the injection site and decrease the
tendency for the agent to diffuse away or be carried away by the
bloodstream prior to uptake by the neuron. However, in some
applications, such as SPECT and PET imaging, it may, in fact, be
desirable to encourage such washout by the bloodstream to minimise
radiation dose to the muscle after a brief period of uptake by the
nerves innervating that muscle.
[0154] For investigative work involving animals, wheat germ
agglutinin (WGA) can be used to provide specificity for active
uptake and transport. However, there are a wide range of nerve
adhesion molecules which can be used to cause selective and active
adsorptive endocytosis into nerves. This class of nerve adhesion
molecules includes:
[0155] 1) Anti-synaptosomal monoclonal (and non-monoclonal)
antibodies which are purified or generated based upon their
affinity for nerve membranes. These can be made by using crude
nerve homogenates and then testing for endocytotic efficacy in
cultured neuroblasoma cells or by direct measurement of uptake of
radiolabeled forms after intramuscular injection in laboratory
animals. These agents may involve entire antibodies of, preferably,
the fragment of the antibody responsible for recognition without
the F.sub.c region. Similar considerations apply for antibodies to
dopamine-beta-hydroxylase.
[0156] 2) Various growth factors such as nerve growth factors,
epidermal growth factors, insulin-related growth factors and other
proteins and peptides in this functional category which are known
to have or discovered to have efficacy at causing the neuronal
uptake of themselves and of other agents with which they are
conjugated.
[0157] 3) Lectins of various sorts which are proteins having a high
degree of affinity for particular carbohydrate and other types of
cell surface markers. This is meant to include both plant lectins
as well as various endogenous vertebrate, mammal, or human lectins
as are known or may be discovered.
[0158] 4) Fragments of neurotrophic viruses such as Herpes simplex,
pseudorabies virus or poliovirus or proteins or other markers from
the viral coat responsible for their highly efficacious uptake and
transport. These fragments-or inactivated whole viral particles, or
cloned and produced copies of the crucial proteins are of
particular interest for trans-neuronal transport.
[0159] 5) Fragments of bacterial toxins such as the B-chain of
cholera toxin and non-toxic fragments of tetanus toxin such as the
C fragment as well as modified versions or cloned portions of other
safely administered proteins with high neural affinity.
[0160] 6) A wide variety of peptides and small proteins such as
endorphins, vasoactive intestinal polypeptide, calcitonin
gene-related peptide, cholecystokinin, substance P, somatostatin,
and neuropeptide Y or the relevant portions of such peptides for
the encouragement of neuronal uptake and transport.
[0161] 7) Enzymes which are selectively endocytosed for synaptic
recycling purposes such as acetylcholinesterase and dopamine beta
hydroxylase or portions of these enzymes which are effective at
inducing neuronal uptake.
[0162] 8) Various cell adhesion molecules including peptides,
proteins and various simple and complex carbohydrates which are
effective at promoting neuronal uptake and transport of conjugated
pharmaceutical agents.
[0163] 9) Neurotransmitters and neurotransmitter analogs such as
GABA, D-aspartate, dopamine, norepinephrine, serotonin, and
benzodiazepine drugs which can be so constructed as to maintain
their efficacy in promoting transport after the conjugation to
pharmaceutically useful agents.
[0164] Two optimal nerve adhesion molecules for most applications
are transferrin and .beta.-nerve growth factor depending on whether
primarily mixed or primarily sensory nerves are to be imaged;
however, various other molecules may be optimal for particular
sorts of pharmaceutical tasks.
[0165] These proteins and other nerve adhesion molecules can be
attached to the therapeutics, prophylactic or diagnostic moiety or
to the coating (e.g. dextran coating) thereof by various methods
including a periodate oxidation reaction carried out under mild
conditions which does not afford the oxidation state of ferrite
crystals or the binding affinity of the targeting molecule. Other
binding reactions include carbodiimide binding, glutaraldehyde
binding, biotin/avidin linking, or noncovalent coating with the
molecule of interest. Metal oxide particles provide a core of
useful size which can be securely coated by a variety of types of
agents under mild, non-denaturing conditions--they are therefore
useful as a particle seed even when their special metallic or
magnetic properties are not relevant to the task at hand.
[0166] All of the above conditions may be optimized so as to use
this new drug administration method to efficiently deliver a
specially designed pharmaceutical to a specific and preselected
location within the nervous system. The sites will include
peripheral nerves along their continuous lengths, sensory ganglia,
autonomic ganglia, spinal cord, and brainstem or the olefactory
tract system upon intranasal administration. When the route of
administration is intrathecal, or topically upon the brain during
surgery or in the brain during open surgery or during stereotactic
surgical procedures, then a wide range of sites within the central
nervous system can be reached. In these cases of direct application
in the brain, it may be the purpose of the agent to be transported
to a surgically inaccessible site by axonal transport from a
surgically accessible site, or in any case to a site different from
the site of application. This may also be used to demonstrate a
successful incorporation of grafted neural tissue which will become
capable of transporting included agents from the graft to distant
sites. It will occasionally be helpful to achieve transport with a
small molecule such as an amino acid or protein labelled with e.g.
a positron emitting nuclide; however, in many of the applications,
the preferred type of agent will include a particle which is up to
50. nanometers in diameter and may contain many atoms, crystal
subunits, or molecules of the active ingredient for diagnosis or
therapy.
[0167] The exact relation between particle core size and resulting
T.sub.2 relaxivity per microgram or iron is dependent on a variety
of factors in the particle synthesis and upon the biochemical
environment in which the particle must exist prior to imaging. Shen
in SMRM 10:871 (1991) reports a relation between relaxivity and
crystal size which greatly favours the use of the largest possible
particle. Relaxivity data on particles produced by the inventor
showed values of up to 4.0.times.10.sup.5 sec.sup.-1M.sup.-1 which
are greater than those reported by Shen and are consistent with
this trend. Thus, an optimal relaxation effect per amount of iron
to be cleared by the nerve may be achieved with the largest
particles endocytosed and transported efficiently.
[0168] When more diffuse effects are desired, an injection into the
bloodstream or into the cerebro-spinal fluid can be used to cause
uptake by a specific type of neuron wherever it contacts the fluid.
However, for specific delivery to an individual peripheral nerve,
the intramuscular technique will be preferred.
[0169] The usefulness of the intramuscular administration derives
from the fact that individual axons which enter a muscle divide
multiply and fan out to innervate numerous individual muscle cells
making up a `motor unit`. Such a motor unit is a group of muscle
cells which fire simultaneously whenever their shared axon delivers
a depolarization signal. Due to the dictates of muscle energetics
and control mechanisms, the cells of a unit are generally scattered
through a muscle rather than concentrated in a single area. Thus an
injection at any one site will usually be near the axon termini of
a large number of different motor units (see FIG. 7). This presents
the injectate with a relatively large axon terminal surface area
and provides access to a mix of axons distributed evenly through
the incoming nerve. Additionally, because all muscles have a rich
sensory supply to muscle spindle tension, length, and rate
receptors, distributed through the muscle, there is simultaneous
access to sensory and to motor fibers (see FIG. 8).
[0170] When I.sup.125-NGF was injected in muscle, high intraneural
concentrations were achieved because of uptake by the various types
of sensory endings in the spindle organs of the muscle.
[0171] Intramuscular injections with intraneuronal pharmaceuticals
present no greater risk of nerve injury than any other sort of
routine intramuscular injection. However, it is well documented
that any fine axonal branch which is injured causes no lasting
muscle impairment. This is because of the short-distance of
regrowth and the tendency for axon terminal branches to recolonize
any denervated muscle cell. It should be noted that although injury
to some axonal branches may occur, the distribution of tracer in
nerve and in cell bodies upon histologic inspection of the spinal
cord is consistent with uptake by intact cells rather than by any
important contribution from uptake by injured cells.
[0172] There are a wide variety of clinical uses for an imaging
method that labels nerves, traces connections. and assesses
pathologic changes in neurons. Axonal transport continues in
crushed or compressed nerves with accumulation of transported
substances both proximal and distal to the site of injury and so
could be used to mark the location of the pathology--inside or
outside the spinal cord. Experiments conducted by the inventor
confirm the accumulation of .sup.125I-labelled WGA distal to
compression and ligation sites at concentrations just moderately
greater than in distal nerve but much greater than in proximal
nerve, with relative concentration ratios compatible with image
based location of injury sites.
[0173] Imaging of axon transport may be achieved using agents in
which various nerve adhesion molecules including proteins capable
of inducing active endocytosis by axon termini are bound to coated
mixed metal spinel particles or other particles or diagnostic
markers optimised for the various techniques discussed herein or to
such small molecules or detectable nuclides as are suitable to the
imaging method of choice. After administration by an appropriate
route, such as intramuscular injection, the compounds are caused to
enter and to travel along within the nerve axons. The alteration in
imaging contrast progresses along the nerve from the axon terminus
towards the cell body (or vice versa) unless it is impeded by a
nerve compression or crush. In an incomplete injury, the tracer
molecule will accumulate at the blockage point. The rate of
accumulation will be affected by such phenomena as turnaround
transport at the compression site and by depression of rates of
transport in more severe compressions. In general, the
administration of the agent is intended to produce a change in
relative concentration of the agent which will distinguish axon
contrast proximal and distal to the site.
