U.S. patent application number 12/661398 was filed with the patent office on 2010-09-16 for molecular targeting agents.
Invention is credited to Paloma H. Giangrande, James O. McNamara, Michael K. Schultz.
Application Number | 20100234450 12/661398 |
Document ID | / |
Family ID | 41669208 |
Filed Date | 2010-09-16 |
United States Patent
Application |
20100234450 |
Kind Code |
A1 |
Schultz; Michael K. ; et
al. |
September 16, 2010 |
Molecular targeting agents
Abstract
The present invention relates to aptamer conjugates comprising a
metal chelating group and to methods of using these aptamers.
Inventors: |
Schultz; Michael K.; (Iowa
City, IA) ; McNamara; James O.; (Iowa City, IA)
; Giangrande; Paloma H.; (Iowa City, IA) |
Correspondence
Address: |
VIKSNINS HARRIS & PADYS PLLP
P.O. BOX 111098
ST. PAUL
MN
55111-1098
US
|
Family ID: |
41669208 |
Appl. No.: |
12/661398 |
Filed: |
March 16, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US09/53023 |
Aug 6, 2009 |
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12661398 |
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61087652 |
Aug 9, 2008 |
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61155288 |
Feb 25, 2009 |
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Current U.S.
Class: |
514/44R ;
536/23.1 |
Current CPC
Class: |
C12N 15/115 20130101;
A61K 31/7088 20130101; C12N 2310/3519 20130101; C12N 2320/32
20130101; C12N 15/1135 20130101; C12N 2310/344 20130101; C12N
2310/14 20130101; C12N 2310/16 20130101; A61K 47/554 20170801; A61K
47/549 20170801; C12N 2310/322 20130101; A61P 35/00 20180101; C12N
2310/3533 20130101; C12N 2310/322 20130101 |
Class at
Publication: |
514/44.R ;
536/23.1 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; C07H 21/00 20060101 C07H021/00; A61P 35/00 20060101
A61P035/00 |
Claims
1. A conjugate comprising a nucleic acid aptamer that is not more
than 45 nucleotides in length comprising the nucleic acid sequence
5'-n.sub.1n.sub.2n.sub.3CGGAUCAGCn.sub.4n.sub.5n.sub.6GUUUA-3' (SEQ
ID NO:1), linked to one or more chelating groups, wherein each
n.sub.x can be present or absent, wherein when present each n.sub.x
represents any nucleotide.
2. The conjugate of claim 1, wherein nucleotides
n.sub.1n.sub.2n.sub.3 and n.sub.4n.sub.5n.sub.6 are present and
hybridize to form a stem structure.
3. The conjugate of claim 1, wherein the nucleic acid aptamer
molecule comprises the nucleic acid sequence
5'-AUGCGGAUCAGCCAUGUUUA-3' (SEQ ID NO:2).
4. The conjugate of claim 1, wherein the nucleic acid aptamer
molecule comprises the nucleic acid sequence
5'-n.sub.an.sub.bn.sub.cn.sub.dn.sub.1n.sub.2n.sub.3CGGAUCAGCn.sub.4n.sub-
.5n.sub.6GUUUAn.sub.en.sub.fn.sub.gn.sub.h-3' (SEQ ID NO:3).
5. The conjugate of claim 4, wherein nucleotides
n.sub.1n.sub.2n.sub.3 and n.sub.4n.sub.5n.sub.6 are present and
hybridize to form a first stem structure and nucleotides
n.sub.an.sub.bn.sub.cn.sub.d and n.sub.en.sub.fn.sub.gn.sub.h are
present and hybridize to form a second stem structure.
6. The conjugate of claim 1, wherein the nucleic acid aptamer
molecule comprises the nucleic acid sequence
5'-GACGAUGCGGAUCAGCCAUGUUUACGUC-3' (SEQ ID NO:4).
7. The conjugate of claim 1, wherein the nucleic acid aptamer
molecule comprises the nucleic acid sequence
5'-n.sub.10n.sub.11n.sub.12n.sub.13n.sub.14n.sub.an.sub.bn.sub.cn.sub.dn.-
sub.1n.sub.2n.sub.3CGGAUCAGCn.sub.4n.sub.5n.sub.6GUUUAn.sub.en.sub.fn.sub.-
gn.sub.hn.sub.15n.sub.16n.sub.17n.sub.18n.sub.19n.sub.20n.sub.21-3'
(SEQ ID NO:6).
8. The conjugate of claim 7, wherein nucleotides
n.sub.1n.sub.2n.sub.3 and n.sub.4n.sub.5n.sub.6 are present and
hybridize to form a first stem structure, nucleotides
n.sub.an.sub.bn.sub.cn.sub.d and n.sub.en.sub.fn.sub.gn.sub.h are
present and hybridize to form a second stem structure, and
nucleotides n.sub.10n.sub.11n.sub.12n.sub.13n.sub.14 and
n.sub.16n.sub.17n.sub.18n.sub.19n.sub.20 are present and hybridize
to form a third stem structure.
9. The conjugate of claim 1, wherein the nucleic acid aptamer
molecule comprises the nucleic acid sequence aptamer A10-3.2
(5'-GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCU-3' (SEQ ID NO:5)).
10. The conjugate of claim 1, wherein a linker links the aptamer to
the one or more chelating groups.
11. A pharmaceutical composition comprising the conjugate of claim
1 and a pharmaceutically acceptable carrier.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of prior PCT
Application No. PCT/US2009/053023 filed Aug. 6, 2009, which claims
the benefit of U.S. Provisional Application No. 61/087,652 filed
Aug. 9, 2008 and U.S. Provisional Application No. 61/155,288 filed
Feb. 25, 2009.
BACKGROUND OF THE INVENTION
[0002] Worldwide, cancer affects approximately 10 million people
each year. Approximately 22 million people are living with cancer
and almost 7 million people die worldwide from cancer each year.
The most common cancers include cancers of the lung, breast,
colon/rectum, stomach, liver, prostate, cervix, esophagus, and
bladder. The elderly tend to be the highest population for new
incidence, as more than 75% of all new cancer cases are diagnosed
in people over the age of 60. With the aging population, incidence
is expected to increase each year. Prostate cancer is the most
common cancer in men and the second leading cause of cancer death
in men, behind lung cancer. Approximately one in six men in the
U.S. will contract prostate cancer in his lifetime. Approximately
80% of prostate cancers are diagnosed in men over 65 years of age,
and, due to the lack of symptoms, 75% of first-time patients over
65 are diagnosed with advanced-stage prostate cancer. Worldwide,
more than 680,000 men are diagnosed annually and over 29,000 men in
the U.S. will die as a result of prostate cancer this year alone.
Prostate cancer characteristically spreads to the bone. The
therapeutic regimen for disease that is localized in the prostate
bed (local disease) can be drastically different from the
prescribed therapy for metastatic prostate cancer.
SUMMARY OF THE INVENTION
[0003] Targeted molecular imaging and therapy provides precise
delivery of diagnostic and therapeutic agents to the site of
prostate cancer, which provides a critically-needed approach to
improve outcomes for prostate cancer patients.
[0004] One embodiment the invention provides a conjugate of the
invention, which is a ribonucleic acid (RNA) aptamer that is not
more than 45 nucleotides in length comprising the nucleic acid
sequence
5'-n.sub.1n.sub.2n.sub.3CGGAUCAGCn.sub.4n.sub.5n.sub.6GUUUA-3' (SEQ
ID NO:1) linked to one or more chelating groups, wherein each
n.sub.x can be present or absent, wherein when present each n.sub.x
represents any nucleotide, or a pharmaceutically acceptable salt
thereof.
[0005] In one embodiment the invention provides a method for
delivering a therapeutic metal or an imagable metal to a cell
having a prostate specific membrane antigen (PMSA) receptor,
comprising contacting the cell with a conjugate of the
invention.
[0006] In one embodiment the invention provides a pharmaceutical
composition comprising a conjugate of the invention and a
pharmaceutically acceptable diluent or carrier.
[0007] In one embodiment the invention provides a method for
treating a patient having cancer comprising administering a
conjugate of the invention to the patient.
[0008] In one embodiment the invention provides a method for
determining whether a patient has cancer (i.e., diagnosing a
patient) comprising administering a conjugate of the invention that
comprises an imagable metal to the patient and determining whether
the patient has cancer. Imagable metals may be radionuclides for
nuclear medicine imaging by positron emission tomography (PET) or
single photon emission computed tomography (SPECT) or metals
suitable for magnetic resonance imaging (MRI). These techniques may
be combined with computed tomography to form dianostic imaging
known as PET/CT, SPECT/CT, PET/MRI or other combination of imaging
technique. Imaging techniques may be used for diagnosis or for
measuring the response to therapy of any kind. For example, because
certain conjugates of the invention are targeted to the prostate
specific membrane antigen (PMSA) receptor and include an imagable
metal, detection of a relatively higher level of the conjugate can
be used to diagnose a patient as having prostate cancer or to
determine the effectiveness of a therapeutic regimen of any
type.
[0009] In one embodiment the invention also provides a conjugate of
the invention for use in therapy.
[0010] In one embodiment the invention provides the use of a
conjugate of the invention for treating cancer.
[0011] In one embodiment the invention provides a conjugate of the
invention for use in the prophylactic or therapeutic treatment of
cancer (e.g. a solid sarcoma, carcinoma, or prostate cancer).
[0012] The invention also provides in certain embodiments novel
intermediates and processes disclosed herein that are useful for
preparing conjugates of the invention synthetic processes described
in the schemes and Examples herein.
[0013] In certain embodiments, the present invention provides a kit
comprising the conjugate or the pharmaceutically acceptable salt
thereof as described above, packaging material, and instructions
for administering the conjugate or the pharmaceutically acceptable
salt thereof to an animal to detect or treat prostate cancer. In
certain embodiments, the kit further comprises an imageable metal
or a therapeutic metal.
BRIEF DESCRIPTION OF DRAWINGS
[0014] This patent or application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawing(s) will be provided by
the Office upon request and payment of the necessary fee.
[0015] FIGS. 1A-1C Data confirming synthesis and purity of
1-amino-3-azidopropane (2): (A) Reaction of 1-amino-3-bromopropane
(1) with sodium azide; (B) Infrared absorbance of 2098.4 cm.sup.-1
(theoretical 2100 cm.sup.-1) for the azide; (C) 1H NMR spectrum
confirms structure of 1-amino-3-azido propane.
[0016] FIG. 2 illustrates a scheme for the preparation of DOTAzide.
Synthesis of DOTAzide (6) from precursor DOTA-NHS (5) and
1-amino-3-azidopropane (2). Includes t-butyl ester protecting
groups (*). Confirmed by ESI-MS and MALDI-TOF.
[0017] FIGS. 3A-3B Confirmational analysis of the preparation of
t-butyl protected DOTAzide (6): (A) Atomic mass determined by
Electron Spray Ionization Mass Spectrometry=654.35; and (B) by
LC-MS=655.36. Theoretical atomic mass 654.84. Minor peaks in A can
be explained by ionization of t-butyl groups by analytical
technique. No trace of starting material (5) could be detected
using these techniques, indicating that the synthetic reaction
proceeds to completion.
[0018] FIG. 4 illustrates a scheme for the preparation of DOTAzide
conjugation to phenyl acetylene. DOTAzide conjugation to phenyl
acetylene: (6) azide modified DOTAzide tether (DOTA ring not
shown); (3) phenyl acetylene; and (7) DOTAzide-phenyl acetylene
conjugate showing triazole ring (*) and DOTA ring with t-butyl
protecting groups and showing theoretical position of .sup.68Ga.
Molecular mass of construct (7) (without .sup.68Ga) confirmed in
our laboratory by MALDI-TOF at 758 and LC MS 756 (theoretical
757.7).
