U.S. patent application number 16/732618 was filed with the patent office on 2020-06-25 for gel formulations for guiding radiotherapy.
This patent application is currently assigned to DANMARKS TEKNISKE UNIVERSITET. The applicant listed for this patent is DANMARKS TEKNISKE UNIVERSITET NANOVI RADIOTHERAPY APS. Invention is credited to Morten Albrechtsen, Thomas Lars Andresen, Rasmus Irming Jolck.
Application Number | 20200197541 16/732618 |
Document ID | / |
Family ID | 50819731 |
Filed Date | 2020-06-25 |
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United States Patent
Application |
20200197541 |
Kind Code |
A1 |
Andresen; Thomas Lars ; et
al. |
June 25, 2020 |
GEL FORMULATIONS FOR GUIDING RADIOTHERAPY
Abstract
The present invention describes an X-ray contrast composition
for local administration, wherein the X-ray contrast composition
exhibits contrast properties and wherein at least 60% of an
administrated amount of said X-ray contrast composition remains
more than 24 hours within 10 cm from an injection point when the
X-ray contrast composition is administrated to a human or animal
body.
Inventors: |
Andresen; Thomas Lars;
(Vanlose, DK) ; Jolck; Rasmus Irming; (KGS.
Lyngby, DK) ; Albrechtsen; Morten; (Charlotenlund,
DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DANMARKS TEKNISKE UNIVERSITET
NANOVI RADIOTHERAPY APS |
Kgs. Lyngby
Kgs. Lyngby |
|
DK
DK |
|
|
Assignee: |
DANMARKS TEKNISKE
UNIVERSITET
Kgs. Lyngby
DK
NANOVI RADIOTHERAPY APS
Kgs. Lyngby
DK
|
Family ID: |
50819731 |
Appl. No.: |
16/732618 |
Filed: |
January 2, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14892811 |
Nov 20, 2015 |
10561746 |
|
|
PCT/EP2014/060673 |
May 23, 2014 |
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16732618 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 51/00 20130101;
A61K 49/1806 20130101; A61K 49/0485 20130101; A61N 5/10 20130101;
A61K 49/0438 20130101; A61K 49/0457 20130101; A61P 35/00 20180101;
A61K 49/04 20130101; A61B 6/481 20130101; A61K 49/0447
20130101 |
International
Class: |
A61K 49/04 20060101
A61K049/04; A61N 5/10 20060101 A61N005/10; A61K 51/00 20060101
A61K051/00; A61K 49/18 20060101 A61K049/18; A61B 6/00 20060101
A61B006/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 24, 2013 |
SE |
1350637-3 |
Claims
1. An imaging contrast composition for local administration,
wherein the imaging contrast composition exhibits contrast
properties and wherein at least 60% of an administrated amount of
said imaging contrast composition remains more than 24 hours within
10 cm from an injection point when the imaging contrast composition
is administrated to a human or animal body.
2. The imaging contrast composition according to claim 1, wherein
the imaging contrast composition is an X-ray contrast composition,
and wherein the X-ray contrast composition is a liquid before
administration and having the ability to transform into a gel after
administration.
3. (canceled)
4. The imaging contrast composition according to claim 2, wherein
the X-ray contrast composition is a liquid before administration
into a human or animal body that increases in viscosity by more
than 1,000 centipoise (cP) after administration into a human or
animal body.
5. (canceled)
6. The imaging contrast composition according to claim 2, wherein
the X-ray contrast composition has a viscosity of less than 10,000
centipoise (cP) at 20.degree. C.
7. The imaging contrast composition according to claim 2, wherein
the X-ray contrast composition comprises an X-ray contrast agent
that is part of the X-ray contrast composition and said X-ray
contrast agent is an organic sub stance.
8. The imaging contrast agent according to claim 2, wherein the
X-ray contrast agent comprises one or more natural polymers,
synthetic polymers, oligomers, lipids, saccharides, disaccharides,
polysaccharides, peptides or any combination thereof, or wherein
the X-ray contrast agent comprises one or more iodinated polymers,
oligomers, lipids, saccharides, disaccharides, polysaccharides,
peptides, or a derivative or a combination thereof.
9-13. (canceled)
14. The imaging contrast composition according to claim 2, wherein
the X-ray contrast composition exhibits gel-formation in response
to a temperature in the range of 35 to 40.degree. C., in response
to hydration, in response to an ion-concentration in the range of 1
.mu.M to 500 mM, in response to a pH in the range of 6 to 8 and or
in response to contacting with an initiator.
15-18. (canceled)
19. The imaging contrast composition according to claim 2, wherein
the X-ray contrast composition also comprises, radioactive
compounds, paramagnetic compounds, fluorescent compounds or
ferromagnetic compounds, or any mixture thereof, and/or wherein the
X-ray contrast composition also comprises at least one
pharmaceutical substance.
20-24. (canceled)
25. The imaging contrast composition according to claim 2, wherein
the X-ray contrast composition comprises poly(ethylene
glycol-b-caprolactone) (PEG-PC1), sucrose acetate isobutyrate
(SAIB), poly(D,L-lactic acid), or poly(lactic-co-glycolic acid)
(PGLA), or a combination thereof.
26. The imaging contrast composition according to claim 2, wherein
the X-ray contrast composition comprises sucrose acetate
isobutyrate (SAIB) or a derivative thereof.
27. The imaging contrast composition according to claim 2, wherein
the X-ray contrast composition comprises an iodinated derivate of
sucrose acetate isobutyrate (SAIB) or an iodinated derivate of
sucrose acetate isobutyrate (SAIB) doped into sucrose acetate
isobutyrate (SAIB).
28. (canceled)
29. The imaging contrast composition according to claim 2, wherein
the X-ray contrast composition comprises an iodinated derivate of
sucrose acetate isobutyrate (SAIB) solubilized in a mixture of
ethanol and sucrose acetate isobutyrate (SAIB).
30. The imaging contrast composition according to claim 2, wherein
the X-ray contrast composition comprises an iodinated derivate of
sucrose acetate isobutyrate (SAIB) and contains a pharmaceutical
substance or particle that contains a pharmaceutical substance.
31. (canceled)
32. The imaging contrast composition according to claim 2, wherein
the X-ray contrast composition comprises an iodinated derivate of
sucrose acetate isobutyrate (SAIB) solubilised in a mixture of
ethanol and sucrose acetate isobutyrate (SAIB) and contains a
pharmaceutical substance or a particle that contains a
pharmaceutical substance.
33. (canceled)
34. A kit comprising a syringe, a needle used for injection into a
body or surgical related procedures, such as but not limited to
biopsy adapted to the open end of said syringe, and a composition
according to claim 1.
35. The imaging contrast composition according to claim 1, for use
in radio therapy, imaging, diagnostics, treatment and/or quality
rating of radio therapy, for use as a tissue marker and/or for use
as a controlled drug release composition.
36-40. (canceled)
41. The imaging contrast composition according to claim 1, wherein
the imaging contrast composition is parenterally administered to a
predetermined location of the body of said mammal, and wherein an
X-ray image of at least a part of the body of the mammal including
the predetermined location is recorded.
42. A method of recording an X-ray image of the body of a mammal,
comprising the steps of a. providing an X-ray contrast composition
comprising an organic X-ray agent in a gel-forming system; b.
administering the X-ray contrast composition to a predetermined
location of the mammal, and c. recording X-ray-based images of at
least a part of the body which comprises the predetermined
location.
43. A method of joint radiotherapy and X-ray imaging of a target
tissue in a mammal, comprising the steps of a. providing an X-ray
contrast composition comprising an organic X-ray agent in a
gel-forming system; b. administering the X-ray contrast composition
to a predetermined target tissue of the mammal, c. recording
X-ray-based images, of at least a part of the body which comprises
the target tissue, thereby providing a definition of the target
tissue, and d. using the definition of the target tissue obtained
in c) to direct external beam radiotherapy to the target
tissue.
44. A method for directing local administration of a
pharmaceutically active agent to a target tissue in a mammal,
comprising the steps of a. providing an X-ray contrast composition
comprising an organic X-ray agent in a gel-forming system; b.
administering the X-ray contrast composition to a predetermined
target tissue of the mammal, c. recording X-ray-based images, of at
least a part of the body which comprises the target tissue, thereby
providing a definition of the target tissue, and d. using the X-ray
contrast composition in b) to further comprise an active
pharmaceutical agent for delivery of an active pharmaceutical agent
to a predetermined target tissue of the mammal.
45. The method according to claim 43, wherein the target tissue
comprises undesirably growing cells.
46-47. (canceled)
48. The method according to claim 42, wherein the X-ray contrast
composition comprises the feature of exhibiting contrast properties
and wherein at least 60% of an administrated amount of the X-ray
contrast composition remains more than 24 hours within 10 cm from
an injection point when the X-ray contrast composition is
administrated to a human or animal body, and wherein the X-ray
contrast composition is a liquid before administration and has the
ability to transform into a gel after administration.
49. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to improved formulations for
guiding radiotherapy.
TECHNICAL BACKGROUND
[0002] Every year more than 12 million people are diagnosed with
cancer worldwide and over 7.5 million people die from cancer each
year. These numbers are expected to increase because of population
growth and due to the lifestyle in the Western world. Radiotherapy
is an important part of modern cancer treatment and more than 50%
of cancer patients receive radiotherapy at least once. Modern
radiotherapy relies on advanced high precision planning, treatment
equipment and imaging techniques (such as, e.g., computed
tomography (CT), positron-emission tomography (PET) and magnetic
imaging resonance (MRI)) in order to deliver high radiation doses
to a precisely defined target in patients.
[0003] One of the main difficulties in external beam radiotherapy
is that both tumors and the surrounding tissue move significantly
and unpredictably during radiotherapy; both within each single
treatment, and during the whole course of radiotherapy, lasting
usually 5-7 weeks. These movements can be dramatic (e.g. several cm
within seconds) and may be caused by various factors such as
respiration, bladder- and bowel filling, air passing colon, tumor
shrinkage and set-up variation of the patient. One way of
minimizing this problem is the implantation of markers in or
adjacent to the tumor allowing frequent imaging and treatment
adaptation. So far, markers have been inserted using long and thick
needles, a complicated procedure with a significant risk of
complications, which is limiting the practical usefulness of
markers in radiotherapy.
[0004] Ideally, a tissue marker should enable tracking of tumor
movement; be visible on several image modalities; be visible for an
extended period (e.g., at least 4 weeks); be non-toxic; and be easy
to insert.
[0005] Various attempts have been made for improvements within the
field of radiotherapy. EP1006935 describes a composition for
controlled release of a substance WO9403155 describes a hydrogel
composition prepared from a backbone bonded to a cross-linking
agent. The hydrogels may be loaded with therapeutic drugs and
diagnostic labels, including X-ray contrast imaging agents for
disease diagnostics and treatment. US20120065614 discloses a hybrid
system for bio imaging. Gold is bound into a matrix comprising a
hydrogel or polymer or similar. In U520100297007 a substantially bi
concave shaped nanoparticle is disclosed, the nanoparticle
comprising an aqueous inner core and a hydrophilic outer shell
comprising an amphiphilic polymer.
[0006] Furthermore, US2009110644 discloses a nanoparticle
consisting of a polymer which is a metal chelating agent coated
with a magnetic metal oxide, wherein at least one active agent is
covalently bound to the polymer. In the documents US20100290995 and
US2005036946, radio-opaque biodegradable compositions are disclosed
by modifying terminal groups of synthetic and natural biodegradable
polymers such as polylactones with iodinated moieties and in
SE403255 a contrast agent is disclosed that comprises a polymer
comprising hydroxy- and/or carboxy- and/or amino groups further
comprising X-ray contrast giving iodo-substituted aromatic groups.
Further yet, the document WO9519184 discloses air encapsulating
micro particles formed by ionotropically gelling synthetic
polyelectrolytes such as poly(carboxylato-phenoxy)phosphazene,
poly(acrylic acid), poly(methacrylic acid) and methacrylic acid
copolymers (Eudragit's) by contact with multivalent ions such as
calcium ions.
[0007] There are several drawbacks to the current clinical practice
using solid markers and the methods described in the documents
above. Installation of solid markers is invasive due to the large
dimension of the solid implant which may cause severe complications
limiting is usefulness in radiotherapy. By combining gel-forming,
low-viscosity solutions with solid particles and/or organic X-ray
contrast agents (or other imaging modalities) injectable gels can
be formulated with fine-tuned properties as these can be modified
by multiply parameters with respect to the gel forming solution and
the contrast agents used. The solid particles can, besides
contributing to the overall contrast of the system, also carry
pharmaceutical substances and control their release in a controlled
manner.
[0008] One aim of the present invention is to provide new
formulations comprising gel-forming, low-viscosity systems that are
easy to administer parenterally, and wherein the present invention
provides good visualization by one or multiple imaging modalities,
including X-ray imaging.
SUMMARY OF THE INVENTION
[0009] X-ray imaging of a locally administered reference marker is
achieved by use of an X-ray contrast composition, wherein the X-ray
contrast composition exhibits contrast properties and wherein at
least 60% of an administrated amount of said X-ray contrast
composition remains more than 24 hours within 10 cm from an
injection point when the X-ray contrast composition is
administrated to a human or animal body.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The formulation is preferably in the form adapted for
parenteral administration, and should preferably consist of
pharmaceutically acceptable constituents. The formulation which as
such has a comparable low viscosity is intended for injection in
the body of a human or animal, where after the formulation becomes
more viscous, e.g. it goes through a sol-gel transition (liquid to
gel) or forms a amorphous glass matrix, due to the presence of the
gel-forming system. It is preferred that the viscosity of the
formulation after injection in the body of a human or animal
increases by at least 50%, such as at least 80%, such as at least
100%, or at least 150%, or at least 200%, or at least 300%, or at
least 500%, or at least 750%, or at least 1000%, or at least
10,000%, or that the formulation becomes essentially solid
(non-viscous).
[0011] The formulation is preferably adapted for injection via a
thin needle used for injection into a body or surgical related
procedures, such as but not limited to biopsy. The viscosity of the
hydrogel or gel-forming formulation before injection can be any
suitable viscosity such that the formulation can be parenterally
administered to a patient.
[0012] Exemplary formulations include, but are not limited to,
those having a viscosity (prior to administration/injection) lower
than 10,000 centipoise (cP), e.g. lower than 2,000 cP, such as 10
to 2,000 cP, such as 20 to 1,000 cP, such as 150 to 350 cP, such as
400 to 600 cP, such as 600 to 1,200 cP or such as 1,000 to 2,000
cP, or 10 to 600 cP, or 20 to 350 cP, at 20.degree. C.