[0174] The rate of progression of the contrast particles is
determined in part by their size and by the particular protein used
to provide their specificity. Attachment to nerve growth factor as
opposed to other cell adhesion molecules will take advantage of
specialized transport pathways. Various specific neural pathways
can be studied by using e.g. antibodies to the opiate receptor or
to other receptor/neurotransmitter systems or even to non-receptor
synaptosomal antigens as the protein portion. However, for a given
adhesion protein, the rate of progression of the particles along
the axons will be altered in various neuropathies and other
diseases affecting nerves. Intramuscular injection of 10 to 100
microliters of concentrated particles (5-20 mg Fe/ml is achieved by
use of Amicon Centriprep-30 concentrators or by reconstitution
after freeze drying) is adequate to cause transport which can then
be imaged. Much smaller injections can be made into central nervous
system tissues under stereotactic or image based guidance in order
to observe transport between central structures or even for drug
delivery. Also, using proteins which encourage transneuronal
transport, a peripheral muscle injection can be used to cause
transport along the spinal cord and so help to diagnose the
severity of spinal cord injuries.
[0175] The various types of inorganic particles described herein
can all be attached to nerve adhesion molecules for the study and
evaluation of the nervous system by means of axon transport. These
materials include 10 to 50 nm ferrous ferrite dextran coated
particles, resonant tuned superparamagnetic particles, enhancement
agents for Overhauser MRI (see for example WO-A-91/12024), multiple
nuclide particles for MR spectroscopic and multiple nuclide MR
imaging, positron emitting particles, gamma emitting particles for
SPECT, proteins or small molecules labelled with positron or gamma
emitting nuclides, shielded positron emitting particles, high Z
substituted particles for CT X-ray contrast with poly-energetic or
selective mono-energetic imaging.
[0176] Additional types of agents for imaging include paramagnetic
metal chelates of polychelants (e.g. polylysine gadolinium-DTPA 40
which uses the macromolecular/particulate aspects of uptake to
introduce groups of paramagnetic nuclei (40 Gd atoms per molecule)
(see EP-A-305320, EP-A-357622,.EP-A-355097, EP-A-331616,
WO-A-90/12050 and WO-A-90/13256)), liposomes containing
superparamagnetic or paramagnetic MR contrast compounds, and
air-containing albumin spheres typically used for ultrasound
contrast which can introduce susceptibility based MR contrast
effects into nerve with a minimum of foreign material to digest.
Also, fluorescein or other biocompatible fluorescent molecules
conjugated to a nerve adhesion molecule or conjugated to dextran
coated ferrites can be injected to permit confirmation of nerve
location by a neurosurgeon during an operation. Spinal Root
Compression from Herniated Lumbar Disk: The axon transport of
ferrites or other particulate agents according to the invention can
be used instead of myelographic X-ray, unenhanced X-ray CT
(computed tomography), electromyography (EMG), nerve conduction
velocity (NCV) studies and somatosensory evoked potential (SSEP) to
evaluate back and leg pain to check for sciatica. The patient
receives a very small intramuscular injection of the agent at their
doctor's office one to three days prior to the imaging session. The
agent then travels up the nerve, and a moderate contrast change
develops in the nerve along the path of transport. However, if
there is any compression of the nerve, the contrast agent piles up
"upstream" of the obstruction. An imaging study is then
obtained.
[0177] The resulting scan would show the precise location of the
nerve root compression, and, by the amount of contrast agent piled
up at the nerve compression site compared to the amount that
passes, the severity of the compression could be assessed.
[0178] Unlike myelography, there is no lumbar puncture, no need for
hospitalization, and if the MR version of the imaging agent is
used, no need for any X-ray exposure. A single study shows the
surrounding anatomy, confirms the actual nerve compression rather
than related nearby problems that might or might not cause actual
compression, and demonstrates the physiologic effect of the
compression through demonstration of interference with axonal
transport. This is particularly helpful in MR imaging since the
nerve is often compressed against bone and the bone itself does not
show well on MR. The use of MRI in the diagnosis of sciatica has
been greatly hindered heretofore because this imaging technique
reveals herniated disks in up to 60% of normal asymptomatic
individuals. What is needed is a means of showing both that there
is a herniated disk and that it is actually causing a nerve
compression since surgical decision making requires knowledge of
both of these findings. There is no special risk of failing to
diagnose far lateral disc herniations, and there is no need for
uncomfortable and unreliable EMG, NCV, or SSEP studies.
[0179] Cervical Radiculopathy: Nearly identical arguments apply to
the condition call "cervical radiculopathy" in which an
intervertebral disk or bony spur in the neck pinches a spinal nerve
causing hand, arm, shoulder and neck pain. Myelography in this
condition is even more dangerous since it involves placing a needle
in the high cervical spinal canal. A nerve injury from a needle in
the lumbar region will only exacerbate sciatica, but a spinal cord
injury from a cervical puncture can cause death or
quadruplegia.
[0180] By making myelography unnecessary in the assessment of
sciatica and cervical radiculopathy there would result a very large
overall reduction in procedure costs and radiologist's time, and a
saving of tens of thousands of hospital admission days.
[0181] Nerve Entrapment Syndromes: There are a wide variety of
nerve compression syndromes of which the most well known is carpal
tunnel syndrome. In that particular condition, a gradual thickening
of ligaments in the wrist causes pain, muscle wasting, numbness and
weakness in the hands affecting hundreds of thousands of patients
each year. There are some eight or ten other similar conditions
affecting various nerves in various locations about the body
(thoracic outlet, supracondylar/struthers ligament, anterior
interosseous and posterior interosseous/arcade of Frohnse, cubital
tunnel/ulner palsy, ulnar compression in the wrist/Guyon's canal,
suprascapular, meralgia paraesthetica/lateral femoral cutaneous,
saphenous, peroneal, and tarsal nerve compression syndromes).
[0182] These conditions are exceedingly difficult to confirm. The
only reliable method for carpal tunnel is EMG and many of the other
conditions must be inferred from the clinical examination of the
patient with subsequent "blind" surgical exploration. In fact many
depressed patients filling up the waiting lists at pain clinics and
consuming a variety of non-efficacious medications actually have
easily correctable nerve compressions. These compressions cannot be
treated, however, because they can not be reliably diagnosed or
located.
[0183] The axonal tracer method is exceedingly well suited to the
diagnosis of all manner of nerve compression syndromes. These
patients rarely have complete denervation, so axonal transport
still functions distal to the compressional point. This is a realm
where the competing method is EMG (a three month waiting list for
this painful test is common in the UK) or, in many cases where
there is no existing method at all for confirming a clinical
suspicion without surgical-exploration.
[0184] A related area of use concerns cranial nerve compressing
responsible for trigeminal neuralgia, Glossopharyngeal neuralgia
Torticollis, hemi-facial spasm, Vertigo/Meniere's Disease, and even
essential hypertension due to Vagal compression.
[0185] Incontinence and Impotence: Another extremely common problem
which is exceedingly difficult for the physician to evaluate is
urinary incontinence and bladder dysfunction. It is en important to
determine whether there is any failure of the nerves involved in
distinction from a mechanical failure. This is currently an
extremely difficult problem. However, a few carefully placed
injections would permit imaging studies capable of identifying a
variety of treatable causes. A similar set of problems also arises
occasionally in the evaluation of male impotence.
[0186] Localization of Nerve Bruises and Lacerations: The muscles
of the face are operated by a single nerve which is unfortunately
subject to severe bruising or even laceration at several points
during traumatic facial injury. A clinician is often presented with
a patient who, after a blow to the face, has a risk of irreparable
corneal abrasion because he cannot close his eye, a distorted and
grotesque fixed facial droop, and an ongoing drool from the corner
of a mouth he cannot elevate. The problem is that there is no way
to locate the exact site of injury along the complex course of the
nerve as it travels among bones, muscles, glands, arteries, and
other structures. If there is only a bruise to the nerve, then it
will recover on its own over months with no intervention required,
but if the nerve is actually lacerated, it must be reconnected
surgically on an urgent basis to minimise the risk of retraction
requiring subsequent nerve grafting.
[0187] Unfortunately, until now there has been no way to learn how
severe the injury is. Because of the impact on the patient's life
if the need to repair is not appreciated, one might advocate
surgical exploration and direct inspection in all cases. However,
because the exact location of injury could not be ascertained, this
would require unacceptable incisions at multiple locations on the
face and throat with danger to a variety of uninjured structures
along the course of the nerve.
[0188] This is a frustrating clinical problem, and there is no
current method to locate or assess such an injury. Because axonal
transport continues for several days even after the nerve is
actually cut, the agents described herein would dramatically alter
the situation. An injection of tracer into facial muscle could be
undertaken immediately in the emergency department and imaging
would be possible within hours because of the small nerve transport
distances involved.
[0189] Such an investigation could show that contrast agent still
passed the compression point--so that only a bruise was
responsible--or, even if it could not prove the severity, it would
show the surgeon precisely where to look via a tiny incision.
Similar considerations apply for traumatic nerve injuries at
various locations around the body as well-as to the problem of
distinguishing between spinal nerve root avulsion and brachial
plexus injury.
[0190] Assessment of Spinal Cord Injury: In spinal cord injury, it
is often difficult to distinguish deficits due to direct damage to
the cord from effects of nearby root compressions. Studies of cord
injury per se could be approached in several different ways. An
injection into muscle would label the motorneuron cell body, and
transneuronal transport would then introduce tracer into descending
corticospinal neurons. By injecting in transversospinalis back
muscles, the initial transport distance could be reduced to no more
than two centimeters. A second approach relies on the
multisegmental distribution of motorneurons projecting to back
muscles. An injection would cause labeling of cell bodies in intact
cord with a cutoff in areas where neuron cell bodies were crushed
or injured. A third method would be to rely on proprioceptive
sensory neurons many of which do not synapse at the level of entry
into cord, but project up to the medulla before reaching the first
synapse. For this approach, injection of an intervertebral joint
capsule might be effective.
