Note: Descriptions are shown in the official language in which they were submitted.
CA 02208566 1997-06-23
W ~96tl9242 PC~rnUS94~I4776
DESCRIPTION
COMPLEXES OF DERMATAN SULFATE ANG ~RIJGS, GIVING IMPROVED PHARMAcoKINETIcs
BACKGR~UND OF THE lNVl!;N-l lON
Until recently, the localization of intravascular
drugs :in body tissues has depended on chemical
partit.ioning across microvascular barriers into the
tissue compartments of multiple body organs. This
resulted in only 0.01~ to 0.001~ of the injected dose
actual:ly reaching the intended targets. Approximately 20
years ago, drugs were entrapped in liposomes and
~icrospheres. This modified the initial biodistributions
and redirected them to phagocytes in the
reticuloendothelial organs: liver, spleen and bone
marrow.
In 1978, the present inventor and coworkers (Widder,
et al., Proc. Am. Assn. Cancer Res., V. 19, p 17 (1978))
develo~Sed a means to co-entrap drug plus magnetite in
microspheres which could be injected intravenously and
localized magnetically in the tissue compartments o~
nonreticuloendothelial target organs (e.g., lung and
brain). Magnetic capture was accomplished by selective
dragging of the particles through the vascular
endothelium into normal tissues and tissue tumors
. 35 positioned adjacent to an extracorporeal magnet of
suffici~nt strength (0.5 to 0.8 Tesla) and gradient (0.1
Tesla/mm). Although this technique was highly efficient
. CA 02208~66 1997-06-23
-- W O96/19242 PCTrUS94114776
~. , .
.
~ .
~ - 2 -
. - - : ~. ....................................................................... '
~ ' and deposited between 25~ and 50~ of an injected dose in
... . .
--~ the desired target tissue, it was also a very complicated
~- - approach which had the following major disadvantages: 1)
~:- restriction of use to specialized medical centers; 2)
permanent disposition of magnetite in target tissue; 3)
~, b ' focal overdosing of drug due to inhomogeneity of the
=.~ -~ . capturing magnetic field; and 4) application to a very
~ limited number of therapeutic agents. In the process of
- ~ studying magnetic targeting, however, it was learned that
~; 10 slow (controlled) release of toxic drugs from entrapment-
~r '' , ' type carriers (microspheres) protected the normal cells
within the local tissue environment from drug toxicity
-- ~ and still gave effective treatment of tumor cells and
- '~ microorganisms.
~ --- 15
=~ When monoclonal antibodies became generally
available for animal and clinical research, it was hoped
-- that antibody-drug conjugates would limit the
- biodistribution of toxic agents and cause them to become
.~ i 20 deposited in foci of disease (tumors and infections)
,
~ ~ which were located across the microvascular barrier
within target tissues. Unfortunately, most monoclonal -~
., .
~ antibodies were (and are still) obtained from mice,
-- making them immunologically foreign to human recipients.
- 25 Conjugation of drugs at therapeutically relevant
~ - substitution ratios makes the monoclonal antibody
-- - derivatives even more foreign and impairs their binding
;~ ~ specificities. Hence, antibody-drug conjugates are
, cleared substantially by the liver, as are liposomes.
-~ 30 Importantly, their localization in most solid tumors is
~ - even further impaired by the presence of a partially
-,-~. intact microvascular barrier which separates the tumor
~ ~ tissue (interstitium) from the bloodstream. This allows
- - only about 1~ to 7~ (at best) of the injected dose to
35 reach nonreticuloendothelial targets. Selected lymphomas .
~- and leukemias provide exceptions to this rule because of
- a greater natural breakdown of this vascular barrier.
. , =,
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
a However, for the vast majority of solid tumors and
infecti3ns, a general-purpose method is still needed to
deliver drugs efficiently across microvascular barriers
in a depot (controlled release) form.
Su~h a form of drugs is necessary in order to
protect the normal vascular endothelium, organs, tissue
cells from the toxic effects of drugs, protect the drug
from en~othelial and tissue metabolism during transit,
and make the drug bicavailable at a controlled
therapeltic rate selectively within the target tissues
and tissue lesions, including solid tumors.
Ac.ive endothelial transport has been demonstrated
for small molecules (e.g., glucose and insulin), however,
no studies other those that of the present inventor have
shown such transport for larger molecules or molecules
carried in a cargo format. Present examples show that
transendothelial migration of macromolecular conjugates
and noncovalent paired-ion formulations of drugs and
diagnos_ic agents with sulfated glycosaminoglycan, having
a combined size of between about 8,000 daltons and about
500 nanometers, are accelerated by the inclusion of
sulfatel~ glycosaminoglycans, and in particular, dermatan
sulfate.s, which bind multiply to receptors or antigens
which are either synthesized by disease-induced
endothe.Lium or are synthesized at other sites but become
selectively bound to the induced endothelial receptors at
sites o~ disease. (Ranney, Biochem. Pharmacology, V. 35, .
No. 7, 1?p. 1063-1069 (1986)).
- . The present invention describes improved novel
composi_ions, carriers, agents and methods of in vivo use
which g.ive improved selectivity, efficacy, uptake
~ 35 mechanism and kinetic-spatial profiles at sites of
disease~ It further describes compositions, agents and
methods of use for improved selectivity, sensitivity,
. CA 02208~66 1997-06-23
= W O96/19242 PCT~US94/14776
.
-- 4
-
-~- uptake mechanism and kinetic-spatial profiles of in vivo
selective drug localization, accumulation and action at
- sites of disease, including but not limited to solid
~ - 1
~ ~ tumors. Novel compositions are prepared by (a) unique
5 non-covalent chemical binding, further enhanced by (b)
-~- - physical stabilization. Other compositions are prepared
~ by covalent chemical binding. Binding is of cationic or
-- chemically basic metal chelators to carriers comprising
==' g
~ anionic or chemically acidic saccharides, sulfatoids and
: - 10 glycosaminoglycans, typically and advantageously of a
' hydrophilic or essentially completely hydrophilic nature.
Binding of the active and carrier may also be by a
- combination of non-covalent, physical, and covalent
means. Non-covalent binding can be carried out by means
15 including but not limited to admixing cationic or basic
drugs and metal chelates at appropriate ratios with
anionic or acidic saccharide carriers, thereby forming
. ~ .
strong solution-state and dry-state paired-ion complexes
- - and salts, respectively, based principally on
~ ~ ~ 20 electrostatic binding of cationic (basic) group or groups
-~ - of the metal chelator to anionic (acidic) group or groups
of the acidic carrier. Such binding may be further
- stabilized by hydrogen bonds and physical factors,
~ including but not limited to concentration, viscosity,
: 25 and various means of drying, including lyophilization.
--- . Carrier substances useful in this invention may~-= include, but are not limited to natural and synthetic,native and modified, anionic or acidic saccharides,
~~ 30 disaccharides, oligosaccharides, polysaccharides and
~ glycosaminoglycans (GAGs) and in particular, dermatan
- sulfates. It will be apparent to those skilled in the
~= art that a wide variety of additional biologically
compatible, water-soluble and water dispersable, anionic
~ 35 carrier substances can also be used. Due to an absence
= - of water-diffusion barriers, favorable initial
-~- - biodistribution and multivalent site-binding properties,
.
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
oligome;-ic and polymeric, hydrophilic and substantially
completely hydrophilic carrier substances are included
among the preferred carriers for agents to be used for
treatinc~ tumors, cardiovascular infarcts and other types
of local disease. However, it will be apparent to those
skilled in the art that amphoteric and hydrophobic
carriers may be favored for certain therapeutic
applications. Drugs and metal chelators mos~ useful in
this in~ention include those which contain cationic,
basic ard basic-amine groups for binding to the carrier,
and which are effective to treat local disease conditions
either directly or indirectly, including by chelation of
metals c.nd metal ions, transition elements and ions, and
lanthanide series elernents and ions. It will be apparent
to those skilled in the art that essentially any single
atomic element or ion amenable to chelation by a
cationic, basic and amine-containing chelator, may also
be usefu.l in this invention.
For purposes of this invention, a cationic or basic
metal chelator is defined and further distinguished from
a metal-ion complex as follows: a cationic or basic metal
chelator comprises an organic, covalent, bridge-ligand
molecule, capable of partly or entirely surrounding a
single metal atom or ion, wherein the resulting formation
constant of chelator for appropriate metal or ion is at
least about 10l4. A chelator is further defined as
cationic or basic if it or its functional group or groups
which confer the cationic or basic property, and which
include but are not limited to an amine or amines, is
(are) conpletely or essentially completely electrophilic,
positiveLy charged or protonated at a typical pH employed
for formulation. A formulation pH is characteristically
selected to closely bracket the range of physiologic pH
'~ 35 present .in mammalian vertebrates. This typically
includes, but is not limited to a pH in the range of pH 5
to 8. Al-nines may include primary, secondary, tertiary or
~ i CA 02208~66 l997-06-23
.
~~- ' W O 96/1~242 PCTrUS94/14776
- quaternary amines or combinations thereof on the metal
~ chelator. Herein, and as specified, a hydrophilic
~i- carrier is defined as a substance which is water soluble,
~-~ partitions into the water phase of aqueous-organic
solvent mixtures, or forms a translucent aqueous
=~ solution, complex, aggregate, or particulate dispersion
under the conditions employed for formulation. A carrier
is further defined as being anionic or acidic if it is
completely or nearly completely nucleophilic, or if its
~ 10 functional group or groups are capable of interacting
- -~- with cationic, basic or amine metal chelators, is (are)
~- ~ completely or nearly completely negatively charged,
anionic or ionized at the pH employed for formulation.
Such anionic and acidic groups include, but are not
limited to sulfates, phosphates and carboxylates, or
- -- combinations thereof on the carrier.
,
- - Novel agent compositions include, but are not
;~ i limited to the classes of cationic or basic, typically
basic-amine metal chelator actives, or metal chelator
~ actives including the chelated metal or metal ion,
-=., -t wherein these actives are further bound to anionic and
acidic carriers comprising natural or synthetic carriers,~
~- - including but not limited to hydrophilic anionic or
-:-, 25 acidic, natural or synthetic, native, modified,
~ '~ derivatized and fragmented, anionic or acidic
-- - saccharides, oligosaccharides, polysaccharides,
- - sulfatoids, and glycosaminoglycans (GAGs).
,
~Y~-; - 30 Anionic and acidic saccharide and glycosaminoglycan
--f ~ ~ carriers may contain monomeric units comprising glucose,
- ~ glucuronic acid, iduronic acid, glucosamine, galactose,
- galactosamine, xylose, mannose, fucose, sialic acid,
pentose, and other naturally occurring, semi-synthetic or
~ - 35 synthetic monosaccharides or chemical derivatives
; ~ . thereof, comprising amine, sulfate, carboxylate, sialyl,
~ phosphate, hydroxyl or other side groups.
:
,
CA 02208~66 1997-06-23
WO96/19242 PCT~S94/14776
Glycosarlinoglycans (G~Gs) comprise essentially the
carbohyclrate portions of cell-surface and tissue matrix
proteog].ycans. They are derived from naturally occurring
proteog]ycans by chemical separation and extraction; and
in certain instances, by enzymatic means [Lindahl et al.
(1978), incorporated herein by reference]. They include,
but are not limited to those of the following types:
heparin, heparan sulfate, dermatan sulfate, chondroitin-
4-sul~ate, chondroitin-6-sulfate, keratan sulfate,
syndecar., and hyaluronate, and over-sulfated, hyper-
sulfatec, and other chemical derivatives thereo~, as
described further below.
The strongly acidic, sulfated glycosaminoglycans
include all of those classes listed just above, except
for hyaluronate, which contains only the more weakly
acidic carboxylate groups and not sul~ate groups.
Natural sources of glycosaminoglycans include, but are
not limited to: pig and beef intestinal mucosa, lung,
spleen, pancreas, and a variety o~ other solid and
parenchymal organs and tissues.
Sulfatoids comprise a second class cf su_fated
sacchari~e substances which are derive~ prlr.c ally bu~
not exclusively from bacterial and non-mammalian sources.
Sulfatoi~s are typically of shorter chain length and
lower molecular weight than glycosaminoglycans, but may
be synthetically modified to give (a) longer chain
lengths, (b) increased sulfation per unit saccharide, (c)
various other chemical side groups, or (d) other
properties favorable to the desired ligand-binding
property and site-selective binding, uptake and
accumulation property (or properties) in vivo. Sucrose
and othe:- short-chain oligosaccharides may be obtained
from natural and synthetic sources.
- ; CA 02208~66 1997-06-23
'~ . W O96/19242 PCTrUS94114776
=. , ,
~ 8 -
'.
--- These oligosaccharides can be rendered anionic or- ' acidic by chemical or enzymatic derivatization with
.... . .
~ - carboxylate, phosphate, sulfate or silyl side groups, or
-~-- combinations thereof, at substitution ratios of up to
~ - 5 about eight anionic or acidic substituent groups per
~. -~ disaccharide unit. Modified glycosaminoglycans may be
-
derived from any of the types and sources of nativeglycosaminoglycans described above, and include: (1)
glycosaminoglycan fragments, further defined as
glycosaminoglycans with chain lengths made shorter than
the parental material as isolated directly from natural
sources by standard ion-exchange separation and solvent
fractionation methods; (2) glycosaminoglycans chemically
modified to decrease their anticoagulant activities,
thereby giving "non-anticoagulant" (NAC) GAGs, prepared
typically but not exclusively by (a) periodate oxidation
followed by borohydride reduction; (b) partial or
complete desulfation; and (c) formation of non-covalent
divalent or trivalent counterion salts, principally
including but not limited to salts of the more highly
acidic sulfate functional groups, with principally but
not exclusively: calcium, magnesium, manganese, iron,
gadolinium and aluminum ions.
For purposes of this invention, a special class of
such solution complexes and salts includes those strong
complexes and salts formed by electrostatic or paired-ion
association between the acidic or sulfate groups of
, -.
. - acidic saccharide or glycosaminoglycan carrier, and the
basic or cationic group or groups of the metal chelator
. ;~ = or metal chelator including metal, as described above.
Dejrivatized acidic saccharides and glycosaminoglycans are
. = typically prepared by derivatization of various chemical
side groups to various sites on the saccharide units.
This may be performed by chemical or enzymatic means.
.
. -
CA 02208~66 l997-06-23
W O96/19242 PCT/US94rl4776
En:.ymatic means are used in certain instances where
highly selective derivatization is desired. Resulting
chemica: and enzymatic derivatives include, but are not
limited to acidic saccharides and glycosaminoglycans
derivat.zed by: (1) esterification of (a) carboxylate
groups, (b) hydroxyl groups, and (c) sulfate groups; (2)
oversuliation by nonselective chemical or selective
enzymati.c means; (3) acetylation, and (4) formation of
various other ligand derivatives, including but not
limited to (a) addition of sialyl side groups, (b)
addition of fucosyl side groups, and (c) treatment with
various carbodiimide, anhydride and isothiocyanate
linking groups, and (d) addition of various other
ligands.
If and when present, sulfa~e and sialyl side groups
may be present at any compatible position of saccharide
monomer, and on any compatible position of
glycosaminoglycan monomers [Lindahl et al. (1978),
incorpoY-ated herein by reference]. Certain of the
resulting derivatized acidic saccharides and
glycosaminoglycans may have desired alterations of
anticoagulant activities, site-localization patterns,
clearance and other biological properties. As one
example of this relationship between certain classes of
glycosaminoglycans and biological properties, dermatan
sulfates with a native sulfate/carboxylate ratio
preferably in the range of from 0.7:1 to 1.8:1, more
preferably between 0.9:1 and 1.5:1 and typically 1:1, are
reported to have relatively low binding to normal
endothelial cells, avcid displacement of endogenous
~heparan sulfate from endothelial-cell surfaces, have
. relatively high selectivity to induced endothelia at
sites of disease, including thrombus, and have rapid
~- 35 plasma clearance, principally by the renal route; whereas
heparins and oversulfated dermatan sulfates with higher
sulfate/,_arboxylate ratios of between 2:1 and 3.7:1, are
. CA 02208~66 1997-06-23
- W O 96/19242 PCTrUS94/14776
-- 1 0
. reported to have relatively higher binding for both
normal and induced endothelia, to displace relatively
more endogenous endothelial heparan sulfate, and to clear L
.:: more slowly than dermatans [Boneu et al. (1992),
- ;~
~- 5 incorporated herein by reference].
~ .
As newly described and used in the present
invention, the dermatan sulfate class of
glycosaminoglycans, and especially the new special class
of dermatan sulfates which contain selectively
oversulfated oligosaccharide sequences, have the further
unique advantages of higher potency combined with very
low toxicity as carrier substances of associated or bound
actives (i.e., dermatan sulfate-actives, DS-actives).
This is related to their (a) relatively low
sulfate/carboxylate ratios which range between 0.7:1 and
1.8:1, most preferably lying between 0.9:1 and 1.5:1, and
most typically being 1:1; (b) very low anticoagulant
activities -- related to very low factor Xa and USP
heparin activity plus negligible binding to antithrombin
III; (c) very low or absent platelet-aggregating, and
hence thrombocytopenia-inducing properties -- related to
their relatively low S03-/COO- ratios in combination with
a modal molecular weight of less than about 45,000
daltons and preferably less than about 25,000 daltons;
(d) essentially complete absence of in vivo metabolism;
and (e) very rapid blood and body clearance, all as
further described below. These properties result in an
extremely high in vivo safety profile with an absence of
bleeding, metabolism and in vivo residua in normal
tissues and organs. These properties and their resulting
safety profiles clearly distinguish the dermatan sulfates
from all other classes of glycosaminoglycans (~,AGs) and r
other classes of acidic saccharides, oligosaccharides,
polysaccharides and sulfatoid substances (taken together, .~
comprising acidic and anionic saccharide substances), and
they provide uniquely surprising and unexpected
.
.
CA 02208~66 1997-06-23
W O96/19242 c PCT/US941I4776
advanta!3es for dermatan sulfates over these other classes
of acidic and anionic saccharides. Most particularly,
the dermatan sulfates show these surprising and
unexpec ed advantages over other glycosaminoglycan
polysul:~ates, with SO3-/COO- ratios in the range of
between 2:1 and 3.7:1 and sulfur contents of greater than
or equa:L to 10~ (weight basis -- indicative of their much
higher sulfate contents). Also, most particularly, the
new special class of dermatan sulfates (as described at
length below), which is enriched for selectively
oversuli-.ated oligosaccharide sequences without comprising
oversuliated or polysulfated molecules overall throughout
the enti.re chain length (the latter being characterized
by SO3-/COO- ratios greater than or equal to 2.0:1 and
sulfur c'ontents greater than or equal 10~), have the
further surprising and unexpected advantage of more
strongly binding to the selectively induced receptors of
endothelium, tissue matrix and target-cells at sites of
disease (including tumors) by means of the complementary,
selecti~ely oversulfated oligosaccharide sequences of
these new special dermatan sulfates. Hence, these new
special dermatan sulfates exhibit surprisingly and
unexpectedly more potent site localization and site-
targeting potencies than would otherwise be expected
based on their moderately low overall S03-/COO- ratio and
sulfation and on their related extremely low cellular and
systemic toxicity properties and side-effect profiles.
In a special case unique to the present invention,
derivatization of the acidic saccharide and
glycosaminoglycan carriers may be accompanied by the
basic metal chelator itself. Although the general
classes ~f carriers described above are particularly
suitable to the present invention, it will be apparent to
those skilled in the art that a wide variety of
additional native, derivatized and otherwise modified
carriers and physical formulations thereof, may be
~_r.' '.' CA 02208~66 1997-06-23
~.= W O96/19242 PCTrUS94/14776
, ~
~ . ~
~ 12 -
j~ . .
particularly suitable for various applications of this
invention. As one representative example, the source and
~ -~ type of glycosaminoglycans, its chain length and
: sulfate/carboxylate ratio can be optimized to (1) provide~= 5 optimal formulation characteristics in combination with
=-- different small and macromolecular diagnostic agents and
-~ drugs; (2) modulate carrier localization on diseased
- versus normal endothelium; (3) minimize dose-related side
' effects; (4) optimize clearance rates and routes of the
;- 10 carrier and bound diagnostic and therapeutic actives.
~~ Non-covalent formulations of active and carrier
r afford markedly higher active-to-carrier ratios than
- those possible for covalent chemical conjugates. In the
~- - 15 present invention, non-covalent binding affords a minimum
. ~
~ - of 15~ active to total agent by weight [active / (active~~ ~ carrier), w/w]; typically greater than about 30~ (w/w);:-- ' preferably at least about 50~ (w/w); and frequently
~
between about 70-99~ (w/w). Covalent binding
s 20 characteristically limits the percent active to (a) less
: than about 12~ for non-protein small and polymeric
- - ~ carriers, (b) less than about 7~ fo- pep~iae ar.d prote -.
~~ carriers, including antibodies, and ~-) les~ ,han abo~
0.5-2.0~ for antibody fragments. Th~s li,..~a..-n is
based on the number of functional groups avallable or.
~ :~ carrier molecules which are useful in agent formulatio
=~- and in vivo site localization.
.. ~ ~
- It will be apparent to those skilled in the art that
- 30 covalent active-carrier agent compositions of low
- -- substitution ratio may be useful for certain in vivo
: applications of typically narrow range, and that non-
- covalent active-carrier agent compositions of high
~~ ~ substitution ratio may be useful for other in vivo
~ 35 applications of typically broader range. Generally, but -'
not exclusively, non-covalent agents may be particularly
~ useful for the ma~ority of diagnostic imaging
': ' ,, .
CA 02208566 1997-06-23
W O 96tl9242 PCTrUS94~I4776
applica~ions and for most therapeutic applications,
wherein high total-body and site-localized doses are
needed, and rapid clearance of the non-localized fraction
of admi~listered agent is desired in order to accelerate
plasma clearance and to achieve low background levels ~or
purposes of maximizing image contrast and minimizing
systemic drug toxicity.
The~se properties of the present formulations
represent additional substantial improvements over prior,
non-selective and covalently conjugated active-carrier
agents. The resultin~ agents are broadly useful for: (a)
site-se]ective drug localization, including tumors,
infectic~ns and cardiovascular disease with an acute
endothelial induction; (b) MRI contrast and spectral
enhancement, Ultrasound contrast enhancement, and X-Ray
contrast enhancement, where relatively high administered
doses may be favored or required; (c) Nuclear Medical or
Radionuclide imaging and therapy, where enhanced
clearance of the non-targeted dose may be favored or
required: and (d) certain high-dose, extended-release or
sustained-effect therapy may be favored or required.
Such therapeutic agents include but are not limited to
those useful at a broad variety of organ sites and
medical indications, for the treatment of: (a) acute
vascular ischemia, acute infarct, acute vascular damage,
shock, h~potension, restenosis, tumors and tumor
angiogen~sis and parenchymal-cell or other pathological
proliferations; and (b) the following classes of disease:
vascular, parenchymal, mesenchymal, endothelial, smooth
muscle, striated muscle, adventitial, immune,
inflamma_ory, bacterial, fungal, viral, degenerative,
neoplast.ic, genetic and enzymatic.
.
. 35 MRI contrast enhancement and drug therapy are
important: indications for which high payload and
controlled release of active agents are important unique
~-- CA 02208~66 1997-06-23
: :
~ W O 96/19242 PCTrUS94/14776
':
- 14 -
'~ advantages in addition to site selective localization
- (see below).
For purposes of this invention, potentially
therapeutic metal ions generally useful for trans
chelation at sites of disease may include divalent and
trivalent cations selected from the group consisting of:
iron, manganese, chromium, copper, aluminum, nickel,
gallium, indium, gadolinium, erbium, europium, dysprosium
- 10 and holmium. Chelated metal ions generally useful for
radionuclide imaging and compositions and uses, and in
- radiotherapeutic compositions and uses, may include
~- metals selected from the group consisting of:
- phosphorous, sulfur, gallium, iodine, germanium, cobalt,
calcium, rubidium, yttrium, technetium, ruthenium,
rhenium, indium, tin, iridium, platinum, thallium,
; strontium and samarium. Metal ions useful in neutron-
capture radiation therapy may include boron and others
with large nuclear cross sections. Metal ions useful in
Ultrasound contrast and X-Ray contrast compositions and
~ uses may, provided they achieve adequate site
; concentrations, include any of the metal ions listed
above, and in particular, may include metal ions of
atomic number at least equal to that of iron.
- For purposes of this invention, agents for
therapeutic composition and uses in chelating internal
body iron, copper or both, in order to make these metals
unavailable locally (1) which are typically required for
- 30 neovascularization, or (2) which cause and amplify local
: tissue injury [Levine (1993), incorporated herein by
reference], include the carrier with basic metal chelator
in one or both of the following forms: (a) carrier plus
chelator without metal ion; and (b) carrier plus chelator
with metal ion added and chelated in the composition at a
- formation constant lower or equal to that of the internal
body metal which is to be chelated by metal ion exchange
CA 02208~66 1997-06-23
W O96/19242 PCTAUS94~4776
into th~ respective basic metal chelator of the
composition (see below). Such weakly chelated metal ions
of the c~omposition may include one selected from the
group consisting of: calcium, manganese, magnesium,
chromium, copper, zinc, nickel, iron, gallium, indium,
aluminur,l, cobalt, gadolinium or other exchangeable ion.
Metal i~ns useful for inclusion in compositions for other
therapeutic uses may include the divalent and trivalent
cations selected from the group consisting of magnesium,
manganese, chromium, zinc and calcium, iron, copper and
aluminum. It will be obvious to those skilled in the art
that various ones of the preceding metal ions can be used
in combination with basic metal chelators, for
alternat.ive indications than those specified above, and
that met.al ions other than those listed above may, under
certain conditions, be useful in the uses and indications
listed above.
The compositions described in this invention give
surprising and unexpected improvements of performance and
use which include:
(1) retained high association of active plus
carrier during in vitro dialysis and in
vivo targeting;
(2) selective binding of the active plus carrier to
~ induced endothelia at sites of disease;
(3) following intravenous administration, very
rapid (2-7 min) localization at the
. diseased site, due to rapid selective
~~ . endothelial binding, envelopment and
~ extravasation of the carrier plus metal
~ 35 chelator across disease-induced endothelia
(including histologically non-porous
endothelia);
'~ CA 02208566 1997-06-23
W O96/19242 PCTrUS94/14776
.
.- - 16 -
; (4) widespread uptake throughout the diseased tissue
site;
(5) sustained retention (multiple hours to days)
within the diseased site in combination
with
..
~ . (6) rapid plasma clearance (minutes) of the non-
. = targeted fraction;
- - 10
~ = ~7) moderately slower, polymeric backdiffusion
rates into the plasma, affording prolonged
disease-site retention;
,. :
(8) capacity to selectively treat and image solid
tumors or acute vascular and myocardial
~- infarcts at body sites, as well as at
brain and central nervous system sites,
with substantially improved selectivity
and sensitivity, including small tumor
-
- metastases, in liver, lung and other organ
sites.
Diagnostic and drug enhancement can be made to occur
. 25 by a number of mechanisms, the principal ones being:
1. Effective TARGETING to tissue sites of disease;
2. STABILIZATION during both storage and plasma
transit;
: 3. Prolonged RETENTION at the site of disease, giving a markedly increased area under the curve at the tissue
site;
4. RAPID CLEARANCE of the non-TARGETED fraction,
thereby reducing background signal in imaging.
CA 02208~66 1997-06-23
WO 96119242 PCT~IJS94J14776
applicat~ions and reducing normal organ exposure and
systemic toxicity in therapeutic applications.
Five further significant advantages of the present
compositions and uses are:
1. Simple formulations of active and carrier;
2. Stabilization of diagnostic and therapeutic
actives on the shelf and during plasma transit;
3. Rapid site localization and sustained site
retention;
4. Rapid clearance of the non-targeted fraction;
5. Availability of low toxicity carbohydrate and
glycosaminoglycan carriers from natural sources
and, where needed, modification or
derivatization by straightforward synthetic
means.
Acidic or anionic saccharides a-.d g'~co~a~inoglyca..--
have unique mechanisms of site loca' za i_-. a~.~ reten~,s.
25 in vivo. They bind to the body's endothelial
determinants which are selectively induced on the
microva~cular barrier by underlying tissue disease.
Previous approaches to site targeting were directed at
antigenic determinants. However, because these
determinants are typically located in sequestered sites
within the tissues, in other words at sites across the
endothelial barrier and not within the bloodstream and
not on its endothelial surface, carriers and agents
injected into the bloodstream had no ef~ective means to
~, 35 recognize and localize in the region of these target
antigens. Stated another way, previous approaches
ignored the major problem of inappropriate carrier
- CA 02208~66 1997-06-23
W O96/19242 PCTrUS94/14776
,
- 18 -
~ distribution which resulted from its failure to recognize
- the vascular access codes required for efficient
~- extravasation at disease sites. Hence, these carriers
failed to effectively load the relevant tissue sites with
effective concentrations of their bound actives.
.