[0013] Alternative formulations include, but are not limited to,
those having a viscosity (prior to administration/injection) lower
than 10,000 centipoise (cP), e.g. lower than 2,000 cP, such as 10
to 2,000 cP, such as 20 to 1,000 cP, such as 150 to 350 cP, such as
400 to 600 cP, such as 600 to 1,200 cP or such as 1,000 to 2,000
cP, or 10 to 600 cP, or 20 to 350 cP, at 5.degree. C.
[0014] When referred to herein, the (dynamic) viscosity is measured
at the specified temperature in accordance with the method
described in ASTM D7483.
[0015] Hydrogels, gels or amorphous glass matrixes may be formed
either through covalent bond formation or ionic- or hydrophobic
interactions. Physical (non-covalent) cross-links may result from
complexation, hydration, hydrogen bonding, desolvation, Van der
Waals interactions, ionic bonding, combinations thereof, and the
like, and may be initiated by mixing two precursors that are
physically separated until combined in situ, or as a consequence of
a prevalent condition in the physiological environment, including
temperature, pH, ionic strength, combinations thereof, and the
like. Chemical (covalent) cross linking may be accomplished by any
of a number of mechanisms, including free radical polymerization,
condensation polymerization, anionic or cationic polymerization,
step growth polymerization, electrophile-nucleophile reactions,
combinations thereof, and the like. FIGS. 1-6 illustrate exemplary
hydrogel and/or gel-forming and/or amorphous glass matrix systems
that can be used in the present invention.
[0016] The hydrogel, gel or amorphous glass matrix forming
compositions may be loaded with organic x-ray agents such as
iodinated polymers or sugars and nanoparticles or submicron
particles either prior to or during gel formation, such as when the
formulation is in a sol-state or in transition to the gel-state,
e.g., by diffusion into the hydrogel composition. These x-ray
agents or particles may either be entrapped in the gel matrix
without any chemical cross-linking, or they may be bonded,
non-covalently or covalently, to the backbone or cross-linking
agent of the hydrogel, gel or amorphous glass matrix. The organic
x-ray agents may be one component in the gel and the particles
another component, where the particles are either a contrast agent
for imaging by x-ray, MRI, PET, SPECT, fluorescence or ultrasound,
and/or contain pharmaceutical agents. Pharmaceutical agents may be,
but not limited to, radiosensitzers, chemotherapeutics or hormones.
MRI agents such as gadolinium may be a component in the gel forming
systems. Pharmaceutical agents can furthermore be covalent or
non-covalently embedded in the hydrogel, gel or amorphous glass
matrix.
[0017] After injection, the formulation typically provides a well
defined assembly of x-ray contrast agents which provides contrast
in e.g. X-ray imaging, and which may serve as a marker, thus,
enabling tracking of tumor movement during e.g. radiotherapy or
surgical procedures.
[0018] US2001/0142936 discloses covalently linked hydrogels
particles in the micrometer range (10 .mu.m-500 .mu.m) with/without
radiopaque agents for use of conformal filling of surgical sites
with optional imaging in order to ensure that the implants are
positioned correctly. The present invention offers several
advantageous features as it exploits organic x-ray contrast agents
that may be in combination with nano-sized particles combined with
a gel forming injectable liquid. Nano-sized particles exhibit
low/no sedimentation rate due to the effects of Brownian motion
which is problematic for micrometer sized particles. Furthermore,
dividing the particles and the gel forming solution into two
components enables control over particle diffusion, release etc.
within the gel which is advantageous for controlling the overall
properties of the formulation. US2011/0142936 is built on the
invention that swelling of the gel will increase the distance
between normal and tumor tissue by injecting into iatrogenic
("medically produced") spaced. The present invention aims at
infiltrate tissue with minimal impact on the shape and position of
the target tissue typically being a cancer. Furthermore, the
intention of the present invention is to infiltrate tissue with
minimal change in size and location why swelling is for this
invention a disadvantage. This in contrast to US2001/0142936
[0019] In the context of the present invention, a "marker" or
"tissue marker" is a detectable agent or composition which does not
move, or stays substantially in the same position, for several days
or weeks once it has been administered or implanted into a specific
site or tissue of a mammalian body. A tissue marker can, for
example, comprise one or more X-ray contrast agents, radioactive
compounds, paramagnetic compounds, fluorescent agents, or other
detectable agents.
[0020] In the context of the present invention, a "gel" is defined
as a carrier matrix in which the detectable agent (contrast agent)
is dispersed and/or dissolved within. The term "gel" includes
systems such as hydrogels, gels or amorphous glass matrixes which
upon injection into a human or an animal increases viscosity due to
chemical and/or physical stimulus.
[0021] An "imageable tissue marker" or "imageable marker" comprises
a detectable agent in a form and/or a sufficient amount to allow
for detection of the tissue marker by an external imaging modality
if administered or implanted into a mammalian body. Exemplary
external imaging modalities include, but are not limited to, X-ray
imaging, CT imaging, MRI, PET imaging, single photon emission
computed tomography (SPECT) imaging, nuclear scintigraphy imaging,
ultrasonography imaging, ultrasonic imaging, near-infrared imaging
and/or fluorescence imaging. Some examples of the brand names and
types of different image techniques are e.g. ExacTrac.RTM.
(BrainLAB), Cone Beam (e.g. Vairan) and OBI (e.g. On-Board
Imager.RTM. Varian).
Contrast Agents
[0022] Contrast may be achieved using organic x-ray contrast
agents, such as radiopague agents such as iodinated compounds,
which may be combined with chelators of MRI agents such as
gadolinium, and/or combined with chelators of PET imaging agents
such as copper-64, which may further be combined with solid
inorganic particles. Chelators may be DOTA, EDTA, or DTPA and
chelators will be non-covalently embedded or covalently conjugated
to the gel-forming components. The combined contrast agents should
preferably be visible by at least CT imaging.
[0023] Preferred contrast agents are iodinated compounds such as
polymers or sugar molecules such as derivatives of glucose or
sucrose or other oligosaccharides. Solid particles may comprise, or
consist of, one or more X-ray contrast agents, i.e., compounds that
are able to block or attenuate X-ray radiation. Such compounds
include transition metals, rare earth metals, alkali metals, alkali
earth metals, other metals, as defined by the periodic table. A
metal or alkali metal may appear in non-oxidized or any of the
existing oxidation states for the metal. These oxidation states
include monovalent cations, divalent cations, trivalent cations,
tetravalent cations, pentavalent cations, hexavalent cations and
heptavalent cations.
[0024] In one embodiment, the one or more X-ray contrast agents are
selected from Iodine (I), gold (Au), bismuth (Bi), gadolinium (Gd),
iron (Fe), barium (Ba), calcium (Ca) and magnesium (Mg). In a
particular embodiment, the detectable compound comprises one or
more compounds selected from the group of gold (Au) and bismuth
(Bi). The one or more X-ray contrast agents are typically present
in metal form, in alloy form, in oxide form or in salt form.
[0025] It should be understood that besides iodinated compounds
which provides a useful contrast for X-ray imaging, the formulation
may also include solid particles that are visible by X-ray imaging
or other imaging modalities than X-ray imaging. In one embodiment,
the solid-particles are furthermore visible by MR and/or PET
imaging, or by other imaging modalities.
[0026] In a particular embodiment, the gel-forming composition may
further comprise a radioactive or paramagnetic compound for one or
more imaging modalities such as MRI, PET imaging, SPECT imaging,
nuclear scintigraphy imaging, ultrasonography imaging, ultrasonic
imaging, near-infrared imaging and/or fluorescence imaging.
[0027] In some interesting embodiments, the formulation according
to any one of the preceding claims, contain solid particles that
comprise one or more radioactive, paramagnetic or ferromagnetic
particles.
Moreover, individual particles may comprise two or more types of
compounds which are visible in different imaging modalities.
[0028] Said radioactive compounds may comprise isotopes of Copper
(.sup.61Cu, .sup.64Cu, and .sup.67Cu), Indium (.sup.111In)
Technetium (.sup.99mTc), Rhenium (.sup.186Re, .sup.188Re), Gallium
(.sup.67Ga, .sup.68Ga), Strontium (.sup.89Sr), Samarium
(.sup.163Sm), Ytterbium (.sup.169Yb), Thallium .sup.201Tl),
Astatine (.sup.211At), Lutetium (.sup.177Lu), Actinium
(.sup.225Ac), Yttrium (.sup.90Y), Antimony (.sup.119Sb), Tin
(.sup.117Sn, .sup.113Sn) Dysprosium (.sup.159Dy), Cobalt
(.sup.56Co), Iron (.sup.59Fe), Ruthenium (.sup.97Ru, .sup.103Ru),
Palladium (.sup.103Pd), Cadmium (.sup.115Cd), Tellurium
(.sup.118Te, .sup.123Te), Barium (.sup.131Ba, .sup.140Ba),
Gadolinium (.sup.149Ga, .sup.151Gd), Terbium (.sup.160Tb), Gold
(.sup.198Au, .sup.199Au), Lanthanum (.sup.140La), Zirconium
(.sup.89Zr) and Radium (.sup.223Ra, .sup.224Ra), wherein said
isotope of a metal radionuclide may appear in any of the existing
oxidation states for the metal. These oxidation states include
monovalent cations, divalent cations, trivalent cations,
tetravalent cations, pentavalent cations, hexavalent cations and
heptavalent cations.
[0029] Said paramagnetic or ferromagnetic compounds may also be
selected from the group of Scandium (Sc), Yttrium (Y), Lanthanum
(La), Titanium (Ti), Zirconium (Zr), Hafnium (Hf), Vandium (V),
Niobium (Nb), Tantalum (Ta); Chromium (Cr), Molybdenium (Mo),
Tungsten (W), Manganese (Mn), Technetium (Tc), Rhenium (Re), Iron
(Fe), Ruthenium (Ru), Osmium (Os), Cobalt (Co), Rhodium (Rh),
Iridium (Ir), Nickel (Ni), Palladium (Pd), Platinum (Pt), Copper
(Cu), Silver (Ag), Gold (Au), Zinc (Zn), Cadmium (Cd), Mercury
(Hg), the lanthanides such as Lathanum (La), Cerium (Ce),
Praseodymium (Pr), Neodymium (Nd), Promethium (Pm), Samarium (Sm),
Europium (Eu), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy),
Holmium (Ho), Erbium (Er), Thulium (Tm), Ytterbium (Yb), Lutetium
(Lu)) and the actinides such as Actinium (Ac), Thorium (Th),
Protactinium (Pa), Uranium (U), Neptunium (Np), Plutonium (Pu),
Americium(Am), Curium (Cm), Berkelium (Bk), Californium (Cf),
Einsteinium(Es), Fermium (Fm), Mendelevium (Md), Nobelium (No) and
Lawrencium (Lr), wherein said paramagnetic or ferromagnetic
compounds may appear in any of the existing oxidation states for
the metal. These oxidation states include monovalent cations,
divalent cations, trivalent cations, tetravalent cations,
pentavalent cations, hexavalent cations and heptavalent
cations.
[0030] Said one or more radioactive, paramagnetic or ferromagnetic
compounds may be covalently linked to gel-forming components or the
nano-sized particles or non-covalently associated with the
gel-forming components or nano-sized particles.
[0031] In one embodiment, the gel-forming components or nano-sized
particles further comprise one or more fluorophore compounds for
near infrared fluorescence imaging. Said compounds may comprise a
fluorescent proteins, peptides, or fluorescent dye molecules.
Common classes of fluorescent dyes include xanthenes such as
rhodamines, rhodols and fluoresceins, and their derivatives;
bimanes; coumarins and their derivatives such as umbelliferone and
aminomethyl coumarins; aromatic amines such as dansyl; squarate
dyes; benzofurans; fluorescent cyanines; carbazoles;
dicyanomethylene pyranes, polymethine, oxabenzanthrane, xanthene,
pyrylium, carbostyl, perylene, acridone, quinacridone, rubrene,
anthracene, coronene, phenanthrecene, pyrene, butadiene, stilbene,
lanthanide metal chelate complexes, rare-earth metal chelate
complexes, and derivatives of such dyes. Typical fluorescein dyes
include 5-carboxyfluorescein, fluorescein-5-isothiocyanate and
6-carboxyfluorescein; examples of other fluorescein dyes can be
found, for example, in U.S. Par. Nos. 6,008,379, 5,750,409,
5,066,580, and 4,439,356. The species may also include a rhodamine
dye, such as, for example, tetramethylrhodamine-6-isothiocyanate,
5-carboxytetramethylrhodamine, 5-carboxy rhodol derivatives,
tetramethyl and tetraethyl rhodamine, diphenyldimethyl and
diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101
sulfonyl chloride (sold under the tradename of TEXAS RED), and
other rhodamine dyes. The species may alternatively include a
cyanine dye, such as, for example, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5,
Cy. Or IRDye 800CW, IRDye 680LT, Qdot 800 nanocrystal, Qdot 705
nanocrystal or porphyrazine compounds
[0032] In another embodiment, the nano-sized particles further
comprise or consist of one or more gasses encapsulated in lipid,
polymer or inorganic based particles for ultrasonography imaging.
Said gasses may comprise air, sulphur halides such as sulphur
hexafluoride or disulphur decafluoride; fluorocarbons such as
perfluorocarbons; fluorinated (e.g. perfluorinated) ketones such as
perfluoroacetone; and fluorinated (e.g. perfluorinated) ethers such
as perfluorodiethyl ether. Representative perfluorocarbons, which
may for example contain up to 7 carbon atoms, include
perfluoroalkanes such as perfluoromethane, perfluoroethane,
perfluoropropanes, perfluorobutanes (e.g. perfluoro-n-butane,
optionally in a mixture with other isomers such as
perfluoro-iso-butane), perfluoropentanes, perfluorohexanes and
perfluoroheptanes; perfluoroalkenes such as perfluoropropene,
perfluorobutenes (e.g. perfluorobut-2-ene) and perfluorobutadiene;
perfluoroalkynes such as perfluorobut-2-yne; perfluorocycloalkanes
such as perfluorocyclobutane, perfluoromethylcyclobutane,
perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes,
perfluorocyclopentane, perfluoromethylcyclopentane,
perfluorodimethylcyclopentanes, perfluorocyclohexane,
perfluoromethylcyclohexane and perfluorocycloheptane; and mixtures
of any of the foregoing, including mixtures with gases such as
nitrogen, carbon dioxide, oxygen etc, but not limited to those. In
another embodiment, contrast in achieved using small organic iodine
containing compounds. Said small organic iodine containing
compounds includes commercial available iodinated contrast agents
such as diatrizoate (marketed e.g. under the trade name
Gastrografen.TM.), ionic dimers such as ioxaglate (marketed e.g.
under the trade name Hexabrix.TM.), nonionic monomers such as
iohexol (marketed e.g. under the trade name Omnipaque.TM.),
iopamidol (marketed e.g. under the trade name Isovue.TM.), iomeprol
(marketed e.g. under the trade name Iomeron.TM.) and the non-ionic
dimer iodixanol (marketed under the trade name and Visipaque.TM.)