[0191] Experiments done by the inventor have demonstrated transport
of radiolabelled tracer up the spinal cord after intramuscular
injection. This has been most successful with small molecule
imaging agents such as transneuronally transported WGA and
potentially with tetanus toxin fragments, either of which can be
labelled with Iodine.sup.124.
[0192] Myelography is considered dangerous immediately after spinal
cord injury since the changes in spinal fluid pressure that result
from the lumbar puncture can make the spinal cord injury worse.
Further, it often fails to reveal details of the site of injury
since swelling will tend to exclude dye from the area of injury.
MRI is sometimes used, but the results are difficult to interpret
since the extra tissue water due to swelling often overwhelms other
imaging information.
[0193] A variety of other more rare spinal cord conditions might
also be better studied by application of this technique. These
include congenital anomalies of the spinal cord which lead to
tethering and stretching of the spinal nerves, and also a variety
of inflammatory or neuritic conditions such as transverse myelitis
which may affect axonal transport in the spinal cord.
[0194] Evaluation of Neuropathies: Another clinically important
aspect of axonal transport concerns peripheral neuropathies such as
occur in diabetes. These are conditions which involve dysfunction
of nerve metabolism rather than actual mechanical impingement.
Diabetic neuropathy afflicts hundreds of thousands of diabetic
patients. A common outcome is loss of sensation resulting in sores
and ulcers of the feet and legs (occasionally becoming so severe as
to set the stage for gangrene or to require amputation), difficulty
with balance and loss of the ability to walk. It is difficult to
learn how to treat this condition because there is no way to
accurately follow its course and no early warning of its onset. A
diagnostic agent according to the present invention could be
injected into muscle and its rate of progress evaluated with MRI or
other imaging techniques. In this fashion, a diagnosis could be
made, and the severity assessed. Such a technique would be a
tremendous boon to research in diabetes and might help set the
stage for progress towards some medical treatment in the
future.
[0195] This problem as well as a variety of other clinical entities
such as amyotrophic lateral sclerosis--which causes profound, even
lethal muscle weakness, Alzheimer's disease--which condition is
exceedingly difficult to diagnose, and neurologic deficits after
shearing type head injuries are all thought to involve disorders of
axonal transport or related aspects of axon cytoskeletal function.
An imaging study that assesses quantity and rate of transport can
permit diagnosis as well as follow the progress of remissions and
would also be quite helpful in research.
[0196] Oncology: Neuropathy is a complicating concern in the
management and treatment of cancer. This is because neuropathies
that are due to the cancer itself may be confused with neuropathies
caused by chemotherapy. Tumours or metastases of tumours can cause
neuropathy by direct mechanical nerve compression, however a number
of cancers seem to cause neuropathy by paraneoplastic phenomena
which are not entirely understood. A wide variety of
chemotherapeutic agents also cause neuropathies and this sometimes
is the key limiting factor on maximal permissible dose.
[0197] The oncologist is therefore often faced with a dilemma when
a patient develops pain, weakness and paresthesias during the
course of treatment. If the neuropathy is due to tumour
progression, then increased therapy is indicated. However, if the
neuropathy is due to the treatment itself then those drugs must be
abandoned or replaced. An axon tracer imaging technique would be
helpful in identifying nerve and spinal cord compression, in
studying the puzzling paraneoplastic effects, and in the
development, dosing and monitoring of chemotherapeutic agents.
[0198] Epilepsy: Another interesting possibility is a link between
axonal transport and epilepsy. Kainic acid is used to induce a
murine model of pileptic kindling in the hippocampus. It is well
known that this involves increased excitability of the involved
cells. It has been observed that kainic acid also serves to block
retrograde transport of horseradish peroxidase. If there is any
association between epileptic foci and altered axonal transport,
this could lead to a means of imaging epileptic foci as part of,
for example an operation for microelectrode array placement done
several days prior to the definitive epilepsy surgery.
Intra-operative application of the tracer at the first operation
would provide useful information for the second procedure.
[0199] Verification of Denervation: It is sometimes desirable to
denervate a structure. The most common such situation is surgical
vagotomy to treat ulcer disease. In these cases it is essential to
achieve total vagal denervation in order to assure there is no
further gastric acid production. however, because of the complexity
of the vagal innervation it is often difficult to be certain if an
adequate result has been obtained. This may require the continued
use of various kinds of testing for acidity and the continued use
of medications at considerable expense and difficulty for the
patient. A misjudgment may lead to death by internal bleeding or
gastric perforation. Oral administrations of axonal transport
imaging agents will permit repeated assessments of vagal
innervation of the stomach and upper GI tract as these can be
absorbed from the gastric wall if vagal innervation is intact.
[0200] Intraoperative Nerve Identification: There are a number of
situations in which the neurosurgeon is faced with extreme
difficulty in distinguishing nerves from pathological tissues of
roughly similar colour and texture such as tumours or fibrotic fat
pads. This includes surgery for untethering of
lipomyelomeningoceles and surgery for removing acoustic neuromas
where the facial nerve passes through the tumour. In this latter
situation for instance, a fluorescein conjugated or chromophore
conjugated, dextran coated ferrite with a nerve adhesion molecule
conjugated as well may be used according to the invention. An
injection into the facial musculature is done preoperatively and an
MR image is obtained to demonstrate the course of the facial nerve
through or around the tumour. An appropriate ultraviolet or other
light source can be directed towards various areas of the tumour
mass to permit direct visual confirmation of nerve location by the
surgeon intraoperatively.
[0201] Clinical Research Uses: Outside of purely clinical issues,
there are a variety of compelling areas of neurobiology research
where the diagnostic agents of the invention can be used, e.g.
intraoperative research on the neurophysiology and distribution of
speech areas in awake humans. Although neuroanatomical tract
tracing studies have been carried out in monkeys to identify
connections among areas thought to be homologous to human speech
areas, human studies are needed. An axonal tracer with an MRI
detectable label might permit tract tracing studies in humans in
conjunction with the intraoperative recordings.
[0202] Therapeutic Uses: It is often necessary to administer drugs
whose intended site of action is in the spinal cord or dorsal root
ganglia (DRG). However, in the past there has been no easy way of
safely administering such drugs near their site of action. While
injections are commonly used to achieve local drug effects in
muscle or in joints, it is exceedingly hazardous to introduce a
needle percutaneously and blindly into the vicinity of the spinal
cord or sensory ganglia. Therefore, in order to achieve
pharmacologically efficacious doses of various drugs at these
locations it has been necessary to give very high systemic doses by
intravenous and oral routes, or by intramuscular routes wherein the
actual delivery of the drug depended upon vascular uptake from the
injection site in order to achieve the best available distribution.
Alternatively, cumbersome procedures in which special catheters are
threaded into place near the spinal cord have been undertaken with
attendant dangers of spinal cord injury. There has never been any
route except by whole body vascular distribution to deliver any
drugs to ganglia.
[0203] However, by taking advantage of the drug distributions
achievable by axonal transport where particulates are used, a
dramatic change in pharmaceutical practice can be achieved. When
the desired drug is trapped in a polymer, liposome, or protein
nanosphere with an attached nerve adhesion molecule, it becomes
possible to carry out an intramuscular injection of an extremely
small amount at a location to which the desired part of the nervous
system is connected by a peripheral nerve. Most of the injectate
will stay in the muscle at the site of injection while the drug
particles are ingested by nerve endings and transported to the
ganglionic or spinal cord. sites toward which treatment is to be
directed.
[0204] Liposomes with phosphatidlycholine and
phosphatidylethanolamine (e.g. as described by Grant in Mag. Res.
Med. 11:236-243 (1989)), derivatized for attachment of a nerve
adhesion molecule can be prepared in the necessary size range for
efficient uptake and neuronal transport. In this fashion, a wide
variety of hydrophilic and hydrophobic drugs can be packaged for
delivery direct to their intended neural sites of action while
minimizing systemic dose.
[0205] The particles yield a large amplification of the uptake
process since many drug molecules are ingested by the cell with
each event and helps minimize spread of the drug away from the
injection site.
[0206] While there are a number of well known methods for producing
particulate drug carriers, many of these are not suitable for
intraneural drug delivery. Many of the known techniques are not
applicable because they result in the production of particles which
are far too large to permit access to the axon terminus in general
and synaptic cleft in particular as needed to promote neural
uptake. For instance, many polylactic acid particles can only be
produced in sizes near 100 microns which is three orders of
magnitude too large. Liposomes similarly tend to be too large
unless they are produced as small unilamellar vesicles (SUV) which
require harsh physical and chemical conditions for synthesis.
[0207] Polycyanoacrylate/vinyl particulates (many of which are
termed "latex" microspheres or nanoparticles) can be readily
produced in the appropriate size range, but require the use of
organic solvents and other treatments which are destructive to many
biological molecules of therapeutic interest. Albumin or other
protein microspheres of useful size can be produced from sonicated
emulsions, but these require denaturing by heating over 100.degree.
C. or chemical crosslinking to achieve stability and this similarly
limits the-range of potential pharmaceuticals.