[0019] FIG. 5 illustrates the structure of the
5'-/5-hexynyl-phosphoramidite and 5'-/5-amino-phosphoramidite,
bearing the terminal alkyne and amine functional groups that are
available for conjugation to chelator groups. The alkyne function
is reactive with azide functionalized chelator moieties such as
DOTAzide by cycloaddition (click chemical reaction) to the azide
functional group on the DOTAzide molecule. The 5'-amine terminus is
reacted with amine-reactive functionalized chelators, such as
isothiocyanato- or esterified chelators. The example shows the
5'-/5-hexynyl-phosphoramidite conjugated to the 5' end of a DNA
oligo showing: (A) the alkyne functional group; (B) the reactive
site on the phosphoramidite; and (C) phosphodiester linkage of
post-synthetic 5' conjugation to DNA and RNA.
[0020] FIG. 6 is ESI-MS spectrum showing the mass peak for
DOTA-conjugated 20mer DNA oligonucleotide sequence (SEQ ID NO:7),
with an observed mass 6444.6 (theoretical 6442.9) and include Ca,
and showing an impurity resulting from addition of
tris-hydroxypropyltriazolyl copper stabilizer to the
DOTA-conjugated oligo.
[0021] FIG. 7 shows the structure of difluoromethylene cyclooctyne
modified phosphoramidite and Cu-catalyst-free click chemical
reaction with DOTAzide.
[0022] FIGS. 8A-8C: Schema of the preparations of DOTA- and
NOTA-conjugated RNA aptamer A10-3.2. In this case, a two-step
process was used starting with an alkyne modified RNA aptamer as
starting material: (A) An amine terminal RNA (A1) was prepared by
click chemical reaction of the 5'-alkyne modified RNA with
1-amino-3-azidopropane followed by desalting and careful removal of
excess Cu using free DOTA; (B) NOTA-modified RNA (B1) was prepared
by reacting the A1 with the p-NCS-Bn-NOTA; DOTA-modified RNA was
prepared by reacting A1 with the active NHS ester of DOTA to afford
B2; (C) Confirmation of the preparation of NOTA-RNA (C1,
theoretical mass 13,510; observed 13,509.5, 85% purity) and
DOTA-RNA (C2, theoretical mass 13,13.574.6; observed 13,575.5, 82%
purity) by ESI mass spectrometry following HPLC purification. Soft
ionization of ESI results in secondary peaks with mass values
indicating association of sodium (Na) and small amounts of iron
(Fe) with the macrocyclic rings. Na is not expected to interfere
with radiolabeling, while Fe will need to be removed in subsequent
preparations.
[0023] FIGS. 9A-9C: Illustration of synthesis and confirmation of
the preparation of NOTA and DOTA modified PSMA RNA aptamer by mass
spectrometry. FIG. 9A Schema of the preparations of DOTA- and
NOTA-conjugated RNA aptamer A10-3.2 and FIGS. 9B and 9C
confirmation by ESI mass spectrometry. In this case, an 5'-amine
modified RNA aptamer is conjugated to p-SCN-Bn-NOTA and
p-SCN-Bn-DOTA via an amine nucleophilic route in a single step.
Purification is performed by dialysis spin filtration using a 10 k
molecular weight cutoff filter. A 50 eq. excess of
2,2',2''-(2-(4-isothiocyanatobenzyl)-1,4,7-triazonane-1,4,7-triyl)tria-
cetic acid (p-SCN-Bn-NOTA) (FIG. 9B) or
2,2',2'',2'''-(2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane--
1,4,7,10-tetrayl)tetraacetate (p-SCN-Bn-DOTA) (FIG. 9C) is added to
a solution containing a 5'-amine modified PSMA-RNA aptamer in
sodium bicarbonate at pH .about.9 and reacted for 5 hours at
40.degree. C., followed by purification by spin filter
centrifugation (5.times.) in high purity water (molecular weight
cutoff 10 kD), and confirmation of the product purity by
electrospray ionization mass spectrometry.
[0024] FIGS. 10A, 10B, and 10C: Demonstration of the radiochemical
purity achievable by reacting imagable radiometals .sup.64Cu (FIG.
10B), .sup.68Ga (FIG. 10A) with a NOTA conjugated RNA aptamer and
.sup.111In (FIG. 10C) with a DOTA conjugated RNA aptamer. For these
representative figures, between 200 and 400 MBq of radioactivity
was added to a buffer solution containing between 3.5 and 15 nmoles
of NOTA- or DOTA-modified PSMA RNA aptamer and reacted for 15-60
minutes at 70.degree. C. to 100.degree. C. Final purification was
performed by spin filter filtration to achieve radiochemical purity
of >98%.
[0025] FIGS. 11A-11C: Representative cell binding assays of the RNA
aptamer radiolabeled with .sup.111In and .sup.64Cu when contacted
with cells that express PSMA and cells that do not express PSMA.
FIG. 11A shows binding of the A10-3.2 aptamer labeled at the 5' end
with 32P to 22Rv1(1.7) PSMA-positive prostate cancer cells with a
KD of 106 nM. FIG. 11B shows binding of 111In-DOTA-3.2 to
22Rv1(1.7) cells with a KD of 119 nM. The binding capacity (Bmax)
to a PSMA negative cell line, 22Rv1(wmr), is much lower. FIG. 11C
shows binding of 64Cu-NOTA-3.2 to 22Rv1(1.7) cells with a KD of 131
nM. Addition of the chelator DOTA or NOTA to the A10-3.2 aptamer
had minimal effect on its affinity for PSMA-expressing cells.
[0026] FIGS. 12A and 12B: PET imaging study of the biodistribution
of [.sup.68Ga]-NOTA-PSMA-Aptamer in a xenograft (subcutaneous,
22RV1 right flank) nude mouse model of prostate cancer (injected
dose: 422 .mu.Ci (16 MBq) in 50 .mu.L sterile isotonic saline,
specific activity 11 MBq nmole.sup.-1. A static image was acquired
at 120 minutes, indicating a accumulation in the right flank tumor
in this coronal slice with excellent tumor:background (8:1)
conspicuity. Transaxial and Sagittal slices are at the center of
the maximum intensity of the tumor indicated in the coronal slice
of the image shown.
[0027] FIGS. 13A-13C: These figures demonstrate the in vivo
affinity and specificity of the 111In-DOTA-PSMA-RNA aptamer to a
PSMA-expressing prostate cancer tumor. Virtually no accumulation of
the radiolabeled aptamer was observed in a xenograft PSMA-negative
tumor, demonstrating specificity for PSMA. FIG. 13A shows a
bioluminescent imaging (BLI) experiment to confirm the presence of
the PSMA-positive xenograft, which also expresses luciferase. The
PSMA-positive tumor is on the animal's right side, while a
PSMA-negative xenograft is on the animal's left side (asterisk).
The animal is in the prone position. FIG. 13B shows a fusion
SPECT/CT image taken at 24 hours post injection of 1 mCi of
111In-DOTA-PSMA-RNA aptamer with the PSMA-positive tumor marked by
the white arrow. The PSMA-negative tumor on the left of the animal
is marked by an asterisk. The PSMA-positive tumor measured
3.times.3 mm, while the PSMA-negative tumor was much larger at
approximately 1.times.2 cm. FIG. 13C shows a close-up of a
computerized reconstruction of the SPECT/CT image, clearly showing
that our agent binds the PSMA-positive tumor on the right, but not
the PSMA-negative tumor on the left (asterisk).
[0028] FIG. 14. Flow cytometry was used to confirm expression of
PSMA in the 22Rv1(1.7) cells used for in vitro binding experiments
and in vivo xenograft experiments. The population of cells labeled
with a fluorescent PE-conjugated primary antibody against PSMA is
shown in gray, compared with the population of unlabeled cells
(unshaded).
DETAILED DESCRIPTION OF THE INVENTION
[0029] The chemical structure of the invention can be described by
conventions used commonly in the field of use as a molecular
targeting agent that is composed of four parts: (1) an aptamer
portion that is designed to bind to a cell surface receptor (e.g.,
PSMA); (2) a metal chelator that is designed to bind metals used
for imaging and therapy (e.g., .sup.68Ga for PET imaging, or stable
Gd for MRI); (3) a linker that is a chemical entity that is used to
connect the chelator and aptamer portions of the invention
together; and (4) a radiometal or stable metal that is used for
imaging or therapy using the aptamer as the targeting vector in
vivo. The linker not only connects the chelator and aptamer
portions together, but also can be used to optimize the in vivo
characteristics of the entire molecular targeting agent (e.g,
biodistribution and pharmacokinetics).
[0030] Molecular targeting refers to the development of ligands
that are engineered to bind to a specific molecular target, e.g., a
cell-surface receptor. Examples of targeting mechanisms (often
referred to as "targeting vectors" or "targeting mechanisms")
include monoclonal antibodies (mAbs), peptides, antisense DNA
oligonucleotides (oligos), and more recently RNA aptamers
(aptamers) and peptide-nucleic acid hybrids (PNAs). For in vivo
imaging using metals and radiometals and for in vivo targeted
radionuclide therapy applications, the addition of a chelator
moiety to the targeting mechanisms results in a "bi-functional"
ligand that enables the molecular targeting mechanism to bind a
contrast-agent metal (e.g., gadolinium for magnetic resonance
imaging, MRI) or radionuclide (e.g., gallium-68 (68Ga) for positron
emission tomography, PET), or in some cases by connecting a light
active moiety to the targeting vector for optical imaging or
photodynamic therapy, while maintaining high affinity for the in
vivo molecular target. A similar approach has been employed for
specific targeting of therapeutic radionuclides (e.g., alpha and
beta emitters) to the site of cancerous tissue, while sparing
healthy cells in vivo. To be effective, a chelator-modified
bifunctional ligand (bioconjugate) must maintain affinity for the
molecular target, while also affording a highly stable
metal-chelating complex with the contrast-agent metal or
radionuclide.
[0031] One example of an effective chelator, which has demonstrated
effectiveness in formation of extremely stable metal-chelator
complexes, particularly with tri-valent metals and radionuclides,
is the well-studied metal chelator
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).
DOTA derivatives have been synthesized for conjugation to mAbs and
peptides. Recent developments of DOTA derivatives involve
approaches for conjugation of peptides for radionuclide imaging and
therapy in nuclear medicine. DOTA-modified peptides can be
synthesized in solution or on a solid support, attaching the DOTA
residue to a free amine or at a carboxy terminus of the resin-bound
peptide. The usual synthetic approach involves using
electrophilic/nucleophilic reactions with protected DOTA
derivatives to prevent side reactions with the activated carboxylic
acid groups of the DOTA ring. Examples of useful DOTA derivatives
employed for these applications include protected and unprotected
derivatives of the DOTA-tris-t-butyl ester, and the isothiocyanate
functionalized p-NCS-Bz-DOTA. In alternative approaches,
DOTA-derivatized amino acids or N-terminus reactions have been used
to add the DOTA moiety at the conclusion of automated peptide
synthesis. Because the active esters and other reactive chelator
precursors described above are highly reactive (particularly in
water), these reactions are generally carried out in organic
solvents and the active precursors must be dissolved immediately
before use to ensure that the precursors are chemically-available
in the reactive form at the time of introduction of the targeting
vector to which they are to be attached. Other examples of
chelators that are useful for binding radiometals and metals for
imaging and therapy include: NOTA and TETA.
[0032] A particularly promising new class of targeting mechanisms
is modified ribonucleic acids (RNAs) that are selected to bind with
high affinity to specific cell surface receptors that are
overexpressed or amplified in specific cancer cell types in vivo.
