Note: Descriptions are shown in the official language in which they were submitted.
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_
CELLULAR AND SERUM PROTEIN ANCHORS
FOR DIAGNOSTIC IMAGING
INTRODUCTION
Technical Field
The field of this invention is the non-invasive diagnostic imaging
of the mammalian vascular space.
Background
It is frequently desirable to non-invasively image the mammalian
vascular space for such purposes as detecting abnormalities in blood flow,
measuring cardiac function or for visualizing anatomical structures of the
10 circulatory system. For example, in the case of certain disorders of or
injuries to the vascular system which affect blood flow, one may wish to
detect and visualize abnormal bleeding or, alternatively, the presence of
thromboses. One may also wish to measure the effect of certain vascular
disorders on cardiac efficiency and ventricular output. Additionally, non-
15 invasive diagnostic imaging of anatomical structures of the mammalianvascular system may allow for the early detection of developmental
abnormalities or various lesions, e.g., tumors, associated with the vascular
system.
Present methodologies for non-invasively imaging the
20 mammalian vascular system include such diagnostic techniques as
positron emission tomography ~PET), computerized tomography (CT),
single photon emission computerized tomography (SPECT), magnetic
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resonance imaging~~MRI), nuclear magnetic imaging (NMI), fluoroscopy,
uitrasound, etc. However, while these techni~ues are extremeiy useful for
a variety of different applications, they often provide far less than the
desired utility, particularly when one wishes to preferentially image only a
5 single or limited number of specific cell types or anatomical structures
associated with the mammalian vascular space or to image the vascular
space over an extended period of time.
There is, therefore, substantial interest in providing novel
methods for enhancing the ability to preferentially image specific cell types
10 or compartments of the mammalian vascular space and to diagnostically
image the mammalian vascular space over an extended period of time.
SUMMARY OF THE INVENTIQN
Methods and compositions are provided for non-invasive
15 imaging of a anatomical compartment by employing bifunctional reagents
capable of covalently bonding to proteins present on the membrane of
circulating blood cells or proteins in the plasma of the mammalian vascular
system. The methods allow for monitoring the mammalian vascular
compartment over an extended period of time. The reagent compositions
20 of the present invention are bifunctional anchor molecules that have a
functional group capable of activation which, when activated, forms a
covalent bond with a reactive functionality on a target protein present in
the mammalian vascular system, thereby "anchoring" the molecule to that
target protein. The bifunctional anchor molecules of the present invention
25 are also conjugated, either directly or indirectly, to a diagnostic agent of
interest which provides the ability to diagnostically and non-invasively
image the mammalian compartment.
The applications of the subject invention encompass MRI, CT,
PET, SPECT imaging, detection of blood flow, abnormal bleeding,
30 thromboses and vascular infiammation, vessel imaging, measuring cardiac
efficiency and/or visualizing anatomical structures of the circulatory
system.
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DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods and compositions are provided for non-invasive
imaging and diagnosis of anatomical compartments, particularly the
5 vascular compartment. The methods comprise covalently bonding a
~ diagnostic agent of interest to a protein or proteins present in the
mammalian vascuiar system, wherein the diagnostic agent becomes bound
to a long-lived protein or proteins present in the vascular system, thereby
aliowing one to diagnostically image the mammalian compartment space
over an extended period of time and/or wherein the diagnostic agent is
preferentially bound to a specific protein or limited number of proteins
present in the vascular system, thereby enhancing the ability to
diagnostically image only a specific compartment. The covalent bonding
is achieved by administering to the vascular system of a mammalian host
from one to two compounds, including at least a first compound
comprising a bifunctional anchor molecule having an activated functional
group capable of forming covalent bonds with reactive functionalities on a
vascular protein or proteins, which is linked either to a diagnostic agent of
interest or to a first binding member of a specific binding pair.
By administering the first compound to the vascular system of a
mammalian host, the activated functional group wiil covalently bond to
reactive functionalities on a protein or proteins present in the vascular
system, thereby creating a population of functionalized vascular proteins.