[0208] Metal oxide particles are particularly convenient as drug
carriers. They readily form stable colloids of appropriate size and
can be coated by a wide variety of molecules. When the particles
are prepared by adding a strong base to the metal chlorides in
saturated dextran, a strong bond between the particle and the
dextran is formed. Subsequently, some proteins can be covalently
bound to the dextran molecules. However, this type of synthesis
precludes the use of any drugs which cannot tolerate the strong
acidity of the metal salts or the rapid shift to a strong alkali
typically used in the precipitation reaction.
[0209] The predominant solution to this problem has been to
precipitate the particles with no coating, and then apply the
coating at a later step under more mild conditions. However,
whether NaOH or NH.sub.4OH is used, and whether the metal salts are
added to the alkali or vice versa, uncoated particles always
aggregate and despite high power sonication of the resuspended
pellets, it is difficult to produce a stable colloid.
[0210] Precipitation by addition to ammonia is more effective than
using NaOH because of a clear peptizing effect reconfirmed in
experiments conducted by the inventor. Such uncoated ammonia
precipitates become stable for centrifugal ultrafiltration even
without sonification. This preparation is sufficiently reactive as
to permit reliably stable but non-covalent binding of a wide range
of molecules. In one series of preparations made by the inventor,
coatings were made with alpha-cyclodextrin which is capable of
binding relatively non-polar drugs and is well known as a drug
vehicle. Further, particles were prepared which were first used to
adsorb tritiated dexamtethasone and then to adsorb WGA. These
particles were finally coated with dextran or bovine serum albumin
to cover unused reactive sites on the particle surface.
Preparations of this sort were subjected to repeated washing,
ultrafiltration, and N-acetylglucosamine affinity purification.
These steps demonstrated a very slow release of the labelled
dexamethasone, but also showed that the affinity purified particles
carried with them a high concentration of bound dexamethasone.
[0211] Particles of this sort can be used to introduce this helpful
steroid drug into neurons for delivery to areas of spinal
cord-injury. As has also been shown by the inventor, such particles
are extruded from the neuron into the endbneurial space and so are
then placed in a position to properly interact with and activate
cell surface glucocorticoid receptors at otherwise inaccessible
locations inside the blood brain barrier.
[0212] An entirely novel means of producing metal oxide particles
under mild conditions suitable for delicate proteins and peptides
has also been developed by the inventor. This method is based on
the tendency of mixed iron salts to commence precipitation when the
pH is raised above 4.0. Thus, rather than raising the pH of the
solution to pH 9, 10 or 11 as is done in all previously known
methods, the new method involves precipitating the crystals at
physiological pH in biochemically tolerable buffer solutions.
[0213] Thus viewed from a further aspect the invention provides a
method of producing a physiologically. tolerable particulate metal
oxide, said method comprising precipitating said oxide from a
biologically tolerable, physiological pH buffered solution,
optionally containing a coating agent whereby coated particles are
formed.
[0214] For instance, a strong buffer such as 1 molar HEPES pH 7.4
is prepared with the desired coating molecules in the buffer
solution. The mixed metal salt solution, either with or without
dextran, is then added to the buffer solution in dropwise fashion.
This method has resulted in the production of stable coated
colloids produced at physiological pH. This new method of
production of these crystals greatly broadens the range of
pharmaceuticals which can be included in the particle coat of these
10-100 nm particles for delivery by intraneural or other
routes.
[0215] Treatment of HTLV-I Associated Myelopathy: A focal infection
of the spinal cord associated with HTLV-1 (Human T-Cell Leukaemia
Virus, type 1) in which there is involvement of the phagocytic
microglia and oligodendrocytes as well as of neurons has presented
a very forbidding picture for any possible treatment options. What
few anti-viral agents have been available have poor penetration of
the blood brain barrier and so must be administered intravenously
in very high concentration to achieve potentially therapeutic dose
levels in the spinal cord. Similarly, intrathecal administration
into the CSF allows uncontrolled spread of the anti-viral agent
throughout the CNS, affecting various sites of reduced blood brain
barrier (e.g. median eminence of the hypothalamus, pineal gland,
and area prostrema) more than the infected site. Further,
intrathecally administered drugs tend to be swept out of the CSF
into the blood stream over a relatively rapid time course compared
to what is required for anti-viral therapy.
[0216] However, because of the focal nature of the lesion, it is
quite easy to identify the dermatome supplied by the involved
location of the spinal cord. Therefore, by. undertaking
intramuscular injections in neck/back muscles as well as in muscles
placed at greater distances from the spinal cord, it is possible to
set up a continuous inflow of specific medication into the CNS
neurons, and by transcellular transport, into the surrounding
oligodendrocytes and microglia. The active agents used can include
small molecules conjugated to a nerve adhesion molecule, small
liposomes or other types of nanoparticles carrying antiviral drugs.
The administration of steroids by the same route can also help to
reduce injury due to inflammatory components of the disease.
[0217] Where transport in the affected dermatome is severely
impaired by extensive cell death, injections on the contralateral
side of the body and at dermatomes below and above those affected
will still bring the drugs into a relatively high concentration at
close proximity to the lesion and usefully across the blood brain
barrier.
[0218] Pain: Among the most common of all tasks faced by the
physician or surgeon is the treatment of a severe localized pain
whose duration will probably be only a few days but which will
cause considerable distress and discomfort to the patient. This
might be from a bone fracture, ankle sprain, a surgical incision,
abscess, severe back muscle spasm, exacerbation of arthritis,
dental extraction, burn, or tumour among other problems. The
available pharmaceutical options at present fall into four main
categories, an oral drug such as aspirin or acetaminophen which
acts at the site of injury by altering the effects of prostacyclin
related pain mediators, a range of oral or locally injected
anti-inflammatory drugs of both steroidal and non-steroidal
composition, locally injected anaesthetics such as marcaine or
lignocaine which reduce neural activity, and the systemic
administration of opiates or opiate analogs by oral, intramuscular
or intravenous routes. There are a few extraordinary treatments
most commonly undertaken. in cancer patients such as the placement
of continuous infusion morphine cannulas near the spinal cord but
these are little used because of difficulty in preventing infection
and the complexity of placing them. Further, such treatments cannot
be used for pain in the arm or neck because free opiates in spinal
fluid of the upper spinal cord may reach the medulla and cause
respiratory arrest.
[0219] If opiate or anti-inflammatory drugs are included in
particles, bound to nerve adhesion molecules, and then injected in
the dermatome/myotome which is involved in the pain, then an
extremely efficient distribution can be achieved. This permits the
extended administration of an opiate at an extremely high
concentration in the dorsal root ganglion and dorsal root entry
zone of the spinal cord at-the involved level, while resulting in
negligible systemic levels of the drug. For opiates, this avoids
tolerance, sedation, respiratory depression and addiction and
provides for steady long term administration over days after a
single injection. For steroids and non-steroidal anti-inflammatory
drugs, this will help reduce the risk of gastric irritation and
internal bleeding as well as the other side effects of
steroids.
[0220] Some types of chronic pain syndromes are remarkably
resistant to all standard pain medications including the various
opiates and opiate derivatives. The prototype for this sort of pain
is trigeminal neuralgia and indeed the sine qua non of this
syndrome is that the pain can be relieved only by anticonvulsant
medications such as carbamazepine. This sort of chronic pain is
often viewed as a kind of focal sensory epilepsy. The problem in
treating such pain is that there is often great difficulty in
achieving adequate doses at the dorsal root entry zone of the
spinal cord or the trigeminal nuclei in the brainstem without
causing unacceptable systemic side effects. By use of
pharmaceutical agents according to the invention, agents such as
carbamazepine can be delivered via an intraneural route after
intramuscular injection or intradermal injection.
[0221] Spinal Cord Injury: Although acute spinal cord injury has
long proven very difficult to treat, it has recently been
appreciated that extremely high doses of methylprednisolone given
intravenously over the first 24 to 48 hours post-injury can be
significantly beneficial to eventual neurological outcome. Of
course, the actual site of action is at the injury location in the
spinal cord, but the dose is limited by the extremely large amounts
of steroid which must be distributed to the rest of the body for no
useful purpose.
[0222] These steroids can be incorporated into liposomes or other
particles of appropriate size, conjugated to a nerve adhesion
molecule and injected intramuscularly in back or neck muscles at
the level of sensory or motor loss. When this is done, they are
transported directly into the nervous system at a continuing flow
and arrive precisely at the site of injury. Wherever actual nerve
compression or injury has occurred, the transported agent will
accumulate and automatically achieve particularly high
concentrations. Drug leaking out of torn or inflamed cells, and
extruded into the nervous tissue around the affected neurons will
act on other injured tissues in the vicinity. This will also assure
good delivery into the spinal cord grey matter even when the
fracture has caused venous congestion which is slowing vascular
delivery to the most affected areas.
[0223] Certain pharmaceutical agents useful in the method according
to the invention are already known--others may be produced by
methods analogous to those used for producing the known agents,
e.g. for particulate agents: obtaining particles of a matrix
material comprising a physiologically active agent or diagnostic
marker; optionally coating said particles with a physiologically
tolerable optionally biodegradable coating material, e.g. a natural
or synthetic polymer or derivative thereof such as latex,
polylactic acid, proteins, albumin, polysaccharides, starches,
dextrans, polymerized sugar alcohols, etc (see for example
EP-A-184899 (Jacobsen)); and conjugating said particle (optionally
via coupling to a said coating, optionally after appropriate
derivatization thereof e.g. to provide a binding site or to block
excess binding sites) to a nerve adhesion molecule, preferably with
a NAM: particle ratio of up to 10, especially up to 5 more
especially up to 2 and most preferably about 1; optionally
separating NAM-conjugated particles so formed from unconjugated
particles, preferably by size separation, especially preferably by
repeated size separation followed by at least one affinity
separation; optionally sterilising the NAM-conjugated particles, if
desired after formulation thereof with a pharmaceutical carrier and
optionally with further conventional pharmaceutical excipients,
e.g. viscosity enhancing agents, pH regulators, osmolality
adjusting agents, etc.