: Acidic or anionic saccharides, including
glycosaminoglycans, dermatan sulfates and the new special
dermatan sulfates, localize at target sites by binding
first to complementary receptors on disease-site vascular
- endothelium, induce very rapid (ca. 3-minute)
extravasation of the carrier and associated active agent,
and then widely permeate throughout the underlying tissue
- matrix, forming a depot reservoir of the carrier-agent
selectively at the site of disease (including tumors --
= even at sites up to several hundred micrometers distant
- from the typically irregularly spaced and perfused
microvessels within the tumor matrix), and thirdly, bind
to complementary receptors on the final target cells
(including tumor cells), leading to induced tumor-cell
internalization of GAG-actives (including DS-actives)
(see Examples below). The new special class of dermatan
sulfates (described just above and more extensively
- below) appears to perform this complementary binding
- 25 function via their selectively enriched oversulfated
saccharide sequences, which correlate with an enriched
heparin cofactor II activity of at least about 220 U/mg,
and which appear to bind the positively charged, cationic
' and/or structurally complementary receptors or lectins
that are selectively induced on disease-site endothelium,
~ tissue matrix and target cells (including in tumors).
For the new dermatan sulfates, these binding and
targeting functions and potencies occur without either
the overall high sulfation/polysulfation or the incumbent
35 toxicity and safety disadvantages thereof (as otherwise ~~
~ described herein).
.= ~
CA 02208~66 1997-06-23
W O96J19242 F' PCTnUS94/14776
-- 19 --
Th~ biological address of a disease site can be
viewed in a fashion similar to that of a postal address,
wherein effective carrier substances must (1) first
recognize the "state" address of the signal endothelium
induced by proximal tissue disease; (2) next extravasate
and loa~ the "city' address of the extracellular tissue
matrix ~ith locally effective doses of the diagnostic and
therapeutic actives; and (3) finally bind and load the
~street" address of the target cells and antigens.
Previous approaches to site delivery have attempted to
recogni~e the "street" address without first recognizing
the "st~te" and "city" addresses.
Th- reason that acidic saccharide and sulfated
glycosaninoglycan systems work substantially better than
previous antigen-reccgnition approaches, is that they
recogni~e the newly induced signals which the body uses
to attract and target white blood cells into sites of
tissue ,lisease. When disease strikes at a local SitQ, it
initiates a cascade of local mediators within the tissue
matrix and at the endothelial-blood interface which
signal L-he blood cells and central body systems that
inflammatory and immune cells are required within the
tissue site. These mediators include cytokines,
chemoat~ractants, cytotoxins, induced cell-surface
adhesions, selections and integrins, and various tissue-
derived and blood-borne, soluble and cell-surface
procoagulants. White cell accumulation begins within
minutes and continues over days to weeks, depending on
the natllre, severity and persistence of local disease and
the continued generation of tissue mediators and trans-
endothe:lial signals.
-
As has now been reported and reviewed in detail
[Ranney ~1990); Ranney (1992); Bevilaqua et al. (1993);
Bevilaqua et al. (1993); Travis (1993); Sharon et al.
(1993), all incorporated herein by reference], tumors,
= - CA 02208~66 1997-06-23
~-- W O 96tl9242 PCTrUS94/14776
;
.
infarcts, infections, inflammatory diseases, vascular
- disorders, and other focal diseases, characteristically
induce the release of such host mediators, or cytokines,
from resident macrophages and local tissue matrix. In
- 5 certain diseases, alien mediators such as bacterial
lipopolysaccharides (LPS), viral RNA, and tumor-derived
inducers, including EMAP II, and chemoattractants may
also be released. Although additional mediators remain
- to be elucidated, the principal ones have now been
= 10 defined and include: interleukin 1 (IL-1), tumor necrosis
: factor (TNF), vascular endothelial growth factor/vascular
permeability factor (VEGF/VPF), transforming growth
;-- factor beta (TGF-beta), Lipopolysaccharide (LPS), single
and double stranded nucleotides, various interferons,
: 15 monocyte chemoattractant protein (MCP), interleukin 8
-- (IL-8), interleukin 3 (IL-3), interleukin 6 (IL-6),
tumor-derived inducers and chemoattractant peptides (as
above), various prostaglandins and thromboxanes. Certain
ones of the preceding mediators induce the local
generation and release of metalloproteinases, and these
~~ - in turn, expose latent tissue binding sites, including
intact and partially cleaved integrins, RDGS peptides,
laminin, collagen, fibronectin, and cell-surface core-
protein components of glycosaminoglycans.
Cytokines, including VEGF/VPF and monocyte
- chemoattractant protein (MCP); and tissue
metalloproteinases and proteolytic tissue matrix
fragments, directly induce the local endothelium to
- 30 become adhesive for circulating white blood cells,
;; including neutrophils, monocytes and lymphocytes. The
. induced endothelial adhesive molecules (adhesins)
~ include: P-selectin (gmp-140), E-selectin (ELAM-1),
=~ intercellular cell adhesion molecule (ICAM-1), vascular
35 cell adhesion molecule (VCAM-1), inducible cell adhesion .~
~; molecule, (INCAM-110), von Willebrand's factor (vWF,
Factor VIII antigen) (see below for disease states which
CA 02208~66 l997-06-23
W O 96/19242 PCT~USg4/14776
activate these respective types of endothelial adhesins).
Additional cascades become activated which indirectly
amplify endothelial adhesiveness. These include (1)
coagulat.ion factors, especially fibronectin, tissue
factor, thrombin, fibrinogen, fibrin, and their split
product~" especially ~ibronectin split products and
fibrinopeptide A; (2) platelet-derived factors: platelet
activating factor (PAF), glycoprotein IIb/IIIa complex;
(3) whit-e-cell (a) L-selectin, and (b) integrins,
including VLA-4 (very late antigen 4); and (4) numerous
complemcnt factors.
The preceding pathologic processes and signals are
involvec, directly or indirectly as follows, in the
binding and site localization of acidic carriers,
including acidic saccharides (AC) and glycosaminoglycans
(GAGS) ~Note that in the following outline, potential
tissue binding sites are designated as "GAGs" and "ACs").
1. Local tissue diseases induce local cytokines
and mediators, as described above. In
particular, it is reported recently that the
cytokine, vascular endothelial growth
factor/vascular permeability factor (VEGF/VPF),
is selectively induced by many or most tumors
- of human and animal origin [Senger et al.
(1994), incorporated by reference herein] and
is a 34-42 kDa heparin-binding and GAG-binding
glycoprotein that acts directly on endothelial
cells by way of specific endothelial receptors
[Jakeman e t al . (1993), incorporated by
reference herein], to cause endothelial
- activation and induce additional new
endothelial receptors which can bind GAGS (see
below). VE~F/VPF is a chemically basic growth
factor which is quite highly selective for
endothelial cells versus fibroblasts and other
~' ; CA 02208~66 1997-06-23
- W O96/19242 PCTrUS94114776
-= - 22 -
cell types [Senger et al. (1994); Nicosia et
~ al. (1994), incorporated by reference herein].
- - It appears to be a key growth factor for f
stimulating the long-term endothelial
- 5 angiogenesis in many or most human and animal
--- tumors, and in AIDS-associated Kaposi's sarcoma
[Connolly et al. (1989); Weindel et al. (1992),
both incorporated by reference herein]. In
~ certain instances, VEGF/VPF may also be
important for the more transient and
anatomically restricted angiogenic processes of
- wound healing and vascular restenosis [Senger
- . ~ et al. (1994); Miller et al. (1994); Nicosia et
al. (1994); Berse et al. (1992), all
incorporated by reference herein]. VEGF/VPF
- and platelet-derived growth factor, PDGF-BB,
, are reported recently to be the only species of
: the group of basic, GAG-binding growth factors
- which have significant angiogenic potency in
- 20 vitro, i . e., ones which are directly active in
= the absence of in vivo cofactors [Nicosia et
al. (1994), incorporated by rere~e~.ce hereir.].
The effects of VEGF/VPF are i~h ~_~ei by
antibodies directed agains. certa_.. peptides c.
the external surface of the molecu'e [Sioussa~
et al. (1993), incorporated by reference
- herein], and importantly, such inhibition
- suppresses the growth of animal tumors in vivo[Kim et al. (1993), incorporated by reference
herein]. Hence, VEGF/VPF both provides and
induces receptor targets for binding of GAG
: carrier substances in tumors and potentially in
- other pathologic lesions.
,~
2. These cytokines and mediators induce tissue ~'
chemoattractants, including VEGF/VPF, MCP
(Yamashiro et al., 1994) and IL-8, which
...:
CA 02208~66 1997-06-23
WO 96/19242 PCT~US9~/14776
- 23 -
comprise a family of arginine-rich, 8Kd,
heparin-binding proteins reported to bind
GAGs/ACs t~uber et al. (1991), incorporated by
reference herein];
3. The cytokines and mediators of No. 1, above,
induce the local endothelium to express P-
selectin, the vascular cell adhesion molecular
(VCAM-1), inducible cell adhesion molecule
(INCAM-110), and von Willebrand's factor (vWF,
Factor VIII antigen), which are reported
binding determinants for GAGs/ACs [Bevilaqua et
al. (1993); Bevilacqua et al. (1993)]; P-
selectin is reported to bind GAGs [sevilacqua
et al. (1993)];
4. Integrins, including but not limited to VLA-4,
are induced on circulating white blood cells,
including lymphocytes, during various disease
processes (see below); VLA-4 has a distinct
binding site on the (induced) endothelial
selectin, VCAM-1 (see No. 3, above);
fibronectin, which is abundantly present in
~ plasma and also available from tissue sites,
has a distinct and separate binding site on
VLA-4 [Elices et al . (1990)]; since fibronectin
has specific binding sites for GAGs/ACs
[Bevilaqua et al . (1993)], these amplification
steps provide a strong additional mechanism for
site locali3ation of GAGs/ACs;
5. The chemoattractants, MCP and IL-8, lymphocyte
- integrin, VLA-4, platelet factor, PAF, and
coagulation factors, thrombin, fibronectin and
others, diffuse from local tissue and blood,
respectively, bind to the induced endothelial
selections, and amplify adhesiveness and
CA 02208~66 l997-06-23
W O96/19242 PCTrUS94/14776
- 24 -
activation at the initial endothelial P-
selectin sites for GAGs/ACs [Elices et al.
(1990); Lorant et al . (1993)];
6. Tissue metalloproteinases become activated and
expose new binding sites for GAGs/ACs in the
r tissues which underlie the activated
endothelia. These new tissue binding sites
include as follows [Ranney (1990); Ranney
(1992); Travis (1993); Bevilaqua et al.
(1993)]:
a. fibronectin fragments;
- 15 b. collagen fragments;
C . l~m; n; n fragments;
d. RGDS peptides;
e. Exposed core proteins of GAGs;
.
7. White blood cells are attracted to the site,
become activated and release additional
- 25 proteolytic enzymes, thereby amplifying No. 6
and increasing the exposure of binding sites
for GAGs/ACs in the tissue matrix.
8. GAG/AC carriers selectively bind the induced
and exposed determinants listed in Nos. 1-7,
- above, giving immune-type iocalization which is
specific for induced binding sites (lectins) at
the lectin-carbohydrate level characteristic of
white-cell adhesion;
9. In cases where the carrier substance has
multivalent binding to the target binding
.-' , .
CA 02208~66 1997-06-23
W O96119242 PCT~US94/14776
substance, including for example, cases in
which the carrier is an acidic oligosaccharide
or polysaccharide or an acidic
glycosaminoglycan, multivalent binding of the
endothelial surface induces rapid extravasation
of the carrier and bound active, and results in
substantially increased loading o~ the
underlying tissue matrix, relative to that
achieved by antibodies, liposomes, and
monovalent binding substances, such as hormones
and monovalent-binding sugars;
10. Adhesion of GAGs/ACs to induced and exposed
tissue binding sites, reduces plasma
backdiffusion of GAGs/ACs and their bound
actives, thereby giving sustained retention
within the tissue site;
11. Controlled release of the diagnostic or drug
activity from carriers comprising GAGs/ACs
occurs gradually within the dlseased site,
thereby resulting in targeted controlled
release;
12O Tumor cells, microbial targets and damaged
cellular targets within the tissue site, may
selectively take up the GAG/AC plus bound
diagnostic or drug active, based respectively,
on: induced tumor anion transport pathways,
microbial binding sites for GAGs/ACs, and
proteolytically exposed cell-surface core
. proteins [Ranney 07/880,660, 07/803,595 and
- - 07/642,033] -- Fe uptake by hepatomas, Cr4S
uptake by prostatic adenocarcinomas; [Kjellen
: 35 et al. (1977)]
- CA 02208~66 1997-06-23
W O 96/19242 PCTAUS94/14776
- 26 -
. .
_ 13. In cases where the carriers are hydrophilic or
- essentially completely hydrophilic, these
- carriers cause their bound actives to migrate
= (permeate) deeply into and throughout the tumor
mass even at microanatomic sites distant from
the tumor's typically irregularly spaced
-- ~ microvessels; and also to migrate out
- - (permeate) into a small rim of normal tissue
- - around each focus of disease, typically
.
comprising a rim about 30-75 um thick; however,
~ such carriers and/or their associated active
-~;= substances (diagnostics or therapeutics)
-~ undergo selective uptake (internalization) by
- - abnormal cells within tissue site and
- 15 preferentially avoid uptake by normal cells
within the site, thereby giving:
;~
:~ a. In cases of diagnostic imaging
~ applications: very sharp definition of
the boundary between tumors or infarcts
and the surrounding normal tissues;
.
b. In cases of therapeutic applications:
- - 25 (1) protection against spread of disease
at the rim;
.
(2) relative protection of normal cells
~ - within and adjacent to the site of
- 30 disease, from uptake of cytotoxic
drugs.
.
14. In the case of hydrophilic carriers, including
- but not limited to GAGs/ACs, the non-targeted
- 35 fraction of active is cleared rapidly and non- -
. toxically, thereby minimizing:
.
~. :
CA 02208~66 1997-06-23
W O 96/19242 PCT~US94/14776
a. in imaging uses, background signal
intensity;
b. in all uses:
(1) normal organ exposure; and
(2) systemic side effects.
Regarding the above outline, the tumor-selective GAG-
binding cytokines, VEGF/VPF and MCP, are now known to be
present in all three of the following microanatomic
locations: tumor-cell surface, tumor extracellular
matrix, and local tumor neovascular endothelium. Hence,
these cytokines provide receptor targets for GAG-agents
at all t:hree of the key tumor sites: tumor endothelium,
tumor e.{tracellular matrix, and tumor cells proper. The
presence of these cytokines selectively on tumor
endothe:lium, allows for site-selective binding of
intravascularly administered GAG-agents to tumor
microvesJsels and very rapid (ca. 3-minute) selective
extravas,ation of GAG-agents across the VEGF/VPF-
"permeabilized" endothelium. Note: such
"permeahilization" is recently shown to actually (a)
comprise rapid transport by vesicular endosomes which are
markedly enlarged (over the standard 120nm Palade
vesicles characterizing normal endothelium) and markedly
increased in number (over normal vascular endothelium)
[Senger et al. (1993), incorporated by reference herein];
and (b) comprise anatomically non-porous vascular
endothe:ium, as assessed by macromolecular and
particu:.ate markers of true microfiltration porosity.
The presence of VEGF/VPF and MCP cytokines on tumor cell
surfaces may account for selective tumor-cell
: 35 interna:ization of GAG-agents, as shown in certain of the
Examples below. Importantly, the presence of these
cytokines plus the GAG-binding peptides of No. 6 (above)
CA 02208~66 1997-06-23
W O96/19242 PCTrUS94/14776
- 28 -
in the large extracellular volumes of the tumor matrix,
accounts in part, for the large tumor-tissue reservoirs
of GAG-associated agents (including metal chelates) which
are observed by MRI contrast enhancement (see Examples
below). The relatively slow (ca. 7-hour) backdiffusion
- of such agents into the bloodstream, further corroborates
the presence of such extracellular tissue-matrix
- receptors. Importantly, the combination of: (1)
prolonged tumor retention of ~ag-agents as an
~ 10 extracellular reservoir (depot); (b) tumor-cell
internalization of a portion of this extracellular agent;
and (c) very rapid blood and body clearance of the non-
targeted portion, provides the following surprising and
- unexpected advantages for in vivo imaging (including MRI
contrast enhancement) and therapy: (a) enhanced tumor
selectivity; (b) prolonged, high "areas under the curve"
(AUC's) in tumor; (c) short, low AUC's in blood; (d)
~: minimization of local and systemic toxicities.
- Additionally, involving the above outline, the following
(A) cytokines and mediators; and (B) selections,
integrins and adhesins are reported to be induced by
various disease states in addition to that reported for
~ tumors, above [Bevilaqua et al. (1993)]. Representative
non-oncologic induction also occurs as follows.
A. Cytokines and mediators.
1. MCP: Experimental autoimmune encephalomyelitis
~~ in mice [Ransohoff et al . (1993)];
2. IL-8: Neovascularization: [Strieter et al.
~- (1992)];
. .
:. 3. PAF: Reperfused ischemic heart [Montrucchio et
al . (1993)].
.;
= B. Selections, Integrins and Adhesins.
CA 02208~66 l997-06-23
W O 9611~242 PCTrUS9~/14776
- 29 -
1. ELAM-1:
a. Liver portal tract endothelia in acute and
chronic inflammation and allograft
rejection [Steinho:Ef et al . (1993)];
b. Active inflammatory processes, including
acute appendicitis [Rice et al . (1992)].
2. VCAM-l:
a. Simian AIDS encephalitis [Sasseville et
al . ( 1 9 9 2)].
b. Liver and pancreas allograft rejection
[Bacchi et al . (1993)].
3. INCAM-110: Chronic inflammatory diseases,
including sarcoidosis [Rice et al . (1991)].
4. Integrin, beta 1 subunit cell adhesion
receptor: inflammatory joint synovium [Nikkari
et al. (1993)].
It is a~parent from the above, that broad categories and
many specific types of focal tissue disease may be
~ . addressed by the carriers and actives of the present
inventicn, both for diagnostic and therapeutic uses,
including tumors, cardiovascular disease, inflammatory
disease, bacterial ancl viral (AIDS) infections, central
nervous system degenerative disorders, and allograft
. rejection. It will also be obvious to those skilled in
- the art, that numerous additional disease states may be
selectively addressed by the carriers disclosed in this
' 35 invention.
= CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
- 30 -
The site selectivity of glycosaminoglycans (GAGs)
appears to mimic an immune mechanism at the level of
white-cell targeting rather than antibody targeting.
Because antibodies have extremely high specificities,
they characteristically miss major subregions of disease
foci (typically as great as 60~ of tumor cells are
nonbinding). Recently, one of the GAG-binding
determinants of endothelial P-selectin has been
identified as sialyl Lewis x. Others are in the process
~ 10 of identification. Notably, the available nonvalent
- oligosaccharides specific for sialyl Lewis x suffer from
- two critical problems:
1. They are exceedingly expensive materials,
available only by synthetic or semi-synthetic
means, and hence, are not cost effective;
2. They do not bind effectively at diseased sites
under in vivo conditions, apparently due to the
inability as monomeric binding substances to
displace endogenous interfering substances
which are pre-bound at these sites.
..
There are two apparent benefits of the relatively
broader range of GAG specificities and redundancy of GAG
binding sites present on diseased endothelium, tissue
matrix and cells:
1. GAGs allow a broader range of tumors and
diseases to be targeted than that possible with
antibodies (which typically target only a
subset of histologic types -- even within a
given class of tumor, and hence, are typically ~'
ineffective from both a medical and
cost/development standpoint);
CA 02208~66 1997-06-23
WO 96119242 PCI'~IIS94~I4776
2. GAGs are projected to be effective over a
greater time interval, from early onset of
disease to progression and regression.
Despite the broader targeting specificity of GAGs over
antibodies, their favorable clearance and avoidance of
uptake ]~y normal cells reduce systemic and local
toxicit:es, even though more than one type of disease
site mav undergo targeted accumulation of the
diagnosl;:ic/drug within its extracellular matrix.
The polymeric and multivalent binding properties of GAGs
both are very important for optimal site localization of
the attached diagnostic/drug. GAG molecular weights of
general].y ca. 8,000 to 45,000 MW, preferably 10,000 to
23,000 ~ and more preferably 13,000 to l9,0oO MW, are
important in order to:
1. Restrict initial biodistribution of the
diagnostic/drug to the plasma compartment and
thereby maximize the quantity of agent
available for site targeting;
2. Displace endogenous interfering substances
which are pre-bound to diseased endothelium;
3. Induce active endothelial translocation of the
GAG-diagnostic/drug into the underlying tissue
matrix;
4. Afford rapid clearance and markedly reduced
side effects of the attached actives.
.
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS9~/14776
' ~ '
- 32 -
SUMMARY OF THE lNv~NllON
In all of the following descriptions, the
paramagnetic metal-ion chelates and images obtained
therewith, are intended to be demonstrative of agent
localizations in sites of disease, including in tumor
~ ~ sites, and to be generally reflective of the disease-site
- levels, distributions and residence times, as well as of
--- the blood and organ clearance patterns and kinetics, all
of which may be useful in interpreting the targeting,
localization, accumulation, cellular internalization, and
.
blood and body clearance of therapeutic agents, including
-~ agents useful in treating tumors, infection,
-- cardiovascular diseases and other local sites of disease,
as described herein.
The present invention encompasses novel agents
; comprising cationic or chemically basic, amphoteric or
hydrophobic therapeutic agents, including peptides,
~- 20 polypeptides and proteins, and metal chelators and metal-
- ion chelates in association with hydrophilic carriers of
anionic or chemically acidic saccharides, sulfatoids and
glycosaminoglycans. In certain embodiments of the
invention, the agents also comprise chelated metals and
metal ions. The binding of the metal chelators to the
carriers is stabilized by covalent or non-covalent
chemical and physical means. In some embodiments, novel
non-covalently bound compositions give uniquely high
payloads and ratio of metal chelator to carrier, ranging
~ 30 from a low of about 15~ metal chelator by weight, to a
: characteristic range of 70~ to 90~ metal chelator by
weight. Specific embodiments comprise deferoxamine,
ferriox~m-ne, iron-basic porphine, iron-
triethylenetetramine, gadolinium DTPA-lysine, gadolinium
N-methyl-1,3-propanediamine (N-MPD)-DTPA, gadolinium
DOTA-lysine and gadolinium with basic derivatives of
. porphyrins, porphines, expanded porphyrins, Texaphyrins
CA 02208~66 1997-06-23
W O 96/19242 PCT~US9~1I4776
- 33 -
- and sapEhyrins as the basic or cationic metal chelators,
which are in turn, bound to acidic or anionic carriers,
including one or more of acidic or anionic saccharides,
and including sulfated sucrose, pentosan polysulfate,
dermatan sulfate, essentially purified dermatan sulfate
with a ulfur content of up to 9~ (w/w) and with
selective oligosaccharide oversulfation, heparan sulfate,
beef heparin, porcine heparin, non-anticoagulant
heparin~, and other native and modified acidic
saccharides and glycosaminoglycans.
Methods of magnetic resonance image (MRI) contrast
enhancement are a particular embodiment of the present
inventicn which confirm very rapid, carrier-mediated,
site-selective i~ vivo localization and sustained site
retention of metal-chelator compositions, based on stable
binding of the metal chelator and carrier during in vivo
plasma transit, allowi.ng site localization following
intrave~ous administration. Rapid and selective
endothelial-site binding, facilitated rapid extravasation
into uncerlying tissue sites, site accumulation,
sustained site retention, together with rapid clearance
of the ~on-site-localized fraction are also demonstrated
by the use of the compositions of the present invention
in the ~elective MRI contrast enhancement of tumors and
cardiovascular infarcts.
Surprising and unexpected improvements of
selectivity, mechanism of localization and cellular
uptake, and MRI contrast sensitivity are shown for metal
chelates having standard paramagnetic potencies. Further
advantages of the use of the compositions and methods of
- the present invention are delineated in the examples
(infra) including special histologic staining evidence
which ccnfirms the site-selective endothelial binding,
extrava~ation, tissue matrix accumulation and cellular
uptake mechanism. Selective localization and MRI imaging
CA 02208~66 l997-06-23
W O 96/19242 PCTrUS94/14776
- 34 -
- efficacy are also shown to occur when paramagnetic metal
chelator actives are administered in carrier-bound form
but not in free form.
In its most general embodiment, the present
invention is an agent comprising a chelator for metal
ions, said chelator having a cationic group and being
bound to an anionic, hydrophilic carrier. In alternate
: embodiments, the chelator for metal ions which has a
cationic group is bound to an anionic, hydrophilic
carrier by non-covalent electrostatic binding. And, in
certain alternate embodiments the invention comprises an
agent comprising a basic chelator for metal ions, said
chelator having a cationic group and being covalently
bound to an anionic, hydrophilic carrier. In the
; particular embodiments of the invention in which the
chelator is not covalently bound to the carrier, the said
chelator may be basic.
In certain embodiments of the present invention, the
agent which comprises a chelator for metal ions and
having a cationic group bound to an anionic hydrophilic
carrier may further comprise a chelated metal ion, and in
particular it may further comprise a paramagnetic metal
ion. The agents of the present invention, in particular
those which comprise the chelator for metal ions non-
covalently bound to the carrier may be further defined as
being at least about 15 weight percent chelator.
Preferably, the chelator has a formation constant for
- 30 paramagnetic metal ions of at least about 1014.
.
. Those agents of the present invention which comprise
a metal ion will preferably comprise a metal ion selected
from the group consisting of iron, manganese, chromium,
copper, nickel, gadolinium, erbium, europium, dysprosium
. and holmium. In certain embodiments, the agents of the
present invention may even comprise a metal ion selected
CA 02208~66 1997-06-23
W O96~19242 PCT~US94114776
_ from th~ group consisting of boron, magnesium, aluminum,
gallium, germanium, zinc, cobalt, calcium, rubidium,
yttrium, technetium, ruthenium, rhenium, indium, iridium,
platinun~, thallium, samarium, tin and strontium. In
certain embodiments, non-radioactive or radioactive
phosphorous, sulfur or iodine may be bound directly to
the car~ier (below). It is understood that other metal
ions which are functionally equivalent to the listed
metal ic;ns are also included and would ~all within the
scope a~d spirit of the presently claimed invention.
In certain preferred embodiments of the invention,
the agents comprise a carrier wherein said carrier is an
acidic saccharide, oligosaccharide, polysaccharide or
glycosa~inoglycan. The carrier may also be an acidic
glycosa~inoglycan or sulfatoid. In particular, the
carrier may be, but is not limited to heparin, desulfated
heparin, glycine-conjugated heparin, heparan sulfate,
dermatan sulfate, essentially purified dermatan sulfate
with a sulfur content of up to 9~ (w/w) and with
selective oligosaccharide oversulfation, hyaluronic acid,
pentosan polysulfate, dextran sulfate, sulfated
cyclodextrin or sulfated sucrose.
In certain embodiments of the invention, the
chelator is a chelator of iron ions. Preferably the
chelator is a hydroxamate, and more preferably it is
deferoxanine. In certain preferred embodiments the
chelator together with the metal ion is ferrichrome,
ferrioxaLnine, enterobactin, ferrimycobactin or
ferrichr~sin. In a particularly preferred embodiment,
the chel~tor is deferoxamine, the carrier is heparin, or
a heparin fragment and the agent further comprises
iron(III). In an alternate embodiment, the chelator is
'~ 35 deferoxanine and the carrier is dermatan sulfate or a
dermatan sulfate fragment and the agent may further
comprise chelated iron(III).
CA 02208~66 1997-06-23
W O96/19242 PCTAUS94114776
. .
- 36 -
In a certain embodiment, the invention may also
comprise deferoxamine bound to a carrier selected from
the group consisting of heparin, heparan sulfate,
dermatan sulfate, essentially purified dermatan sulfate
with a sulfur content of up to 9~ (w/w) and with
selective oligosaccharide oversulfation or chondroitin
sulfate, and may further comprise a metal ion. The
agents of the present invention may also comprise a
- chelator which is a porphine, porphyrin, sapphyrin or
texaphyrin and which may further comprise a metal ion,
and preferably an iron ion or a gadolinium ion.
In a particularly preferred embodiment the agent of
the present invention may comprise a chelator which is
5,10,15,20-Tetrakis(1-methyl-4-pyridyl)-21H,23-porphine,
a carrier which is heparin and a chelated iron ion. In
- certain embodiments, the chelator may also be a
polyaminocarboxylate or macrocyclic, and preferably a
basic or amine derivative of
diethylenetriaminetetraacetate, or more preferably a
basic or amine derivative of 1,4,7,10-
tetraazacyclododecane-N,N',N",N"'-tetraacetate (DOTA).
In the agents of the present invention, the carrier may
also be defined further as being complementary to
~ 25 endothelial determinants selectively induced at disease
sites.
In a certain embodiment, the present invention is an
image-enhancing agent or spectral-enhancing agent to
enhance images arising from induced magnetic resonance
signals, the agent comprising ferrioxamine covalently
conjugated to heparin by 1-ethyl-3-(3-
: dimethylaminopropyl) carbodiimide, N-ethoxycarbonyl-2- ~-
ethoxy-1,2-dihydroquinoline, or carbonyldiimidazole.