If the first compound comprises a bifunctional anchor molecule linked to a
2~ first binding member of a specific binding pair, a second compound
comprising a reciprocal second binding member joined to a diagnostic
agent of interest is administered to the vascular system at any time during
the lifetime of the functionalized vascular proteins. After administration,
the second binding member will bind to the first binding member, thereby
anchoring the diagnostic agent of interest to the functionalized vascular
protein or proteins. The first binding molecule can also represent a
composite of both the binding compound and the diagnostic agent.
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Imaging of the diagnostic agent. present in the host's vascular system can
then be accomplished.
Bifunctional anchor molecules comprise an active functional
group capable of covalently bonding to a reactive functionality on long-
lived proteins present in the mammalian vascular system, the diagnostic
agent of interest or the first binding member of a specific binding pair and
a linker to ioin the above described moieties. Long-lived vascular proteins
have an in vivo half-life of at least about 1 Z hours, preferably at least
about 48 hours, more preferably at least about 5 days. As such, bonding
of the bifunctional anchor molecule to such proteins allows for imaging of
the vascular space over an extended period of time of at least about 6
hours, preferably at least about 24 hours and more preferably at least
about 3 days. Examples of long-iived proteins present in the mammalian
vasculature include serum albumin, ferritin, immunoglobulin, a,-
microglobulin, o~2-macroglobulin, a-, ~- or y-globulin, thyroxine binding
protein, steroid binding proteins. Specific protein targets include as part of
cells include the surface membrane proteins of erythrocytes, particularly
glycophorin A and C, T or B cell surface proteins, such as CD3, CD4,
CD5, CD8, CD28, CD34, B7, p28, CTLA-4, Thy1, LFA1, slgE, slgM,
llb/lllA, leukocyte surface membrane proteins, IL-2 receptor, integrins,
serum albumin, immunoglobulins, particularly IgG and IgM,
apolipoproteins, such as LDL, HDL and VLDL, endotheiial cell surface
proteins, including integrins, adhesion proteins, etc., or the like.
The reactive functionalities available on vascular proteins for
covalent bond formation with the bifunctional anchor molecule are
primarily amino, carboxyl and thiol groups. While any of these may be
used as the target for the reactive functional group of the bifunctional
anchor molecule, for the most part, bonds to amino groups will be
employed, particularly with the formation of amide bonds.
To form amide bonds, one may employ a wide variety of active
carboxyl groups as the reactive functional group of the bifunctional anchor
molecule, particularly esters, where the hydroxyl group is physiologically
.
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acceptable at the le~els required. Whiie a number of different hydroxyl
groups may be employed, the most convenient will be N- ~
hydroxysuccinirnide and N-hydroxy sulfosuccinimide, although other
alcohols, which are functional in the vascular environment may also be
employed. In some cases, special reagents find use such as diazo, azido,
carbodiimide anhydride, hydrazine, or thiol groups, depending on whether
the reaction is in vivo or in vitro, the target, the specificity of the anchor,
and the like.
The two moieties of the anchor may be joined by a bond ( 0
atoms in the chain) or a linker which linker is convenient, is physiologically
acceptable at utilized doses and fills the requirements of the bifunctional
anchor molecule, such as being stable in the vascular system, effectively
presenting the diagnostic agent of interest or first binding member,
allowing for ease of chemical manipulation, and the like, may be
employed. The linker may be aliphatic, alicyclic, aromatic or heterocyclic,
or combinations thereof, and the selection will be primarily one of
convenience. The linker may be substituted with heteroatoms including
nitrogen, oxygen, sulfur, phosphorus. Groùps which may be employed
include alkylenes, arylenes, aralkylenes, cycloalkylenes, and the like.
Generally, the linker of from 1-30, usually 1-10, more usually of
from 1-6 atoms in the chain, where the chain will include carbon and any
of the heteroatoms indicated above. For the most part, the linker will be
straight chain or cyclic, since there normally will be no benefit from side
groups. The length of the linker will vary, particularly with the nature of
the diagnostic agent of interest or the first binding member, since in some
instances, the diagnostic agent of interest or the first binding member may
have a chain or functionality associated with it. The length of the linker
may be used to provide for flexibility, rigidity, polyfunctionality,
orientation, or other characteristics for improved function of the
bifunctional anchor molecule. The linker may also provide for preferential
bonding to a given protein epitope or sequence present in the vasculature
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as compared to oth~T proteins epitopes or se~uences present in the
vasculature .