[0224] The matrix material used may be an inorganic matrix, e.g. a
metal oxide, or an organic matrix, e.g. a polymer such as a
cross-linked starch or dextran, and it may serve as a carrier for
the physiologically active agent or diagnostic marker or it may
itself serve as the active agent or marker, as would for example be
the case with superparamagnetic ferrite crystals.
[0225] Incorporation of the agent or marker within a carrier matrix
can be achieved by conventional techniques, for example by
co-precipitation, by steeping a porous matrix material to
impregnate it with the desired agent or marker, by exposing the
agent to ultrasonically suspended, uncoated metal oxide particles,
or by means of the buffered precipitation technique described
herein.
[0226] The matrix particles should desirably be relatively
uniformly dimensioned, e.g. within the ranges discussed above, and
this may be achieved for example by conventional screening or
particle precipitation techniques. Monodisperse particles will be
preferred.
[0227] Where the agent used according to the invention is
non-particulate it may again be produced by conventional
techniques, e.g. by binding a desired nuclide directly or via a
chelant molecule to a NAM or by binding a chromophore or
fluorophore or a physiologically active molecule to a NAM,
optionally and indeed preferably so as to provide a biodegradable
bonding which will permit liberation of the physiologically active
agent after endocytosis.
[0228] For axonal delivery of therapeutic or prophylactic
substances, in certain cases it may be desirable in the method of
the invention to select physiologically active substances which
occur naturally in neurons or which are analogues of such naturally
occurring substances.
[0229] Viewed from a further aspect the invention provides a
process for the preparation of a particulate pharmaceutical agent
according to the invention which process comprises conjugating a
NAM to an optionally coated particulate physiologically active or
diagnostically marked substance.
[0230] Viewed from a yet still further aspect the invention
provides a process for the preparation of the physiologically
tolerable marked metal oxides, metal sulphides or alloys of the
invention which comprises precipitating a said metal oxide or
sulphide from a solution containing a positron emitter nuclide and
preferably also containing an element having high positron
affinity, and if desired reducing said precipitate.
[0231] Viewed from a yet still further aspect the invention also
provides a process for the preparation of the modified spinel and
garnet particles according to the invention which process comprises
precipitating di and trivalent metal ions of ionic radii such as to
permit crystals of spinel or garnet structure to form, said
precipitation being from a solution containing scandium, a
radioactive yttrium isotope, a sixth period metal, a high MR
receptivity nuclide or an element having a desired therapeutic or
prophylactic activity.
[0232] In these particle precipitation processes according to the
invention the physiologically active, marker, or high positron
affinity elements to be incorporated into the particles may
themselves be in solution or alternatively they may be in fine
"seed" crystals which become included in the precipitating
particles. For administration in vivo, the dosages used will
clearly depend upon a wide range of factors such as the patient's
weight, the specificity of the NAM (for NAM-conjugated agents), the
nature of the imaging or visualization modality (e.g. ultrasound,
MRI, CT, PET, scintigraphy, etc) where the agent is to be used to
assist surgery or diagnostic investigations, the nature of the
physiologically active or diagnostic marker component of the
pharmaceutical agent, the extent or severity of the injury or
ailment that is being investigated or treated, the distance over
which axonal transport is required, etc. The appropriate dosage
however can readily be determined taking these factors into
account.
[0233] However the intramuscular administration according to the
invention of NAM-targetted agents offers the possibility of very
efficient and very specific delivery. Thus taking the example of a
PET contrast agent, to fill the volume of a peripheral nerve one
might require 0.1 microCurie, i.e. 0.01 pCi/ml. The injection site
might commence with 50 .mu.Ci in.a 10 ml volume of muscle (5
.mu.Ci/ml), but Within 24 hours the injectate of a small molecule
tracer with minimal affinity for muscle would distribute in about
50 litres of blood and extracellular fluid space yielding a
concentration of 0.001 .mu.Ci/ml and thus even reconcentration in
for example liver-or kidney would not overwhelm the signal from the
nerve. For MRI, NAM-targetted 10K dextran-coated superparamagnetic
ferrous ferrites, e.g. incorporating Zn(II) and Mn(II) in normal
spinel inclusions to enhance-magnetization and optionally Co or Mn
but much more preferably Sc doped to permit detection/verification
by MRS, may conveniently be administered intramuscular as 10-100
.mu.l doses containing 5-20 mg Fe/ml, e.g. produced by a
Centriprep-30 concentrator. 10000 MW dextran may be replaced by
1500 MW or more preferably 6000 MW dextran.
[0234] The invention is illustrated in more detail by the following
Example of diagnostically marked ferrites.
EXAMPLE
[0235] Ferrite particle synthesis can be efficiently carried out in
less than 24 hours. The chloride salts of the metals with the
positron nuclide. (if desired) at specific activities of 1 .mu.Ci-1
mCi/mM Fe (370 MBq-3.7 GBq/pM) of-2+ and 3+ oxidation state metal
(both metal salts may be stable Fe.sup.56) are dissolved in a
saturated or supersaturated solution of 1,500 to 10,000 MW dextran
preferably 10,000 MW in a ratio near Mt(II)1.0:Fe(III) 2.0 at a
concentration of 0.2 to 1.0 molar and at a temperature of
0-60.degree. C. depending upon the final particle size distribution
desired but preferably at 50.degree. C. and where Mt is the
divalent cation of a transition metal or of a mix of transition
metals. Typical starting amounts are 540 mg FeCl.sub.3, 230 mg
FeCl.sub.2, 3 gm Dextran 10K, in 4.5 ml of dH.sub.2O. The dextran
solution should be heated only briefly to avoid recrystalization or
sludging.
[0236] Trivalent cations (such as Sc(III)) may be used in low
ratios if they are stoichiometrically balanced with monovalent
metal salts, preferably LiCl. The ferrites are precipitated by
addition of 5 to 10%, preferably 7.5% aqueous solution of NH.sub.3
to reach a pH of 9 to 12 and preferably.pH 11 (about 15 ml added to
7.5 ml of dextran/metal salt solution. This solution can be heated
to 60.degree. C. prior to adding it to the metal/dextran
solution.
[0237] By emitting LiCl in the precipitation reactions with
.sub.25Mn.sup.52, and .sub.26Fe.sup.52, any chromium decay product
is unlikely to remain at a II oxidation state and so will tend to
be excluded from the crystal, resulting in a sort of final
purification at time of synthesis which effectively increases the
specific activity of the nuclide and helps preserve maximal
possible magnetic saturation of the ferrites.
[0238] A variety of sizes of dextrans can be used, for example
ranging from 1.5K to 40K MW although the -10K dextrans have proven
most reliable in these syntheses. Changes in outer coating also
effect the tumbling behaviour of the particles and this can have an
effect on some resonant behaviour of the particles and on their
interactions with water molecules. It is also possible to coat the
particles with non-metabolizing latex from for example
cyanoacrylate monomers to alter their rate of processing through
the cells. Other biodegradable coatings such as polylactic acid or
even protein/albumin coats can be applied. A shift in average
crystal core size towards smaller size can be produced by lowering
the temperature of the synthetic reaction or elevating the pH.
However, a variety of separation techniques may then be required to
trim the size distribution to select the desired size range.
[0239] Additionally, the spinel crystal can be constituted. of
mixed metals in various amounts in order to achieve various
specific optimizations. Mixed spinels including various useful
transition series metals, and even some lanthanide metals can be
made by adding the metal chloride powders directly to the saturated
dextran solution prior to alkali precipitation. The product of the
reaction is centrifuged 2 times at 1,000 g.times.10 minutes and one
time at 1,500 g.times.10 minutes to remove particulates which are
discarded in the precipitate. The resulting suspension is passed
through a 2.5 cm x 40 cm column of Sephadex
G-25M/150.RTM.(Pharmacia) equilibrated in 0.1M NaAcetate buffer pH
6.5 in order to remove free metal ions, particulates, ferrous
hydrous oxides, chloride and ammonia.
[0240] The Sephadex eluant is then passed through successively
finer microfilters. Two passes through a 0.22 micron nylon filter
are followed by two passes through a 0.1 micron nylon filter. The
third filtration is slow but can be accomplished with 100 mm or 47
mm diameter filters on a suction funnel using a 50 nm filter such
as Millipore.RTM.VMWP-04700 Cellulose MF filters although nylon
filters are preferable. The speed and general success of this step
are highly dependent on the initial precipitation conditions--being
most efficacious with smaller particle size distribution. These
filtrations may also be accomplished with centrifugal filters.
[0241] This is cleared, desalted, and size trimmed product is then
concentrated with a centriprep-30.RTM. (Amicon) ultrafilter, at
1,500 g for 45 minutes, to achieve a final volume of five to seven
ml. The sample is then applied-to a 2.5 cm.times.25 cm column of
Sephacryl-200 R (Pharmacia) equilibrated with 0.1M NaAcetate buffer
pH 6.5 with elution by the same buffer. This traps dextran and
small ferrous hydrous oxides while letting. the particles pass in
the excluded, unfractionated volume. The late tail of this fraction
should be discarded as it contains much of the hydrous oxide. The
resulting eluant is concentrated to 4 ml with a Centriprep-30
concentrator (1,500 g for 15 minutes) for conjugation.