Alternatively, the invention is a spectral-enhancing
agent to enhance images arising from induced magnetic
resonance signals, the agent comprising
-
CA 02208~66 1997-06-23
WO 96119242 PCT/US94/14776
Gd(III)diethylenetriaminepentaacetate covalently
conjugated to one of heparin, dermatan sul~ate or
essentially purified dermatan sulfate with a sulfur
content of up to 9~ (w/w) and with selective
oligosaccharide oversulfation. In another alternative,
the inve~ntion is an agent for in vivo imaging, the agent
compris:ng a basic chelator for metal ions and chelated
metal ion, said chelator being bound by non-covalent
electrostatic binding to a hydrophilic carrier selected
from the~ group consisting of heparin, desulfated heparin,
glycine-conjugated heparin, heparan sul~ate, dermatan
sulfate, essentially purified dermatan sulfate with a
sulfur content of up to 9~ (w/w) and with selective
oligosaccharide oversulfation, hyaluronic acid, pentosan
polysul~ate, dextran sulfate, sulfated cyclodextrin or
sul~atecl sucrose. The agent for enhancing body imaging
preferably comprises deferoxamine, chelated Fe(III) and a
glycosaminoglycan carrier bound to said deferoxamine and
more preferably the glycosaminoglycan carrier is dermatan
sulfate, and/or the Fe(III) is a radiopharmaceutical
metal ion, and most preferably the radiopharmaceutical
metal ion is 59iron or 67gallium.
In an alternate preferred embodiment, the invention
is an acent for enhancing body imaging, the agent
comprising diethylenetriaminepentaacetate-lysine or
N-methyl.-1,3-propanediamine DTPA, chelated Gd(III) and a
glycosaminoglycan carrier bound to said
diethylenetriaminepentaacetate-lysine. Alternatively,
the invention is an agent for enhancing body imaging, the
agent ccmprising DOTA-lysine, chelated Gd(III) and a
~ glycosaminoglycan carrier bound to said 1,4,7,10-
. tetraazacyclododecane-N,N',N",N"'-tetraacetate-lysine
(DOTA-l~-sine). In a particular embodiment, the invention
is an asent comprising ferrioxamine bound by non-covalent
electro~tatic binding to dermatan sulfate or essentially
purifiec dermatan sulfate with a sulfur content of up to
CA 02208~66 1997-06-23
W096119242 PCT~S94/14776
- 38 -
9% (w/w) and with selective oligosaccharide
- oversulfation.
In an additional preferred embodiment, the invention
is an agent for enhancing body imaging, including MRI
imaging and spectral shift, the agent comprising
gadolinium (III) chelated to N-methyl-l,3-propanediamine-
diethylenetriaminepentaacetate (N-MPD-DTPA), the N-MPD-
DtPA being bound or in association most preferably by
paired-ion or other non-covalent means or alternatively
preferably bound by covalent means to a
glycosaminoglycan, preferably dermatan sulfate, and most
preferably the new special class of dermatan sulfate, and
most preferably the new special class of dermatan
sulfates containing selectively oversulfated
oligosaccharide sequences.
It is understood that any of the agents of the
present invention as described in the above paragraphs or
in the appended claims may be defined further as being in
a combination with at least one of a buffer, saccharide,
sulfated saccharide, or salt, to produce an osmotic
strength suitable for parenteral administration, and as
being an aqueous solution or a lyophilized or dry
preparation suitable for aqueous reconstitution having
the desired osmotic strength, and wherein said agent is
aseptic or sterile.
Another embodiment of the invention is a method of
enhancing magnetic resonance images or spectra in
vertebrate animals comprising administering to said
animal an effective amount of an agent of the invention
which comprises the metal ion chelator, the ca~rier as
described and a paramagnetic ion. In particular, the
invention is a method of enhancing in vivo images arising
from induced magnetic resonance signals, comprising the
- steps of administering to a subject an effective amount
;
-
CA 02208~66 1997-06-23
W O 96119242 PCTIUS94/14776
- 39 -
_ of an aqent of the present invention which comprises a
paramagnetic ion, exposing the subject to a magnetic
field a~ld radiofrequency pulse and acquiring an induced
magnetic resonance signal to obtain a contrast effect.
In an alternative embodiment, the invention is a
method of enhancing i.n vivo images, comprising the steps
of administering to a sub~ect an effective amount of an
agent oi~ the present invention which comprises a chelated
metal ic~n, exposing the body to ultrasound or X-rays and
measuring signal modulation to obtain a contrast effect.
In another embodiment, the invention is a method of
obtaining in vivo body images comprising administering to
a subjec~t an effective amount of an agent of the
inventic~n which comprises a metal ion wherein the metal
ion is a radioisotope and measuring scintigraphic signals
to obta:n an image.
In another embodiment, the invention includes
composit:ions and methods for delivering, localizing and
retaining therapeutic actives selectively to sites of
local d:sease, while clearing the non-targeted dose
effectively, rapidly and/or non-toxically from the body,
so as to minimize local and systemic toxicities and side
effects These therapeutic actives and methods of
treatments may be for any type and location of local
disease site, provided it has a vascular or other access
route, and that it has any form of induced vascular
receptors, adhesins, sr other signals capable of
recognit-.ion by the carrier substances described herein.
In part:.cular, such actives for tumor treatment may
include but are not limited to: doxorubicin, adriamycin,
taxol, vincristine, vinblastine, bleomycin, idarubicin,
~- 35 epirubicin, and also to amsacrine, azacitidine,
dideoxy-nosine, dihydro-5-azacytidine, ethanidazole,
ethiofos, methotrexate, misonidazole, porfiromycin,
CA 02208~66 1997-06-23
- W O 96/19242 PCTrUS94/14776
- 40 -
pyrazoloacridinek, terephthalamidine, taxotere and other
taxane derivatives, topotecan, trimetrexate, N-formyl-
met-leu-phe-lys, arginine bradykinin, poly-L-lysine,
other chemoattractants, biological response modifiers,
cytokines, interferons, lymphokines and other agents
useful in treating tumors or neoplastic disease, with any
of the above used singly or in combination. Further, in
- particular, such actives and methods for treating
infection may include but are not limited to: gentamicin,
~ 10 amikacin, tobramycin, and other amine, basic, basic
peptide, basic polypeptidic, hydrophobic or amphoteric
antibiotics or agents for treating bacterial, fungal,
- mycobacterial, viral or other microbial or
microbiological diseases.
The present invention may be described in certain
: embodiments as a drug carrier composition comprising a
drug in combination with essentially purified dermatan
~ sulfate with a sulfur content of up to 9~ (w/w) and with
- 20 selective oligosaccharide oversulfation, wherein said
composition has a non-embolizing size of less than about
500 nm. In certain embodiments the dru~ m~ be a
chelator. In certain embodiments th~ com~ en ma~
have a size of less than about 250 r.m. ~r.e ~~'~_, carr e
- 25 composition may also be defined further where n bindin-
= to disease induced endothelia causes the endothelia to
totally or partially envelop bound drug carrier
- composition in less than 10 to 15 minutes, and wherein
said essentially purified dermatan sulfate with sulfur
~ 30 content of up to 9~ (w/w) and with selective
- oligosaccharide oversulfation, contains Ido-GalNAc4SO3,
: and further comprises IdoA2SO3-GalNAc4SO3 and
IdoAGalNAc4,6SO3.
-
The drug carrier composition of the present ~-
~ invention may also be defined in certain embodiments as
being a nanoparticle, and the drug may preferably by an
CA 02208~66 1997-06-23
W O 96/19242 PCT~US94/I4776
- 41 -
- oncotherapeutic drug. The oncotherapeutic drug is
preferahly selected from the group consisting of
adriamycin, doxorubicln, epirubicin, daunorubicin,
idarubicin or salts thereof, with doxorubicin being the
most pr~ferred. The oncotherapeutic drug may
alternat.ively be selected from the group consisting of
bleomycin, taxol, taxotere, vinblastine and vincristine,
amsacrir.e, azacytidine, dideoxyinosine, dihydro-5-
azacytidine, ethanidazole, ethiofos, methotrexate,
misonizadole, porfiromycin, pyrazoloacridinek,
terephtk.alamidine, taY.otere, topotecan, trimetrexate and
carbopla.tin or salts thereof.
A particular embodiment of the present invention is
a drug carrier composition comprising a drug selected
from the group consisting of doxorubicin, epirubicin,
daunoru~icin and idarubicin in combination with
essentially purified dermatan sulfate with a sulfur
content of up to 9~ (w/w) and with selective
oligosaccharide oversulfation, wherein said composition
has a non-embolizing size of less than about 500 nm,
preferably less than 250 nm, most preferably less than 25
nm.
Embodiments of the present invention also include
drug carrier compositions wherein the drug is an
antiinfective (antiviral, antimicrobial, antifungal or
antitubercular) drug, with gentamycin, tobramycin or
amikacin being preferred, or the drug is a biological
response modifier (modifying an endogenous biological
response), preferably a biologically active peptide or
polypept.ide, and more preferably a white cell
chemoattractant, bradykinin or poly-L-lysine. The white
cell chel-noattractant is preferably N-formyl-met-leu-phe-
: 35 lys (SEQ ID NO:1).
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
..
; - 42 -
The drug carrier compositions of the present
invention may be further defined as being in a
pharmaceutically acceptable solution suitable for
intravascular or other parenteral injection, and may be
-- 5 formed by paired-ion charge interactions, amphoteric or
hydrophobic interactions between the carrier and drug.
In certain embodiments, the present invention is a
method of treating an animal for tumors responsive to an
oncotherapeutic agent, the method comprising the steps of
preparing a drug carrier composition comprising an
oncotherapeutic drug in combination with essentially
purified dermatan sulfate with a sulfur content of up to
- 9~ (w/w) and with selective oligosaccharide
oversulfation, wherein said carrier has a non-embolizing
size of 500 nm or less; containing said drug carrier
composition in a pharmaceutically acceptable carrier; and
administering the drug carrier composition in the
pharmaceutically acceptable carrier to an animal. In the
preferred drug carrier compostions and methods, it is
- understood that the drug is in a controlled release form
and preferably wherein binding of a sample of said drug
carrier composition to disease induced endothelia
produces an induction of the endothelia to totally or
partially envelop the bound sample in less than 10 to 15
minutes.
In embodiments involving treatment of various
diseases, the drug carrier composition may be
administered by selected arterial perfusion, to obtain
high-efficiency uptake in proximal target organs, tissues
or tissue lesions, or it may be administered
intravenously to obtain semiselective, medium-efficiency
uptake in tissue lesions located at widely distributed
systemic sites.
CA 02208~66 l997-06-23
W O 96/19242 PCT/US9~I4776
- 43 -
_ The present invention is also a method of treating
an animal for tumors responsive to an oncotherapeutic
agent, the method comprising the steps of preparing a
drug carrier composition comprising an oncotherapeutic
drug in combination with essentially purified dermatan
sulfate with a sulfur content of up to 9~ (w/w) and with
selective oligosaccharide oversulfation, wherein said
carrier has a non-embolizing size of 500 nm or less;
containing said drug carrier composition in a
pharmac~utically acceptable carrier; and administering
the dru!~ carrier composition in the pharmaceutically
acceptable carrier to an animal; wherein said
oncothe:rapeutic drug is selected from the group
consist.ing of adriamycin, doxorubicin, epirubicin,
daunorubicin, idarubicin, bleomycin, taxol, taxotere,
vinblasl~ine and vincristine or salts thereof.
Alt:;ernatively, the invention may be described as a
method of treating an animal for tumors responsive to an
oncotherapeutic agent, the method comprising the steps of
preparing a drug carr.ier composition comprising an
oncotherapeutic drug in combination with essentially
purified dermatan sulfate with a sulfur content of up to
9~ (w/w) and with selective oligosaccharide
oversulfation, wherein said carrier has a non-embolizing
size of 500 nm or less; containing said drug carrier
composition in a pharmaceutically acceptable carrier; and
administering the drug carrier composition in the
pharmaceutically acceptable carrier to an animal; wherein
said oncotherapeutic drug is selected from the group
consisting of amsacrine, azacytidine, dideoxyinosine,
. dihydro-5-azacytidine, ethanidazole, ethiofos,
~ r ~ ' methotrexate, misonizadole, porfiromycin,
~ pyrazolo~cridinek, terephthalamidine, taxotere,
- 35 topotecan, trimetrexate and carboplatin or salts thereof.
CA 02208~66 1997-06-23
W O96/19242 PCTrUS94/14776
=
- 44 -
In certain of the methods of treatment of the
present invention, the binding of a sample of said drug
carrier composition to disease induced endothelia may
produce an induction of the endothelia to totally or
partially envelop the bound sample in less than 10 to 15
minutes, or the drug carrier composition is administered
by selected arterial perfusion, to obtain high-efficiency
uptake in proximal target organs, tissues or tissue
lesions, or the drug carrier composition is intravenously
administered to obtain semiselective, medium-efficiency
uptake in tissue lesions located at widely distributed
systemic sites.
In another embodiment, the invention is a method of
treating vascular disease, comprising administering to a
subject a therapeutically effective amount of an agent of
the present invention, and preferably an agent which
comprises a metal ion.
~ m; nl stration of the composition of the present
invention may involve any mode of administration
resulting in contact of the therapeutic agent with the
target tumor, disease site or site of infection. This
may include intravenous, intraarterial, intracisternal,
intraperitoneal, oral or other administration modes.
The compositions or formulations of the present
invention may be prepared dissolved or dispersed in a
pharmaceutically acceptable carrier or diluent or in any
other pharmaceutically acceptable form. Such
pharmaceutically acceptable formulations wil~ generally
comprise an effective amount of the compositions, such as
- doxorubicin:essentially purified dermatan sulfate in a
pharmacological preparation.
o
The phrases "pharmaceutically or pharmacologically
acceptable" refers to molecular entities and compositions
that do not produce an adverse, allergic or other
untoward reaction when administered to a human. As used
CA 02208~66 1997-06-23
W O96119242 PCT~US94~14776
- 45 -
_ herein, "pharmaceutically acceptable carrier" includes
any and all solvents, dispersion media, coatings,
antibac_erial and antifungal agents, isotonic and
absorpt;~on delaying agents and the like. The use of such
media and agents ~or pharmaceutical active substances is
well kn~wrL in the art. Except insofar as any
convent:ional media or agent is incompatible with the
active :Lngredient, its use in the therapeutic
composilions is contemplated. Supplementary active
ingredients can also be incorporated into the
composilions.
The compositions of the present invention may thus
be formulated for parenteral administration, such as for
intravenous, subcutaneous or intramuscular injection; for
oral administration, where the compositions may be
formulat:ed into tablets, caplets, or other solids; and
the compositions may also be formulated into time release
capsules and any other form of pharmaceutical currently
used, including cremes, lotions, mouthwashes, inhalents
and the like, depending upon location of targeted sites.
BRIEF DI-'SCRIPTION OF THE DRAWINGS
The following drawings and figures are presented t-
illustrcLte preferred embodiments of the present inventio-.
and thei.r uses in MRI contrast enhancement. These
examples are purely illustrative, and do not in any way
delimit the full scope of the present invention.
FIC~. lA is a control infrared spectrum of
diethylenetriaminetetraacetate (DTPA) substrate (see
Example 3).
FIG. lB is a conkrol infrared spectrum of L-
lysine.FrCl substrate (see Example 3).
CA 02208~66 l997-06-23
- W O 96/19242 PCT~US94/14776
46 -
FIG. lC is a control infrared spectrum of a physical
mixture of these DTPA and L-lysine.HCl substrates without
any chemical covalent linkage of the two substrates (see
- Example 3).
FIG. lD is the experimental infrared spectrum of L-
lysine covalently conjugated to DTPA by l-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC) linkage (see
- Example 3). Note the changes (height, width and loss of
splitting) in signature peaks in the range of 1250-1700
wavenumbers, which indicate covalent conjugate formation.
For the following Figures (2-13), the dermatan
sulfate carrier is of the new special class of dermatan
sulfates with selectively oversulfated oligosaccharide
sequences but without overall oversulfation (S03-/COO-
ratio = 1:1 and sulfur content = 6.3 wt ~; supplied by
Opocrin S.P.A., Corlo Di Formigine, Italy, as "435
type").
FIG. 2A, FIG. 2B, FIG. 3A, FIG. 3B, FIG. 4A, FIG.
4B, FIG. 4C, FIG. 4D and FIG. 8A, FIG. 8B and FIG. 8C
show T1-weighted MRI images (TR/TE = 800/45, 550/23 and
600t45) performed at 1.0 and 1.5 Tesla, before (Pre) and
after (Post) intravenous (i.v.) injection of
~ Ferrioxamine:Dermatan Sulfate Selective Paramagnetic
Contrast Agent, prepared as in Examples 2 and 5, and
injected i.v. at a Ferrioxamine dose of 0.155 mmol/Kg
into Fisher 344 female rats, with syngeneic breast
- 30 adenocarcinoma inoculated previously into the liver, such
that tumor diameters at the time of imaging are between
1.0 cm and 2.5 cm.
FIG. 2A. Precontrast image of liver (tumor not
conspicuous).
=
CA 02208~66 1997-06-23
W 0961~9242 PCT~US94~14776
- 47 -
_ FI(J. 2B. Liver image at 7 min postinjection (MPI)
of the ~elective Paramagnetic Contrast Agent,
Ferrioxamine:Dermatan Sulfate (0.155 mmol/Kg) i.v.,
showing marked contrast enhancement of tumor in right
lobe of liver, very sharp tumor boundaries against
surroun~ling liver, and discretely demarcated darker
central region of tumor necrosis -- allowing tumor
perfusion and function to be spatially resolved and
assesse~l within different, very small anatomical
subregi~)ns.
FIG. 3A. Precontrast image of liver (tumor is
present but not conspicuous).
FIG. 3B. Liver image at 7 MPI of Ferrioxamine
Active ~lone (without any Dermatan Sulfate Carrier).
Note that acute contrast enhancement is only very slight
or nonexistent. This differs markedly from the
pronounced tumor enhancement seen in Fig. 2B; and it
indicates that binding of the Ferrioxamine active by the
Dermatan Sulfate carrier is a requirement for tumor-site
localizction and tumor uptake of Ferrioxamine active.
FI~. 4A. Precontrast T1 image (TR/TE = 800/45) of
liver (breast tumor is present but not conspicuous).
FI~. 4B. Liver image at 21 MPI of
Ferrioxamine:Dermatan Sulfate Selective MRI Contrast
Agent. Note the marked enhancement of main tumor mass
and distinct tumor borders. Also note the small, 2-mm,
bright enhancement of tumor metastasis in left lobe of
liver. This metastasis is completely non-visualized in
the Precontrast Tl images.
i~ 35 FIG. 4C. Liver image at 30 MPI of
Ferrioxanine:Dermatan Sulfate Selective MRI Contrast
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
- 48 -
Agent. Note the sustained enhancement of main tumor and
- metastasis.
FIG. 4D. Liver image at 42 MPI of
Ferrio~m;ne:Dermatan Sulfate Selective MRI Contrast
- Agent. Note: continued strong enhancement of main tumor
and metastasis at prolonged post-contrast interval, at
high, sustained sensitivity, and with continued
delineation of tumor boundaries in both nodules
(selectivity), plus delineation of the very small non-
perfused region centrally within the 2-mm liver
metastasis.
FIG. 5. Region-of-interest (ROI) analyses of MRI
image intensities from a tumor animal analogous to that
shown in FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D. Upper
line = tumor ROI's; Lower line = liver ROI's; time points
= Precontrast; and 12, 27, 44 and 64 MPI of
Ferrioxamine:Dermatan Sulfate Selective MRI Contrast
Agent. Note the Intensity Ratios of Tumor to Liver are:
(a) at the Peak time of 12 MPI = 11:1; (b) as an average
over the 27-64 MPI = 3.2:1 -- both (a) and (b)
additionally indicating very good selectivity for tumor
versus liver. Intensity fades only very gradually with
time, unlike the kinetics reported for Gd:DTPA, which are
very rapid and have a tl/2 at the site of ca. 12-20 min
(images not shown).
FIG. 6. Special histologic stain (heated
ferroferricyanide reaction) of formalin-fixed section of
syngeneic breast adenocarcinoma excised from liver
-. inoculation site of Fisher 344 female rats: Outer Tumor
Rim 7-10 MPI of Ferrioxamine:Dermatan Sulfate Selective
MRI Contrast Agent. Note selective staining for
35 ferrioxamine iron (a) strongly positive on and within ~t
tumor endothelium, (b) strongly positive in the
subendothelia, (c) moderately positive in the
CA 02208~66 1997-06-23
W O96/19242 PCT~US9~14776
- 49 -
_ extraceLlular matrix of tumor, and (d) lightly to
moderately positive within tumor intracellular sites.
FII~. 7A. Same tumor, stain, conditions, and post-
contras- time as FIG. 6, except tissue section is taken
from Central Tumor, 7-10 MPI of Ferrioxamine:Dermatan
Sulfate Selective MRI Contrast Agent. Significant
staininq positivity is present at all sites as in FIG. 6.
FIC~. 7B. Identical to FIG. 7A, except a different
animal with identical type and site of breast tumor, 7-
10 MPI clfter i.v. Ferrioxamine Active Alone at a
Ferrioxamine dose identical to FIG. 6 and FIG. 7A. Note
the complete absence of staining positivity. This
correlat:es directly with the results of MRI imaging with
the ful: Agent (Active bound to Carrier) versus that with
Active Z~lone (Active in free form) -- (refer to FIG. 2A
and FIG 2B versus 3).
FIG. 8A. Tl-weighted (TR/TE = 600/45) image of Lung
Field iIL rat with primary liver breast tumor. Note that
the lung metastases (2-mm to 3-mm nodules) are only
faintly conspicuous Precontrast.
FIG. 8B. Lung Field of same rat at 12 MPI. Note the
marked improvement in sensitivity of tumor detection
(conspicuity) due to selective, bright enhancement of the
lung metastases. Also note the sharpness of tumor
boundaries.
FIG. 8C. Same Lung Field at 17 MPI -- showing
sustained enhancement and sustained sharpness of tumor
~ boundaries. By comparison, the rapid diffusion rates of
Gd:DTPA lead to rapidly fuzzy boundaries at early times;
and thereby also decrease the sensitivity of detecting
pulmonary metastases.
CA 02208~66 1997-06-23
W O96/19242 PCTrUS94/14776
.
- 50 -
FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG.
10A, FIG. 10B, FIG. 10C, FIG. 10D and FIG. 10E show T1-
weighted MRI images (TR/TE = 250/8) performed at 4.7
Tesla, before (Pre) and after (Post) intravenous (i.v.)
injection of Ferrioxamine:Dermatan Sulfate Selective
Paramagnetic Contrast Agent (FIG. 9A, FIG. 9B, FIG. 9C,
FIG. 9D, FIG. 9E) prepared as in Examples 2 and 5, and
injected i.v. at an Iron(III) dose of 0.155 mmol/Kg;
compared to Gadolinium DTPA dimeglumine (FIG. 10A, FIG.
- 10 10B, FIG. 10C, FIG. 10D, FIG. 10E), injected i.v. at a
Gd(III) dose of 0.100 mmol/Kg; each of these agents being
administered to Copenhagen rats with syngeneic AT-1
prostate adenocarcinoma inoculated into previously
prepared skin pouches [Hahn et al . (1993)], such that
tumor diameters at the time of imaging are between 1.0 cm
and 2.5 cm.
FIG. 9A. Precontrast image for
Ferrioxamine:Dermatan Sulfate Selective Contrast Agent.
FIG. 9B. 7 MPI of Ferrioxamine:Dermatan Sulfate,
liquid form at a ferrioxamine concentra~ c-. c~ ~.166
mmol/mL. Note the strong enhanceme~ r e- ~_m and
-Vascular array which fans out from t.~ m~~ Fe~cle.
FIG. 9C. Same as FIG. 9B, except 20 MPI. Note the
sustained, discrete enhancement of elements in FIG. 9B.
FIG. 9D. Same as FIG. 9C, except 40 MPI. Note the
sustained contrast and delineation of Outer Rim.
FIG. 9E. Same as FIG. 9D, except 60 MPI. Note the
onset of contrast fading.
35 FIG. 10A. Precontrast image for Gd:DTPA dimeglumine ~~
Nonselective Contrast Agent.
CA 02208~66 1997-06-23
WO 96/19242 PCTWS9~t4776
_ FI~J. lOB. 7 MPI of Gd:DTPA dimeglumine. Note that
the Outer Rim is not well delineated, even at this very
early post-contrast interval.
FI~ lOC. Same as FIG. lOB, except 20 MPI. Note
the mar]~ed early contrast fading overall, with some agent
sequest:~ation seen at the central, poorly perfused
(cystic regions of tumor (as is typically reported for
Gd:DTPA when used for imaging at body sites).
FIG. lOD. Same as FIG. lOC, except 40 MPI. Note
that enhancement is nearly reverted to background levels.
FI(,. lOE. Same as FIG. lOD, except 60 MPI. No
residua:L contrast, except for central cystic regions.
FIG. llA, FIG. llB, FIG. llC and FIG. llD show T1-
weightecl MRI ECG-gated cardiovascular images performed at
0.5 Tes..a, before (Pre) and after (Post) rapid
intravenous (i.v.) in:Eusion of Ferrioxamine:Dermatan
Sulfate Selective Paramagnetic Contrast Agent prepared as
in Examples 2 and 5, and injected i.v. at an Iron(III)
dose of 0.155 mmol/Kg into German Shepherd dogs with
acute, '1O-min myocardial infarcts (ligature of proximal
left anterior descending coronary artery) followed by
reperfusion for ca. 90 minutes prior to contrast agent
infusion.
FIC,. llA. Precontrast image.
FIC,. llB. 7 MPI, showing strong enhancement of
infarct by Ferrioxamine:Dermatan Sulfate Agent, and in
particu]=ar delineating the boundary of the infarct --
putatively the boundary of the marginal zone. Note the
~- 35 central darker region -- putatively the irreversible
central infarct zone.
CA 02208~66 l997-06-23
W O 96/19242 PCTrUS94/14776
- 52 -
- FIG. llC. 20 MPI, showing sustained strong
- enhancement and zones as above.
- FIG. llD. 40 MPI, same as FIG. llC, except filling
in of central zone; absence of significant overall
-~ contrast fading. NOTES: (1) injection of Ferrioxamine
Agent Alone at 0.155 mmol/Kg, gives no detectable
enhancement (images not shown); (2) infarct sizes and
: positions are documented by double dye infusion methods
- 10 immediately after imaging.
: FIG. 12A, FIG. 12B, FIG. 12C and FIG. 12D show MRI
4.7 Tesla, T1-weighted images of Copenhagen rats with the
AT-1 prostate tumor model (as in FIG. 9A, FIG. 9B, FIG.
-~ 15 9C, FIG. 9D, FIG. 9E, FIG. 10A, FIG. 10B, FIG. 10C, FIG.
~~ 10D and FIG. 10E), but rats are injected i.v. with
Ferriox~m;ne:Dermatan Sulfate Selective Contrast Agent in
the lyophilized (versus liquid) form, and the Agent is
~-~ reconstituted with water just prior to administration at
a higher concentration of 0.415 mmol/mL Fe(III) and
administered at the usual dose of 0.155 mmol of Fe(III)
~-- per Kg.
.
FIG. 12A. Precontrast image for
Ferrioxamine:Dermatan Sulfate Selective Contrast Agent.
FIG. 12B. 7 MPI of Ferrioxamine:Dermatan Sulfate,
lyophilized reconstituted to a Fe(III) concentration of
0.415 mmol/mL. Note the very strong enhancement of the
entire Outer Rim of tumor.
.
-- FIG. 12C. Same as FIG. 12B, except 20 MPI. Note
the sustained, very strong enhancement and delineation of
Outer Rim.
FIG. 12D. Same as FIG. 12C, except 40 MPI. Note
the sustained very strong enhancement of Outer Rim with
CA 02208~66 1997-06-23
WO 961'1~242 PCT/17S9~14776
- 53 -
_ the Central Tumor now also starting to enhance brightly.
Also no e there is virtually no contrast fading at 40
minutes.
s FI~. 13A, FIG. 13B, FIG. 13C, FIG. 13D show MRI 4.7
Tesla, '~1-weighted images of Copenhagen rats with the AT-
1 prostate tumor model (as in FIG. 12A, FIG. 12B, FIG.
12C and FIG. 12D), but rats are injected i.v. with
Gd(III);DTPA-Lys:Dermatan Sulfate Selective Contrast
Agent in liquid form pre-concentrated to 0.415 mmol/mL
Gd(III) and administexed at the usual dose of 0.155 mmol
of Gd(III) per Kg.
FIC.. 13A. Precontrast image for Gd(III):DTPA-
Lys:Dermatan Sulfate Selective Contrast Agent.
FIG. 13B. 7 MPI of Gd(III):DTPA-Lys:Dermatan
Sulfate, at 0.415 mmol/mL. Note the exceedingly strong
enhancement of the entire Outer Rim as well as Central
Tumor. This is consistent with the higher paramagnetic
potency of Gd:DTPA chelate, R1 = 4.3 [mmol.sec]-1,
relative to ferrioxamine chelate, R1 = 1.5-1.8
[mmol.sec]-1.
FIG. 13C. Same as FIG. 13B, except 20 MPI. Note
the sust~ined, very strong absolute enhancement Outer
Rim. Also note additionally strong enhancement of the
central vascular array (as differentiated from cystic
sequestr~tion).
FIG. 13D. Same as FIG. 13C, except 40 MPI. Note
sustained enhancement of Outer Rim, with overall
enhancement just beginning to fade at 40 minutes, but
absolute enhancement rem~;n;ng as bright or brighter in
all regi~ns relative to Ferrioxamine:Dermatan Sulfate.