A large number of small synthetic bifunctional otganic
compounds comprising an appropriate activatable or activated functional
5 group are available for joining the activated functional group to the
diagnostic agent of interest or to the first binding member of a specific
binding pair. Illustrative compounds include: azidobenzoyl hydrazine, N-~4-
(p-azidosalicylamino~butyl]-3'-[2'-pyridyldithio)propionamide, bis-
sulfosuccinimidylsuberate, dimethyl adipimidate, disuccinimidyl tartrate, N-
10 y-maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl-4-
azidobenzoate, N-succinimidyl [4-azidophynyl]-1,3'-dithiopropionate, N-
succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde, succinimidyl 4-
~N-maleimidomethyl]cyclohexane- 1 -carboxylate.
The linker joining the activated functional group and the
15 diagnostic agent of interest or the first binding member may be oligomeric
in nature and possess a high non-covalent affinity for a specific protein
present in the mammalian vasculature as compared to other proteins
present in the mammalian vasculature. Such oligomeric anchor molecules
allow one to direct diagnostic agents of interest to specific targets, cells
20 and/or proteins, in the vasculature, thereby allowing one to preferentially
enhance the diagnostic signal in a particular anatomical compartment.
Preferably, oligomeric anchor molecules which find use will
have the capability of preferentially bonding to reactive functionalities on a
specific protein present in the vascular system. ~ef~rential bonding
25 means that the anchor molecule exhibits some preferential bonding to the
vascular protein of interest as against other proteins present in the
vascular environment. The preference for bonding the specified protein
target will normally be at least about 1.5, preferably at least about 2
times, and may be 5 times or more as compared to random bonding in the
30 absence of the oligomer.
The oligomeric linker of the bifunctional anchor molecule may
be an oligopeptide, oligonucleotide, combinations thereof, or the like.
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Generally, the nurrrber of monomeric units in an oligomeric linker will be
from 4 to 12, more usually from 4 to 8 and preferably from 5 to 8. The
monomer units may be naturally occurring or synthetic, generally being
from about 2 to 30 carbon atoms, usually from about 2 to 18 carbon
atoms and preferably from about 2 to 12 carbon atoms.
If the linker is an oligopeptide, the amino acid monomers may
be naturally occurring or synthetic. Conveniently, the L-a-amino acids will
be used, although the D-enantiomers may also be employed.
The amino acids employed will preferentially be free of reactive
functionalities, particularly reactive functionalities which would react with
the activated functional group or diagnostic agent of interest attached to
the oligomeric linker. Therefore, the amino acids which are used will
usually be free of reactive amino, guanidino and carboxy groups,
frequently being free of hydroxy and thiol groups. Of particular interest
are the naturally occurring amino acids having hydrocarbon side chains
including alanine (A), glycine (G), proline ~P), valine (V), phenylalanine (F),
isoleucine (I) and leucine (L) or uncharged polar amino acids like
methionine (M).
The amino acid monomers of an oligopeptide linker may also be
synthetic. Thus, any unnatural or substituted amino acids of from 4 to
30, usually from 4 to 20, carbon atoms may be employed. Of particular
interest are the synthetic amino acids ~-alanine and y-aminobutyrate or
functional group protected amino acids such as 0-methyl-substituted
threonine (T), serine (S), tyrosine (Y), or the like.
Amino acids which find use may have the carboxyl group at a
site other than the terminal carbon atom, may have the amino group at a
site other than the a-position or may be substituted with groups other
than oxy, thio, carboxy, amino or guanidino.
.,
Synthetic amino acids may also be monosubstituted on
nitrogen. N-substituted amino acids which find use will have an N-
substituent of from about 1 to 8, usually 1 to 6 carbon atoms, which may
be aliphatic, alicyclic, aromatic or heterocyclic, usually having not more
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than about 3 heteroatoms, which may include amino, either tertiary or
quaternary, oxy, thio, and the like.