[0242] The particle sample-in a volume of 4 ml is oxidized adding
slowly 1 ml of 20 mM NaIO.sub.4 at 23.degree. C. This mixture is
reacted while stirring (non-magnetic stirring only) for 60 minutes
in the dark.
[0243] The periodation reaction is halted by passing the sample
through two PD-10 Sephadex G-25M/150 columns equilibrated with 20
mM NaBorate buffer pH 8.5, concentrating with a Centriprep-30
ultrafilter to 1-2 ml then passing the sample through a third PD-10
column of Sephadex G-25M/150 to completely remove any unreacted
periodate. The final volume is brought up to 4 ml with borate
buffer.
[0244] A protein solution is prepared having 2-10 mg of antibody,
lectin, growth factor, or other selective adhesion molecule
dissolved in 1 ml of 20 nM NaBorate buffer, pH 8.5. Where possible,
blocking molecules to protect the active/recognition site should be
added at this point if the blocker will not be bound by the
periodate activated dextran. For example, adding 1 mM
CaCl.sub.2/MnCl.sub.2-helps protect the binding site on some
lectins. This solution is then added to the particle solution,
mixed, and allowed to incubate for 4 to 12 hours depending upon the
molecule involved and the number of adhesion molecules desired per
particle. The reaction is quenched by the addition of 200
microliters of 0.5M glycine with an additional two hours of
incubation.
[0245] The covalent bonds are then reduced by the addition of 0.5
ml of 0.25M NaBH.sub.4 with allowance for the generation of H.sub.2
gas. After one hour of reaction, the mixture is passed through
three PD-10 columns of Sephadex G-25M/150 equilibrated with 20 mM
HEPES buffer at a pH of 7.4 to remove glycine, NaBH.sub.4 and
H.sub.2, then concentrated to a 1-2 ml volume with a Centriprep-100
concentrator (500 g for 60 minutes) to clear unbound adhesion
molecule and smaller, unconjugated particles. This product is then
applied to a 1.6. x 35 cm column of Sephacryl 200 and eluted with
20 mM HEPES buffer at pH 7.4. This column run further
removes-unbound targeting molecules and traps any newly formed
hydrous oxides. The eluant is collected and concentrated with a
Centriprep-100 concentrator at 500 g.times.30 minutes to achieve a
final volume of 4 ml.
[0246] The four ml of reaction product are then applied to a 4 ml
column of affinity ligand Sepharose 6B with divinyl sulfone links
(such as Sigma A2278 for some lectins) equilibrated with 20 mM
HEPES buffer pH 7.4. It is preferable to avoid conditions normally
intended to maximize binding as this may make it impossible to
elute the specific fraction. The column is then washed extensively
with four to five volumes of buffer and then a 2 ml volume of 1
molar affinity eluant in the same buffer is applied. This elutes
the active fraction in a fairly sharp band.
[0247] The specific fraction is collected and passes through a
PD-10 Sephadex G-25M/150 column to help clear affinity eluant and
then concentrated to 1 mL with a small volume Centricon-30
centrifugal concentrator (1,500 g.times.20 minutes). This product
is passed through a second PD-10 column and the final output then
concentrated to a volume of 300 to 500 microliters with a
Centricon-30 concentrator (1,500 g.times.60 minutes). The final
product is then sterilized by 0.22 or 0.1 micron filtration using a
Costar 1 ml centrifugal microfilter and stored for use.
[0248] For axon transport studies, small injections of 100-200
microliters with 0.5 to 1 mg of particles (at 0.5 to 10 mCi (18.5
to 370 MBq) for PET) are made into muscle with subsequent study
with positron emission tomography, X-ray CT, magnetic resonance
imaging, or magnetic resonance heteronuclear spectroscopy one to
five days after administration as indicated. For tumour evaluation,
unlabelled and cold ferrite conjugated irrelevant antibody is
administered intravenously followed by intravenous administration
of 0.5 to 10 mCi (18.5 to 370 MBq) of positron ferrite/antibody
complex. Studies can then be undertaken for tumour evaluation two
to five days after intravenous administration with any or all of
PET, CT, MRI or MRS.
[0249] FIG. 23 shows MR images obtained with .sup.52Mn doped
ferrite particles obtained in a similar manner. A phantom was
prepared using a 2 cm polyacrylamide gel containing the particles
with a mm channel of polyacrylamide gel containing the particles at
about 25 times higher concentration. This phantom thus mimics the
occurrence of a nerve in surrounding tissue (e.g. a leg). The
concentrations were selected to simulate the results of the
Fe.sup.59-WGA-ferrite study of FIG. 14. Additionally a syringe with
a 3 mm diameter was taped to the outside of the gel-containing 2 cm
diameter universal tube. As shown in FIG. 23, using a low
resolution multiwire proportional position emission tomography
(MUPPET), camera it was possible to distinguish the "nerve" from
both the "leg" and the syringe about 1 cm away.
[0250] The ferrite particles-can be obtained by similar procedures,
e.g.:
[0251] a) Following a method analogous to that of Molday (J.
Immunol. Meth. 52:353-367 (1982)) metal chloride powder is added
directly to a supersaturated 10K dextran solution prior to
precipitation with NH.sub.4OH. Particle size separation is effected
on Sephacryl 1000 with subsequent density gradient
centrifuging.
[0252] b) The ferrite particles are synthesized by a modification
of the method of Molday (supra) which can be efficiently carried
out in less than 24 hours. The chloride salts of the metals with
the positron nuclide at specific activities of 10-100 mCi/.mu.M
(370 MBq-3.7 GBq/pM) of 2+oxidation state metal are dissolved in a
supersaturated solution of 10,000 MW dextran in a ratio near Mt(II)
1.0:Fe(III) 2.0 at a concentration of 0.5:1.0 molar and at a
temperature of 20-60.degree. C. depending upon the-final particle
size distribution desired and where Mt is the divalent cation of a
transition metal or of a mix of transition metals. The ferrites are
precipitated by addition of 8% aqueous solution of NH.sub.3 to
reach a pH of 11 (about 4 ml added to 2 ml of dextran/metal salt
solution), centrifuged at 1,000 g to remove particulates, separated
and concentrated with a Centriprep-30 (Amicon) concentrator at
2,000 g for collection of small particles in the filtrate when
desired.
[0253] The products of this concentration/separation step, either
filtrate (reconcentrated with Centriprep-10 concentrator) or
retentate, are passed through a preparative column of Sephadex
G-25M.(150).equilibrated in 0.1 M NaAcetate buffer pH 6.5 at least
four times the volume of the applied sample in order to remove free
metal ions, chloride and ammonia.
[0254] This desalted sample is again concentrated with a
Centriprep-30-concentrator (2,500 g for one hour) to a 3-4 ml
volume then passed through a 2.5 cm x 25 cm column of Sephacryl-300
(Pharmacia) equilibrated with 0.1M NaAcetate buffer pH 6.5 with
elution by 0.1M NaAcetate/0.15M NaCl buffer pH 6.5 and 0.15M NaCl
to separate unbound dextran, and the resulting fraction
concentrated to 4 ml with a Centriprep-30 concentrator (2,500 g for
15 minutes) and activated by reacting with 1 ml of 20 mM NaIO.sub.4
at 23.degree. C. while stirring (non-magnetic stirring only) for 60
minutes in the dark.
[0255] The periodation reaction is halted by passing the ferrite
sample through a Sephadex G-25M (150) column equilibrated with 20
mM NaBorate buffer pH 8.5, concentrating with Centriprep-30 to 1-2
ml then passing the sample through a second column of Sephadex
G-25M(150) to completely remove any unreacted periodate. The
protein solution of 2-10 mg of antibody, lectin, growth factor, or
other selective adhesion molecule dissolved in 1 ml of 20 mM
NaBorate buffer pH 8.5 is then added to the ferrite solution,
mixed, and allowed to incubate-for 4 to 12 hours depending upon the
molecule involved and the number of adhesion molecules desired per
ferrite particle. The reaction is quenched by the addition of 200
microliters of 0.5M glycine with additional two hours of
incubation.
[0256] The covalent bonds are then reduced by the addition of 0.5
ml of 0.25M NaBH.sub.4 with allowance for the generation of H.sub.2
gas. After one hour of reaction, the mixture is passed through a
column of Sephadex G-25M(150) equilibrated with 20 mM HEPES buffer
at a pH of 7.4 to remove NaBH4 and H.sub.2, concentrated to a 1-2
ml volume with a Centriprep-30 concentrator (2,500 g for 30
minutes) and applied to a 1.5 cm.times.40 cm column of
Sephacryl-300 equilibrated-with 20 mM HEPES buffer pH 7.4 for
subsequent elution with 20 mM HEPES/0.15M NaCl buffer pH 7.4 in
order to remove unbound adhesion molecules and passaged into. 0.1M
phosphate buffer pH 7.4 via Sephadex G-25M for administration.
[0257] The resulting fraction can then be concentrated to a 1 ml
volume with a Centriprep-30 concentrator for use or further
purified with affinity chromatography and subsequent concentration
when necessary. Reconstitution after freeze drying can also be used
if desired.