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
: '
- 54 -
FIG. 14A, FIG. 14B, FIG. 14C and FIG. 14D show MRI
4.7 Tesla, Tl-weighted images of Copenhagen rats with the
AT-1 prostate tumor model (as in FIG. 13A, FIG. 13B, FIG.
13C and FIG. 13D), but rats are injected i.v. with
Ferrioxamine Selective Contrast Agent, wherein the Active
is non-covalently bound to Oversulfated Dermatan Sulfate,
the Agent lyophilized and reconstituted with water just
prior to administration at a concentration of 0.332
mmol/mL Fe(III) and administered at the usual dose of
0.155 mmol of Fe(III) per Kg.
FIG. 14A. Precontrast.
FIG. 14B. 7 MPI.
FIG. 14C. 20 MPI.
FIG. 14D. 40 MPI. Note the equivalent to slightly
greater enhancement of Tumor Rim and greater definition
of the vascular array at all times, in relation to
Ferrioxamine bound to Native Dermatan Sulfate (above).
- FIG. 15A, FIG. 15B, FIG. 15C and FIG. 15D show MRI
4.7 Tesla, T1-weighted images of Copenhagen rats with the
AT-1 prostate tumor model (as in FIG. 13A, FIG. 13B, FIG.
13C and FIG. 13D), but rats are injected i.v. with
Ferrioxamine Selective Contrast Agent, wherein the Active
is non-covalently bound to Oversulfated Chondroitin
Sulfate, the Agent lyophilized and reconstituted with
water just prior to administration at a concentration of
0.332 mmol/mL Fe(III) and administered at the usual dose
o~ 0.155 mmol of Fe(III) per Kg.
FIG. 15A. Precontrast.
FIG. 15B. 7 MPI.
CA 02208~66 l997-06-23
W O96/19242 PCT~US94/14776
- 55 -
FIG. lSC. 20 MPI.
..
FIG. 15D. 40 MPI. Note the moderately greater
enhancement of Tumor Rim and greater definition of the
vascula~ array at 7 MPI, and the only slightly greater
enhance~ent at the two later times, in relation
Ferrioxamine bound to Native Dermatan Sulfate (above).
FIG. 16A, FIG. 16B, FIG. 16C and FIG. 16D show MRI
4.7 Tesla, T1-weighted images of Copenhagen rats with the
AT-1 prostate tumor model (as in FIG. 13A, FIG. 13B, FIG.
13C and FIG. 13D), bu~ rats are injected i.v. with
Ferrioxamine Selective Contrast Agent, wherein the Active
is non-covalently bound to a non-anticoagulant GAG,
Heparan Sulfate, the Agent lyophilized and reconstituted
with water just prior to administration at a
concent~ation of 0.332 mmol/mL Fe(III) and administered
at the usual dose of 0.155 mmol of Fe(III) per Kg.
FIG. 16A. Precontrast.
FIG. 16B. 7 MPI.
FIG. 16C. 20 MPI.
FIG. 16D. 40 MPI. Note the very homogeneous
enhancement of Outer Rim and Central Tumor at virtually
all post-contrast times, in relation to the differential
Rim enhancement achieved by essentially all of the other
GAG carriers. This property may be useful in certain
diagnostic and/or therapeutic applications.
FIG. 17A is a control infrared (IR) spectrum of
gadolini~m diethylenetriaminepenaacetate (Gd:DTPA) (see
Example 21).
CA 02208~66 1997-06-23
W O96/19242 PCTAUS94/14776
- 56 -
FIG. 17B is a control IR spectrum of N-methyl-1,3-
propanediamine (MPD) (see Example 21).
t
FIG. 17C is a control IR spectrum of a mixed (and
dried) solution of the individual chemical components,
Gd:DTPA and MPD (1:1 molar ratio).
FIG. 17D is the experimental IR spectrum of MPD
covalently conjugated at a 1:1 molar ratio to DTPA (as
described in Example 21). Note the change in the height
and splitting of the signature peak at 1400 wavenumber,
and the change in the height and configuration of the
broader stretching bands at 3300-3600 wavenumbers, which
are indicative of covalent conjugate formation.
.- FIG. 18A shows a T2-weighted MRI scout image (TR/RE
= 2100/85) of the liver regions of Fisher 344 female rats
- with syngeneic breast adenocarcinomas inoculated
previously into the liver, such that tumor diameters at
~ 20 the time of imaging are between 1.0 and 2.5 cm, with the
image acquired at 1.0 Tesla, just before performing the
T-l weighted series of images (shown below). This T2
image is performed in order to identify the approximate
locations of 2 tumor nodules tright posterior liver) and
1 tumor infiltrate (central liver region), all tumor
growths being confirmed at necropsy by gross visual
inspection.
FIG. 18B, FIG. 18C, FIG. 18D, FIG. 18E and FIG. 18F
show T-l weighted images (TR/TR = 800/45) performed at
1.0 Tesla, before (Precontrast) and at various minutes
after intravenous (i.v.) injection (Post-contrast, MPI)
of Gd:MPD-DTPA:dermatan sulfate selective contrast agent,
prepared as in Examples 21 and 22, and injected per
Example 25, at a dose of 0.155 mmol/Kg into Fisher 344
female rats with syngeneic breast adenocarcinomas
inoculated previously into the liver, such that the tumor
CA 02208~66 1997-06-23
W O 96/19242 PCTnUS94~14776
- 57 -
- diameters at the time of imaging are between 1.0 and 2.5
cm.
FI~. 18B. Tl Precontrast image of liver (tumor not
conspicuous).
FI~. 18C. Tl liver image a 7 MPI, Gd:MPD-
DTPA:dermatan sulfate selective contrast agent (0.155
mmol/Kg), showing extremely strong contrast enhancement
of 2 solid tumor nodules (right posterior liver) and 1
irregular tumor infiltrate (central liver region), in the
identical locations as those indicated by the T2-weighted
scout image (FIG. 18A), but with much better definition
- of the tumor margins and much higher contrast gradients
at the tumor margins. Note the moderately smaller size
of tumor nodules and improved definition of the central
tumor infiltrate, both due to an absence in the Tl mode
of T2 imaging artifacts, namely an additional rim
(corona) of water outside the actual tumor margin, which
appears in the T2 pu]se mode but not in the preferred Tl
mode.
FIG. 18D and FIG. 18E. Tl Liver image at 20 and 40
MPI, Gd.:MPD-DTPA:dermatan sulfate selective contrast
agent (0.155 mmol/Kg), showing continued very marked
contra~t enhancement of the 2 solid tumor nodules (right
~ posterior liver) and the 1 irregular tumor in~iltrate
(central liver region), with continued very highly
demarcatea tumor margins and essentially no contrast
fading.
. FIG. 18F. Tl Liver image at 20 and 40 MPI, showing
contin~ed very marked contrast enhancement of the 2 solid
~ tumor nodules (right posterior liver) and 1 irregular
tumor infiltrate (central liver region), with only a very
slight degradation in the sharpness of tumor margins over
40 MPI, only a very minimal decrease (fading) of tumor
CA 02208~66 1997-06-23
W O 96/19242 PCT~US9~/14776
- 58 -
contrast intensity in the 2 solid nodules (right
posterior liver), a further brightening of the tumor
infiltrate tcentral liver region), and a very slight
background brightening of surrounding uninvolved liver.
FIG. l9A, FIG. l9B, FIG. l9C, FIG. l9D and FIG. l9E
show T1-weighted images at 4.7 Tesla (TR/TE = 250/8) of
Copenhagen rats with syngeneic AT-1 prostate
adenocarcinomas inoculated into previously prepared skin
pouches [Hahn et al. (1993)], and imaged at diameters of
- 1.0-2.5 cm.
FIG. l9A. Precontrast image for Gd:MPD-
DTPA:dermatan sulfate selective contrast agent, showing
- 15 only the tumor and superficial back fat and back muscle,
because a surface coil is used and not a whole body coil.
FIG. 19B. Post-contrast image, 7 MPI i.v. of
- Gd:MPD-DTPA:dermatan sulfate selective contrast agent,
liquid form. Note the extremely strong enhancement of
the entire tumor mass and the extremely strong gradient
at the boundary between tumor and underlying normal
- tissue (image right).
.
FIG. l9C. Post-contrast image, 20 MPI i.v. of
= Gd:MPD-DTPA:dermatan sulfate selective contrast agent,
liquid form. Note the extremely strong enhancement of
- the entire tumor mass and the extremely strong contrast
- gradient at the boundary between tumor and underlying
normal tissue. Contrast has decreased slightly in the
central tumor region, such that the tumor neovascular
~- . array is now very well visualized.
FIG. l9D and FIG. lgE. Post-contrast image, 40 and
35 60 MPI, of Gd:MPD-DTPA:dermatan sulfate selective .
= contrast agent, liquid form. Note the still very strong
enhancement of the tumor, and particularly the retention
CA 02208~66 1997-06-23
W O96/19242 PCT~US94/14776
- 59 -
of an extremely strong contrast gradient at the boundary
between tumor and underlying tissue. Contrast intensity
in the c~ntral tumor and outer rim (image left, away from
the anim~l) has decreased moderately, apparently due to
progressive tumor accumulation in these regions, of such
a high local concentration of the highly potent Gd:MPD-
DTPA:dernatan sulfate [Rl = 7.8 (mmol.sec)~l], that T2*
effects ~re starting to produce competitive darkening of
the central and outer tumor regions (image left; see also
Example ~6). The basal rim (image right), is relatively
protected from this T2* darkening artifact, due to more
rapid ba_kdiffusion of the agent into plasma at this
basal si_e. Hence, moderately lower doses are indicated.
FIG. 20 shows a special histochemical stain
(microwave augmented Prussian blue metal-ion stain) of
AT-1 prostate adenocarcinoma (from Copenhagen rat), with
the tumor tissue removed at 60 MPI just following the
completion of MRI imaging, freshly frozen, sectioned and
stained as above and as in Example 26 and FIG. 6 and FIG.
7. Note the selective staining positive for Gd(III)
metal iOll as follows: (a) very strongly positive within
almost a:Ll tumor cells (tumor intracellular sites); (b)
strongly positive at tumor-cell nuclei -- ~or many but
not all t:umor cells (e.g., see tumor cells underlying
grid mar~;er "9" and directly to the left of grid marker
"10" at mage left); (c) moderately positive neovascular
endothelial cells (e.g., see directly above grid marker
"8" at image top -- appearing as "railroad tracks": and
directly under grid marker "2"); and (d) weakly positive
to negative in subendothelial and extracell~lar matrix
sites (- the spaces between tumor cells and endothelial
ribbons). The low 60-minute staining of extracellular
matrix mcy result from either or both of: (a) a more
- 35 diffuse distribution of metal ions at 60 minutes (versus
7 minute~ in FIG. 6 and FIG. 7A), diffuse metal ions
being mo~-e difficult to visualize (due to their smaller
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
- 60 -
optical staining niduses); or (b) plasma backdiffusion of
a portion of the initially localized metal. These
findings of metal-ion positivity in tumor endothelium,
tumor matrix, tumor cells proper and tumor-cell nuclei,
provide the basis for selectively localizing MRI and
radionuclide diagnostic and therapeutic agents, and
indeed, other types of active substances.
FIG. 21A. Frozen 8-micron thick section of prostate
adenocarcinoma (outer 2/3 of tumor of ca. 4 cm diameter),
excised from its host rat (Copenhagen strain, syngeneic)
3 hours after intravenous injection of 5 mg/Kg
doxorubicin:DS (doxorubicin in association with
essentially purified dermatan sulfate, 435 Type, Opocrin)
at a weight ratio of 60:40 (doxorubicin to dermatan
sulfate), and fluorescence microscopy performed using a
rhodamine-type filter to elicit direct fluorescence of
the doxorubicin drug substance (see also Example 29).
Note the very bright direct drug fluorescence in almost
all tumor cells which are packed into a relatively dense
sheet throughout the tissue section. This is indicative
of high tumor-cell internalization of the doxorubicin
drug substance. Note also the endothelial cytoplasmic
and nuclear positivity, indicative that endothelium, as
well as tumor cells proper, constitute a target of
doxorubicin:DS (but not of standard doxorubicin -- see
below).
FIG. 21B. Section of same tumor as in FIG. 21A, but
in a subregion with looser clusters of tumor cells which
are located and arranged around an endothelial stalk
(oriented horizontally across the image). Note the very
bright staining of almost all cells, plus the strikingly ~ ~
bright fluorescence of doxorubicin now localized at
35 nuclear sites, as well as in the tumor-cell cytoplasm. '
Also note the strong fluorescence of endothelial cells
and endothelial-cell nuclei.
CA 02208~66 1997-06-23
W O96/19242 PCT~US94/14776
- 61 -
FIG. 21C. Section of the same tumor type and size,
but from a different Copenhagen rat, which was sacrificed
3 hours after intravenous injection of 5 mg/Kg of
standard doxorubicin (Adriamycin PFS liquid). Note the
dense sheets of cells at upper right and the looser
clusters at lower left -- all of which exhibit markedly
lower fl~orescence (indicative of overall tumor and
intracellular drug levels), as well as the general lack
of fluorDscence in and around the large tumor microvessel
(image center) and no identifiable fluorescence in the
tumor-cell nuclei. The finding latter is strongly
suggesti~e of lower drug levels at a key intracellular
site and target of drug action, namely the nucleus and
nuclear DNA.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention involves nontoxic,
biodegradable small molecules, particles or microspheres
(less than about 0.2-100 micrometers (um) in size) and
microaggregates (1-200 nanometers, nm) comprising
endothelial-binding substances and in particular,
~ dermatan sulfate. These substances induce the following
serial steps upon intravenous injection of particles into
test rodents: 1) endothelial bioadhesion; 2) rapid (2-
minute) endothelial envelopment (partial or total) of the
substances; 3) a facilitated (accelerated) migration of
intact drug-carrier particles across microvessels into
the tissle compartment; (which is largely complete within
10 to 20 minutes of injection); and 4) delayed release of
drug from a microsphere formuiation of envelopment
carrier ~hich is known to correlate with controlled
bioavailability of drug within the target tissue (lesion)
- 35 i~ vivo.
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94114776
- 62 -
The examples presented herein include compositions
of matter serving as formulation carriers for efficient,
nonmagnetic drug localization in normal and diseased
tissues, including microspheres or nanospheres comprising
a special class of dermatan sulfate as described herein
which binds to the complementary heparins and heparan
sulfates present on normal endothelium throughout the
body.
This invention is not considered to be constrained
by prior art involving the formulation of microcarrier
- matrices from any of the presently proposed materials
providing that the said materials were not previously
recognized and documented in vivo as undergoing multiple
endothelial binding and inducing rapid endothelial
envelopment, and producing accelerated extravasation of
macromolecules, microaggregates and microparticles in
either the first microvascular bed encountered, or
potentially (as proposed) semiselectively at foci of
disease following systemic intravenous administration.
~- Endothelial-envelopment carriers may be formulated
and stored in either the dry or fluid state, to which may
be added, for example, pharmaceutically acceptable
appropriate stabilizers, osmotic agents, colorings,
flavorings and physiologic solutions which render them
appropriate for intravascular and intracavitary
injection. The present invention is envisioned as most
particularly applying to the vascular targeting phase of
any future device (see below) which is developed for the
~; efficient first-step transit across the external body
barriers (e.g., gastrointestinal tract; oral, nasal
rectal, bladder or vaginal mucosa; skin, cornea or
sclera).
~ The present disclosure documents that drug carriers
which comprise microencapsulation spheres with surface
CA 02208~66 1997-06-23
WO 96119242 PCT/US94/14776
-- 63
; adhesion properties were selectively taken up into
tissues by endothelia]. bioadhesion and by induced
transenc.othelial migration, into the tissue interstitium.
The present application additionally documents that drugs
controlled by such carriers, are deposited in selected
target tissues, such as lung, in exact proportion to the
deposition of drug carriers. It is now further
establi~hed that soluble drug-carrier complexes (as well
as forma.lly microencapsulated drugs) give comparable
tissue u.ptake of drugs, under conditions in which the
drug alcne is not taken up. It is now further
established that the same and similar carriers are taken
up by t~e transepithelial route in the lungs,
gastroin.testinal tract and bladder. Finally, it is
establi~hed that the same and similar carriers undergo
prefereDtial lesional concentration in tumors and niduses
of pulmonary infection.
The unique aspect of drug carrier technologies
establi~hed by the present application are that these
novel ca.rriers afford high-efficiency tissue uptake and
localization of drugs, in particula-, wher. ~he drugs are
controlled by nonembolizing (less than 3-4 ~ carriers.
The carriers are preferably of a non-embol_zlr.g size o'
less than 500 nm and more preferably less than about 25C
nm. Otk.er unique features are that these carriers a) are
formulated of water-soluble, biocompatible and
biodegradable materia].s, and b) afford widespread
percolation throughout tissue interstitium (and lesional
gels) in. a fashion which is not possible for hydrophobic
~ carrier~ (e.g., liposomes). Finally, the carriers of the
principal embodiments interact with their initial sites
of cellu.lar uptake (endothelial and epithelial cells)
based on. carbohydrate-carbohydrate binding and they do so
in such a fashion as to produce multivalent binding,
which leads to an induced, active endothelial (or
epithelial) envelopment and transendothelial (or
CA 02208~66 1997-06-23
W O 96/19242 PCT~US94/14776
- 64 -
transepithelial) transport of both the carriers and drugs
controlled by the carriers. This preferably involves
transcytosis (process occurring across one endothelial or
- epithelial cell) or may involve endothelial (epithelial)
migrational overgrowth of the carriers, leading to
envelopment.
- In the practice of preferred embodiments of the
invention, multivalent binding to cells (or adjacent
matrix substances) must occur, in order to induce active
extravasation (or epithelial transport) of the drug-
carrier couple, wherein such transport is significantly
accelerated relative to that obtained for uncoated
(uncontrolled) particles or drug-carrier complexes; this
acceleration being of such a degree that transcellular
transport of nonembolizing as well as embolizing
particles (complexes) is completed within twelve minutes
-- of endothelial/epithelial contact (typically in less than
5 minutes), under in vivo conditions of microvascular
= 20 blood flow and/or cavitary fluid flow, air flow, or
enteric flow (in microvessels, bladder, lungs, bowel, or
other body cavities, respectively).
The carriers must control the delivery of multiple
(at least two) molecules of drug, in order to distinguish
them from naturally transported simple hormones,
proteins, peptides, and hybrid conjugates of two low-
molecular-weight drugs.
- 30 Although the preferred embodiment describes a
surface coating of dermatan sulfate, alternative carriers
(and surface coatings and drug-complexing agents), such
as dermatan sulfate fragments, heparin fragments,
tridodecyl methylammonium chloride heparin, hereinafter
referred to as TDMAC heparin, and other
.glycosaminoglycans (GAG's), and preferably the new class
of dermatan sulfates with a sulfur content of up to 9~
~.-
CA 02208~66 1997-06-23
W O96119242 PCTAUS94~14776
- 65 -
; (w/w) ar.d with select.ive oligosaccharide oversulfation,
also serve to bind to constitutive and induced heparin
cofactor II. The 8-12 unit fragment of dermatan sulfate
binds heparin cofactor II without activating it. Unlike
native heparin, neither dermatan sulfate nor its 8-12
unit frcgment inhibits the constitutive endothelial
surface coagulant, antithrombin III. This is also true
of the ~horter, semisynthetic fragments of heparin.
Hence, cermatan sulfate and the short fragments of both
heparin and dermatan sulfate, are envisioned as having
even lec~s anticoagulant activity than does native heparin
(whose ninimal anticoagulant activities are still
acceptably low in this regard, when the heparin is
incorporated into drug microspheres and complexes).
Encothelial uptake is described for a new physical
formulation, namely a macromolecular complex between
dermatar. sulfate and doxorubicin (an antitumor drug). It
is understood in the present application that doxorubicin
is preferably provided as doxorubicin HCl in order to
promote ion pair binding or complexation with the sodium
salt fo~m (preferably) of dermatan sulfate. Therefore,
doxorubicin and doxorubicin HCl are used interchangeably
when deccribing the active agent in the drug carrier
compositions of the present disclosure. Selective high-
efficie~cy uptake of this drug and carrier complex is
documented in the present application following
administration of the complexed agent. The absence of
endothelial injury by dermatan sulfate-doxorubicin is
also documented. This novel result established the
rationale for reformulating existing drugs using dermatan
sulfate and related kits (as devices), which can be
performed by hospital pharmacists on-site, ~ust prior to
drug ad~inistration. This new approach can allow
'- 35 localized tissue (lesional) uptake of drugs controlled by
nonembolizing carriers, as follows:
CA 02208~66 1997-06-23
- W O 96119242 PCTrUS94/14776
' .
- 66 -
- a) by intravenous administration to the lungs
(high efficiency delivery) and systemic
lesional sites (moderate efficiency delivery);
or
b) by selective arterial perfusion to liver,
- kidney, brain, pelvis, extremities and other
- body sites (high efficiency delivery).
-
The present application describes that secondary
tissue percolation of these hydrophilic drug-carriers
occurs in normal target tissues for dermatan sulfate-
coated microspheres (interstitium, lymphatic and
epithelial). In the present application, additional
examples are presented, which establish the general
principal that, unlike the situation for lipid
- microemulsions, liposomes and other hydrophobic carriers,
the present hydrophilic spheres percolate extensively
through the interstitium of a tumor and the lesional gel
of a spontaneous pneumonitis, to reach both the outer
spreading rims and the inner necrotic cores of these
lesions. This provides new rationale for improved
lesional penetration, cellular (microbial) access and
uptake of drug carriers, and their entrapped (controlled)
drugs. It is envisioned as allowing improved drug access
- to tumor cells and microorganisms lying in sequestered
sites.
The present invention describes new entrapments of
substances such as:
.
a~ doxorubicin HCl; and
b) other antitumor drugs such as taxol,
vincristine or peptide onco agents that are '
~m~n~hle to coating with dermatan sulfate and
its derivatives.
CA 02208~66 1997-06-23
W 096119242 PCT~US9~/14776
- 67 -
r The present invention includes formulations which
employ additional detergents as excipients for preparing
the inte~rnal drug nanoparticles, nanoemulsions, or other
interna:ly entrapped, controlled-release subcapsules,
complexes or agents for formulation and entrapment of the
interna] drug emulsions. Such detergents include:
a) preferably, sodium deoxycholate;
b) alternatively, cholesterol, TWEEN 80,
zwitterionic detergents, or other biocompatible
nonionic, polysulfated or positively charged
detergents, as needed to formulate stable drug
emulsions.
The present invention teaches that cancers (and
drugs) can potentially be treated (and localized) in an
improvec fashion by using the described technology. The
bioadheEion carriers set forth in the present application
are envisioned as being preferred for the delivery of
drugs which are highly toxic (certain antitumor drugs);
drugs which are highly labile; agents which experience
inappropriate biodistribution or poor tissue access due
to their large molecular size or the presence of
disseminated, competing receptors in the body; and anti-
adhesion pharmaceuticals (as depot formulations, for the
prevention of cancer-cell metastasis, prophylaxis of
atherosclerosis, and inhibition of white-cell and
platelet adhesion to vascular endothelium).
The present invention includes the use of additional
methods for matrix stabilizing and controlling the
release ~f drugs. These include addition of thickening
agents, ~uch as polylactic and polyglycolic acids,
' 35 polyamin~acids, poly-L-lysine, polyethyleneimine,
glycerol, polyglycerols or polyalchols (with or without
heating or chemical reaction), polyethylene oxides,
CA 02208~66 l997-06-23
W O96/19242 PCTrUS94/14776
- 68 -
biodegradable poloxamers or poloxamines (pluronics or
tetronics), poly-COOH compounds (polycarbols), or
polyamines.
Additional methods of microparticle formulation are
envisioned as including (particularly for the purposes of
product scale-up): preferably, extrusion of matrix
(and/or surface) components through single (and/or
coaxial), sonified or air-stream-fractured micro-orifices
(single or multiport); alternatively, aerosolization
using hybrid, homogenization-spray drying apparatus.
The present invention includes additional methods of
extracting the solvents used for phase emulsification and
simultaneously crystallizing the matrices, surfaces
and/or entrapment materials): preferably, hexanes;
alternatively, ethanol or methanol.
Additional methods of sterilization (and/or particle
sizing) of the final (or subfinal) preparations, include:
preferably, for heat-stable agents: autoclaving at 120~C
for 10-20 minutes; preferably, for heat-lab _- agents:
submicron filtration of complexes an~ na~ a-~;~les; an~;
irradiation of particles larger thar.
alternatively, ultrasonification.
The many innovative teachings of the present
invention will be described with particular reference to
the presently preferred embodiments, wherein these
innovative teachings are advantageously applied to the
particular issues of in vivo T1-Type MRI image contrast
enhancement by site-selective localization and sustained
site retention of paramagnetic metal chelates according
to optimal spatial and kinetic profiles at the site,
while simultaneously enhancing clearance and minimizing
toxicity of the non-localized dose fraction. However, it
should be understood that this principal embodiment is
CA 02208~66 1997-06-23
W O96/19242 PCT~US94/14776
- 69 -
only one example of the many advantageous uses of the
innovative teachings herein. For example, the various
types of innovative compositions and methods disclosed
herein can alternatively be used to selectively localize
and enhance clearance of radionuclide imaging agents, X-
ray contrast agents, ~ltrasound-acoustic image enhancing
agents and a wide spectrum of therapeutic agents which
are base~ on site delivery of metal chelates and in si tu
chelatio~ of endogenous body metals. Of special interest
to the therapeutic agents and uses embodied herein, are
actives and indications useful in oncotherapy,
cardiova~cular infarcts, restenosis, atherosclerosis,
acute th~ombosis, microvascular disease, inflammation,
and any other tissue diseases which have as part of their
development or progression, a vascular component amenable
to binding, adhesion, transport and/or modulation by the
novel teachings, compositions and uses described herein.
Hence, il- will be obvious to those skilled in the art,
that numerous additional compositions and uses are
uniquely enabled by the present invention. The following
examples are presented to illustrate preferred
embodiments of the present invention, their uses in MRI
contrast enhancement. These examples are purely
illustrative, and do not in any way delimit the full
scope of the present invention.
The present invention specifically describes the
preparation and utilization of novel contrast agents for
magnetic resonance imaging. These novel contrast agents
consist cf paramagnetic metal chelates, as distinguished
from metal-atom complexes, wherein the presently
disclosec chelates are bound to glycosaminoglycans (GAG).
. - Binding cf the metal complex to the GAG may take the form
of, but is not limited to, electrostatic interactions
(ion-paired), hydrogen~bonding, Van der Waals
interactions, covalent linkages, or any combination of
these interactions. Following parenteral administration
CA 02208~66 1997-06-23
W O 96/19242 PCT~US94/14776
- 70 -
of these metal complex-glycosaminoglycan formulations,
the technology described herein utilizes a biocompatible
carrier molecule to deliver an associated biologically
active substance to sites of vascular injury.
The present invention provides substantially
improved MRI image and spectral enhancement compositions
and methods, whereby the capacity of MRI hardware systems
to detect tumors, cardiovascular diseases, and other
diseases with a vascular or endothelial adhesive
component are greatly enhanced. These improvements are
presently accomplished by introducing a chelated
paramagnetic metal ion selectively into tissue sites of
interest, inducing selective (local) modulation of Tl-
Type, paramagnetic relaxation of water protons or otherdiffusible nuclei present within the site which are
susceptible to orientation by fixed and gradient magnetic
fields and to pulsed re-orientation by radiofrequency
fields of appropriate resonant frequencies, thereby
giving rise to detectable modulations of induced magnetic
resonance signals, in the forms of either image contrast
enhancement or spectral enhancement.
Past disclosures (Ranney: US serial No. 07/880,660,
filed May 8, 1992, US Serial No. 07/803,595 filed April
3, 1992, and US Serial No.07/642,033 filed January 1,
1991] have dealt with the binding of magnetic agents
which required: (a) magnetic potencies greater than that
of the most potent single metal ion, gadolinium(III); (b)
intramolecularly coupled, polyatomic metal-atom complexes
stabilized by non-bridged ligands which have a stronger
potential for chemical and physical instability than the
stably, bridged-ligand chelated metal ions disclosed
herein; and (c) divalent cationic charge on the
"superparamagnetic" active for binding to anionic
carriers, versus the presently disclosed requirement for
only a monovalent cationic charge on paramagnetic metal
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
- 71 -
chelato:~ actives. It is understood, that for MRI uses,
the metal chelator will also comprise an appropriate
paramagnetic metal ion, for example, preferably iron(III)
or gado:linium (III), however, for certain other
diagnoslic and therapeutic compositions and uses, the
present:y disclosed metal chelators may either comprise
or avoid an appropriate metal ion. For the presently
preferred MRI applications, basic metal chelators and
metal c]lelators with electrophilic properties at
formulal;;ion pH's are preferred, for example, ferriox~mine
[Crumbliss, 1991], basic or amine derivatives of the
polyaminocarboxylate chelator,
diethylenetriaminepentaacetate (DTPA), and basic or amine
derivat:Lves of the macrocyclic chelator, 1,4,7,10-
tetraazacyclododecane-N,N',N",N"'-tetraacetate (DOTA) [~i
et al. :L993; Brechbiel et al. 1986] . In certain
instances and with certain potent carriers bound to these
and related actives, site localization may be so
pronounced that the inherent potency ( in vi tro
paramagIletic Rl) o~ the paramagnetic metal ion may not be
crucial to obtaining optimal site-localized image
contrast- or spectral enhancement effects. Hence, the
present invention discloses pronounced T1 image contrast
effects for the basic metal chelate, ferrioxamine, which
by virtue of chelated Fe(III) ions, has a potency, or R1
relaxiv-ty, of about 1. 6-1. 8 [mmol.sec]-1.