Oligopeptide linkers are usually constructed by employing
standard Merrifield solid phase synthetic techniques using an automated
peptide synthesizer, standard protection chemistry (e.g., t-boc or f-moc
chemistry) and resins (e.g., 4-methyl benzhydryl amine). Other synthetic
techniques, however, such as liquid phase oligopeptide synthesis may also
find use.
If the oligomeric linker is an oligonucleotide, either naturally
occurring or synthetic nucleotide monomers may be employed.
Particularly, for synthetic nucleotides, the phosphate or sugar groups may
be modified where phosphate may be substituted by having the oxygen
atoms replaced with sulfur or nitrogen, the phosphate group may be
replaced with sulfonate, amide etc., the ribose or deoxyribose may be
replaced with 5 to 6 carbon atom sugars such as arabinose, fructose,
glucose, or the like, and the purines and pyrimidines may be modified by
substitution on nitrogen, with alkyl or acyl, may employ different ring
structures, may have nitrogen replaced by oxygen, or vice versa, and the
like.
Once synthesized, an available functional group on the
oligomeric linker may be activated so as to be able to covalently bond to a
reactive functionality present on a vascular protein in the environment in
which the reaction is to occur. The activated functional group may be
present at any position on the oligomeric linker, but will usually be
proximal to one or the other terminus. Conveniently, a member of the
oligomer may carry the activated functional group, such as on an aspartate
or glutamate moiety.
For activation of a carboxyl group on the oligomeric linker, one
may use a wide variety of anhydride or ester leaving groups, where the
leaving group may have oxygen or sulfur bonded to carbonyl. In instances
where one is interested in using the oligomeric anchor molecule in vivo,
one may selec~ the leaving group to be physiologically acceptable.
. . .
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Compounds which~nay be used to activate the carboxyl functional group
include carbodiimides, phenois, thiophenols, benzyl alcohols, N-hydroxy
imides, etc.
If the oligomeric linker is synthesized on a solid support, a
5 functional group on the oligomer may be activated and the activated
oligomer subsequently liberated from the solid support. Alternatively, the
oligomer may be liberated from the support, thereby providing an available
functional group for activation, and the functional group subsequently
activated.
Bifunctional anchor molecules, whether comprising an
oligomeric or non-oligomeric linker, are also coupled, either directly or
indirectly, to a diagnostic agent of interest which imparts the ability to
diagnosticaliy image the mammalian vascular space. Preferably, the
diagnostic agent is such that it does not react with the activated
15 functional group of the bifunctional anchor molecule nor is it affected
when the functional group of the anchor molecule is activated. Thus,
diagnostic agents which find use generally do not react with reactive
carboxyl and amino groups.
Diagnostic agents may be attached directly to the linker of the
20 bifunctional anchor molecule, where it may be attached at any convenient
site, or attached either directly or indirectly to the second binding member
of a specific binding pair. Direct attachments are via a chemical bond.
However, if the diagnostic agent is indirectly attached to the second
binding member, it may be attached through a bond or an appropriate
25 linking group. Depending upon the nature of the diagnostic agent
employed, the linking group between the second binding member and the
diagnostic agent may provide for displacement in solution of the diagnostic
agent from the second binding member by having a relatively long
hydrophilic linking group having heteroatoms in the chain or as side
30 groups, e.g. oxy, amino, oxo, carboxylate, etc. The linking group may be
an amino acid or oligopeptide of from 2 to 6 amino acids, polyoxyalkylene
of from 1 to 10 units, where alkylene is of from 2 to 3 carbon atoms, a
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sugar or the like. The particular iinking group employed will usually
depend on the nature of the diagnostic agent and its function.
Diagnostic agents which find use include those that commonly
serve as probes for known diagnostic imaging techniques such as PET,
SPECT, CT, MRI, NMI, fluoroscopy, angiography, or the like. Diagnostic
agents of interest include contrast agents, radioisotopes of such elements
i di (l~ including 123l 125l 131l, etc., barium ~Ba), gadolinium (Gd)~
technetium (Tc), including 99Tc, phosphorus (P), including 31p, iron ~Fe),
manganese (Mn), thallium (Tl), chromium (Cr), including 5lCr, carbon (C),
including 1lC, or the like, fluorescently labeled compounds, etc.
