U.S. patent application number 12/801980 was filed with the patent office on 2010-11-25 for novel compositions magnetic particles covered with gem-bisphosphonate derivatives.
This patent application is currently assigned to Guerbet.. Invention is credited to Claire Corot, Marc Port, Isabelle Raynal, Olivier Rousseaux.
Application Number | 20100297025 12/801980 |
Document ID | / |
Family ID | 32406319 |
Filed Date | 2010-11-25 |
United States Patent
Application |
20100297025 |
Kind Code |
A1 |
Port; Marc ; et al. |
November 25, 2010 |
Novel compositions magnetic particles covered with
gem-bisphosphonate derivatives
Abstract
The invention relates to a composition comprising acid magnetic
particles (p) based on an iron compound, the acid magnetic
particles (p) being complexed by one or more gem-bisphosphonate
compounds, of formula I: X-L-CH(PO.sub.3H.sub.2).sub.2 (I) in
which: L represents an organic group connecting the X group to the
gem-bisphosphonate group --CH(PO.sub.3H.sub.2).sub.2; X represents
a chemical group capable of reacting with a biovector; all or some
of the X groups of the particles optionally being coupled to a
biovector. The invention relates also to a process for the
preparation of the compositions and their use, in particular as
contrast products for Magnetic Resonance Imaging (MRI).
Inventors: |
Port; Marc; (Deuil La Barre,
FR) ; Corot; Claire; (Lyon, FR) ; Raynal;
Isabelle; (Saint Maur des Fosses, FR) ; Rousseaux;
Olivier; (Senlis, FR) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W., SUITE 600
WASHINGTON
DC
20004
US
|
Assignee: |
Guerbet.
|
Family ID: |
32406319 |
Appl. No.: |
12/801980 |
Filed: |
July 6, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10739400 |
Dec 19, 2003 |
7780953 |
|
|
12801980 |
|
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|
|
Current U.S.
Class: |
424/9.32 ;
977/773 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
29/00 20180101; B82Y 5/00 20130101; A61K 49/1842 20130101; A61P
35/00 20180101 |
Class at
Publication: |
424/9.32 ;
977/773 |
International
Class: |
A61K 49/10 20060101
A61K049/10 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 20, 2002 |
FR |
02 16410 |
Claims
1-49. (canceled)
50. In a method of medical magnetic resonance imaging, for the
diagnosis or monitoring of a patient condition, comprising
administering a composition to a patient, the improvement wherein
the composition comprises acid magnetic particles (p) based on an
iron compound, the acid magnetic particles (p) being complexed by
one or more gem-bisphosphonate compounds of formula I:
X-L-CH(PO.sub.3H.sub.2).sub.2 (I) in which L represents an organic
group connecting the X group to gem-bisphosphonate group
--CH(PO.sub.3H.sub.2).sub.2 and X represents a chemical group
capable of reacting with a biovector and all or some of the X
groups of the particles optionally being coupled to a
biovector.
51. The method of claim 50, in which all or some of the X groups
are coupled to a biovector.
52. The method of claim 50, in which L is selected from the group
consisting of aliphatic, alicyclic, aromatic, alicyclic-aliphatic,
aromatic-aliphatic, and -L.sub.1-NHCO-L.sub.2 where L.sub.1 and
L.sub.2 are identical or different and individually represent an
aliphatic, alicyclic, aromatic, alicyclic-aliphatic, or
aromatic-aliphatic group, and wherein each of L, L.sub.1, and
L.sub.2 is optionally substituted by a methyl, hydroxyl, methoxy,
acetoxy, or amido group or a chlorine, iodine, or bromine atom.
53. The method of claim 50, in which L represents an optionally
substituted phenylene group, and wherein the X and
gem-bisphosphonate groups are in the ortho, meta, or para
position.
54. The method of claim 50, in which L represents an aliphatic
group.
55. The method of claim 50, in which L represents a group
--(CH.sub.2).sub.p, where p is an integer from 1 to 5.
56. The method of claim 50 in which L represents
L.sub.1-CONH-L.sub.2, where L.sub.1 and L.sub.2 are either
identical or different and individually represent an aliphatic,
alicyclic, aromatic, alicyclic-aliphatic or aromatic-aliphatic
group, it being optionally possible for the groups to be
substituted by methyl, hydroxyl, methoxy, acetoxy, amido group or a
chlorine, iodine or bromine atom.
57. The method of claim 50, in which L represents a group
--(CH.sub.2).sub.m--CONH--(CH.sub.2).sub.n where m and n
individually represent an integer from 0 to 5.
58. The method of claim 50, in which X is selected from the group
consisting of --COOH, --NH.sub.2, --NCS, --NH--NH.sub.2, --CHO,
maleimidyl, alkylpyrocarbonyl, acylazidyl, iminocarbonate,
vinylsulfuryl, pyridyldisulfuryl, haloacetyl, dichlorotriazinyl,
and halogen.
59. The method of claim 50, in which X represents --COOH or
--NH.sub.2.
60. The method of claim 50, in which all or some of the compounds
of formula (I) have the formula (Ia)
HOOC--(CH.sub.2).sub.2--CH(PO.sub.3H.sub.2).sub.2 (Ia).
61. The method of claim 50, in which the particles (p) are, on
average, complexed on at least 90% of their protonated sites by
compounds of formula (I).
62. The method of claim 50, in which the protonated sites are
complexed by at least two gem-bisphosphonate compounds of formula
(I) having a different structure.
63. The method of claim 50, in which the protonated sites are
complexed by at least two compounds of formula (I) having an
identical structure.
64. The method of claim 50 in which all or some of the magnetic
particles (p) are composed of iron hydroxide, iron hydroxide
hydrate, ferrite, mixed iron oxides, or a mixture thereof.
65. The composition of claim 50, in which all or some of the
magnetic particles (p) are composed of a ferrite.
66. The method of claim 50, in which all or some of the magnetic
particles (p) are composed of maghemite, magnetite, or a mixture
thereof.
67. The method of claim 50, in which the particles (p) are
superparamagnetic.
68. The method of claim 50, in which between 1 and 70% of the X
groups of compounds of formula (I) are coupled to a biovector.
69. The method of claim 50, in which the X groups of compounds of
formula (I) are coupled to at least two different biovectors.
70. The method of claim 50, in which the biovector is selected from
proteins, optionally recombinant or mutated, glycoproteins,
lectins, biotin, vitamins, phospholipids, folic acid derivatives,
antibodies or antibody fragments, peptides and derivatives thereof,
monosaccharides or polysaccharides, avidin, streptavidin, receptor
substrates or inhibitors, steroids and analogs thereof,
oligonucleotides, ribonucleic acid sequences, deoxyribonucleic acid
sequences, hormones or hormone-like substances, amino acids and
derivatives, organic molecules with pharmacological activity, amino
alcohols, integrin-targeting agents and MMP-targeting agents.
71. The method of claim 50, in which the biovector represents a
peptide derivative.
72. The method of claim 50, in which the biovector represents an
antifolic or folic acid derivative.
73. The method of claim 50, in which the biovector represents a
phospholipid derivative.
74. The method of claim 50, in which the biovector represents a
phospholipid derivative selected from the group consisting of a
phosphatidylserine derivative, phosphatidylethanolamine derivative,
phosphatidylcholine derivative, phosphatidylinositol derivative,
sphingomyelin derivative, or ceramide derivative.
75. The method of claim 50, in which the biovector represents a
protein, optionally recombinant or mutated.
76. The method of claim 50, in which the biovector represents an
antibody or an antibody fragment which is optionally
functionalized.
77. The method of claim 50, in which the biovector represents an
optionally functionalized pharmacophore.
78. The method of claim 50, in which the biovector is selected from
the amino alcohols of formula (II) ##STR00026## in which R.sub.1
and R.sub.2 are identical or different and represent an aliphatic
hydrocarbon chain comprising from 2 to 6 carbon atoms, preferably
substituted by 6 to 10 hydroxyl groups, or else by 4 to 8 hydroxyl
groups when R.sub.1 and/or R.sub.2 is interrupted by an oxygen
atom.
79. The method of claim 50, in which the biovector is selected from
aminopolyethylene glycols.
80. The method as claimed in claim 78, in which the biovector is
selected from the amino alcohols of general formula (II)
##STR00027## in which R.sub.1 represents a group
--(CH.sub.2)--(CHOH).sub.4--CH.sub.2OH or
--(CH.sub.2)--CHOH--CH.sub.2OH and R.sub.2 represents a group
--CH.sub.2--(CHOH).sub.4--CH.sub.2OH.
81. The method of claim 50, in which the X groups form a covalent
bond with the biovector, of type --CONH--, --COO--, --NHCO--,
--OCO, --NH--CS--NH--, --C--S--, --N--NH--CO--, --CO--NH--N--,
--CH.sub.2--NH--, --N--CH.sub.2--, --N--CS--N--,
--CO--CH.sub.2--S--, --N--CO--CH.sub.2--S--,
--N--CO--CH.sub.2--CH.sub.2--S, --CH.dbd.NH--NH--,
--NH--NH.dbd.CH--, --CH.dbd.N--O-- or --O--N.dbd.CH--, and also the
formulae below ##STR00028##
82. The method of claim 50, in which the particles complexed and
coupled to the biovector have an overall hydrodynamic diameter of
between 5 nm and 200 nm.
83. The method of claim 50, in which the concentration of the
particles (p) in the composition is between 2 .mu.mol L.sup.-1 and
4 mol L.sup.-1, expressed as total number of moles of metal
ions.
84. The method of claim 50, wherein the composition further
comprises one or more pharmaceutically acceptable vehicles and/or
additives.
85. The method of claim 50, wherein the composition is sterile.
86. The method of claim 50, for the diagnosis or monitoring of a
pathology selected from the group consisting of cardiovascular
disease, cancer, inflammatory disease, and degenerative disease.
Description
[0001] This is a divisional of Ser. No. 10/739,400, filed, Dec. 19,
2003, the disclosure of which is incorporated by reference
herein.
[0002] The invention relates to compositions of magnetic particles
covered with molecules comprising, on the one hand, a
gem-bisphosphonate group bonded to the surface of the particle and,
on the other hand, a reactive group capable of covalently bonding a
targeting entity called a biovector. The invention relates also to
their process of preparation and their use in particular as
contrast products for nuclear Magnetic Resonance Imaging (MRI).
[0003] MRI permits the visualisation of internal organs in living
beings and therefore constitutes a powerful guide and auxiliary in
diagnostics, medical treatment and surgery.
[0004] The administration of contrast products helps to improve the
resolution of the images obtained by that technique and the
precision of diagnosis.
[0005] Thus, the contrast effect may be accentuated by the
presence, in the environment of the organs subjected to
examination, of various magnetic species, for example paramagnetic,
ferromagnetic or superparamagnetic species.
[0006] Paramagnetic substances comprise specific metals, such as
iron, manganese, gadolinium in the ionic or organometallic state.
Ferromagnetic contrast substances generally comprise magnetic
aggregate particles of micrometric or sub-micrometric size, that is
to say, not less than 100-200 nm, for example particles of
ferrites, including in particular magnetite (Fe.sub.3O.sub.4),
maghemite (.gamma.-Fe.sub.2O.sub.3) and other magnetic mineral
compounds of transition elements which behave like permanent
magnets. Superparamagnetic particles are normally very small
particles of ferrite, including in particular magnetite
(Fe.sub.3O.sub.4), maghemite (.gamma.-Fe.sub.2O.sub.3) and other
magnetic mineral compounds of transition elements, having a size of
less than approximately 100-150 nm. Unlike ferromagnetic particles,
superparamagnetic particles no longer behave like small autonomous
magnets owing to the fact that their size is smaller than a
critical value, that is to say, they are aligned in a preferential
direction only when they are subjected to an external magnetic
field. Superparamagnetic particles advantageously have a higher
effective density compared with ferromagnetic particles.
[0007] In order to obtain colloidal solutions of magnetic
particles, which are also referred to as ferrofluids hereinafter
and which are stable in a physiological medium, it is necessary to
condition the surface of the magnetic particles.
[0008] Generally, the magnetic particles used in medical imaging
are covered with macromolecules, such as carbohydrates, such as
dextran, proteins, such as albumin, or synthetic polymers, such as
methacrylates and organosilanes. The syntheses enabling that type
of particle to be obtained are generally carried out in accordance
with the so-called <<de Molday>> process (Reference:
Robert S. Molday and D. Mackenzie; J. of Immunological Methods
(1982), 52, pp. 353-367) and require laborious and expensive
purification processes.
[0009] In many biomedical applications of magnetic particles, it is
necessary to couple the particles to a molecule of specific
affinity, so that the particles bond specifically to the target
cells and to tissue. This recognition is often ensured by the
formation of an affinity complex between a ligand (biovector) fixed
to the particle and a receptor at the surface of the target cell or
of a tissue. This molecule of specific affinity may be either
adsorbed directly at the surface of the particles or bonded by a
covalent bond.
[0010] In general, the covalent grafting of the specific affinity
molecule onto a macromolecule requires prior chemical activation,
for example by oxidation, in order to generate fixation sites on
the macromolecule.
[0011] However, the number of fixation sites generated on the
macromolecule is generally difficult to control and, consequently,
the later grafting of a specific affinity molecule cannot be
controlled.
[0012] The control of the amount of specific affinity molecule
grafted is important for modulating the affinity of the final
solution of particles relative to the target, but also for
modulating the pharmacokinetics, the biodistribution and optionally
the metabolism, the excretion and innocuousness of these particles.
In addition, from an industrial point of view, it is advantageous
to be able to minimise the amount of specific affinity molecule,
the cost of which is generally very high.
[0013] Furthermore, the reactive groups at the surface of the
macromolecule that have not reacted with the specific affinity
molecule are capable of bringing about toxic reactions in vivo.
[0014] Likewise, the grafting of a specific affinity molecule by
chemisorption is generally difficult to control from a
physico-chemical point of view and, moreover, is much less stable
in a biological medium. In addition, chemisorption brings about an
equilibrium with a free fraction of the specific affinity molecule.
This free fraction is detrimental to MRI contrast.
[0015] Magnetic particles covered with low-molecular-weight organic
molecules have also been described to date.
[0016] The document J. of Coll. And Interf. Science 2001, 238, pp.
37-42 teaches that the covering of magnetic particles with organic
molecules comprising a bisphosphonate portion enables objects that
are small (less than 15 nm) and stable over a wide pH range to be
obtained.
[0017] However, the measurement of the hydrodynamic size of the
particles covered with bisphosphonate compounds by PCS (Photon
Correlation Spectroscopy) described in that document requires a
prior filtration step on 100-nm filters in order to remove the
aggregates and to isolate only the particles of small hydrodynamic
size. Therefore, that measurement does not reflect the real
hydrodynamic size of the particles obtained, which is in fact much
larger.
[0018] In addition, the purification of magnetic particles covered
with gem-bisphosphonate compounds by the process described in that
document requires the use of a step of dialysis or treatment by
resins, which is found to be laborious, poorly reproducible and
very difficult to exploit industrially. It will be remembered that
gem-bisphosphonates are --CH(PO.sub.3H.sub.2).sub.2 groups.
[0019] That process therefore permits control neither of the
grafting of gem-bisphosphonate compounds to the surface of magnetic
particles nor of the hydrodynamic size of the resulting particle,
without having recourse to laborious purification processes.
Furthermore, that document neither describes nor suggests coupling
specific targeting entities to particles covered with
bisphosphonate compounds, and in particular gem-bisphosphonates,
for specific biomedical applications, which makes their use highly
unsuitable for this targeting.
[0020] The hydrodynamic diameter, in the context of the present
invention, refers to the diameter of the particle in solution
surrounded by its hydration layer, as determined by light diffusion
according to the PCS technique (Photon Correlation Spectroscopy:
reference: R. Pecora--J. of Nanoparticle Research (2000), Vol. 2,
pp. 123-131).
[0021] The publication WO 97/01760 describes magnetic particles
covered with dimercaptosuccinic acid (DMSA) that are coupled to a
specific targeting entity, annexin, via the thiol groups of
DMSA.
[0022] However, in normal pH and ionic strength conditions, the
thiol groups of DMSA that are present at the surface of the
particle have a tendency to oxidise and to form disulphide bridges,
which leads to the formation of aggregates. The result is a large
hydrodynamic size and a high degree of polydispersion.
[0023] The grafting of annexin also requires, beforehand,
regeneration of the thiol groups by reduction of the disulphide
bridges.
[0024] Finally, the grafting of annexin is poorly controlled.
Therefore, after coupling, it is found to be necessary to
deactivate the residual thiol groups by reaction with BSA (Bovine
Serum Albumin) in order to avoid toxic reactions in vivo or the
later aggregation of the particles obtained.
[0025] There is therefore still a need to obtain particles that are
efficient in respect of a specific diagnosis and that, at the same
time, exhibit a low degree of polydispersion and are stable.
[0026] It has now been found that it is possible to control both
the grafting of functionalised gem-bisphosphonate compounds onto
magnetic particles and the coupling of the resulting particles to
biovectors thanks to the combined use of the gem-bisphosphonate
compounds and acid magnetic particles. This double control has the
advantage of permitting, in particular, the control of the
hydrodynamic size of the resulting particles, namely the acid
magnetic particles (p) grafted by gem-bisphosphonate compounds,
regardless of whether or not they are coupled to biovectors. The
invention thus relates in particular to:
[0027] the acid particles onto which the gem-bisphosphonates are
grafted, those particles, once grafted, being capable of binding to
at least one biovector;
[0028] the particles grafted by gem-bisphosphonates and coupled to
at least one biovector.
[0029] In this context, it has been found that the process for
obtaining such magnetic particles comprises a limited number of
steps and is both simple to implement and reproducible.
Furthermore, it does not require laborious purification processes
and is characterised by very good yields.
[0030] In the context of the present invention, "acid magnetic
particle" means magnetic particles of the same type as those
present within so-called "acid" ferrofluids, which are known from
the prior art but the association of which with gem-bisphosphonates
has been neither described nor suggested.
[0031] These "acid" ferrofluids, like the "alkaline" ferrofluids
have been widely described in the literature, in particular in the
publication Massart et al., C. R. Acad. Sc. Paris, t. 291, Jul. 7,
1980, Serie C and IEE, Transactions on Magnetics, 1981, vol.
MAG-17, n.sup.o 2, p. 1247.
[0032] The acid magnetic particles of acid ferrofluids are
generally described as having protonated hydroxyl sites at the
surface, in an acid medium.
[0033] In contrast, the magnetic particles of alkaline ferrofluids
are described as generally having sites complexed by OH.sup.- ions
in a basic medium.
[0034] This representation in terms of sites in fact corresponds to
a model which has also been confirmed by various works showing that
acid and base magnetic particles behave like weak polyacids and/or
polybases.
[0035] Advantageously, the inventors have shown that, unlike
"alkaline" ferrofluids, "acid" ferrofluids are very stable and do
not exhibit a particle aggregation phenomenon, which results in a
better control of the hydrodynamic size of the precursor (that is
to say, of the non-grafted particle) and subsequently of the
particles grafted by gem-bisphosphonate compounds, then, where
appropriate, of the said particles coupled to biovectors, without a
laborious purification step.
[0036] Surprisingly, the inventors have also shown that it is
possible to obtain very good covering of the magnetic particles by
gem-bisphosphonate compounds, it being possible for each protonated
hydroxyl site at the surface of the acid magnetic particle to be
potentially grafted by a molecule of gem-bisphosphonate complexing
agent. This interaction advantageously enables the surface of the
particle to be masked homogeneously, which in particular prevents
later recognition of the particle by the immune system.
[0037] In particular, the inventors have also found a process
enabling the hydrodynamic size of acid magnetic particles to be
reduced and consequently enabling magnetic particles of very small
size to be obtained. Advantageously, the controlled grafting of
these particles by gem-bisphosphonate agents, optionally followed
by likewise controlled coupling with biovectors, permits the
production of final magnetic particles of small hydrodynamic size
without a laborious purification step, and therefore ultimately
provides a robust, reproducible and therefore industrially
applicable preparation process.
[0038] The inventors have also studied the complexing of acid
magnetic particles (p) by compounds comprising, on the one hand, an
X group capable of covalently bonding a biovector and, on the other
hand, a diphosphonate group in place of the gem-bisphosphonate
group.
[0039] They have thus obtained stable particles, which is already a
marked improvement over the prior art, but coupling to a biovector
is significantly less efficient than with a gem-bisphosphonate. In
particular, when a diphosphonate compound
HOOC--CH.sub.2--N(CH.sub.2(PO.sub.3H.sub.2)).sub.2 is used, for
reasons which are not entirely clear, the particles have a rate of
coupling with the biovector which is twice as low as with a
gem-bisphosphonate. The result is therefore that the cost of
producing magnetic particles grafted by diphosphonates and coupled
to biovectors would be much higher.
[0040] The object of the present invention is therefore to propose
compositions of magnetic particles grafted in a controlled manner
by gem-bisphosphonate derivatives and optionally coupled in a
controlled manner, via those derivatives, to species of the
specific targeting entity type (biovector).
[0041] More specifically, the object of the invention is also to
provide compositions of that type that are stable in suspension, in
particular in a biological medium, and that advantageously have a
controlled hydrodynamic size, especially a small hydrodynamic
size.
[0042] The object of the present invention is also to propose a
process for the preparation of those compositions.
[0043] According to a first aspect, the invention relates to a
composition comprising acid magnetic particles (p) based on an iron
compound, the acid magnetic particles (p) each being complexed by
one or more gem-bisphosphonate compounds, of formula (I):
X-L-CH(PO.sub.3H.sub.2).sub.2 (I)
[0044] in which: [0045] L represents an organic group connecting
the X group to the gem-bisphosphonate group
--CH(PO.sub.3H.sub.2).sub.2; [0046] X represents a chemical group
capable of reacting with a biovector; all or some of the X groups
of the particles (p) optionally being coupled to a biovector.
[0047] The person skilled in the art will understand the expression
"each being complexed" to mean that the composition may also
comprise magnetic particles (p) not complexed by compounds of
formula (I), this being principally for random distribution reasons
and/or owing to the process for the preparation of the composition,
which process is described hereinafter.
[0048] The composition is in the form of a stable aqueous solution
of nanoparticles.
[0049] In these compositions, the rate of complexing of the
compound (I) on the particles is higher than 70%, and preferably
higher than 80, 90, 95%, it being understood that the expression
"rate of complexing" in the present description refers preferably
to the amount of compounds of formula (I), in relation to the
amount of protonated sites present on the acid magnetic particles
(p).
[0050] It is in particular preferable for the acid magnetic
particles (p) to be complexed on at least 90% of their protonated
sites by compounds of formula (I), which advantageously leads to a
good covering of the particle, capable in particular of ensuring
good stability of the particle, preventing recognition of the
particle by the immune system and making the optional later
coupling with a biovector more efficient.
[0051] Magnetic Particle (P)
[0052] In the context of the present description, "particles based
on an iron compound" means particles, all or some of which are
constituted by an iron derivative, generally comprising iron (III),
generally an iron oxide or hydroxide.
[0053] As a general rule, some or all of the acid magnetic
particles (p) are composed of iron hydroxide; hydrated iron oxide;
ferrites; mixed iron oxides, such as mixed iron oxides of cobalt,
nickel, manganese, beryllium, magnesium, calcium, barium,
strontium, copper, zinc, or platinum; or a mixture thereof.
[0054] In the context of the present invention, the term "ferrite"
means iron oxides of the general formula [xFe.sub.2O.sub.3,
yMO.sub.z], where M denotes a metal magnetisable under the effect
of a magnetic field, such as Fe, Co, Ru, Mg, Mn, it optionally
being possible for the magnetisable metal to be radioactive.
[0055] Preferably, the acid magnetic particles (p) of the
compositions of the invention comprise a ferrite, especially
maghemite (.gamma.Fe.sub.2O.sub.3) and magnetite (Fe.sub.3O.sub.4),
or also mixed ferrites of cobalt (Fe.sub.2CoO.sub.4) or of
manganese (Fe.sub.2MnO.sub.4).
[0056] In this context, very particular preference is given to acid
magnetic particles (p), all or some of which, and preferably most
of which, are composed of maghemite or magnetite or of a mixture
thereof.