Alternatlvely basic metal chelates of Gd(III) ma,vbe
expectecl under certain but not all in vivo conditions, to
have a potentially greater relaxivity, due to its greater
3 0 in vi trc~ R1 of about 4.0-4.3 [mmol.sec]-1 when chelated by
~ DTPA, and potentially moderately higher when chelated by
DOTA [Geraldes et al . 1985], and as hlgh as R1 2 7.5
~ ~ . [mmol.se~c]~1 when Gd(III) is chelated to certain DTPA
~ derivati.ves, including N-methyl-1,3-propane diamine-DTPA
as one preferred embodiment of a group of preferred DTPA-
amine and DTPA-basic ~erivatives which can both (a) allow
accelerated water diffusion and relaxation above that of
CA 02208~66 l997-06-23
W O 96/19242 PCTrUS94/14776
- 72 -
DTPA; and (b) bind non covalently to acidic saccharides,
including, preferably, glycosaminoglycans. Alternative
metal ions may preferably include the divalent or
:trivalent cations, manganese, chromium and dysprosium;
and less preferably, those ions of copper, nickel,
erbium, europium, and holmium.
Preferred chelators of the present invention include
those with a formation constant of at least about 1014 for
-10 strongly paramagnetic metal ions disclosed above, and
including a basic or cationic group. These chelators
preferably include ferrioxamine, basic or amine
-derivatives of DOTA, DTPA, porphines, porphyrins,
sapphyrins or texaphyrins, which can preferably chelate
Fe(III) and most preferably chelate Gd(III), as well as
bind by principally paired-ion (electrostatic) means to
the acidic groups of acidic carriers. For example,
certain texaphyrins have an expanded macrocyclic ring
which may, in certain instances, stably chelate Gd(III)
[Sessler et al . ' 065; Sessler et al . ' 720; Sessler et al .
~498, incorporated by reference herein]. Whereas
- texaphyrins and sapphyrins are not exemplified in the
- present invention, it will be obvious to those skilled in
- the art, from the references cited just above, and from
the presently disclosed and exemplified Fe(III) chelator,
~ 5,10,15,20-Tetrakis(1-methyl-4-pyridyl)-21-23-porphine,
-~ that the related texaphyrins and sapphyrins and their
- basic, cationic and amine derivatives, as well as the
presently disclosed porphine derivative and its analogues
~ -30 and basic, cationic and amine derivatives, would be
- included under the disclosures and teachings of the
present invention, and may be used in combination with
the presently disclosed acidic carriers. There are
hybrid considerations of, among others: (a) paramagnetic
potency of the metal chelate; (b) binding stability to
the acidic carrier; and (c) formulation compatibility;
and (d) biocompatibility and clearance in vivo.
.
CA 02208~66 1997-06-23
WO 96/19242 PCT/US94~14776
-- 73
Hydrophilic chelators and carriers are usually, but not
always preferred, due to their typically favorable
formulai~ion properties (absence of aggregation),
biodistribution properties (absence of generalized
binding to hydrophobic plasma and cell-membrane
constituents during the process of localization) ; and
clearance plus toxicity advantages. Alternative
chelato:rs may include the hydroxamates, ferrichrome,
enterobactin, ferrimycobactin, ferrichrysin, and their
basic o:- amine derivatives, all derivatives being defined
as subsumed under the parent chelators listed above.
Preferred carriers include monomeric, oligomeric and
polymeric substances which contain or comprise anionic or
acidic groups defined at the pH's used for formulation.
These typically contain or comprise groups of
carboxy:l.ate, and more preferably, the even more strongly
acidic groups of phosphate, and most preferably, sulfate.
Preferred carriers include, but are not limited to an
acidic saccharide, oligosaccharide, polysaccharide,
glycosarl~inoglycan or sulfatoid, typically of bacterial or
semi-synthetic origin, or derivatives, modi~lcations or
fragment-.s of the preceding substances, a ~ de~ined here -.
as beinq subsumed under the names o~ t"e Fa-e~~
substances and categories. Hence, preferred carriers
include the following: heparin, desulfated heparin,
glycine--conjugated heparin, heparin sulfate, dermatan
sulfate, chondroitin sulfate, pentosan polysulfate, and
sulfatecl sucrose, including sucrose octasulfate, and any
derivati.ve, modification or modified form thereof, with
the most preferred being the essentially purified
dermatan sulfate as described herein. Less preferably
for typlcal MRI formulations and uses, are included the
carriers of sulfated cyclodextrin, dextran sulfate and
~- 35 hyaluroric acid, although any of these may be
particularly suitable for certain specific diagnostic or
therapeutic formulations and uses.
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
- 74 -
In all cases reported and tested, non-covalent
binding of the basic amine chelator to the acidic carrier
gives payloads of active agent which are markedly higher
than those afforded by covalent conjugation. For
example, preferred basic chelators, ferrioxamine and
Gd(III) DTPA-lysine, and most preferred, N-methyl-1,3-
propane diamine-DTPA (N-MPD-DTPA), are bound to their
acidic glycosaminoglycan carriers at weight ratios of ~
70~. Alternative covalent active-carrier conjugates may
be preferred in certain instances, and preferred examples
thereof are shown for MRI applications.
Specific embodiments of the present invention which
have been tested in vivo, include, but are not limited to
the presently exemplified preferred embodiments of: (a)
deferoxamine, (b) ferrioxamine, (c) Gd(III):DTPA-lysine,
(d) N-methyl-1,3-propane diamine-DTPA, and (e) other
basic metal chelates bound most preferably by non-
covalent means, and also preferably by covalent means, as
exemplified below, to acidic glycosaminoglycans,
including preferably, dermatan sulfate, essentially
purified dermatan sulfate having a sulfur content of up
to 9~ (w/w) and with selective oligosaccharide
oversulfation, heparan sulfate, and heparin, which
include by definition, any derivative or modification
thereof, including oversulfation and modification
undertaken to reduce anticoagulant activities or provide
improved site binding, enhanced clearance or other
desired formulation or in vivo properties. In
particular, however, the preferred carrier substances
from the standpoint of low toxicity and optimal safety
margins at the higher doses which typify MRI contrast
agent administrations, are the dermatan sulfates with
relatively low S03-/COO- ratios of preferably between
0.7:1 and 1.8:1, most preferably between 0.9:1 and 1.5:1,
and typically 1:1; and additionally with relatively low
sulfur content of preferably less than 9~ (w/w), most
CA 02208~66 1997-06-23
W O96119242 PCTrUS94/14776
preferably between 4~ and 7~ (w/w/), and typically 6.3-
6.4~ (w/w); and the most preferred carrier substances
under the high-dose a~ministration conditions employed
just above, comprise the new special class of dermatan
sulfates with oversulfation of only selected
oligosaccharide sequences but without overall
oversulfation of the entire molecule (as described and
defined above). Alternative preferred Agents obvious
from the present disclosures, to those skilled in the
art, may induce arginine and histidine basic derivatives
of DTPA nd DOTA, and also of the various texaphyrins,
sapphyrins, porphines, porphyrins, EHPG, and by
definition, most preferably for MRI applications,
comprising their Gd(III) and Fe(III) metal-ions, and also
preferably comprising their alternative paramagnetic
metal iO]l chelates, as disclosed above.
The carrier substance most preferably used in the
present :invention is the new class of essentially
purified dermatan sulfates, enriched in uronic (L-
iduronici acid content and, in addition to its major
monosulfated disaccharide sequence, (Ido-GalNAc4SO3), also
characterized by an oligosaccharide sequence with a
selectively high degree of sulfation, including the
oversulfated saccharide sequences, (IdoA2SO3-GalNAc4SO3)
and (Ido~GalNAc4, 6SO3) (as assessed by disaccharide
analysis and as uniquely correlated with 1H and 13C
magnetic resonance spectra~, enriched in heparin cofactor
II activi.ty, preferably greater than 220 Units/milligram,
but low i.n factor Xa and antithrombin III activity and in
overall anticoagulant activity (preferably less than 10
and most preferably less than 5~ of standard heparin by
~ USP anticoagulant assay), low in S03-/COO- ratio,
preferabl.y in the range of 0.7:1 to 1.8:1 and most
preferably in the range of 0.9:1 to 1.5:1, and low in
sulfur content, preferably less than 9~ and most
preferably in the range of 4 to 7~; and preferably having
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
a modal molecular weight of between 10,000 and 23,000
daltons, and most preferably between 13,000 and 19,000
daltons -- the lower end of this molecular weight bracket
generally being important in order for the carrier to be
highly retained within the vascular compartment of normal
organs after intravenous administration; and the higher
end of this molecular weight bracket generally being
important for effective disease site binding and uptake,
while still affording the very rapid blood clearance by
the renal route, which is important for rapidly achieving
low blood imaging backgrounds and low body residua at
early post-injection times.
The dermatan sulfates of the preceding paragraph
may, in one case, be prepared by the methods of: (a)
grinding and treating animal organs or tissues, including
beef mucosa, swine skin or lung, and preferably for
certain of the present uses, beef mucosa, with
proteolytic enzymes including papain, at pH 5 to 7 for
the shortest possible time to remove proteins; (b)
passage over a strong anion (basic) exchange resin
including a macroreticular styrene-divinylbenzene matrix
functionalized with quaternary ammonium groups and having
a particle size range of 0.3 to 1.3 mm; (c) eluting the
sulfated polysaccharides with a neutral salt solution
between of 0.7 and 2.0 molarity; (d) crystallization of
the dermatan sulfate as a low-solubility salt of a
~ bivalent or trivalent metal including copper, iron and
calcium, and preferably copper; (e) reconversion to
sodium salt via cation exchange resin including chelex
~ 100 type (Bio-Rad 143-5852); selectively enriching for
the oversulfated oligosaccharide sequences (above) by
~ . chromatography on a strongly basic anion exchange resin
~ functionalized with quaternary ammonium groups, wherein
the resin typically has a particle size of less than or
equal to 10 micrometers and a cross-linkage of 2-8~; (f).
concentrating the eluate by reverse osmosis; and (g)
CA 02208~66 1997-06-23
W 096119242 PCT~US9~/14776
lyophili~zing the resulting liquid to form a fine white
powder. One example of the above dermatan species, which
is not i.ntended in any way to limit the scope of the
present invention, comprises a subspecies of these
dermatar. sulfates (sulphates), as described [Mascellani,
et al. ~ro 93/05074 (1993), incorporated herein by
referenc:e; Mascellanit et al. (1994), incorporated herein
by reference]. One of most preferred example of this
subspecies o~ dermatan sulfate is the Type 435 beef
mucosal dermatan sulfate (sulphate) manufactured and
supplied by Opocrin S.P.A., Via Pacinotti, 3, I-41040
Corlo Di Rormigine, Italy. It has a modal molecular
weight of approximatel.y 17,500 to 18,000 daltons, as
determined by charge suppressed molecular sieve
chromatography with W absorbance analysis, and a sulfur
content of approximatelv 6.2 to 6.6~ weight percent --
this low sulfur content occurring despite the selective
enrichment in these dermatan sulfates of certain
oligosac-haride sequences with a high degree of
sulfatio~, including the oversulfated saccharide
sec~ences, (IdoA2SO3-GalNAc4SO3) and (IdoAGalNAc4, 6SO3)
whose en~ichment correlates wlth high heparin cofactor II
activity.
In the descriptions of the two preceding paragraphs,
(a) enrichment for uronic (L-iduronic) acid content plus
the preceding 2,4-disulfated disaccharide sequences in
combination with (b) the preferred molecular weights in
the range of 10,000 to 23,000 and most preferably 13,000
to 19,000 daltons, and (c) low S03-/COO- ratio,
corresponding to a low overall sulfur content, typically
~. in the range of 4.5 to 7~ by weight, correlates with the
surprising and unexpected advantages of: (a) in vivo
potency of rapid disease-site binding, localization,
uptake and deep penetration, e.g., of tumor endothelium,.
tumor ext-.racellular matrix and tumor cells; and (b) low
side effects of induced platelet aggregation,
CA 02208~66 l997-06-23
W O 96/19242 PCT~US94/14776
anticoagulation and bleeding -- which are
characteristically induced by the more highly sulfated
and/or longer-chain (higher molecular weight) carriers,
including sulfated, oversulfated and polysulfated
glycosaminoglycans and natural and synthetic sulfated,
oversulfated and polysulfated polysaccharides and
sulfatoids -- most specifically those with a sulfur
content of 10~ or greater, and those with a USP heparin-
type anticoagulant activity ranging from 15 to 145 USP
units per milligram or greater.
The preferred dermatan sulfates (above) and the most
preferred new special dermatan sulfate subspecies,
essentially purified as prepared by the special processes
described above, when used as site-selective diagnostic
or drug carrier substances, are clearly distinguished
from all of the previous, older dermatan sulfates, i.e.,
those (a) not having the special structures described
above; (b) not prepared according to the above isolation
and purification processes; or (c) not prepared by such
alternative processes as would give comparable enrichment
of the preferred oligosaccharide sequences an~ selective
sulfations described above. These preferre~ essential ~,
purified dermatan sulfates are also ciearl~
distinguished, when used as above, from all of the prio-
older dermatan sulfates in that they are not only
structurally different, but they are also essentially
free of the contaminating heparins, heparan sulfates and
heparinoids which bind normal endothelium, undergo
various degrees of in vivo metabolism, and interfere with
~ rapid and complete blood and body clearance [Dawes,
.et al. (1989), incorporated herein by reference]. It
will be further obvious to those skilled in the art, that
~ the new special dermatan sulfates described above, are,
when used as site-selective diagnostic or drug carrier
substances, even more distantly distinguished from the
non-dermatan sulfate classes of glycosaminoglycans,
CA 02208~66 1997-06-23
WO 96/19242 PCT/US94/14776
-- 79 -
namely: (a) chondroitin sulfates A and C -- which do not
share the uronic (L-iduronic) acid sugars of dermatan
sulfate tWalton, et al ., US Patent 4,489,065; Maeda,
et al . 1993), both incorporated herein by reference];
(b) hepcrin -- which does share uronic (L-iduronic) acid
structure but which has high anticoagulant activity and
high bir.ding to normal endothelium [Cremers, et al.
(1994); Kalishevskaya, et al. (1988), both incorporated
by refe~ence herein]; (c) hyaluronic acid -- which is a
non-sulfated glycosaminoglycan; (d) all of the
polysulfated glycosamlnoglycans and oversulfated
sulfatoids, e.g., bacterial polysulfates including
pentosan polysulfate ~- all of which characteristically
have sulfur contents of 10~ or greater that create
significant in vivo safety issues due to polysulfate-
induced platelet aggregation and cell membrane
perturbation/lysis, or act as cofactors for such cellular
lysis and which can affect normal body cells as well as
tumor cells and other pathological cells/organisms, such
as that specifically described as direct toxic cofactor
"opening~ of tumor cells produced by chondroitin
polysulfate, resulting from chondroitin polysulfate-
induced nembrane damage [Landsberger (1984)]. Hence, the
new special dermatans preferred in the present invention
are ones which do not themselves cause significant direct
cellular or membrane damage, but instead induce rapid (3-
to 7-mimlte) selective binding of disease-site
endothelium, rapid (10 to 5-minute) endothelial cell
transporl_, tumor uptake, deep matrix permeation and
tumor-ce:l internalization of the attached diagnostic or
drug act:;ve without the dermatan sulfate carrier itself
or alone damaging either the intermediate (e.g.,
- endothel:Lal) or final (e.g., tumor) target cells.
This new special class of dermatan sulfate is
clearly clistinguished from chondroitin sulfate Types A
and C by its high content of L-iduronic (uronic) acid
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94114776
- 80 -
relative to the low or absent content in chondroitin
sulfates A and C; and by its relatively lower modal
molecular weight, most typically less than 25,000 daltons
versus the chondroitin sulfates A and C, which typically
equal or exceed 25,000 daltons modal molecular weight.
The relatively lower molecular weight of the new special
dermatan sulfates has at least three surprising and
unexpected advantages when used as a carrier substance
for bound or associated active substances: (a) very rapld
blood clearance of the carrier and active, predominantly
by the renal route, with a blood t 1/2 of typically about
20 to 120 minutes, increasing only very gradually as a
function of increasing dose; (b) minimal to absent in
vivo metabolism -- in major contrast to standard
heparins, heparan sulfates and chondroitin sulfates A and
C -- thereby giving extremely low residual in vivo
deposition or retention of the carrier material; and (c)
maximal, rapid vascular egress across disease-site
endothelium -- including across induced and
"permeabilized" endothelium, e.g., induced by Vascular
Endothelial Growth Factor/Vascular Permeability Factor
(VEGF/VPF) for maximal disease-site and tumor access,
uptake and tumor-cell internalization of the bound or
associated active substance.
Whereas, this new class of dermatan sulfates has
been recognized as useful for conferring antithrombosis
in the absence of (heparin-type) anticoagulant activity
and bleeding side effects, it has not previously been
recognized, nor would it be obvious to one skilled in the
art, that this new special class of dermatan sulfates
could confer the surprising and unexpected advantages of
acting as a highly potent and effective in vivo carrier
of noncovalently or covalently bound amine or chemically
basic chelators or metal chelates, furthermore, to
selectively localize them in sites of disease, including
tumors, across non-permeabilized as well as
CA 02208~66 1997-06-23
W O 96/19242 PCTAUS9~/14776
"permea~ilized" vascular endothelium and simultaneously
to promcte very rapid clearance of the non-targeted
fraction of carrier plus active, highly preferentially by
the renal route, in a fashion which increases only very
gradually with increasing dose -- thereby conferring not
only reduced side effects and low in vivo retention, but
also the additional advantages of: (a) very low imaging
backgrounds at very early times post-injection upon
intravenous administration for the purpose of in vivo
contrast enhancement by associated paramagnetic metal
chelate; and (b) pronounced capacity for dose escalation
with acceptable safety. These surprising and unexpected
advantages are particularly important for use in
paramagn~tic enhancement of in vivo magnetic resonance
images (~RI) because of low sensitivity of the imaging
equipmen~ and detection method, and hence, the need for
injecting high intravenous doses of paramagnetic metal
chelate (typically in the range of 0.1 to 0.3 mmol/kg) in
order to deposit sufficient local-site concentrations of
paramagnetic agent (ca. 50-100 micromolar). This further
emphasizes the advantage of using a carrier material,
including the new special dermatan sulfates, which can
preferable allow a noncovalent method of binding the
active to the carrier, and hence, can enable a high
quantity of active to be bound per unit of carrier,
preferab:y greater than 70~ (weight ~ of active to
[active -- carrier]) versus typically 7 to 12~ (w/w) for
most covalently bound active-polymer systems, including
antibody systems. Hence, the self-assembling,
noncovalent formulation (as well as covalent formulation)
properties of the new special dermatan sulfates provide
an additional surprising and unexpected advantage of
- minimizing the quantity of dermatan sulfate carrier
required to administer and selectively localize an
effective in vivo dose of paramagnetic chelate.
CA 02208~66 1997-06-23
WO96/19242 PCT~S94/14776
- 82 -
The present invention describes the preparation and
utilization of a novel MRI contrast agent for enhancement
of solid tumors and cardiovascular infarcts. The
contrast agents consist of cationic or basic paramagnetic
metal complexes in association with strongly acidic,
including polysulfated carriers, and including preferably
glycosaminoglycans. It would be obvious to those skilled
in the art that any acidic glycosaminoglycan can be used.
Of the paired-ion systems described below, most notable
are those consisting of ferrioxamine with
glycosaminoglycans, DTPA-lysine with glycosaminoglycans,
N-methyl-1,3-propanediamine-DTPA with glycosaminoglycans,
and most preferably, N-methyl,3-propanediamine-DTPA with
the new special subspecies of dermatan sulfates described
above.
In one particularly preferred embodiment,
- essentially purified dermatan sulfate (435 Type of 17,000
to 19,000 modal MW, with selectively oversulfated
oligosaccharides and a heparin cofactor II activity at
least about 220 U/mg, Opocrin), is used in noncovalent
(or covalent) association with the following oncology
actives to localize them in sites of disease and
facilitate their clearance them from the rest of the
body: doxorubicin, adriamycin, taxol, vincristine,
vinblastine, bleomycin, idarubicin, epirubicin,
amsacrine, azacitidine, dideoxyinosine, dihydro-5-
~ azacytidine, ethanidazole, ethiofos, methotrexate,
misonidazole, porfiromycin, pyrazoloacridinek,
terephthalamidine, taxotere and other taxane derivatives,topotecan, trimetrexate, carboplatin, N-formyl-met-leu-
- phe-lys, arginine bradykinin, poly-L-lysine, other
chemoattractants, biological response modifiers,
cytokines, interferons and lymphokines.
In another particularly preferred embodiment,
essentially purified dermatan sulfate (435 Type of 17,000
CA 02208~66 199i-06-23
WO 96/19242 PCT~US94/14776
- 83 -
to 19,0()0 modal MW, with selectively oversulfated
oligosa~charides and a heparin cofactor II activity at
least a~out 220 U/mg, Opocrin), is used in noncovalent
(or covc~lent) association with the following anti-
infectives: gentamicin, amikacin, tobramycin, and otheramine, hasic, basic peptidic, basic polypeptidic,
hydrophobic or amphoteric antibiotics or bacterial,
fungal, mycobacterial, viral or other microbial or
microbicilogical diseases.
It is envisioned that alternative diagnostic and
therapeutic compositions and applications may be carried
out using compositions substantially similar to those
disclosed above. For example, alternative metal ions may
be chelated for purposes of metal-ion exchange at the
site. Hence, the present formulations may contain or
comprise metal ions of manganese, aluminum, germanium,
zinc, cobalt, calcium, platinum, or others.
Alternatively, for purposes of radiation and radionuclide
therapy, such compositions may contain or comprise metal
ions of .~oron, cobalt, rubidium, yttrium, technetium,
rutheniun, rhenium, indium, iridium, thallium, samarium
or others. Specifically, and in some cases preferably,
59Fe and s7Ga [Hashimoto et al. 1983; Janoki et al. 1983]
may be substituted as radionuclide forms of the non-
radioact:ive metal ions, for purposes of nuclear medical
imaging of tumors, thrombi, and other biomedical imaging
purposes a
30The preceding discussion is presented to specify
~major aspects of the invention and their use in in vivo
~ diagnost c and therapeutic applica~ions, however, to
- . those ski.lled in the art many additional and related
compositi.ons and methods of use will be obvious from this
precedinc discussion and are encompassed by the present
inventlor .
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
- 84 -
T~iBLE 1
Advantages of Metal Ion Chelator and
Anionic, Hydrophilic Carrier
Techn~'c~y Scle~ MRI Antibodies PEG Li~.oso,lles
Agent
Property
Drug Payload High * 60- Very Low 5% Low 10-30% Low 15-20%
90%; **
77.5%
Locali~dlion in Yes Very Low No No
Tissue Sites
Selectivity Broad Immune Narrow Immune None None
(CHO lectin) (Ab-antigen~
Time to Target Very Rapid Slow (several Slow Very Slow
(several mins) hrs) (many hrs) (hrs-days)
Time to Clear Rapid Very Slow Very Slow Extremely
Plasma & Body Slow IRES)
Applications Broad (Tissue Narrow Narrow Narrow (RES)
Sites) (Inlravdscular~ (Enzymes)
*preferred
** most preferred
The following examples are included to demonstrate
preferred embodiments of the invention. It should be
appreciated by those of skill in the art that the
techniques disclosed in the examples which follow
represent techniques discovered by the inventor to
function well in the practice of the invention, and thus
can be considered to constitute preferred modes for its
practice However, those of skill in the art should, in
light of the present disclosure, appreciate that many
changes can be made in the specific embodiments which are
disclosed and still obtain a like or similar result
without departing from the spirit and scope of the
invention.
CA 02208~66 l997-06-23
WO 96119242 PCT~US94~14776
-- 85 --
In all of the following Examples, except as
otherwi~3e stated, all references to dermatan sulfate and
native cLermatan sulfate refer to the new special class of
dermatan sulfates witn oversulfation of only selected
5 oligosaccharide sequences but without overall
oversulfation (hypersulfation) of the entire molecule (as
described and defined herein), and in particular refer to
the new special "435 Type" of dermatan sulfate as
supplieci by Opocrin S.P.A., Via Pacinotti, 3, I-41040
Corlo Di Formigine, Italy.
EXAMPLE 1
Preparation of Deferoxamine Free Base
and Use in Formulation of Ferrioxamine
The free base of deferoxamine is used in certain
instances, in order to minimize the residual salt content
present in final formulations which utilize deferoxamine
as a basic metal chelator. In these instances,
deferoxa~ine is precipitated out of aqueous salt
solutions by the addition o~ 2 N KHC03, as previously
reported [Ramirez et al. (1973), incorporated by
referenci~ herein]. A saturated solution of deferoxamine
(320 mg/-nL at 25~C) is prepared by dissolving 4.0 g of
deferoxamine mesylate salt in 12.5 mL of pharmaceutical-
grade wai_er. The solution is cooled to 4~C in an ice
bath and 2.5 mL of 2.0 N KHC03 added. The glass container
is scratched with a stainless steel spatula to initiate
precipitation. The precipitate is collected by
centrifugation, washed repeatedly with ice cold water,
and filtered. The crude deferoxamine free base is
purified by sequential recrystallization from hot
~- methanol The resulting pure deferoxamine free base is
dried uncler a stream of nitrogen. The infrared spectrum
of the deferoxamine as prepared is consistent with that
referenced above.
- CA 02208~66 l997-06-23
W O 96/19242 PCTrUS94/14776
- 86 -
Ferrio~mlne is formulated from the deferoxamine
free base by addition of ferric chloride at
stoichiometric molar ratios of Fe(III) to deferoxamine
free base. This results in chelated iron and minimizes
- 5 residual mesylate and chloride ions.
EXAMPLE 2
Preparation of Ferrioxamine-Iron (III) Chelate
Batch quantities of the Fe(III) chelate of
deferox~m;ne are prepared as follows. Deferoxamine
mesylate (methanesulfonate) (Ciba-Geigy Limited, Basel,
Switzerland), 390 g, is dissolved in pharmaceutical-grade
water. Alternatively, the chloride salt of deferoxamine
may be used. A highly purified slurry of ferric iron in
- the form of Fe(O)OH (13.44~ w/v of Fe(O)OH particles,
Noah Technologies Corporation, San Antonio, Texas), 372.9
g is suspended in 1899 mL of water and added to the
deferoxamine with constant stirring. The resulting
suspension is heated to 60~ C for between 1 and 24 hours
and the pH adjusted periodically to between 6.5 and 7.9
by addition of 0.10 N NaOH. Formation of the
ferrioxamine complex is evidenced by development of an
intense, deep reddish-brown color to the solution.
Stoichiometric chelation of Fe(III) with deferoxamine is
confirmed by in-process W-Visible absorbance
spectroscopy at 430 nm, against stoichiometrically
chelated ferrioxamine standards. The batch solution is
cooled to room temperature and centrifuged at 4500 rpm
(~2500 g) for 15 minutes to remove any unreacted or
aggregated Fe(O)OH. This final batch volume is adjusted
as desired, typically to a final volume of 2600 mL. Any
remaining trace amounts of unreacted Fe(O)OH are removed
. and the solution also made aseptic, by passing the
- 35 supernatant through a 0.22 ~m Millipore GV-type filter in
: a Class 100 l~m; n~r flow hood. The resulting batch is
stored at 4~C in an autoclaved, sealed glass container
CA 02208~66 1997-06-23
W O 96119242 PCT~US94/14776
- 87 -
until further use (see Examples below). The final
concentration of ferrioxamine (DFe) is determined once
again by W-Visible absorbance spectrophotometry at 430
nm. The R1=1.6 (mmol.sec)-l, based on ICP-AA measurement
of Fe(I~I).
EXAMPLE 3
Preparation of the Basic Amine Chelator:
Diethylenetriaminepentaacetate-Lysine (DTPA-Lys)
DTFA, 500 mg, is dissolved in 20 mL of
pharmaceutical-grade water and heated to 60~C. L-Lysine
hydrochloride powder, 931 mg, is added with constant
stirring until dissolved. Alternatively, N-epsilon-t-
BOC-L-lysine can be used to prevent reaction of the
carbodiimide intermediate at the lysine epsilon amino
group (see below), and when used, is dissolved in
dimethylformamide:water (50:50, w/v). The solution pH is
adjusted to 4.75 by addition of 0.1 N HCl. The water-
soluble _arbodiimide, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide HCl (EDC), 732.5 g, is
dissolve~ in 2 mL water and its pH also adjusted as
above. This EDC solution is added dropwise to the DTPA +
lysine s,~lution mixture (above) over 1 hour at 22OC with
constant stirring and periodic adjustment of pH to 4.75,
and the reaction allowed to proceed to completion over 2
more hou:rs. When N-epsilon-t-BOC-L-lysine is used (see
above), the N-epsilon-t-BOC group is hydrolyzed at this
step, by acidification with hydrochloric acid to a pH of
between :.0 and 2.0, and stirring for 30-60 min. The pH
is readjusted to 4.75 as needed, and the reaction
solution is concentrated down to 5 mL by rotary
- evaporat:Lon at 60~C, and the DTPA-lysine (DTPA-Lys)
derivative is precipitated by addition of 3 volumes of
r 35 ethanol. Note: under these conditions, the ethanol:water
ratio used, maintains the solubility of all individual
substrates (above). The resulting precipitate is
CA 02208~66 1997-06-23
WO96/19242 PCT~S94/14776
- 88 -
~ harvested by centrifugation at 2,500 x g, washed with
ethanol, re-centrifuged, and dried over a stream of dry
nitrogen. Covalent conjugation of lysine to DTPA is
confirmed by infrared (IR) spectroscopy. The resulting
reaction product has a faint yellow color.