Additional examples of small organic iodine containing compounds
includes the ones disclosed in WO2009/071605, EP1186305, EP686046,
EP108638, EP0049745, EP0023992, WO2003080554, WO2000026179,
WO1997000240, WO9208691, U.S. Pat. Nos. 5,3804,892, 4,239,747,
3,763,226, 3,763,227 and 3,678,152, but not limited to those. In
another interesting embodiment, the said small organic iodine
containing compounds includes iodinated derivates of sucrose
acetate isobutyrate (SAIB). In contrast to what is disclosed in for
example EP1006935, where a composition for controlled release of a
substance is disclosed which composition comprises SAIB, this
specific embodiment according to the present invention aims at
providing a stable contrast agent embedded in SAIB-gel. Examples of
such iodinated derivates of sucrose acetate isobutyrate (SAIB) are
illustrated in FIG. 7, but not limited to those. Such compounds may
be used alone or in combination with solid particles to achieve an
injectable gel visible by at least CT imaging. In one specific
embodiment of the invention the hydration sensitive gel forming
component is sucrose acetate isobutyrate (SAIB) a hydrophobic
component composed of sucrose (the scaffold) which has been
acylated with isobutyrate and acetate. Preferred scaffolds of this
invention are monosaccharides, disaccharides or trisaccharides. A
particularly preferred dissacharide scaffold is sucrose, however,
the alcohol containing scaffold may be derived from a polyhydroxy
alcohol having from about 2 to about 20 hydroxy groups and may be
formed by esterifying 1 to 20 polyol molecules. Suitable alcohol
moieties include those derived by removing one or more hydrogen
atoms from: monofunctional C1-C20 alcohols, difunctional C1-C20
alcohols, trifunctional alcohols, hydroxy-containing carboxylic
acids, hydroxy-containing amino acids, phosphate-containing
alcohols, tetrafunctional alcohols, sugar alcohols,
monosaccharides, and disaccharides, sugar acids, and polyether
polyols. More specifically, alcohol moieties may include one or
more of: dodecanol, hexanediol, more particularly, 1,6-hexanediol,
glycerol, glycolic acid, lactic acid, hydroxybutyric acid,
hydroxyvaleric acid, hydroxycaproic acid, serine, ATP,
pentaerythritol, mannitol, sorbitol, glucose, galactose, fructose,
maltose, lactose, glucuronic acid, polyglycerol ethers containing
from 1 to about 10 glycerol units, polyethylene glycols containing
1 to about 20 ethylene glycol units. Additionally, any
oligosaccharide containing from 3 to about 6 monosaccharides may be
used as the scaffold in the present invention. In general, the
scaffold esters of the invention can be made by reacting one or
more alcohols, in particular one or more polyols, which will form
the alcohol moiety of the resulting esters with one or more
carboxylic acids, lactones, lactams, carbonates, or anhydrides of
the carboxylic acids which will form the acid moieties of the
resulting esters. The esterification reaction can be conducted
simply by heating, although in some instances addition of a strong
acid or strong base esterification catalyst may be used.
Alternatively, an esterification catalyst such as stannous
2-ethylhexanoate or activation reagents such as
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC),
N,N'-Dicyclohexylcarbodiimide (DCC),
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and the like can be used.
[0033] The acyl groups forming the acyloxy substituents of the
invention may be any moiety derived from a carboxylic acid. More
particularly, the acyl groups of the compositions of the invention
may be of the RCO-, where R is optionally oxy-substituted alkyl of
2-10 carbon atoms which may be linear or branched hydrocarbons with
one or more functional groups present in the chain. Using
carboxylic acids and/or polyols of different chain length and using
carboxylic acids having oxy-substitution allows control of the
degree of hydrophilicity and of the solubility of the resulting
ester. Such materials are sufficiently resistant to dissolution in
vivo that they are able to form stabile hydrophobic gels which may
encapsulate the said contrast agents of the present invention. The
gels may further comprise a pharmaceutical agent in combination
with the contrast agent.
Coating of Solid Particles
[0034] The solid particles may further comprise a variety of other
components.
[0035] Useful solid particles include uncoated or coated metal
particles, uncoated or coated solid metal salts, as well as
liposomes, polymersomes, dendrimers, water-soluble cross-linked
polymers, and micelles comprising such solid particles. As used
herein, a solid particle which is "coated" comprises a shell or
surface coating around a solid core material. The shell or surface
coating can be attached to the core material covalently,
non-covalently, or by a mixture of covalent and non-covalent bonds.
Exemplary shell or surface coatings are described herein. In one
embodiment, the solid particle comprises a polymer surface coating
non-covalently or covalently attached to the particle core surface.
The polymer may be a homopolymer, a copolymer, block copolymer, or
a graft copolymer, or a dendrimer-type copolymer of synthetic or
natural origin, but not limited to those. Typically, the polymer
coating comprises polyethylene glycol (PEG), typically with a PEG
molecular weight from 2,000 to 70,000 Daltons, such as 5,000
Daltons; dextrans, typically with a molecular weight between 2,000
and 1,000,000 Daltons; and/or hyaluronic acid, typically with a
molecular weight between 2,000 and 1,000,000 Daltons. The polymers
are typically combined as block copolymers in such a way that the
overall polymer structure in negatively charged, allowing
electrostatic interactions with a positively charged nano-sized
particle surface to achieve efficient coating. In a particular
embodiment, the solid particles comprise conjugated PEG.sub.1000,
PEG.sub.2000, PEG.sub.3000, PEG.sub.5000 or PEG.sub.10000, i.e.,
PEG preparations having an average molecular weight of
approximately 1,000, 2,000, 3,000, 5,000 and 10,000 Daltons,
respectively, but not limited to those. In an additional
embodiment, the solid particles comprise conjugated
PNIPAM.sub.1000, PNIPAM.sub.2000, PNIPAM.sub.3000, PNIPAM.sub.5000
or PNIPAM.sub.10000, i.e., PNIPAM preparations having an average
molecular weight of approximately 1,000, 2,000, 3,000, 5,000 and
10,000 Daltons, respectively, but not limited to those. In one
embodiment, the solid particles comprise a shell or surface coat
comprising a lipid layer such as a lipid monolayer and/or one or
more lipid bilayers, and a particle core comprising an inorganic
particle. Surface-coating lipids for the purpose of the present
invention, and include, for example, fatty acids, neutral fats,
phosphatides, glycolipids, ceramides, sphingoglipids, aliphatic
alcohols, and steroids. Specific, non-limiting examples of solid
particles are gold nano-sized particles synthesized with a PEG
coating or PEGylated gold nanorods as described in WO 2007/129791
and Kim et al 2007 [Invest. Radiol., 2007, 42, 797-806],
polymer-coated bismuth sulphide nano-sized particles as described
in Rabin 2006 [Nat. Mater., 2006, 5, 188-122], calcium phosphate
liposome core-shell nanocomposites, dendrimers of PAMAM with
entrapped gold nano-sized particles for CT imaging as described in
Haba et al. 2007 [Langmuir, 2007, 23, 5243-5246] and Kojima et al
2010 [Bioconjugate Chem., 2010, 21, 1559-1564] and other solid
particles comprising X-ray contrast agents known in the art. In a
specific embodiment of the present invention, the shell of the
nano-sized particle comprises
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) "A", cholesterol
"B", and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy
(polyethylene glycol)-2000] (DSPE-PEG-2000) "C", and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy
(polyethylene glycol)-2000]-TATE (DSPE-PEG-2000-RGD) "D" with the
molar ratio A:B:C:D, wherein A is selected from the interval 45 to
65, B is selected from the interval 35 to 45, C is selected from
the interval 5 to 13, D is selected from the interval 0 to 3, and
wherein A+B+C+D=100.
[0036] Coating of the solid particles can be exploited to introduce
the desired chemical and/or physical properties to the colloid
particles. Properties such as hydrophobicity/hydrophilicity,
particle charge, hydrodynamic diameter and stability in various
environments such as high/low salt concentrations, organic
solvents, reductive environments and heat, among others, can be
controlled by choosing the correct surface coating material. These
properties, introduced to the solid particles by the surface
coating, are important factors to control in order to tune the
overall behavior of the X-ray contrast composition described
here.
[0037] The amount of contrast agent comprised within the
gel-forming composition including an embedded the nano-sized
particles according to the present invention may be quantified by
the weight percent of the contrast agent relative to the total
weight of the gel-forming system including an embedded nano-sized
particle, excluding any water comprised by the nano-sized particle,
by defining the weight percent of the contrast agent relative to
the weight of the shell of the nano-sized particle, or by
quantifying the size of the contrasting agent within the prepared
nano-sized particles. The latter can be measured by conventional
methods in the art, such as cryo-transmission electron microscopy
or dynamic light scattering.
Shape and Size
[0038] The nano-sized particles according to the present invention
can be quasi spherical, spherical or non-spherical such as
rod-shaped. Suitable nanoparticles include those having a size up
to 50 .mu.m, preferably up to 5 .mu.m. Preferably, the nano-sized
particles according to the present invention are of a size in the
range of 1 to 1000 nm, such as 2 to 10 nm, or such as 10 to 100 nm,
such as 10 to 80 nm, such as 10 to 50 nm, such as 10 to 20 nm, such
as 10 to 15 nm, or such as 15 to 20 nm, or such as 20 to 50 nm, or
such as 50 to 80 nm, or such as 80 to 110 nm, or such as 110 to 140
nm, or such as 140 to 170 nm, or such as 170 to 200 nm or such as
200 to 220, or such as 220 to 250 nm, or such as 250 to 280 nm, or
such as 280 to 310 nm, or such as 310 to 340 nm, or such as 340 to
370 nm, or such as 370 to 400 nm, or such as 400 to 420, or such as
420 to 450 nm, or such as 450 to 480 nm, or such as 480 to 500 nm,
or such as 500 to 1000 nm. The size may according to the present
invention be measured in terms of the diameter, length or width,
including the number average diameter, length or width. In a
preferred embodiment, the nano-sized particles in the composition
of the present invention have a number average diameter in the
range of 10 nm to 150 nm, such as 10 to 100 nm, such as 10 to 80
nm, such as 10 to 50 nm, such as 10 nm to 30 nm, such as 10 to 20
nm, or such as 30 nm to 40 nm, or such as 40 nm to 50 nm, or such
as 50 nm to 60 nm, or such as 60 nm to 70 nm, or such as 70 nm to
80 nm, or such as 90 nm to 100 nm, or such as 100 nm to 110 nm, or
such as 110 nm to 120 nm, or such as 120 nm to 130 nm, or such as
130 nm to 140 nm, or such as 140 nm to 150 nm. Controlling the
shape and the size of the nano-sized particles may have significant
influence on the stability of the nano-scale colloidal suspensions
as well as the in vivo fate of the particles. In a preferred
embodiment, the nano-sized particles in the composition of the
present invention have a number average diameter in the range of 10
nm to 100 nm. Such nano-sized particles exhibit low/no
sedimentation rate due to the effects of Brownian motion. In
another preferred embodiment, the nano-sized particles in the
composition of the present invention have a number average diameter
<10 nm.
[0039] Such particles may be cleared, after degradation of the
hydrogel, by e.g. renal filtration with subsequently excretion into
the urine, which may prevent prolonged tissue retention and/or thus
lower the risk of toxicity.
The Organic Gel-Forming System
[0040] Suitable gel-forming components include, but are not limited
to, those composed of organic constituents such as derivatized
saccharides such as esterified saccharides, derivatized polyols
such as esterified polyols, polymers, lipids, peptides, proteins,
low molecular weight gelators and non-water soluble high-viscosity
liquid carrier materials as well as combinations hereof.
[0041] The saccharides and polyols gel forming sysemts may be
sucrose acetate isobutyrate (SAIB) a hydrophobic component composed
of sucrose (the scaffold) which has been acylated with isobutyrate
and acetate. Preferred scaffolds of this invention are
monosaccharides, disaccharides or trisaccharides. A particularly
preferred dissacharide scaffold is sucrose, however, the alcohol
containing scaffold may be derived from a polyhydroxy alcohol
having from about 2 to about 20 hydroxy groups and may be formed by
esterifying 1 to 20 polyol molecules. Suitable alcohol moieties
include those derived by removing one or more hydrogen atoms from:
monofunctional C1-C20 alcohols, difunctional C1-C20 alcohols,
trifunctional alcohols, hydroxy-containing carboxylic acids,
hydroxy-containing amino acids, phosphate-containing alcohols,
tetrafunctional alcohols, sugar alcohols, monosaccharides, and
disaccharides, sugar acids, and polyether polyols. More
specifically, alcohol moieties may include one or more of:
dodecanol, hexanediol, more particularly, 1,6-hexanediol, glycerol,
glycolic acid, lactic acid, hydroxybutyric acid, hydroxyvaleric
acid, hydroxycaproic acid, serine, ATP, pentaerythritol, mannitol,
sorbitol, glucose, galactose, fructose, maltose, lactose,
glucuronic acid, polyglycerol ethers containing from 1 to about 10
glycerol units, polyethylene glycols containing 1 to about 20
ethylene glycol units. Additionally, any oligosaccharide containing
from 3 to about 6 monosaccharides may be used as the scaffold in
the present invention. In general, the scaffold esters of the
invention can be made by reacting one or more alcohols, in
particular one or more polyols, which will form the alcohol moiety
of the resulting esters with one or more carboxylic acids,
lactones, lactams, carbonates, or anhydrides of the carboxylic
acids which will form the acid moieties of the resulting esters.
Such systems are known to form biodegradable, amorphous
carbohydrate glass matrixes upon hydration due to solvent induced
phase separation.
[0042] The polymer may be a homopolymer, a copolymer, block
copolymer, or a graft copolymer, or a dendrimer-type copolymer of
synthetic or natural origin.
[0043] Specific examples of suitable monomers may include: Lactide,
glycolide, N-vinyl pyrrolidone, vinyl pyridine, acrylamide,
methacrylamide, N-methyl acrylamide, hydroxyethyl methacrylate,
hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl
acrylate, methacrylic acid and acrylic acid having an acidic group,
and salts of these acids, vinyl sulfonic acid, styrenesulfonic
acid, etc., and derivatives having a basic group such as
N,N-dimethylaminoethyl methacrylate, N,N-diethylaminoethyl
methacrylate, N,N-dimethylaminopropyl acrylamide, salts of these
derivatives, etc. Other monomers may include: acrylate derivatives
and methacrylate derivatives such as ethyl acrylate, methyl
methacrylate, and glycidyl methacrylate; N-substituted alkyl
methacrylamide derivatives such as N-n-butyl methacrylamide; vinyl
chloride, acrylonitrile, styrene, vinyl acetate, lactones such as
-caprolactone, lactames such as -caprolactame and the like.