[0057] The acid magnetic particles (p) of the compositions
according to the invention have at their surface hydroxyl sites
protonated in an acid medium, which correspond more precisely to
the species Fe--OH.sub.2.sup.+ resulting from a very strong
interaction between an Fe.sup.3+ ion at the surface of the particle
and an acid water molecule (H.sub.3O.sup.+). These protons may be
regarded as constituting the particle according to the works of J.
Lyklema et al., Materials Science Research, 1984, 17, pp. 1-24. The
percentage of protonated sites on an acid particle is typically
from 1.3 to 2.5% (mole of sites/mole of iron).
[0058] According to a particularly preferred variant, the acid
particles (p) are superparamagnetic.
[0059] The magnetic particles (p) then have a hydrodynamic diameter
of from 5 to 300 nm, preferably from 5 to 60 nm, and more
preferably from 5 to 30 nm.
[0060] Preferably, the composition of the invention is a suspension
of acid magnetic particles (p) which advantageously has, where
appropriate, a concentration suitable for the formation of a stable
colloidal solution (stable aqueous suspension of nanoparticles),
that is to say, in general, a concentration of magnetic particles
(p) of from 2 .mu.moles L.sup.-1 to 4 moles L.sup.-1, expressed as
the total number of moles of metal ions.
[0061] Compounds of Formula (I)
[0062] The compounds of formula (I) according to the invention
comprise a gem-bisphosphonate group which ensures that the compound
of formula (I) is fixed to the surface of the acid magnetic
particle.
[0063] More precisely, the inventors have demonstrated that each
gem-bisphosphonate group interacts with a single protonated
hydroxyl site at the surface of the acid particle.
[0064] The compounds of formula (I) of the invention comprise a
linker (L) enabling the gem-bisphosphonate group and the reactive
entity X, capable of ensuring the covalent grafting of a biovector,
to be connected and/or spaced from one another.
[0065] Preferably, the linker L represents a divalent group.
[0066] Preferably, the linker L is selected from:
[0067] an aliphatic; alicyclic; alicyclic-aliphatic; aromatic;
aromatic-aliphatic group, it optionally being possible for the
aliphatic, alicyclic and aromatic groups to be substituted by a
methyl, hydroxy, methoxy, acetoxy, amido group, or a chlorine,
iodine or bromine atom;
[0068] a group -L.sub.1-NHCO-L.sub.2 where L.sub.1 and L.sub.2 are
either identical or different and represent an aliphatic;
alicyclic; aromatic; alicyclic-aliphatic or aromatic-aliphatic
group, it optionally being possible for the said groups to be
substituted by a methyl, hydroxy, methoxy, acetoxy, amido group or
a chlorine, iodine or bromine atom.
[0069] An aliphatic group in the context of the present invention
means a linear or branched hydrocarbon chain preferably comprising
from to 16 carbon atoms, and even more preferably from 1 to 6
carbon atoms. Examples thereof are, in particular, methyl, ethyl,
propyl, isopropyl, butyl, tert-butyl, isobutyl, pentyl and hexyl
radicals.
[0070] The term "alicyclic" means a cyclic hydrocarbon chain
preferably comprising from 3 to 8 carbon atoms. By way of example,
mention will be made in particular of cyclopropyl and
cyclohexyl.
[0071] The term "aromatic" represents a mono- or polycyclic
aromatic hydrocarbon group preferably comprising from 5 to 20
carbon atoms, and even more preferably from 6 to 18. Examples
thereof are, in particular, phenyl and 1- or 2-naphthyl radicals.
According to one particular variant, an "aromatic" group in the
context of the invention may incorporate one or more hetero atoms,
such as sulphur, oxygen or nitrogen. In this particular case, the
"aromatic" group denotes a mono- or polycyclic heteroaromatic
group.
[0072] The "aliphatic-alicyclic" and "aliphatic-aromatic" groups
represent aliphatic chains corresponding to the above-mentioned
definition that are substituted by an alicyclic or aromatic group,
respectively, such as defined above. In particular, benzyl may be
mentioned by way of example of an aliphatic-aromatic group.
[0073] According to a preferred variant, L represents an optionally
substituted phenylene group, it being possible for the X and
gem-bisphosphonate groups to be in the ortho, meta or para
position.
[0074] According to one particularly preferred embodiment, L
represents a substituted or unsubstituted aliphatic group, and more
preferably a group --(CH.sub.2).sub.p--, where p is an integer from
1 to 5.
[0075] According to another preferred embodiment, L represents a
group L.sub.1-CONH-L.sub.2 and more preferably a group
--(CH.sub.2).sub.n--NHCO--(CH.sub.2).sub.m-- where n and m
represent an integer from 0 to 5.
[0076] The X end of the gem-bisphosphonate compound of formula (I)
is chosen in such a manner that it is capable of reacting and
forming a covalent bond with a group present on the biovector. For
more information concerning these coupling processes reference may
be made in particular to the work Bioconjugate techniques, Greg T.
Hermanson, 1995, Publisher: Academic, San Diego, Calif.
[0077] There may be mentioned as preferred X groups, in
particular:
[0078] --COOH,
[0079] --NH.sub.2, --NCS, --NH--NH.sub.2, --CHO, alkylpyrocarbonyl
(--CO--O--O-alk), acylazidyl (--CO--N.sub.3), iminocarbonate
(--O--C(NH)--NH.sub.2), vinylsulphuryl (--S--CH.dbd.CH.sub.2),
pyridyldisulphuryl (--S--S-Py), haloacetyl, maleimidyl,
dichlorotriazinyl, halogen,
[0080] the groups --COOH and --NH.sub.2 being especially
preferred.
[0081] The term <<alk>> in the context of the present
description means a C.sub.1-C.sub.6 alkyl radical, the term
<<Py>> for its part meaning a pyridyl radical.
[0082] The maleimidyl radical denotes a cyclic radical of
formula:
##STR00001##
[0083] The dichlorotriazinyl radical denotes a radical of
formula:
##STR00002##
[0084] Among the halogen groups, mention may in particular be made
of chlorine, bromine, fluorine and iodine, chlorine and bromine
being particularly preferred.
[0085] In the context of the present invention, " haloacetyl "
means an acetyl radical CH.sub.3--CO--, one of the hydrogen atoms
of which is substituted by a halogen atom, said halogen atom being
as defined above.
[0086] Preferably, X represents a group --COOH or --NH.sub.2 and L
represents a substituted or unsubstituted aliphatic group, better
still a group --(CH.sub.2).sub.p--, where p is an integer from 1 to
5.
[0087] The compound of formula (Ia)
HOOC--(CH.sub.2).sub.2--CH(PO.sub.3H.sub.2).sub.2 (Ia)
is most particularly preferred.
[0088] According to another preferred embodiment, L represents a
group L.sub.1-CONH-L.sub.2 and more preferably a group
--(CH.sub.2).sub.p--NHCO--(CH.sub.2).sub.m-- where n and m
represent an integer from 0 to 5 and X represents --COON or
--NH.sub.2.
[0089] Of course, also falling within the context of the invention
is the coupling of the X group and of the biovector in an indirect
manner, that is to say via a homobifunctional or heterobifunctional
reagent. By way of illustration the homo-bifunctional reagent,
glutaraldehyde may be suitable for carrying out the coupling, for
example, of a group X=--NH.sub.2 with a group --NH.sub.2 of the
biovector.
[0090] According to a preferred variant of the invention, the X
groups form a covalent bond L.sub.3 with the biovector, of type
[0091] --CONH--, --COO--, --NHCO--, --OCO--, --NH--CS--NH--,
--C--S--, --N--NH--CO--, --CO--NH--N--, --CH.sub.2--NH--,
--N--CH.sub.2--, --N--CS--N--, --CO--CH.sub.2--S--,
--N--CO--CH.sub.2--S--, --N--CO--CH.sub.2--CH.sub.2--S--,
--CH.dbd.NH--NH--, --NH--NH.dbd.CH--, --CH.dbd.N--O-- or
--O--N.dbd.CH--, or corresponding to the formulae below:
##STR00003##
[0092] According to one particular embodiment, it is possible to
envision coupling two biovectors on the same X group via a bond of
type:
##STR00004##
[0093] The protonated sites of the acid magnetic particles (p)
according to the invention can be complexed by at least two
compounds of formula (I) having identical or different
structures.
[0094] According to a preferred embodiment, the magnetic particles
(p) are complexed by one or more molecules of two compounds of
formula (I) having different structures, in particular having
different X groups. This advantageously permits the subsequent
grafting of biovectors having groups capable of reacting with
different X groups.
[0095] According to a preferred embodiment, all or some of the X
groups of the gem-bisphosphonate compound are coupled to a
biovector.
[0096] The invention thus also relates to a composition (which can
be used as a contrast product for MRI) comprising a stable aqueous
suspension of acid nanoparticles (p) in which at least 90% of the
protonated sites are complexed by one or more identical or
different gem-bisphosphonate compounds of formula (I):
X-L-CH(PO.sub.3H.sub.2).sub.2 (I)
[0097] in which all or some of the X groups are coupled to at least
one biovector.
[0098] The expression "all or some" means that not all the X groups
of the gem-bisphosphonate compound are necessarily coupled to a
biovector, but that the degree of coupling is sufficient for the
desired diagnostic specificity to be obtained with the biovectors
used. It is also specified that if several X groups and/or several
biovectors are used, the X groups and/or the biovectors can be
identical or different.
[0099] The particle thus vectorized is written:
(BRANCH).sub.r-ACID PARTICLE (III)
[0100] in which r represents the number of branches grafted per
particle and BRANCH is defined by:
[0101] [((BIOVECTOR)-L.sub.3).sub.s-L-CH(PO.sub.3H.sub.2).sub.2],
L.sub.3 being a covalent bond as defined above resulting from the
coupling of the X group of the compound of formula (I) and of the
biovector, optionally via a homobifunctional or heterobifunctional
reagent; s represents the number of biovectors coupled per X group,
and can in particular be equal to 1, 2 or 3.
[0102] Biovector
[0103] In the context of the present invention "biovector"
preferably means a specific targeting entity (generally a ligand),
that is to say having a specific affinity for a given target tissue
and/or biological receptor and/or transporter and/or biological
matrix.
[0104] There are many receptor/ligand affinity couples, and the
invention as defined may be readily applied by the person skilled
in the art to various ligands capable of forming an affinity pair
with a target receptor, such as the antibody/antigen,
lectin/polysaccharide, biotin/avidin or nucleic acids (+ and -
strand) couples, it being understood that the ligand may be coupled
via a covalent bond either directly to the X end of the compound of
formula (I), or indirectly via a bifunctional reagent.
[0105] By extension, in the context of the present description, the
term "biovector" can also cover organic molecules having a
pharmacological and/or cytotoxic activity or, more generally,
pharmacophores useful in methods of therapeutic or prophylactic
treatment.
[0106] In the context of the present invention, the term
"pharmacophore" covers organic molecules with pharmacological
and/or cytotoxic activity, and also structural analogs thereof, the
latter possibly having a pharmacological and/or cytotoxic
activity.
[0107] In the context of the present description, a "biovector"
denotes, by extension, biocompatible molecules which are markedly
hydrophilic in nature, such as amino alcohols.
[0108] As preferred biovectors, mention can in particular be made
of proteins, optionally recombinant or mutated, glycoproteins,
lectins, biotin, vitamins, pteroic or aminopteroic derivatives,
antifolic and folic acid derivatives, antibodies or antibody
fragments, peptides and derivatives thereof, monosaccharides or
polysaccharides, avidin, (membrane or nuclear) receptor substrates
or inhibitors, phospholipids, steroids and analogs thereof,
oligonucleotides, ribonucleic acid sequences, deoxyribonucleic acid
sequences, hormones or hormone-like substances, amino acids,
organic molecules with pharmacological activity, pharmacophores,
proteins, optionally recombinant or mutated, antibodies or antibody
fragments, amino alcohols, phospholipid derivatives,
pharmacophores; antifolic and folic acid derivatives,
integrin-targeting agents or MMP-targeting agents being
particularly preferred.
[0109] The biovectors, including in particular the antibodies and
the pharmacophores, can be optionally functionalized so as to be
able to react with the X groups and to form a covalent bond,
preferably of type
[0110] --CONH--, --COO--, --NHCO--, --OCO--, --NH--CS--NH--,
--C--S--, --N--NH--CO--, --CO--NH--N--, --CH.sub.2--NH--,
--N--CH.sub.2--, --N--CS--N--, --CO--CH.sub.2--S--,
--N--CO--CH.sub.2--S--, --N--CO--CH.sub.2--CH.sub.2--S--,
--CH.dbd.NH--NH--, --NH--NH.dbd.CH--, --CH.dbd.N--O-- or
--O--N.dbd.CH--, or corresponding to the formulae below:
##STR00005##
[0111] The term "folic acid derivatives and antifolic derivatives"
denotes molecules obtained by functionalization of at least one
chemical group of folate or of pteroic acid such that they may form
a covalent bond with the X groups, in particular of the type such
as those mentioned above. By way of example of functionalization,
mention may in particular be made of etherification of a hydroxyl
group, esterification or amidation of a carboxylate group, or the
modification and/or introduction of substituents on the
(2-aminopyrimidine) structural motif) and more particularly on the
(2-aminopyrimidine) structural motife present in the aminopterinic
and pteroic derivatives. By way of preferred examples of folate
derivatives, mention may more particularly be made of those
described in the article by M. P Costi--Current Drug Targets
(2001), vol 2, p. 135-166.
[0112] By way of preferred examples of "phospholipid" derivatives,
mention may be made of phosphatidylserine,
phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol
or sphingomyelin derivatives or ceramide derivatives, the term
"derivative" here meaning that the molecules are functionalized in
such a way that they are capable of reacting covalently with the X
groups described above, in particular with X=--COON or
--NH.sub.2.
[0113] In the context of the present description, "peptide
derivative" means a peptide derived from amino acids which may or
may not be natural, the native structure of which has been
chemically modified by functionalization of certain chemical
groups, in such a way that the resulting peptide is capable of
reacting covalently with the X groups, for example by modification
of a peptide bond. By way of indication, a peptide derivative can
denote a polypeptide, a pseudo-peptide, a retropeptide, a
peptidomimetic, a cyclic peptide, enzyme-inhibiting peptides,
agonist peptides or antagonist peptides. By way of preferred
examples, mention may be made of the functionalized peptides used
in nuclear medicine (Reference: S. Liu and D. Scott Edwards; Topics
in Current Chemistry (2002), 222, p 259-278) and Okarvi, Nuclear
Medicine Communication, 1999, 20: 1093-1112) and the inhibitors or
substrates of proteases, in particular of metalloproteases.
[0114] A particular class of biovectors are the highly hydrophilic
molecules such as amino alcohols or amino polyethylene glycols.
These amino alcohols are particularly useful for obtaining final
magnetic particles which are sufficiently hydrophilic in terms of
aqueous stability and of biocompatibility, and also for modulating
the pharmacokinetics and the biodistribution.
[0115] Among the amino alcohols, preference is particularly given
to those corresponding to formula (II) below:
##STR00006##
[0116] in which R.sub.1 and R.sub.2 are identical or different and
represent an aliphatic hydrocarbon chain comprising from 2 to 6
carbon atoms, preferably substituted by 6 to 10 hydroxyl groups, or
else by 4 to 8 hydroxyl groups when R.sub.1 and/or R.sub.2 is
interrupted by an oxygen atom.
[0117] In this context, the amino alcohols for which R.sub.1
represents a group --(CH.sub.2)--(CHOH).sub.4--CH.sub.2OH or
--(CH.sub.2)--CHOH--CH.sub.2OH and R.sub.2 a group
--CH.sub.2--(CHOH).sub.4--CH.sub.2OH are generally preferred.
[0118] Among the aminopolyethylene glycols, preference is
particularly given to the compounds of formula (II) in which
R.sub.1 and R.sub.2, which are identical or different, represent H,
an alkyl group or a polyethylene glycol chain of formula
--CH.sub.2--(CH.sub.2--O--CH.sub.2).sub.k--CH.sub.2OR.sub.3 in
which k ranges from 2 to 100, and R.sub.3 is selected from H, alkyl
or --(CO)alk, the term "alkyl" or "alk" denoting a linear or
branched aliphatic hydrocarbon group having approximately from 1 to
6 carbon atoms in the chain.
[0119] Examples of aminopolyethylene glycols are in particular the
compounds O-(2-aminoethyl)-O'-methylpolyethylene glycol 1100,
O-(2-aminoethyl)-O'-methylpolyethylene glycol 2000 and
O-(2-aminoethyl)-O'-methylpoly-ethylene glycol 750.
[0120] According to a particular variant of the invention, where
appropriate, biovectors which are different in nature are coupled
via the X groups to the surface of magnetic particles (p). In this
context, it is most particularly preferred to couple two different
biovectors to each acid magnetic particle (p).
[0121] According to one embodiment, a "mixed" coupling is carried
out between an amino alcohol and a biovector which is different in
nature, that is to say which exhibits either a specific affinity
for a given target, or a pharmacological and/or cytotoxic activity,
so as to modify the biodistribution of the final product.
[0122] It is preferred that 1 to 100% of the X groups of the
gem-bisphosphonate compounds be coupled to at least two identical
or different biovectors. By way of example, 30% of the X groups can
be coupled to a biovector B1, 30% to a biovector B2 and 30% coupled
to a biovector B3, B1, B2 and B3 being different.
[0123] While it is possible to envision coupling 100% of the X
groups present at the surface of the acid magnetic particles (p) to
biovectors, a coupling yield which limits the increase in
hydrodynamic size of the final particle, while at the same time
making it possible to ensure effective specific targeting, is
generally preferred.
[0124] Thus, according to a preferred embodiment of the invention,
1 to 70%, in particular 1 to 50%, of the X groups of the compounds
of formula (I) are coupled, and in particular when the biovectors
are identical.
[0125] According to a variant of the invention which is also
preferred, when the biovectors are different, in particular when it
involves an amino alcohol and a biovector different in nature, 1 to
100% of the X groups of the compounds of formula (I) are then
coupled.
[0126] More generally, a mixed coupling can, for example, be used
with biovectors which are different but capable of specifically
targeting the same pathology, such as peptides having different
sequences which are capable of targeting various metalloproteases
(MMPs) overexpressed in a pathological cancerous or cardiovascular
region.
[0127] Various biovectors, which can particularly be used in the
context of the invention are now described more precisely in three
fields: [0128] A) cardiovascular [0129] B) oncology [0130] C)
inflammatory and degenerative diseases.
[0131] A) Cardiovascular Field and Atheroma Plaque at Risk
[0132] Various biological systems/mechanisms present in
cardiovascular diseases, in particular those associated with
atheroma plaques, are preferred targets for the contrast or
therapeutic agents according to the invention, in particular the
system involving metalloproteases (MMPs), the thrombus system, the
annexin V system.
[0133] It is known that matrix metalloproteinases (MMPs) or
matrixins, are enzymes which have the property of degrading the
protein components of the extracellular matrix. This extracellular
matrix which surrounds cells and tissues consists of proteins such
as collagen. The MMPs are classified into 3 groups: gelatinases
(type IV collagenases), stromelysins and interstitial
collagenases.
[0134] MMPs are overexpressed in atheroma plaques. In the
cardiovascular field, many studies indicate that MMPs are involved
in the remodeling of the extracellualr matrix in the plaque. With
regard to atheroma plaques, in a nonexhaustive manner, at least
eight MMPs are overexpressed therein:
TABLE-US-00001 Enzyme MMP reference Interstitial collagenase MMP-1
Gelatinase A MMP-2 Gelatinase B MMP-9 Stromelysin-1 MMP-3
Matrilysin MMP-7 Macrophage elastase MMP-12 Collagenase-3 MMP-13
Membrane-type-MMP MT-MMP-1 MMP-14
[0135] MMP1 and MMP3 are particularly described in Johnson J et
al., Activation of Matrix-degrading Metalloproteinases by Mast Cell
Proteases in Atherosclerotic Plaques, Arterioscl. Thromb. Vasc.
Biol. 1998; 18: 1707-1715.
[0136] The invention thus relates to vectorized nanoparticles of
formula (III) in which the biovector is an agent capable of
inhibiting at least one MMP, in particular an MMP inhibitor, for
applications in the cardiovascular field. According to a preferred
embodiment, the inhibitor is a derivative of Ilomastat or a peptide
as exemplified later. Preference will also be given to MMP
inhibitors selected from those described in Current Medicinal
Chemistry, 2001, 8, 425-474; Chem. Rev, 1999, 99, 2735-2776. Use
may in particular be made of MMP inhibitors referred to as TIMPs,
recalled in DDT vol 1, N.sup.o 1, January 1996, Elsevier Science,
16-17; Bioconjugate Chem, 2001, 12, 964-971
[0137] For the thrombus, it is known that:
[0138] the GpIIb/IIIa receptor is expressed on activated platelets
(it is already used in therapeutics as a target for antiplatelet
agents);
[0139] fibrin is associated with thrombosis.
[0140] More precisely, as regards the thrombus, the involvement of
GpIIb/IIIa glycoproteins on activated platelets has been
demonstrated. The platelets are anuclear fragments of bone marrow
megakaryocytes which play a pivotable role in the processes of
atherosclerosis and of thrombosis. The most commonly used
conventional antiplatelet medicinal products are aspirin,
ticlopidine and clopidogrel. Knowledge of the molecular mechanisms
resulting in platelet aggregation has made it possibile to develop
a new family of molecules directed against the platelet receptor
fibrinogen, the integrin GPIIb/IIIa. The major advantage compared
to the antagonists mentioned above is that the final step of
platelet activation is blocked, independently of the route of
activation of the platelets. Since platelet-platelet interaction is
critical for formation of the thrombus, binding of fibrinogen
(which forms bridges between the platelets) to the GP IIb/IIIa
complex is a key event in hemostasis and thrombosis. When the
platelets are activated, the GP IIb/IIIa glycoprotein receptors
which are at the surface of the platelet membranes undergo a
modification of their spatial conformation and can then bind
molecules of fibrinogen soluble in the plasma, and calcium. The
fibrinogen is linked between the platelets via Ca.sup.2+-fibrinogen
bonds forming a network in which the blood cells will be trapped.
The thrombin, by converting the fibrinogen to fibrin, tightens the
mesh of this net. This aggregation will lead to the formation of a
thrombus in the damaged area. Many studies have therefore been
carried out in order to identify, on the GP IIb/IIIa receptor, the
ligand interaction sites. The GP IIb/IIIa complex is an important
membrane-bound heterodimeric glycoprotein complex in platelets
(approximately 50 000 copies per platelet).
[0141] At least two series of peptides, corresponding to amino acid
sequences naturally present in human fibrinogen, are known to
inhibit the binding of adhesion macromolecules to the GPIIb/IIIa
receptor: the sequence Arg-Gly-Asp (RGD) and the
Lys-Gln-Ala-Gly-Asp-Val gamma chain.
[0142] The sequence Arg-Gly-Asp (RGD) was initially identified as
the adhesion sequence of fibronectin, an integrin which plays an
important role in platelet-platelet and platelet-vessel
interactions after its release by platelets. This sequence is also
present in fibrinogen, von Willebrand factor and vitronectin (role
in fibrinolysis and binding to vessels).
[0143] The GPIIb/IIIa complex recognizes this sequence which
inhibits the binding of fibronectin, of fibrinogen, of von
Willebrand factor and vitronectin on platelets. All these ligands
contain at least one RGD sequence; while fibrinogen contains two
thereof per half-molecule. In vivo, fibrinogen is the main ligand
due to its high concentration in the plasma.
[0144] The invention thus relates to vectorized particles
(III):
[0145] in which the biovector is capable of targeting the
GpIIb/IIIa receptor;
[0146] in which the biovector is capable of targeting fibrin, in
particular through peptides selective for the fibrin monomers in
order to differentiate the fibrin from the soluble fibrinogen
molecule.
[0147] The invention relates in particular to products (III) in
which the biovector comprises an RGD motif, for cardiovascular
applications.
[0148] Use may, for example, be made of peptides described in WO
2001/9188, U.S. Pat. No. 6,521,211, U.S. Pat. No. 6,403,056,
Seminars in nuclear medicine, 1990, 52-67, Nucear Medicine and
Radiology, 28,2001,515-526, the apcitide Acutect from the company
Diatide.