EXAMPLE 4
Preparation of the Gadolinium(III)
Metal Chelate of DTPA-Lys: gadolinium:DTPA-Lys
[Gd(III):DTPA-Lys]
The gadolinium(III) chelate of DTPA-Lys, namely
Gd(III):DTPA-Lys, is prepared by dissolving a known
quantity of DTPA-Lys in water and adding a stock solution
of gadolinium chloride, prepared at 0.l-l.0 M, as needed,
until a stoichiometric quantity of Gd(III) has been
added. The pH is adjusted to 7.0 by addition of l.0 N
- NaOH. Alternatively, gadolinium oxide can be added and
the reaction mixture stirred for 24 hours. In the case
of gadolinium oxide, neutralization with l.0 N NaOH is
not needed. Each batch of Lys-DTPA conjugate is pre-
titrated and the final chelation product checked for
stoichiometric addition of Gd(III), using a standard
xylenol orange titration method [Lyle et al. (1963)], and
further confirmed by quantitative ICP atomic absorption
spectroscopy for gadolinium. The resulting Gd(III):DTPA-
Lys is precipitated by addition of ethanol (3 volumes per
volume of water), and the precipitate collected by
centrifugation. This precipitate is rewashed with
ethanol and centrifuged (as above), washed with acetone
- plus centrifuged, and the collected precipitate dried
over a stream of dry nitrogen. The resulting product
continues to have the same faint yellow color as noted in -'
Example 3. The Rl of aqueous product = 4.2(mmol.sec)-
based on ICP-AA measurement of Gd(III).
CA 02208~66 1997-06-23
W 096/19242 PCT~US94~14776
- 89 -
EXAMPLE 5
Preparation of Paired-ion Agents of
Ferrioxamine bound to Dermatan Sulfate Carriersi and
Ferrioxamine to Depc~lymerized Dermatan Sulfate Carrier
Ferrioxamine:dermatan sulfate paired-ion agents are
prepared by mixing appropriate ratios of the water
solutions of ferrioxamine (see Example 2, above) with
either: (a) dermatan sulfate of modal MW between
approximately 5,000 daltons and 45,000 daltons (Opocrin,
S.p.A., Modena, Italy, 435 type from beef mucosa modal
MW=18,00~ daltons; and Scientific Protein Laboratories,
Waunake, Wisconsin, from porcine mucosa, modal MW=19,600
daltons); or (b) depolymerized dermatan sulfate of modal
MW betwe(~n approximately 2,000 daltons and 5,000 daltons
(Opocrin S.p.A., Modena, Italy, 370 type from beef
mucosa, depolymerized from 435 type starting material).
A range of ratios of ferrioxamine to dermatan sulfate are
prepared between a low of 1:99 (wt ~) of
ferrioxamine:dermatan sulfate or depolymerized dermatan
sulfate; and a high of 30:70 (wt ~) of ferrioxamine:
dermatan sulfate or depolymerized dermatan sulfate).
Using 0.~ to 1.0 N NaOH, the pH of the mixture is
adjusted to between 5.5 and 8, the mixture is stirred
continuously for 0.5 to 72 hours and the pH re-adjusted
between 5.5 and 8, and typically to 7.5. This
ferrioxamine:dermatan mixture is passed through a 0.22 ~m
filter to remove any residual insoluble iron oxides and
hydroxides, and to render the liquid agent aseptic. The
aseptic agent is stored either as a liquid at 4~C, or as
a lyophilized powder (see below). Further processing is
carriecl out on the liquid, by filling into glass vials
- and autoclaving at 120~C for 15 minutes. Alternatively,
further ~rocessing is carried out on the liquid by
filling into glass vials, freezing at -50~C, and
lyophilization to give an aseptic lyophilized powder.
The lyophilized vials are reconstituted by adding sterile
CA 02208~66 1997-06-23
- W O 96/19242 PCT~US94/14776
-- 90 --
water and hand mixing for 1 to 5 minutes, to give a
reconstituted liquid of desired concentration which is
ready for injection. The resulting concentrations of
ferris~m;ne and dermatan sulfate are measured and vial
quantities confirmed by standard reverse-phase HPLC and
macromolecular size exclusion HPLC methods, respectively.
Multiple batches of Ferrioxamine:Dermatan Sulfate
Agent have been prepared. In vi tro test results for a
representative batch are as follows:
ferrioxamine:dermatan sulfate ratio: 77.5:22.5 (w/w);
solubility of agent, 550 mg/mL; water:octanol partition,
17,600 (+ 2,750):1; concentration of ferrioxamine, 0.166
mmol/m~; concentration of dermatan sulfate, 32 mg/mL;
molecular weight of dermatan sulfate, Opocrin type 435,
MN = 18,000 daltons; sulfate/carboxylate ratio of
- dermatan sulfate, 1.0 + 0.15; ferrioxamine and dermatan
purities, nominal i 10~; pH, 6.5-7.9; viscosity, 3.8-4.2
centipoise; osmolality, 475-525 mOsm/Kg; Rl, 1.5-1.8
[mmol.sec]-l; oversized particles, within USP guidelines
for small-volume parenterals; Anticoagulant activity,
less than 4.5 U.S.P. Units/mg (modified USP XXII assay);
binding of ferrioxamine active to dermatan carrier, at
least 92~ retained (dialysis for 3 hours against 200
volumes, 500 daltons molecular weight cutoff).
In vitro stability of Ferrioxamine:Dermatan Sulfate
Agent under accelerated conditions, indicate the
following. (a) The liquid form is stable, by the
preceding physicochemical and HPLC parameters for longer
than 6 months at 4~C, 22~C and 40~C; is slightly unstable
at 2 to 6 months at 60~C, and degrades significantly
within 1 to 3 days at 80~C. (b) The liquid form can be
autoclaved as above, with less than 3~ degradation of
ferrioxamine. (c) The lyophilized form is stable with
respect to all parameters (above), including oversized
CA 02208~66 1997-06-23
WO 96/19242 PCT/US94~14776
- .91
particle~s; and is projected to be stable over storage
periods of multiple years.
EXAMPLE 6
Preparation of Paired-ion Agents of
Ferrioxamine bound to Heparin
Ferrio~m~ne:dermatan sulfate paired-ion agents are
prepared by mixing appropriate ratios of water solutions
of ferriox~m;ne (as in Example 5, above) with (a) beef
lung heEJarin of modal MW between approximately 8,000
daltons; and (b) porcine heparin of modal MW between
approximately 10,000 daltons and 20,000 daltons. A range
of ratios of ferrioxamine to heparin or heparin fragment
are preF~ared between a low of 1:99 (wt/wt) of
ferrioxamine:heparin or heparin fragment; and a high of
30:70 (wt ~) of ferrioxamine:fragment. Using 0.1 to 1.0
N NaOH, the pH of the mixture is adjusted to between 5.5
and 8, the mixture is stirred continuously for 0.5 to 72
hours and the pH re-adjusted between 5.5 and 8. This
ferrioxamine:heparin mixture is passed through a 0.22 ~m
filter to remove any residual insoluble iron oxides-
hydroxides and render the liquid agen~ asept~_. The
aseptic agent is stored at 4~C. As inG' cate~, ~urthe~-
processing is carried out by filling the aseptic liquicin glass vials, followed by freezing and lyophilizing, te
render t~e agent as an aseptic lyophilized powder. The
lyophilized vials are reconstituted by adding sterile
water anl hand mixing for l to 5 minutes, to give a
reconsti~uted liquid of desired concentration which is
ready for injection. The resulting concentrations of
ferrioxanine and heparin are measured and vial quantities
- confirmed by standard reverse-phase HPLC and
macromolecular size exclusion HPLC methods, respectively.
~ .
~: =
CA 02208~66 1997-06-23
W O 96/19242 PCT~US94/14776
- 92 -
EXAMPLE 7
Preparation of Non-anticoagulant Heparin Carrier
by glycine Derivatization
The anticoagulant activity of heparin can be reduced
to almost negligible activity by derivatizing its
carboxylate groups with glycine residues as reported
~Danishefsky et al. (1971); Danishefsky et al. (1972)].
This non-anticoagulant heparin (Nac-heparin) can then be
utilized as a modified glycosaminoglycan carrier.
According to one present method of glycine conjugation,
0.75 g of heparin is weighed into a 100 mL beaker and
dissolved in 25 mL of pharmaceutical-grade water.
Glycine, 0.75 g, is added and the pH of the resulting
solution adjusted to 4.75 with 0.10 N HCl. l-ethyl-3-(3-
dimethylaminopropyl)carbodiimide HCl (EDC), 0.75 g, is
weighed into a separate vial, solubilized by adding a
minimum amount of water, and the pH adjusted to 4.75 with
0.10 M HCl. Aliquots of the EDC solution are added to
the mixture of glycine-glycosaminoglycan over a one hour
period. After each addition of EDC, the pH is adjusted
to maintain it at 4.75. After addition of all EDC, the
reaction is allowed to proceed for an additional two
hours with constant stirring and periodic pH adjustment.
The glycine-heparin conjugate (Gly-HEP) is then
precipitated by addition of 3 volumes of absolute
ethanol. The precipitate is collected by centrifugation
at 4500 rpm (~ 2500 x g) for 15 minutes; and washed three
times with 20-mL aliquots of ethanol with re-
centrifugation.
. , s
CA 02208~66 1997-06-23
W ~ 96/19242 PCTAUS94/14776
- 93 -
_ EX~MPLE 8
Preparation of Paired-ion Agents o~ Ferrioxamine bound to
Glycosaminoglytans, Modified and Derivatized
Glycosaminoglycans of: heparan sulfate,
5non-anticoagulant heparin
oversulfated dermatan sulfate
chondroitin sulfate, oversulfated chondroitin sulfate
and the bacterial Sulfatoid, pentosan polysulfate
10Ferrioxamine paired-ion agents are prepared with
various glycosaminoglycan carriers by mixing appropriate
ratios of water solutlons of ferrioxamine (as in Example
5, above) with the following glycosaminoglycans: (a)
heparan sulfate of MN = 8,500 daltons; (b) non-
15anticoagulant heparin SPL, ++ of MN = 10,500 daltons; (c)
oversulfated dermatan sulEate of MN = 19,000 daltons; (d)
chondroitin sulfate of MN = 23,400 daltons; (e)
oversulfated chondroitin sulfate of MN = 14,000 daltons;
and (f) pentosan polysulfate of MN = 2,000 daltons. The
ratios cf ferrioxamine to glycosaminoglycan and sulfatoid
carriers are prepared to give a payload of [77.5:22.5 ~
(w/w) of ferrioxamine to carrier] (adjusted) by a scaling
factor of [(mEq sulfates/mg of carrier as above) / (mEq
sulfates/mg of beef lung heparin*)]. Using 0.1 to 1.0 N
NaOH, the pH of the mixture is adjusted to between 5.5
and 8, the mixture is stirred continuously for 0.5 to 72
hours an~ the pH re-adjusted between 5.5 and 8. This
ferrioxamine:heparin mixture is passed through a 0.22 ~m
filter to remove any residual insoluble iron oxides-
hydroxides and render the lit~uid agent aseptic. Theaseptic agent is stored at 4~C. As indicated, further
processing is carried out by filling the aseptic liquid
-in glass vials, followed by freezing and lyophilizing, to
render the agent as an aseptic lyophilized powder. The
lyophilized vials are reconstituted by adding sterile
water and hand mixing :Eor 1 to 5 minutes, to give a
reconstil.uted liquid of desired concentration which is
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
- 94 -
ready for injection. The resulting concentrations of
ferriox~m;ne and heparin are measured and vial quantities
confirmed by standard reverse-phase HPLC and
macromolecular size exclusion HPLC methods, respectively.
Although not prepared in the present application, it
is apparent that by combining the teaching of the present
Example with those of previous disclosures 07/880,660,
07/803,595, and 07/642,033, ferrioxamine complexes can be
similarly prepared with additional acidic saccharides,
including sucrose octasulfate and sulfated cyclodextrins;
with additional glycosaminoglycans, including keratan
sulfate and hyaluronate; and with additional sulfatoids,
including the bacterial sulfatoid, dextran sulfate.
* For beef lung heparin, mEq SO3~/g carrier = 4.4.
EXAMPLE 9
Preparation of Paired-ion Agents of
Gd(III):DTPA-Lys bound to Dermatan Sulfate Carrier
Gd(III):DTPA-Lys:Dermatan Sulfate paired-ion agents
are prepared by mixing the water solutions of
Gd(III):DTPA-Lys with dermatan sulfate of modal MW
between approximately 5,000 daltons and 45,000 daltons
(as in Example 5, above), and in particular, dermatan
sulfate of MN = 18,000 (Opocrin, S.p.A., Modena, Italy,
435 type), to form a final solution ratio of 75:25~ (w/w)
of the Gd(III):DTPA-Lys active to the Dermatan Sulfate
carrier. Several stable Agent variations of the
resulting liquid have been prepared, wherein the
concentration of Gd(III):DTPA-Lys ranges from 0.166 to
0.415 mmol/mL, and the respective concentration of
dermatan sulfate ranges from 35 to 87.5 mg/ml. The T1
relaxivity (R1) of Gd (III):DTPA-Lys = 4.2.
CA 02208~66 1997-06-23
W O96119242 PCT~US94/I~7~6
- 95 -
E~MPLE 10
P:-eparation of a Basic Iron-porphine Chelate;
and Paired-ion Binding to Heparin
The soluble, tetra-basic porphine, 5,10,15,20-
tetrakis(1-methyl-4-pyridyl)-21H-23-Hporphine, 40 mg as
the tetra-p-tosylate salt, is refluxed with Fe(II)
chloride, 30 mg, for 2 hours in 20 mL of
dimethylformamide. Evidence of iron complexation is
observed in the form of a red to dark green color.
Solvent was removed by evaporation, the solid product
dissolved in water. The pH is adjusted to 7.5 to
insolubilize excess ferric iron, followed by filtration
of the iron-porphine product. A 2 mg/mL solution of
iron-porphine complex and ca. loo~ product yield is
confirmed by inductively coupled plasma atomic
absorption. A comparable reaction in water gives ca. 70
yield.
This iron-porphine complex is added to beef lung
heparin lissolved in water, ca. 8 Kd, at ratios ranging
from 1:20 to 20:1 (iron-porphine:heparin). This resulted
in clear solutions without precipitates. Binding of
iron-porphine to heparin is nearly 100~ as evaluated by
dialysis against water for 16 hours, using bags with
molecular weight cutoffs of 3.5 Kd and 12 Kd. Iron-
porphine alone is nearly completely dialyzed. W-Visible
spectrophotometric titration-indicates maximum binding
occurs al; a molar ratio of 18:1 (iron-porphine:heparin).
Since the beef lung heparin used is known to have
approximately 18 available strongly acidic (sulfate)
groups per mole (and per heparin chain), these results
indicate strong ionic interaction and stable (to
dialysis' binding of the basic tetraamine porphine
~~ 35 complex t:o the sulfate groups of heparin.
.
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94114776
- 96 -
EXAMPLE 11
Preparation of a Basic
Triethylenetetraamine-iron Chelate;
and Paired-ion Binding to Heparin and Sucrose Octasulfate
Soluble complexes of triethylenetetraamine and
iron(III) are formed by dissolving 1.0 g of
triethylenetetraamine.2HCl (Syprine~) (Merck, West Point,
PA) in water and adding a 1:1 mole ratio of iron chloride
under acidic conditions (pH = 2) to give a clear yellow
solution. Using 0.1 N NaOH, the pH is adjusted to 6.8,
giving a red solution indicative of iron complexation.
This solution develops a feathery red precipitate,
indicative of intermolecular aggregation of the iron-
triethylenetetraamine complex.
(a) To this resulting aqueous dispersion of complexis added beef lung heparin, to give final complex-to-
heparin ratios of between 95:5 and 5:95 (by weight). At
a ratio of 65:35 (complex:heparin) and higher ratios of
heparin, heparin completely solubilizes the complex.
This apparent solubilization is indicative c~ paired-ic~.
binding between triethylenetetraamine-ir_-. a..- heparir..
(b) To the aqueous dispersion of
triethylenetetraamine-iron complex is added sucrose
octasulfate (SOS), to give final complex-to-SOS ratios o~
between 95:5 and 5:95 (by weight). At a ratio of 65:35
(complex:SOS) and higher ratios of SOS, SOS causes the
dispersion to become very much finer, indicative of
paired-ion binding between triethylenetetraamine-iron
: complex and SOS. The absence of complete clarification
of this SOS paired-ion system relative to that with
- heparin (above), is due to the much higher density of
sulfates on SOS relative to heparin, which confers
substantially increased intermolecular hydrogen bonding
on the SOS system.
CA 02208~66 1997-06-23
W O96119242 PCT~US94/I4776
- 97 -
Allhough not directly exemplified, it will be
apparenl: that polyamines with the homologous series
CXHX~yNx-z~ which also form stable complexes with Iron(III),
can also be used in place of triethylenetetraamine-iron
complex and SOS in the present. invention.
Preparation of Covalent Conjugates of Deferoxamine
Glycosaminoglycan Carriers
Su~strates with electrophilic amine groups may be
covalently conjugated reagents to nucleophilic
carboxylate groups of acidic carriers, acidic saccharides
and acidic glycosaminoglycans as reported [Danishefsky et
al. (1971); Danishefsky et al. (1972); Janoki et al.
1983); Axen (1974); Bartling et al. (1974); Lin et al.
(1975)]. The coupling reagents described in these
references activate carboxylate groups toward
nucleophilic attack. The mechanism involves ~ormation of
an activa~ed intermediate resulting from reaction of the
coupling reagent with the carboxylate residues on the
carrier. The intermediate undergoes nucleophilic attack,
typically by an amine functional group. This results in
formation of a stable covalent conjugate, typically via
an amide bond between the active and the carrier.
Examples 12, 13, and 14 (below) describe the synthesis of
ferrioxamine-heparin covalent conjugates, wherein the
ferrioxamine is covalently bound to heparin via three
differenl. coupling reagents.
EXAMPLE 12
Preparation of a Covalent Ferrioxamine-Heparin Conjugate
by i-ethyl-3-(3-dimethylaminopropyl) Carbodiimide
- (EDC) Linkage
A~ueous ferrio~m;ne, 2.0 g, as prepared in Example
1, is adjusted to pH 4.75 by addition of 0.10 M HCl.
Beef-lung heparin (Hepar-Kabi-Pharmacia, Franklin, OH),
CA 02208~66 1997-06-23
- W O 96119242 PCT~US94/14776
- 98 -
0.75 g, is dissolved 5.0 mL of pharmaceutical-grade water
and added to the ferrio~m;ne with constant stirring.
The pH of the resulting solution is re-adjusted to 4.75
with 0.10 M HCl. The water-soluble carbodiimide, 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide HC1 (EDC), 2
g, is weighed into a scintillation vial, solubilized in a
minimum amount of water, and the pH adjusted to 4.75 with
0.10 M HCl. Aliquots of EDC solution are pipetted into
the mixture of ferrioxamine-heparin over a one hour
period. After each addition of EDC the 0.10 M HCl is
added to maintain the pH at 4.75. After addition of all
EDC, the reaction is allowed to proceed for an additional
two hours with constant stirring. The ferrioxamine-
heparin conjugate is precipitated by addition of 3
volumes of absolute ethanol. This precipitate is
collected by centrifugation at 4500 rpm (~ 2500 x g) for
15 minutes and washed three times with 20 mL aliquots of
ethanol plus centrifugation. The complex is further
purified by redissolving in water and re-precipitating
with 3 volumes of ethanol plus centrifugation. The final
product is collected and dried over nitrogen.
Ferrioxamine derivatization of heparin is confirmed by
W-visible absorbance spectroscopy of the ferrioxamine
chelate at 430 nm and heparin analysis by size-exclusion
HPLC chromatography.
EXAMPLE 13
Preparation of a Covalent Ferrioxamine-Heparin Conjugate
by N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)
Linkage
Beef-lung heparin (Hepar-I~abi-Pharmacia, Franklin,
OH), 0.50 g, is weighed into a 3-necked 100 mL round
bottom flask fitted with an inlet and outlet for N2 purge.
Anhydrous dimethylformamide (DMF), 20 mL, is added with
constant stirring and the resulting suspension warmed to
50~C under a constant flow of nitrogen. A 30 mole excess
CA 02208~66 1997-06-23
W O 96/19242 PCr~US94~14776
463.,i mg) of N-ethoxycarbonyl-2-ethoxy-1,2-
dihydroc,uinoline (EEDQ) is added and the resulting
suspension stirred at 50~C for 3 hours. The activated
EEDQ-activated heparin is collected by centrifugation at
4500 rp~ (~ 2500 x g) for 10 minutes. The pellet is
washed repeatedly with anhydrous DMF and then 3 times
with acetone. The activated intermediate is dried under
a stream o~ nitrogen.
An aliquot of ferrioxamine solution containing 766.3
mg of the iron complex, as prepared in Example 1, is
pipetted into a 50 mL beaker and diluted to 25 mL with
anhydrou= DMF. In a separate 50 mL beaker, a known
amount o EEDQ-activated heparin is suspended in 50 mL of
anhydrous DMF with constant stirring. The DMF solution
of ~erriox~m;ne is pipetted slowly into the EEDQ-heparin
suspensi~n over a 5 minute period. The resulting
suspension is stirred continuously ~or 3 hours at 40~C.
After cooling to room temperature, the final product is
collecte(l by centrifugation, washed three times with
anhydrou~ DMF, washed three times with acetone, and dried
under nitrogen. Con~irmation of conjugate formation is
performed as in Example 12.
EXAMPLE 14
Preparation of a Covalent Ferrioxamine-Heparin Conjugate
by Carbonyldiimidazole (CDI) Linkage
An activated intermediate of beef-lung heparin
(Hepar-Kabi- Pharmacia, Franklin, OH) is prepared by
weighing 3.0 g of heparin into a 50 mL round bottom flask
and adding 25 mL of anhydrous dimethylformamide (DMF)
1 with constant stirring. Carbonyl- diimidazole (CDI),
608.1 mg, (10 mole excess relative to heparin) is weighed
into a separate vial and dissol~ed in 20 mL o~ anhydrous
DMF. The DMF solution of CDI is added to the DMF-heparin
suspension and stirred at 30~C for one hour. The CDI-
CA 02208~66 l997-06-23
W O 96/19242 PCTrUS9~/14776
- 100 -
activated heparin is collected by centrifugation, washed
repeatedly with acetone to remove unreacted CDI and
residual DMF, and dried under nitrogen.
,
- 5 The deferoxamine-heparin conjugate is prepared by
weighing 1.0 g of the CDI-activated heparin into a 50 m~
round bottom flask and suspending this in 25 mL of
anhydrous DMF. Deferoxamine, 250 mg, prepared as in
Example 1, is weighed into a separate round bottom flask
and dissolved in 20 mL of anhydrous DMF. The
deferoxamine free base solution is added slowly to the
CDI-heparin suspension and stirred continuously for 16
hours at 75~C. The deferoxamine-heparin conjugate is
collected by centrifugation at 4500 rpm (~ 2500 x g) for
15 minutes, washed repeatedly with anhydrous DMF, washed
repeatedly with acetone, and dried under nitrogen. The
resulting product is dissolved in water, and its
concentration determined by W-Visible spectroscopy. A
stoichiometric quantity of aqueous FeCl3 is added and the
resulting solution adjusted gradually to pH 6.5 and
stirred for 2 hours. This results in a deep brown-red
product. This ferrioxamine-heparin conjugate is
separated from any residual substrates and intermediates
by dialysis through a 2,000 MW cutoff bag against 150
volumes of water. The retentate is collected and
concentrated by rotary evaporation. Confirmation of
derivatization is performed as in Examples 12 and 13.
EXAMPLE 15
Preparation of a Covalent Heparin-
Diethylenetriaminepentaacetate Conjugate (DTPA-heparin)
DTPA-functionalized carriers are prepared in aqueous
media from the reaction of diethylenetriàminepentaacetic
dianhydride (cDTPAA; Calbiochem-Bhering Corp.) and a
molecule containing a nucleophilic functional group.
Beef-lung heparin (Hepar-Kabi-Pharmacia, Franklin, OH),
CA 02208~66 1997-06-23
W O 96/19242 PCT~US94~I4776
-- 101 --
1.5 g, is dissolved in 75.0 mL of 0.05 M HEPES buffer and
the pH adjusted to 7.Q with 0.10 M NaOH. cDTPAA, 4.5 g
(~ 100 mole excess relative to heparin), is weighed out
and divided into 20 equal (225 mg) aliquots. An aliquot
of cDTPA~ is added to the heparin solution every 3-5
minutes lntil all cDTPAA has been added. The pH of the
solution is monitored continuously throughout cDTPAA
addition and maintained at pH 7.0 with 0.10 M NaOH.
After ad~ition of the last aliquot of cDTPAA, the
solution is stirred for an additional 30 minutes. The
DTPA-heparin solution is dialyzed through 1000 MW bags
against L50 volumes to remove non-conjugated DTPA. The
resulting conjugate is concentrated by nitrogen-
evaporat:ion at 37~C and stored at 4~C.
EXAMPLE 16
Preparat.ion of Gadolinium(III) and Iron(III) Chelates of
DTPA-heparin Covalent Conjugate
The DTPA-heparin conjugate of Example 15 is further
prepared in the form of paramagnetic metal chelates of
the DTPA group with gadolinium(III) or Fe(II~ , by
pipettincg the required volume of DT~A-hepar~ nto a 1
mL Erlenmeyer flask, adding a 1.5-~o- 0 mo_~ excess o~
the paramagnetic metal ion oxide, as Gd.03 or Fe(O)OH, a~.-
stirring for 24 to 36 hours at 37~C to obtain
.solubiliz;ation of the metal oxides sufficient for
complete occupancy of the DTPA groups. The residual
metal oxides are precipitated by centrifugation at 4500
rpm (~ 2500 g), and the product separated from unreacted
metal oxides by filtration through a Millipore 0.22 ~m
GV-type filter, followed by dialysis against 150 volumes.
- The concentrations of chelated metal ion and heparin are
determined by inductively coupled plasma (ICP) and size-
'- 35 exclusion HPLC, respectively. In the case of Gd(III),
stoichiometric chelation is also confirmed by standard
xylenol orange titration [Lyle et al. (1963)].
CA 02208~66 1997-06-23
W O 96/19242 PCT~US94/14776
- 102 -
EX~iMPLE 17
Toxicity Studies of Ferrioxamine:Dermatan Sulfate,
435 Type
Acute intravenous Toxicity Studies with 14-day
recovery and necropsy are performed in male and female
rats and male and female dogs. At standard i.v.
injection rates of 0. 075 mmol/Kg/min., significant signs
generally occur only after 5-12.5 times the effective
imaging dose of 0.155 mmol/Kg. The LD50 is much greater
than 4.5 mmol/Kg and is limited by technical aspects of
tail-vein infusion. At this rate, some rats can be
infused with 10 mmol/Kg without untoward effects. At an
artificially accelerated i.v. injection rate of 0.080
mmol/Kg, deaths in rats can be obtained, and the LD50 is
between 2.5 and 3.0 mmol/Kg. Terminal necropsy reveals
no abnormalities in any rats after i.v. injection of 2.2,
3.0 and 4.5 mmol/Kg tn =5 males and 6 females per dose
level).
A pyramid acute i.v. toxicity study is performed in
dogs at escalating doses of 0.5, 1. 2 and 2.25 mmol/Kg and
an infusion rate of 0. 012 mmol/Kg/min in protocol
studies. An acute symptom complex of hypotension can be
obtained, which is minimal and reversible. No deaths
occurred and terminal necropsy at 14 days revealed no
abnormalities (n = 2 males and 2 females, all
administered each of the three dose levels, with a 72-
hour rest interval).
CA 02208~66 1997-06-23
W O96/19242 PCT~US94/14776
- 103 -
EXAMPLE 18
Ferrioxamine:Dermatan Sulfate Selective Contrast Agent:
MR:- Imaging of Lactating Breast Adenocarcinomas
in Syngeneic Fisher 344 Female Rats;
5Plus Correlation with Special Histochemical Studies
As shown in FIG. 2A, FIG. 2B, FIG. 3A, FIG. 3B, FIG.
4A, FIG. 4B, FIG. 4C and FIG. 4D, T1-weighted MRI images
(TR/TE - 800/45 and 550/23) are performed at 1.0 and 1.5
Tesla, before (Pre) and after (Post) intravenous (i.v.)
injection of Ferrioxamine:Dermatan Sulfate, 435 type
Selectiv~ Paramagnetic Contrast Agent (Example 5), at a
Ferrioxamine dose o~ 0.155 mmol/Kg into Fisher 344 female
rats, wi_h syngeneic breast adenocarcinomas inoculated by
trocar into the livers, such that tumor diameters at the
time of :imaging are between 1.0 cm and 2.5 cm. Tumors
are not ~onspicuous on standard T1-weighted Precontrast
images. Following injection of Ferrioxamine:Dermatan
Sulfate i~gent, the tumors (a) become rapidly and markedly
enhanced at an early post-injection time (7 mins) (FIG.