Additional examples of suitable monomers include alkylene oxides
such as propylene oxide, ethylene oxide and the like, but not
restricted to any of these specific examples.
[0044] On the other hand, specific examples of polymeric blocks to
be combined with (or bonded to) the above-mentioned monomers may
include: methyl cellulose, dextran, polyethylene oxide,
polypropylene oxide, polyvinyl alcohol, poly N-vinyl pyrrolidone,
polyvinyl pyridine, polyacrylamide, polymethacrylamide, poly
N-methyl acrylamide, polyhydroxymethyl acrylate, polyacrylic acid,
polymethacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic
acid, and salts of these acids; poly N,N-dimethylaminoethyl
methacrylate, poly N,N-diethylaminoethyl methacrylate, poly
N,N-dimethylaminopropyl acrylamide, and salts of these, poly
lactic-co-glycolic acid, polycaprolactone and combinations hereof,
but not limited to those. The lipid may be any phospholipid
including one or more of a sterol such as cholesterol, and
cholestanol, a fatty acid having a saturated or unsaturated acyl
group having 8 to 22 carbon atoms and an antioxidant such as
alpha-tocopherol. Examples of the phospholipids include, for
example, phosphatidylethanolamines, phosphatidylcholines,
phosphatidylserines, phosphatidylinositols, phosphatidyl-glycerols,
cardiolipins, sphingomyelins, ceramide phosphorylethanolamines,
ceramide phosphorylglycerols, ceramide phosphorylglycerol
phosphates, 1,2-dimyristoyl-1,2-deoxyphosphatidylcholines,
plasmalogens, phosphatidic acids, and the like, and these may be
used alone or two or more kind of them can be used in combination.
The fatty acid residues of these phospholipids are not particularly
limited, and examples thereof include a saturated or unsaturated
fatty acid residue having 12 to 20 carbon atoms. Specific examples
include an acyl group derived from a fatty acid such as lauric
acid, myristic acid, palmitic acid, stearic acid, oleic acid and
linoleic acid. Further, phospholipids derived from natural products
such as egg yolk lecithin and soybean lecithin can also be used.
Also suitable are, for example, di- and tri-glycerides,
1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP),
1-N,N-dimethylaminodioleoylpropane (DODAP),
1-oleoyl-2-hydroxy-3-N,N-dimethylamino-propane,
1,2-diacyl-3-N,N-dimethylaminopropane,
1,2-didecanoyl-1-N,N-dimethylamino-propane,
3-beta-[n-[(N',N'-dimethylamino)ethane]-carbamoyl]-cholesterol
(DC-Chol), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium
bromide (DMRIE),
1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium bromide
(DORI), and the like, but not limited to those.
[0045] A "peptide" or "polypeptide" refers to a string of at least
two .alpha.-amino acid residues linked together by chemical bonds
(for example, amide bonds). Depending on the context, the term
"peptide" may refer to an individual peptide or to a collection of
peptides having the same or different sequences, any of which may
contain only naturally occurring .alpha.-amino acid residues,
non-naturally occurring .alpha.-amino acid residues, or both. The
peptide may exhibit self-assembling properties, for example,
peptide amphiphiles, and peptides with .beta.-sheet or
.alpha.-helical forming sequences. The peptides may include D-amino
acids, L-amino acids, or combinations thereof. Suitable,
naturally-occurring hydrophobic amino acid residues which may be in
the self-assembling peptides include Ala, Val, Ile, Met, Phe, Tyr,
Trp, Ser, Thr and Gly. The hydrophilic amino acid residues may be
basic amino acids (for example, Lys, Arg, His, Orm); acidic amino
acids (for example, Glu, Asp); or amino acids that form hydrogen
bonds (for example, Asn, Gln). Degradation of L-amino acids
produces amino acids that may be reused by the host tissue.
L-configured amino acid residues occur naturally within the body,
distinguishing peptides formed from this class of compounds from
numerous other biocompatible substances. L-configured amino acids
contain biologically active sequences such as RGD adhesion
sequences. The amino acid residues in the self-assembling peptides
may be naturally occurring or non-naturally occuring amino acid
residues. Naturally occurring amino acids may include amino acid
residues encoded by the standard genetic code, amino acids that may
be formed by modifications of standard amino acids (for example
pyrrolysine or selenocysteine), as well as non-standard amino acids
(for example, amino acids having the D-configuration instead of the
L-configuration). Although, non-naturally occurring amino acids
have not been found in nature, they may be incorporated into a
peptide chain. These include, for example,
D-alloiso-leucine(2R,3S)-2-amino-3-methylpentanoic: acid,
L-cyclopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid.
Self-assembling peptides used in accordance with the disclosure may
vary in length so long as they retain the ability to e.g.
self-assemble to an extent useful for one or more of the purposes
described herein. Peptides having as few as two .alpha.-amino acid
residues or as many as approximately 50 residues may be suitable.
In embodiments, .alpha.-amino acid analogs can be used. In
particular, .alpha.-amino acid residues of the D-form may be used.
Useful peptides may also be branched. One or more of the amino acid
residues in a self-assembling peptide may be functionalized by the
addition of a chemical entity such as an acyl group, a carbohydrate
group, a phosphate group, a farnesyl group, an isofarnesyl group, a
fatty acid group, or a linker for conjugation. This functional
group may provide for inter-peptide linkages, or linkages between
the peptide and the hydrogel or hydrogel precursor. For example,
the hydrophobic portion of an amphiphilic peptide may be
functionalized with acetylene groups. Alternatively, either or both
ends of a given peptide may be modified. For example, the carboxyl
and/or amino groups of the carboxyl- and amino-terminal residues,
respectively, may be protected or not protected. Examples of self
assembling peptides include the ones disclosed by Nagai, et al. [J.
Controlled Release, 2006, 115, 18-25], Schneider et al. [PLoS ONE,
2008, 1, 1-8] and Hartgerink et al. [PNAS, 2002, 99,
5133-5138].
[0046] The protein is not particularly limited and may have a
molecular weight from 5-500 kDa, such as 20-200 kDa. It may be of
natural origin or human engineered protein expressed in accessible
biological expression systems such as e.g. yeast, mammalian, and
bacterial expression systems. Preferably, is has a responsive
domain such as .alpha.-helical coiled-coil or leucine zipper
domain--but not limited to those, which upon external or internal
stimuli results in hydrogel formation which structurally respond to
changes in e.g. pH, temperature, and ionic strength. Examples of
such proteins include the ones disclosed by Banta et al. [Annu.
Rev. Biomed. Eng., 2010, 12, 167-86].
[0047] The low molecular weight gelators include any molecule with
molecular weight from 100-4,000 Daltons, such as 250-1,000 Daltons
with an amphiphilic structure capable of forming a hydrogel.
Specific, non-limiting examples of low molecular weight gelators as
described in WO 2008/102127 A2, Chem. Rev., 2004, 104, 1201-1217
and Eur. J. Org. Chem., 2005, 3615-3631.
[0048] The non-water soluble high-viscosity liquid carrier
materials include, but are not limited to, sucrose acetate
isobutyrate, stearate esters such as those of propylene glycol,
glyceryl, diethylaminoethyl, and glycol, stearate amides and other
long-chain fatty acid amides, such as N,N'-ethylene distearamide,
stearamide MEA and DEA, ethylene bistearamide, cocoamine oxide,
long-chain fatty alcohols, such as cetyl alcohol and stearyl
alcohol, long-chain esters such as myristyl myristate,
behenyerucate, glyceryl phosphates, acetylated sucrose distearate
(Crodesta A-IO), and the like.
[0049] The gel of the present invention having biodegradability and
sol-gel phase transition which depends on pH, temperature,
ion-concentration, enzymatic activity, electric field or
hydration.
[0050] The composition of the solvent (dispersion medium) should
not be particularly limited, and examples include, for example, a
buffer such as phosphate buffer, citrate buffer, and
phosphate-buffered physiological saline, physiological saline, a
medium for cell culture and biocompatible organic solvent such as
ethanol, ethyl lactate, propylene carbonate, glycofurol,
N-methylpyrrolidone, 2-pyrrolidone, propylene glycol, acetone,
methyl acetate, ethyl acetate, methyl ethyl ketone, benzyl alcohol,
triacetin, dimethylformamide, dimethylsulfoxide, tetrahydrofuran,
caprolactam, decylmethylsulfoxide, oleic acid,
1-dodecylazacycloheptan-2-one and the like. Although the
formulation can be stably dispersed in these solvents (dispersion
media), the solvents may be further added with a saccharide
(aqueous solution), for example, a monosaccharide such as glucose,
galactose, mannose, fructose, inositol, ribose and xylose,
disaccharide such as lactose, sucrose, cellobiose, trehalose and
maltose, trisaccharide such as raffinose and melezitose, and
polysaccharide such as .alpha.-, .beta.-, or .gamma.-cyclodextrin,
sugar alcohol such as erythritol, xylitol, sorbitol, mannitol, and
maltitol, or a polyhydric alcohol (aqueous solution) such as
glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene
glycol, ethylene glycol, diethylene glycol, triethylene glycol,
polyethylene glycol, ethylene glycol mono-alkyl ether, diethylene
glycol mono-alkyl ether and 1,3-butylene glycol. Additives may
furthermore be selected from the group consisting of bioavailable
materials such as amiloride, procainamide,
acetyl-beta-methylcholine, spermine, spermidine, lysozyme, fibroin,
albumin, collagen, transforming growth factor-beta (TGF-beta), bone
morphogenetic proteins (BMPs), fibroblast growth factor (bFGF),
dexamethason, vascular endothelial growth factor (VEGF),
fibronectin, fibrinogen, thrombin, proteins, dexrazoxane,
leucovorin, ricinoleic acid, phospholipid, small intestinal
submucosa, vitamin E, polyglycerol ester of fatty acid, Labrafil,
Labrafil M1944CS, citric acid, glutamic acid, hydroxypropyl,
isopropyl myristate, Eudragit, tego betain,
dimyristoylphosphatidyl-choline, scleroglucan, and the like;
organic solvents such as cremophor EL, ethanol, dimethyl sulfoxide,
and the like; preservatives such as methylparaben and the like;
sugars such as starch and derivatives thereof, sugar-containing
polyols such as sucrose-mannitol, glucose-mannitol, and the like;
amino acids such as alanine, arginine, glycine, and the like;
polymer-containing polyols such as trehalose-PEG; sucrose-PEG,
sucrose-dextran, and the like; sugar-containing amino acid such as
sorbitol-glycine, sucrose-glycine, and the like; surfactants such
as poloxamer of various molecular weights, Tween 20 Tween 80,
Triton X-100, sodium dodecyl sulfate(SDS), Brij, and the like;
sugar-containing ions such as trehalose-ZnSO.sub.4,
maltose-ZnSO.sub.4, and the like; and bio-acceptable salts such as
silicate, NaCl, KCl, NaBr, NaI, LiCl, n-Bu.sub.4NBr, n-Pr.sub.4NBr,
Et.sub.4NBr, Mg(OH).sub.2, Ca(OH).sub.2, ZnCO.sub.3,
Ca.sub.3(PO.sub.4).sub.2, ZnCl.sub.2,
(C.sub.2H.sub.3O.sub.2).sub.2Zn, ZnCO.sub.3, CdCl.sub.2,
HgCl.sub.2, CaCl.sub.2, (CaNO.sub.3).sub.2, BaCl.sub.2, MgCl.sub.2,
PbCl.sub.2, AlCl.sub.2, FeCl.sub.2, FeCl.sub.3, NiCl.sub.2, AgCl,
AuCl, CuCl.sub.2, sodium tetradecyl sulfate,
dodecyltrimethyl-ammonium bromide, dodecyltrimethylammonium
chloride, tetradecyltrimethyl-ammonium bromide, and the like, but
not limited to those.
[0051] In one embodiment of the present invention, the content of
the additive is from 1.times.10.sup.-6-30 wt %, preferably
1.times.10.sup.-3 to 10 wt %, based on the total weight of the gel
forming component(s).
[0052] A preferred injectable medical gel-forming system can have
one or more, preferably all, of the following features:
[0053] (1) In order to be injectable, the system should be in a sol
state before administration. The sol state should be of
sufficiently low viscosity--typically lower than 10,000 cP,
preferably lower than 2,000 cP, at 20.degree. C. (or alternatively
lower than lower than 10,000 cP, preferably 2,000 cP, at 5.degree.
C.)--to allow for small needle head to alleviate the patient
discomfort and simplify insertion procedure.
[0054] (2) Gelation via either chemical cross-linking, physical
association or hydration starts to happen or is complete after
injection.
[0055] (3) The gels should be biodegradable or gradually
dissolvable within a controlled time period, and the products
should be cleared/secreted through normal pathways.
[0056] (4) The polymer itself and the degradable products should be
biocompatible. Likewise, if additives are added, such as
cross-linking agents, initiators etc. these should also be
biocompatible.
[0057] (5) The gel could potentially have cell/tissue-adhesive
properties.
[0058] (6) The gel should not result in adverse effects such as
immune response, e.g. inflammation.
[0059] It should be understood, that the gel-forming system should
preferably be biocompatible, i.e. does not stimulate a severe,
long-lived or escalating biological response to the formulation
when injected into a mammal, in particular a human. To facilitate
metabolism of the gel scaffold, degradable linkages can be included
through the use of polylactide, polyglycolide,
poly(lactide-co-glycolide), polyphosphazine, polyphosphate,
polycarbonate, polyamino acid, polyanhydride, and
polyorthoester-based building blocks, among others. Additionally,
small molecule crosslinking agents containing similar hydrolyzable
moieties as the polymers such as carbonates, esters, urethanes,
orthoesters, amides, imides, imidoxy, hydrazides, thiocarbazides,
and phosphates may be used as building blocks. Additionally,
polyglycolide diacrylate, polyorthoester diacrylate and
acrylate-substituted polyphosphazine, acrylate-substituted
polyamino acid, or acrylate-substituted polyphosphate polymers can
be used as degradable building blocks. Methacrylate or acrylamide
moieties can be employed instead of acrylate moieties in the above
examples. Similarly, small molecules containing a hydrolyzable
segment and two or more acrylates, methacrylates, or acrylamides
may be used. Such degradable polymers and small molecule building
blocks may be functionalized with acrylate, methacrylate,
acrylamide or similar moieties by methods known in the art.
[0060] In order to be injectability, the system should be in a sol
state before administration. The sol state should be of
sufficiently low viscosity to allow for small needle head to
alleviate the patient discomfort and simplify insertion procedure.
Gelation via either chemical cross linking or physical association
starts to happen or is complete after injection.