[0258] c) The ferrite particles are synthesized by a modification
of the method of Molday (supra) which can be efficiently carried
out in less than 24 hours. The chloride salts of the metals with
the positron nuclide (if desired) at specific activities of 10-100
mCi/.mu.M (370 MBq-3.7 GBq/.mu.M) of 2+ and 3+ oxidation state
metal (both metal salts may be stable Fe.sup.56) are dissolved in a
supersaturated solution of 1,500 to 10,000 MW dextran preferably
6,000MW in a ratio near Mt(II)1.0 :Fe(III)2.0 at a concentration of
0.2 to 1.0 molar and at a temperature of 20-60.degree. C. depending
upon the final particle size distribution desired but preferably at
50.degree. C. and where Mt is the divalent cation of a transition
metal or of a mix of transition metals. Typical starting amounts
are 540 mg FeCl3, 230 mg.FeCl.sub.2, 3 gm Dextran 10K, in 4.5 ml of
dH.sub.2O. The dextran solution should preferably be heated only
briefly to avoid recrystallization or sludging. Trivalent cations
(such as V[III]) may be used in low ratios if they are
stoichiometrically balanced with monovalent metal salts, preferably
LiCl. The ferrites are precipitated by addition of 5 to 10%,
preferably 7.5% aqueous solution of NH.sub.3 to reach a pH of 9 to
12 and preferably pHil (about 4 ml added to 2 ml of dextran/metal
salt solution).
[0259] A variety of sizes of dextrans can alternatively be used,
ranging from 1.5K to 40K MW although the OK dextrans have proven
most reliable in these syntheses.
[0260] Additionally, the spinel crystal can be constituted of mixed
metals in various amounts in order to achieve various specific
optimizations. Mixed spinels including various useful transition
series metals, and even some lanthanide metals can be made by
adding the metal chloride powders directly to the saturated dextran
solution prior to alkali precipitation.
[0261] The product of the precipitation reaction is centrifuged 3
times at 1,000 g to remove particulates which are discarded in the
precipitate. The resulting suspension is passed through a
preparative column of Sephadex G-25M/150.RTM. (Pharmacia)
equilibrated in 0.1M NaAcetate buffer pH 6.5 at least five times
the volume of the applied sample in order to remove free metal
ions, chloride and ammonia.
[0262] This cleared and desalted product is then concentrated with
a Centriprep-100.RTM. (Amicaon) ultrafilter, at 1,500 g for two
hours, resuspended and again concentrated to a 4 ml volume. This
yields good clearance of particles below 5 nm and of unbound
dextran into the filtrate for discard and this is the preferred
method for the superparamagnetic agent.
[0263] When a range of particle sizes including smaller particles
are to be processed this concentration step is done with a
Centriprep-30 concentrator. In this case, the unbound dextran will
have to be removed by applying the sample as a 3-4 ml volume to a
2.5 cm.times.25 cm column of Sephacryl-200 (Pharmacia) equilibrated
with 0.1M NaAcetate buffer pH 6.5 with elution by 0.1M
NaAcetate/0.15M NaCl buffer pH 6.5 and 0.15M NaCl. The resulting
fraction concentrated to 4 ml with a centriprep-30 concentrator
(2,500 g for 15 minutes) for conjugation.
[0264] When only very small particles are desired, the initial
concentration is done with a Centriprep-100 ultrafilter, but it is
the filtrate which is then processed further. This filtrate is
reconcentrated three times with a Centriprep-30 ultrafilter to
clear the dextran.
[0265] When primarily larger particles (in the-50 to 300 nm range)
are desired, the desalted, ultrafiltered sample is concentrated
with a Centriprep-100 concentrator (2,500 g for one hour) to a 4 ml
volume and then applied to a 2.5 cm x 25 cm column of Sephacryl-400
R (Pharmacia) equilibrated with 0.1M NaAcetate buffer pH 6.5 with
elution by 0.1M NaAcetate/0.15M NaCl buffer pH 6.5 and 0.15M NaCl.
The resulting fraction concentrated to 4 ml with a Centriprep-30
concentrator (2,500 g for 15 minutes) for conjugation.
[0266] Particularly for the intraneural agents, it is preferable
for the particles to be less than 50 nm in diameter. Therefore, the
Centriprep 100 or other product from step 3 is passed through first
0.2 micron and then 0.1 micron Nalgene.RTM. nylon microfilters. The
resulting product is then concentrated to a 2 ml volume and applied
to a 2.5 cm.times.50 cm column of Sephacryl-1000.RTM. (Pharmacia)
for size fractionation. Particles in the later fractions are
collected for further processing.
[0267] The particle sample in a volume of 4 ml is oxidized adding
slowly 1 ml of 20 mM NaIO.sub.4 at 23.degree. C. This mixture is
reacted while stirring (non-magnetic stirring only) for 60 minutes
in the dark.
[0268] The periodation reaction is halted by passing the sample
through a Sephadex G-25M (150) column equilibrated with 20 mM
NaBorate buffer pH 8.5, concentrating with a Centriprep-30
ultrafilter to 1-2 ml then passing the sample through a second
column of Sephadex G-25M(150) to completely remove any unreacted
periodate. The protein solution of 2-10 mg of antibody, lectin,
growth factor, or other selective adhesion molecule dissolved in 1
ml of 20 mM NaBorate buffer pH 8.5 is then added to the particle
solution, mixed, and allowed to incubate for 4 to 12 hours
depending upon the molecule involved and the number of adhesion
molecules desired per particle. The reaction is quenched by the
addition of 200 microliters of 0.5M glycine with an additional two
hours of incubation.
[0269] The covalent bonds are then reduced by the addition of 0.5
ml of 0.25M NaBH.sub.4 with allowance for the generation of H.sub.2
gas. After one hour of reaction, the mixture is passed through a
column of Sephadex G-25M(150) equilibrated with 20 mM HEPES buffer
at a pH of 7.4 to remove NaBH.sub.4 and H.sub.2, concentrated to a
1-2 ml volume with a Centriprep-100 concentrator (1,500 g for 60
minutes) to clear unbound adhesion molecule and smaller,
unconjugated particles. This product can then be passaged into 0.1
M phosphate buffer pH 7.4 via Sephadex G-25M for administration, or
further purified by affinity chromatography on non-porous beads or
Nalgene.RTM. affinity membranes.
[0270] The resulting fraction can then be diluted to 20 ml in
sterile buffer and passed through a 0.2 micron or preferably 0.1
micron microfilter to assure sterilization. The final product is
concentrated to a 1 ml volume with a Centriprep-100 concentrator
for use. Reconstitution after freeze drying can also be used to
achieve desired concentrations for some preparations.
[0271] Alternatively the product of the precipitation reaction is
centrifuged 2 times at 1,000 g.times.10 minutes and one time at
1,500 g.times.10 minutes to remove particulates which are discarded
in the precipitate. The resulting suspension is passed through a
2.5 cm.times.40 cm of Sephadex G-25M/150.RTM. (Pharmacia)
equilibrated in 0.1M NaAcetate buffer pH 6.5 in order to remove
free metal ions, particulates, ferrous hydrous oxides, chloride and
ammonia. The Sephadex eluant is then passed through successively
finer microfilters. Two passes through a 0.22 micron nylon filter
are followed by two passes through a 0.2 micron nylon filter. The
third filtration is slow but can be accomplished with 100 mm or 47
mm diameter filters on a suction funnel using a 50-nm filter such
as Millipore.RTM. VMWP-04700 Cellulose MF filters.
[0272] This cleared, desalted, and size trimmed product is then
concentrated with a Centriprep-30.RTM. (Amicon) ultrafilter, at
1,500 g for 45 minutes, to achieve a final-volume of five to seven
ml. The sample is then applied to a 2.5 cm.times.25 cm colum of
Sephacryl-200.RTM. (Pharmacia) equilibrated with 0.1M NaAcetate
buffer pH 6.5 with elution by the same buffer. This traps dextran
and small ferrous hydrous oxides while letting the particles pass
in the excluded, unfractionated volume. The late tail of this
fraction should be discarded as it contains much of the hydrous
oxide. The resulting eluant is concentrated-to 4-ml with a
Centriprep-30 concentrator (1,500 g for 15 minutes) for
conjugation.
[0273] The particle sample in a volume of 4 ml is oxidized adding
slowly 1 ml of 20 mM NaIO.sub.4 at 23.degree. C. This mixture is
reacted while stirring (non-magnetic stirring only) for 60 minutes
in the dark.
[0274] The periodation reaction is halted by passing the sample
through two PD-10 Sephadex G-25M/150 columns equilibrated with 20
mM NaBorate buffer pH 8.5, concentrating with a Centriprep-30
ultrafilter to 1-2 ml then passing the sample through a third PD-10
column of Sephadex G-25M/150 to completely remove any unreacted
periodate. The final volume is brought up to 4 ml with borate
buffer.