These RNAs are commonly referred to as aptamers and represent a new
class of molecular targeting mechanism with ideal characteristics
for delivery of radionuclides and other stable contrast metals for
imaging and therapy of cancer and other diseases. However, facile
post-synthesis modifications that include a chelator moiety are
required to produce aptamers with bifunctional capability. The
addition of a chelator can provide bifunctional
aptamer-radionuclide bioconjugates, which are not only useful to
enable further study of molecular targets and cellular pathways,
but also have translational value as clinically viable molecular
imaging agents for diagnosing and confirming diagnosis of disease,
measuring response to therapy to evaluate therapeutic approaches,
and monitoring of disease progression for treatment planning. Thus,
preparation of bioconjugate bifunctional aptamers can lead to the
development of radiopharmaceuticals for human use. Ideally, the
chemical conditions for preparation of chelator-aptamer
bioconjugates will be relatively mild, be possible in aqueous and
mild organic solvent conditions and, importantly, be highly
regioselective in character of conjugation reaction, such that side
reactions can be circumvented to rapidly produce highly pure
bifunctional ligands.
[0033] An embodiment of the invention described herein is an
optimized RNA-based therapeutic reagent for the detection or
treatment of prostate and other solid sarcomas and carcinomas. This
reagent comprises four basic components, an RNA aptamer (a
structural, synthetic RNA); a metal chelating group; a chemical
linker to connect the chelator and RNA aptamer; and a radionuclide,
stable metal that is used to image the location of the bioconjugate
in vivo. In certain embodiments, the chelator can be a light active
species that can be used for optical imaging, or photodynamic
therapy of cancer. The aptamer portion of the reagent serves as a
targeting moiety by binding specifically to a cell surface receptor
(e.g., prostate specific membrane antigen; PSMA) expressed on
cancer cells (e.g., prostate cancer cells).
Aptamer Portion
[0034] Aptamers are single stranded oligonucleotides that can
naturally fold into different 3-dimensional structures, which have
the capability of binding specifically to biosurfaces, a target
compound or a moiety. The term "conformational change" refers to
the process by which a nucleic acid, such as an aptamer, adopts a
different secondary or tertiary structure. The term "fold" may be
substituted for conformational change.
[0035] Aptamers can be used as molecular targeting agents with
applications in imaging and treatment of disease. Post-synthetic
modifications can be employed to optimize the in vivo stability and
pharmacokinetics of these molecular constructs. One particularly
attractive innovation is the introduction of oligonucleotides and
RNA aptamers with post-synthetic modifications that enable
radiolabeling of the targeting mechanisms for in vivo imaging by
positron emission tomography (PET) and single photon emission
computer tomography (SPECT). The same or similar molecular
modifications can be made to enable radionuclide therapy, by
employing a therapeutic radionuclide such as .sup.90Y, .sup.213Bi,
.sup.177Lu, .sup.211At, .sup.210Po, .sup.223Ra, or other suitable
radionuclide and using the RNA aptamer as the targeting vector in
the same manner as for imaging applications.
[0036] Aptamers have advantages over more traditional affinity
molecules such as antibodies in that they are very stable, can be
easily synthesized, and can be chemically manipulated with relative
ease. Aptamer synthesis is potentially far cheaper and reproducible
than antibody-based diagnostic tests. Aptamers can be produced by
solid phase chemical synthesis, an accurate and reproducible
process with consistency among production batches. An aptamer can
be produced in large quantities by polymerase chain reaction (PCR)
and once the sequence is known, can be assembled from individual
naturally-occurring nucleotides and/or synthetic nucleotides.
Aptamers are typically stable to long-term storage at room
temperature, and, if denatured, aptamers can easily be renatured, a
feature not shared by antibodies. Furthermore, aptamers have the
potential to measure concentrations of ligand in orders of
magnitude lower (parts per trillion or even quadrillion) than those
antibody-based diagnostic tests. These characteristics of aptamers
make them attractive for diagnostic applications.
[0037] Aptamers are typically oligonucleotides that may be single
stranded oligodeoxynucleotides, oligoribonucleotides, or modified
oligodeoxynucleotide or oligoribonucleotides. The term "modified"
encompasses nucleotides with a covalently modified base and/or
sugar. For example, modified nucleotides include nucleotides having
sugars which are covalently attached to low molecular weight
organic groups other than a hydroxyl group at the 3' position and
other than a phosphate group at the 5' position. Thus modified
nucleotides may also include 2' substituted sugars such as
2'-O-methyl-; 2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl;
2'-fluoro-; 2'-halo or 2-azido-ribose, carbocyclic sugar analogues
a-anomeric sugars; epimeric sugars such as arabinose, xyloses or
lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.
[0038] Modified nucleotides are known in the art and include, by
example and not by way of limitation, alkylated purines and/or
pyrimidines; acylated purines and/or pyrimidines; or other
heterocycles. These classes of pyrimidines and purines are known in
the art and include, pseudoisocytosine; N4, N4-ethanocytosine;
8-hydroxy-N6-methyladenine; 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil; 5-bromouracil;
5-carboxymethylaminomethyl-2-thiouracil; 5-carboxymethylaminomethyl
uracil; dihydrouracil; inosine; N6-isopentyl-adenine;
1-methyladenine; 1-methylpseudouracil; 1-methylguanine;
2,2-dimethylguanine; 2-methyladenine; 2-methylguanine;
3-methylcytosine; 5-methylcytosine; N6-methyladenine;
7-methylguanine; 5-methylaminomethyl uracil; 5-methoxy amino
methyl-2-thiouracil; .beta.-D-mannosylqueosine;
5-methoxycarbonylmethyluracil; 5-methoxyuracil;
2-methylthio-N-6-isopentenyladenine; uracil-5-oxyacetic acid methyl
ester; psueouracil; 2-thiocytosine; 5-methyl-2 thiouracil,
2-thiouracil; 4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic
acid methylester; uracil 5-oxyacetic acid; queosine;
2-thiocytosine; 5-propyluracil; 5-propylcytosine; 5-ethyluracil;
5-ethylcytosine; 5-butyluracil; 5-pentyluracil; 5-pentylcytosine;
and 2,6,-diaminopurine; methylpsuedouracil; 1-methylguanine;
1-methylcytosine.
[0039] The aptamers of the invention are synthesized using
conventional phosphodiester linked nucleotides and synthesized
using standard solid or solution phase synthesis techniques which
are known in the art. Linkages between nucleotides may use
alternative linking molecules. For example, linking groups of the
formula P(O)S, (thioate); P(S)S, (dithioate); P(O)NR'2; P(O)R';
P(O)OR6; CO; or CONR'2 wherein R is H (or a salt) or alkyl (1-12C)
and R6 is alkyl (1-9C) is joined to adjacent nucleotides through
--O-- or --S--.
[0040] In certain embodiments of the present invention, the aptamer
portion binds to Prostate-Specific Mediated Antigen (PSMA). A PSMA
aptamer of 71 nucleotides (A10-Plk1) (Lupold et al., Cancer Res.
62(14):4029-33 (2002)) and a PSMA aptamer of 39 nucleotides
(A10-3.2) have effective binding activity. In certain embodiments,
additional modifications are made to the aptamer portion.
Additional modifications to the aptamer portion include 2'O-methyl
modification of the pyrimidines. In other embodiments, all of the
nucleotides in the aptamer are 2'O-methyl modified. Alternatively,
the pyrimidines, or all the nucleotides, may be modified with 2'
fluoros (both pyrimidines and purines). Additional modifications to
the nucleotides in the aptamer include large molecular weight
conjugates like pegylation, lipid-based modifications (e.g.,
cholesterol) or nanoparticles (e.g., PEI or chitosan) to improve
the pharmacokinetic/dynamic profile of the chimera.
[0041] Prostate-specific membrane antigen (PSMA) is a type-II
transmembrane protein that comprises an intracellular part, a
transmembrane part, and an extracellular domain that is expressed
extracellularly on prostate cancer cells (and other solid tumors,
such as renal cancer cells) and the endothelial cells of new blood
vessels that supply most other solid tumors. However, it has also
been shown to be present at low levels in the brain, kidneys (brush
border of proximal tubes) and liver. One advantage of targeting
PSMA is that it is a transmembrane protein, and is not secreted.
The truncated PSMA aptamer can be used as a tool to target prostate
cancer as well as the vasculature of all solid sarcomas and
carcinomas. It has been previously shown that PSMA expression is
elevated in malignant prostate disease as well as tumor
vasculature.
[0042] In certain embodiments, modifications are introduced into
the stem sequence in the aptamer. Different nucleotides can be used
as long as the structure of the stem is retained.
[0043] Certain embodiments of the invention provide a conjugate of
the invention, which is a conjugate comprising a nucleic acid
aptamer that is not more than 45 nucleotides in length comprising
the nucleic acid sequence
5'-n.sub.1n.sub.2n.sub.3CGGAUCAGCn.sub.4n.sub.5n.sub.6GUUUA-3' (SEQ
ID NO:1), linked to one or more chelating groups, wherein each
n.sub.x can be present or absent, wherein when present each n.sub.x
represents any nucleotide, or a pharmaceutically acceptable salt
thereof. In certain embodiments, each of the n.sub.x nucleotides
can be present or absent. In certain embodiments, the nucleic acid
molecule includes a sufficient number of n.sub.x nucleotides so as
to form the first, second and/or third stem structures. In certain
embodiments the nucleic acid molecule is not more than 45
nucleotides in length, e.g., from 15-45 nucleotides in length,
e.g., 39 nucleotides in length.
[0044] In certain embodiments nucleotides, n.sub.1n.sub.2n.sub.3
and n.sub.4n.sub.5n.sub.6 are present and hybridize to form a stem
structure.
[0045] In certain embodiments, the nucleic acid molecule includes
the nucleic acid sequence 5'-AUGCGGAUCAGCCAUGUUUA-3' (SEQ ID
NO:2).
[0046] In certain embodiments, the nucleic acid molecule includes
the nucleic acid sequence
5'-n.sub.an.sub.bn.sub.cn.sub.dn.sub.1n.sub.2n.sub.3CGGAUCAGCn.sub.4n.sub-
.5n.sub.6GUUUAn.sub.en.sub.fn.sub.gn.sub.h-3'(SEQ ID NO:3).
[0047] In certain embodiments, nucleotides n.sub.1n.sub.2n.sub.3
and n.sub.4n.sub.5n.sub.6 are present and hybridize to form a first
stem structure and nucleotides n.sub.an.sub.bn.sub.cn.sub.d and
n.sub.en.sub.fn.sub.gn.sub.h are present and hybridize to form a
second stem structure.
[0048] In certain embodiments, the nucleic acid molecule includes
the nucleic acid sequence 5'-GACGAUGCGGAUCAGCCAUGUUUACGUC-3' (SEQ
ID NO:4).
[0049] In certain embodiments, the nucleic acid molecule includes
the nucleic acid sequence
5'-n.sub.10n.sub.11n.sub.12n.sub.13n.sub.10n.sub.an.sub.bn.sub.cn.sub.dn.-
sub.1n.sub.2n.sub.3CGGAUCAGCn.sub.4n.sub.5n.sub.6GUUUAn.sub.en.sub.fn.sub.-
gn.sub.hn.sub.15n.sub.16n.sub.17n.sub.18n.sub.19
n.sub.20n.sub.21-3' (SEQ ID NO:6). In certain embodiments, n.sub.21
is U. In certain embodiments, n.sub.21 is absent.
[0050] In certain embodiments, nucleotides n.sub.1n.sub.2n.sub.3
and n.sub.4n.sub.5n.sub.6 are present and hybridize to form a first
stem structure, nucleotides n.sub.an.sub.bn.sub.cn.sub.d and
n.sub.en.sub.fn.sub.gn.sub.h are present and hybridize to form a
second stem structure, and nucleotides
n.sub.10n.sub.11n.sub.12n.sub.13n.sub.14 and
n.sub.15n.sub.16n.sub.17n.sub.18n.sub.19 are present and hybridize
to form a third stem structure.
[0051] In certain embodiments, the nucleic acid molecule includes
the nucleic acid sequence
5'-GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCU-3' (SEQ ID NO:5).