[0061] Preferred properties of the gel-forming system include one
or more of the following:
[0062] The gel-forming system may form a hydrogel. Hydrogels are
comprised of cross-linked polymer networks that have a high number
of hydrophilic groups or domains. These networks have a high
affinity for water, but are prevented from dissolving due to the
chemical or physical bonds formed between the polymer chains. Water
penetrates these networks causing swelling, giving the hydrogel its
form. Fully swollen hydrogels have some physical properties common
to living tissues, including a soft and rubbery consistency, and
low interfacial tension with water or biological fluids. The
elastic nature of fully swollen or hydrated hydrogels can minimize
irritation to the surrounding tissues after implantation. A low
interfacial tension between the hydrogel surface and body fluid
minimizes protein adsorption and cell adhesion, which reduces the
risk of an adverse immune reaction. Many polymers used in hydrogel
preparations (e.g. polyacrylic acid (PAA), PHEMA, PEG, and PVA)
have mucoadhesive and bioadhesive characteristics that enhance drug
residence time and tissue permeability. This adhesive property is
due to inter-chain bridges between the hydrogel polymer's
functional groups and the mucus glycoproteins, which can help
enhance tissue specific binding.
[0063] Preferably, before in vivo administration, the gel-forming
system according to the invention is a flowable solution. The
organic x-ray contrast agent, such as iodinated SAIB derivatives as
illustrated in FIG. 7 or other iodinated polymers, and solid
inorganic particles can, for example, be added to the gel-forming
system simply by mixing before injection. Once injected, the
gel-forming system rapidly gels under physiological conditions. An
injectable matrix can thus be implanted in the human body with
minimal surgical procedure. After gelation in situ, the matrix can
provide a reference marker for imaging and image-guided
radiotherapy.
[0064] A number of activators or conditions can be used to trigger
this transition upon injection, either externally applied or in
response to the tissue micro-environment. Examples of this include
gelation as a response to pH, temperature, ion-concentration,
enzymatic activity, electric field and hydration (FIG. 1). In
relation to the invention it is relevant to be able to tune the
mechanical stability within the tissue to allow for single
injections.
Gel-Forming System in Response to Temperature Change
[0065] In one embodiment, the gel-forming system undergoes
gel-formation in response to a temperature in the range of
10-65.degree. C., preferably in the range 35-40.degree. C.
[0066] The favored thermosensitive material might exhibit an
inverse sol-gel transition. The term "inverse" here means that
gelation occurs upon heating instead of cooling. Exemplary
biodegradable or bioabsorbable thermogelling polymers are shown in
FIG. 2. According to the origin of materials, thermogelling
hydrogels can be classified into natural (or seminatural) polymeric
systems and synthetic polymeric systems. The polymers in the former
system include cellulose, chitosan, xyloglucan, gelatin etc. and
their derivatives. The polymers in the latter class include some
polyethers, block copolymers of polyethers and biodegradable
polyesters, synthetic polypeptides, and other polymers (FIG.
2).
[0067] Other examples of such gel-forming systems are those
described in; i) Eur. J. Pharm. Biopharm., 2004, 57, 53-63, ii)
Chem. Soc. Rev., 2008, 37, 1473-1481, iii) Adv. Drug Deliv. Rev.,
2010, 62, 83-99, iv) Macromol. Biosci., 2010, 10, 563-579, v) J.
Controlled Release, 2005, 103, 609-624, vi) Expert Opin. Ther.
Patents, 2007, 17, 965-977, vii) Appl. Microbiol. Biotechnol.,
2011, 427-443, viii) Science, 1998, 281, 389-392, ix) Eur. J.
Pharm. Biopharm. 2008, 68, 34-45, x) Biomacromolecules, 2002, 4,
865-868, xi) Colloids and Surfaces B: Biointerfaces, 2011, 82,
196-202, xii) Biomacromolecules, 2010, 11, 1082-1088, xiii) Adv.
Eng. Mater., 2008, 10, 515-527, xiv) Eur. J. Pharm. Biopharm.,
2004, 58, 409-426, xv) Adv. Drug Deliv. Rev., 2002, 54, 37-51, xvi)
Biomater., 2004, 25, 3005-3012, xvii) J. Biomed. Mater. Res., 2000,
50, 171-177, xviii) xix) WO 2007/064252, xx) WO 2009/150651, xxi)
WO 2007/064152, xxii) WO 99/07416, xxiii) Park K., Shalaby W. S.
W., Park H., Biodegradable hydrogels for drug delivery. Basel:
Technomic Publishing Co., Inc., 1993. ISBN 1-56676-004-6, Print,
xxiv) Biomedical polymers and polymers therapeutics, Ed. Chiellini
E., Sunamoto J., Migliaresi C., Ottenbrite R. M., Cohn D., New
York, Kluwer Academic Publishers, 2002, ISBN 0-30646472-1,
Print--and references herein, but not limited to those.
[0068] In one interesting embodiment the thermo sensitive polymer
is poly(ethylene glycol)-b-poly(propylene glycol)-b-poly(ethylene
glycol) (PEG-PPG-PEG, Pluronic.RTM. or Poloxamer) or derivates
hereof. By controlling the PEG/PPG composition, the molecular
weight and the concentration, reversible gelation can occur at
physiological temperature and pH.
[0069] In another interesting embodiment the thermo sensitive
polymer is chitosan. Chitosan can be a thermally sensitive, pH
dependent, gel-forming system by the addition of polyol salts (e.g.
.beta.-glycerophosphate, GP). These formulations possess a neutral
pH, remain liquid at or below room temperature, and form monolithic
gels at body temperature. The stability of the sol at room
temperature and the gelation time increase as the chitosan degree
of deacetylation decreases [Int. J. Pharm., 2000, 203, 89-98]. The
gelation for these chitosan-based systems occurs by the combination
of charge neutralization, ionic and hydrogen bonds and, as the main
driving force, hydrophobic interaction factors. Additionally, such
systems are highly compatible with biological compounds and can be
used to inject in vivo biologically active growth factors and cells
[Biomater., 2000, 21, 2155-2161].
[0070] In one very interesting embodiment the thermo sensitive
polymer is poly(caprolactone-b-ethylene glycol-b-caprolactone)
(PCL-PEG-PCL), poly(ethylene glycol-b-caprolactone- ethylene
glycol) (PEG-PCL-PEG) or poly(ethylene glycol-b-caprolactone)
(PEG-PCL). This family of block co-polymers can be tuned to be free
flowing solutions at room temperature and strong biodegradable gels
at body temperature. Such polymers are highly biocompatible having
showed very little toxicity with a maximum tolerance dose of 25
g/kg body weight by subcutaneous administration [J. Pharm. Sci.,
2009, 98, 4684-4694] and have been found stabile in vivo for more
than 4 weeks [Tissue Eng. 2006, 12, 2863-2873].
[0071] In another interesting embodiment the thermo sensitive
polymer is poly(ethylene glycol-b-[DL-lactic acid-co-glycolic
acid]-b-ethylene glycol) (PEG-PLGA-PEG) triblock copolymers.
PEG-PLGA-PEG (33 wt %) is a free-flowing sol at room temperature
and become a gel at body temperature. The gel showed good
mechanical strength and the integrity of gels persisted longer than
1 month [J. Biomed. Mater. Res., 2000, 50, 171-177]. Additional
examples includes poly(N-isopropylacrylamide)-g-methylcellulose
copolymer as a reversible and rapid temperature-responsive sol-gel
hydrogel. By tuning the methylcellulose content gelation
temperature, gelation time and mechanical strength can be
controlled [Biomater., 2004, 25, 3005-3012].
Gel-Forming System in Response to Change in Ion-Strength
[0072] In another embodiment, wherein the gel-forming system
undergoes gel-formation in response to change in ion-strength in
the range of 1 .mu.M-500 mM--preferably in the range of 1-50 mM or
50-200 mM.
[0073] Non-limiting examples of such gel-forming systems include
those illustrated in FIG. 3 and those described in i) Int. J.
Pharm. 1989, 57, 163-168, ii) J. Controlled Release, 1997, 44,
201-208, iii) J. Am. Chem. Soc., 2001, 123, 9463-9464, iv) J.
Controlled Release, 2003, 86, 253-265, v) Biomater., 2001, 22,
511-521, xi) Park K., Shalaby W. S. W., Park H., Biodegradable
hydrogels for drug delivery. Basel: Technomic Publishing Co., Inc.,
1993. ISBN 1-56676-004-6, Print xii) Biomedical polymers and
polymers therapeutics, Ed. Chiellini E., Sunamoto J., Migliaresi
C., Ottenbrite R. M., Cohn D., New York, Kluwer Academic
Publishers, 2002, ISBN 0-30646472-1, Print; and references cited
therein.
[0074] One intriguing example of such a gel-forming system is that
of alginate. Alginic acid is an unbranched binary copolymer of 1-4
glycosidically linked L-guluronic acid (G) and its C-5 epimer
D-mannuronic acid (M). The proportion as well as the distribution
of the two monomers determines to a large extent the physiochemical
properties of alginate.
[0075] In one embodiment, the gel-forming system is based on an
aqueous solution of an alginate. Alginates are a family of linear
polysaccharides, which, in aqueous solutions, can gel after
addition of multivalent cations. The use of alginate as an
immobilizing agent in most applications rests in its ability to
form heat-stable strong gels which can develop and set at room
temperatures. It is the alginate gel formation with calcium ions
which has been of interest in most applications. However, alginate
forms gels with most di- and multivalent cations. Monovalent
cations and Mg.sup.2+ ions do not induce gelation while ions like
Ba.sup.2+ and Sr.sup.2+ will produce stronger alginate gels than
Ca.sup.2+. The gel strength depends on the guluronic content and
also of the average number of G-units in the G-blocks. Gelling of
alginate occur when divalent cations takes part in the interchain
binding between G-blocks giving rise to a three-dimensional network
in the form of a gel (FIG. 1). The alginate gel as an
immobilization matrix is sensitive to chelating compounds such as
phosphate, lactate and citrate, presence of anti-gelling cations
such as Na.sup.+ or Mg.sup.2+. To avoid this gel beads may be kept
in a medium containing a few millimolar free calcium ions and by
keeping the Na.sup.+/Ca.sup.2+ ratio less than 25:1 for high G
alginates and 3:1 for low G alginates. An alternative is also to
replace Ca.sup.2+ with other divalent cations with a higher
affinity for alginate. There has been found a correlation between
mechanical gel strength and affinity for cations. It has been found
that gel strength may decrease in the following orders:
Pb.sup.2+>Cu.sup.2+.dbd.Ba.sup.2+>Sr.sup.2+>Cd.sup.2+>Ca.sup.-
2+>Zn.sup.2+>Co.sup.2+>Ni.sup.2+ However, in applications
involving immobilization of living cells toxicity is a limiting
factor in the use of most ions, and only Sr.sup.2+, Ba.sup.2+ and
Ca.sup.2+ are considered as nontoxic for these purposes. Alginate
gels have been found stable in a range of organic solvents.
[0076] Since the gel-inducing factor is added before injection,
slow physical gelation is required in order to avoid syringe jam.
To combat this, calcium ions can be slowly released from, e.g.,
CaSO.sub.4 powder after the powder has been added to a sodium
alginate aqueous solution [J. Biomater. Sci., Polym. Ed., 1998, 9,
475-487]. In another interesting embodiment co-injection of the
gel-inducing factor and the aqueous alginate solution using a
double syringe results in rapid gelation in the tissue of interest
thus avoiding syringe jam. Another interesting embodiment is Gellan
gum (Gelrite.RTM., FIG. 3)--a high molecular weight polysaccharide
(500 kDa) produced by the microbe Sphingomonas elodea. Gellan gum
is consists of four linked monosaccharides, including one molecule
of rhamnose, one molecule of glucuronic acid and two molecules of
glucose. It forms gels when positively charged ions (i.e., cations)
are added. Thus, the properties of the gel can be controlled by
manipulating the concentration of potassium, magnesium, calcium,
and/or sodium salts.
[0077] In another interesting embodiment the ion-strength sensitive
gel-forming system is a peptide such as H-(FEFEFKFK).sub.2--OH
(FEK16) which is known to self-assemble into .beta.-sheet
structures in an ionic-strength dependent manner [J. Am. Chem.
Soc., 2001, 123, 9463-9464]. FEK16 has been found to be highly
soluble in pure H.sub.2O but form self-assembled hydrogels at
concentrations >10 mg/mL in the presence of mM concentrations of
NaCl, KCl, and CaCl.sub.2.
Gel-Forming System in Response to Change in pH
[0078] In still another embodiment, the gel-forming system
undergoes gel-formation in response to changes in pH. Optionally,
the gel-forming system undergoes gel-formation in response to a
combined change in pH and temperature, such as a pH in the range of
6-8 and a temperature in the range of 35 to 40.degree. C.
[0079] Non-limiting examples of such gel-forming systems are
illustrated in FIG. 4, and include those described in i) Macromol.
Biosci., 2010, 10, 563-579, J. Controlled Release, 2001, 73,
205-211, Topics in tissue engineering--Smart Polymers, Vol. 3,
2007, Chapter 6, iv) Adv. Drug Delivery Rev., 2010, 62, 83-99, v)
J. Controlled Release, 2003, 86, 253-265 vi) Biodegradable
hydrogels for drug delivery. Basel: Technomic Publishing Co., Inc.,
1993. ISBN 1-56676-004-6, Print, vii) Biomedical polymers and
polymers therapeutics, Ed. Chiellini E., Sunamoto J., Migliaresi
C., Ottenbrite R. M., Cohn D., New York, Kluwer Academic
Publishers, 2002, ISBN 0-30646472-1, Print, and references cited
therein.
[0080] The pH of the formulation (before injection) is preferably
in the range of pH=2-10, optionally in a range selected from 4-6,
6-8 and 8-9.
[0081] The properties of pH responsive hydrogels are highly
depending on the pK.sub.a of the ionizable moiety, the hydrophobic
moieties in the polymer backbone, their amount and distribution.
When ionizable groups become neutral--non-ionized- and
electrostatic repulsion forces disappear within the polymer
network, hydrophobic interactions dominate. The introduction of a
more hydrophobic moiety can offer a more compact conformation in
the uncharged state and a more accused phase transition. The
hydrophobicity of these polymers can be controlled by the
copolymerization of hydrophilic ionizable monomers with more
hydrophobic monomers with or without pH-sensitive moieties, such as
2-hydroxyethyl methacrylate, methyl methacrylate and maleic
anhydride.
[0082] An example of a gel-forming system responsive to pH changes
is that which employs the pH-sensitive property of chitosan
solutions at low pH. Once injected into the body, these polymer
solutions face different environmental pH conditions and form gels.