Generally, it will be satisfactory to have the diagnostic agent of
interest bonded directly to the bifunctional anchor molecule, thereby doing
away with the need to administer a second compound comprising a
second binding member attached to the diagnostic agent of interest.
1~ However, in certain situations it may be preferable to attach the diagnostic
agent of interest to the bifunctional anchor molecule (which is covalently
bonded to a vascular protein or proteins) indirectly, i.e., through the
association between a first binding member of a specific binding pair with
its reciprocal second binding member. Such situations include where it is
useful to covalently bond the bifunctional anchor to vascular proteins in
vivo and then periodically administer small amounts of the second binding
member/diagnostic agent complex which is cleared from the vascular
system, e.g., for use of diagnostic agents which are toxic when
maintained at continuously high concentrations in vivo.
When the diagnostic agent of interest is bonded to the
bifunctional anchor molecule indirectly, the first binding member which is
joined to the bifunctional anchor molecule will generally be a small
molecule, where the molecule is likely to minimize any immune response.
Thus, for the most part, the first binding member will be haptenic, usually
30 below about 1 kD and generally more than about 100 D, preferably less
than about 600 D. Any physiologically acceptable molecule may be
employed, where there is a convenient reciprocal second binding member.
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Thus, of particular Tnterest is biotin, where avidin or streptavidin may be
the reciprocal second binding member, but other molecules such as metal
chelates, molecules mimicking a natural epitope or receptor or antibody
binding site, also may find use, where the reciprocai second binding
5 member may be an antibody or a fragment thereof, particuiarly a Fab
fragment, an enzyme, a naturally occurring receptor, or the iike. Thus, the
first binding member may be a ligand for a naturally occurring receptor, a
substrate for an enzyme, or a hapten with a reciprocal receptor.
The first binding member will naturally be found at low
10 concentration, if at all, in the host vascular system, so there will be little if
any competition between the first binding member and naturally occurring
compounds in the vascular system for binding to the reciprocal second
binding member. The reciprocal second binding member is such that it
should not bind to compounds which it may encounter in the vascular
15 system of the host.
The reciprocal second binding member of the specific binding
pair will be determined by the nature of the first binding member
employed. As already indicated, the second binding member may take
numerous forms, particularly as binding proteins, such as immunoglobulins
20 or fragments thereof, particularly Fab, Fv, or the like, particularly
monovalent fragments, naturally occurring receptors, such as surface
membrane proteins, enzymes, other binding proteins, such as avidin or
streptavidin, or the like. Generally, the affinity of the second binding
member for its reciprocal first binding member will be at least about 1 o-6,
25 more usually about 1 o-8, e.g., binding affinities normally observed for the
binding of monoclonal antibodies to their specific binding entities. Of
particular interest is avidin and streptavidin, although other receptors of
particular interest include receptors for steroids, LH, TSH, FSH, or their
agonists, as well as sialic acid and viral hemagglutinins, and superantigens.
30 The second binding member will usually be a macromolecule, generally of
at least about 5 kD, more usually of at least about 10 kD and usually less
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than about 160 k~3-j preferably Jess than about 80 kD, which may be
mono- or divalent in binding sites, usually monovalent.
The vascular protein or proteins chosen as targets for reaction
with the bifunctional anchor molecule will depend upon the indication
desired. Thus, depending upon the vascular target or targets chosen, the
diagnostic agent can be bonded to long-lived proteins and dispersed
substantially throughout the entire vasculature, in which case the
indication of choice will be diagnostic imaging of the vascular system over
an extended period of time, or preferentially localized to specific areas of
the vascular system, in which case the indication of choice will be the
preferential diagnostic imaging of a specific anatomic compartment, either
with or without imaging over an extended period of time. The target may
be fixed or mobile; that is substantially fixed in position, as in the case of
endothelial cells, or mobile in the vascular system, i.e., not having a fixed
situs for an extended period of time, generally not exceeding 5, more
usually, one minute. Target cells and proteins may have a substantially
uniform or variant distribution in the vascular system, where the target
may preferentially localize or be concentrated in particular compartments,
as compared to the vascular system or other anatomic compartments.