[0052] In certain embodiments, the nucleic acid molecule consists
essentially of the nucleic acid sequence
5'-GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCU-3' (SEQ ID NO:5).
[0053] In certain embodiments, the nucleic acid molecule consists
of the nucleic acid sequence
5'-GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCU-3' (SEQ ID NO:5).
[0054] In certain embodiments, a molecular entity (such as
polyethylene glycol PEG) is conjugated to the RNA at the 5'-end,
the 3' end or at any other nucleic acid constituent on the aptamer.
The molecular extension is added to confer some in vivo behavioral
characteristic that improves the performance of the molecular
targeting agent comprised by the chelator, linker, and aptamer.
Examples of extensions include PEG groups with molecular weights
ranging from 200 Da to 50,000 Da. Other possible extensions include
the addition of peptide extensions comprised of amino acids, or
other molecular structures that are designed to confer specific in
vivo behavior.
[0055] In certain embodiments, the molecular extension is
conjugated to the chelator that has been connected to the aptamers
by the linker.
Metal Chelating Group
[0056] The disclosed compounds can be prepared using reagents that
are readily available. A general procedure for the preparation of a
polyamine polycarboxylic acid derivatives such as DOTA can be
illustrated by preparation of
1,4,7,10,13,16-hexaazacyclohexadecane-N,N',N'', N''', N'''',
N''''', N''''''-hexaacetic acid derivatives in mg quantities as
described in Deal et al., J. Med. Chem., 1999, 42, 2988-2992. The
hexaester
1,4,7,10,13,16-hexakis(tert-butoxycarbonylmethyl)-1,4,7,10,13,1-
6-hexaazacyclootadecane is prepared by reacting the free base
([18]aneN6 500 mg, 1 eq) in 25 mL anhydrous acetonitrile with the
addition of 2 g sodium carbonate (10 eq) and 2.3 mL (8 eq)
tert-butyl bromoacetate. The reaction is complete in about 5-10
days under inert atmosphere at room temperature. The desired
product is purified by chromatographic means and solvents
evaporated. The active ester (with a single useful leaving group)
that can be used for conjugation to various molecular linkers (such
as in the preparation of the DOTAzide compound) is then prepared by
reacting the fully carboxylated species with 1 eq. N-hydroxy
succinimide in dicyclohexyl carbodiimide (DCC) to afford an
N-hydroxysuccinimide active ester, followed by purification by
chromatographic methods, evaporation of solvents, and storage for
use.
Linking Groups and Linkers
[0057] Chemistries that can be used to link the aptamers and metal
chelating groups are known in the art, such as disulfide linkages,
amino linkages, covalent linkages, etc. Additional linkages and
modifications can be found on the world-wide-web at
trilinkbiotech.com/products/oligo/oligo_modifications.asp. In
certain embodiments, the present invention provides conjugates that
comprise a nucleic acid molecule is operably linked to one or more
metal chelating groups either directly (e.g., through a covalent
bond) or through a linking group (i.e., a linker). The nature of
the linker can be important, not only as a means to connect the
chelator to the targeting vector, but also as a means to modify the
pharmacokinetics and biodistribution of the bifunctional
bioconjugate. It is also critical that the linker not interfere
with the ability of the conjugate compound to function for its
intended use, e.g., as a therapeutic or imaging agent. The metal
chelating group or the linker can be linked to the compound at any
synthetically feasible position on the compound that does not
interfere with binding of the bioconjugate to the molecular target
(e.g., PSMA).
[0058] In the present invention, a linker is a molecular tether
that connects the chelator group to the targeting mechanism or
small molecule through any number of functional group combinations
that afford facile conjugation chemistry. In certain embodiments,
the linker connects an azide or alkynyl or cyclooctyne functional
group to a macrocyclic polyamino ring or other molecular chelator
entity such as ethylene diaamine tetraacetic acid that is intended
to complex strongly a radioactive or stable metal for the purpose
of delivery of the bound radionuclide or stable metal in vivo for
imaging or therapy of disease, so as to reveal expression of a
protein. In certain embodiments, the linker connects an azide or
alkynyl functional group to a nucleic acid molecule. The azide
functional group at the terminus of the linker is intended for
applications in conjugation of the molecular construct described
above to an alkyne-functionalized molecule, such as a peptide,
oligonucleotide, or ribonucleic acid aptamer, or peptide-aptamer
construct (PNA) by click chemical techniques as described in
Sharpless et al., Angew. Chem. Int. Ed. 2001, 40, 2004-2021. In
certain embodiments, the linker incorporates a primary amine,
activated carboxy ester, or other reactive group, that can be used
to conjugate the chelator or small molecule to the targeting vector
for delivery of a radionuclide or other small molecule for imaging
or therapy. The conjugate is intended to deliver, e.g., via
injection, the complete pharmacologic agent (including a
radioactive or stable metal, chelator and targeting mechanism as a
single combined-complete pharmaceutical agent) into the body of a
mammal, e.g., human or other animal the radioactive or stable
metals described above to a specific protein, mRNA, cell surface
receptor, integrin, or other cellular or physiologically derived
molecular construct or potentially some other molecular
construct.
[0059] In certain embodiments, the linker is connected to the
macrocyclic ring or linear chelator through an amide, thiourea
bond, conjugation through Staudinger ligation, or any number of
other conjugation strategies known in the art and is useful as
molecular tethers designed to modify the pharmacokinetics and
biodistribution of the final molecular composition of matter for
the purpose of optimizing specific delivery of the radioactive
metal or stable metal to a specific physiological location (such as
a specific cancerous tumor or the location of volatile plaque in
veins or arteries or blood vessels of the mammal, e.g., human (or
in an animal) for research, imaging, therapy or other medically or
research related reason, such as is described in Smith-Jones et
al., Nuclear Medicine and Biology, 24:761-769, 1997 and Garrison et
al., Bioconjugate Chemistry, September; 19(9):1803-12 (2008, Epub
Aug. 20, 2008).
[0060] In certain embodiments, the linker includes (1) a repeating
polyethylene glycol (PEG) tether of molecular weight from
approximately 200 to 20,000 daltons; (2) an alkyl repeating tether
of the form (--CH.sub.2--).sub.n where n can vary from 2 to about
1200; (3) an aromatic insertion, for example a benzyl group with
appropriate functional terminus to allow for effective conjugation
to the azide and the secondary amine on the macrocyclic ring; (4)
an amino acid, deoxyribonucleic and ribonucleic acid or other
insertion such as a glycine residue and (5) any combination of
aromatic, alkyl, PEG, and amino acid or nucleic acid groups that
constitutes an adequately stable or engineered cleavable sequence
that is used to connect the chelator to the azide group for
conjugation to a molecular targeting mechanism such as an RNA
aptamer.
[0061] Exemplary linking groups include:
##STR00001##
[0062] A: alkylene linkers
##STR00002##
[0063] B: ether or ether/alkylene linkers
##STR00003##
[0064] C: polyethylene glycol linkers
##STR00004##
[0065] D: aromatic linkers
##STR00005##
[0066] E: glycine/aromatic linkers
##STR00006##
[0067] F: glycine/alkyl/aromatic linkers
[0068] and combinations of A-F or repeating units of A-F. Linkers
can also consist of peptide-like amino acid sequences, PNA
sequences in combination with or in addition to the specific linker
sequences shown here.
[0069] In certain embodiments, the linking group includes an
alkylene that can have from 2 to about 1200 carbon atoms, such as
from 2 to about 50 carbon atoms, from 2 to about 20 carbon atoms,
or even from about 2 to 5 carbon atoms. In certain embodiments, the
linker comprises an alkylene or alkenylene group having or
(C.sub.6-C.sub.10)aryl; where the alkylene or alkenylene groups can
be optionally interrupted by one or more heteroatom groups or an
aromatic rings; where the heteroatom groups include --NR.sup.5--,
--O--, or --S--; each R.sup.5 is hydrogen, (C.sub.1-C.sub.6)alkyl,
(C.sub.6-C.sub.10)aryl, substituted aryl, or absent; an amino acid,
peptide, deoxyribonucleic or ribonucleic acid residue; or any
combination of two or more of (a), (b), or (c); each X is
independently --(CH.sub.2).sub.m--NR.sup.1--; n is 0, 1, 2, or 3; m
is 2, or 3; each R' is independently
--(CH.sub.2).sub.i--COOR.sup.2, where i is 0, 1, or 2; each R.sup.2
is independently, hydrogen, or a protecting group for carboxyl
groups; and each j is independently 1 or 2; or a pharmaceutically
acceptable salt thereof.
[0070] The carbon atom content of various hydrocarbon-containing
moieties is indicated by a prefix designating a lower and upper
number of carbon atoms in the moiety, i.e., the prefix C.sub.i-j
indicates a moiety of the integer "i" to the integer "j" carbon
atoms, inclusive. Thus, for example, C.sub.1-7alkyl refers to alkyl
of one to seven carbon atoms, inclusive.
[0071] Specifically, (C.sub.1-C.sub.6)alkyl can be methyl, ethyl,
propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl,
or hexyl; (C.sub.3-C.sub.6)cycloalkyl can be cyclopropyl,
cyclobutyl, cyclopentyl, or cyclohexyl;
(C.sub.3-C.sub.6)cycloalkyl(C.sub.1-C.sub.6)alkyl can be
cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,
cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl,
2-cyclopentylethyl, or 2-cyclohexylethyl; (C.sub.1-C.sub.6)alkoxy
can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy,
sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; C.sub.2-C.sub.20
alkenyl, can be an olefinically unsaturated branched or linear
group having from two to twenty carbon atoms and at least one
double bond. Typically, C.sub.2-C.sub.20 alkenyl groups include,
but are not limited to, vinyl, 1-propenyl, 2-propenyl,
1,3-butadienyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl,
1-hexenyl, 2-hexenyl, heptenyl, octenyl and the like;
(C.sub.2-C.sub.6)alkynyl can be ethynyl, 1-propynyl, 2-propynyl,
1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 1-hexynyl,
2-hexynyl, heptynyl, octynyl and the like;
(C.sub.1-C.sub.6)alkanoyl can be acetyl, propanoyl or butanoyl;
halo(C.sub.1-C.sub.6)alkyl can be iodomethyl, bromomethyl,
chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl,
2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl;
hydroxy(C.sub.1-C.sub.6)alkyl can be hydroxymethyl, 1-hydroxyethyl,
2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl,
1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl,
1-hydroxyhexyl, or 6-hydroxyhexyl; (C.sub.1-C.sub.6)alkoxycarbonyl
can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl,
isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or
hexyloxycarbonyl; (C.sub.1-C.sub.6)alkylthio can be methylthio,
ethylthio, propylthio, isopropylthio, butylthio, isobutylthio,
pentylthio, or hexylthio; (C.sub.2-C.sub.6)alkanoyloxy can be
acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy,
or hexanoyloxy; aryl can be phenyl, indenyl, or naphthyl; and
heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl,
isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl,
tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its
N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its
N-oxide). In certain embodiments, the linking group is
(C.sub.3)alkylene.