One example is mucoadhesive pH-sensitive chitosan/glyceryl
monooleate (C/GMO) in situ gel system which consisted of 3% (w/v)
chitosan and 3% (w/v) GMO in 0.33 M citric acid. Chitosan is
normally insoluble in neutral or alkaline pH. However, in dilute
acids (pH.ltoreq.5.0), it becomes soluble due to the protonation of
free amino groups on the chitosan chains (RNH.sub.3.sup.+). The
solubility of chitosan in acidic medium also depends on its
molecular weight. Acidic solutions of chitosan when exposed to
alkaline pH or body biological pH lose this charge and form viscous
gels. Chitosan and GMO both own mucoadhesive property which has
been applied in drug delivery system. Positive charges on the
chitosan backbone may give rise to a strong electrostatic
interaction with mucus or a negatively charged mucosal surface.
Gel-Forming System in Response to Enzymatic Activity
[0083] In still another embodiment, the gel-forming system
undergoes gel-formation in response to enzymatic activity.
[0084] Non-limiting examples of such gel-forming systems are
illustrated in FIG. 5 and include those described in i) Tissue
Eng., 2006, 12, 1151-1168, Biomater. 2001, 22, 453-462, Biomater.,
2002, 23, 2703-2710, iv) Colloids Surf., B, 2010, 79, 142-148, v)
Biomacromolecules, 2011, 12, 82-87, vi) Macromolecules 1997, 30,
5255-5264, vii) Biodegradable hydrogels for drug delivery. Basel:
Technomic Publishing Co., Inc., 1993. ISBN 1-56676-004-6, Print,
viii) Biomedical polymers and polymers therapeutics, Ed. Chiellini
E., Sunamoto J., Migliaresi C., Ottenbrite R. M., Cohn D., New
York, Kluwer Academic Publishers, 2002, ISBN 0-30646472-1, Print,
and references cited therein.
[0085] The enzyme or its origin is not particularly limited. I can
be added prior, during or after injection of the gel forming
system, thus function as a trigger molecule to induce gel
formation. It may be encapsulated in an e.g. liposomes etc. which
upon exposure to an internal or external stimuli releases the
enzyme.
[0086] Additionally, the enzyme might be present in the injected
tissue, either as a natural tissue component, or as an up-regulated
enzyme due to the pathophysiological conditions at the site of
injection.
[0087] In one embodiment, the enzyme triggered gel-forming system
is based on caseins, a group of phosphoproteins with a molecular
weight in the range from 20 kDa to 30 kDa. Such system can be
turned into a hydrogel by addition of microbial transglutaminase
(MTGase), a natural tissue enzyme, at physiological temperature and
pH [Colloids Surf., B, 2010, 79, 142-148].
[0088] Another interesting example of a gel forming system based on
enzymatic activation is based on Schiff base formation of lysine
rich peptides due to activation by either lysyl oxidase or plasma
amine oxidase [Biomacromolecules, 2011, 12, 82-87].
[0089] Oxidation of -amino groups of lysine by either lysyl oxidase
or plasma amine oxidase results in aldehyde formation which readily
forms a Schiff base with an additional E-amino group of lysine
resulting in hydrogel formation.
Gel-Forming System in Response to an Initiator
[0090] In still another embodiment, the gel-forming system
undergoes gel-formation in response to contact with an initiator,
e.g. a molecule or irradiation which results in gel formation by
cross linking the gel forming system by the means of a covalent
chemical bond.
[0091] Non-limiting examples of such gel-forming systems are
described in i) U.S. Pat. No. 5,410,016, ii) J. Controlled Release,
2005, 102, 619-627, iii) Macromol. Res., 2011, 19, 294-299, iv)
Polym. Bull. 2009, 62-699-711, v) J. Biomater. Sci., Polym. Ed.,
2004, 15, 895-904, and references cited therein.
[0092] In one embodiment the gel forming system is cross linked by
photoinitiation by free radical generation, most preferably in the
visible or long wavelength ultraviolet radiation. The preferred
polymerizable regions are acrylates, diacrylates, oligoacrylates,
methacrylates, dimethacrylates, oligomethoacrylates, or other
biologically acceptable photopolymerizable groups. Useful
photoinitiators for the above mentioned system which can be used to
initiate by free radical generation polymerization of the macromers
without cytotoxicity and within a short time frame, minutes at most
and most preferably seconds. Preferred dyes as initiators of choice
for visible light initiation are ethyl eosin,
2,2-dimethoxy-2-phenyl acetophenone, other acetophenone
derivatives, and camphorquinone. In all cases, cross linking are
initiated among macromers by a light activated free-radical
polymerization initiator such as 2,2-dimethoxy-2-phenylacetophenone
or a combination of ethyl eosin and triethanol amine, for
example.
[0093] In another embodiment the gel forming system is cross linked
by hetero- or homo bifunctional linkers such as e.g.
dithiothreitol, glutaraldehyde, diphenylmethanebismaleimide,
dissucinimidyl suberate, bis(sulfosuccinimidyl) suberate, dimethyl
adipim and the like, but not limited to those. An example of such a
gel forming system is multiacrylate PEG-based polymers which have
been reported to form a hydrogel upon addition of the initiator DTT
[J. Controlled Release, 2005, 102, 619-627]. The properties the gel
could be fine tuned by controlling the size of the polymer and the
amount of initiator added and the gel could be formed under
physiological temperature and pH. An additional example of such a
system is hydrogel formation by chemically cross-linking an
hyaluronic acid (HA) derivative with a hydrazide moiety and another
HA derivative with an aldehyde, thus, forming a slowly hydrolysable
hydrazone bond [Eur. J. Pharm. Biopharm., 2008, 68, 57-66]. This
method has the advantage of allowing in situ cross-linking without
the use of initiators, cross-linking chemicals, or extra equipment
for cross-linking such as a light source.
Gel-Forming System in Response to Hydration
[0094] In still another embodiment, the gel-forming system
undergoes gel-formation in response to hydration. Example of such
gel-forming systems are those is selected from; i) WO 2006/075123,
Adv. Drug Delivery Rev., 2001, 47, 229-250, US 2007/0092560--and
references herein, but not limited to those. Formulations composed
of neutral diacyllipids and/or tocopherols and/or phospholipids
solubilized in biocompatible, oxygen containing, low viscosity
organic solvent may form a liquid crystalline phase structure upon
hydration, e.g. contact with an aqueous fluid such as
extra-vascular fluid, extracellular fluid, interstitial fluid or
plasma, but not limited to those. Other systems include non-water
soluble high-viscosity liquid carrier materials such as sucrose
acetate isobutyrate (SAIB). Such a system may be mixed with solid
particles described in the present invention followed by parental
injection, thus functioning as a injectable contrast agent which
that can be visualized by one or multiple imaging modalities,
including X-ray imaging.
Gel-Forming Systems with Cross Linking Groups
[0095] In still another embodiment, any of the afore mentioned
gel-forming systems, are further functionalized by introducing one
or more cross-linkable groups such as acrylate, methacrylate,
acrylamide, methacrylamide, vinyl ether, styryl, epoxide, maleic
acid derivative, diene, substituted diene, thiol, alcohol, amine,
hydroxyamine, carboxylic acid, carboxylic anhydride, carboxylic
acid halide, aldehyde, ketone, isocyanate, succinimide, carboxylic
acid hydrazide, glycidyl ether, siloxane, alkoxysilane, alkyne,
azide, 2'-pyridyldithiol, phenylglyoxal, iodo, maleimide,
imidoester, dibromopropionate, and halo acetates, such as
bromoacetate, but not limited to those.
Gel-Forming Systems with Chelating Groups
[0096] In an additional embodiment, the gel-forming system is
comprised of a chelating agent that is known to chelate ions. Any
ion chelating agent now known or later discovered may be used in
the articles of the present invention. Examples of metal ion (e.g.,
Gd.sup.3+ or Cu.sup.2+) chelating agents include, but are not
limited to, expanded porphyrins and porphyrin-like derivatives,
DOTA, DTPA, AngioMARK.TM. (a backbone-functionalized DTPA chelate),
DTPA-BMA (a neutral bis-methyl amide derivative of DTPA), and
HP-DO3A (a DOTA-like macrocyclic compound wherein one chelate arm
is replaced with a hydroxylpropyl group). Additional chelates
include, but are not limited to, DPDP (TeslaScan.TM.) and
Deferoxamine (e.g. Fe.sup.3+ and Zr.sup.4+).
Other Constituents of the Formulation
[0097] The formulation may further include other constituents, such
as .alpha.-, .beta.-, and/or .gamma.-cyclodextrins and any derivate
hereof. Such constituents may form guest/host complexes with the
gel forming system and the nano-sized particles, thus, both aiding
in the gel formation and possible alter the particle leakage
profile [Adv. Drug Delivery Rev., 2008, 60, 1000-1017]. In one very
interesting embodiment the gel forming system is based on
PEG-PHB-PEG triblock copolymers, .alpha.-cyclodextrin and PEG
coated solid nano sized particles. In such a formulation,
.alpha.-cyclodextrin may form inclusion complexes with both the PEG
blocks of the PEG-PHB-PEG triblock copolymers and the PEG coated
solid nano sized particles which, combined with hydrophobic
interactions between the PHB middle block, forms a strong hydrogel
with enhanced retention of solid nano sized particles due
.alpha.-cyclodextrin interactions which thus altering the particle
leakage profile.
[0098] The formulation may further comprise compounds or polymers
which are visible in imaging modalities other than X-ray
imaging.
[0099] In one embodiment, the formulation further comprises an
iodine-containing polymer, e.g. polyvinylpyrrolidone-iodine
(PVP-I), or one selected from i) Polym. Chem., 2010, 1, 1467-1474,
US 3852341, US 4406878, iv) US 5198136, v) Biomedical polymers and
polymers therapeutics, Ed. Chiellini E., Sunamoto J., Migliaresi
C., Ottenbrite R. M., Cohn D., New York, Kluwer Academic
Publishers, 2002, ISBN 0-30646472-1, Print, and references cited
therein. Such polymers can be added to the gel forming components
prior to gelation and function as contrast agent in vivo. Such
polymers may additionally or alternatively be covalently bound to
the one or more of the gel forming components or adhered to the
particles of the present invention.
[0100] In one specific embodiment, the formulation consist of
SAIB/6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose
(8)/EtOH. The said combination enables the formation of stabile
injectable formulations with very high iodine content which may be
used to provide good visualization by one or multiple imaging
modalities, including X-ray imaging. High iodine contents (high
HU-contrast) is especially important for less sensitive imagining
techniques such as e.g. fluoroscopy among others. The iodine
concentration of the said formulation consisting of
SAIB/6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH
can be fine tuned by varying the weight percent (w %), as defined
by the weight of the atom/molecule giving x-ray contast such as
iodoine divided by the total weight of the material composition
times 100, of 6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose
(8) added to the matrix. The elemental composition of
6,6'-(2,4,6-triiodophenoxy)-acetoxy-isobutyric-Sucrose (8) is; C,
34.96; H, 3.61; I, 42.62; O, 18.81, based on this, the overall
iodine content (w %) in various formulations can be calculated:
SAIB/6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH
(75:5:20) equals 2.13 w %/2.67 w % iodine before/after injection
(diffusion of EtOH out of the formulation after injection causes an
increases the w % of iodine);
SAIB/6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH
(70:10:20) equals 4.26 w %/5.33 w % iodine before/after injection;
SAIB/6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH
(60:20:20) equals 8.52 w %/10.66 w % iodine before/after injection;
SAIB/6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH
(55:25:20) equals 10.65 w %/13.32 w % iodine before/after
injection;
SAIB/6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH
(45:35:20) equals 14.92 w %/18.65 w % iodine before/after
injection;
SAIB/6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH
(30:50:20) equals 21.30 w %/26.64 w % iodine before/after
injection.
[0101] An increase in iodine concentration of the formulation can
directly be correlated to the observed contrast in Hounsfield units
(HU). The following contrast (HU) was observed at different
energies; 80-, 100-, 120- and 140 kV, all 200 mAs, 2 mm (col
40.times.0.6 mm) for the following formulations; a)
SAIB/6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH
(70:10:20) (4.26 w %/5.33 w % iodine before/after injection) 2500
HU (80 kV), 1800 HU (100 kV), 1500 HU (120 kV) and 1300 HU (140
kV); b) SAIB/6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose
(8)/EtOH (55:25:20) (10.65 w %/13.32 w % iodine before/after
injection) 5000 HU (80 kV), 4500 HU (100 kV), 3500 HU (120 kV) and
3000 HU (140 kV); c)
SAIB/6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH
(30:50:20) (21.30 w %/26.64 w % iodine before/after injection)
10500 HU (80 kV), 8800 HU (100 kV), 6200 HU (120 kV) and 5900 HU
(140 kV).
[0102] The gel-forming formulation may further comprise
pharmaceutical agents including prodrugs (in short "drugs"; broadly
interpreted as agents which are able to modulate the biological
processes of a mammal). Examples of pharmaceutical active agents
include small drugs, plasmid DNA (e.g. for gene therapy), mRNA,
siRNA, carbohydrates, peptides and proteins. Specific examples of
pharmaceutical agents include; a) chemotherapeutic agents such as
doxorubicin, mitomycin, paclitaxel, nitrogen mustards, etoposide,
camptothecin, 5-fluorouracil, etc.; b) radiation sensitizing agents
such as gemcitabine and doranidazole, porphyrins for photodynamic
therapy (e.g. visudyne) or 10B clusters or 157Gd for neutron
capture therapy; c) peptides or proteins that modulate apoptosis,
the cell cycle, or other crucial signaling cascades; d) Anti
inflammatory drugs, such as methylprednisolone hemisuccinate,
.beta.-methasone; e) Anti anxiety muscle relaxants such as
diclofenac, pridinol; f) Local anesthetics such as lidocaine,
bupivacaine, dibucaine, tetracaine, procaine; g) Analgesics such as
opiods, non-steroidal anti-inflammatory drugs (NSAIDs); h)
Antimicrobial medications such as pentamidine, azalides; i)
Antipsychotics such as chlorpromazine, perphenazine; j) The
antiparkinson agents such as budipine, prodipine, benztropine
mesylate, trihexyphenidyl, L-DOPA, dopamine; k) Antiprotozoals such
as quinacrine, chloroquine, amodiaquine, chloroguanide, primaquine,
mefloquine, quinine; l) Antihistamines such as diphenhydramine,
promethazine; m) Antidepressants such as serotonin, imipramine,
amitriptyline, doxepin, desipramine; n) Anti anaphylaxis agents
such as epinephrine; o) Anticholinergic drugs such as atropine,
decyclomine, methixene, propantheline, physostigmine; p)
Antiarrhythmic agents such as quinidine, propranolol, timolol,
pindolol; q) Prostanoids such as prostaglandins, thromboxane,
prostacyclin, but not limited to those. These drugs can be
formulated as a single drug or as a combination of two or more of
the above mentioned drugs in its active form or as a prodrug.