[0149] B) Oncology Field
[0150] The specific imaging obtained by virtue of the present
invention is aimed at obtaining, according to one embodiment,
labeling of neoangiogenesis, specific targeting of tumor cells or
alterations in the extracellular matrix, not obtained with known
techniques. Various biological systems (or mechanisms) associated
with tumor development are preferred targets for the contrast or
therapeutic agents according to the invention: the system involving
compounds capable of binding to folate receptors, the system
involving MMPs, the system involving growth factors involved in
angiogenesis.
[0151] According to one embodiment, the invention relates to
products (III) in which the biovector is a derivative capable of
targeting a folate receptor, this biovector being capable of
providing specific recognition of tumor cells. Use will in
particular be made, as biovector, of folic derivatives of formula
(E) written: BIOVECTOR:
##STR00007##
[0152] in which:
[0153] a) G1 is selected independently from the group consisting
of: halo, R.sub.f2, OR.sub.f2, SR.sub.f3, NR.sub.f4R.sub.f5;
preferably, G1 is NH.sub.2 or OH;
[0154] b) G2 is selected independently from the group consisting
of: halo, R.sub.f2, OR.sub.f2, SR.sub.f3, and
NR.sub.f4R.sub.f5;
[0155] c) G3 and G4 represent divalent groups selected
independently from the group consisting of --(R.sub.f6')C.dbd.,
--N.dbd., --(R.sub.f6')C(R.sub.f7')-, --N(R.sub.f4')-;
[0156] preferably, G3 is --N.dbd. (folic acid) or --CH-- (compounds
described later: CB3717, raltitrexed, MAI) when the ring comprising
G3 is aromatic, and G3 is --NH-- or --CH2- (compounds described
later: AG-2034, lometrexol) when the ring comprising G3 is
nonaromatic;
[0157] preferably, G4 is --CH-- or --C(CH3)- when the ring
comprising G3 is aromatic, and --CH2- or --CH(CH3)- when the ring
comprising G3 is nonaromatic;
[0158] e) G5 is absent (compound pemetrexed) or selected from
--(R.sub.f6')C.dbd., --N.dbd., --(R.sub.f6')C(R.sub.f7')-,
--N(R.sub.f4')-;
[0159] f) The ring J is a possibly heterocyclic aromatic 5- or
6-membered ring, it being possible for the atoms of the ring to be
C, N, O, S;
[0160] vii) G6 is N or C (compound described later:
3-deaza-ICI-198,583);
[0161] h) K1 and K2 are selected independently from the group
consisting of --C(Z.sub.f)--, --C(Z.sub.f)O--, --OC(Z.sub.f)--,
--N(R.sub.f4'')-, --C(Z.sub.f)--N(R.sub.f4),
--N(R.sub.f4'')-C(Z.sub.f), --O--C(Z)--N(R.sub.f4'')-,
--N(R.sub.f4'')-C(Z.sub.f)--O--,
N(R.sub.f4'')-C(Z.sub.f)--N(R.sub.f5'')-, --O--, --S--, --S(O)--,
--S(O)2-, --N(R.sub.f4'')S(O)2-, --C(R.sub.f6'')(R.sub.f7'')-,
--N(C.ident.CH)--, --N(CH.sub.2--C.ident.CH)--, C1-C12 alkyl and
C1-C12 alkoxy; in which Zf is O or S; preferably, K1 is
--N(R.sub.f4'')- or --C(R.sub.f6'')(R.sub.f7'')- with R.sub.f''4,
R.sub.f6'', R.sub.f7'' being H; A2 possibly being covalently bonded
to an amino acid;
[0162] i) R.sub.f1 is selected from the group consisting of: H,
halo, C1-C12 alkyl and C1-C12 alkoxy; R.sub.f2, R.sub.f3, R.sub.f4,
Rf4', R.sub.f4'', R.sub.f5, R.sub.f5''', R.sub.f6'' and R.sub.f7''
are selected independently from the group consisting of: H, halo,
C1-C12 alkyl, C1-C12 alkoxy, C, --C, 2 alkanoyl, C, --C, 2 alkenyl,
C1-C12 alkynyl, (C1-C12 alkoxy)carbonyl and (C, --C, 2
alkylamino)carbonyl;
[0163] j) R.sub.f6 and R.sub.f7 are selected independently from the
group consisting of: H, halo, C1-C12 alkyl, C1-C12 alkoxy; or
R.sub.f6 and R.sub.f7 together form O.dbd.;
[0164] k) R.sub.f6' and R.sub.f7' are selected independently from
the group consisting of: H, halo, C1-C12 alkyl, C1-C12 alkoxy; or
R.sub.f6' and R.sub.f7' together form O.dbd.;
[0165] l) Lf is a divalent linkage which includes, where
appropriate, a natural amino acid or a natural poly(amino acid),
bonded to K2 or to K1 via its alpha-amino group via an amide
bond;
[0166] m) n, p, r and s are independently 0 or 1.
[0167] The formula (E) includes the tautomeric forms, for example
compounds in which G1 is OH, SH or NH.
[0168] For the compounds of the invention in which at least one of
the groups K1, K2, R.sub.f1, R.sub.f2, R.sub.f3, R.sub.f4,
R.sub.f4', R.sub.f4'', R.sub.f5, R.sub.f5'', R.sub.f6, R.sub.f7'',
R.sub.f6, R.sub.f7, R.sub.f6' and R.sub.f7' comprises a hydrogen
atom or an alkyl, alkoxy, alkylamino, alkanoyl, alkenyl, alkynyl,
alkoxycarbonyl or alkylaminocarbonyl group, the group preferably
comprises 1 to 6 carbon atoms (C1-C6), more preferably 1 to 4
carbon atoms (C1-C4).
[0169] As regards MMPs in the oncology field, it is known that MMPs
have two distinct functions:
[0170] a) they contribute to tumor dissemination by destroying the
extracellular matrix;
[0171] b) they create an environment which promotes the growth and
the angiogenesis of primary tumors and metastasized tumors.
[0172] The MMPs expressed in the main human tumors are in
particular the following:
[0173] breast cancer: MMP-1,2,3,7,9,11,13,14;
[0174] colorectal cancer: MMP-1,2,3,7,9,11;
[0175] lung cancer: MMP-2,3,7,9,11,14;
[0176] prostate cancer: MMP-1,2,3,7,9.
[0177] There is a frequent correlation between the state of tumor
progression and the level of expression of MMPs. In general,
malignant tumors express a greater amount of MMP than benign
tumors.
[0178] The invention thus relates to products (III) in which the
biovector is an MMP inhibitor, for applications in the oncology
field. According to preferred embodiments, use will be made of
inhibitors selected from those described in Current Medicinal
Chemistry, 2001, 8, 425-474; Chem. Rev, 1999, 99, 2735-2776. Use
may in particular be made of MMP inhibitors referred to as TIMPs,
recalled in DDT vol 1, N.sup.o 1, January 1996, Elsevier Science,
16-17; Bioconjugate Chem, 2001, 12, 964-971.
[0179] As regards angiogenesis, studies have shown that
angiogenesis is a prognostic factor in diverse tumors, in
particular breast cancer, kidney cancer, prostate cancer, colon
cancer and brain cancer, and also melanoma.
[0180] The invention thus relates to products (III) in which the
biovector is capable of targeting an angiogenesis marker.
[0181] It has been shown that certain endothelial growth factors
are tumor specific. Endothelial cells, which constitute the inner
vessel wall, show no natural tendency to proliferate in normal
adults. On the other hand, in pathological situations, for example
during the development of tumors or the formation of metastases,
the increased needs in oxygen and in nutritive supplies are
transported by an increase in local irrigation. The tumors thus
derive, to their benefit, a new vascular network, a process known
as angiogenesis.
[0182] Vascular endothelial cell growth factor (VEGF) is a powerful
and selective angiogenic growth factor. It acts by stimulating at
least three receptors on the extracellular membrane: VEGFR-1
(Flt-1), VEGFR-2 (Flk-1/KDR) and NP-1. VEGFs receptors belong to
the large RTK (tyrosine-kinase receptor) family. These proteins of
the integrin family have an extracellular region capable of binding
ligands, a transmembrane domain and a cytoplasmic region carrying
the tyrosine kinase activity. In the case of VEGFRs, the kinase
domain is interrupted by a short sequence specific to each
receptor.
[0183] The invention thus relates to products (III) in which the
biovector is an agent capable of binding to angiogenic receptors
present at the surface of endothelial cells.
[0184] Use may in particular be made of biovectors (in particular
peptides obtained by phage display) described in documents WO
01/012809, WO 01/83693, WO 02/057299 (peptides of 8 amino acids
targeting VEGFR3); Nuclear Medicine Communications (1999), 20,
Pharmacological Review, 179, 206, 2000; Journal of Biological
Chemistry, 2000, 275, 13588-13596; Journal of Biological Chemistry,
2002, 277, 45, 43137-43142; J. Mol. Biol, 2001, 316, 769-787;
Biochemistry, 1998, 37, 17754-17772; Chem. J. Biochem. Mol. Biol,
2000, 16, 803-806. Use may in particular be made of (i) inhibitors
of the VEGF-related enzyme activity, such as quinazoline,
aminothiazole or anthranilamide compounds, (ii) compounds which are
VEGF antagonists, in particular small molecules such as
dibenzothiophenes and molecules of documents JP2001-353075,
JP2000-395413, antibodies. Use may also be made of biovectors of
document U.S. Pat. No. 6,610,269, which describes many agents for
targeting receptors overexpressed in the case of antiogenesis.
[0185] It is also known that the integrin .alpha.v.beta.3 is hardly
expressed in the vascular system, but is overexpressed in tumor
cells (final-stage glioblastoma, ovarian carcinoma melanoma).
.alpha.v.beta.3 takes part in angiogenesis at various stages:
.alpha.v.beta.3 regulates endothelial cell adhesion to the matrix,
it transmits signals to the cell nuclei and is a receptor which is
pro-angiogenic by cooperating with the endothelial cell growth
factor receptor (VEGFR-2, flk). .alpha.v.beta.3, acting with
membrane-type metalloproteinase-1 (MT1-MMP), is responsible for
activation of the metalloproteinase-2 of the matrix at the surface
of the cells. The blocking of this receptor, but also of
.alpha.v.beta.5, with RGD peptides or with antibodies induces
apoptosis of the cells. .alpha.v.beta.5, like .alpha.v.beta.5, is
involved especially in the proliferative phase of angiogenesis.
Many matrix proteins have a common sequence: Arg-Gly-Asp (referred
to as RGD), which has been identified as the site of interaction
with certain integrins. A large number of antiogenesis inhibitors
comprising this RGD sequence have thus been developed.
[0186] The invention thus relates to products (III) in which the
biovector is an RGD peptide suitable for applications in
oncology.
[0187] For the RGD peptides targeting .alpha.v.beta.3, the
inventors prefer biovectors for inhibiting angiogenesis exhibiting
a high affinity for .alpha.v.beta.3 (in order to prevent binding of
matrix proteins) but a low affinity for .alpha.IIb.beta.3. In fact,
it has been shown that .alpha.IIb.beta.3 antagonists can cause
adverse bleeding problems. The inventors prefer in particular
antagonists having a cyclic RGD peptide sequence which is more
stable to enzyme degradation and better affinity and selectivity,
the conformation of the peptide being maintained in a favored
position due to rotation restriction.
[0188] More precisely, after a structure-activity analysis of the
RGD peptides, the inventors prefer most particularly, in order to
be under conditions favorable to maintaining affinity for
.alpha.v.beta.3, cyclic RGD peptides in order to prevent
degradation by enzymes (exopeptidase and endopeptidase), with quite
a short ring in order for it to be more rigid (5-amino acid ring
preferable to a 6-amino acid ring), with an amino acid in the D
configuration. The distance between the .beta.-carbons of aspartic
acid and of arginine is particularly less than 6.6 .ANG. in order
to have greater selectivity for .alpha.v.beta.3 than for
aIIb.beta.3 (the site of which is large than that of
.alpha.v.beta.3). A hydrophobic amino acid close to the aspartic
acid also promotes better selectivity. Such a structure is optimal
for correct exposure of the hydrophobic and hydrophilic groups in
the receptor site. The inventors prefer in particular the peptide
cyclo(Arg-Gly-Asp-Dphe-Val), also referred to as cyclo(RGDfV), the
lower-case letters indicate a D configuration for the corresponding
amino acid. It exhibits an IC.sub.50 of nanomolar (2-2.5 nM). This
peptide is particularly advantageous for the side amine group of
lysine which can react with an FR derivative. The synthesis thereof
is described in X. Dai, Z. Su, J. O. Liu; Tetrahedron Letters
(2000), 41, pp 6295-6298.
##STR00008##
[0189] It permits the coupling to USPIOs (functionalized particles)
which are in particular COOH--, squarate- or
isothiocyanate-functionalized. The inventors have had to overcome
the technical difficulties associated with the choice of protection
of the amino acids, and with the choice of the coupling medium.
[0190] Use may also be made of compounds having a conformation with
restricted flexibility, these peptides having good affinity and
selectivity for .alpha.v.beta.3:
[0191] Peptides in which the RGD motif is placed between two
cysteines, the activity for .alpha.v.beta.3 increasing, for example
(cyclo(CRGDC));
[0192] peptides in which the RGD group is not in the ring, but is
bordered by two rings, at least one of which was formed by a
disulfide bridge between 2 cysteines (WO 02/26776).
[0193] Use may also be made of:
[0194] polypeptide biovectors chosen such that they interact in
vivo with at least one enzyme, in particular MMP, this interaction
resulting in stronger binding with a target protein;
[0195] biovectors comprising an enzyme cleavage site, the cleavage
resulting in a conformational modification;
[0196] biovectors derived from antibodies, such as LM609 (Nature
Medicine, vol 4, 5, May 1998, 623)
[0197] biovectors which mimic RGD peptides, having a structure of
quinolone or benzodiazepine type.
[0198] The invention also relates to products (III) in which the
biovector is a peptidomimetic of the RGD peptide, suitable for
applications in oncology. Use may be made of: [0199] peptides with
substitution of one or two peptide bonds by thioamide bonds (CSNH)
or by keto methylene groups (COCH.sub.2) provided that the
substitutions do not induce any important conformation change;
[0200] peptides (EMD 121974, also called Cilengitide:
cyclo(RGDf-N(Me)V), which exhibit an N-methylation of each of the
amino acids of the cyclic peptide (RGDfV), this peptide exhibiting
very high affinity (IC.sub.50=0.58 nM) and very great selectivity
(1500 times greater than for .alpha.IIb.beta.3);
[0200] ##STR00009## [0201] peptides having the following sequence:
cyclo(Xaa-Yaa-GD-Mamb), the Mamb group being N-aminomethylbenzoic
acid. It is demonstrated that DXaa-N-MeArg peptides are very active
antagonists of .alpha.IIb.beta.3, whereas LXaa-Arg antagonists are
selective for .alpha.v.beta.3;
[0201] ##STR00010## [0202] the cyclic peptide (RGDD(tBuG)Mamb)
exhibiting very high affinity for .alpha.v.beta.3 (IC.sub.50=0.6 nM
versus 14 .mu.m for .alpha.IIb.beta.3) due to the hydrophobic
nature of the amino acid neighboring the aspartic acid and to the
Mamb group which makes the peptide less flexible in aqueous
solution; [0203] the peptide XJ735 (DUPONT) having the group:
cyclo(ARGD-Mamb). This makes it possible to inhibit the binding of
fibrinogen to the .alpha.v.beta.3 receptor (IC.sub.50=70 nM) but
does not block other integrins (.alpha.v.beta.5, .alpha.5.beta.1);
[0204] peptides obtained by introducing various groups in place of
Dphe-Val (or fV) into the reference peptide RGDfV.
##STR00011##
[0205] These groups were synthesized in order to mimic the
.beta.II' folding, but also to reduce the flexibility in the
.gamma.-rotation region. The most active agent is the
cyclo(RGD-(R)-ANC) (IC.sub.50=0.8 nM). It is also possible to
introduce therein sugars X in place of these two amino acids (fv)
in the sequence cyclo(RGDX) (Lohof E. et al; Angew. Chem. Int. Ed.
Engl. (2000), 39 (15), pp. 2761-2764); [0206] peptides with
introduction of a double bond into the ring (Kawaguchi, et al,
Biochemical and Biophysical Research Communications (2001), 288(3),
pp. 711-717);
[0206] ##STR00012## [0207] peptides described in
Chemlibrary.bri.nrc.ca.
[0208] The invention also relates to products (III) in which the
biovector is a nonpeptide molecule targeting the integrin
.alpha.v.beta.3, suitable for applications in oncology. Use may be
made of the compounds in the following table, the nanomolar
affinity of which has been demonstrated.
##STR00013##
and other compounds of document WO 01/97861
##STR00014##
[0209] and other compounds of document WO 99/52896
##STR00015##
and other compounds from the document Lohof et al., Angew. Chem.
Int. Ed. Eng 2000, 39(15), pp. 2761-2764
##STR00016##
and other compounds based on acetyithiophene of document WO
00/00486,
##STR00017##
(and the compounds SG256, SM256 and XJ735 from Dupont
Pharmaceuticals)
##STR00018##
and analogs from the document WO 00/03973
##STR00019##
and other compounds with a pyrrothine group.
[0210] Use may also be made of biovectors mentioned in documents
U.S. Pat. No. 6,537,520 (biovectors targeting .alpha.v.beta.3
overexpressed during angiogenesis) and WO 01/198294 (indazole
core). In certain cases, use may be made of several different
biovectors in the same compound in order to increase the chances of
attaining the same target, for example an RGD peptide and a
benzodiazepine for .alpha.v.beta.3. In addition to the biovectors
of the RGD type or functional equivalents mentioned above, use may
be made of the following MMP-inhibiting compounds, in the knowledge
that the detailed description gives experimental protocols which
make it possible to finalize an in vitro or in vivo screening:
[0211] peptides, peptidomimetics, functionally equivalent
nonpeptides, which are effective in diagnostic or therapeutic
terms, selected from commercially available inhibitors, in
particular in the 2002 catalogs of Bachem, Amersham; [0212]
inhibitors described in documents WO 01/60416, WO 01/60820, WO
2001/92244, EP 558 635, EP 663 823; [0213] inhibitors of the type
such as hydroxamates, pyrrolidine hydroxamates, bicyclic
hydroxamates, cyclobutyl hydroxamates, succinyl hydroxamates,
sulfonamide hydroxamates, alanine hydroxamates, as described in
documents EP 793 641, EP 766 665, EP 740 655, EP 689 538, EP 575
844, EP 634 998, WO 99/29667, EP 965 592, EP 922 702, WO 99/52889,
WO 99/42443, WO 01/60416 (Dupont; in particular of formulae Ia and
Ib); [0214] phosphinic acid-based inhibitors as described in
documents EP 725 075, U.S. Pat. No. 5,679,700, WO 98/03516, EP 716
086, WO 2000 74681, WO 2000 04030; [0215] cyclic imide-based
inhibitors (U.S. Pat. No. 5,854,275); [0216] tricyclic
sulfonamide-based inhibitors (WO 2000 06561); [0217] oxobutyric
acid-based inhibitors; [0218] derivatives of TIMPS (Bioconjugate
Chem, 2001,12, 964-971).
[0219] Still in the oncology field, compounds (III) in which the
biovector is based on phosphonates or on bisphosphonates are very
useful for cancer of bone tissues. These compounds are also useful
for diseases related to bone problems, due to problems of immunity
(autoimmune diseases such as rheumatoid arthritis), to metabolic
diseases (osteoporosis etc.) infectious diseases.
[0220] In these compounds, the biovector is, for example, a
biovector described in document WO 02/062398. Use may also be made
of a bisphosphonate described in U.S. Pat. No. 6,534,488 or U.S.
Pat. No. 6,509,324, a commercially available bisphosphonate such as
etidronate, clodronate, pamidronate, alendronate, ibandonate, YH
592 or EB-1053, and the like.
[0221] C) Field of Inflammatory and Degenerative Diseases
[0222] Biovectors which are ligands for receptors located on
macrophages, such as SRA receptors (scavenger receptors) or Fc
receptors (US 2002/58284), will in particular be targeted.
[0223] The progression of atherosclerosis involves the capture of
LDLs and then the oxidation thereof in plaques. The phagocytosis of
these oxidized LDLs by macrophages is mediated by a set of
receptors referred to as scavenger receptors (SRs). This family of
membrane-bound proteins therefore plays a major role in the gradual
conversion of macrophages to foam cells. SRs are membrane-bound
surface proteins capable of binding senescent cells and also
chemically or biologically modified lipoproteins. The main SR
groups are classified into various classes:
[0224] 1/ class A SRs: type I, type II and MARCO
[0225] 2/ class B SRs: type I, type II and CD36
[0226] 3/ class D SRs: CD68
[0227] 4/ class E and F SRs, "lectin-like": LOX-1
[0228] 5/ recent unclassified SRs: SR-PSOX.
[0229] As recalled in document US A 2002/0127181 and in De Winther
et al., ATVB 2000; 20: 290-297; Kunjathoor et al., J Biol. Chem.
2002; 277: 49982-49988, the SRA receptor is overexpressed by
macrophages in cardiovascular diseases (atherosclerosis, atheroma
plaque, coronary artery disease, thrombosis, ischemia, myocardial
infarction etc.). The use of products according to the invention
for targeting SRA associated with these pathologies is part of the
invention.
[0230] The invention thus also covers, for the diagnosis and/or
treatment of inflammatory diseases, particles complexed by
compounds of formula (I) associated:
[0231] 1) with SR-targeting biovectors, in particular:
[0232] 1a) biovectors described in documents U.S. Pat. No.
6,255,298, U.S. Pat. No. 6,458,845, WO 00/06147, WO 00/03704;
[0233] 1b) modified lipoproteins, in particular acetylated LDLs
(acLDL; Gurudutta et al., Nucl. Med. Biol. 2001 28: 235-24) and
oxidized LDLs (oxLDL);
[0234] 1c) AcLDL, OxLDL and LPS ligands described in Esbach et al.,
Hepatology, 199318: 537; De Rijke et al., J. Biol. Chem, 1994, 269:
824; Van Oosten et al., Infect. Immun. 1998, 66: 5107; Bijsterbosch
et al., Nucleic Acids Res. 1997 25: 3290; Biessen et al, Mol.
Pharmacol. 53: 262, 1998;
[0235] 1d) peptides screened by phage display, capable of binding
to the SR-AI receptor;
[0236] 2) with folate receptor-targeting biovectors (these folate
receptors are overexpressed in activated macrophages) for use in
pathologies involving macrophage activation;
[0237] 3) with peptides for targeting amyloid plaques leading in
particular to Alzheimer's disease (for example, described in WO
01/74374, U.S. Pat. No. 6,329,531);
[0238] 4) with biovectors of the CSF type (GCSF, GM-CSF, etc.)
described, for example, in U.S. Pat. No. 6,491,893;
[0239] 5) with antibodies or antibody fragments which target
receptors overexpressed on macrophages (CD68, MRP8-14, etc.).
[0240] In the field of inflammatory diseases, the inventors have in
particular used phosphatidylserine or a derivative of
phosphatidylserine as a biovector for use in the diagnosis of
macrophage-related diseases.
[0241] Phosphatidylserine (PS) is a membrane phospholipid located
mainly on the inner face of the cell on the cytoplasmic side. Its
overall negative charge stabilizes its polarity and prevents it
from diffusing across the plasma membrane. PS serves as a
recognition signal for the macrophage. The nature of this signal is
still unknown (direct recognition, charge density, multiple
receptors, inducible single receptor). According to the latest
studies published, it is thought to be a direct interaction between
PS and PS receptor (PSR) after induction of this receptor at the
surface of certain macrophages present in the region undergoing a
rupture of homeostasis. In some macrophage cells, PS also interacts
with the scavenger receptors via their site of attachment for
anionic phospholipids. PS is expressed on the inner face of the
membrane of all viable cells. In the event of cell suffering, or at
the beginning of the apoptotic process, a membrane-bound
translocase causes the PS to flip onto the extracellular face of
the cell. This extracellular expression constitutes a recognition
signal for macrophages, which recognize and phagocytose the
suffering cell, thus avoiding a local inflammation.