[0072] In certain embodiments, the linking group comprises an amino
acid residue, peptide deoxyribonucleic or ribonucleic acid residue,
or any combination thereof. In certain embodiments, the linking
group is an amino acid residue or a peptide having from one to four
amino acid groups in the chain. The term "amino acid", includes the
residues of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys,
Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, and Val) in Dextrorotary or Levorotary
stereoisomeric forms, as well as unnatural amino acids (e.g.
phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline,
and gamma-carboxyglutamate; hippuric acid,
octahydroindole-2-carboxylic acid, statine,
1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,
ornithine, citruline, alpha-methyl-alanine,
para-benzoylphenylalanine, phenylglycine, propargyiglycine,
sarcosine, and tert-butylglycine). The term also comprises natural
and unnatural amino acids (Dextrorotary and Levorotary
stereoisomers) bearing a conventional amino protecting group (e.g.
acetyl or benzyloxycarbonyl), as well as natural and unnatural
amino acids protected at the carboxy terminus (e.g. as a
(C.sub.1-C.sub.6)alkyl, phenyl or benzyl ester or amide; or as an
.alpha.-methylbenzyl amide). Other suitable amino and carboxy
protecting groups are known to those skilled in the art (See for
example, Greene, T. W.; Wutz, P.G.M., Protecting Groups In Organic
Synthesis; second edition, 1991, New York, John Wiley & sons,
Inc, and documents cited therein). An amino acid can be linked to
the remainder of a compound of formula (I) through the carboxy
terminus, the amino terminus, or through any other convenient point
of attachment, such as, for example, through the sulfur of
cysteine.
[0073] In certain embodiments, R.sup.2 is a protecting group. In
certain embodiments, each R.sup.2 is independently hydrogen,
--C(CH.sub.3).sub.3, or --CH.sub.2Ph and each i is independently 0
or 1. In certain embodiments, each R.sup.2 is hydrogen, or
--C(CH.sub.3).sub.3, and each i is 1. In certain embodiments,
R.sup.2 is --C(CH.sub.3).sub.3, and each i is 1.
[0074] In certain embodiments, the alkylene in the linking group
optionally comprises one or more aryl groups or a polyethylene
glycol (PEG) polymer having a weight average molecular weight of
about 200 to about 20,000, such as a molecular weight of about 200
to 1000. In certain embodiments, the PEG polymer has a weight
average molecular weight of about 200 to about 500.
[0075] In certain embodiments, the alkylene groups can have from 2
to about 50 carbon atoms, such as from about 2 to 20 carbon atoms,
or from 2 to about 10 carbon atoms. In certain embodiments, the
alkylene group has from 2 to about 5 carbon atoms. In certain
embodiments, the alkylene group may optionally contain an aryl
group.
[0076] In another embodiment of the invention the linking group has
a molecular weight of from about 20 daltons to about 400
daltons.
[0077] In another embodiment of the invention the linking group or
linker has a length of about 5 angstroms to about 300
angstroms.
[0078] In another embodiment of the invention the linking group
separates the nucleic acid molecule and the metal chelating group
and a P(.dbd.Y.sup.1) residue by about 5 angstroms to about 200
angstroms, inclusive, in length
[0079] In another embodiment of the invention the linking group is
a divalent, branched or unbranched, saturated or unsaturated,
hydrocarbon chain, having from 2 to 25 carbon atoms, wherein one or
more (e.g. 1, 2, 3, or 4) of the carbon atoms is optionally
replaced by (--O--), and wherein the chain is optionally
substituted on carbon with one or more (e.g. 1, 2, 3, or 4)
substituents selected from (C.sub.1-C.sub.6)alkoxy,
(C.sub.3-C.sub.6)cycloalkyl, (C.sub.1-C.sub.6)alkanoyl,
(C.sub.1-C.sub.6)alkanoyloxy, (C.sub.1-C.sub.6)alkoxycarbonyl,
(C.sub.1-C.sub.6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo
(.dbd.O), carboxy, aryl, aryloxy, heteroaryl, and
heteroaryloxy.
[0080] In another embodiment of the invention the linking group is
of the formula W-A wherein A is (C.sub.1-C.sub.24)alkyl,
(C.sub.2-C.sub.24)alkenyl, (C.sub.2-C.sub.24)alkynyl,
(C.sub.3-C.sub.8)cycloalkyl, (C.sub.6-C.sub.10)aryl or a
combination thereof, wherein W is --N(R)C(.dbd.O)--,
--C(.dbd.O)N(R)--, --OC(.dbd.O)--, --C(.dbd.O)O--, --O--, --S--,
--S(O)--, --S(O).sub.2--, --N(R)--, --C(.dbd.O)--,
--N(R)C.dbd.N(R)--N(R)--, --C(R).dbd.N(R)--, --S(O).sub.M2--N(R)--,
--N(R)--S(O).sub.M2--, or a direct bond; wherein each R is
independently H or (C.sub.1-C.sub.6)alkyl. In certain embodiments,
the linker is --CH.sub.2--C(.dbd.O). In certain embodiments, each A
is alkylene of 1 to 10 carbons.
[0081] In certain embodiments, each linker is a divalent radical
formed from a peptide. In certain embodiments, each linker is a
divalent radical formed from an amino acid. In another embodiment
of the invention the linking group is a divalent radical formed
from a peptide. In another embodiment of the invention the linking
group is a divalent radical formed from an amino acid. In certain
embodiments of the invention, the linking group is a divalent
radical formed from poly-L-glutamic acid, poly-L-aspartic acid,
poly-L-histidine, poly-L-ornithine, poly-L-serine,
poly-L-threonine, poly-L-tyrosine, poly-L-leucine,
poly-L-lysine-L-phenylalanine, poly-L-lysine or
poly-L-lysine-L-tyrosine.
[0082] In certain embodiments, the linking group is of the formula
W--(CH.sub.2).sub.n wherein, n is between about 1 and about 10; and
W is --N(R)C(.dbd.O)--, --C(.dbd.O)N(R)--, --OC(.dbd.O)--,
--C(.dbd.O)O--, --O--, --S--, --S(O)--, --S(O).sub.2--,
--C(.dbd.O)--, --N(R)--, --N(R)C.dbd.N(R)--N(R)--,
--C(R).dbd.N(R)--, --S(O).sub.M2--N(R)--, --N(R)--S(O).sub.M2--, or
a direct bond; wherein each R is independently H or
(C.sub.1-C.sub.6)alkyl. In certain embodiments, each linker is
methylene, ethylene, or propylene.
[0083] In another embodiment of the invention the linking group is
methylene, ethylene, or propylene.
[0084] In another embodiment of the invention the linking group is
attached to the phosphonate group through a carbon atom of the
linker.
[0085] In certain embodiments, the linking group is L.sub.a,
wherein L.sub.a comprises (a) a repeating polyethylene glycol (PEG)
polymer having an average molecular weight of about 200 to about
20,000; (b) an alkylene or alkenylene group having from 2 to about
1200 carbon atoms, or (C.sub.6-C.sub.10)aryl; where the alkylene or
alkenylene groups can be optionally interrupted by one or more
heteroatom groups or an aromatic rings; where the heteroatom groups
include --NR.sup.5--, --O--, or --S--; each R.sup.5 is hydrogen,
(C.sub.1-C.sub.6)alkyl, (C.sub.6-C.sub.10)aryl, substituted aryl,
or absent; or (c) an amino acid, peptide, deoxyribonucleic or
ribonucleic acid residue, or a combination thereof.
Biologically Active Conjugates of Aptamers and Metal Chelating
Groups
[0086] In certain embodiments the present invention provides a
conjugate comprising a nucleic acid aptamer linked to one or more
metal chelating groups or a pharmaceutically acceptable salt
thereof. The present invention employs these conjugates for
targeted in vivo delivery of radionuclides and other metals and
therapeutic entities such as described above for imaging and
therapy of prostate cancer by targeting the PSMA protein expressed
in prostate cancer, or by targeting PSMA for other cancers, which
express this receptor in the neovasculature of early stage
disease.
[0087] In addition, the biologically active conjugates, e.g.,
bifunctional aptamer-radionuclide bioconjugates described here, are
useful for the study of molecular targets and cellular pathways,
such as the development of neovasculature of cancerous tumors, but
targeting PSMA. In their preferred form, the bifunctional
bioconjugates can be prepared using mild or highly regioselective
conjugation techniques or both mild or highly regioselective
reaction conditions.
[0088] For in vivo imaging applications, a chelator is often added
chemically to the aptamer to enable labeling of the resulting
bifunctional ligand. Bifunctional refers to the characteristic that
these chelator-modified aptamers bind a contrast metal or
radionuclide strongly, while maintaining high affinity for the in
vivo molecular target of interest (e.g., a cell surface receptor).
These bifunctionals are often used a contrast-agent metal (e.g.,
gadolinium for magnetic resonance imaging, MRI) or radionuclide
(e.g., gallium-68 (.sup.68Ga) for positron emission tomography,
PET).
[0089] An identical approach can be employed for specific targeting
of therapeutic radionuclides (e.g., alpha and beta emitters) to the
site of cancerous tissue, while sparing healthy cells in vivo. To
be effective, the chelator-modified bifunctional ligand (the
conjugate molecule) should maintain affinity for the molecular
target, while also affording a highly stable metal-chelate complex
with the contrast-agent metal or radionuclide.
[0090] In certain embodiments, one or more metals are chelated to
the one or more chelating groups.
[0091] In certain embodiments the conjugate has formula I:
M-L-APT (I)
wherein:
[0092] M is a metal chelating group;
[0093] L is absent or is a linking group; and
[0094] APT is a nucleic acid aptamer that is not more than 45
nucleotides in length comprising the nucleic acid sequence
5'-n.sub.1n.sub.2n.sub.3CGGAUCAGCn.sub.4n.sub.5n.sub.6GUUUA-3' (SEQ
ID NO:1), wherein each n.sub.x can be present or absent, wherein
when present each n.sub.x represents any nucleotide.
[0095] In certain embodiments the nucleic acid aptamer has a 5'-end
and a 3'-end and is operably linked through the 5'-end or 3'-end to
one or more metal chelating groups.
[0096] In certain embodiments the metal chelating group has the
formula II:
##STR00007##
[0097] wherein:
[0098] each X is independently --(CH.sub.2).sub.m--NR.sup.1--; m is
2 or 3; n is 0, 1, 2, or 3;
[0099] each R.sup.1 is independently
--(CH.sub.2).sub.i--COOR.sup.2;
[0100] each i is independently 0, 1, or 2;
[0101] each R.sup.2 is independently, hydrogen, or a protecting
group for carboxyl groups; and
[0102] each j is independently 1 or 2;
[0103] or a salt thereof.
[0104] In certain embodiments the conjugate has formula III:
##STR00008##
wherein:
[0105] APT is; a nucleic acid aptamer molecule having a 5'-end and
a 3'-end wherein the aptamer molecule is operably linked to L'
through the 5'-end or the 3'-end, and wherein the nucleic acid
molecule is not more than 45 nucleotides in length comprising the
nucleic acid sequence
5'-n.sub.1n.sub.2n.sub.3CGGAUCAGCn.sub.4n.sub.5n.sub.6GUUUA-3' (SEQ
ID NO:1), wherein each n.sub.x can be present or absent, wherein
when present each n.sub.x represents any nucleotide;
[0106] L' is a linking group;
[0107] L'' is a linking group;
[0108] each X is independently --(CH.sub.2).sub.m--NR.sup.1--; m is
2 or 3; n is 0, 1, 2, or 3;
[0109] each R.sup.1 is independently
--(C.sub.1-12).sub.i--COOR.sup.2,
[0110] each i is independently 0, 1, or 2;
[0111] each R.sup.2 is independently, hydrogen, or a protecting
group for carboxyl groups; and
[0112] each j is independently 1 or 2;
[0113] or a salt thereof.
[0114] In certain embodiments, L'' is
-L.sub.a-C(.dbd.O)--CH.sub.2--, wherein L.sub.a comprises a
repeating polyethylene glycol (PEG) polymer having an average
molecular weight of about 200 to about 20,000 (e.g., about 200 to
about 1000); an alkylene or alkenylene group having from 2 to about
1200 carbon atoms, such as an alkylene group having from 2 to about
50 carbon atoms and optionally one or more aryl groups, or an
alkylene group having from 2 to about 20 carbon atoms, or an
alkylene group having from 2 to 5 carbon atoms (e.g.,
(C.sub.3)alkylene), or (C.sub.6-C.sub.10)aryl; where the alkylene
or alkenylene groups can be optionally interrupted by one or more
heteroatom groups or an aromatic rings; where the heteroatom groups
include --NR.sup.5--, --O--, or --S--; each R.sup.5 is hydrogen,
(C.sub.1-C.sub.6)alkyl, (C.sub.6-C.sub.10)aryl, substituted aryl,
or absent; or an amino acid, peptide, deoxyribonucleic or
ribonucleic acid residue, or a combination thereof. In certain
embodiments, L.sub.a is an amino acid residue or peptide having
from one to four amino acid groups in the chain.