2A, FIG. 2B); (b) display very sharp tumor boundaries
against surrounding liver (FIG. 2A, FIG. 2, FIG. 4A, FIG.
4B, FIG. 4C and FIG. 4D), and discretely demarcated,
darker central region of tumor necrosis (FIG. 2A, FIG.
2B) (allowing tumor perfusion and function to be
spatially resolved and assessed within different, very
small anatomical subregions); (c) exhibit sustained
contrast for longer than 64 minutes postinjection (MPI)
(FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, MRI images; FIG. 5,
quantitative region-of interest, ROI, analysis) with
continued very well defined tumor borders at prolonged
imaging intervals. Correlation of these MRI images with
microwave augmented iron stains of the ~reshly excised, 7
MPI tumors, indicate that tumor-site localization of the
'- 35 Ferriox~m;ne active occurs only when it is bound (non-
covalently) to carrier (FIG. 6 and FIG. 7A) and not when
administered in free form (Active alone) (FIG. 3A, FIG.
CA 02208~66 1997-06-23
W O 96/19242 PCTnUS94/14776
- 104 -
3B). As shown in FIG. 8A, FIG. 8B and FIG. 8C, lung
- metastases of the liver tumor are rapidly and sensitively
enhanced in very small 2-mm to 3-mm nodules at an early
post-contrast interval; and this enhancement of the tumor
at lung sites is also sustained for a prolonged period
with high sensitivity plus retention of very sharp tumor
boundaries against normal lung. The sustained intervals
shown in FIG. 8A, FIG. 8B and FIG. 8C are much longer
than those typically reported for Gd:DTPA dimeglumine
contrast enhancement at body organ sites.
EXAMPLE 19
Ferrioxamine:Dermatan Sulfate Selective Contrast Agent:
MRI Imaging of Prostate AT-1 Carcinomas
in Syngeneic Copenhagen Rats and
Comparison with Gd(III)DTPA
As shown in FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG.
9E, FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D and FIG. 10E,
T1-weighted MRI images (TR/TE - 250/8) performed at 4.7
Tesla, before (Pre) and after (Post) intravenous (i.v.)
injection of Ferrioxamine:Dermatan Sulfate, 435 type
Selective Paramagnetic Contrast Agent prepared as in
Examples 2 and 5, and injected i.v. at an Iron(III) dose
of 0.155 mmol/Kg (FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D,
FIG. 9E); compared to Gadolinium DTPA dimeglumine,
injected i.v. at a Gd(III) dose of 0.100 mmol/Kg (FIG.
10A, FIG. 10B, FIG. 10C, FIG. 10D, FIG. 10E); each of
these agents being administered to Copenhagen rats with
syngeneic AT-1 prostate adenocarcinomas inoculated into
previously prepared skin pouches [Hahn, et al. ], such
. that tumor diameters at the time of imaging are between
1.0 cm and 2.5 cm. Ferrioxamine:Dermatan Sulfate A
p~oduces a rapid large enhancement of the Outer Rim of
tumor and also of the Vascular Array which fans out from
the tumor pedicle which carries a high majority of the
tumor vasculature. Sustained contrast and delineation of
CA 02208~66 1997-06-23
W O96119242 PCT~US94/14776
- 105 -
these e].ements remaints present through kinetic time
points of 40 minutes. By comparison, following Gd:DTPA
dimeglunline, the outer rim is not well delineated, even
at the ~~arliest post-contrast interval (7 MPI). Marked
early cc~ntrast fading occurs overall in the tumor at 20
MPI, and some agent sequesters in the central, poorly
perfusecL (cystic) regions of tumor (as is typically
reportec. for Gd:DTPA when used for imaging at body
sites). At 40 MPI, enhancement reverts to essentially
background levels, and at 60 MPI, there is no residual
contrast, except for central cystic regions.
EXAMPLE 20
MRI Contrast Enhancement of Acute Dog Myocardial Infarcts
by Ferrioxamine:Dermatan Sulfate
As shown in FIG. llA, FIG. 11s, FIG. llC and FIG.
llD, T1-weighted MRI ECG-gated cardiovascular images are
performe~ at 0.5 Tesla, before (Pre) and after (Post)
rapid intravenous (i.v.) infusion of
Ferrioxamine:Dermatan Sulfate, 435 type Selective
Paramagnetic Contrast Agent injected i.v. at an Iron(III)
dose of ~.155 mmol/Kg into German Shepherd dogs with
acute, 90-min myocardial infarcts (ligature of proximal
left anterior descending coronary artery) followed by
reperfus:Lon for ca. 90 minutes prior to contrast agent
infusion~ At 7 MPI, Ferrioxamine:Dermatan gives strong
enhancement of the infarct zone, and in particular
distingu:ishes the outer boundary of the infarct, which
represent:s the putative marginal zone of the infarct
amenable to potential recovery, from the central darker
region, vthich represents the putative irreversible
central infarct. Sustained strong enhancement and zonal
demarcation is present through 40 MPI. Ferrioxamine
injected without carrier at 0.155 mmol/Kg, gives no
detectible enhancement. In these studies, infarct sizes
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
-
- 106 -
and positions are documented by double dye infusion
performed immediately after MRI imaging.
EXAMPLE 21
- 5Comparison of MRI Tumor-imaging Potency In Vivo
with Ferrioxamine Active Bound to
~ Various Sulfated Glycosaminoglycans
Based on low anticoagulant activity, safety and
- 10 projected site-localization potential, certain
alternative glycosaminoglycan carriers and certain
alternative physical forms of the resulting Selective MRI
Contrast Agents are compared for their relative in vivo
potencies of carrier-mediated tumor localization of bound
Ferrioxamine. Because of its high spatial resolution and
capacity to detect subtle ~uantitative differences in
agent localization, the AT-l prostate tumor model of
Example 19 is used.
CA 02208566 1997-06-23
W O96/19242 PCTrUS9~/14776
- 107 -
Table 2
Dose Relative
FIG. [metal]
Agent Form LiquidlLyo mmollkg Potency
No. mmollmL
(scale of 1-6)
19 Gd;MPD DTPA Liquid 0.332 0.155 7
Dermatan-SO3'
43b type
12 Ferrioxamine Lyo 0.415 0.155 4.0
Delnl~Lan-S03'
435 type
13 Gd:DTPA-Lys Liquid 0.415 0.155 6
Der~llalan SO3'
435 type '
14 Ferlioxamine Lyo 0.332 0.155 4.0-4.5
Oversulfated
Den~atan-SO3
Ferrioxamine Lyo 0.332 0.155 5
Oversulfated
Chondroitin-SO3'
16 Ferrioxamine Lyo 0.332 0.155 3.5
Hep~ran Sulfate
~ Ferr oxamine Lyo 0.332 0.155 1.5
D~ dldn
Sulf.3te-'
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
- 108 -
Table 2 (Continued)
Carriers of shorter chain length than the
glycosaminoglycans, namely pentosan polysulfate, are
: 5 found to be less potent (typically only 2/6 on the scale
above) and remain at the tumor site for intervals of less
than about 20 minutes, whereas the GAGs shown in the
table above, are much more potent and have considerably
longer tumor site localization intervals. In comparing
these carriers, there is a slight-to-moderate trend
towards increased carrier potency based on carrier
sulfate charge density.
Lyo = Lyophilized powder form
~ 15 S03- = Sulfate (e.g. dermatan S03- = dermatan sulfate)
beef mucosa, purified, 18,000 daltons
porcine mucosa, 19,600 daltons
CA 02208~66 1997-06-23
WO96/19242 PCT~S94/~4776
- 109 -
EXAMPLE 22
Preparation of a N-Methyl-1,3
Propanediamine Derivative of DTPA
(MPD DTPA) and Chelation
5 with Gadolinium (III)
The diethylenetriamine-pentaacetic acid anhydride
(DTPA anhydride) solution is prepared by adding 180 ml of
anhydro~s dimethylformamide (DMF) into a 250 ml round
bottom flask. The flask is fitted with a side arm
addition funnel and contains a magnetic stir. While the
DMF is stirring vigorously, 5 g (14 mmol) of DTPA
anhydride (Sigma Chemical Co.) is added in 0. 5 g portions
over one hour. The resulting suspension is warmed to
60~C to 15 minutes or until the solution clears. The
flask is removed from the heat and placed in an ice bath
until the solution has equilibrated to 4~C.
The MPD-DTP~ derivative is prepared by mixing 15 ml
of DMF with 1.46 ml (14 mmol) of N-methyl-1,3
propanediamine (Sigma Chemical Co.) in the addition
funnel. The MPD-DMF mixture in the side arm addition
funnel i~s added to the cold (4~C), vigorously stirring
DTPA anhydride solution, dropwise. A white precipitate
forms th:~oughout the addition. The suspension is allowed
to stir overnight at room temperature. The MPD-DTPA
derivative is collected by centrifugation at 2500g for 10
minutes and washed repeatedly with acetone (5 x 300 ml).
The product at this stage, in concentrated solution
has a pH of 3.5, additional purification re~uires a
solution pH of 7Ø The product MPD-DTPA derivative is
dissolvecL in water and the pH is adjusted to 7 with 5 N
NaOH. The product is lyophilized for 16 hours to
r 35 dryness. The lyophilized material is dissolved in a
minimum amount (40 ml) of warm (50~C) methanol for 15
minutes, cooled to room temperature, and precipitated
; CA 02208~66 1997-06-23
W O 96/19242 PCT~US94/14776
- 110 -
with 10 volumes of acetone. The precipitate is collected
by centrifugation at 2500g for 10 minutes. This material
is again dissolved in warm methanol for 15 minutes,
precipitated with 10 volumes of acetone and collected by
5 centrifugation at 2500xg. The precipitate is washed
repeatedly with acetone, dried under nitrogen and stored
in a vacuum desiccator.
Formation of the MPD-DTPA conjugate is confirmed by
10 infrared (IR) Spectroscopy (see FIG. 17A, FIG. 17B, FIG.
17C) and HPLC chromatograph. HPLC characterization is
carried out using a cation exchange column (Dionex IonPac
CS14, 4x250 mm, 8 micrometer, carboxylic acid) with a
mobile phase consisting of 20 mM methanesulfonic acid in
acetonitrile-water (99:1) at pH 1.8 and with W detection
at 220nm. This gives well separated, chromatographically
pure (exceeding 9996 purity) peaks for: (a) DTPA at 3.7
minutes; (b) N-methyl-1,1-propanediamine (20:1 molar
ratio of MPD to DTPA required for detection, due to low
W absorbance of MPD) at 8.4 minutes; (c) the solution
mixture of DTPA (or hydrolyzed DTPA anhydride) with MPD
(1:1 molar ratio) at 3.7 minutes (only DTPA detected and
MPD, due to very low extinction coefficient of MPD); and
(d) MPD-DTPA conjugate (1:1 molar ratio) at 15.6 minutes.
The product purity of (d) is greater than 93~ by HPLC
absorbance at 220 nm.
The chelating capacity of N-Methyl-1,3-
propanediamine-DTPA (MPD-DTPA) is determined by titrating
a small aliquot with 0.1 M GdCl3 5H2O in 1 M ammonium
acetate (pH 5.5) buffer, using Xylenol Orange (5~, w/v)
as the colorimetric indicator of endpoint. Based on this
titration, a stoichiometric quantity of 1 M GdCl3 5H2O is
added to a batch quantity of N-MPD-DTPA as follows: the
bulk MPD-DTPA is dissolved in a minimum amount of water
(ca. 300 mg/ml), lM GdCl3 5H2O is to the added while
vigorously stirring, and the pH is adjusted from <4.0 to
CA 02208~66 l997-06-23
W O 96/19242 PCT~US94/14776
,. 7.0 with 5 N NaOH. The average chelating capacity is
about 2,~ (by weight), with slight variation based on the
extremely hygroscopic nature of the dry chelator.
EXAMP~E 23
Preparation of Paired-Ion Formulation
of Gadolinium:MPD-DTPA:Dermatan Sulfate
The paired-ion formulation of gadolinium(Gd):MPD-
DTPA:der~atan sulfate (using the new, special 435 Type
dermatan sulfate, Opocrin) is prepared over a range of
weight ratios ~rom 10:1 to 1:10 of Gd:MPD-DTPA to
dermatan sulfate, and is particularly prepared at one of
the preferred ratios of 60~ Gd:MPD-DTPA to 40~ dermatan
sulfate (w/w)(= a mole ratio of 43:1). These paired-ion
formulat:ons are prepared by dissolving the desired
amount o.- dermatan sulfate at a concentration of 400
mg/ml and stirring in the Gd:MPD-DTPA as prepared in
Example ~2. This results in a hydrophilic, completely
clear solution without any detectable molecular
aggregates by laser light scattering analysis (Nicomp
Instrument). Strong paired-ion binding between GdMPD-
DTPA and dermatan sulfate is confirmed and evaluated by
dialysis through a 500 MW cutoff bag for 3 hours, 150
volumes, and is assessed by ICP atomic absorption
analysis of the retained Gd (mass balance = 95~). Very
strong paired-ion binding is indicated by 73~ retention
of Gd within the bag for the Gd:MPD-DTPA:dermatan sulfate
formulation prepared at 60:40~ (Gd:MPD-DTPA to dermatan
sulfate); compared to the much lower 23~ retention within
the bag for Gd:DTPA:dermatan sulfate when prepared at the
same molar ratio of Gd:DTPA to dermatan sulfate.
.
Quantification of dermatan sulfate is performed by
- 35 assessing the decrease in W absorbance at 620 nm which
occurs upon binding of the extremely strong binding
(displacing) cationic dye, Azure A, as previously
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94/14776
-
- 112 -
described [Klein et al. (1982: Grant et al. (1984), both
incorporated by reference herein].
The R1 potencies (T1 relaxivities) of (a) Gd:MPD- ~
DTPA alone and (b) the 60:40~ (w/w) paired-ion
~ formulation of Gd:MPD-DTPA:dermatan sulfate, are
evaluated using an IBM PC20 Minispectrometer, and both
are determined to be 7.8 mmol~1s~1 (based on parallel
determinations of Gd concentration by ICP atomic
absorption). The equality of Rl's for the Gd chelate
alone and Gd chelate bound to dermatan sulfate, indicate
that binding of the chelate to dermatan sulfate does not
interfere with water diffusion and paramagnetic
relaxation. Furthermore, the absence of R1 prolongation
lS indicates an absence of increase in rotational
correlation time, and hence, further corroborates that
the size of the Gd:MPD-DTPA-dermatan sulfate molecular
complex is relatively small (likely less than about
50,000-60,000 daltons). This further confirms a basis
for the surprising and unexpected advantages of high
tumor accessibility across even the relatively more
(anatomically and filtration) intact portions of tumor
neovascular endothelium, and also the very rapid renal
clearance, both of which are observed in intact animals
(see below). This result correlates with the absence of
detectible molecular aggregates by laser light scattering
(above). The remarkably high Rl of this new formulation
is repeated multiple times and appears to correlate with
enhanced water diffusion of the new Gd:MPD-DTPA conjugate
(and also for the full dermatan sulfate product) in
relation~to Gd:DTPA with the MPD side group (R1 = ca. 4
[mmol. sec]~1. The stability Kd of Gd:MPD-DTPA is greater
than 10~7.
CA 02208~66 1997-06-23
W ~961~9242 PCTAUS94/14776
- 113 -
E~MPLE 24
Ac~te Murine Toxicity of Paired-Ion Formulation
of Gadolinium:MPD-DTPA:Dermatan Sulfate
One of the formulations of EXAMPLE 22, Gd:MPD-
DTPA:dermatan sulfate (at a 60 :40 wt ~ of Gd:MPD-DTPA to
dermatan sulfate; 435 Type dermatan sulfate, Opocrin) was
tested for acute toxicity by intravenous tail-vein
injecticn into 20-gram, male Balb/c mice (n = 6). When
injecticns were performed over 10-12 minutes, the average
LD50 = 11.0 mmol/kg (of Gd and chelator), with 3 mice
surviving at an average of 9.9 mmol/kg and 3 mice dying
at an average of 12.2 mmol/kg. When injections were
performed more rapidly, over a 2-3 minute interval, the
LD50's were moderately lower in dose. These results
compare favorably to those of Gd:DTPA (dimeglumine), for
which LD~0 = 4.0 mmol/kg.
EXAMPLE 25
Acute Blood Clearance of Radiolabeled
Paired-Ion Formulations of:
67Ga-labeled Deferoxamine:Dermatan Sulfate; and
lllIn-labeled MPD-DTPA:Dermatan Sulfate
In order to assess if dermatan sulfate carriers
could confer their own very rapid and complete blood
clearance properties to attached active substances
(including non-covalently bound chelates), the
formulations of Examples 2, 5, 21 and 22 (above) are
modified such as to bind the radioactive single-photon-
emitting (SPECT) metals, 67Ga or lllIn, in place of the
non-radicactive metal ions, Fe(III) or Gd(III).
For the 67Ga experiments, approximately 1.55 umole
of deferoxamine (DFo)-dermatan sulfate (77.5:22.~ wt ~;
DS Type 435, Opocrin) is labeled with approximately 800
uCi of 67Ja, by converting the 67Ga from a chloride to a
CA 02208~66 1997-06-23
W O 96/19242 PCT~US94/14776
- 114 -
citrate form and incubating it for 10 min at room
temperature with DFo:dermatan sulfate at pH 5.5-6.5,
injecting Copenhagen-strain rats intravenously in the
tail vein with 0.39 umoles of DFo:dermatan sulfate to
which is chelated ca. 200 uCi of 67Ga, obtaining serial
gamma camera images over a l-hour interval (and again at
24 and 48 hours), and analyzing the heart, upper
abdominal region and pelvic regions of interest (ROI's)
for blood, liver and renal clearances, respectively. The
blood clearance tl/2 average = 18 minutes, with a very
rapid tl/2 alpha component of 8 minutes plus a tl/2 beta
component of 35 minutes. No liver clearance is observed
at all. Renal clearance is very rapid, accounting for
all of the discernable clearance and leading to rapid
bladder activity. There is no significant residual
activity in the snout, skeletal axis or regions of bone
or bone marrow. In a control experiment, injection of
67GaDFo alone (without dermatan sulfate) also results in
very rapid blood clearance, however, a significant
fraction of the agent (ca. 30~) cleared quite rapidly
(10-30 minutes) into the liver and bowel, producing high
organ backgrounds in the liver and colon.
In a separate experiment wherein the Copenhagen rats
had AT-l prostate adenocarcinomas (1.0-4.5 cm in
diameter) implanted in the back of the neck, the tumors
become very rapidly (ca. 5 minutes) active (bright) with
radionuclide agent, and the tumor counts per pixel exceed
those of the blood and liver at all times after 15
minutes of injection, resulting in rapid, sensitive
detection of the tumors. This corroborates the MRI
imaging results in the same tumor model (Example 19).
~
In another experiment, the dose of DFo:dermatan
sulfate is increased 100X from 1.55 umole/kg to 155
umol/kg (0.155 mmol/kg) while maintaining the dose of
radionuclide constant at 200 uCi per rat, in order to
CA 02208~66 1997-06-23
W O 96/19242 PCTAUS9~/14776
- 115 -
assess t-he effects of MRI doses, dose augmentation and
potentially therapeutic doses, on clearance half times.
By visual assessment, clearance is very nearly identical
to the 100-fold lower dose of agent (above), with only a
very minimal, ca. 5-mi.nute prolongation.
In a further separate experiment, lllIn is converted
to the a~etate form at pH 5.5-6.5, used to radiolabel
MPD-DTPA:dermatan sulfate (60:40 wt ~ MPD-DTPA:dermatan
sulfate, 435 Type, OpGcrin). Clearance times and organ
clearance patterns (renal versus liver) are comparable to
those of 67GaDFo:dermatan sulfate (above); and when
tested, l_umor uptake is also rapid and distinct.
The~e surprising and unexpected advantages of: (a)
very rap:~d clearance over a 100-fold (or greater) dose
eschelat:on, for two different actives non-covalently
bound (by paired-ion binding) to dermatan sulfate; and
(b) avoidance of liver and bowel clearance in the
presence but not the absence of dermatan sulfate carrier,
provide major advantages for low MRI and radionuclide
imaging backgrounds in the blood and especially
additionally, in the critical and difficult body regions
of liver and mid-abdomen. Upon bladder catheterization,
the pelvic region is also observed without substantial
background interferences. Additionally, significant
therapeutic regimens are enabled because of the only very
gradual increase in blood and body clearance times with
major dose increments of at least 2 orders of magnitude.
These clearance properties, coupled with the selective
(tumor) uptake properties shown in this Example and
above, provide even further surprising and unexpected
- advantages for augmenting the differential between
selectivily versus body residual and systematic toxicity.
=
CA 02208~66 l997-06-23
W 096/19242 PCT~US94/14776
- 116 -
EXAMPLE 2 6
- Gadolinium:N-methyl-1,3,propanediamine-
DTPA:Dermatan Sulfate
(Gd:MPD-DTPA:DS) Selective Contrast Agent: MRI Imaging of
Lactating Breast Adenocarcinomas in Syngeneic Fisher
344 Female Rats
As shown in FIG. 25A, FIG. 25B, FIG. 25C, FIG. 25D,
FIG. 25E AND FIG. 25F, T1-weighted MRI images (TR-TE =
800/45) are performed at 1.0 Tesla, before (Pre) and
after (Post) intravenous (i.v.) injection of Gd:MPD-
DTPA:DS (DS = 435 Type, Opocrin) at a dose of 0.155
mmol/kg into Fisher 344 female rats with syngeneic breast
adenocarcinomas inoculated by trocar into the livers, as
in Example 18 (above). A T2 scout image (TR/TE =
2100/85) is performed in advance of the T1 image contrast
series, in order to identify the approximate location(s)
of tumor nodule(s) (FIG. 18A). This reveals 2 solid
tumor nodules (right posterior liver) and one irregular
tumor infiltrate (central liver region), all tumor sites
subsequently being confirmed by gross visual inspection.
These nodules are unidentifiable in the T1 (800/45)
Precontrast (Pre) image (FIG. 18B), however following
injection of Gd:MPD-DTPA:DS, all three tumor nodules: (a)
- 25 become rapidly and exceedingly strongly enhanced at an
early post-injection time of 7 minutes (FIG. 18C); (b)
display rapid and prolonged (through 60 minutes) sharp
tumor boundaries against the surrounding uninvolved liver
(FIG. 18C, FIG. 18D, FIG. 18E), and exhibit prolonged
(sustained) contrast through 60 minutes (FIG. 18F), with
only a very slight degradation of the contrast gradient
at the tumor boundaries at 60 minutes postinjection
(MPI). In this animal model, the MRI contrast
enhancement produced by Gd:MPD-DTPA:DS, is markedly
greater (more potent on a dose basis) than that produced
by the ferrioxamine:dermatan sulfate agent of Example 18;
and is slightly to moderately greater (more potent on a
-
CA 02208~66 l997-06-23
W O 96/19242 PCT~US94/14776
- 117 -
dose basis) than that produced by Gd:DTPA-lysine:dermatan
sulfate (prepared per Examples 3, 4 and 9; see also FIG.
13A, FIG. 13B, FIG. 13C, FIG. 13D, Example 21 and Table 2
for relative potency); both of the preceding agents
containing less potent metal chelates, namely, with Rl~s
of 1.6 and 4.2, respectively, compared to an R1 of 7.8
[mmol.sec]-l for Gd:MPD-DTPA:DS of the present Bxample.
Also, the images of the present Example show all the
~ollowing, surprising and unexpected advantages over
Gd:DTPA (dimeglumine), as well as over all the reported
liver-specific T1 and T2 contrast agents: (a) uptake by
tumor proper without substantial uptake by the
surrounding uninvolved liver; (b) enhanced tumor
selectivity and sensitivity; (c) prolonged as well as
immediat:e tumor uptake, for improved clinical flexibility
of multl-site and multi-image acquisition without
contrast. fading or need for multiple contrast-agent
injections; (d) improved contrast sharpness and
brightness gradient at the tumor boundaries, for improved
tumor staging and improved detection of small tumors; (e)
improvec. detection of small metastases; and (f) improved
detecticn of small invasive outgrowths, for enhanced
prognostic and therapeutic monitoring information. Note
~ that there is a minor blood-pool enhancement in the
surrounding normal liver at all post-contrast times,
strongly suggesting that an even lower dose than 0.155
mmol/kg ~ould be highly effective, indicated and
appropriate for optimal T1 imaging of Gd:MPD-DTPA:DS.
This is :because the Gd:MPD-DTPA chelate is substantially
more pot,-nt [R1 = 7.8 (mmol.sec)-1] than all of the others
described herein, and hence, gives more of T2* darkening,
as well as T1 brightening effects, per micromole of agent
- deposited in the tumor (see Example 26 for co~roboration
of this ~ffect).
= --
CA 02208~66 l997-06-23
W O 96/19242 PCT~US9~/14776
. - 118 -
- EXAMPLE 27
Gadolinium:N-methyll-1,3,propanediamine-
DTPA:Dermatan Sulfate
(Gd:MPD-DTPA:DS) Selective Contrast Agent: MRI Imaging of
Prostate AT-1 Adenocarcinomas in Syngeneic
Copenhagen Rats;
Plus Correlation with Special Histochemical Stain
As shown in FIG. 20A, FIG. 20B, FIG. 20C, FIG. 20D,
AND FIG. 20E, T1-weighted images (TR/Te = 250/80) are
performed at 4.7 Tesla, before (Pre) and after (Post)
intravenous (i.v.) injection of 0.155 mmol/Kg [Gd(III)
dose] of the Gd:MPD-DTPA:DS (DS = 435 Type, Opocrin)
selective contrast agent, as prepared in Examples 21 and
22, into AT-1 prostate adenocarcinomas grown in skin
pouches of syngeneic Copenhagen rats (described and
referenced in Example 18). Gd:MPD-DTPA:DS produces a
rapid, extremely strong T1 contrast enhancement of the
entire tumor at 7 minutes (FIG. 20B) and 20 minutes ~FIG.
20C) post-injection (MPI), and a continued strong
contrast enhancement of the tumor at 40 MPI (FIG. 20D)
and 60 MPI (FIG. 20E), especially at the tumor rim and
most especially at the basal tumor rim (where tumor host
staging is typically assessed). Upon further
experimental evaluation, the apparent moderate contrast
darkening of central tumor regions which appears at 40
and 60 MPI, actually represents an overconcentration of
the agent within these tumor regions, leading to T2*
effects, which compete with the strong T1 brightening
effects and artifactually darken the T1 contrast in these
central tumor regions. This T2* artlfact is detected and
assessed by utilizing a T2 pulse sequence of TR/TE -
2500/250, (= selectively sensitive to T2* effects) and
observing substantial contrast darkening at the more
delayed post-contrast times. Hence, the very high R1 of
Gd:MPD-DTPA:DS (relative to all of the preceding agents),
in combination with an injected dose of 0.155 mmol/Kg,
CA 02208~66 l997-06-23
W O 96/19242 PCT~US94~14776
- 119 -
togethe:~ with the very marked tumor uptake of agent and
the paramagnetic response characteristics of the TR/TE =
250/80 pulse sequence at a 4.7 Tesla field, leads to an
overly high local paramagnetic activity within the tumor
5 as Gd:MPD-DTPA:DS acc~mulates over time, especially in
the central regions of the tumor. The rim, and
especia].ly the basal rim, is relatively protected from
this T2t darkening artifact, due to more rapid
backdiffusion of agent into plasma at this basal site.
10 The preceding results and considerations lead to the
conclusion that a lower dose than 0.155 mmol/Kg is
indicated for optimal T1 imaging with Gd:MPD-DTPA:DS,
because the Gd:MPD-DTPA chelate is a substantially more
potent I1 paramagnetic active than all of the others
15 described herein. Note that in Example 25, there appears
to be a slight overdose, as evidenced by the very
slightly enhanced blood-pool background in the uninvolved
liver surrounding the 3 liver tumor nodules.
Nevertheless, these nodules are still exceptionally well
20 visualized at all post-contrast ~imes (7-60 MPI).
Correlation of these MRI images with a m_crowave
augmente,~ Prussian blue stain for Gd(III) me.al ion is
performed (as described in Example 18~, for t;~e Gd(III'
25 of Gd:MPi~-DTPA:DS which becomes localized in the oute~
2/3 of the tumor mass excised at 60 MPI (and freshly
frozen for sectioning and staining). (See FIG. 20).