[0275] The protein solution of 2-10 mg of antibody, lectin, growth
factor, or other selective adhesion molecule dissolved in 1 ml of
20 mM NaBorate buffer-pH 8.5. Where possible, blocking molecules to
protect the active/recognition site should be added at this point
if the blocker will not be bound by the periodate activated
dextran. For example, adding 1 mM CaCl.sub.2/MnCl.sub.2 helps
protect the binding site on some lectins. This solution is then
added to the particle solution, mixed, and allowed to incubate for
4 to 12 hours depending upon the molecule involved and the number
of adhesion molecules `esired` per particle. The reaction is
quenched by the addition of 200 microliters of 0.5M glycine with an
additional two-hours of incubation. The covalent bonds are then
reduced by the addition of 0.5 ml of 0.25M NaBH.sub.4 with
allowance for the generation of H.sub.2 gas. After one hour of
reaction, the mixture is passed through three PD-10 columns of
Sephadex G-125M/150 equilibrated with 20 mM HEPES buffer at a pH of
7.4 to remove glycine, NaBH.sub.4 and H.sub.2, then concentrated to
a 1-2 ml volume with a Centriprep-100 concentrator (500 g for 60
minutes) to clear unbound adhesion molecule and smaller,
unconjugated particles. This product is then applied to a
1.6.times.35 cm column of Sephacryl 200 and eluted with 20 mM HEPES
buffer at pH 7.4. This column run further removes unbound targeting
molecules and traps any newly formed hydrous oxides. The eluant is
collected and concentrated with a Centriprep-100 concentrator at
500 g.times.30 minutes to achieve a final volume of 4 ml.
[0276] The four ml of reaction product are then applied to a 4 ml
column of affinity ligand Sepharose 6B with divinyl sulfone links
(such as Sigma A2278 for some lectins) equilibrated with 20 mM
HEPES buffer pH 7.4. It is preferable to avoid conditions normally
intended to maximize binding as this may make it impossible to
elute the specific fraction. The column is then washed extensively
with four to five volumes of buffer and then a 2 ml volume of 1
molar affinity eluant in the same buffer is applied. This elutes
the active fraction in a fairly sharp band.
[0277] The specific fraction is collected and passes through a
PD-10 Sephadex G-25M/150 column to help clear affinity eluant and
then concentrated to 1 ml with a small volume Centricon-30
centrifugal concentrator (1,500 g.times.20 minutes). This product
is passed through a second PD-10 column and the final output then
concentrated to a volume of 300 to 500 microliters with a
Centricon-30 concentrator (1,500 g.times.60 minutes). The final
product is then sterilized by 0.22 or 0.1 micron filtration using a
Costar 1 ml centrifugal microfilter and stored for use.
[0278] The invention is also further illustrated by the
accompanying drawings already mentioned above in which:
[0279] FIG. 1 is a diagram of one face of a spinel crystal
demonstrating the position of 1) oxygen atoms, 2) "A" sites for
metal ions and 3) "B" sites for metal ions.
[0280] FIG. 2 is a table of data on various elements discussed
herein arranged in the form of the periodic table. Data shown for
various elements include radioisotope disintegration pattern, half
life and energy profile; magnetic resonance receptivity relative to
hydrogen, Larmor frequency at 4.7 Tesla, typical oxidation state
and ionic radios, and characteristic positron affinity.
[0281] FIG. 3 is a schematic demonstration of the benefits of
spinel moderated emitters (SMPE) showing 4) a targeting protein in
free solution with a ch elated positron emitting atom undergoing
radioactive disintegration, 5) a targeting protein bound to the
surface of an SMPE particle, 6) a coated SMPE particle with
positron ionization tracks marked including one positron proceeding
to annihilation before exiting the particle, 7) the paths followed
by the two annihilation photons, 8) the correlation angle (nearly
180.degree.) between the two photon paths, 9) the ionization track
of a positron travelling in water, and 10) the site of a
matter/anti-matter annihilation reaction between an electron and
the exhausted positron occurring at some distance from where the
initial disintegration took place.
[0282] FIG. 4 is a CT X-ray showing a polyacrylamide gel. phantom
within which several gel channels are doped with different contrast
agents.
[0283] FIG. 5 is a schematic diagram of a portion of a human torso
depicting 18).the conus medullaris at the lower termination of the
spinal cord and 19) a motor axon which is part of a single cell
nearly three feet in length.
[0284] FIG. 6 shows the anatomy of the spinal roots with 20) a
dorsal root ganglion containing sensory neurons, 21) the dorsal
containing sensory axons, 22) the dorsal root entry zone, 23) the
dorsal ramus carrying motor and sensory fibers to the back muscles,
24) the ventral ramus carrying motor and sensory fibers to the
limbs and anterior portion of the body, 25) the ventral root
carrying motor axons, and 26) the ventral grey matter of the spinal
cord containing motor neuron cell bodies.
[0285] FIG. 7 depicts a motor unit omprising 27) one of several
muscle cells which fire in unison, 28) a muscle made up of many
motor units such as the one shown, and 19) a motor axon supplying
the motor unit.
[0286] FIG. 8 demonstrates 27) a myocyte or muscle cell upon which
19) a motor axon terminates and 29) a muscle spindle with 30)
sensory endings as well as intrafusal motor innervation not
shown.
[0287] FIG. 9 is a two part schematic of a peripheral nerve
including 31) the epineurium sheath, 32) a fascicle, 33) the
endoneurial space of a fascicle, 34) the perineurium surrounding
the endoneurial space and which is the site of the blood/nerve
barrier to small molecule diffusion, 19) a motor axon seen in cross
section, 35) a schwann cell-surrounding an enlarged single axon,
36) a mitochondrion within the axon seen in cross section, 37) the
axolemma or membrane of the axon, 38) a microtubule within the axon
and dimension marks indication the 20 micron diameter of the motor
axon.
[0288] FIG. 10 shows an axon in longitudinal section with 35) a
schwann cell sheath, 39) a Node of Ranvier between two schwann
cells, and 38) microtubules within the axon (19).
[0289] FIG. 11 depicts the mechanics underlying axonal transport
based on a relatively stationary microtubule (38) with 40) one of a
series of molecules of dynein and 41) one of a series of molecules
of kinesin, in which 42) a lipid vesicle is being transported in
43) a retrograde direction toward the cell body and-44) the
anterograde direction.
[0290] FIG. 12 demonstrates some of the parts of an axon terminus
with 38) a microtubule, 42) a vesicle, 36) a mitochondrion, 46) the
muscle cell membrane, 47) a 20 to 50 nm dextran coated ferrite
particle, 48) the synaptic cleft, 49) a cell surface receptor, 50)
a cell surface marker or antigen, 51) a vesicle containing an
internalized group of receptor ligand complexes, and 45) the
diameter of the axon which is 2 to 10 microns.
[0291] FIG. 13 is a graph depicting the results of the
.sup.125I-WGA distribution study in which the vertical axis gives
counts per minute per gm of tissue normalized by dividing by the
cpm/gm of blood for the individual animal in the series, various
tissues are displayed along the long Y axis and lines 1 through 8
reflect the results from the different animals with varying
treatment/survival times and doses. DRG signifies spinal roots and
dorsal root ganglia and demonstrates concentrations 5-10 times
higher than any other tissue except the muscle and lymph nodes near
the injection site which are not shown.
[0292] FIG. 14. is a graph depicting the results of a .sup.59Fe
WGA-dextran magnetite distribution study after intramuscular
injection. Counts per minute/gm tissue show concentration in distal
and proximal ipsilateral peripheral nerve which are 50 to 100 times
higher than in any other tissue excluding the muscle and lymph
nodes at the injection site which are not shown.
[0293] FIG. 15 is a graph showing results of T.sub.2 measurements
upon polyacrylamide gels doped with varying concentration of
various preparations of dextran coated magnetite. The white arrow
indicates a T.sub.2 of 30 msec which would be a 40% reduction from
normal T.sub.2 of nerve and the black arrow indicates
concentrations of ferrite particles achieved in nerve equivalent
and greater than 40 micrograms/ml. The concentrations in nerve are
up to ten times higher than the amounts required to reduce T.sub.2
in gel below 30 msec.
[0294] FIG. 16 demonstrates the general arrangements for in vivo MR
microscopy of an intact nerve in the leg of experimental animal
showing the position before being moved into the MR magnet. Note
that the leg is perpendicular to the long axis of the magnet.
[0295] FIG. 17 enlarges the view of FIG. 16 to show a surface coil
(52) placed around an incision line on the skin of the thigh.
[0296] FIG. 18 shows a cross section through the thigh with 53) the
sciatic nerve and 54) the tibial nerve approaching an implanted
cuff.
[0297] FIG. 19 demonstrates the silastic cuff (55) with a central
channel (56) for the tibial nerve and three surrounding channels
for various doped polyacrylamide gels used to standardize image
contrast. The central channel is about 1 mm in diameter.
[0298] FIG. 20 includes photographs of the tibial nerve in the
silastic cuff from 57) the uninjected leg and 58) the injected
side. The nerve is in the central channel and is darker than the
lower gel channels in the injected leg but brighter in the
uninjected side.
[0299] FIG. 21 is an electron micrograph of the tibial nerve of a
rabbit collected three days after intramuscular injection with
WGA-dextran magnetite. The photograph shows the thick myelin sheath
and, within the axon, small particles and large vesicles associated
with the particles.
[0300] FIG. 22 is a blow up of a portion of FIGS. 21 to
195,000.times.. This demonstrates small ferrite particles along the
microtubules as well as somewhat larger particles within two
vesicles being transported. FIG. 23 demonstrates the results of a
positron emission tomography trial with .sup.52Mn dextran coated
ferrite particles. There is a "nerve gel" one millimeter in
diameter (59) cast within a larger "leg gel" where the ratio of
concentrations of the positron emitting ferrites is 25:1
(nerve:leg), a relation reflecting the results of earlier
distribution trials. The diameter of the test tube is about 2.2 cm
(60) and there is a 1 milliliter syringe taped to the outside which
also contains concentrated 52Mn Ferrite. Cross sectional images
(61) show ready distinction between the two high concentration
sources and this is demonstrated in two dimensional format in 62.
Seen from anteriorly, the two sources can still be
distinguished.
* * * * *