[0242] The inventors have prepared a contrast agent bonded to PS or
derivative, intended to be actively taken up by macrophages in
order to image these various pathologies. Several chemical
technical difficulties have been overcome in order to prepare the
following PS biovectors, the chelate being coupled to the free
NH.sub.2 group:
##STR00020##
[0243] In order to completely control the structure of the final
products, it has been necessary to prepare correctly functionalized
PS derivatives. The presence of an amino acid residue on the polar
portion of the PS is a source of further difficulties which had to
be controlled by performing chemistry to protect/deprotect the free
amine and acid groups, and/or by using chemical methods compatible
with these groups. The groups which protect the amino acid residue
are the conventional groups used in amino acid chemistry (Boc, tBu,
Z, Bn, etc.). The groups preferred to provide the bond between the
PS and the superparamagnetic particle are NH.sub.2, COOH and
SH.
[0244] The inventors have also prepared vectorized products in
which the phosphatidylserine lacks at least one of the fatty
chains, the affinity not being altered in an interfering
manner.
[0245] In addition to those already mentioned above in the present
application in the fields, use may also be made of:
[0246] 1) The biovectors described in documents WO 01/97850
(targeting VEGF receptors and angiopoietin), U.S. Pat. No.
6,372,194 (polymer such as polyhystidine), WO 2001/9188
(fibrin-targeting polypeptide), WO 01/77145 (integrin-targeting
peptide), WO 02/26776 (.alpha.v.beta.3 integrin-targeting peptide),
WO 03/062198 (MMP metalloprotease-targeting peptides), WO 99/40947
(peptides targeting, for example, the KDR/Flk-I receptor, including
R--X--K--X--H and R--X--K--X--H, or the Tie-1 and 2 receptors), WO
02/062810 and Muller et al, Eur. J. Org. Chem, 2002, 3966-3973
(sialyl Lewis glycosides), WO 02/40060 (antioxidants such as
ascorbic acid), U.S. Pat. No. 6,524,554 (targeting of tuftsin), WO
02/094873 (targeting of G-proteinreceptors GPCRs, in particular
cholecystokinin), U.S. Pat. No. 6,489,333 (integrin antagonist and
guanidine mimetic combination), U.S. Pat. No. 6,511,648 (quinolone
targeting .alpha.v.beta.3 or .alpha.v.beta.5), USA 2002/0106325, WO
01/97861 (benzodiazepines and analogs targeting integrins),
biovector for targeting integrin, including .alpha.5.beta.1, WO
01/98294 (imidazoles and analogs), WO 01/60416 (MMP inhibitors, in
particular hydroxamates), WO 02/081497 (.alpha.v.beta.3-targeting
peptides such as RGDWXE), WO 01/10450 (RGD peptides), U.S. Pat. No.
6,261,535 (antibodies or antibody fragments (FGF, TGFb, GV39, GV97,
ELAM, VCAM, inducible with TNF or IL)), U.S. Pat. No. 5,707,605
(targeting molecule modified by interaction with its target), WO
02/28441 (amyloid-deposit targeting agents), WO 02/056670
(cathepsin-cleaved peptides), U.S. Pat. No. 6,410,695 (mitoxantrone
or quinone), U.S. Pat. No. 6,391,280 (epithelial-targeting
polypeptides), U.S. Pat. No. 6,491,893 (GCSF), US 2002/0128553, WO
02/054088, WO 02/32292, WO 02/38546, WO 2003/6059, U.S. Pat. No.
6,534,038, WO 99/54317 (cysteine protease inhibitors), WO 0177102,
EP 1 121 377, Pharmacological Reviews (52, n.sup.o 2, 179; growth
factors PDGF, EGF, FGF, etc.), Topics in Current Chemistry (222, W.
Krause, Springer), Bioorganic & Medicinal Chemistry (11, 2003,
1319-1341; .alpha.v.beta.3-targeting tetrahydrobenzazepinon
derivatives).
[0247] 2) Angiogenesis inhibitors, in particular those tested in
clinical trials or already commercially available, especially:
[0248] antiogenesis inhibitors involving FGFR or VEGFR receptors,
such as SU101, SU5416, SU6668, ZD4190, PTK787, ZK225846, azacycle
compounds (WO 00/244156, WO 02/059110);
[0249] angiogenesis inhibitors involving MMPs, such as BB25-16
(marimastat), AG3340 (prinomastat), solimastat, BAY12-9566,
BMS275291, metastat, neovastat;
[0250] angiogenesis inhibitors involving integrins, such as SM256,
SG545, EC-ECM-blocking adhesion molecules (such as EMD 121-974, or
vitaxin);
[0251] medicinal products with a more indirect mechanism of
antiangiogenesis action, such as carboxiamidotriazole, TNP470,
squalamine, ZD0101;
[0252] the inhibitors described in document WO 99/40947, monoclonal
antibodies very selective for binding to the KDR receptor,
somatostatin analogs (WO 94/00489), selectin-binding peptides (WO
94/05269), growth factors (VEGF, EGF, PDGF, TNF, MCSF,
interleukins); VEGF-targeting biovectors described in Nuclear
Medicine Communications,1999, 20;
[0253] the inhibitory peptides of document WO 02/066512.
[0254] 3) Biovectors capable of targeting receptors: CD36, EPAS-1,
ARNT, NHE3, Tie-1, 1/KDR, Flt-1, Tek, neuropilin-1, endoglin,
pleiotrophin, endosialin, Axl., alPi, a2ssl, a4P1, a5pl, eph B4
(ephrin), laminin A receptor, neutrophilin 65 receptor, OB-RP
leptin receptor, CXCR-4 chemokine receptor (and other receptors
mentioned in document WO 99/40947), LHRH, bombesin/GRP, gastrin
receptors, VIP, CCK, Tln4.
[0255] 4) Biovectors of the tyrosine kinase inhibitor type. 5)
Known inhibitors of the GPIIb/IIIa inhibitor selected from: [0256]
(1) the fab fragment of a monoclonal antibody for the GPIIb/IIIa
receptor, Abciximab (ReoPro.TM.) [0257] (2) small peptide and
peptidomimetic molecules injected intravenously, such as
eptifibatide (Integrilin.TM.) and tirofiban (Aggrastat.TM.)
[0258] 6) Peptides which are fibrinogen receptor antagonists (EP
425 212), peptides which are IIb/IIIa receptor ligands, fibrinogen
ligands, thrombin ligands, peptides capable of targeting atheroma
plaque, platelets, fibrin, hirudin-based peptides, guanine-based
derivatives which target the IIb/IIIa receptor.
[0259] 7) Other biovectors or biologically active fragments of
biovectors known to the person skilled in the art as medicinal
products, with antithrombotic action, anti-platelet aggregation
action, action against atherosclerosis, action against restenosis,
and/or anticoagulant action.
[0260] 8) Other biovectors or biologically active fragments of
biovectors which target .alpha.v.beta.3, described in combination
with DOTA in patent U.S. Pat. No. 6,537,520, selected from the
following: mitomycin, tretinoin, ribomustin, gemcitabine,
vincristine, etoposide, cladribine, mitobronitol, methotrexate,
doxorubicin, carboquone, pentostatin, nitracrine, zinostatin,
cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole,
fotemustin, thymalfasin, sobuzoxane, nedaplatin, cytarabine,
bicalutamide, vinorelbine, vesnarinone, aminoglutethimide,
amsacrine, proglumide, elliptinium acetate, ketanserin,
doxifluridine, etretinate, isotretinoin, streptozocin, nimustine,
vindesine, flutamide, drogenil, butocin, carmofur, razoxane,
sizofilan, carboplatin, mitolactol, tegafur, ifosfamide,
prednimustine, picibanil, levamisole, teniposide, improsulfan,
enocitabine, lisuride, oxymetholone, tamoxifen, progesterone,
mepitiostane, epitiostanol, formestane, alpha-interferon,
alpha2-interferon, beta-interferon, gamma-interferon, colony
stimulating factor-1, colony stimulating factor-2, denileukin
diftitox, interleukin-2, leutinizing hormone releasing factor.
[0261] 9) Some biovectors which target specific types of cancer,
for example peptides which target the ST receptor associated with
colorectal cancer, or the tachykinin receptor.
[0262] 10) Biovectors which use phosphine-type compounds.
[0263] 11) Biovectors for targeting P-selectin, E-selectin; for
example the 8-amino acid peptide described by Morikawa et al, 1996,
951, and also various sugars.
[0264] 12) Annexin V or biovectors which target apoptotic
processes.
[0265] 13) Any peptide obtained by screening technologies such as
phage display, optionally modified with unnatural amino acids
(http//chemlibrary.bri.nrc.ca), for example peptides derived from
phage display libraries: RGD, NGR, CRRETAWAC, KGD, RGD-4C,
XXXY*XXX, RPLPP, APPLPPR.
[0266] 14) Other known peptide biovectors for targeting atheroma
plaques, mentioned in particular in document WO 2003/014145.
[0267] 15) Vitamins.
[0268] 16) Ligands for hormone receptors, including hormones and
steroids.
[0269] 17) Opioid receptor-targeting biovectors.
[0270] 18) TKI receptor-targeting biovectors.
[0271] 19) LB4 and VnR antagonists.
[0272] 20) Nitriimidazole and benzylguanidine compounds.
[0273] 21) Biovectors recalled in Topics in Current Chemistry, vol.
222, 260-274, Fundamentals of Receptor-based Diagnostic
Metallopharmaceuticals, in particular: [0274] biovectors for
targeting peptide receptors overexpressed in tumors (LHRH
receptors, bombesin/GRP, VIP receptors, CCK receptors, tachykinin
receptors, for example), in particular somatostatin analogs or
bombesin analogs, optionally glycosylated octreotide-derived
peptides, VIP peptides, alpha-MSHs, CCK-B peptides; [0275] peptides
selected from: cyclic RGD peptides, fibrin-alpha chain, CSVTCR,
tuftsin, fMLF, YIGSR (receptor: laminin).
[0276] 22) Oligosaccharides, polysaccharides and ose derivatives,
Glu-targeting derivatives.
[0277] 23) Biovectors used for products of the smart type.
[0278] 24) Markers of myocardial viability (tetrofosmin and
hexakis-2-methoxy-2-methylpropyl isonitrile).
[0279] 25) Sugar and fat metabolism tracers.
[0280] 26) Ligands for neurotransmitter receptors (D, 5HT, Ach,
GABA, NA receptors).
[0281] 27) Oligonucleotides.
[0282] 28) Tissue factor.
[0283] 29) Biovectors described in WO 03/20701, in particular
PK11195, a ligand for the peripheral benzodiazepine receptor.
[0284] 30) Fibrin-binding peptides, in particular the peptide
sequences described in WO 03/11115.
[0285] 31) Amyloid plaque aggregation inhibitors described in WO
02/085903.
[0286] 32) A biovector of the fluorochrome type which can be used
in optical imaging.
[0287] Where appropriate, the length of the linker L of the
compound (I) will be varied in order to optimize the
biodistribution of the compound (III) and/or to promote its
specific recognition by the biological target.
[0288] Final Particles
[0289] In the context of the invention, the final particles,
complexed and optionally coupled to the biovector(s), the overall
hydrodynamic diameter of which is between 5 nm and 200 nm,
preferably between 5 and 60 nm, are preferred.
[0290] The compositions of magnetic particles (p), optionally
coupled to the biovectors, are generally in the form of an aqueous
suspension, optionally in the presence of a water-miscible organic
solvent such as DMSO, acetone or methanol, biocompatible
suspensions being particularly preferred.
[0291] According to a preferred variant of the invention, the
compositions of acid magnetic particles (p) complexed and
optionally coupled to biovectors comprise one or more
pharmaceutically acceptable vehicle(s) and/or additive(s). In this
context, sterile compositions are particularly preferred.
[0292] Process for Preparing the Particles
[0293] According to another aspect, a subject of the invention is a
process for the preparation of acid magnetic particles (p) in which
at least 90% of the protonated sites are complexed by one or more
identical or different gem-bisphosphonate compounds of formula (I),
it being possible for the X groups to be coupled to at least one
biovector, said process comprising bringing an acid solution of
acid magnetic particles (p) based on an iron compound into contact
with compounds of formula (I) as defined above in sufficient
amount, and recovering the complexed particles obtained.
[0294] According to another aspect, a subject of the invention is a
process for the preparation of a composition of magnetic
nanoparticles complexed by compounds (I) and covered with
biovectors, comprising the successive steps consisting in:
[0295] i) bringing an acid solution of acid magnetic particles (p)
based on an iron compound into contact with compounds of formula
(I) as defined above in sufficient amount, and recovering the
complexed particles obtained;
[0296] ii) coupling all or some of the X groups of the compounds of
formula (I) complexed at the surface of the magnetic particles (p)
with biovectors.
[0297] Preferably, step i) comprises the following steps i1) and
i2):
[0298] i1) preparing an acid solution of acid magnetic particles
(p) based on an iron compound;
[0299] i.2) bringing the solution from i.1) into contact with
compounds of formula (I) as defined above in sufficient amount, and
recovering the complexed particles obtained.
[0300] According to a preferred embodiment of the process according
to the invention, the acid ferrofluid used in step i) comprises
magnetic particles based on ferrite, preferably on maghemite or on
magnetite.
[0301] Generally, step i) is carried out in aqueous medium,
optionally in the presence of a water-miscible solvent, such as
DMSO, acetone or methanol, at an acid pH, preferably a pH of 1 to
3.
[0302] Without wishing to be limited to any theory, during the step
consisting of the complexing of magnetic particles (p), the
initially protonated sites of the magnetic particles (p) interact
with the gem-bisphosphonate end of the compounds of formula (I) via
the expulsion of protons as described in particular in the
publication by N. Fauconnier et al.: Prog. Colloid Polym. Sci.
(1996), 100, pp. 212-216. The protonated sites of the acid magnetic
particles (p) can therefore be considered as "points of attachment
or points of bonding" of the gem-bisphosphonate groups of the
compounds of formula (I) to the surface of the magnetic
particle.
[0303] According to a particularly preferred embodiment, the
procedure is carried out in the presence of an amount ranging from
1 to 3 molar equivalents relative to the number of moles of
hydroxyl sites protonated at the surface of the particle (p), so as
to complex at least 70%, and preferably more than 90%, of the
protonated sites of the magnetic particle (p). This represents, in
general, approximately 40 to 200 compounds of formula (I) per
particle, depending on the size thereof, the number of surface
protonated sites being proportional to the surface of the acid
magnetic particle (p).
[0304] Advantageously, the evaluation of the degree of grafting
which defines the ratio of the number of moles of compounds of
formula (I) to the number of moles of protonated sites of the acid
magnetic particle (p) can be readily determined by conventional
methods such as C, P and Fe elemental analysis. Specifically, the
acid magnetic particle (p), before grafting of the compound (I),
comprises no phosphorus atom or carbon atom. Now, after grafting of
the compounds (I) to the surface of the particle, determination of
the phosphorus/iron ratio makes it possible to determine the number
of moles of compound (I) grafted onto the acid magnetic particle
(p).
[0305] Moreover, the number of protonated sites per magnetic
particle is accessible according to conventional techniques known
to the person skilled in the art, such as conductimetry or
acid-based assays.
[0306] By virtue of these analytical tools, it is possible to
adjust the amount of compound of formula (I) according to the
degree of grafting that it is desired to ultimately obtain.
[0307] Advantageously, this process therefore makes it possible to
control the grafting of the magnetic particle (p) with the
compounds of formula (I).
[0308] Against all expectations, particularly advantageously
compared to the prior art which did not use acid particles, the
inventors have also shown that, unlike the case of grafting onto
alkaline particles, the acid magnetic particle (p) grafted with
compounds of formula (I) is most commonly stable within a pH of
from 3 to 12, in particular at physiological pH, which constitutes
a major advantage for carrying out the subsequent coupling of
biovectors, including in particular of proteins or antibodies,
without degrading them or denaturing them.
[0309] This property was not evident given that, in general, the
grafting of a complexing agent, whatever it is, to the surface of a
particle modifies the surface charge of the particles and therefore
their stability range as a function of pH and the ionic
strength.
[0310] Unexpectedly, the inventors have also observed very good
selectivity between the gem-bisphosphonate group and the reactive X
group: only the gem-bisphosphonate groups are complexed at the
surface of the magnetic particle.
[0311] Advantageously, unlike the prior art, isolation of the
complexed magnetic particles does not require any laborious
purification steps, for example using dialysis or treatment on a
resin.
[0312] After complexation, the acid magnetic particles (p)
complexed by the compounds of formula (I) form a flocculate which
can be, according to a preferred embodiment, isolated from the
reaction medium by simple magnetic separating.
[0313] Prior to step (ii), the recovered flocculate is washed
several times with water so as to remove the excess compounds of
formula (I) which have not reacted.
[0314] At the end of step (i), the recovered flocculate is
generally suspended in an aqueous solution having a pH of between 6
and 7, which results in disaggregation of the flocculate
(peptization) and the production of a colloidal solution.
[0315] According to a preferred embodiment, a prior step is carried
out consisting in suspending the flocculate formed in step (i) in
an aqueous solution having a basic pH of between 10 and 11, before
returning to a pH of between 6 and 7. This treatment at basic pH is
in fact aimed at reinforcing the bonding of the gem-bisphosphonate
compound to the surface of the acid magnetic particle (p).
[0316] According to an advantageous aspect, the acid magnetic
particles (p) complexed by the gem-bisphosphonate compounds,
obtained at the end of step (i), are relatively non-polydispersed
and exhibit a "hydrodynamic size" profile very similar to the
starting acid ferrofluid. This complexing step does not therefore
produce any phenomenon of aggregation, which makes it possible to
avoid any addition filtration step.
[0317] According to another advantageous aspect, the magnetic
particles complexed by the gem-bisphosphonate compounds are
obtained with very good iron yields, generally between 70 to
95%.
[0318] The acid magnetic particles (p) grafted with compounds of
formula (I) are advantageously stable at physiological pH in the
presence, optionally, of a solvent, which makes it possible to
carry out the coupling of the biovectors directly in ferrofluid
medium.
[0319] The prior art did not in any way suggest grafting
gem-bisphosphonate compounds onto acid particles.
[0320] According to another advantageous aspect, the reactive X
group of the compound of formula (I) grafted to the surface of the
particle has very good reactivity with respect to the biovectors,
and makes it possible to obtain high coupling yields. This
therefore makes it possible to decrease the losses of biovectors
used and to thus decrease the cost of the specific magnetic
particle ultimately obtained.
[0321] Another particularly advantageous aspect is the
determination of the degree of coupling of the biovector to the
magnetic particle (p) grafted with compounds of formula (I). In
fact, the composition, determined by elemental analysis (C, P, Fe),
of the magnetic particle (p) grafted with compounds of formula (I)
makes it possible to exactly determine the phosphorus/iron ratio,
and the elemental analysis of the final particles onto which a
biovector is grafted makes it possible in the same way to determine
the nitrogen/phosphorus or carbon/phosphorus ratio, which makes it
possible to calculate the number of biovectors on each particle.
This type of analysis is completely impossible on the magnetic
particles obtained by "Molday" synthesis, coated with polymers such
as dextran to which specific affinity molecules have been grated,
due to the polydispersed nature of the coating polymer.
[0322] Step (ii) consists in bringing the magnetic particles (p)
complexed by gem-bisphosphonate compounds into contact with a
sufficient amount of biovector, generally in ferrofluid medium,
with stirring at ambient temperature for a few hours, the amount of
biovector used being fixed as a function of the degree of coupling
that it is desired to obtain.
[0323] At the end of step ii), a filtration step can be carried out
in order to separate the magnetic particles (p), coupled with
biovectors, from the possible biovectors which have not
reacted.
[0324] According to a preferred variant, the acid magnetic
particles (p) used in step (i) are obtained according to a process
comprising the steps consisting in:
[0325] a) preparing a colloidal solution of acid ferrofluid;
[0326] b) treating the solution obtained in step a) with a nitric
acid solution;
[0327] c) isolating the acid flocculate obtained in step b);
[0328] d) carrying out a peptization.
[0329] Steps a) to c) correspond, in this variant, to steps i.1)
and i.2) described above. The preparation of the colloidal solution
of acid ferrofluid carried out in step a) is well known and
described in many references. Mention may in particular be made to
patent FR 7918842 and to the publications C. R. Acad. Sc. Paris
(Jul. 7, 1980), t. 291--series C and IEE Transactions on Magnetics,
1981, vol. MAG-17, n.sup.o 2, p. 1247.
[0330] By way of indication, "acid" and "alkaline" ferrofluids are
generally obtained by bringing an aqueous solution containing
Fe.sup.2+ and Fe.sup.3+ salts in suitable proportions into contact
with a basic solution, in general aqueous ammonia.
[0331] The peptization or disaggregation of the gelatinous
precipitate obtained with an acid solution, generally of perchloric
or nitric acid, results in an "acid" ferrofluid being obtained,
whereas peptization with a solution of tetramethylammonium
hydroxide (TMAOH) leads to "alkaline" ferrofluids being
obtained.
[0332] "Alkaline" ferrofluids are not suitable for the invention in
that the process for obtaining them is such that their hydrodynamic
size cannot be readily controlled, and that, after grafting of the
compounds of formula (I), they require long and laborious
purification steps such as dialysis or filtration on resins. In
addition, since TMAOH is toxic, the biomedical application of the
resulting alkaline ferrofluids means that further treatments aimed
at removing all traces of TMAOH must be carried out.
[0333] On the other hand, it is possible to envision, in the
context of the present invention, the use of "acid" ferrofluids
resulting from the conversion of "alkaline" ferrofluids according
to processes described in the literature, in particular in the
following publications: R. Massart, V. Cabuil (Journal de
Chimie-Physique [Journal of Chemistry-Physics], 1987, 84, n.sup.o
7-8, p. 967-973); R. Massart, C R Acad. Sc. Paris, t. 291 (Jul. 7,
1980), series C1.
[0334] According to a preferred embodiment of the process according
to the invention, the acid ferrofluid used in step a) comprises
magnetic particles based on ferrite, preferably on maghemite or on
magnetite.
[0335] In this context, the acid ferrofluids based on ferrite
resulting from further treatment with a solution of
Fe(NO.sub.3).sub.3 are particularly preferred. This further
treatment, carried out at the end of step a), makes it possible to
carry out a core oxidation of the magnetic particles (p) based on
ferrite, and advantageously to decrease the phenomena of cellular
toxicity associated with the presence of Fe.sup.2+ ions. In
addition, this treatment makes it possible to increase the
stability of the final ferrofluid solution obtained at the end of
step d).
[0336] Advantageously, the treatment used in step b) makes it
possible to control the hydrodynamic size of the magnetic particles
obtained in step a).
[0337] Thus, the inventors have discovered, unexpectedly, that the
size of the magnetic particles obtained in step a) can be decreased
provided that the concentrations and duration of treatment with
nitric acid are adjusted.
[0338] Preferably, use is made in step b) of a nitric acid solution
having a concentration of between 1.5 and 4 mol/L. In particular,
in this case, the treatment is applied for a period ranging
generally from 1 hour to 48 hours, and preferably for a period at
least equal to 2 H, and preferably less than 24 H.
[0339] At the end of this treatment, the flocculate obtained is
isolated in step c) advantageously by simple magnetic separation
and washed several times with an organic solvent such as
acetone.
[0340] Finally, the flocculate isolated in step c) is suspended in
a volume of aqueous solvent, optionally in the presence of a
water-miscible organic solvent allowing peptization, that is to say
disaggregation of the flocculate and the production of a colloidal
solution of acid ferrofluid.
[0341] The invention is also aimed at the compositions, as such,
comprising acid magnetic particles (p) complexed by compounds of
formula (I) and coupled to biovectors, which can be obtained by the
process comprising steps (i) and (ii).
[0342] According to another aspect, a subject of the invention is a
composition, as such, comprising the acid magnetic particles (p)
complexed by compounds of formula (I), which can be obtained in
step (i) of the process according to the invention.
[0343] Also included in the invention are the compositions
comprising a mixture of these magnetic particles with the magnetic
particles coupled to biovectors.