This shows strongly positive histochemical staining of
almost all tumor cells, with a significant number of the
30 tumor cells having positive staining of the nucleus as
well (i.e., nuclear localization of the metal-ion
marker). This very strong staining of nearly all tumor
r cells at 60 minutes, compared to the lighter staining of
fewer nun~ers of (breast) tumor cells at 7 minutes
-- 35 (Example 18), and the additional nuclear localization
seen here at 60 minutes but not in the (breast) tumor at
7 minuteC (Example 18), strongly suggests that tumor-cell
CA 02208~66 1997-06-23
W O96/19242 PCTrUS94114776
- 120 -
internalization proceeds over a 1-hour interval, and
likely over the entire interval of time during which the
dermatan-sulfate bound metal chelates remain at
significant concentrations within the extracellular
matrix is initially and rapidly loaded via local
microvessels, by extremely rapid and selective
extravasation across tumor-induced neovascular MRI
endothelium -- see text above for tumor-selective
induction and endothelial localization of GAG-binding
receptors, including VEGF/VPF and others. The surprising
and unexpected advantage of endothelial localization
observed here for malignant prostate tumor, was also
observed in Example 18 for malignant breast tumor. This
corroborates the surprising and unexpected finding of
Example 18 above, that tumor-induced neovascular
endothelium, as well as tumor cells proper, are targets
for binding, pumping, extravasation and tumor-cell
internalization of the dermatan sulfate-bound (including
non-covalently bound) classes of MRI contrast agents, and
indeed for other active agents similarly bound to
dermatan sulfates and GAGs. These findings of tumor
- endothelium, tumor matrix, tumor cell and nuclear
localizations and accumulations, further provide the
basis for selectively localizing therapeutic agents,
whether metal chelates or other types of active
substances.
EXAMPLE 28
Preparation of Doxorubicin Formulation as a Paired-Ion
30Complex with Essentially Purified Dermatan Sulfate
Essentially purified dermatan sulfate (435 Type,
- Opocrin, modal MW = 18,000 daltons) is dissolved in water
at 10 mg/ml, and a 4 mg/ml solution of high-purity
35 doxorubicin (Meiji Seika Kaisha, Ltd., Japan) is added .
dropwise while vigorously stirring, to give a 60:40 (w/w)
ratio of doxorubicin to dermatan sulfate. (Other ratios
CA 02208~66 1997-06-23
WO96/19242 PCT~S94/14776
- 121 -
are also tested between 10:90 and 90:10 (w/w) doxorubicin
to dermc~tan sulfate.) The mixture is homogenized by
sonication for 8 minutes at 4~C, using a macroprobe
sonicator (Heat Systems). This effectively reduces the
doxorubicin:dermatan sulfate complex to its limit (small)
size of 11 nanometers, as assessed by laser light
scattering (Nicomp sy8tem). The res~lting liquid is
filtered through a 0.22 um low binding filter (Millipore,
Millex GV), 3 mL of a 500 mg/mL solution of saccharose
(Boehrin~er Mannheim) is added and stirred, then 1.5 mL
o~ a 10 ~g/mL solution of polyethylene glycol (Hoechst,
mean MW = 3,350 dalto~s) is added, the resulting solution
is sonicated once again (as above) and again filtered
through 0.22 um GV filters for asepsis, filled into
vials, a~d either saved as a liquid or frozen and
lyophili~ed over a 17-hour primary drying cycle at
appropriate shelf and chamber temperatures and
conditions, to give a well-formed, brick-red cake with
ca. 2.0~ residual water (Karl Fisher method). The vials
are stoppered and sealed. For use, the cakes are
resuspended with sterile water (hand shaking for 15
seconds) at 2 mg/ml. The resulting Liquid is completely
translucent and red-orange, with a pH of 6.8-7.1, an
osmolalit:y of ca. 210 mOsm/Kg, and a Zeta potential of -
38 to -40 mV, indicating the presence of strongly bound
dermatan sulfate in slight molar excess (the doxorubicin
itself having a positive Zeta potential, due to its sugar
amine grcup, which is effective to bind the sulfates of
dermatan sulfate by strong paired-ion binding). The
lyophilized cakes are stable (in both doxorubicin and
dermatan components) for long intervals at room
temperature as well as 4~C (doxorubicin analysis by HPLC:
C-18 Lichrosphere; mobile phase: acetonitrile:water
(50:50), 20mM phosphoric acid + 5mM sodium
- 35 dodecylsulfate, pH 2.3; dermatan sulfate analysis by
HPLC: TSK molecular sieve; mobile phase: 0.2M sodium
sulfate f~r charge suppression, with Opocrin dermatan
CA 02208~66 1997-06-23
W O 96/19242 PCT~US94/14776
- 122 -
molecular weight standards of 1,800-17,250 daltons). The
reconstituted cakes meet USP specifications for oversized
particles above 10 and 25 um (by Hyac-Royco laser
analysis).
Adriamycin (Adria Laboratories source of
doxorubicin) is also similarly prepared in paired-ion
couples with dermatan sulfate (Opocrin, as above).
EXAMPLE 29
Preparation of Doxorubicin Formulation as a Paired-Ion
Complex with Beef Lung Heparin
Beef lung heparin (Hepar-Kabi-Pharmacia) is
solubilized, mixed with high-purity doxorubicin, over the
ranges of 90:10 to 10:90 ratios (w/w,
doxorubicin:heparin) and at one of the optimal ratios,
namely 60:40 (w/w), and then subjected to the additional
steps as described in EXAMPLE 28. A visually clear, 0.22
um filterable liquid results. However, this liquid has a
larger limit size of 25 nanometers by laser light
scattering, and the lyophilized cake is considerably more
resistant to rapidly homogeneous reconstitution
(requiring ca. 1-2 hours).
EXAMPLE 30
Preparation of Taxol Nanoparticle Formulations
Coated with Essentially Purified Dermatan Sulfate
and with Beef Lung Heparin
These formulations are prepared in two steps, first
by solubilizing and preparing the taxol in lecithin or
lecithin-cholesterol nanodispersions, and second, by
interacting the lecithin-coated nanodispersions with
dermatan sulfate (435 Type, Opocrin) or beef lung heparin
(Hepar-Kabi-Pharmacia) to produce and stabilize the final
nanodispersions by paired-ion interaction at the
CA 02208~66 1997-06-23
WO 9~/19242 PCT/US9~1/14776
- 123 -
nanoparticle surface, with binding of the
glycosaminoglycan sulfate groups to the highly basic
nitrogen groups of lecithin. Taxol (Sigma Chemical Co.,
St. Lou:Ls) is dissolved in methylene chloride, egg yolk
lecithill in chloroform, and alternatively, soy lecithin +
cholesterol in methylene chloride. The solubilized
components are placed in a round bottom flask and the
solvents evaporated under vacuum for 30 minutes,
resulting in formation of a thin-film of taxol-lecithin
(in one case, also with cholesterol). After sufficient
drying, water is added (under nitrogen), the ingredients
hydratec~ for 2 hours and probe sonicated ~or lo minutes
at 4~C lHeat Systems). Dermatan sulfate, or
alternat;ively beef lung heparin, is added over a range of
2-6~ (w/'w, to lecithin) and the mixture probe sonicated
for 1 minute at 4~C. The resulting nanoparticles are
observec by optical microscopy, and surface
glycosaminoglycan (GAG) is confirmed by addition of the
cationic dye, Azure A, which turns rapidly purple and
produce~ particle aggregation upon binding to the surface
GAG (uncoated formulations with lecithin only, =
negative). The size and quantity of drug in dermatan
sulfate-coated and heparin-coated nanoparticles are
assessed by microfiltration and W absorption analysis
for solubilized taxol (230 nm), and size is further
confirmed by laser light scattering (Nicomp).
Formulations containing 4~ glycosaminoglycans are
optimal. Representative results for the quantities of
taxol present before filtration ("None") and remaining
after filtration through various pore sizes (5.0, 0.45 um
and 0.22 um) are shown in Table 3 for dermatan sulfate
formulations.
-
CA 02208566 1997-06-23
W O96/19242 PCTrUS94114776
- 124 -
Table 3
Relative (~) Drug R~;n;ng after Filtration
Filter Taxol sulfate
with Lecithin Taxol + ~ecithln
without DS gg olk Lecithin Cholesterol
None 100~ 100~ 100
5.0 um 90~ 98~ 97
0.45 um 14~ 55~ 48
0.22 um -- 23~ 15
The results of these analyses indicate that both
lecithins interact effectively with dermatan sulfate and
beef lung heparin, to form a glycosaminoglycan surface
coating which stabilizes the nanoparticulate dispersion
of taxol. However, for the optimal type of lecithin (egg
yolk), the resulting nanoparticle size (by laser light
scattering (see Table 4)) is smaller for dermatan sulfate
(column a) than for heparin (column c), and only dermatan
sulfate allows taxol to be formulated, ~ e~-_i and
obtained at acceptable recoveries t~-oua'l a.. ase?~ic
cutoff filter (0.45 um), giving an asept__, ~ ravenous ~.
acceptable nanodispersion of taxol without the need for
the standard taxol solubilizer, cremofor, and without i~s
incumbent toxic and acute allergic side effects.
=
CA 02208~66 1997-06-23
WO 96/19242 PCT/US9~1477
- 125 -
Table 4
Comparison of Nanoparticle Size
by Laser Light Scattering (No filtration)
]~ermatan Sulfate Coating Heparin Coating
(a) (b) (c)
+ E,~g Yolk + Soy Lecithin + Egg Yolk
~e,-ithin and Cholesterol Lecithin
10.8 nm (98.7~)31.4 nm (40.1~)45.5 nm (63.6~)
10281.8 nm (1.3%)234.1 nm (18.4~)235.4 nm (36.4~)
908.4 nm (41.5~)
This s~aller nanoparticle size with dermatan sulfate
versus beef lung heparin, is similar to that observed for
doxorubi_in (Example 28). This collaborates the
additional surprising and unexpected formulation
advantages (above) of dermatan sulfate over heparin.
EXAMPLE 31
Pre~aration of Vincristine Paired-ion Formulation
with Essentially Purified Dermatan Sulfate (435 Type)
Vincristine (Sigma Chemical Co., St. Louis) is
dissolved in water and mixed with dermatan sulfate at
ratios o:- between 90:10 and 30:70 (w/w) drug to dermatan
sulfate. This results in clear solutionsj with an
optimal ratio occurring at 30-40~ (w/w) of drug, with
particles being undetectable (by laser light scattering).
This result, in combination with retention of the paired-
ion form, but not the drug alone, inside a 500 MW cutoff
dialysis bag, is indicative of a strong paired-ion
- formation between the amine group of vincristine and the
sulfate groups of dermatan sulfate.
-
CA 02208~66 1997-06-23
W O 96/19242 PCTrUS94114776
: - 126 -
EXAMPLE 32
Preparation of the Amine-containing
Antibiotic Anti-infectives, Amikacin,
Gentamicin and Tobramycin, as Paired-ion Formulations
with Essentially Purified Dermatan Sulfate (435 Type)
- and with Beef Lung Heparin
.
Amikacin, gentamicin and tobramycin (all obtained
from Sigma Chemical Co., St. Louis) are dissolved in
water and mixed with either dermatan sulfate (435 Type)
or beef lung heparin (Hepar-Kabi-Pharmacia) at ratios of
between 90:10 and 30:70 (w/w) drug to glycosaminoglycan.
For the optimal range of 30-50~ (w/w), strong paired-ion
complexes form between drug and both glycosaminoglycans,
as evidenced by laser light scattering (Nicomp), in the
100-200 nanometer range for beef lung heparin; or by 500
MW cutoff dialysis retention of the smaller dermatan
sulfate formulations (undetectable to ca. 10 nanometers
by laser light scattering).
EXAMPLE 33
Paired-ion Formulations of Basic Peptides
with Essentially Purified Dermatan Sulfate (435 Type)
The basic, white-cell chemoattractant, and
inflammatory peptides, (a) N-formyl-met-leu-phe-lys
(acetate), (b) arginine bradykinin (arg-pro-pro-gly-phe-
ser-pro-phe-arg) and (c) poly-L-lysine (all 3 from Sigma
Chemical Co., St. Louis), are dissolved in water and
mixed with essentially purified dermatan sulfate (435
Type, Opocrin) at ratios of 90:10 to 10:90 (w/w of active
substance to dermatan sulfate). Strong paired-ion
binding occurs at optimal ratios of ca. 60:40 (active
substance to dermatan sulfate), as evidenced by laser
light scattering, retention in a 500 MW cutoff dialysis
bag, and in the case of (a) N-formyl-met-leu-phe-lys,
CA 02208~66 1997-06-23
W 096/19242 PCT/US94/14776
- 127 -
fluorescence enhancement at an emission wavelength of 518
nm (Wit]l excitation = 257 nm).
These basic peptides, in iormulation with
essentially purified dermatan sulfate, provide a novel
means fc~r site-selective localization, accumulation,
retentic)n and action of biomodulatory peptides at sites
of tumors and/or infections, in order to recruit and
activate endogenous or transfused white blood cells for
the purposes of local therapy, under conditions where the
systemically circulating free form of agent could not be
tolerated, due to mar~ed system-wide inflammatory side
effects. Hence, these new dermatan-sulfate formulations
provide surprising and unexpected advantages for in vivo
tumor and anti-infective therapies.
EXAMPLE 34
In Vitro Tests of Paired-Ion Doxorubicin:Dermatan Sulfate
20Formulation and Standard Doxorubicin
for Comparative Activity against Wild-type and
Doxo:-ubicin-resistant Human Breast Carcinoma Cells
The paired-ion doxorubicin:dermatan sulfate
(essentially purified dermatan sulfate, 435 Type,
Opocrin) of Example 28 (= doxorubicin:DS), is compared to
standard doxorubicin liquid (Adria Laboratories), for
tumor-ce:Ll killing potency in a clonigenic assay, using
the following human breast cancer cell lines: parent MCF-
7 cell line and adriamycin/doxorubicin-resistant MCF-7
cell line. For each group, five serial dilutions of each
formulat.on were mixed on day 0, with an appropriate
number oi cells, growth medium and 14C-labelled glucose,
the mixture then injected into serum vials pre-gassed
: 35 with 5~ C~2~ and the vials incubated at 37~C (with cells
continuously exposed to drug). The ~uantity of 14C-
glucose C~~2 produced in control and drug-treated vials was
CA 02208~66 1997-06-23
W O96/19242 PCTrUS94/14776
- 128 -
determined using a Bactec machine (Bactec Corp.) on Days
6, 9 and 12 of incubation, and the data recorded and
analyzed as percent survival values, IC50 and IC90
values, with percent survival values greater than 100
normalized to 100~ against control cells (incubated
without drug). Results are shown in Table 5 for the day
of peak counts (day 9):
Table 5
Cell Line & Test Substance IC50 (uM) IC90 (uM)
A. Parent MCF-7 Line
1. Doxorubicin:DS0.01-0.02 0.05-0.06
2. Standard doxorubicin 0.02 0.06
(adriamycin liquid)
B. Doxorubicin-resistant MCF-7 Line
1. Doxorubicin:DS 0.81-0.893.15-13.33
2. Standard doxorubicin 22.28not achieved
- (adriamycin liquid)
20 The finding of comparably low IC50 and IC90
concentrations for the Parent MCF-7 line, but very
different IC50 and IC90 concentrations for the
Doxorubicin-resistant MCF-7 line, with the doxorubicin-
dermatan sulfate formulation able to overcome (or bypass)
resistance but standard doxorubicin unable to do so,
strongly suggests that the dermatan sulfate combination
overcomes the multi-drug resistant phenotype and may do
so by bypassing the Pgp (P glycoprotein) pump. This
might be predicted by the net negative charge of the
dermatan sulfate formulation. Notwithstanding the
specific mechanism, this result provides additionally
important, surprising and unexpected advantages of the
dermatan sulfate anti-tumor formulations, and in
particular, doxorubicin dermatan sulfate doxorubicin, and
35 most particularly, the essentially purified doxorubicin ~-
dermatan sulfate formulation (Example 28) in the
treatment of tumors and neoplastic disease. Note, these
CA 02208~66 1997-06-23
WO 96119242 PCT/lJS94~14776
- 129 -
test as~;ays were performed by Donna Degen, M.S., and
Daniel rj. Von Hoff, M.D., of the Cancer Therapy and
Research Center, Institute for Drug Development, San
~ Antonio, Texas.
EXAMPLE 35
Z~cute In Vivo Toxicity Tests of Paired-Ion
Doxorubicin:Dermatan Sulfate
Male Balb/c mice (n = 4/group) are injected
intravenously with 22.5 mg/Kg of either the paired-ion
doxorubicin:dermatan sulfate (essentially purified
dermatan sulfate, 435 Type, Opocrin) of Example 28 (=
doxorubicin:DS), or standard doxorubicin liquid (Adria
Laboratories) and then housed in cages with filter tops
and observed for the day of death. Note that 22.5 mg/Kg
is the r;-ported LD90 in mice for standard doxorubicin,
and it was chosen in order to minimize the time needed to
observe the test endpoint; lower doses might be expected
to widen any differences which may be observed in the
present protocol. The days of death are: doxorubicin:DS:
modal day = 6, mean = 5.5 + 0.9 SE; standa-~ dsxorubiciz:
modal day = day 5, mean = 4.8+ o.s cE .~e~._e,
doxorubicin has an acute murine tox_c- ~- h .~ S at
least comparable to and trending towards superior ove~
standard doxorubicin, although the differences in this
present test are not statistically significant. This
result, ~n combination with the advantages of overcoming
adriamycin/doxorubicin resistance in human tumor cells
(Example 34) and localizing more effectively in vivo, on
a constarst-dose basis, in animal tumors and tumor
intracel~ular sites (see Example 36, below), provide
-~ further ~urprising and unexpected advantages of the
dermatan sulfate anti-tumor formulations, and in
particular, doxorubicin dermatan sulfate doxorubicin and
essentially purified doxorubicin dermatan sulfate
CA 02208~66 l997-06-23
W O 96/19242 PCTrUS94/14776
- 130 -
(Example 28) in the treatment of tumors and neoplastic
disease. 7
EXAMPLE 36
Acute In vivo Tumor Localization and Tumor-cell
Internalization
of Paired-Ion Doxorubicin:Dermatan Sulfate
Compared to Standard Doxorubicin
The paired-ion doxorubicin:dermatan sulfate
(essentially purified dermatan sulfate, 435 Type,
Opocrin) of Example 28 (= doxorubicin:DS) and standard
doxorubicin liquid (Adria Laboratories) are injected at 5
mg/kg of doxorubicin i.v., into Copenhagen rats with AT-l
prostate carcinomas grown in a skin pouch (to mimic
growth at deep organ sites). The rats are sacrificed at
3 hours after injection, the tumors and major organs
removed, the cut tumors & organ pieces placed in OCT
polymer and frozen at 4~C, and cryostat sections cut at 8
um and coverslipped. Fluorescence microscopy is
performed by exciting the sections using a rhodamine-type
bandpass filter (at ca. 485nm -- in order to selectively
excite doxorubicin) and assessing direct doxorubicin
fluorescence (at an emission wavelength greater than
530nm) to determine the following:
~ relative tumor drug levels;
~ depth and homogeneity of drug penetration into
tumor mass, at sites both proximal to and more
distant from tumor microvessels;
~ tumor targets, i.e., endothelium cells as well as
- tumor cells proper;
~ normal organ fluorescence, as predictor
of clearance and potential toxicities (see Table 6).
CA 02208~66 1997-06-23
WO 96/19242 PCTfUS9 ~ 776
- 131 -
Table 6
Summary of results in Tumor (on intensity scale of O to 4+):
Doxorubicin:
essentially
Property Adriamycin PFS Puri~ied Delll,dl~
Sulfate
Overall flu~sc~l~ce 0-1+ 3-4+
Macropl~arl"acology:
~ Nea~ capill~lies l+ (113 of regions) 4+ (nearly all regions~
O (213 of regions)
Awey from capillaries O (most regions) 2-3+ (ca. 81l0 of
regions~
~ Fluo ~scence at invading edge 2+ 4+
Cellular Ph3r",acology:
~ Tum~r-cell fluor~cence l+ 3+
~ N~clear 1 + 2-3 +
~ End~thelial fluor~s~ence 0 3+
~ N Iclear 0 2-3 +
See FIG. 21A for doxorubicin:DS -- dense sheet of
tumor ce:ls, with very bright fluorescence in almost all
tumor ce]ls (= tumor-cell internalization) and in
neovascu].ar endothelia. See FIG. 21B for doxorubicin:DS
-- looser clusters of tumor cells on an endothelial
stalk. Looser tumor-cell clusters are most likely to be
in growth phase or division (compared to the more dense
tumor-cell sheets of FIG. 21A). Note the very bright
staining of almost all cells, plus the strikingly bright
nuclear fluorescence of doxorubicin now localized at this
site, as well as in the cytoplasm. Also note the strong
fluorescence of endothelial cells and endothelial-cell
~ nuclei. See FIG. 21C for standard doxorubicin (dense
sheets of cells at upper right and looser clusters at
lower left) -- all with markedly lower fluorescence and
CA 02208~66 1997-06-23
W O96/19242 PCTrUS94/14776
- 132 -
general lack of fluorescence in and around tumor
- microvessel (image center) and in tumor-cell nuclei.
Fluorescence intensities and pattern in other major
organs, indicate that the clearance of doxorubicin:DS is
shifted away from the kidneys (relative to standard
~ doxorubicin) and caused to clear via the liver in an
; F accelerated fashion (relative to standard doxorubicin),
into the bile. No increment in cardiac or splenic red
pulp is observed (which might be predictive of toxicities
at these sites, and which are the major sites of toxicity
for standard doxorubicin), and the fluorescence levels at
these two sites, if anything, is slightly lower for
doxorubicin:DS than for standard doxorubicin.
These results indicate a markedly higher tumor
localization, depth and breadth of tumor extracellular
matrix penetration, tumor-cell internalization and
nuclear migration of doxorubicin (its key cellular site
of action) when the drug i9 formulated as doxorubicin:DS
(but not as doxorubicin alone); and they further indicate
surprising and unexpected uptake by induced tumor
endothelia and endothelial nuclei (for doxorubicin:DS).
These surprising and unexpected advantages, taken
together with those of the preceding Examples (29-35),
clearly and completely distinguish the present
formulation of doxorubicin with essentially purified
dermatan sulfate, and other oncology and non-oncology
therapeutic (and diagnostic) actives, when in association
with the essentially purified dermatan sulfates of the
present invention.
CA 02208566 l997-06-23
W O 96/19242 PCTrUS91/14776
- 133 -
REFERE~CES
-
Axen (1974) Prostaglandins, 5(1):45.
Bacchi et al. (1993) Am. J. Pathol., 142:579.
Bartlin~ et al. (19 7 4) Biotechnology and Bioengineering,
16:1425~
Berse e~ al. tl992), Molecular Biology of the Cell,
3:211-2:~0.
Bevilac~lua et al. (199 3) .J. Clin. Invest., 91:91.
Bevilaqua et al. (July 10, 1993) Congress of Intl. Soc.
of ~hrombosis and Hemostasis.
Boneu ei al. (199 2) Heparin and Related Polysaccharides,
Plenum ~~ress, NY, pg. 237.
Brechbiel et al. (1986) Inorg. Chem., 25:2772-2781.
Connolly et al. (1989) J. Clin. Invest., 84:1470-1478.
Cremers et al. (1994) International ~ournal of
Pharmaceutics, 110:117-125.
Crumblics et al. (1991), H~NDBOOK OF MICROBIAL IRON CHELATES,
Chapter 7, CRC Press.
Danishefsky et al. ( 1972) Thrombosis Research, 1: 173.
Danishefsky et al. (1971) Carbohydrate Research, 16:199.
~. 35 Dawes et al. (1989) Thrombosis and Haemostasis,
62(3):945-949.
-
CA 02208~66 l997-06-23
W O 96/19242 pcTrus94ll4776
- 134 -
Elices et al. (1990) Cell, 60:577-584.
Geraldes et al. (1985) Proc. Soc. Mag. Res. Med., 2:860.
Grant et al. (1984) Analytical Biochemistry, 137:25-32.
Hahn et al. (1993) Mag. Res. Imaging, 11:1007.
Hashimoto et al. (1983) ,J. Nucl. Med. 24:123.
Huber et al. (1991) Science, 254:99-102.
Jakeman et al. (1992) .J. Clin. Invest., 89:244-253.
Janoki et al. 1983) Int. J. Appl. Radiat. Isot.,
34(6):871.
Kalishevskaya et al. (1988) Eksp. Onkol., 10(4):59-62.
Kim et al. (1993) Nature, 362:841-844.
Kjellen et al. (1977) Biochem. and ~3ioph~s. F~es. Comm.,
74:126-133.
Klein et al. (1982) Analytical Biochemistr~-, 124:59-64.
Landsberger (1987) U.S. Patent No. 4,710,493.
Levine (1993) FASEB Journal, 7:1242.
Li et al. (1993) Bioconjugate Chem. 4:275-283.
Lin et al. (1975) Analytical Biochemistry, 63:485.
Lindahl and Hook (1978) Ann. Rev. Biochem., 47:385. ,-
Lorant et al. (1993) .J. Clin. Invest., 92:559.
CA 02208566 1997-06-23
W O96/19242 PCT~US94114776
- 135 -
Lyle et al. (1963) Talanta, 10:1177.
, .
Maeda et~ al. (1993) Anti-Cancer Drugs, 4 :167-171.
-
Mascel1ani et al. (1994) Thromb. Res., 74(6):605-615.
Mascel1clni et al. (1993) International Patent Application
Wo 93/0~;074.
Miller et al. (1994) Am. J. Pathol., 145:574-584.
Montrucchio et al. (1993) Am. J. Pathol., 142:471.
Munro et al. (1992) Am. J. Pathol., 141:1397.
Nicosia et al. (1994) Am. J. Pathol., 145:1023-1029.
Nikkari et al . (1993) Am. ~. Pathol., 143:1019.
Ramirez ~t al. (1973) J. Macromol. Sci-Chem., A7(5):1035.
Ranney (:'990) U.S. Patent No. 4,925,678.
Ranney (:992) U.S. Patent No. 5,108,759.
Ranney, Patent Application SN 07/642,033.
Ranney, E~atent Application SN 07/803,595.
Ranney, E~atent Application SN 07/880,660.
Ransohoff et al. (1993) FASEB Journal, 7:592.
.
Rice et al. (1991) Am. J. Pathol ., 138:385.
Sasseville et al. (1992) Am. ~. Pathol., 141:1021.
- ~ =
CA 02208566 l997-06-23
W O96/19242 PCTrUS91/14776
- 136 -
Senger et al. (199 3) Cancer and Metastasis Reviews,
12:303-324.
Sessler et al~, U.S. 5,159,065.
Sessler et al., U.S. 5,252,720.
Sessler et al., U.S. 4,935,498.
Sharon et al. (January 1993) Scientific American, pg. 83.
Sioussat et al. (1993) Arch. Biochem. Biophys., 301:15-
20.
Steinhoff et al. (1993) Am. J. Pathol. 142:481.
Strieter et al. (1992) Am. ~J. Pathol., 141:1279.
Travis (1993) Science, 260:906.
Weindel et al. (1992) BBRC, l83(3):1167-1174.
Yamashiro et al. (1994) Am. ~. Pathol., 145:856-867.
CA 02208566 1997-06-23
W O 96/19242 PCT~US94/14776
- 137 -
SEQUENCE LISTING
(1) GENE~RAL INFORMATION:
(i) APPLICANT:
(A) NAME: ACCESS PHARMACEUTICALS, INC.
(B) STREET: 2600 N. Stemmons Freeway, Suite 210
(C) CITY: Dallas
(D) STATE: TEXAS
(E) COUNTRY: UNITED STATES OF AMERICA
(F) POSTAL (ZIP) CODE: 75207
(ii) INVENTORS: RANNEY, David F.
(iii) TITLE OF INVENTION: IN VIVO AGENTS COMPRISING
CATIONIC DRUGS, PEPTIDES
AND METAL CHELATORS WITH
ACIDIC SACCHARIDES AND
GLYCOSAMINOGLYCANS, GIVING
IMPROVED SITE-SELECTIVE
LOCALIZATION, UPTAKE
MECHANISM, SENSITIVITY AND
KINETIC-SPATIAL PROFILES,
INCLUDING TUMOR SITES
(iv~ NUMBER OF SEQUENCES: 2
(v' CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Arnold, White & Durkee
(B) STREET: P. O. Box 4433
. (C) CITY: Houston
~ (D) STATE: Texas
(E) COUNTRY: USA
(F) ZIP: 77210
(vi) COMPUTER READABLE FORM:
CA 02208~66 l997-06-23
- W 096/19242 PCTrUS94/14776
- 138 -
(A) MEDIUM TYPE: Floppy disk
(B) COM~ ~K: IBM PC compatible
(C~ OPERATING SYSTEM: PC-DOS/MS-DOS/ASCII
(D) SOFTWARE: WordPerfect 5.1
(vii) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: Unknown
(B) FILING DATE: Concurrently herewith
(C) CLASSIFICATION: Unknown
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: HODGINS, DANIEL S.
(B) REGISTRATION NUMBER: 31, 026
(C) REFERENCE/DOCKET NUMBER: RANN024PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (512) 418-3000
(B) TELEFAX: (713) 789-2679
- (C) TELEX: 79-0924
(2) INFORMATION FOR SEQ ID NO 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
. (ix) FEATURE:
(A) NAME/KEY: Modified-site .~
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "Xaa = ~-
N-formyl-met"
CA 02208566 1997-06-23
wo 96/19242 PCTJUS94/14776
- 139 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Xaa Leu Phe Lys
(2) INFORMATION FOR SEQ ID NO 2:
(i) SÉQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii~ MOLECULE TYPE: peptide
(xi SEQUENCE DESCRIPTION: SEQ ID NO:2:
Arg Pro Pro Gly Phe Ser Pro Phe Arg
1 5