[0115] In certain embodiments, L' has a molecular weight of from
about 20 daltons to about 400 daltons. In certain embodiments, L'
has a length of about 5 angstroms to about 300 angstroms. In
certain embodiments, L' is a divalent, branched or unbranched,
saturated or unsaturated, hydrocarbon chain, having from 2 to 25
carbon atoms, wherein one or more of the carbon atoms is optionally
replaced by (--O--), and wherein the chain is optionally
substituted on carbon with one or more substituents selected from
(C.sub.1-C.sub.6)alkoxy, (C.sub.3-C.sub.6)cycloalkyl,
(C.sub.1-C.sub.6)alkanoyl, (C.sub.1-C.sub.6)alkanoyloxy,
(C.sub.1-C.sub.6)alkoxycarbonyl, (C.sub.1-C.sub.6)alkylthio, azido,
cyano, nitro, halo, hydroxy, oxo (.dbd.O), carboxy, aryl, aryloxy,
heteroaryl, and heteroaryloxy. In certain embodiments, L' is of the
formula W-A wherein A is (C.sub.1-C.sub.24)alkylene,
(C.sub.2-C.sub.24)alkenylene, (C.sub.2-C.sub.24)alkynylene,
(C.sub.3-C.sub.8)cycloalkylene, (C.sub.6-C.sub.10)aryl or a
combination thereof, wherein each W is --N(R)C(.dbd.O)--,
--C(.dbd.O)N(R)--, --OC(.dbd.O)--, --C(.dbd.O)O--, --O--, --S--,
--S(O)--, --S(O).sub.2--, --N(R)--, --C(.dbd.O)--,
--N(R)C.dbd.N(R)--N(R)--, --C(R).dbd.N(R)--, --S(O).sub.M2--N(R)--,
--N(R)--S(O).sub.M2--, or a direct bond; wherein each R is
independently H or alkyl of 1 to 10 carbons. In certain
embodiments, A is alkylene of 1 to 10 carbons. In certain
embodiments, L' is a divalent radical formed from an amino acid or
a peptide. In certain embodiments, L' is a divalent radical formed
from poly-L-glutamic acid, poly-L-aspartic acid, poly-L-histidine,
poly-L-ornithine, poly-L-serine, poly-L-threonine, poly-L-tyrosine,
poly-L-leucine, poly-L-lysine-L-phenylalanine, poly-L-lysine or
poly-L-lysine-L-tyrosine.
[0116] In certain embodiments, L' is of the formula
W--(CH.sub.2).sub.n wherein, n is between about 1 and about 10; and
W is --N(R)C(.dbd.O)--, --C(.dbd.O)N(R)--, --OC(.dbd.O)--,
--C(.dbd.O)O--, --O--, --S--, --S(O)--, --S(O).sub.2--,
--C(.dbd.O)--, --N(R)--, --N(R)C.dbd.N(R)--N(R)--,
--C(R).dbd.N(R)--, --S(O).sub.2--N(R)--, --N(R)--S(O).sub.2--, or a
direct bond; wherein each R is independently H or
(C.sub.1-C.sub.6)alkyl. In certain embodiments, L' is methylene,
ethylene, or propylene.
[0117] In certain embodiments, the aptamer portion is operably
linked to its azido group or its alkynyl group by means of a linker
prior to the formation of the conjugate compound. In certain
embodiments the metal chelating group is linked to its azido group
or its alkynyl group by means of a linker prior to the formation of
the conjugate compound. The structure of each linker in the
conjugate compound is not critical as long as it does not interfere
with the intended function of the conjugate compound.
Metals to Chelate to Metal Chelating Group
[0118] In certain embodiments the metal chelating group is chelated
to a metal. In certain embodiments, the metal is an imageable metal
or a therapeutic metal. In certain embodiments the imageable metal
is a radionuclide, such as an alpha or beta-emitting radionuclide.
Exemplary radionuclides include gallium-68 (68Ga, half life
t.sub.1/2=68 min), yttrium-90 (.sup.90Y, t.sub.1/2=64 hours),
lutetium-177 (177Lu, t.sub.1/2=6.63 days), bismuth-213 (.sup.213Bi,
t.sub.1/2=46 minutes), indium-111 (.sup.111In, t.sub.1/2=2.8 days);
gallium-67 (.sup.67Ga, t.sub.1/2=3.3 days); copper-64 (64Cu,
t.sub.1/2=12.7 hours), actinium-225 (.sup.225Ac, t.sub.1/2=10
days), or radium-223 (.sup.223Ra, t.sub.1/2=11.4 days). Other
radionuclides include: Cr-51, Ir-192, Sc-44, Co-55, Cu-61, Cu-67,
Mn-51, Mn-52, Pb-203, Po-210, Phosphorus-32 and the like. In
certain embodiments, the metal is a therapeutic metal.
Methods of Making Conjugates
[0119] Processes for preparing conjugates or for preparing
intermediates useful for preparing conjugates are provided as
further embodiments of the invention. Intermediates useful for
preparing conjugates are also provided as further embodiments of
the invention.
[0120] In certain embodiments, the present invention provides a
method of covalently linking the nucleic acid aptamer as described
above to the metal chelating group as described above by forming a
reaction mixture of a corresponding aptamer comprising an alkynyl
group with a corresponding metal chelating group comprising an
azido group; or forming a reaction mixture of a corresponding
aptamer comprising an azido group with a corresponding metal
chelating group comprising an alkynyl group, wherein the reaction
mixture is formed under conditions permitting a 1,3-dipolar
cycloaddition reaction to occur between the azido and the alkynyl
groups to covalently link the nucleic acid aptamer to the metal
chelating group. In certain embodiments, the forming of the
reaction mixture occurs at about room temperature. In certain
embodiments, the reaction mixture further comprises an agent which
catalyzes a 1,3-dipolar cycloaddition reaction. In certain
embodiments, the agent is copper. In certain embodiments, the
reaction mixture is devoid of copper.
[0121] In the preparation of molecular targeting agents for imaging
and therapy, the aptamer is conjugated with a chelator for housing
the radionuclide or metal used as the contrast agent for the
imaging modality (PET, SPECT, MRI or other). Many methods have been
applied to these conjugation techniques. For applications prior to
the invention of RNA aptamers for molecular targeting, the chemical
conditions could be relatively "harsh" (highly acidic or basic,
high temperatures, organic solvents, etc.). For example, in the
preparation of a chelator modified peptides and antibodies, a
deprotection step is employed where protecting groups (such as
tertiary-butyl groups connected to the carboxylic acids of
macrocyclic, polyamine, polycarboxylic acids that comprise the
basic structure of many chelator groups) are removed from the
chelator moiety to render the construct useful as a
bifunctional.
[0122] The need for these approach arises from two problems: (1) in
order for the conjugation reaction to be regioselective (add the
chelator at the precise location necessary to maintain the
targeting behavior of the aptamer, the carboxylic acid groups are
protected by the presence of the protecting groups (e.g., the
t-butyl groups); and (2) the t-butyl groups must be removed to
preserve the binding affinity chelation behavior of the chelator
group for the metal of interest. The conditions for the
deprotection step (as an example of harsh conditions that peptides
(amino acid constructs) and antibodies (larger amino acid sequences
that peptides) are known in the art to generally be highly acidic
(acid hydrolysis at pH conditions generally less than 2) or highly
basic conditions (pH values generally greater than 9). These
reaction are also generally slow (hours to days to completion),
which is less than optimum for economical manufacturing. In
addition, for RNA aptamers, these harsh conditions are intolerable
and lead to unacceptable degradation of the RNA construct. Thus, an
approach was needed for conjugation of chelators to RNA molecules
that was rapid, regioselective, and could be applied under mild
conditions in high chemical yield.
[0123] In certain embodiments, aptamers (such as the PSMA targeted
RNA aptamer of the invention disclosed herein), peptides,
antibodies, antibody fragments, PNAs, or other molecular targeting
entity, are conjugated to a metal chelating group by means of
"Click Chemistry." "Click Chemistry" is a term for a synthesis
method developed by Nobel laureate chemist Karl Barry Sharpless
(Scripps Research Institute) in 2001. It is a copper-catalyzed
azide-alkyne reaction that makes it possible for certain chemical
building blocks to "click" together in an irreversible linkage.
Briefly, an azido group is operably linked to the aptamer and an
alkynyl group is linked to the metal chelating group (or an alkynyl
group is linked to the aptamer and an azido group is operably
linked to the metal chelating group). The two components are
combined under conditions amendable to Click Chemistry linkage.
General methods for performing Click Chemistry are well known in
the art. Kolb et al., Angew. Chem. Int. Ed. 40:2004-2021 (2001); Ju
et al., US Patent Application Publication No. 2005/0032081; Kolb et
al., US Patent Application Publication No. 2006/0263293; Kolb et
al., US Patent Application Publication No. 2006/0269942.
Alternative methods of "Click Chemistry" can be performed without
the use of a copper catalyst. Sletten et al., Org. Lett.
10:3097-3099 (2008); Codelli et al., J. Am. Chem. Soc.
130:11486-11493 (2008); Johnson et al., Chem. Commun. 2008,
3064-3066.
[0124] Prior to the current disclosure, despite numerous
disclosures of chelator precursors with numerous descriptions
functional groups for conjugation to targeting vectors such as
peptides and antibodies, no mention is made to the need for or the
synthesis of an azide of a chelator for conjugation to a molecular
targeting agent. It is likely that the need for such a compound was
not perceived given the chemical robustness of peptides,
antibodies, antibody fragments, and PNAs. On the other hand, the
advent of the use of RNA aptamers for molecular targeted created a
need for innovation in preparing these new bifunctional
bioconjugates. The click chemical approach seemed to provide the
chemical conditional requirements for adding chelators to RNA.
Unfortunately, no azide of a chemical chelator was available or had
been described previously in the literature and would need to be
conceived and carried forward to enable the reaction as a method
for conjugating chelators to RNA aptamers.
[0125] To begin the development of the an azide modified chelator
for click chemical conjugation to a molecular targeting vector, a
synthetic route for the preparation of a prototype
azide-functionalized chelator derivative and the popular chelator
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, known as
DOTA was used to develop an azide chelator as described
earlier.
##STR00009##
[0126] The resulting compound (termed DOTAzide) allows for the
conjugation of the DOTA chelating moiety to nucleic acids,
peptides, and other targeting vectors that have been modified with
an alkyne-functional group. In one embodiment, the conjugation
reaction is catalyzed by the addition of ascorbate or other
suitable reducing agent, Cu(I) (or other suitable metal) catalyst
and tris-hydroxypropyltriazolylamine (or other suitable redox
stabilizer) as a Cu(I) stabilizer if required). The DOTAzide
molecular construct is provides an azide-functionalized tether that
allows for simple conjugation of the chelator to oligonucleotides
and RNA aptamers by click chemistry methods. This allows for the
preparation of radiolabeled aptamers for imaging and therapy of
disease, particularly cancer through radiolabeling techniques. In
one embodiment, the method employs the DOTAzide (or other azide
modified chelator as described below) for copper-free click
chemical conjugation using a cyclooctyne-modified functional group
on the RNA or other targeting vector that is modified in such a way
to enable fast click chemical conjugation that is promoted by ring
strain. The cyclooctyne functional group enables the click chemical
reaction using potential energy associated with ring strain at the
carbon-carbon triple bond. The kinetics of the reaction is enhanced
by addition of a difluorosubstitution at a carbon position that is
adjacent to the carbon-carbon triple bond of the cyclooctyne
functional group. This combination of ring strain and difluoro or
other suitable electron withdrawing group promote the click
reaction to be used as an efficient conjugation step for addition
of the azide modified chelator in the absence of the copper
catalyst. This is an important embodiment because the copper will
need to be removed from the system prior to use as a
pharmaceutical. Copper free click chemistry reduces the number of
steps required for preparation of the bifunctional
bioconjugate.