[0344] Applications
[0345] The invention also relates to contrast products for medical
magnetic resonance imaging, which comprise the compositions of acid
magnetic particles (p) optionally coupled to biovectors, optionally
combined with a pharmaceutically acceptable vehicle and with
pharmaceutically acceptable additives.
[0346] When the particle is not coupled to a biovector, the X group
of the gem-bisphosphonate compound is selected such that it does
not induce a toxic reaction in vivo. In this context, it is
particularly preferred for X to represent --COON or --NH.sub.2. In
the absence of coupling to a biovector, the compositions of
magnetic particles (p) complexed with the compounds of formula (I)
may, by way of example, be used as a constrast agent for
angiography by MRI, and also for other MRI applications.
[0347] The compositions of magnetic particles (p) complexed with
compounds of formula (I) coupled to biovectors are particularly
useful for specific magnetic resonance imaging (MRI), in particular
the specific magnetic resonance imaging of an organ or of a
pathology.
[0348] In the absence of coupling to a biovector, the compositions
of magnetic particles (p) complexed with the compounds of formula
(I) may, by way of example, be used as a contrast agent for
angiography by MRI, and also for other nonspecific MRI
applications.
[0349] The relaxivities r.sub.1 and r.sub.2 of a magnetic contrast
product give a measure of its magnetic efficiency and make it
possible to assess its influence on the signal recorded.
[0350] In this context, the inventors have shown that the
compositions of magnetic particles (p) complexed by the
gem-bisphosphonate compounds exhibit very advantageous relaxivities
r.sub.1 and r.sub.2, making it possible to obtain a large increase
in the proton relaxation rates (R.sub.1=1/T1 and R.sub.2=1/T2).
This effect on the relaxation rates then makes it possible to
obtain a very good contrast in MRI, in the targeted areas.
[0351] When the magnetic particles are coupled to biovectors, in
particular to ligands specific for a given target biological
receptor, the compositions of the invention may be used as contrast
agents for specific imaging by MRI, in particular for
characterization and therapeutic monitoring in many pathologies: by
way of indication, mention may be made of cardiovascular diseases
(atheroma plaque imaging), cancers (tumors, metastases),
inflammatory and degenerative diseases (multiple sclerosis,
rheumatoid arthritis, Alzheimer's disease).
[0352] Thus, according to another aspect, a subject of the
invention is a method of diagnosis or of therapeutic monitoring in
which a composition comprising acid magnetic particles (p)
optionally coupled to at least one biovector is administered to a
patient, which makes it possible to visualize an organ or a
pathology in the patient by magnetic resonance.
[0353] By way of example, the final particle compositions for which
the biovector used is a folic acid derivative, a metalloprotease
inhibitor or an antibody, for example the anti-CEA or anti-mucin
type, may be particularly useful as an MRI contrast agent in
oncology for the detection and characterization of tumors and for
obtaining an overall picture of cancer extention (metastases).
[0354] Similarly, the compositions of final particles to which are
coupled biovectors such as phosphatidylserine derivatives make it
possible to target inflammatory and degenerative diseases, atheroma
plaques or stress.
[0355] Similarly, the compositions of final particles to which are
coupled biovectors such as peptides containing the RGD
(arginine-glycine-aspartic acid) sequence make it possible to
target integrins expressed during angiogenesis and in thrombotic
and inflammatory phenomena. They may therefore be used to monitor,
by MRI, the effectiveness of anti-angiogenic treatment in
cancerology or anti-thrombotic treatment in cardiology.
[0356] In general, the use of biovectors such as monoclonal
antibodies or fragments thereof (Fab, Fab'.sub.2, sFv) make it
possible to achieve high specificity in vivo for targeting
pathological areas where the receptors corresponding to the
antibodies used are overexpressed.
[0357] Advantageously, it has been shown that the relaxivities of
the particles (p) complexed by compounds of formula (I) and coupled
with a biovector are not altered in comparison with the
relaxivities of the intermediate without biovector. In addition, it
has been shown that the effectiveness of these compositions does
not depend on the nature of the biovector coupled to the particles
(p) complexed by compounds of formula (I).
[0358] The compositions of magnetic particles which may or may not
be coupled to biovectors are also useful for nuclear medicine,
using a radioactive iron isotope during synthesis.
[0359] According to another aspect, the compositions of acid
magnetic particles (p) coupled to biovectors according to the
invention can be used for delivering a medicinal product by
magnetic targeting. In this context, the magnetic particles (p) are
coupled to biovectors having pharmacological and/or cytotoxic
properties which can be optionally released under the action of a
radiofrequency field in the targeted area.
[0360] A subject of the invention is thus a method of therapeutic
treatment in which a composition comprising acid magnetic particles
(p) coupled to at least one biovector, in combination with a
pharmaceutically acceptable vehicle, is administered to a patient
requiring such a treatment.
[0361] The compositions of acid magnetic particles (p) coupled to
biovectors according to the invention are also useful for the
treatment by hyperthermia of pathological regions. In this context,
a biovector is generally chosen which has a particular affinity for
a receptor present in the targeted area, and a radiofrequency
field, preferably an outside alternating radiofrequency field, is
applied opposite said targeted area.
[0362] The compositions of magnetic particles (p) coupled to
biovectors according to the invention are also useful for cell
labeling, and in particular the labeling of stem cells, which can
be carried out in vitro or ex vivo.
[0363] Thus, according to another aspect, the subject of the
invention is a method for the in vitro detection and/or separation
of cells, comprising: [0364] i) bringing cells into contact with a
composition comprising acid magnetic particles (p) coupled to at
least one biovector capable of labeling the cells; and [0365] ii)
detecting and/or separating the labeled cells obtained.
[0366] Preferably, the biovector is a ligand specific for a
biological receptor expressed by the cells to be labeled.
[0367] In this context a biovector capable of binding specifically
to a stem cell receptor is coupled to the magnetic particles (p).
The stem cells thus labeled, administered to a patient, then make
it possible to follow, under the application of a magnetic field,
the movement, location and survival of the exogenous cells by MRI
visualization. This application is particularly useful for
detecting pathologies related to cell dysfunction.
[0368] A subject of the invention is thus a method for the
diagnosis of pathologies related to cell dysfunction, comprising:
[0369] i) labeling stem cells from a patient according to the
abovementioned method, ex vivo; [0370] ii) administering the
labeled stem cells to said patient; which makes it possible, under
the application of a magnetic field, to follow the movement,
location and survival of the exogenous cells by MRI visualization
and thus to detect pathologies related to cell dysfunction.
[0371] Finally, the compositions of magnetic particles (p) coupled
to biovectors are particularly useful for magnetic cell sorting,
carried out in vitro. In this case, the biovector is generally a
ligand selected so as to differentiate and/or separate the
compounds bearing a receptor for said biovector from those which do
not bear said receptor. By way of example, the biovector may
represent a specific annexin, a ligand capable of binding to
phosphatidylserine (PS) or phosphatidylethanolamine (PE) in the
presence of Ca.sup.2+. In this case, the use according to the
invention makes it possible to differentiate and/or separate the
cells exhibiting an abnormal state, that is to say capable of
binding to the magnetic particles (p) coupled to annexin
biovectors, from the normal cells incapable of binding these
particles.
[0372] The terms "pharmaceutically acceptable" refer to a
composition or a molecular entity which does not produce any
allergic reaction, side effects or adverse effects when it is
administered to an animal or to a human in an appropriate
manner.
[0373] The expression "pharmaceutically acceptable vehicles" can
denote, in the context of the present description, a solvent, a
dispersion medium, antibacterial agents and antifungal agents. The
use of such media and agents falls within the competence of the
person skilled in the art.
[0374] Unless the conventional media or agents are incompatible
with the magnetic particles (p) complexed by compounds of formula
(I), and coupled to biovectors, their use in the diagnostic
compositions can be envisioned. Additional active ingredients can
also be incorporated into the compositions according to the
invention.
[0375] It is understood that, in the methods of treatment of the
invention, the compositions are administered in a therapeutically
effective amount, which can be adjusted by the person skilled in
the art depending on the pathology targeted, the condition of the
patient, his or her weight, his or her age, etc.
[0376] The compositions of the invention are preferably
administered parenterally, or orally, other routes of
administration not, however, being excluded, such as, for example,
administration rectally, administration in the form of an
intravenous injection being particularly preferred.
[0377] When oral administration is envisioned, the compositions of
the invention are in the form of gelatin capsules, effervescent
tablets, bare tablets or coated tablets, sachets, sugar-coated
tablets, oral ampoules or solutes, microgranules or forms with
prolonged or controlled release.
[0378] When parenteral administration is envisioned, the
compositions of the invention are in the form of lyophilizates
ready to be reconstituted or of injectable solutes and suspensions
packaged in ampoules or bottles or prefilled syringes, for slow
venous infusion or intravenous injection as a bolus.
[0379] The forms for oral administration are prepared by mixing the
magnetic particles (p) optionally coupled to biovectors, in
particular to an active substance, with various types of excipients
or of vehicles such as fillers, disintegrating agents, binders,
dyes, flavor enhancers, and the like, and then formulating the
mixture in a gelatin capsule, in particular a controlled-release
gelatin capsules.
[0380] The aqueous solubility and the low osmolality of the
particles of the invention make it possible to prepare isotonic
aqueous solutions of high concentration and acceptable viscosity
for injection.
[0381] The forms for parenteral administration are obtained in the
conventional manner by mixing the magnetic particles (p) optionally
coupled to biovectors with buffers, stabilizers, preserving agents,
solubilizing agents, isotonic agents and suspending agents. In
accordance with known techniques, these mixtures are then
sterilized and then packaged in the form of intravenous injections
or of lyophilizates ready to be reconstituted in a sterile
pharmaceutically acceptable vehicle.
[0382] The solutions can be prepared extemporaneously from the
lyophilized powder containing the magnetic particles (p) optionally
coupled to the biovectors and, optionally, additives and a sterile
solvent, or, due to the great stability of the magnetic particles
coupled to the biovectors in solution, the solutions can be
provided to the radiologist in bottles, ampoules, syringes or
bags.
[0383] For the preparation of suppositories, the magnetic particles
according to the invention are mixed, in a manner known in itself,
with a suitable basic constituent such as polyethylene glycol or
hemisynthetic glycerides.
[0384] The unit doses will depend on the composition of the
magnetic particles (p) coupled to biovectors, on the route of
administration, on the type of diagnosis to be established, and
also on the patient. The unit doses will in general be from 0.01
.mu.mol to 100 .mu.mol of magnetic particles for an individual of
average size (75 kg).
[0385] As demonstrated in the examples, the inventors have
succeeded in obtaining compounds which are both stable and
specific, using, as covering, gem-bisphosphonate compounds. It is
specified that, more broadly, the applicant is studying coverings
capable of binding, firstly, to magnetic particles and, secondly,
to at least one biovector. Various coverings are studied, of
formula Y1-Y2-Y3, Y1 being at least one group capable of coupling
to nanoparticles, Y3 being at least one group capable of
interacting with at least one biovector, Y2, which may be present
or absent, being a is linker between Y1 and Y3. Among Y1s,
phosphonates, phosphates, bisphosphonates and analogs other than
gem-bisphosphonates, hydroxamates or gem-hydroxamates are in
particular studied.
[0386] In the remaining text, examples of preparation of
compositions according to the invention are described by way of
illustration.
[0387] In the examples below, the following generalities are
specified.
[0388] In the following, the abbreviations M, M/L, theoretical M, N
and M/z, ES.sup.+, ES.sup.-, kD and TLC have the following
meanings:
[0389] M or M/L: molar concentration (mol/liter).
[0390] theoretical M: theoretical molecular mass.
[0391] N: normality.
[0392] M/z: mass charge determined by mass spectrometry.
[0393] ES.sup.+: positive-mode electrospray.
[0394] ES.sup.-: negative-mode electrospray.
[0395] TFA: trifluoroacetic acid
[0396] kD: unit of molecular mass (kiloDalton)
[0397] TLC: thin layer chromatography
[0398] Z ave: hydrodynamic diameter measured by PCS
[0399] Poly .sigma.: polydispersity measured by PCS.
[0400] The chemical nomenclature which follows is derived from the
ACD/NAME software (Advanced Chemistry Development Inc, Toronto,
Canada), according to IUPAC rules.
[0401] Total Iron Assay:
[0402] The iron is assayed by atomic absorption spectroscopy
(VARIAN AA10 spectrophotometer) after mineralization with
concentrated HCl and dilution with respect to a standard range of
ferric ions (0, 5, 10, 15 and 20 ppm).
[0403] Particle Size:
[0404] Hydrodynamic Diameter of the Grafted Particle (Z Ave)=PCS
Size:
[0405] Determined by PCS (Malvern 4700 device, laser 488 nm at
90.degree.) on a sample diluted to .about.1 millimolar with water
for injectable preparation, filtered through 0.22 .mu.m.
[0406] PCS=Photon Correlation Spectroscopy--Reference: R. Pecora in
J. of Nano. Res. (2000), 2, p. 123-131.
[0407] Diameter of the Magnetic Particles (p) (Before Grafting)
[0408] Determined by deconvolution of magnetization curves
(measurements taken on a SQUID magnetometer) at various
temperatures. [Reference: R. W. Chantrell in IEEE Transactions on
Magnetics (1978), 14(5), p. 975-977.
[0409] Determination of the Degree of Grafting by
Microanalysis:
[0410] Compound A: expressed in moles per hundred moles of total
iron.
[0411] Other compounds: expressed in moles per hundred moles of
iron and in moles per hundred moles of compound A.
[0412] Structural Analyses:
[0413] By mass spectroscopy (MICROMASS VG Quattro II device) with
an Electrospray source.
[0414] Relaxivity Measurements:
[0415] The relaxation times T1 and T2 were determined by standard
procedures on a Minispec 120 device (Bruker) at 20 Mhz (0.47 T) and
37.degree. C. The longitudinal relaxation time T1 is measured using
an inversion recuperation sequence and the transverse relaxation
time T2 is measured by a CPMG technique.
[0416] The relaxation rates R1(=1/T1) and R2(=1/T2) were calculated
for various concentrations of total metal (ranging from
0.1.times.10.sup.-3 to 1.times.10.sup.-3 mol/L) in aqueous solution
at 37.degree. C. The correlation between R1 or R2 as a function of
concentration is linear, and the slope represents the relaxivity r1
(R1/C) or r2 (R2/C) expressed as (1/second).times.(1/mmol/L), i.e.
(mM.sup.-1.s.sup.-1).
EXAMPLE OF COMPOUNDS OF FORMULA (I)
Example 1
Preparation of Compound A
##STR00021##
[0417] 1) Diethyl-1-[ethoxyphosphoryl]vinyl phosphonate
[0418] 13 g (0.433 mol) of paraformaldehyde and 10 ml (0.097 mol)
of diethylamine are solubilized under hot conditions in 250 ml of
methanol. 24 g (8.67.times.10.sup.-2 mol) of
diethyl[ethoxy(propyl)phosphoryl]methyl phosphonate are then added.
The mixture is brought to reflux for 24 hours. The reaction medium
is concentrated under vacuum. The concentrate is taken up twice
with 250 ml of toluene and is then concentrated under vacuum.
[0419] The oil obtained is solubilized in 125 ml of toluene. 0.14 g
of para-toluenesulfonic acid are added. The mixture is brought to
reflux for 24 hours with a Dean-Stark trap and is then concentrated
to dryness under vacuum.
[0420] The product is extracted with 500 ml of CH.sub.2Cl.sub.2 and
is then washed twice with 250 ml of water. The organic phase is
dried over MgSO.sub.4 and concentrated under vacuum.
[0421] The crude product is purified on 625 g of Merck Geduran.RTM.
silica gel (40-63 .mu.m). Elution: CH.sub.2Cl.sub.2/acetone--50/50
(TLC--SiO.sub.2: Rf=0.45).
[0422] 18.4 g are isolated with a yield of 71%.
[0423] Mass spectrum: M/z=301.4 (ES+) theoretical M=300.2
[0424] C.sup.13 spectrum: (s) 148.8 ppm, (t) 134.8-131.5-128.2 ppm,
(s) 62.2 ppm, (s) 16.7 ppm
[0425] H.sup.1 spectrum: (t) 6.9-6.8-6.6 ppm, (unresolved peak) 3.9
ppm, (t) 1.15 ppm.
2) Diethyl 2-[2.2-bis(diethylphosphoryl)ethyl]malonate
[0426] 1.6 g (0.01 mol) of diethyl malonate, 0.07 g (0.001 mol) of
sodium ethoxide and 3 g (0.01 mol) of
diethyl[ethoxy(propyl)phosphoryl]vinyl phosphonate are stirred for
15 min in 15 ml of ethanol. [TLC: SiO.sub.2; eluent
CH.sub.2Cl.sub.2/acetone 50/50--Rf=0.6].
[0427] 5 ml of a saturated NH.sub.4Cl solution are added to the
ethanolic solution. The mixture is concentrated under vacuum. The
residue is extracted with 30 ml of ethyl acetate and washed twice
with 5 ml of water. The organic phase is dried over MgSO.sub.4 and
is then evaporated to dryness.
[0428] The oil obtained is purified on 200 g of Merck Geduran.RTM.
silica
[0429] (40-63 .mu.m). Elution CH.sub.2Cl.sub.2/acetone 50/50
Rf=0.6
[0430] 3.8 g are isolated with a yield of 82%.
[0431] Mass spectrum: M/z 460.9 (ES+), theoretical M=460.
3) 4,4-Diphosphonobutanoic acid
[0432] 7 g (15.7.times.10.sup.-2 mol) of diethyl
2-[2.2-bis(diethylphosphoryl)ethyl]malonate are brought to reflux
for 8 hours in 350 ml of HCl [5N].
[0433] The brown oil obtained is purified on 60 g of silanized
silica 60 (0.063-0.200 mm) with water elution [HPLC
monitoring].
[0434] 3.6 g are isolated with a yield of 92%.
[0435] Mass spectrum: M/z 249 (ES+), theoretical M=248
[0436] HPLC: column: Hypercarb.RTM. 250.times.4 mm Detection: 202
nm
[0437] Isocratic eluent 99/1: 0.034 N H.sub.2SO.sub.4/CH.sub.3CN
-Tr=8 min
Examples 2 to 7
Preparation of Biovectors
Example 2
Preparation of Compound B
1) Ethyl-2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate
[0438] 9.7 g (0.1 mol) of maleimide are solubilized in 50 ml of
ethyl acetate and 1.1 ml of N-methylmorpholine at 5.degree. C.
[0439] 1.1 ml of ethyl chloroformate are added dropwise. The
mixture is stirred for 1/2 hour. The insoluble material is removed
by filtration. The filtrate is washed with 50 ml of water. The
organic phase is dried over MgSO.sub.4 and is then concentrated
under vacuum. The oil obtained is purified on Merck Geduran.RTM.
silica gel (40-63 .mu.m). Elution: CH.sub.2Cl.sub.2/ACOEt
66/34.
[0440] 16.9 g are isolated with a yield of 50%.
[0441] Mass spectrum: M/z=170.1 (ES+) theoretical M=169
2) tert-Butyl
3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)propylcarbamate
[0442] 0.515 g (2.96.times.10.sup.-3 mol) of compound B1) is
solubilized in 20 ml of saturated NaHCO.sub.3. The solution is
filtered through a filter with a porosity of 0.45 .mu.m and is then
cooled to 0.degree. C.
[0443] 0.5 g (2.96.times.10.sup.-3 mol) of ethylcarbonylmaleimide
in solution in 25 ml of tetrahydrofuran is added dropwise. The
mixture is stirred for 15 minutes.
[0444] 25 ml of tetrahydrofuran are added and the reaction medium
is stirred for 45 min. The pH is adjusted to 6 with qs HCl [1N].
The product is extracted with 2.times.200 ml of ethyl acetate. The
organic phase is dried over MgSO.sub.4 and is then concentrated
under vacuum.
[0445] 737 mg are isolated with a yield of 98%.
3) 1-(3-Aminopropyl)1H-pyrrol-2.5-dione
[0446] 0.7 g (2.75.times.10.sup.-3 mol) of compound B2) is stirred
in 25 ml of HCl [2 N] for 24 hours.
[0447] The solution is concentrated under vacuum. 500 mg are
obtained with a yield of 88%.
[0448] Mass spectrum: M/z=155.1 (ES+) theoretical M=154.1
Example 3
Preparation of Compound C
1-Deoxy-1-[(2,3-dihydropropyl)amino]hexitol
[0449] 173 g (1.9 mol) of 3-amino-1,2-propanediol are solubilized
in 1 liter of methanol. 360 g (2 mol) of glucose are added. The
mixture is stirred for 24 hours at ambient temperature. This
solution is reduced under 20 bar of hydrogen at 60.degree. C. with
50 g of palladium-on-charcoal and 450 ml of water for 6 hours.
[0450] The reaction medium is filtered over clarcel at 35.degree.
C. The filtration liquors are concentrated to a volume of 850 ml.
The concentrate is run into 3 liters of isopropyl alcohol kept at
35.degree. C. with a bath of hot water. The precipitate is filtered
off and dried under vacuum. 272 g are isolated with a yield of
53%.
[0451] Mass spectrum: M/z=256.4 (ES+) theoretical M=253.3
Example 4
Preparation of the Compound D:
N-[(2,3-Dihydroxypropyl)-(2,3,4,5,6-pentahydroxyhexyl)]-2,4,6-tribromo-5--
(glycylamino)isophthalamide
[0452] 255 g (1.9 mol) of compound C are dissolved at 80.degree. C.
in 2.5 liters of 1-methyl-2-pyrrolidinone for 30 minutes. The
reaction medium is cooled to 40.degree. C. before the addition of
67.1 g (0.635 mol) of Na.sub.2CO.sub.3 dried at 170.degree. C. and
of 407.8 g (0.635 mol) of
5-phthalamido(acetamido)-2,4,6-triaminoisophthalic acid chloride,
keeping the temperature below 45.degree. C.
[0453] The medium is maintained at 40.degree. C. for 2 hours and is
then cooled to 20.degree. C., before filtration of the minerals
over Clarcel.
[0454] 610 ml of water are added to the filtrate and the mixture is
heated to 70.degree. C. before the addition of 44.8 ml of hydrazine
hydrate (0.889 mol). The reaction medium is heated at 90.degree. C.
for 2 hours.
[0455] The pH is adjusted to 1 with QS HCl [12 N]. The precipitate
formed is removed by filtration. The filtration liquors are run
into 30 liters of ethanol.
[0456] The product obtained is dissolved in 3.6 liters of water,
the pH is adjusted to 6.5 with QS sodium hydroxide [2N] and the
solution is diafiltered. HPLC monitoring of the purification. HPLC
purity=98.2%.
[0457] HPLC conditions:
[0458] Column: C18 Symetry.RTM. 250.times.4 mm (5 .mu.m).
Detection: 254 nm
[0459] Isocratic eluent: 99/1: H.sub.2SO.sub.4 0.034
N/CH.sub.3CN
[0460] Bromine assays=23.6% (i.e. a purity of 97.2%)
Example 5
Preparation of Compound E:
N-N'-[bis(2,3,4,5,6-pentahydroxyhexyl)]-2,4,6-tribromo-5-(glycylamino)iso-
phthalamide
##STR00022##
[0462] Compound E can be prepared according to the procedure
described in patent: EP 0 922 700 A1.
Example 6
Preparation of Compound F:
(18S)-1-amino-18{[(2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl]amino}be-
nzoyl)amino]-15-oxo-4-trioxa-14-azanonadecan-19-oic acid
##STR00023##
[0464] 9.74 g (0.0214 mol) of
(2S)-2-[(4-{[(2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl]amino}benzoyl-
)amino]-5-methoxy-5-oxopentanoic acid (which can be prepared
according to the procedure in J. Am. Chem. Soc., 1997, 119,
10004-10013) are solubilized in 60 ml of DMSO at ambient
temperature. 235 ml (1.07 mol) de
4,7,10-Trioxa-1,13-tridecanediamine are added. The mixture is
stirred for 48 H at ambient temperature.
[0465] The reaction medium is run into a mixture of 1.5 l of
CH.sub.3CN and 1.5 l of ethyl ether. The precipitate obtained is
filtered off and dried under vacuum.