[0103] Additional examples of antitumor agents include camptothecin
derivatives such as irinotecan hydrochloride, nogitecan
hydrochloride, exatecan, RFS-2000, lurtotecan, BNP-1350,
Bay-383441, PNU-166148, IDEC-132, BN-80915, DB-38, DB-81, DB-90,
DB-91, CKD-620, T-0128, ST-1480, ST-1481, DRF-1042 and DE-310,
taxane derivatives such as docetaxel hydrate, IND-5109, BMS-184476,
BMS-188797, T-3782, TAX-1011, SB-RA-31012, SBT-1514 and DJ-927,
ifosfamide, nimustine hydrochloride, carboquone, cyclophosphamide,
dacarbazine, thiotepa, busulfan, melphalan, ranimustine,
estramustine phosphate sodium, 6-mercaptopurine riboside,
enocitabine, gemcitabine hydrochloride, carmofur, cytarabine,
cytarabine ocphosphate, tegafur, doxifluridine, hydroxycarbamide,
fluorouracil, methotrexate, mercaptopurine, fludarabine phosphate,
actinomycin D, aclarubicin hydrochloride, idarubicin hydrochloride,
epirubicin hydrochloride, daunorubicin hydrochloride, pirarubicin
hydrochloride, bleomycin hydrochloride, zinostatin stimalamer,
neocarzinostatin, mytomycin C, bleomycin sulfate, peplomycin
sulfate, vinorelbine tartrate, vincristine sulfate, vindesine
sulfate, vinblastine sulfate, amrubicin hydrochloride, gefitinib,
exemestan, capecitabine, TNP-470, TAK-165, KW-2401, KW-2170,
KW-2871, KT-5555, KT-8391, TZT-1027, S-3304, CS-682, YM-511,
YM-598, TAT-59, TAS-101, TAS-102, TA-106, FK-228, FK-317, E7070,
E7389, KRN-700, KRN-5500, J-107088, HMN-214, SM-11355, ZD-0473 and
the like.
[0104] Additional examples of radiation sensitizing agents include
magnesium 5,10,15,20-tetrakis(4-sulphophenyl)-porphine
dodecahydrate, PYROA protein (Emericella nidulans), photosan III,
lomefloxacin, cyamemazine, tiaprofenic acid and the like, but not
limited to those.
[0105] The drugs are included in the composition in an amount
sufficient to achieve a desired effect. The amount of drug or
biologically active agent incorporated into the composition depends
upon the desired release profile, the concentration of drug
required for a biological effect, and the desired period of release
of the drug. The biologically active substance is typically present
in the composition in the range from about 0.5 percent to about 20
percent by weight relative to the total weight of the composition,
and more typically, between approximately 1 percent to about 15
percent by weight. Another preferred range is from about 2 percent
to about 10 percent by weight. For very active agents, such as
growth factors, preferred ranges are less than 1% by weight, and
less than 0.0001%.
Viscosity of the Formulation
[0106] The viscosity of the formulation is before the injection
preferably lower than 10,000 cP, in particular lower than 2,000 cP,
at 20.degree. C. Alternatively, the viscosity of the formulation is
before the injection typically lower than 2,000 cP at 5.degree.
C.
[0107] The organic gel-forming system of the formulation is
preferably one which, after injection or under conditions mimicking
those in a human body, forms a gel having a viscosity at 37.degree.
C. in the range of 2,000 to 50,000,000 cP. More particularly, the
viscosity of the hydrogel can be about 2,000 cP, about 5,000 cP,
about 10,000 cP, about 20,000 cP, about 30,000 cP, about 50,000 cP,
about 75,000 cP, about 100,000 cP, about 125,000 cP, about 150,000
cP, about 200,000 cP, about 30,000 cP, about 800,000 cP, about
1,000,000 cP, about 2,000,000 cP, about 5,000,000 cP, about
10,000,000 cP, about 20,000,000 cP, about 30,000,000 cP, about
40,000,000 cP, about 50,000,000 cP, or ranges thereof. Preferably,
the viscosity of the hydrogel after injection (i.e. when present in
the desired location) is above 20,000 cP, e.g. in the range of
20,000 cP to 1,000,000 cP. In particular, the formulation after
injection is preferably essentially solid.
Use of the Formulation
[0108] The present invention also provides the formulation as
defined hereinabove for use in X-ray imaging as a marker of
specific tissue, such as computer tomography (CT), of the body of a
mammal.
[0109] In one interesting embodiment, the formulation is
parenterally administered to a predetermined location of the body
of a human or animal, and wherein an X-ray image of at least a part
of the body of the human or animal including the predetermined
location is recorded.
A Kit Comprising the Formulation
[0110] The present invention further comprises a kit comprising a
syringe, a needle used for injection into a body or surgical
related procedures, such as but not limited to biopsy, adapted to
the open end of said syringe, and a formulation as defined
hereinabove. In one embodiment, the formulation is held in the
interior or said syringe.
[0111] The gel forming system may be provided as a lyophilized
powder, a suspension or a solution. Different components may be
provided in one or more individual vials or pre-mixed in the
interior or said syringe. Exemplary different components include,
but are not limited to, the gel-forming system and the solid
particles, and the formulation and one or more initiators.
[0112] The syringe may consist of a single, a multiple barrel
syringe (e.g. MEDMIX SYSTEMS AG) or a double champer syringe (e.g.
Debiotech S. A.) and the like, but not limited to those. Multiple
barrel syringes and double champer syringes and the like may be
useful for e.g. two components formulations were one component is a
mixture of the gel forming system and the contrast agent(s) and the
other component is an initiator or salt suspension of e.g.
Ca.sup.2+ in the case there the gel forming system is based on
alginate.
[0113] The needle of the syringe can, in some embodiments, be one
suitable for fine-needle biopsies. Non-limiting examples of
syringes and needles for such embodiments are described in U.S.
Pat. No. 7,871,383, U.S. patent publication No. 20040162505, and
references cited therein. Such syringes and needles can
advantageously be used in procedures where a biopsy of a tissue is
to be taken in conjunction with imaging of the same, using a
formulation of the invention. Preferably, the kit has a shelf-life
of at least 6 months, such as at least 12 months when stored at,
e.g., room temperature (typically 18 to 25.degree. C.) or lower
temperatures, such as, e.g., 2 to 10.degree. C., such as about
5.degree. C. The shelf-life can, for example, be determined as the
period wherein the kit can be stored at 25.degree. C., at 80% % RH
and 1 atm. pressure, and where the viscosity is kept within .+-.5%
of the initial viscosity.
A Method of Recording an X-Ray Image of a Body of Animal or
Human
[0114] The present invention also provides a method of recording an
X-ray image of the body of a mammal, comprising the steps of:
[0115] (a) providing a formulation comprising an organic
gel-forming system that is a homogenous liquid before injection
that comprise an organic x-ray contrast agent such as an iodinated
compound detectable by X-ray imaging; [0116] (b) administering the
formulation to a subject, and [0117] (c) recording X-ray-based
images, such as Computed Tomography (CT)--images or 2D X-ray
images.
[0118] In one embodiment, the method is for joint radiotherapy and
X-ray imaging of a target tissue in an individual, wherein the
images in step (c) provides a definition of the target tissue, and
further comprises the step of: [0119] (d) using the definition of
the target tissue obtained in c) to direct external beam
radiotherapy to the target tissue.
[0120] The target tissue is typically one that comprises
undesirably growing cells. In one embodiment, the undesirably
growing cells are tumor cells, such as malignant cells, and the
individual is suffering from or at risk for cancer. In a particular
embodiment, the undesirable growth of cells is associated with lung
cancer, prostate cancer, cervix or ovarian cancer. Other types of
conditions or diseases associated with undesirable cell growth
include extra uterine (ectopic) pregnancy, benign tumors in brain,
such as benign tumors located closely to the optical nerve,
glandule with overproduction of hormone, such as for example
hypothalamus, bone and cartilage in relation with nerve
compression, blood cells which may be killed prior to
transplantation, conditions associated with large tonsils such as
acute tonsillitis or adenoiditis, obstructive sleep apnoea, nasal
airway obstruction, snoring, or peritonsillar abscess or
hyperplasic or angiogenic eye disorders.
[0121] In embodiments where the gel-forming system is one that gels
upon the addition of an initiator, the administration step (a) or
(b) may further comprise mixing with an initiator.
[0122] The formulation according to the present invention may be
administered parenterally, such as by intravenous, intramuscular,
intraspinal, subcutaneous, intraarterial, intracardiac,
intraosseous, intradermal, intracisternal, intrathecal,
intracerebral, transdermal, transmucosal, inhalational, epidural,
sublingual, intravitreal, intranasal, intrarectal, intravaginal or
intraperitoneal administration. The parental administration may be
performed by, e.g., infusion or injection. Typically, the
formulation is administered into, or adjacent to, a predetermined
location, such as a target tissue, optionally in conjunction with a
biopsy of the target tissue.
[0123] The amount of formulation to administer to the mammal or
individual in step (c) can be determined by one of skill in the
art, taking into consideration the nature of the investigation and
the size of the area to be imaged. Typically, at least 100 .mu.L
formulation is administered. In various specific embodiments, the
method comprises administration of between 100 .mu.L and 20 mL,
such as between 200 .mu.L and 10 mL, such as between 200 .mu.L and
2 mL.
[0124] In step (c), an X-ray image is typically recorded of at
least a part of the body of the mammal including the predetermined
location. In particular embodiments, steps (c) and (d) may be
performed simultaneously, so that image-recording and execution of
radiotherapeutic treatment is integrated and performed sequentially
or simultaneously.
Use of the Formulation as a Tissue Sealant
[0125] The present invention also provides the formulation as
defined herein above for use as a tissue sealant, e.g. for needle
canals formed by biopsy in conjunction with an imaging procedure
according to the invention.
[0126] The tissue sealant may include an effective amount of a
hemostatic agent, e.g. an agent selected from coagulation factors,
coagulation initiators, platelet activators, vasoconstrictors and
fibrinolysis inhibitors, e.g. epinephrine, adrenochrome, collagens,
thrombin, fibrin, fibrinogen, oxidized cellulose and chitosan.
Specific Embodiments of the Invention
[0127] As said above, the present invention is in one embodiment an
X-ray contrast composition for local administration, wherein the
X-ray contrast composition exhibits contrast properties and wherein
at least 60% of an administrated amount of said X-ray contrast
composition remains more than 24 hours within 10 cm from an
injection point when the X-ray contrast composition is
administrated to a human or animal body. There are various forms of
injection forms and routes possible, such as, but not limited to,
transcutane injection, using a scope (bronchoscope, gastroscope, or
any other flexible wired systems used to navigate inside a body),
spraying or just adding on a open wound, attached to another such
system, intracranial injection, inside air and fluent filled organs
or cavities (e.g. bladder, stomach), or inside non naturally or
medically created cavities.
[0128] Furthermore, there are various forms of dosing such as, but
not limited to, fast injections (`bolus`), pulling back to needle
while injecting, slowly injection on the site (e.g. less than 5
seconds, 60 seconds, 120 seconds, 5 minutes, 10 minutes or less
than 20 minutes), pulsating the injection, pushing the needle
forward, and pump giving a constant pressure for a defined period.
Furthermore, there are various devices that may be used such as,
but not limited to, needle with 1 or more holes on the side of the
needle forming multiple smaller objects, flexible, multiple chamber
systems. In one embodiment, the present invention has gelating
properties and is a liquid before administration and has the
ability to transform into a gel after administration. In one
specific embodiment, the present invention has gelating properties
and is a homogeneous liquid before administration and has the
ability to transform into a gel after administration. Furthermore,
in one embodiment the present invention is a non-colloidal x-ray
contrast agent as part of a homogeneous liquid x-ray contrast
composition that gels upon injection into a human or animal
subject. In yet another specific embodiment the X-ray contrast
composition is a liquid before administration into a human or
animal body that increases in viscosity by more than 100 centipoise
(cP), such as e.g. more than 1,000, more than 2,000 or more than
5,000 centipoise (cP), after administration into a human or animal
body. According to another specific embodiment of the present
invention the X-ray contrast composition is a liquid before
administration into a human or animal body that increases in
viscosity by more than 10,000 centipoise (cP) after administration
into a human or animal body. In another specific embodiment the
present invention has a viscosity of less than 10,000 centipoise
(cP) at 20.degree. C.
[0129] Furthermore, from one perspective of the present invention,
the X-ray contrast composition comprises an X-ray contrast agent
that is part of the X-ray contrast composition and said X-ray
contrast agent is an organic substance. According to one specific
embodiment, the organic substance is the contrast "agent" and the
X-ray contrast composition comprises alginate and chitosan. In
another specific embodiment the X-ray contrast agent comprises one
or more natural polymers, synthetic polymers, oligomers, lipids,
saccharides, disaccharides, polysaccharides, peptides or any
combination thereof and as mentioned before these may be the
contrast "agent". In yet another specific embodiment of the present
invention the X-ray contrast agent comprises one or more iodinated
polymers, oligomers, lipids, saccharides, disaccharides,
polysaccharides, peptides, or a derivative or a combination
thereof. Further, in one embodiment the X-ray contrast agent is an
inorganic acid or salt, such as chloroauric acid.
[0130] The present invention may in one embodiment comprise
particles for various purposes. One purpose may be an additive
contrast effect; another purpose may be to potentiating the effect
and a third purpose may be as a carrier of e.g. medication or other
substances. According to one specific embodiment of the present
invention, the X-ray contrast composition comprises nanoparticles
comprising gold (Au). In yet another embodiment the X-ray contrast
composition also comprises particles in the size range from 1-1000
nm, such as nanoparticles in the size range from 2 to 500 nm and in
one specific embodiment the nanoparticles comprises gold (Au) as
the prefered X-ray attenuating element. In yet another embodiment,
the X-ray contrast composition comprising nanoparticle that may be
an MRI, PET, ultrasound, fluorescence, radiofrequency, visible
light contrast agent. Furthermore, in one specific embodiment the
nanoparticle is an MRI or PET contrast agent or a combination of
the above mentioned imaging modalities.
[0131] The present invention may in one embodiment comprise solid
particles coated with SH-PNIPAM (MW 3500). By choosing PNIPAM as
the coating material various interesting properties can be
introduced to the particles. PNIPAM is more hydrophobic compared to
e.g. PEG but still water soluble, which enables efficient and
straightforward particle coating in aqueous solution without prior
extraction to organic solvents. Additionally, by having PNIPAM as
the coating material results in a nano composite which can be
lyophilized into a powder without inducing particle aggregation
etc. which is not possible with other polymers e.g. PEG. Having the
solid particles in a powder form is advantageous from multiply
perspectives in terms of increased stability, easy storage and
straight forward formulation procedures. Furthermore, by having
PNIPAM as the only polymer on the solid particles enables the
particles to be suspended in organic solvents such as e.g. EtOH for
a prolonged period of time without aggregation due to the increased
hydrophobicity of the particle introduced by the PNIPAM polymer. By
having PNIPAM attached to the solid particles, as the only polymer
in the formulation, the hydrophobic interactions with the gel
forming solution in terms of e.g. sucrose acetate isobutyrate
(SAIB) is increased resulting in a injectable system with very high
particle retention. Choosing a more hydrophilic coating material
for the particles would induce the release of the solid particles
from the gel matrix which can be an advantage or a disadvantage
depending on the desired properties of the formulation.