The diagnostic agent employed and the vascular protein or
proteins targeted will depend upon whether one wishes to diagnostically
image the anatomic compartment over an extended period of time,
whether one wishes to preferentially image only a specific cell type or
compartment, or both. Applications for covalently bonding a diagnostic
agent of interest to a long-lived vascular protein for diagnostic imaging of
the vascular space over an extended period of time are numerous and
include enhancing the ability to detect abnormalities in blood flow
throughout the entire mammalian vascular system, including the detection
of internal in~ury causing abnormal bleeding or, alternatively, the presence
of thromboses. For example, one may wish to image the vascular space
over an extended period of time to detect the effects of a particular
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treatment while th~y occur, i.e./ detecting the disappearance of an
embolism, the stoppage of internal bleeding, or the like.
Diagnostically imaging the vascular space over an extended
period of time also allows for the detection of various diseases associated
5 with the vascular system, i.e., such as arterial blockage in the heart.
Thus, diagnostically imaging the vascular system over an extended period
of time may be employed to non-invasively detect a consistently reduced
blood flow to the heart. Such a method also provides a means for
quantitatively measuring cardiac efficiency and ventricular output volume
10 over an extended period of time, i.e., during extended periods of exercise,
or the iike.
Other applications for such a method arise from the ability to
non-invasively visualize anatomical structures of the mammalian vascular
system and the effects on those anatomical structures over time of the
15 administration of various drugs, such as vasodilators, vasoconstrictors, or
the like. Such may allow for the early detection of deve~opmental vascular
abnormalities, injuries, or the like.
Additional applications arising from the ability to diagnostically
image the vascular space over an extended period of time include
20 functional assessment of the cardiovascular system as routinely utilized in
nuclear medicine for single measurements.
The first compound and, if required, the second compound, will
usually be administered as a bolus, but may be introduced slowly over
time by transfusion using metered flow, or the like. Alternatively,
25 although less preferable, blood may be removed from the host, treated ex
vivo, and returned to the host. The first and second compounds will be
administered in a physiologically acceptable medium, e.g., deionized
water, phosphate buffered saiine, saline, mannitol, aqueous glucose,
alcohol, vegetable oil, or the like. Usually, a single injection will be
30 employed although more than one injection may be used, if desired. The
first and second compounds may be administered by any convenient
means, including syringe, catheter, or the like. The particular manner of
1 3
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administration willvary depending upon the amount to be administered,
whether a single bolus, sequential, or continuous administration, or the
like. Administration will be intravascular, where the site of introduction is
not critical to this invention, preferably at a site where there is rapid blood
5 flow, e.g., intravenously, peripheral or central vein. The intent is that the
compound administered be effectively distributed in the vascular system
so as to be able to react with target proteins therein.
The dosage of the compound will depend upon whether it
comprises the diagnostic agent of interest and will, therefore, be
10 dependent on the adverse effects of the diagnostic agent of interest, if
any, the time necessary to reduce the unbound concentration of the agent
present in the vascular system, the dosage necessary for successful
diagnostic imaging, the indication being sought, the sensitivity of the
diagnostic agent to destruction by vascular components, the route of
15 administration, and the like. As necessary, the dosage of diagnostic agent
may be determined empirically, initially using a small multiple of the
dosage normally administered, and as greater experience is obtained,
enhancing the dosage. Dosages will generally be in the range of 1 ng/Kg
to 10 mg/Kg, usually being determined empirically in accordance with
20 known ways, as provided for in preclinical and clinical studies.
All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled in the art to
which this invention pertains. All publications and patent applications are
herein incorporated by reference to the same extent as if each individual
25 publication or patent application was specifically and individually indicated to be incorporated by reference.
The invention now being fully described, it will be apparent to
one of ordinary skill in the art that many changes and modifications can be
made thereto without departing from the spirit or scope of the appended
30 claims.
14