[0466] The crude product is solubilized in 100 ml of pyridine [0.1
N] and is purified on 1 kg of silanized silica 60 (0.063-0.200 mm)
with an elution of 9/1 pyridine [0.1 N]--methanol [HPLC
monitoring]. 2.7 g are isolated with a yield of 26% and an HPLC
purity of 97.3%.
[0467] HPLC conditions:
[0468] Column: C18 Symetry.RTM. 250.times.4 mm (5 .mu.m).
Detection: 254 nm
[0469] Isocratic eluent: 99/1: 0.034 N
H.sub.2SO.sub.4/CH.sub.3CN.
Example 7
Preparation of Compound G
L-Propyl-L-leucyl-N.sup.1-hydroxyglycinamide
[0470] 1 g (2.3.times.10.sup.-3 mol) of
1-[(benzyloxy)carbonyl]-L-propyl-L-leucyl-N.sup.1-hydroxyglycinamide
is dissolved in 100 ml of methanol.
[0471] 100 mg of 50% hydrated Pd/C are added to the solution. The
mixture is stirred for 6 hours at ambient temperature under 50 PSI
of hydrogen.
[0472] The catalyst is removed by filtration and the filtrate is
concentrated under vacuum. The product is stored at 4.degree.
C.
[0473] Quantitative yield.
[0474] Mass spectrum: M/z=371.2 (ES-) theoretical M=372.4
Examples 8 and 9
Examples of Preparation of Colloidal Solutions of Acid Magnetic
Particles (p)
Example 8
[0475] A solution of 36 g (0.181 mol) of FeCl.sub.2.4H.sub.2O and
20 ml of HCl at 37% in 150 ml of H.sub.2O is introduced into a
mixture consisting of 3 liters of water and 143 ml (0.302 mol) of
FeCl.sub.3 at 27%. 250 ml of NH.sub.4OH at 25% are introduced
rapidly with vigorous stirring. The mixture is stirred for 30 min.
The liquors are removed by magnetic separation. The ferrofluid is
washed 3 times consecutively with 2 liters of water.
[0476] The nitric ferrofluid is stirred for 15 min with 200 ml of
HNO.sub.3 [2M], and the supernatant is removed by magnetic
separation.
[0477] The nitric ferrofluid is brough to reflux with 600 ml of
water and 200 ml of Fe(NO.sub.3).sub.3 [1M] for 30 min. The
supernatant is removed by magnetic separation.
[0478] The nitric ferrofluid is stirred for 15 min with 200 ml of
HNO.sub.3 [2M], the supernatant being removed by magnetic
separation.
[0479] The nitric ferrofluid is washed 3 times with 3 liters of
acetone, and is then taken up with 400 ml of water. The solution is
evaporated under vacuum until a final volume of 250 ml is
obtained.
TABLE-US-00002 Concentration Z ave Diameter Ms M/L nm Poly .sigma.
SQUID emu/cm.sup.3 4.85 40 nm 0.22 8.5 nm 275
[0480] Ms (emu/cm.sup.3)=magnetization at saturation
[0481] Z ave=Hydrodynamic size by PCS in unimodal mode
[0482] Poly .sigma.: peak polydispersity by PCS
[0483] SQUID=diameter of the nongrafted particle (p) estimated by
deconvolution of magnetization curves measured on a SQUID
magnetometer
Example 9
[0484] 108 g (0.543 mol) of FeCl.sub.2.4 H.sub.2O in 450 ml of
H.sub.2O is introduced into a solution of 4 liters of water and 429
ml (0.906 mol) of FeCl.sub.3 at 27%. 750 ml of NH.sub.4OH at 25%
are introduced rapidly with stirring (1200 rpm). The mixture is
stirred for 30 min. The liquors are removed by magnetic separation.
The ferrofluid is washed twice consecutively with 3 liters of
water.
[0485] The nitric ferrofluid is stirred for 1/4 H with 3 liters of
HNO.sub.3 [2M], and the supernatant is removed by magnetic
separation.
[0486] The nitric ferrofluid is brought to reflux with 1300 ml of
water and 700 ml of Fe(NO.sub.3).sub.3 [1M] for 30 min (600 rpm).
The supernatant is removed by magnetic separation.
[0487] The nitric ferrofluid is stirred for 15 min with 3 liters of
HNO.sub.3 [2M], the supernatant being removed by magnetic
separation.
[0488] The nitric ferrofluid is washed 3 times with 3 liters of
acetone, and is then taken up with 600 ml of water. The solution is
evaporated under vacuum until a final volume of 250 ml is
obtained.
[0489] At this Stage, the Following Characteristics are
Obtained:
TABLE-US-00003 Concentration % yield M/L Z ave (nm) Poly .sigma.
81.8 4.45 31.3 0.21
[0490] Z ave=Hydrodynamic size by PCS in unimodal mode.
[0491] Treatment
[0492] 200 ml of the preceding solution are stirred in 2.4 liters
of HNO.sub.3 for 4 hours. The supernatant is removed by magnetic
separation. The nitric ferrofluid is washed twice with 3 liters of
acetone, and is then taken up with 400 ml of water. The solution is
evaporated under vacuum until a final volume of 250 ml is
obtained.
TABLE-US-00004 Concentration % yield M/L Z ave (nm) Poly .sigma. 77
2.742 23.3 0.20
Examples 10 to 12
Examples of Complexation of the Magnetic Particles (p) by Compounds
of Formula (I)
Example 10
[0493] 50 ml of example 8 at 4.85 M/L are diluted in 3 liters of
water. A solution of 1.3 g (5.24.times.10.sup.-3 mol) of compound A
from Example 1 in 100 ml of water is introduced dropwise. Stirring
is maintained for 30 minutes. The flocculate is isolated by
magnetic separation and is then washed 3 times with 3 liters of
water.
[0494] It is redissolved with 700 ml of water at pH 7.2 with QS
NaOH [1 N]. The final solution is filtered through a 0.22 .mu.m
membrane.
[0495] Iron titer=0.252 M/L PCS size=67.9 nm
[0496] Fe=61.7% mass/mass
[0497] P=1.21% mass/mass
[0498] C=1.04% mass/mass
[0499] Degree of grafting [compound A/Fe]=1.86% mol/mol
Example 11
[0500] 50 ml of example 8 at 4.73 M/L are diluted in 3 liters of
water. A solution of 1.3 g (5.24.times.10.sup.-3 mol) of compound A
from Example 1 in 80 ml of water is introduced dropwise. Stirring
is maintained for 30 minutes. The flocculate is isolated by
magnetic separation and is then washed 3 times with 3 liters of
water.
[0501] It is redissolved with 700 ml of water at pH 11 with QS NaOH
[1N] and then stabilized at pH 7.2 with QS HCl [1N]. The final
solution is filtered through a 0.22 .mu.m membrane.
[0502] Iron titer=0.279 M/L PCS size=40.3 nm Poly .sigma.=0.19
[0503] Fe=63.9% mass/mass
[0504] P=1.38% mass/mass
[0505] C=1.07% mass/mass
[0506] Degree of grafting [compound A/Fe]=1.95% mol/mol
Example 12
[0507] 100 ml of example 9 at 2.742 M/L (PCS size=21.3 nm) are
diluted in 3 liters of water.
[0508] A solution of 1.5 g (6.03.times.10.sup.-3 mol) of compound A
from example 1 in 100 ml of water is introduced dropwise. The
stirring is maintained for 30 minutes. The floculate is isolated by
magnetic separation and is then washed 3 times with 3 liters of
water.
[0509] It is redissolved with 700 ml of water at pH 11 with QS NaOH
[1N] and then stabilized at pH 7.2 with QS HCl [1N]. The final
solution is filtered through a 0.22 .mu.m membrane.
[0510] Iron titer=0.285 M/L PCS size=25.6 nm
[0511] Fe=62.9% mass/mass
[0512] P=1.32% mass/mass
[0513] C=1.22% mass/mass
[0514] Degree of grafting [compound A/Fe]=1.90% mol/mol
[0515] Relaxivities:
[0516] 20 MHz (0.47 T) -37.degree. C.--in aqueous solution
TABLE-US-00005 r.sub.1 r.sub.2 (mM.sup.-1 s.sup.-1) (mM.sup.-1
s.sup.-1) 35 .+-. 2 103 .+-. 5
Examples 13 to 18
Coupling of the Acid Magnetic Particles (p) Complexed by Compounds
of Formula (I), with Biovectors
Example 13
[0517] 36 mg (1.88.times.10.sup.-4 mol) of compound B are dissolved
in 50 ml of example 10 at 0.252 M/L. The pH is adjusted to 5.8 with
QS HCl [1N].
[0518] A drop of triethylamine and 72 mg of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
added to the reaction medium and the mixture is stirred at ambient
temperature for 18 hours.
[0519] The solution is ultrafiltered through a PALL.RTM. stirring
cell having a cutoff threshold of 30 kD. 600 ml of filtrate are
removed in order to obtain a final solution of 35 ml.
[0520] [Fe]=0.212 M/L PCS size=69.2 nm
[0521] Fe=62.8% mass/mass
[0522] P=1.21% mass/mass
[0523] C=2.23% mass/mass
[0524] N=0.48% mass/mass
[0525] Degree of grafting [compound A/Fe]=1.74% mol/mol
[0526] Degree of grafting [compound B/Fe]=1.37% mol/mol
[0527] Degree of grafting [compound B/compound A]=78.6%
Example 14
[0528] 0.193 g (7.6.times.10.sup.-4 mol) of compound C from example
3 is dissolved in 13.55 ml of example 11 at 0.279 M/L. The pH is
adjusted to 6.2.
[0529] 171 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride are added. The mixture is stirred at ambient
temperature for 24 hours. The solution is ultrafiltered through a
PALL.RTM. stirring cell having a cutoff threshold of 30 kD. 500 ml
of filtrate are removed. The retentate is adjusted to 30 ml
[0530] [Fe]=0.155 M/L PCS size=43.7 nm
[0531] Fe=58.4% mass/mass
[0532] P=1.17% mass/mass
[0533] C=2.80% mass/mass
[0534] Degree of grafting [compound A/Fe]=1.80% mol/mol
[0535] Degree of grafting [compound C/Fe]=1.67% mol/mol
[0536] Degree of grafting [compound C/compound A]=93%
Example 15
[0537] 0.358 g (3.78.times.10.sup.-4 mol) of compound D from
example 4 are dissolved in 13.55 ml of example 11 at 0.279 M/L.
[0538] 86 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride are added. The mixture is stirred at ambient
temperature for 24 hours. The solution is ultrafiltered through a
PALL.RTM. stirring cell having a cutoff threshold of 30 kD. 500 ml
of filtrate are removed. The retentate is adjusted to 30 ml
[0539] [Fe]=0.134 M/L PCS size=41.3 nm
[0540] Fe=51.2% mass/mass
[0541] P=1.16% mass/mass
[0542] C=5.79% mass/mass
[0543] Br=3.07% mass/mass
[0544] Degree of grafting [compound A/Fe]=2% mol/mol
[0545] Degree of grafting [compound D/Fe]=1.4% mol/mol
[0546] Degree of grafting [compound D/compound A]=68.4%
Example 16
[0547] 0.853 g (7.6.times.10.sup.-4 mol) of compound E from example
5 are dissolved in 13.55 ml of example 11 at 0.279 M/L. The pH is
adjusted to 6.2.
[0548] 171 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride are added. The mixture is stirred at ambient
temperature for 24 hours. The solution is ultrafiltered through a
PALL.RTM. stirring cell having a cutoff threshold of 30 kD. 500 ml
of filtrate are removed. The retentate is adjusted to 30 ml
[0549] [Fe]=0.132 M/L PCS size=41.6 nm poly .sigma.=0.22
[0550] Fe=52.3% mass/mass
[0551] P=0.77% mass/mass
[0552] C=5.86% mass/mass
[0553] Br=2.65% mass/mass
[0554] Degree of grafting [compound A/Fe]=1.33% mol/mol
[0555] Degree of grafting [compound E/Fe]=1.18% mol/mol
[0556] Degree of grafting [compound E/compound A]=89%
Example 17
[0557] 90 ml of a solution containing 1.35 g/L of compound F from
example 6 are introduced into 66.3 ml of example 12 at 0.285 M/L.
121.7 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride are added. The pH is adjusted to 6.8 and the mixture
is stirred at ambient temperature for 18 hours.
[0558] The reaction medium is ultrafiltered through a PALL.RTM.
stirring cell having a cutoff threshold of 30 kD. The retentate is
adjusted to 100 ml and is then filtered through 0.22 .mu.m.
[0559] [Fe]: 0.213 M/L PCS size=29 nm
[0560] Fe=59.6% mass/mass
[0561] P=1.29% mass/mass
[0562] C=2.67% mass/mass
[0563] N=0.38% mass/mass
[0564] Degree of grafting [compound A/Fe]=1.95% mol/mol
[0565] Degree of grafting [compound F/Fe]=0.28% mol/mol
[0566] Degree of grafting [compound F/compound A]=14.5%
[0567] Relaxivities:
[0568] 20 MHz (0.47 T) -37.degree. C.--in aqueous solution
TABLE-US-00006 r.sub.1 r.sub.2 (mM.sup.-1 s.sup.-1) (mM.sup.-1
s.sup.-1) 36 .+-. 2 105 .+-. 5
[0569] These results show that coupling of the biovector does not
modify the relaxivities of the noncoupled intermediate (example
12).
Example 18
[0570] 100 ml of a solution of example 12 at 0.285 M/L are
ultrafiltered through a PALL.RTM. 30 KD stirring cell. 202 mg of
compound G from example 7 are added to this solution. The pH is
adjusted to 6.1 with QS HCl [0.1 N].
[0571] 203 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride are added and the mixture is stirred at ambient
temperature for 6 hours.
[0572] The reaction medium is ultrafiltered through a PALL.RTM.
stirring cell having a cutoff threshold of 30 kD. 800 ml of
filtrate are removed. The retentate is adjusted to 140 ml and is
then filtered through 0.22 .mu.m.
[0573] [Fe]: 0.223 M/L PCS size=28.4 nm
[0574] Fe=57.9% mass/mass
[0575] P=1.37% mass/mass
[0576] C=3.49% mass/mass
[0577] N=0.89% mass/mass
[0578] Degree of grafting [compound A/Fe]=2.13% mol/mol
[0579] Degree of grafting [compound G/Fe]=1.5% mol/mol
[0580] Degree of grafting [compound G/compound A]=71%
Examples 19 to 21
Comparison of the Magnetic Particles Complexed by a
Gem-Bisphosphonate Compound and Coupled to a Biovector, Derived
from an "Alkaline" Ferrofluid Precursor, with those Derived from an
"Acid" Ferrofluid Precursor
Example 19
Preparation of an " Alkaline " Ferrofluid in TMAOH Medium
[0581] A mixture consisting of a solution of 2.96 ml of FeCl.sub.3
at 27%, and of a solution of 0.6 g of FeCl.sub.2.4H.sub.2O in 40 ml
of water is rapidly run into 200 ml of tetramethylammonium
hydroxide [1 M]. The mixture is stirred for 1 hour at ambient
temperature. The solution is filtered through a 0.22 .mu.m membrane
and kept under nitrogen at 4.degree. C.
[0582] [Fe]: 0.0403 M/L PCS size=68.2 nm Polydispersity
.sigma.=0.48
Example 20
Complexation of the "Alkaline" Ferrofluid from Example 19 by the
Gem-Bisphosphonate Compound from Example 1
[0583] A solution consisting of 416 mg of compound A from example 1
in solution in 5 ml of water is introduced into 25 ml of example
19. The mixture is stirred for 1 hour and then ultrafiltered
through a PALL.RTM. stirring cell having a cutoff threshold of 30
kD.
[0584] 600 ml of filtrate are removed. The retentate is adjusted to
40 ml and is then filtered through a 0.22 .mu.m membrane.
Example 21
Coupling of the Complexed Magnetic Particles from Example 20 with
Compound E from Example 5
[0585] 1.7 g of compound E are introduced into the 40 ml of
solution from example 20. The pH is adjusted to 6.2. 345 mg of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
then added and the mixture is stirred for 24 hours at ambient
temperature.
[0586] The reaction medium is ultrafiltered through a PALL.RTM.
stirring cell having a cutoff threshold of 30 kD.
[0587] 700 ml of filtrate are removed. The retentate is adjusted to
40 ml and is then filtered through 0.22 .mu.m.
[0588] PCS size=60 nm Polydispersity .sigma.=0.60
[0589] Fe=37.9% mass/mass
[0590] P=1.70% mass/mass
[0591] C=12.96% mass/mass
[0592] N=1.84% mass/mass
[0593] Br=6.42% mass/mass
[0594] Degree of grafting [compound A/Fe]=4.04% mol/mol
[0595] Degree of grafting [compound E/Fe]=3.95% mol/mol
[0596] Degree of grafting [compound E/compound A]=97.7%
[0597] Precursors
TABLE-US-00007 N.sup.o Size Z ave (nm) Polydispersity .sigma. Acid
ferrofluid Example 8 40 0.22 Alkaline ferrofluid Example 19 68.2
0.48
[0598] Final molecules (acid or basic ferrofluid+gem-bisphosphonate
compound (Ia)+biovector compound E):
TABLE-US-00008 N.sup.o Size Z ave (nm) Polydispersity .sigma. Acid
process Example 16 41.6 0.22 Alkaline process Example 21 60
0.60
[0599] Conclusions:
[0600] For examples 19 and 21, the inventors have advantageously
demonstrated that, when an alkaline ferrofluid comparable to that
used in document J. of Coll. and Interf., Science, 2001, 238, pp.
37-42 is used, the hydrodynamic size is greater from the precursor
step (alkaline ferrofluid--example 19) and the polydispersity is
high (.sigma.=0.48) compared to the acid ferrofluid (.sigma.=0.22);
this has an influence on the hydrodynamic size of the final product
(after grafting of the compound Ia and of the biovector--compound
E), with an even greater polydispersity (.sigma.=0.6). The
difference in % (compound A/iron) between the two types of final
particles derived respectively from an acid ferrofluid or a basic
ferrofluid indicates the definite formation of a double layer of
compound I (gem-bisphosphonate) when an "alkaline" precursor is
used, which is prejudicial to good stability. In the process
according to the invention, 90% of the sites are grafted with a
compound of formula (I), advantageously forming a monolayer.
Examples 22 to 25
Preparation of Other Biovectors
Example 22
Preparation of Compound H
##STR00024##
[0601] 1) Benzyl
5-{[(2R)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-3-(1H-indol-3-yl)pro-
panoyl]amino}pentylcarbamate
[0602] 15.63 g of
((2R)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-3-(1H-indol-3-yl)propan-
oic acid) are solubilized at ambient temperature in 300 ml of
tetrahydrofuran.
[0603] 10 g of (benzyl-5-aminopentylcarbamate) and then 5.1 ml of
triethylamine are added. The mixture is stirred for 5 minutes at
ambient temperature.
[0604] 5.94 g of hydroxy-1-benzotriazole hydrate and then 8.43 g of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
subsequently added to the reaction medium. The mixture is stirred
for 24 hours at ambient temperature.
[0605] The insoluble material is removed by filtration. The
filtrate is concentrated under vacuum.
[0606] The oil obtained is run into 150 ml of water and is stirred
for 1 hour at ambient temperature.
[0607] The precipitate obtained is filtered, and washed with
vigorous stirring in 100 ml of water for 30 minutes at ambient
temperature.
[0608] The precipitate is filtered off, then washed with 200 ml of
ethyl ether, and then dried under vacuum. 22.78 g are isolated with
a yield of 96.4%.
[0609] Rf=0.94 on silica (MERCK) in CH.sub.2Cl.sub.2/CH.sub.3OH
(8/2)
[0610] HPLC: Waters C18 symmetry column, 5 .mu.m, (250 mm.times.4.6
mm) [0611] Gradient: water-TFA (pH 2.95)/CH.sub.3CN: [0612]
.lamda.=210 nm
TABLE-US-00009 [0612] % water-TFA Time in min. Flow rate (ml/min.)
pH 2.95 % CH.sub.3CN 0 1 98 2 50 1 10 90 55 1 5 95
[0613] Retention time of the expected product: 42 min
[0614] Mass spectrum: m/z=645.3 (ES.sup.+ mode) (theoretical
M=644)
2) Benzyl
5-{[(2R)-2-amino-3-(1H-indol-3-yl)propanoyl]amino}pentylcarbamat-
e
[0615] 20 g of (benzyl
5-{[(2R)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-3-(1H-indol-3-yl)pro-
panoyl]amino}pentylcarbamate) are solubilized at ambient
temperature in 280 ml of tetrahydrofuran.
[0616] 41.4 ml of piperidine and 20 ml of water are then added. The
mixture is stirred for 3 hours at ambient temperature. The reaction
medium is concentrated under vacuum.
[0617] The oil obtained is purified over 1400 g of Merck
Geduran.RTM. silica (40-63 .mu.m). Elution:
CH.sub.2Cl.sub.2/CH.sub.3OH (95/5).
[0618] 12.77 g are isolated with a yield of 97.4%.
[0619] Rf=0.64 on silica (MERCK) in CH.sub.2Cl.sub.2/CH.sub.3OH
(95/5)
[0620] HPLC: Waters C18 symmetry column, 5 .mu.m, (250 mm.times.4.6
mm) [0621] Gradient: water-TFA (pH 2.95)/CH.sub.3CN: [0622]
.lamda.=210 nm
TABLE-US-00010 [0622] % water-TFA Time in min Flow rate (ml/min) pH
2.95 % CH.sub.3CN 0 1 98 2 17 1 66 34 19 1 64 36 50 1 0 100
[0623] Retention time of the expected product: 18.2 min
[0624] Mass spectrum: m/z=423.2 (ES.sup.+ mode) (theoretical
M=422)
3)
Tert-butyl(12R)-12-(1H-indol-3-ylmethyl)-15-isobutyl-3,11,14-trioxo-1-p-
henyl-2-oxa-4,10,13-triazaheptadecan-17-oate
[0625] 9.16 g of (2-(2-tert-butoxy-2-oxoethyl)-4-methylpentanoic
acid), synthe-sized according to the procedure described in (J.
Med. Chem. 1998, Vol 41 (2): p. 209), are solubilized at ambient
temperature in 160 ml of tetrahydrofuran.
[0626] A homogeneous solution made up of 16.81 g (0.039 mol) of
(benzyl
5-{[(2R)-2-amino-3-(1H-indol-3-yl)propanoyl]amino}pentylcarbamate)
in 165 ml of tetrahydrofuran is added all at once, followed by,
successively, 8.3 ml of triethylamine, 6.45 g of
hydroxy-1-benzotriazole hydrate and 9.5 g of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
[0627] The mixture is stirred for 12 hours at ambient temperature.
The insoluble material is removed by filtration. The filtrate is
concentrated under vacuum.
[0628] The oil obtained is purified on 1900 g of Merck Geduran.RTM.
silica (40-63 .mu.m). Elution of the correct product with
CH.sub.2Cl.sub.2/CH.sub.3OH (98/2).
[0629] 24 g are isolated with a yield of 95.3%.
[0630] Rf=0.42 on silica (MERCK) in CH.sub.2Cl.sub.2/CH.sub.3OH
(95/5)
[0631] HPLC: Waters C18 symmetry column, 5 .mu.m, (250 mm.times.4.6
mm) [0632] Gradient: water-TFA (pH 2.95)/CH.sub.3CN: [0633]
.lamda.=220 nm
TABLE-US-00011 [0633] % water-TFA Time in min Flow rate (ml/min) pH
2.95 % CH.sub.3CN 0 1 98 2 30 1 0 100
[0634] Retention time of the expected product: 25.7 min
[0635] Mass spectrum: m/z=635.6 (ES.sup.+ mode) (theoretical
M=634)
4)
(12R)-12-(1H-indol-3-ylmethyl)-15-isobutyl-3,11,14-trioxo-1-phenyl-2-oa-
x-4,10,13-triazaheptadecan-17-oic acid
[0636] 1.4 g of dithioerythritol are added to a solution made up of
100 ml of CH.sub.2Cl.sub.2 and 100 ml of trifluoroacetic acid. 10 g
of
(tert-butyl(12R)-12-(1H-indol-3-ylmethyl)-15-isobutyl-3,11,14-trioxo-1-ph-
enyl-2-oxa-4,10,13-triazaheptadecan-17-oate) are then added. The
mixture is stirred for 30 minutes at ambient temperature and then
concentrated under vacuum.