[0127] For example, positron emitting radionuclide gallium-68
(68Ga) can be labeled to an RNA aptamer that is targeted against a
cell surface protein receptor (such as prostate specific membrane
antigen, PSMA) for in vivo imaging of prostate cancer by PET. While
DOTAzide (or
2,2',2''-(10-(2-(3-azidopropylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclodo-
decane-1,4,7-triyl)triacetic acid) is the first of the
azide-modified chelators to be described, other chelator molecules
can be prepared with the same azide functionality to enable the
click chemical conjugation reaction to be used. Examples of popular
chelators, described in one of many possible reactive forms that
include the azide functional group for click chemistry include:
2,2',2''-(2-(2-(3-azidopropylamino)-2-oxoethyl)-1,4,7-triazonane-1,4,7-tr-
iyl)triacetic acid, referred to as NOTAzide);
10-(2-(3-azidopropylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclotridecane-1,-
4,7-tricarboxylic acid referred to as TETAzide; and 2,2',2'',
2''',2''''-(16-(2-(3-azidopropylamino)-2-oxoethyl)-1,4,7,10,13,16-hexaaza-
cyclooctadecane-1,4,7,10,13-pentayl)pentaacetic acid, referred to
as Hexazide). These compounds, referred to collectively as
"Macrozides" allow radiolabeling with the most popular radiometals
for research applications in targeted molecular imaging and therapy
of peptide-, antibody- and nucleic-acid-based targeting molecules,
such as aptamers. Representative metals used for radionuclide and
metal imaging applications include, but are not limited to
Gd.sup.3+ and other lanthanides and transition metals for MRI, and
radiometals .sup.111In .sup.64Cu, .sup.44Sc and .sup.68Ga for
nuclear imaging (e.g., PET, SPECT), as well as .sup.90Y, .sup.177Lu
and .sup.213Bi for radionuclide-based therapy of cancer. The PSMA
aptamer described herein is effective in binding with high affinity
to prostate cancer cells and expression of PSMA correlates with
progression of disease, making it an excellent molecular target for
in vivo imaging.
[0128] For DOTAzide preparation, high-yield synthesis of the
bifunctional azide-modified chelator was accomplished via
nucleophilic substitution of the N-hydroxysuccinimide group of
precursor tris-1,4,7,10-tetraazacyclododecane-1,4,7-tris(t-butyl
acetate)-10-succinimidyl acetate with 1.1 equivalents of
1-amino-3-azidopropane (THF, -20.degree. C., 12 hours). The
amino-azide precursor is prepared rapidly and in high yield with
inexpensive-readily-available 1-amino-3-bromopropane by
nucleophilic substitution of the bromo group with sodium azide salt
(water, 80.degree. C.). Straightforward purification and removal of
t-butyl protecting groups is performed by reverse-phase HPLC and
application of dilute hydrochloric acid to afford the water soluble
azide derivative DOTAzide. The purified compound can be lyophilized
for storage, is stable in saline solution at standard refrigeration
temperatures, and appears stable at room temperature for extended
periods of time (weeks). DOTAzide affords simple conjugation to
alkyne-functionalized oligonucleotides and RNA aptamers via the
Cu(I) catalyzed cyclo-addition of an azide and alkyne, popularized
as so called "click" chemistry. The resulting triazole-linked
bifunctional conjugate results in a stable
chelator-oligonucleotide(aptamer) bifunctional--ligand that can be
used for targeting in vivo delivery of radionuclides and other
metals for imaging and therapy of cancer and other pathologies.
[0129] The regio-selective click reaction in the absence of the
Cu(I) catalyst is preferential over click chemistry in the presence
of Cu(I). For nuclear medicine applications, particularly in
preparation of short-lived radiopharmaceuticals, streamlining the
preparation and removing the metallic impurity for the synthetic
approach is highly advantageous. The half-life of .sup.68Ga for
example is 68 minutes. Thus, preparation time must be rapid.
[0130] Radiolabeling yields and percent incorporation of .sup.68Ga
are compromised by the presence of non-radioactive metals--even
small quantities can interfere. The right-strain energy of the
smallest possible cyclic alkyne structure (cyclooctyne) promotes
the click reaction. The inventors have developed a right-strain
5'-\5-cyclooctynyl phosphoramidite to enable Cu-free click chemical
addition of chelators to nucleic acids (FIG. 7). The inventors have
been able to prepare a difluoromodified version of cyclooctyne and
confirmed copper-free click chemical conjugation of DOTAzide to the
molecule in high yield in less than 30 minutes at room temperature
in a solution of water and methanol. It is important to note that
the reaction can be undertaken in aqueous conditions with
deprotected DOTAzide and RNA. Elimination of the need for
Cu(I)-catalyst for aptamer-based imaging and therapeutic agents is
advantageous because of the corresponding elimination of the
possibility of metal contamination of drug product and because it
would simplify the quality control and quality assurance testes
that would be required for acceptance for clinical trials.
[0131] In certain embodiments, the complete bioconjugate consisting
of chelator, aptamer, linker, and possibly another imaging
molecular entity can be prepared by nucleophilic substitution or
addition reactions that connect the reactive group of the aptamer,
linkers, and chelators. For example, the inventors have shown that
the active NHS ester of DOTA, and a
para-isothiocyanoato-benzyl-functionalized NOTA can be conjugated
to the aptamer using methods known in the art (FIGS. 8A-8C).
Synthetic Intermediates
[0132] Specific synthetic intermediates can be used to prepare a
conjugate of the invention that comprises a nucleic acid aptamer
molecule linked to a reactive group (e.g. an alkynyl group, an
azido group, an amine group, an isothiocyanato group, or an active
ester of a carboxylic acid). In certain embodiments, the alkynyl
group is linked to the 3' end. In certain embodiments, the alkynyl
group is linked to the 5' end. In certain embodiments, the nucleic
acid molecule is linked to an azido group. In certain embodiments,
the azido group is linked to the 3' end. In certain embodiments,
the azido group is linked to the 5' end. In certain embodiments,
the nucleic acid is linked to an amine group. In certain
embodiments, the amine group is linked to the 3' end. In certain
embodiments, the amine group is linked to the 5' end. Other
reactive groups include reactive groups for Staudinger ligations,
isothiocyanato groups, active esters of carboxylic acids, and many
other such reactive groups that are described and well-known to
persons skilled in the art of the preparation of bifunctional
biological molecules such as bifunctional RNA and DNA aptamers,
peptides, antibodies, affibodies, and PNA's.
[0133] A synthetic intermediate that is useful for preparing a
conjugate of the invention is a compound of formula V:
##STR00010##
[0134] wherein
[0135] L is a linking group:
[0136] each X is independently --(CH.sub.2).sub.m--NR.sup.1--; n is
0, 1, 2, or 3; m is 2, or 3;
[0137] each R.sup.1 is independently
--(CH.sub.2).sub.i--COOR.sup.2; each i is independently 0, 1, or
2;
[0138] each R.sup.2 is independently, hydrogen, or a protecting
group for carboxyl groups;
[0139] each j is independently 1 or 2; and
[0140] R.sub.a is --C.ident.CH, or azido;
[0141] or a salt thereof.
[0142] Another synthetic intermediate that is useful for preparing
a conjugate of the invention is a compound of formula:
##STR00011##
or a salt thereof.
[0143] Another synthetic intermediate that is useful for preparing
a conjugate of the invention is a compound of formula of the
formula:
##STR00012##
or a salt thereof.
[0144] Another synthetic intermediate that is useful for preparing
a conjugate of the invention is a compound of formula:
##STR00013##
or a salt thereof.
[0145] Another synthetic intermediate that is useful for preparing
a conjugate of the invention is a compound of formula:
##STR00014##
or a salt thereof.
[0146] Another synthetic intermediate that is useful for preparing
a conjugate of the invention is a compound of formula:
##STR00015##
Diseases and Conditions Amendable to the Methods of the
Invention
[0147] Worldwide, cancer affects approximately 10 million people
each year. Approximately 22 million people are living with cancer
and almost 7 million people die worldwide from cancer each year.
The most common cancers include cancers of the lung, breast,
colon/rectum, stomach, liver, prostate, cervix, esophagus, and
bladder. The elderly tend to be the highest population for new
incidence, as more than 75% of all new cancer cases are diagnosed
in people over the age of 60. With the aging population, incidence
is expected to increase each year. Prostate cancer is the most
common cancer in men and the second leading cause of cancer death
in men, behind lung cancer. Approximately one in six U.S. men will
contract prostate cancer in his lifetime. Approximately 80% of
prostate cancers are diagnosed in men over 65 years of age, and,
due to the lack of symptoms, 75% of first-time patients over 65 are
diagnosed with Stage C or D, the two most advanced stages of
prostate cancer. Worldwide, more than 680,000 men are diagnosed
annually and over 28,000 U.S. men will die as a result of prostate
cancer this year alone. Prostate cancer characteristically spreads
to the bone. The therapeutic regimen for disease that is localized
in the prostate bed (local disease) can be drastically different
from the prescribed therapy for metastatic prostate cancer.
Targeted molecular imaging and therapy promise to provide precise
delivery of diagnostic and therapeutic agents to the site of
prostate cancer, providing a critically-needed approach to improve
outcomes for prostate cancer patients.
[0148] In certain embodiments of the present invention, a human
patient or mammalian research subject (such as a research rat or
mouse or a larger animal such as a primate) has prostate cancer. In
certain embodiments of the present invention, a human patient or
mammalian research subject (such as a research rat or mouse or a
larger animal such as a primate) has a form of cancer in which the
transmembrane type II protein prostate specific membrane antigen is
present at high concentrations in the cells of that cancer or other
condition which is amenable to imaging or therapy using the
composition of matter of the invention. In certain embodiments,
PSMA is expressed at high concentrations in diseased tissue of the
patient or research subject and the physician or researcher would
like to image or treat the diseased tissue using targeted molecular
imaging or therapy. As used herein, "targeted molecular imaging and
targeted molecular therapy" refers to administration to the patient
nucleic acid material (such as an RNA aptamer) that is designed to
bind specifically with high affinity to an in vivo molecular target
(such as PSMA). In so targeting the diseased tissue or otherwise
targeting the tissue of interest, the administered nucleic acid
material of the present invention delivers a radionuclide. Thus,
the phrase "condition amenable to imaging or therapy of the
invention" embraces conditions such as genetic diseases (i.e., a
disease condition that is attributable to one or more gene
defects), acquired pathologies (i.e., a pathological condition that
is not attributable to an inborn defect), cancers,
neurodegenerative diseases, e.g., trinucleotide repeat disorders,
and prophylactic processes (i.e., prevention of a disease or of an
undesired medical condition).
[0149] A condition amenable to targeted molecular imaging or
therapy can be a genetic disorder or an acquired pathology that is
manifested by abnormal cell proliferation, e.g., cancer. According
to this embodiment, the instant invention is useful for targeted
delivery of radionuclides, metals, or liposomes, nanoparticles, for
the purpose of imaging or delivering a cytotoxic dose of radiation
or other form of therapeutic dose to the tissue of interest (e.g.,
prostate cancer tumors or lesions of prostate cancer). The present
invention can be used to treat a solid sarcoma or carcinoma, such
as is prostate cancer.