[0132] As mentioned previously the present invention may have
gelating properties and the gelling may be initiated by various
factors such as, but not limited to, temperature, hydration,
enzymatic activation, ion concentration and/or pH. In one
embodiment the X-ray contrast composition exhibits gel-formation in
response to a temperature in the range of 35 to 40.degree. C. In
another embodiment the X-ray contrast composition exhibits
gel-formation in response to hydration. In yet another embodiment
the X-ray contrast composition exhibits gel-formation in response
to an ion-concentration in the range of 1 .mu.M to 500 mM, such as
in the range of 1 mM to 200 mM. In one embodiment the ions are
divalent ions, such as calcium ions. In one embodiment the X-ray
contrast composition exhibits gel-formation in response to a pH in
the range of 6 to 8. In yet another embodiment, the X-ray contrast
composition exhibits gel-formation in response to contacting with
an initiator and here an initiator can be many different things
such as, but not limited to, ions, or a chemical reactive compound
that cross link other molecules.
[0133] In one embodiment, the X-ray contrast composition according
to the present invention may comprise radioactive compounds,
paramagnetic compounds, fluorescent compounds or ferromagnetic
compounds, or any mixture thereof.
[0134] As mentioned previously, the X-ray contrast composition may
also act as a carrier of substances such as, but not limited to,
pharmaceutical substances. The substance may be in the composition
or in or coated/linked to the nanoparticles. The substance may also
be other types of additives. Examples of substance could be, but is
not limited to, substances suitable for chemotherapy, gemcitabine,
cisplatin, doxorubicin, doranidazole, hormones or anti-bodies. In
one embodiment the X-ray composition comprise at least one
pharmaceutical substance. In one specific embodiment the X-ray
contrast composition comprises particles in the size range from
1-1000 nm, such as nanoparticles in the size range from 2 to 500 nm
and wherein the particle contains at least one pharmaceutical
substance.
[0135] In one embodiment a polymer may be used to work as a
stabilizer between gel and biological surrounding and therefore,
the X-ray contrast composition may also comprises a molecule that
increase gel stability in the human or animal body, such as an
interfacially active molecule, such as an amphiphilic molecule,
such as an emulsifier. Therefore in one embodiment the X-ray
contrast composition comprises poly(ethylene glycol-b-caprolactone)
(PEG-PCL), sucrose acetate isobutyrate (SAIB), poly(D,L-lactic
acid) (PLA), or poly(lactic-co-glycolic acid) (PGLA), or a
combination thereof. In one embodiment of the present invention
poly(D,L-lactic acid) (PLA) is added to sucrose acetate isobutyrate
(SAIB) gel causing a reduction of burst release of said
encapsulated contents e.g. particles drugs etc. Further, in one
embodiment, the X-ray contrast composition comprises sucrose
acetate isobutyrate (SAIB) or a derivative thereof and in one
specific embodiment of the present invention, the X-ray contrast
composition comprises an iodinated derivate of sucrose acetate
isobutyrate (SAIB). Furthermore in another specific embodiment of
the present invention the X-ray contrast composition comprises an
iodinated derivate of sucrose acetate isobutyrate (SAIB) doped into
sucrose acetate isobutyrate (SAIB). This has been evaluated for
stability and the amount of this iodo-SAIB/SAIB that can be doped
into SAIB, is at least 50 w/w %.
[0136] The iodo-SAIB provides high X-ray contrast. The iodo-SAIB
compound is poorly soluble in ethanol and is a white solid whereas
SAIB is highly soluble in ethanol and is a thick oil. However, a
mixture of ethanol and SAIB can solubilize the iodo-SAIB very
nicely. This means that the SAIB helps solubility of iodo-SAIB,
which is an interesting feature and which provides an injectable
solution which forms a biodegradable, amorphous carbohydrate glass
matrix after administration (through a thin needle, thinner than 20
gauge) that can function as a high contrast X-ray marker. When
injected into mice, the iodo-SAIB/SAIB provides high contrast and
has the desirable stability properties. Furthermore, the gel is
homogeneous. In one embodiment of the present invention the X-ray
contrast composition comprises an iodinated derivate of sucrose
acetate isobutyrate (SAIB) solubilized in a mixture of ethanol and
sucrose acetate isobutyrate (SAIB).
[0137] One way of containing and also storing the composition may
be, held in the interior of a syringe. This indicates a possible
shelf-life of at least 6 months. One embodiment of the present
invention is a kit comprising a syringe, a needle used for
injection into a body or surgical related procedures such as but
not limited to biopsy adapted to the open end of said syringe, and
a composition according to the present invention.
[0138] In one embodiment of the present invention, the X-ray
contrast composition comprises an iodinated derivate of sucrose
acetate isobutyrate (SAIB) and contains a pharmaceutical substance.
In another embodiment the X-ray contrast composition comprises an
iodinated derivate of sucrose acetate isobutyrate (SAIB) and
contains particle that contains a pharmaceutical substance. In yet
another embodiment, the X-ray contrast composition comprises an
iodinated derivate of sucrose acetate isobutyrate (SAIB)
solubilised in a mixture of ethanol and sucrose acetate isobutyrate
(SAIB) and contains a pharmaceutical substance. Furthermore, in one
specific embodiment of the present invention, the X-ray contrast
composition comprises an iodinated derivate of sucrose acetate
isobutyrate (SAIB) solubilised in a mixture of ethanol and sucrose
acetate isobutyrate (SAIB) and contains a particle that contains a
pharmaceutical substance.
[0139] The intended use of the present invention is for radio
therapy or image-guided radiation therapy, but not exclusively,
other uses are thinkable such as, but not limited to, 2D X-ray
scans, for use in imaging, diagnostics, treatment and/or quality
rating of radiation therapy. The present invention may be used as a
tissue marker and/or for use as a controlled drug release
composition.
[0140] In one embodiment the X-ray contrast composition according
to the present invention is for use in administration of an amount
of 0.01-5.0 mL and in one specific embodiment the X-ray contrast
composition is for use in administration wherein the amount is
0.1-1.0 mL. In one embodiment the present invention may be used as
a tissue sealant.
[0141] In one embodiment the X-ray contrast composition according
to the present invention, the X-ray contrast composition is
parenterally administered to a predetermined location of the body
of a mammal, and wherein an X-ray image of at least a part of the
body of the mammal including the predetermined location is
recorded. Further, an embodiment of the invention may comprise a
method of recording an X-ray image of the body of a mammal,
comprising the steps of [0142] a. providing an X-ray contrast
composition comprising an organic X-ray agent in a gel-forming
system; [0143] b. administering the X-ray contrast composition to a
predetermined location of the mammal, and [0144] c. recording
X-ray-based images of at least a part of the body which comprises
the predetermined location. In another embodiment, the invention
comprise a method of joint radiotherapy and X-ray imaging of a
target tissue in a mammal, comprising the steps of [0145] a.
providing an X-ray contrast composition comprising an organic X-ray
agent in a gel-forming system; [0146] b. administering the X-ray
contrast composition to a predetermined target tissue of the
mammal, [0147] c. recording X-ray-based images, of at least a part
of the body which comprises the target tissue, thereby providing a
definition of the target tissue, and [0148] d. using the definition
of the target tissue obtained in c) to direct external beam
radiotherapy to the target tissue.
[0149] Steps (c) and (d) may potentially be performed
simultaneously.
[0150] In another embodiment, the invention comprise a method for
directing local administration of a pharmaceutical agent to a
target tissue in a mammal, comprising the steps of [0151] a.
providing an X-ray contrast composition comprising an organic X-ray
agent in a gel-forming system; [0152] b. administering the X-ray
contrast composition to a predetermined target tissue of the
mammal, [0153] c. recording X-ray-based images, of at least a part
of the body which comprises the target tissue, thereby providing a
definition of the target tissue, and [0154] d. using the X-ray
contrast composition in b) to further comprise an pharmaceutical
agent for delivery of a pharmaceutical agent to a predetermined
target tissue of the mammal. Steps (c) and (d) may potentially be
performed simultaneously.
[0155] In one specific embodiment of the present invention the
target tissue comprises undesirably growing cells and in another
specific embodiment the target tissue comprises tumor cells.
DETAILED DESCRIPTION OF THE DRAWINGS
[0156] FIG. 1. Illustrates various mechanisms of gel-formation
including thermo-, ion-, pH-, enzymatically-, initiator- and
hydration responsive gel-forming systems.
[0157] FIG. 2. Illustrates various thermo responsive gel-forming
systems which can exhibit an inverse sol-gel transition.
[0158] FIG. 3. Illustrates various ion sensitive gel-forming
systems which form gels in high salt concentration.
[0159] FIG. 4. Illustrates various pH sensitive gel-forming systems
which form hydrogels at specific pH intervals. FIG. 5. Illustrates
various enzymatically sensitive gel-forming systems which form
hydrogels in presence of specific enzymes.
[0160] FIG. 6. Illustrates the use of sucrose acetate isobutyrate
(SAIB) as a hydration sensitive gel-forming system. SAIB dissolved
in organic solvent such as ethanol have a low viscosity suitable
for injection trough thin needles. Upon hydration the ethanol
diffuses out of the matrix resulting in a highly viscous
hydrophobic gel suitable for encapsulation of contrast agents.
[0161] FIG. 7. Illustrates various iodo-SAIB derivates which may be
used for x-ray attenuation.
[0162] FIG. 8. Illustrates a synthetic scheme for the synthesis of
2-(2,4,6-triiodophenoxy)acetic acid (3)
[0163] FIG. 9. Illustrates a synthetic scheme for the synthesis of
6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8)
[0164] FIG. 10. Illustrates CT-contrast of iodinated gels with 10-,
25-, or 50 w % (8) ((w % is the weight of the atom/molecule (in
this case iodine) divided by the total weight of the material times
100)) and a negative control containing MQ-H.sub.2O were visualized
in a clinical CT-scanner at different energies; 80-, 100-, 120- and
140 kV, all 200 mAs, 2 mm (col 40.times.0.6 mm).
[0165] FIG. 11. Illustrates AuNP synthesis and characterization. A)
Synthetic scheme for the synthesis of PNIPAM-coated AuNPs using a
seeding approach; B) AuNP characterization by UV-Vis; C) AuNP
characterization by DLS; D) AuNP characterization by
.zeta.-potential.
[0166] FIG. 12. Illustrates the enhanced stability of PNIPAM coated
AuNPs. A) UV-Vis of PNIPAM coated AuNPs before(stock)/after
lyophilization and re-suspension in anhydrous EtOH (concentration
of AuNP in the range of 1.0-5.0 mg Au/mL); B) DLS of PNIPAM coated
AuNPs before(stock)/after lyophilization and re-suspension in
anhydrous EtOH (concentration of AuNP in the range of 1.0-5.0 mg
Au/mL).
[0167] FIG. 13. Illustrates the accumulative release of
PNIPAM.sub.3500- and PEG.sub.5000 coated AuNPs from gels composed
of SAIB/EtOH/PLA (75:20:5)+3.0 w % PNIPAM.sub.3500 or PEG.sub.5000
coated AuNPs.
[0168] FIG. 14. Illustrates a ultrasonography imagee of Formulation
B (SAIB/8/EtOH (55:25:20)) (250 .mu.L) in vitro. Gel present at the
bottom of a glass beaker under water.
[0169] FIG. 15. Illustrates MicroCT images of Formulation B
(SAIB/8/EtOH (55:25:20)) (200 .mu.L) administered by subcutaneous
injection to healthy NMRI mice. A) CT-image recorded 24 h p.i.; B)
CT-image recorded 48 p.i.
[0170] FIG. 16. A) MicroCT image of SAIB/8/EtOH (65:15:20) injected
s.q. in immunocompetent mice; B) MicroCT image of SAIB/8/EtOH
(50:30:20) injected s.q. in immunocompetent mice; C) Ex vivo
visualization of SAIB/8/EtOH (50:30:20) present in the s.q.
compartment 14w p.i. and D) Gel implants composed of SAIB/8/EtOH
(50:30:20) removed after 14w implantation in immunocompetent
mice.
[0171] FIG. 17. A) Series of MicroCT images of SAIB/8/EtOH
(50:30:20) injected s.q. in mice. MicroCT scans recorded with short
time intervals to monitor the gelation kinetics of the iododinated
gel; B) Gelation kinetics of SAIB/8/EtOH (50:30:20) (50 .mu.L)
implanted s.q. in immunocompetent mice and C) 14 w degradation
profiles of iododinated gels composed of SAIB/8/EtOH (65:15:20) or
SAIB/8/EtOH (50:30:20) after s.q. implantation (50 .mu.L).
[0172] FIG. 18. Illustrates a CT-image of Formulation B
(SAIB/8/EtOH (55:25:20)) administrated intratumoral to a companion
dog (American Staffordshire terrier, 9 years, 34 kg) with a mast
cell tumor present between the front legs.
EXAMPLES
Example 1--Iodo-SAIB Gel Formation and CT-Contrast In Vitro
Materials
[0173] Chemicals were purchased from Sigma-Aldrich Inc. (Brondby,
Denmark) unless otherwise stated. 2-(2,4,6-triiodophenoxy)acetic
acid (3) and 6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose
(8) was synthesized in two and four steps, respectively, as
outlined in FIG. 7 and FIG. 8.
Synthesis
[0174] 2-(2,4,6-triiodophenoxy) acetic acid (3).
2,4,6-triiodophenol (1) (10.00 g, 21.2 mmol) was dissolved in dry
DMF (75 mL) under N.sub.2-atmosphere. To this solution, tert-butyl
bromoacetate (4.20 mL, 28.46 mmol) and K.sub.2CO.sub.3 (8.79 g,
63.6 mmol) were added and the stirred overnight at rt. The solvent
was removed in vacou and the remaining yellow oil re-dissolved in
EtOAc (150 mL) and washed with MQ-H.sub.2O (3.times.150 mL). The
organic phase was dried with MgSO.sub.4, filtrated and concentrated
in vacou to give tert-butyl 2-(2,4,6-triiodophenoxy)acetate (2) as
a light yellow oil which was used in the next step without further
purification. 2 was dissolved in CH.sub.2Cl.sub.2 (60 mL) and
trifluoroacetic acid (30 mL) was added. The mixture stirred for 1 h
at rt after which the solvent was removed in vacou