[0637] The oil obtained is purified on 700 g of Merck Geduran.RTM.
silica (40-63 .mu.m). Elution of the correct product with
CH.sub.2Cl.sub.2/CH.sub.3OH (98/2).
[0638] 6.19 g are isolated with a yield of 68%.
[0639] Rf=0.57 on silica (MERCK) in CH.sub.2Cl.sub.2/CH.sub.3OH
(8/2)
[0640] HPLC: C18 Waters symmetry column, 5 .mu.m, (250 mm.times.4.6
mm) [0641] Gradient: water-TFA (pH 2.95)/CH.sub.3CN: [0642]
.lamda.=210 nm
TABLE-US-00012 [0642] Time in Flow rate % Water-TFA min. (ml/min.)
pH 2.95 % CH.sub.3CN 0 1 98 2 30 1 0 100
[0643] Retention time of the expected product: 20.8 min
[0644] Mass spectrum: m/z=579.0 (ES.sup.+ mode) (theoretical
M=578)
5)
Benzyl(8R)-8-(1H-indol-3-ylmethyl)-11-isobutyl-7,10,13-trioxo-16-phenyl-
-15-oxa-6,9,14-triazahexadec-1-ylcarbamate
[0645] 6.11 g of
((12R)-12-(1H-indol-3-ylmethyl)-15-isobutyl-3,11,14-trioxo-1-phenyl-2-oxa-
-4,10,13-triazaheptadecan-17-oic acid) are solubilized at ambient
temperature in 160 ml of tetrahydrofuran. The reaction medium is
cooled to 0.degree. C.
[0646] 1.69 g of O-benzylhydroxylamine hydrochloride, 3 ml de
triethylamine, 1.86 g of hydroxy-1-benzotriazole hydrate and then
2.63 g of 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide
hydrochloride are successively added to the reaction medium and the
mixture is stirred for 24 hours at ambient temperature.
[0647] The insoluble material is removed by filtration. The
filtrate is concentrated under vacuum.
[0648] The oil obtained is run into 200 ml of water. The
precipitate obtained is filtered off, and washed in 100 ml of water
at ambient temperature.
[0649] The precipitate is filtered off and dried under vacuum.
[0650] 5.7 g are isolated with a yield of 79.2%.
[0651] Rf=0.29 on silica (MERCK) in CH.sub.2Cl.sub.2/CH.sub.3CN
(5/5)
[0652] HPLC: C18 Waters symmetry column, 5 .mu.m, (250 mm.times.4.6
mm) [0653] Gradient: water-TFA (pH 2.95)/CH.sub.3CN: [0654]
.lamda.=210 nm
TABLE-US-00013 [0654] Time in Flow rate % water-TFA min (ml/min) pH
2.95 % CH.sub.3CN 0 1 98 2 30 1 0 100
[0655] Retention time of the expected product: 22.7 min
[0656] Mass spectrum: m/z=684.3 (ES.sup.+ mode) (theoretical
M=683)
6)
N.sup.1-[(1R)-2-[(5-aminopentyl)amino]-1-(1H-indol-3-ylmethyl)-2-oxoeth-
yl]-N.sup.4-hydroxy-2-isobutylsuccinamide
[0657] 0.32 g of
(benzyl(8R)-8-(1H-indol-3-ylmethyl)-11-isobutyl-7,10,13-trioxo-16-phenyl--
15-oxa-6,9,14-triazahexadec-1-ylcarbamate) is dissolved in 30 ml of
methanol. A half-spatula of 50% hydrated Pd/C is added to the
solution.
[0658] The mixture is stirred for 4 hours thirty minutes at ambient
temperature under 50 PSI of hydrogen. The catalyst is removed by
filtration over clarcel and the filtrate is concentrated under
vacuum. Quantitative yield.
[0659] The product obtained is purified on 5 g of Merck silanized
silica 60 gel (63-200 .mu.m). Elution of the expected product with
H.sub.2O.
[0660] 0.11 g is isolated with a yield of 51%.
[0661] HPLC: C18 Waters symmetry column, 5 .mu.m, (250 mm.times.4.6
mm) [0662] Gradient: water-TFA (pH 2.95)/CH.sub.3CN: [0663]
.lamda.=220 nm
TABLE-US-00014 [0663] Time in Flow rate % water-TFA min. (ml/min.)
pH 2.95 % CH.sub.3CN 0 1 98 2 30 1 0 100
[0664] Retention time of the expected product: 9.4 min
[0665] Mass spectrum: m/z=460.3 (ES.sup.+ mode) (theoretical
M=459)
Example 23
Preparation of Compound I
1) O-(2-Para-toluenesulfonylethyl)-O'-methylpolyethylene glycol
1100
[0666] 16.2 g of polyethylene glycol monomethyl ether of average
mass 1100 (Fluka.RTM.) are dissolved in 25 ml of dichloromethane.
This solution is cooled to a temperature of 5.degree. C.
[0667] 0.4 g of triethylbenzylammonium chloride, 10.4 ml of an
aqueous 30% sodium hydroxide solution and then 2.94 g of
para-toluenesulfonyl chloride in solution in 15 ml of
CH.sub.2Cl.sub.2 are then added at 5.degree. C.
[0668] The reaction is continued for 24 hours at ambient
temperature. The extraction of the reaction medium produces, after
drying over magnesium and evaporation, 17.2 g of solid. The yield
is 95%.
[0669] TLC: Merck.RTM. SiO.sub.2 eluent: dichloromethane/methanol:
9/1.
[0670] Detection: UV 254 nm and Draggendorf: Rf=0.4 [0671]
Infrared: 1356 cm.sup.-1 (v: R--O--SO.sub.2--R)
2) O-(2-Azidoethyl)-O'-methylpolyethylene glycol 1100
[0672] 12.4 g of
O-(2-paratoluenesulfonylethyl)-O'-methylpolyethylene glycol 1100
are dissolved at ambient temperature in 90 ml of DMF. 2 g of sodium
azide are then introduced. Stirring is maintained for 18 hours at
ambient temperature. Extraction of the reaction medium with
CH.sub.2Cl.sub.2 produces, after evaporation, 4.4 g of compound.
The yield is 40%
[0673] TLC: Merck.RTM. SiO.sub.2 eluent: dichloromethane/methanol:
9/1.
[0674] Detection: UV 254 nm and Draggendorf Rf=0.39 [0675]
Infrared: 2104 cm.sup.-1 (v: N.sub.3)
3) O-(2-Aminoethyl)-O'-methylpolyethylene glycol 1100
[0676] 4.3 g of O-(2-azidoethyl)-O'-methylpolyethylene glycol 1100
and 0.4 g of Pd/C (5%) in solution in 100 ml of ethanol are
introduced into an autoclave. The reaction is carried out under a
hydrogen pressure of 2.5 bar, at 25.degree. C., for 4 hours.
[0677] The catalyst is removed by filtration over celite. After
evaporation, 3.5 g are thus isolated with a yield of 85%.
[0678] TLC: Merck.RTM. SiO.sub.2 eluent: dichloromethane/methanol:
9/1.
[0679] Detection: "Primary amine" specific ninhydrin [0680]
Infrared: 3390 cm.sup.-1 (v NH.sub.2)
Example 24
Preparation of Compound J
1) Tert-butyl
3-{2-12-(3-aminopropoxy)ethoxy]ethoxy}propylcarbamate
[0681] A solution of 5 g di(tert-butyl)dicarbonate dissolved in 50
ml of CH.sub.2Cl.sub.2 is added, by means of a dropping funnel, to
15 g of 3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}-1-propanamine
dissolved in 100 ml of CH.sub.2Cl.sub.2.
[0682] The reaction medium is stirred at ambient temperature for 18
hours and is then half-concentrated so as to be washed with water
(50 ml), dried and concentrated. The solution obtained is filtered
over MERCK Geduran.RTM. silica (40-63 .mu.m) with ethyl acetate and
then methanol.
[0683] 4.7g of product are obtained with a yield of 64%.
[0684] Mass spectrum: M/z=321.3 (ES+) theoretical M=320.4
[0685] TLC: Rf=0.05; Silica SiO.sub.2;
eluent=CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH (93/7/traces)
2)Tert-butyl
16-chloro-15-oxo-4,7,10-trioxa-14-azahexadec-1-ylcarbamate
[0686] 10 g of tert-butyl
3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propyl-carbamate is dissolved
in 50 ml of CH.sub.2Cl.sub.2, in a three-necked flask equipped with
a bath of acetone and ice. 5 g of K.sub.2CO.sub.3 dissolved in 50
ml of water and 5 g of chloroacetyl chloride dissolved in 50 ml of
CH.sub.2Cl.sub.2 are added dropwise and simultaneously, by means of
a dropping funnel. During the addition, the temperature of the
medium is maintained at 0.degree. C.
[0687] When all the reagents have been added, the reaction medium
is maintained with magnetic stirring, at ambient temperature for 1
hour.
[0688] The 2 phases obtained are separated and the chloromethylene
phase is then washed with water until neutral pH, dried over
Na.sub.2SO.sub.4, filtered, and then concentrated.
[0689] 11.2 g of product are obtained with a yield of 90%.
[0690] Mass spectrum: M/z=397.4 (ES +) theoretical M=396.9
[0691] TLC: Rf=0.6; silica SiO.sub.2; eluent=CH.sub.2Cl.sub.2/MeOH
(9/1)
3) Tert-butyl
30-amino-15-oxo-4,7,10,21,24,27-hexaoxa-14,17-diazatriacont-1-ylcarbamate
[0692] 6.8 g de 3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propylamine
are diluted in 50 ml of CH.sub.3CN in the presence of
K.sub.2CO.sub.3. 3.5 g of tert-butyl
16-chloro-15-oxo-4,7,10-trioxa-14-azahexadec-1-ylcarbamate
dissolved in 25 ml of CH.sub.3CN are added to this suspension by
means of a dropping funnel.
[0693] The reaction medium is refluxed for 2 hours and is then
placed at ambient temperature for filtration over sintered glass.
The filtrate is then evaporated to dryness.
[0694] The residue obtained is dissolved in 50 ml of
CH.sub.2Cl.sub.2 in order to perform washes with 4 times 20 ml of
water. The chloromethylene phase is dried over Na.sub.2SO.sub.4,
filtered and concentrated sufficiently to envision direct
purification of Merck Geduran.RTM. silica (40-63 .mu.m) with an
elution of CH.sub.2Cl.sub.2/MeOH (50/50) (retention factor
Rf=0.15).
[0695] 1.8 g are obtained with a yield of 50%.
[0696] HPLC: C18 symmetry column (3.5 .mu.m) 50.times.2.1 mm [0697]
Detector: UV 201 nm [0698] Mobile phase: A: water+TFA (pH 2.8)
[0699] B: CH.sub.3CN
TABLE-US-00015 [0699] Time Flow rate (min.) (ml/min.) % A % B 0 1
98 2 4 1 0 100 5 1 0 100
[0700] The expected product has a retention time=1.8 min.
[0701] Mass spectrum: M/z=581 (ES+) theoretical M=580.7.
4) Tert-butyl
30-[(2-ethoxy-3,4-dioxo-1-cyclobuten-1-yl)amino]-15-oxo-4,7,10,21,24,27-h-
exaoxa-14,17-diazatriacont-1-ylcarbamate
[0702] 0.25 g of tert-butyl
30-amino-15-oxo-4,7,10,21,24,27-hexaoxa-14,17-diazatriacont-1-ylcarbamate
is dissolved in 1.5 ml of CH.sub.2Cl.sub.2. 57.5 .mu.l of
3,4-diethoxy-3-cyclobutene-1,2-dione are added.
[0703] The reaction medium is stirred magnetically for 18 hours at
ambient temperature. This intermediate is not isolated.
[0704] HPLC: C18 symmetry column (3.5 .mu.m) 50.times.2.1 mm [0705]
Detector: UV 201 nm [0706] Mobile phase: A: water+TFA (pH 2.8)
[0707] B: CH.sub.3CN
TABLE-US-00016 [0707] Time Flow rate (min) (ml/min) % A % B 0 1 98
2 4 1 0 100 5 1 0 100
[0708] The expected product has a retention time of 2.30 min.
5) Tert-butyl
17-acetyl-30-[(2-ethoxy-3,4-dioxo-1-cyclobuten-1-yl)amino]-15-oxo-4,7,10,-
21,24,27-hexaoxa-14,17-diazatriacont-1-ylcarbamate
[0709] 40.6 .mu.l of acetic anhydride are added to the reaction
medium obtained in step 4). The reaction medium is stirred at
ambient temperature and is then purified directly on 5 g of Merck
Geduran.RTM. silica (40-63 .mu.m) at atmospheric pressure, with an
eluent of CH.sub.2Cl.sub.2/EtOH (9/1).
[0710] 0.27g of product in the form of an oil is obtained with a
yield of 60%.
[0711] Mass spectrum: M/z=748 (ES+) theoretical M=746.9
6)
1-({2-[(14-acetyl-34,34-dimethyl-16,32-dioxo-4,7,10,21,24,27,33-heptaox-
a-14,17,31-triazapentatriacont-1-yl)amino]-3,4-dioxo-1-cyclobuten-1-yl}ami-
no)-18-[(4-{[(2-amino-4-oxo-3,4-dihydro-6-pteridinyl)methyl]amino}benzoyl)-
amino]-15-oxo-4,7,10-trioxa-14-azanonadecan-19-oic acid
[0712] 0.16 g of tert-butyl
17-acetyl-30-[(2-ethoxy-3,4-dioxo-1-cyclobuten-1-yl)amino]-15-oxo-4,7,10,-
21,24,27-hexaoxa-14,17-diazatriacont-1-ylcarbamate and 0.205 g of
example 6 (compound F) are solubilized in 3 ml of water.
[0713] Drops of water saturated with Na.sub.2CO.sub.3 are added to
the medium so as to obtain a pH=9.2. The medium is stirred
magnetically for 18 hours and is then filtered through Whatmann
paper, neutralized with HCl (1N) and taken up in 15 ml of
CH.sub.2Cl.sub.2.
[0714] The chloromethylene phase is washed with 1 ml of water and
then dried over Na.sub.2SO.sub.4, filtered and evaporated. The
residue is taken up in 10 ml of EtOH in order to be precipitated
from 100 ml of Et.sub.2.
[0715] 0.2 g of crystals (saffron orange color) are obtained with a
yield of 69%.
[0716] HPLC: C18 symmetry column (3.5 .mu.m) 50.times.2.1 mm [0717]
Detector: UV 201 nm [0718] Mobile phase: A: water+TFA (pH 2.8)
[0719] B: CH.sub.3CN
TABLE-US-00017 [0719] Time Flow rate (min) (ml/min) % A % B 0 1 98
2 4 1 0 100 5 1 0 100
[0720] The expected product has a retention time of 2.40 min.
[0721] Mass spectrum: M/z=673 (z=2; ES+) theoretical M=1344.5
7)
1-({2-[(14-acetyl-30-amino-16-oxo-4,7,10,21,24,27-hexaoxa-14,17-diazatr-
iacont-1-yl)amino]-3,4-dioxo-1-cyclobuten-1-yl}amino)-18-[(4-{[(2-amino-4--
amino-4-oxo-3,4-dihydro-6-pteridinyl)methyl]amino}benzoyl)amino]-15-oxo-4,-
7,10-trioxa-14-azanonadecan-19-oic acid
[0722] 0.2 g of
1-({2-[(14-acetyl-34,34-dimethyl-16,32-dioxo-4,7,10,21,24,27,33-heptaoxa--
14,17,31-triazapentatriacont-1-yl)amino]-3,4-dioxo-1-cyclobuten-1-yl}amino-
)-18-[(4-{[(2-amino-4-oxo-3,4-dihydro-6-pteridinyl)methyl]amino}benzoyl)am-
ino]-15-oxo-4,7,10-trioxa-14-azanonadecan-19-oic acid is dissolved
in 2 ml of trifluoroacetic acid at ambient temperature.
[0723] The reaction medium is then precipitated in 20 ml of
Et.sub.2O, filtered and washed thoroughly with ethyl ether.
[0724] 0.18 g of crystals are obtained with a yield of 78%.
[0725] HPLC: C18 symmetry column (3.5 .mu.m) 50.times.2.1 mm [0726]
Detector: UV 201 nm [0727] Mobile phase: A: water+TFA (pH 2.8)
[0728] B: CH.sub.3CN
TABLE-US-00018 [0728] Time Flow rate (min) (ml/min) % A % B 0 1 98
2 4 1 0 100 5 1 0 100
[0729] The expected product has a retention time of 1.9 min.
[0730] Mass spectrum: M/z=1245 (ES+) theoretical M=1244.4
Example 25
Preparation du Compound K
1) tert-butyl
35-[(2-ethoxy-3,4-dioxo-1-cyclobuten-1-yl)amino]-3,6,9,12,15,18,21,24,27,-
30,33-undecaoxapentatriacont-1-ylcarbamate
[0731] 0.8 g of tert-butyl
35-amino-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-pentatriacont-1-ylcarbam-
ate is solubilized at ambient temperature in 4 ml of EtOH. 165
.mu.l of 3,4-diethoxy-3-cyclobutene-1,2-dione are added to this
solution.
[0732] The mixture is stirred at ambient temperature for 18 hours.
After evaporation of the solvent, the residue obtained is dissolved
in 5 ml of CH.sub.2Cl.sub.2 so as to be purified on 30 g of MERCK
Geduran.RTM. silica (4-63 .mu.m) with an eluent made up of
CH.sub.2Cl.sub.2/MeOH (95/5).
[0733] 0.6 g of oil is obtained with a yield of 70%.
[0734] TLC: Rf=0.5; silica SiO.sub.2; eluent=CH.sub.2Cl.sub.2/MeOH
(9/1)
[0735] HPLC: C18 symmetry column (3.5 .mu.m) 50.times.2.1 mm [0736]
Detector: UV 201 nm [0737] Mobile phase: A: water+TFA (pH 2.8)
[0738] B: CH.sub.3CN
TABLE-US-00019 [0738] Time Flow rate (min) (ml/min) % A % B 0 1 98
2 4 1 0 100 5 1 0 100
[0739] The expected product has a retention time of 2.78 min.
[0740] Mass spectrum: M/z=769.5 (ES+) theoretical M=768.9
2)
18-[(4-{[(2-amino-4-oxo-3,4-dihydro-6-pteridinyl)methyl]amino}benzoyl)a-
mino]-1-({2-[(39,39-dimethyl-37-oxo-3,6,9,12,15,18,21,24,27,30,33,38-dodec-
aoxa-36-azatetracont-1-yl)amino]-3,4-dioxo-1-cyclobuten-1-yl}amino)-15-oxo-
-4,7,10-trioxa-14-azanonadecan-19-oic acid
[0741] 0.3 g of tert-butyl
35-[(2-ethoxy-3,4-dioxo-1-cyclobuten-1-yl)amino]-3,6,9,12,15,18,21,24,27,-
30,33-undecaoxapentatriacont-1-ylcarbamate and 0.34 g of example 6
(compound F) are solubilized in 4.5 ml of water and 0.7 ml of
EtOH.
[0742] The pH of the, medium is increased to 9.2 with drops of
Na.sub.2CO.sub.3-saturated water. The reaction medium is stirred at
ambient temperature for 48 hours with the pH maintained at 9.2.
[0743] After evaporation of the ethanol, the aqueous solution
obtained is purified on a "Supervarioflash.RTM." chromatographic
column system with a K026033 cartridge (RP 18; 25-40 .mu.m), with
an eluent of water/CH.sub.3CN (98/2) and a flow rate of 20
ml/min.
[0744] 100 mg of product are obtained with a yield of 25%.
[0745] HPLC: C18 symmetry column (3.5 .mu.m) 50.times.2.1 mm [0746]
Detector: UV (275 nm) [0747] Mobile phase: A: water+TFA (pH 2.8)
[0748] B:CH.sub.3CN
TABLE-US-00020 [0748] Time flow rate (min) (ml/min) % A % B 0 1 98
2 4 1 0 100 5 1 0 100
[0749] The expected product has a retention time of 2.40 min.
[0750] Mass spectrum: M/z=684 (z=2; ES+) theoretical M=1366.5
3)18-[(4-{[(2-amino-4-oxo-3,4-dihydro-6-pteridinyl)methyl]amino}benzoyl)am-
ino]-1-({2-[(35-amino-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacont-
-1-yl)amino]-3,4-dioxo-1-cyclobuten-1-yl}amino)-15-oxo-4,7,10-trioxa-14-az-
anonadecan-19-oic acid
[0751] 0.3 g of
18-[(4-{[(2-amino-4-oxo-3,4-dihydro-6-pteridinyl)methyl]amino}benzoyl)ami-
no]-1-({2-[(39,39-dimethyl-37-oxo-3,6,9,12,15,18,21,24,27,30,33,38-dodecao-
xa-36-azatetracont-1-yl)amino]-3,4-dioxo-1-cyclobuten-1-yl}amino)-15-oxo-4-
,7,10-trioxa-14-azanonadecan-19-oic acid is solubilized in 0.3 ml
of trifluoroacetic acid.
[0752] After stirring for 15 minutes at ambient temperature, the
reaction medium is evaporated and then taken up in 30 ml of
Et.sub.2O.
[0753] After filtration, washing with Et.sub.2O and drying, 0.29 g
of product is obtained.
[0754] HPLC: C18 symmetry column (3.5 .mu.m) 50.times.2.1 mm [0755]
Detector: UV (275 nm) [0756] Mobile phase: A: water+TFA (pH 2.8)
[0757] B: CH.sub.3CN
TABLE-US-00021 [0757] Time Flow rate (min) (ml/min) % A % B 0 1 98
2 4 1 0 100 5 1 0 100
[0758] The expected product has a retention time of 1.87 min.
[0759] Mass spectrum: M/z=1267 (ES+) theoretical M=1266.4
Examples 26 to 37
Coupling of the Acid Magnetic Particles (p) Complexed by Compounds
of Formula (I) with Biovectors
Example 26
[0760] 1) 102.5 mg of compound F from example 6 are introduced into
50 ml of example 12 at 0.285 M/L. 76.6 mg of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
added. The pH is adjusted to 6.2 and the mixture is stirred for 18
hours at ambient temperature.
[0761] The reaction medium is directly ultrafiltered through a
PALL.RTM. stirring cell having a cutoff threshold of 30 kD. The
retentate is adjusted to 65 ml and is then filtered through 0.22
.mu.m, giving solution A.
[0762] Fe=67% mass/mass
[0763] P=1.29% mass/mass
[0764] C=1.95% mass/mass
[0765] N=0.26% mass/mass
[0766] Degree of grafting [compound A/Fe]=1.74% mol/mol
[0767] Degree of grafting [compound F/Fe]=0.17% mol/mol
[0768] Degree of grafting [compound F/compound A]=9.8%.
[0769] 2) 3 g of compound E from example 5 are introduced into 50
ml of solution A obtained in step 1). 600 mg of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
added. The pH is adjusted to 6.2 and the mixture is stirred for 18
hours at ambient temperature.
[0770] The reaction medium is directly ultrafiltered through a
PALL.RTM. stirring cell having a cutoff threshold of 30 kD. The
retentate is adjusted to 40 ml and is then filtered through 0.22
.mu.m.
[0771] [Fe]: 0.289 M/L PCS size=29.6 nm
[0772] Fe=56.6% mass/mass
[0773] P=1.1% mass/mass
[0774] C=7.27% mass/mass
[0775] N=1.1% mass/mass
[0776] Br=2.6% mass/mass
[0777] Degree of grafting [compound A/Fe]=1.75% mol/mol
[0778] Degree of grafting [compound E/Fe]=1.07% mol/mol
[0779] Degree of grafting [compound E/compound A]=61%
Example 27
[0780] 36.5 mg of compound H from example 22 are introduced into 30
ml of example 12 at 0.285 M/L. The pH is adjusted to 6.2 and 18 mg
of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
added. The mixture is stirred for 18 hours at ambient
temperature.
[0781] The reaction medium is directly ultrafiltered through a
PALL.RTM. stirring cell having a cutoff threshold of 30 kD. The
retentate is adjusted to 65 ml and is then filtered through 0.22
.mu.m.
[0782] [Fe]: 0.292 M/L PCS size=32.2 nm
[0783] Fe=66.0% mass/mass
[0784] P=1.48% mass/mass
[0785] C=3.35% mass/mass
[0786] N=0.69% mass/mass
[0787] Degree of grafting [compound A/Fe]=2.02% mol/mol
[0788] Degree of grafting [compound H/Fe]=0.83% mol/mol
[0789] Degree of grafting [compound H/compound A]=41%
Example 28
1) 9H-Fluoren-9-ylmethyl 6-(phosphonooxy)hexylcarbamate
[0790] 1.33 ml of POCl.sub.3 and 1.83 ml of triethylamine are
solubilized in 9 ml of heptane. 3 g of 6-(FMOC-amino)-1-hexanol in
solution in 30 ml of CH.sub.2Cl.sub.2 are introduced dropwise into
the preceding solution, maintaining a temperature of 0.degree.
C.
[0791] The reaction medium is stirred for 1 hour at 0.degree. C.
and then for 18 heures at ambient temperature. 30 ml of water are
added and the mixture is stirred for 2 hours at ambient
temperature.
[0792] The organic phase is washed with water and then concentrated
under vacuum. The resulting solid is taken up with water, filtered
and dried.
[0793] 3 g of product are obtained with a yield of 81% and an HPLC
purity of 60%.
[0794] HPLC: C18 X-TERRA column (5 .mu.m) 250.times.4.6 mm [0795]
Detector: UV 201 nm [0796] Mobile phase: A: solution of ammonium
carbonate, pH=9 [0797] B: CH.sub.3CN
TABLE-US-00022 [0797] Time Flow rate (min) (ml/min) % A % B 0 1 98
2 30 1 2 98
[0798] The expected product has a retention time of 14.1 min.
2) tert-Butyl
15-[(tert-butoxycarbonyl)amino]-1-(9H-fluoren-9-yl)-12-hydroxy-3-oxo-2,11-
,13-trioxa-4-aza-12-phosphahexadecan-16-oate 12-oxide
[0799] 3 g of 9H-fluoren-9-ylmethyl 6-(phosphonooxy)hexylcarbamate
and 3.73 g of Boc-serine-OtBu are dissolved in 54 ml of pyridine. A
solution of 6.49 g of triisopropylbenzenesulfochloride in 42 ml of
pyridine is added dropwise. The mixture is stirred at ambient
temperature for 48 hours.
[0800] 30 ml of water are added and the mixture is stirred for 4
hours and is then concentrated under vacuum. The residue obtained
is taken up in 100 ml of ethyl ether.
[0801] The insoluble material is removed by filtration. The
filtrate is concentrated under vacuum and is then purified on
silica (elution: 95% CH.sub.2Cl.sub.2--5% MeOH).
[0802] HPLC: C18 X-TERRA column (5 .mu.m) 250.times.4.6 mm [0803]
Detector: UV 201 nm [0804] Mobile phase: A: solution of ammonium
carbonate, pH=9 [0805] B: CH.sub.3CN
TABLE-US-00023 [0805] Time Flow rate (min.) (ml/min.) % A % B 0 1
98 2 30 1 2 98
[0806] The expected product has a retention time of 20 min.
3) tent-butyl
3-{[[(6-aminohexyl)oxy](hydroxy)phosphoryl]oxy}-2-[(tert-butoxycarbonyl)a-
mino]propanoate
[0807] 3 g of tert-butyl
15-[(tert-butoxycarbonyl)amino]-1-(9H-fluoren-9-yl)-12-hydroxy-3-oxo-2,11-
,13-trioxa-4-aza-12-phosphahexadecan-16-oate 12-oxide are stirred
in 0.62 ml of piperidine and 3 ml of water for 12 hours at ambient
temperature. The reaction medium is filtered and the crystals
obtained are washed twice with 50 ml of acetonitrile and then
recrystallized from 100 ml of acetonitrile.
[0808] Mass spectrum: M/z=441.3 (ES+) theoretical M=440
4)
16-(tert-butomarbonyl)-13-hydroxy-20,20-dimethyl-13-oxido-4,18-dioxo-1--
phosphono-12,14,19-trioxa-5,17-diaza-13-phosphahenicos-1-ylphosphonic
acid
[0809] 106 mg of tert-butyl
3-{[[(6-aminohexyl)oxy](hydroxy)phosphoryl]oxy}-2-[(tert-butoxycarbonyl)a-
mino]propanoate in solution in 2 ml of water are introduced into a
solution of 50 mg of compound A from example 1 in 4 ml of water.
The pH is adjusted to 6.2 and 60 mg of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
added. The mixture is stirred for 24 hours at ambient
temperature.
[0810] The product obtained is purified on Merck silanized silica
60 (63-200 .mu.m) with elution in a gradient (water/methanol) and
monitoring by HPLC.
[0811] 70 mg are obtained with a yield of 52%.
[0812] Mass spectrum: M/z=671.2 (ES+) theoretical M=670
5)
17-amino-1,1,14-trihydroxy-5-oxo-2-phosphono-13,15-dioxa-6-aza-1.lamda.-
.sup.5,14-diphosphaoctadecan-18-oic acid 1,14-dioxide
##STR00025##
[0814] 60 mg of
16-(tert-butoxycarbonyl)-13-hydroxy-20,20-dimethyl-13-oxido-4,18-dioxo-1--
phosphono-12,14,19-trioxa-5,17-diaza-13-phosphahenicos-1-yl-phosphonic
acid are stirred in 1 ml of TFA for 2 hours.
[0815] The solution is evaporated under vacuum and 46 mg of product
are obtained with a quantitative yield.
[0816] Mass spectrum: M/z=515.1 (ES+) theoretical M=514
[0817] 6) 31 mg of
17-amino-1,1,14-trihydroxy-5-oxo-2-phosphono-13,15-dioxa-6-aza-1.lamda..s-
up.5,14-diphosphaoctadecan-18-oic acid 1,14-dioxide in solution in
10 ml of water are added dropwise to a solution of 1 ml of example
9 (acid ferrofluid) at 2.742 M/L diluted in 100 ml of water. The
mixture is stirred for 20 minutes at ambient temperature and the pH
is adjusted to 7.2.
[0818] The solution obtained is ultrafiltered through a PALL.RTM.
stirring cell having a cutoff threshold of 30 kD. 300 ml of
filtrate are removed so as to obtain a final solution of 10 ml.
[0819] [Fe]=0.262 M/L PCS size=28.1 nm
[0820] Fe=67.6% mass/mass
[0821] P=1.9% mass/mass
[0822] C=3.15% mass/mass
[0823] N=0.57% mass/mass
Example 29
[0824] 1.75 g of compound I from example 23 are dissolved in 30 ml
of example 12 at 0.285 M/L and the pH is adjusted to 6.5. 368 mg of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
added. The mixture is stirred at ambient temperature for 24 hours.
The solution is ultrafiltered through a PALL.RTM. stirring cell
having a cutoff threshold of 30 kD. 800 ml of filtrate are removed.
The retentate is adjusted to 15 ml.
[0825] [Fe]=0.496 M/L PCS size=27.1 nm
[0826] Fe=59.2% mass/mass
[0827] P=1.09% mass/mass
[0828] N=0.32% mass/mass
[0829] C=6.8% mass/mass
[0830] Degree of grafting [compound A/Fe]=1.66% mol/mol
[0831] Degree of grafting [compound I/Fe]=0.97% mol/mol
[0832] Degree of grafting [compound I/compound A]=54%
Example 30
[0833] 0.9 g of the compound O-(2-aminoethyl)-O'-methylpolyethylene
glycol 750 [Amino-PEG750; Fluka] is dissolved in 22 ml of example
12 at 0.285 M/L and the pH is adjusted to 6.5. 280 mg of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
added. The mixture is stirred at ambient temperature for 24 hours.
The solution is ultrafiltered through a PALL.RTM. stirring cell
having a cutoff threshold of 30 kD. 800 ml of filtrate are removed.
The retentate is adjusted to 20 ml.
[0834] [Fe]=0.285 M/L PCS size=24.9 nm
[0835] Fe=53.2% mass/mass
[0836] P=1.30% mass/mass
[0837] N=0.35% mass/mass
[0838] C=5.87% mass/mass
[0839] Degree of grafting [compound A/Fe]=2.2% mol/mol
[0840] Degree of grafting [compound Amino-PEG750/Fe]=1.33%
mol/mol
[0841] Degree of grafting [compound Amino-PEG750/compound
A]=60.3%.
Example 31
[0842] 3.15 g of compound O-(2-aminoethyl)-O'-methylpolyethylene
glycol 2000 [Amino-PEG2000; Fluka] are dissolved in 30 ml of
example 12 at 0.285 M/L and the pH is adjusted to 6.2. 368 mg of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
added. The mixture is stirred at ambient temperature for 24 hours.
The solution is ultrafiltered through a PALL.RTM. stirring cell
having a cutoff threshold of 30 kD. 800 ml of filtrate are removed.
The retentate is adjusted to 25 ml.
[0843] [Fe]=0.290 M/L PCS size=32.2 nm
[0844] Fe=46.3% mass/mass
[0845] P=1.07% mass/mass
[0846] N=0.3% mass/mass
[0847] C=10.3% mass/mass
[0848] Degree of grafting [compound A/Fe]=2.08% mol/mol
[0849] Degree of grafting [compound Amino-PEG2000/Fe]=1.07%
mol/mol
[0850] Degree of grafting [compound Amino-PEG2000/compound
A]=50.4%
Example 32
[0851] 180 mg of compound J from example 24 are introduced into 46
ml of example 12 (concentration: 0.285 mol/L of iron) and the pH is
adjusted to 6.2.
[0852] 50 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride are added to this solution. The mixture is stirred
for 8 hours at ambient temperature. A further 50 mg of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
added to the reaction medium and stirring is maintained for 16
hours.
[0853] The reaction medium is directly ultrafiltered through a
PALL.RTM. stirring cell having a cutoff threshold of 30 kD.
[0854] The retentate is adjusted to 55 ml and is then filtered
through 0.22 .mu.m.
[0855] [Fe]: 0.273 M/L PCS size=72.4 nm
[0856] Fe=62.4% mass/mass
[0857] P=1.16% mass/mass
[0858] C=2.31% mass/mass
[0859] N=0.37% mass/mass
[0860] Degree of grafting [compound A/Fe]=1.68% mol/mol
[0861] Degree of grafting [compound J/Fe]=0.185% mol/mol
[0862] Degree of grafting [compound J/compound A]=11.0%
Example 33
[0863] 750 mg of O-(2-aminoethyl)-O'-methylpolyethylene glycol 750
(Amino-PEG methyl ester 750 .RTM.--Fluka) are introduced into 20 ml
of example 32 at 0.273 M/L of iron and the pH is adjusted to
6.2.
[0864] 200 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride are added to the preceding solution. The mixture is
stirred for 8 hours at ambient temperature. A further 200 mg of
1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride are
then added to the reaction medium and stirring is maintained for 16
hours.
[0865] The reaction medium is directly ultrafiltered through a
PALL.RTM. stirring cell having a cutoff threshold of 30 kD. The
retentate is adjusted to 25 ml and is then filtered through 0.22
.mu.m.
[0866] [Fe]: 0.268 M/L PCS size=69 nm
[0867] Fe=59.7% mass/mass
[0868] P=1.06% mass/mass
[0869] C=6.3% mass/mass
[0870] N=0.65% mass/mass
[0871] Degree of grafting [compound A/Fe]=1.6% mol/mol
[0872] Degree of grafting [Amino-PEG 750/Fe]=1.9% mol/mol
[0873] Degree of grafting [compound J/Fe]=0.19% mol/mol
Example 34
[0874] 1.13 g of example 5 (compound E) are introduced into 20 ml
of example 32 at 0.273 M/L of iron and the pH is adjusted to
6.2.
[0875] 200 mg of 1-(3-dimethylamindpropyl)-3-ethylcarbodiimide
hydrochloride are added to this solution. The mixture is stirred
for 8 hours at ambient temperature.
[0876] A further 200 mg of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
then added to the reaction mixture and stirring is maintained for
16 hours.
[0877] The reaction medium is directly ultrafiltered through a
PALL.RTM. stirring cell having a cutoff threshold of 30 kD. The
retentate is adjusted to 25 ml and is then filtered through 0.22
.mu.m.
[0878] [Fe]: 0.188 M/L PCS size=79 nm
[0879] Fe=55.1% mass/mass
[0880] P=0.91% mass/mass
[0881] C=7.21% mass/mass
[0882] N=1.09% mass/mass
[0883] Br=3.08% mass/mass
[0884] Degree of grafting [compound A/Fe]=1.5% mol/mol
[0885] Degree of grafting [compound E/Fe]=1.3% mol/mol
[0886] Degree of grafting [compound J/Fe]=0.19% mol/mol
Example 35
[0887] 0.29 g of compound K from example 25 is introduced into 74
ml of example 12 (concentration: 0.285 mol/L of iron) and the pH is
adjusted to 6.2.
[0888] 82 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride are added to this solution and the mixture is stirred
for 8 hours at ambient temperature. After 8 hours, a further 82 mg
of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
then added to the reaction medium and stirring is maintained for 16
hours.
[0889] The reaction medium is directly ultrafiltered through a
PALL.RTM. stirring cell having a cutoff threshold of 30 kD. The
retentate is adjusted to 75 ml and is then filtered through 0.22
.mu.m.
[0890] [Fe]: 0.273 M/L PCS size=60.8 nm
[0891] Fe=63.1% mass/mass
[0892] P=1.23% mass/mass
[0893] C=4.1% mass/mass
[0894] N=0.37% mass/mass
[0895] Degree of grafting [compound A/Fe]=1.76% mol/mol
[0896] Degree of grafting [compound K/Fe]=0.22% mol/mol
[0897] Degree of grafting [compound K/compound A]=12.5%
Example 36
[0898] 1 g of O-(2-aminoethyl)-O'-methylpolyethylene glycol 750
(Amino-PEG methyl ester 750.RTM.--Fluka) are introduced into 25 ml
of example 35 (C=0.273 M/L of iron) and the pH is adjusted to
6.2.
[0899] 274 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride are added to this solution. The mixture is stirred
for 8 hours at ambient temperature. After 8 hours, a further 274 mg
of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
then added to the reaction medium and stirring is maintained for 16
hours.
[0900] The reaction medium is directly ultrafiltered through a
PALL.RTM. stirring cell having a cutoff threshold of 30 kD. The
retentate is adjusted to 25 ml and is then filtered through 0.22
.mu.m.
[0901] [Fe]: 0.261 M/L PCS size=59 nm
[0902] Fe=62.4% mass/mass
[0903] P=1.13% mass/mass
[0904] C=7.56% mass/mass
[0905] N=0.8% mass/mass
[0906] Degree of grafting [compound A/Fe]=1.63% mol/mol
[0907] Degree of grafting [Amino-PEG 750/Fe]=1.59% mol/mol
[0908] Degree of grafting [compound K/Fe]=0.22% mol/mol
Example 37
[0909] 1.61 g of example 5 (compound E) are introduced into 25 ml
of example 35 (C=0.273 M/L of iron) and the pH is adjusted to
6.2.
[0910] 274 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride are added to this solution. The mixture is stirred
for 8 hours at ambient temperature. After 8 hours, a further 274 mg
of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are
then added to the reaction medium and stirring is maintained for 16
hours.
[0911] The reaction medium is directly ultrafiltered through a
PALL.RTM. stirring cell having a cutoff threshold of 30 kD. The
retentate is adjusted to 28 ml and is then filtered through 0.22
.mu.m.
[0912] [Fe]: 0.240 M/L PCS size=65.6 nm
[0913] Fe=61.5% mass/mass
[0914] P=1.02% mass/mass
[0915] C=8.0% mass/mass
[0916] N=1.21% mass/mass
[0917] Br=3.8% mass/mass
[0918] Degree of grafting [compound A/Fe]=1.5% mol/mol
[0919] Degree of grafting [compound E/Fe]=1.4% mol/mol
[0920] Degree of grafting [compound K/Fe]=0.22% mol/mol
[0921] Assays for affinity of the acid magnetic particles (p)
complexed by compounds of formula (I) and coupled with biovectors;
for certain biological targets.
[0922] 1/ Assay for Affinity of Example 17 for Folate Binding
Protein (FBP) by the BiaCore Technique
[0923] Reagents
TABLE-US-00024 Products Supplier Reference Folic acid Avogado 14300
FBP Sigma F-0504 Chips CM5 BIAcore Lots 1121065 and 11210007
[0924] HBS buffer: 10 mM Hepes, pH 7.4, 0.15 M NaCl, 4.3 mM EDTA
and 0.005% NP20
[0925] PGM buffer: 100 mM KH.sub.2PO.sub.4, pH 7, 10% glycerol and
4 mM .beta.-mercaptoethanol.
[0926] The immobilization of the FBP was carried out at 1 mg/ml in
the PGM buffer according to the protocols recommended by BIAcore
for coupling to amines. After this, the remaining active
carboxylated groups are saturated with 1M of ethanolamine pH=8. The
amount of FBP attached corresponds approximately to 1.5
ng/mm.sup.2.
[0927] Example 17 was at a concentration of 0.170 mol/L of iron,
which corresponds to 1.9.times.10.sup.-5 mol of particles/L
(considering a crystal of 7.5 nm, i.e. 8900 atoms of
iron/particle).
[0928] The attachment of the folate was followed at four different
concentrations (125, 250, 500 and 1000 .mu.M) whereas that of
example 17 was carried out at 10 .mu.m and at 10 nM (expressed as
mol of particle/L). The association and the dissociation were
studied with a flow at 25 .mu.l/min. The association and
dissociation phases are 5 minutes.
[0929] Results
TABLE-US-00025 Association Dissociation Affinity Products rate ka
rate kd constant KD Folic acid 10.sup.3 M.sup.-1 s.sup.-1 1.6
10.sup.-3 s.sup.-1 1.6 .mu.M Example 17 >10.sup.6 M.sup.-1
s.sup.-1 <10.sup.-6 s.sup.-1 <1 pM
[0930] These results demonstrate that the affinity of example 17
for folate binding protein (FBP) is very high with a lower than
picomolar affinity constant KD (expressed as mol of
particle/L).
[0931] Given that the folic acid receptor (represented here by FBP)
is very highly overexpressed on certain tumor cells (ovarian
cancer, lung cancer, colon cancer and breast cancer) and in
inflammatory sites (in particular in rheumatoid arthritis), example
17 may be advantageously used as a specific MRI contrast agent in
oncology and in inflammatory diseases.
[0932] 2/ Inhibitory Activity of Example 27 for Human
Metalloproteases MMP-1 and MMP-3.
[0933] Example 27 is covered at its surface with a biovector
(example 22: compound H) derived from Ilomastat, a pseudopeptide
acknowledged to be a powerful inhibitor of zinc metalloproteases
(matrix metalloproteinase: MMP).
[0934] The inhibitory activity of example 27 on the human matrix
metalloproteases MMP-1 and MMP-3 was evaluated in vitro. It was
compared with that of example 12 (particle not functionalized with
compound H) and with that of the reference peptide, Ilomastat. The
protocol for measuring the inhibitory effect of the various
products on MMP-1 was as follows: [0935] Principle: evaluation of
the inhibitory effect of product tested on the activity of human
MMP-1 (isolated from human fibroblasts from rheumatoid synovial
fluid), quantified by fluorimetry (.lamda.ex=340 nm, .lamda.em=440
nm; measurement of the formation of
Cys(Me)-His-Ala-Lys-(n-Me-Abz)-NH.sub.2 by cleavage of the
substrate DNP-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys-(n-Me-Abz)-NH.sub.2);
protocol described in the bibliographic reference Bickett et al.
(1993) Anal. Biochem.; 212:58.
[0936] The reference product used in this assay was GM6001
(Ilomastat, Galardin IC.sub.50=1.9.times.10.sup.-9 M).
[0937] More precisely, the test product, or the reference product
or water, was added to a buffer solution, pH=7, containing 7 nM of
MMP-1. The fluorescence intensity of this medium was immediately
evaluated at .lamda.ex=340 nm and .lamda.em=440 nm by means of a
plate reader (GeminiXS, Molecular Devices). This measurement at t=0
serves to verify any possible interference of a product with the
fluorescent detection. The enzyme reaction was then initiated by
adding the substrate
DNP-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys-(n-Me-Abz)-NH.sub.2 at a
concentration of 10 .mu.M. After incubation for 40 min at
37.degree. C., the fluorescence was again evaluated under the same
conditions as previously (t=40 min). The enzyme activity was
determined by subtracting the signal measured at t=0 from the
signal measured at t=40 min. The final result is expressed as
percentage inhibition of the enzyme activity.
[0938] The protocol for measuring the inhibitory effect of the
various products on MMP-3 was as follows:
[0939] Principle: evaluation of the inhibitory effect of the
product tested on the activity of a human MMP-3 (use of a
recombinant enzyme expressed by the sF9 cell line), quantification
by fluorimetry (.lamda.ex=340 nm, .lamda.em=405 nm; measurement of
the formation of Mca-Arg-Pro-Lys-Pro-Tyr-Ala by cleavage of the
substrate Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(DNP)-NH.sub.2
(NFF2)--protocol described in the reference: Nagase et al. (1994)
J. Biol. Chem.; 269:20952.
[0940] The reference product used in this assay was GM6001
(Ilomastat, Galardin IC.sub.50=1.3.times.10.sup.-7 M).
[0941] More precisely, the test product, or the reference product
or water, was added to a buffer solution, at pH=6, containing 6 nM
of MMP-3. The fluorescence of this medium was evaluated at
.lamda.ex=340 nm and .lamda.em=405 nm by means of a plate reader
(GeminiXS, Molecular Devices), after preincubation for 30 min at
37.degree. C. (t=0). This measurement at t=0 serves to verify any
possible interference of a product with the fluorescent detection.
The enzyme reaction was then initiated by adding 10 .mu.m of the
substrate NFF-2. After incubation for 90 min at 37.degree. C., the
fluorescence was again evaluated under the same conditions as
previously (t=90 min). The enzyme activity was then determined by
subtracting the signal measured at t=0 from the signal measured at
t=90 min.
[0942] The final result is expressed as percentage inhibition of
the enzyme activity.
[0943] The results of inhibition of human MMP-1 and human MMP-3 by
example 27 are given in table 1.
TABLE-US-00026 TABLE 1 Concentration % inhibition Concentration of
biovector: of the MMP of iron [M] compound H [M] control value
MMP-1 1.03 .times. 10.sup.-8 0.86 .times. 10.sup.-10 9 1.03 .times.
10.sup.-6 0.86 .times. 10.sup.-8 86 1.03 .times. 10.sup.-4 0.86
.times. 10.sup.-6 103 MMP-3 1.03 .times. 10.sup.-8 0.86 .times.
10.sup.-10 0 1.03 .times. 10.sup.-6 0.86 .times. 10.sup.-8 -1 1.03
.times. 10.sup.-4 0.86 .times. 10.sup.-6 68
[0944] Example 27 inhibits MMP-1 at between 0.87.times.10.sup.-10
and 0.87.times.10.sup.-8 M (expressed as concentration of compound
H), which is in agreement with the experimental value obtained for
the reference peptide--Ilomastat--(IC50=1.9.times.10.sup.-9 M).
[0945] Example 27 inhibits MMP-3 at between 0.87.times.10.sup.-8
and 0.87.times.10.sup.-6 M (expressed as concentration of compound
H), which is in agreement with the experimental value obtained for
the reference peptide--Ilomastat--(IC50=1.3.times.10 .sup.-7
M).
[0946] The results obtained with example 27 on MMP-1 and MMP-3 show
that the particles of iron oxide prepared by the applicant behave
in a manner very similar to the reference peptide (Ilomastat). The
compound of example 27 can be used as an inhibitor with a broad
spectrum with respect to metalloproteases (MMP-1, MMP2, MMP-3,
MMP-7, MMP-9), and also as a specific MRI contrast agent very
useful for imaging pathological regions in which these various MMPs
are overexpressed.
* * * * *