Detection and Imaging Conjugates and Methods
[0150] The present invention provides methods for detecting PSMA in
a sample or in vivo. For example, one can contact a sample with an
aptamer as described herein or the composition as described herein
to form bound PSMA, and detecting the presence or the quantity of
bound PSMA. Alternatively, aptamers or compositions can be
administered in vivo to a patient (e.g. injected intravenously
using a syringe, cathetor, or other medical device). In certain
embodiments, the bound PSMA is detected by means of PCR, nuclear
magnetic resonance, fluorescent capillary electrophoresis, lateral
flow devices, colorimetry, chemiluminescence, fluorescence, western
blots, microarrays, ELISA, radioHPLC, single photon emission
computed tomography (SPECT), or positron emission tomography
(PET).
[0151] In one embodiment for in vivo imaging of prostate cancer,
the present invention involves the administration of the invention
as a radiopharmaceutical for imaging the location of prostate
cancer cells in humans or research mammalian subjects such as rats,
mice, dogs, cats, and primates that have been genetically
engineered to contract or possess prostate cancer cells in vivo. In
this embodiment, the invention consists of (1) an aptamer that is
designed to bind with high affinity to PSMA; (2) a radionuclide for
PET or SPECT imaging; (3) a chelator that binds the radionuclide
tightly; and (4) and a linker that connects the chelator and
aptamer together. In certain embodiments, other molecular entities
are added at various positions on the structure to impart improved
pharmacokinetic or biodistribution properties to the invention that
improve the overall imaging or therapeutic characteristics of the
invention. For example, a polyethylene glycol molecular entity can
be added to the 3'-end of an aptamer in which the 5'-end has been
modified to include the chelator and radionuclide, with the purpose
of the PEG to slow the pharmacokinetics of the aptamer as a means
of improving the overall imaging or therapeutic efficacy of the
complete molecular structure.
[0152] In one embodiment of the present invention, the method also
involves contacting the sample with at least one aptamer to form a
hybridized nucleic acid and detecting the hybridized nucleic acid.
In one embodiment, the detection is by amplification. "Amplifying"
utilizes methods such as the polymerase chain reaction (PCR),
ligation amplification (or ligase chain reaction, LCR), strand
displacement amplification, nucleic acid sequence-based
amplification, and amplification methods based on the use of Q-beta
replicase. These methods are well known and widely practiced in the
art. Reagents and hardware for conducting PCR are commercially
available. In one embodiment of the present invention, at least one
type of aptamer is immobilized on a solid surface.
[0153] The methods of the present invention can be used to detect
the presence of PSMA in a sample.
[0154] According to the methods of the present invention, the
amplification of PSMA present in a sample may be carried out by any
means known to the art. Examples of suitable amplification
techniques include, but are not limited to, polymerase chain
reaction (including, for RNA amplification, reverse-transcriptase
polymerase chain reaction), ligase chain reaction, strand
displacement amplification, transcription-based amplification,
self-sustained sequence replication (or "3SR"), the Q.beta.
replicase system, nucleic acid sequence-based amplification (or
"NASBA"), the repair chain reaction (or "RCR"), and boomerang DNA
amplification (or "BDA").
[0155] The bases incorporated into the amplification product may be
natural or modified bases (modified before or after amplification),
and the bases may be selected to optimize subsequent
electrochemical detection steps.
[0156] Polymerase chain reaction (PCR) may be carried out in
accordance with known techniques. See, e.g., U.S. Pat. Nos.
4,683,195; 4,683,202; 4,800,159; and 4,965,188. In general, PCR
involves, first, treating a nucleic acid sample (e.g., in the
presence of a heat stable DNA polymerase) with one oligonucleotide
primer for each strand of the specific sequence to be detected
under hybridizing conditions so that an extension product of each
primer is synthesized that is complementary to each nucleic acid
strand, with the primers sufficiently complementary to each strand
of the specific sequence to hybridize therewith so that the
extension product synthesized from each primer, when it is
separated from its complement, can serve as a template for
synthesis of the extension product of the other primer, and then
treating the sample under denaturing conditions to separate the
primer extension products from their templates if the sequence or
sequences to be detected are present. These steps are cyclically
repeated until the desired degree of amplification is obtained.
Detection of the amplified sequence may be carried out by adding to
the reaction product an oligonucleotide probe capable of
hybridizing to the reaction product (e.g., an oligonucleotide probe
of the present invention), the probe carrying a detectable label,
and then detecting the label in accordance with known techniques.
Where the nucleic acid to be amplified is RNA, amplification may be
carried out by initial conversion to DNA by reverse transcriptase
in accordance with known techniques.
[0157] Strand displacement amplification (SDA) may be carried out
in accordance with known techniques. For example, SDA may be
carried out with a single amplification primer or a pair of
amplification primers, with exponential amplification being
achieved with the latter. In general, SDA amplification primers
comprise, in the 5' to 3' direction, a flanking sequence (the DNA
sequence of which is noncritical), a restriction site for the
restriction enzyme employed in the reaction, and an oligonucleotide
sequence (e.g., an oligonucleotide probe of the present invention)
that hybridizes to the target sequence to be amplified and/or
detected. The flanking sequence, which serves to facilitate binding
of the restriction enzyme to the recognition site and provides a
DNA polymerase priming site after the restriction site has been
nicked, is about 15 to 20 nucleotides in length in one embodiment.
The restriction site is functional in the SDA reaction. The
oligonucleotide probe portion is about 13 to 15 nucleotides in
length in one embodiment of the invention.
[0158] Ligase chain reaction (LCR) is also carried out in
accordance with known techniques. In general, the reaction is
carried out with two pairs of oligonucleotide probes: one pair
binds to one strand of the sequence to be detected; the other pair
binds to the other strand of the sequence to be detected. Each pair
together completely overlaps the strand to which it corresponds.
The reaction is carried out by, first, denaturing (e.g.,
separating) the strands of the sequence to be detected, then
reacting the strands with the two pairs of oligonucleotide probes
in the presence of a heat stable ligase so that each pair of
oligonucleotide probes is ligated together, then separating the
reaction product, and then cyclically repeating the process until
the sequence has been amplified to the desired degree. Detection
may then be carried out in like manner as described above with
respect to PCR.
[0159] Diagnostic techniques that are useful in the methods of the
invention include, but are not limited to direct DNA sequencing,
pulsed-field gel electrophoresis (PFGE) analysis, allele-specific
oligonucleotide (ASO), dot blot analysis and denaturing gradient
gel electrophoresis, and are well known to the artisan.
[0160] The sample may be contacted with the aptamer in any suitable
manner known to those skilled in the art. For example, the sample
may be solubilized in solution, and contacted with the aptamer by
solubilizing the aptamer in solution with the sample under
conditions that permit binding. Suitable conditions are well known
to those skilled in the art. Alternatively, the sample may be
solubilized in solution with the aptamer immobilized on a solid
support, whereby the sample may be contacted with the aptamer by
immersing the solid support having the aptamer immobilized thereon
in the solution containing the sample.
[0161] In certain embodiments, for small animal imaging of the
efficacy of the aptamer for in vivo imaging, a suitable amount of
the RNA aptamer, in the range of 1-100 nmoles is conjugated to a
chelator by methods described here or by methods that are well
known to those who are skilled in the art. The bifunctional
chelator modified PSMA-targeted aptamer can then be radiolabeled
with radionuclides that are useful for imaging and therapy by
methods that are well known to those that are skilled in the art.
In an exemplary embodiment, a reactive derivative of NOTA can be
conjugated to a 5'-hexynyl-RNA aptamer by a two step process as
described below by first adding 1-amino-3-azidopropane to the
hexynyl functionalized aptamer to obtain a primary amine reactive
function on the aptamer. Following purification by HPLC methods,
the amine modified RNA aptamer can be conjugated to a reactive NOTA
derivative
(2,2',2''-(2-(4-isothiocyanatobenzyl)-1,4,7-triazonane-1,4,7-triyl)triace-
tic acid) to form the bifunctional as described in Example 3. The
final purification is performed by size-exclusion and HPLC
techniques that are well known to those that are skilled in the
art. Following purification, the NOTA-PSMA-Aptamer can be
radiolabeled with a radionuclide that is suitable for imaging by
PET or SPECT or other imaging technique. In an exemplary form, the
NOTA-PSMA-Aptamer is radiolabeled with .sup.68Ga for PET, by
dissolving the NOTA-PSMA-Aptamer in an appropriate solution and
reacting the chelator modified aptamer with .sup.68Ga under
conditions that are well-known to those that are skilled in the
art. The final radiolabeled [.sup.68Ga]-NOTA-PSMA-Aptamer is then
purified by size exclusion and precipitation methods that are well
known to those that are skilled in the art and finally dissolved in
a solution suitable for venous injection of the obtained
radiopharmaceutical.
[0162] For small animal imaging, the final purified
radiopharmaceutical is dissolved in a small volume (100-500 mL) of
sterile isotonic saline or other suitable solution for injection. A
small animal model such as a nude mouse is prepared according to
methods that are well known to those that are skilled in the art
and as exemplified in section "tumor implantation and monitoring
tumor growth" presented here. Such an animal is anesthetized and
injected with the solution containing the radiolabeled aptamer by
methods that are well known to measure the accumulation of
radiolabeled aptamer in the tumor over a time period ranging from a
few minutes to as long as 4 days or longer. The time needed for
accumulation depends on the pharmacokinetics of the radiolabeled
tracer. An image of the accumulation is obtained by use of a
specialized camera system known as a tomograph.
Kits Containing Agents of the Invention
[0163] The present invention provides kits comprising the
conjugates described above, or the pharmaceutically acceptable salt
thereof, packaging material, and instructions for administering the
conjugate or the pharmaceutically acceptable salt thereof to an
animal to detect or treat prostate cancer. In certain embodiments,
the kit further comprises an imageable metal or a therapeutic
metal. In certain embodiments, the imageable metal is a
radionuclide. In certain embodiments, the radionuclide is an alpha
or beta-emitting radionuclide. In certain embodiments, the metal is
Gallium-68 (.sup.68Ga).
Dosages, Formulations and Routes of Administration of the Agents of
the Invention
[0164] In certain embodiments, the agents of the invention are
administered so as to result in a reduction in at least one symptom
associated with a disease. The amount administered will vary
depending on various factors including, but not limited to, the
composition chosen, the particular disease, the weight, the
physical condition, and the age of the mammal, and whether
prevention or treatment is to be achieved. Such factors can be
readily determined by the clinician employing animal models or
other test systems, which are well known to the art.
[0165] In certain embodiments, the present invention is a
pharmaceutical composition comprising a conjugate as described
above and a pharmaceutically acceptable carrier. Pharmaceutical
formulations, dosages and routes of administration for nucleic
acids are generally known in the art.
[0166] The present invention envisions treating a disease, for
example, cancer, in a mammal by the administration of an agent,
e.g., a nucleic acid composition, an expression vector, or a viral
particle of the invention. Administration of the therapeutic agents
in accordance with the present invention may be continuous or
intermittent, depending, for example, upon the recipient's
physiological condition, whether the purpose of the administration
is therapeutic or prophylactic, and other factors known to skilled
practitioners. The administration of the agents of the invention
may be essentially continuous over a preselected period of time or
may be in a series of spaced doses. Both local and systemic
administration is contemplated.
[0167] One or more suitable unit dosage forms having the
therapeutic agent(s) of the invention, which, as discussed below,
may optionally be formulated for sustained release (for example
using microencapsulation), can be administered by a variety of
routes including parenteral, including by intravenous and
intramuscular routes, as well as by direct injection into the
diseased tissue. For example, the therapeutic agent may be directly
injected into the cancer. In another example, the therapeutic agent
may be introduced intramuscularly for viruses that traffic back to
affected neurons from muscle, such as AAV, lentivirus and
adenovirus. In another example, the therapeutic agent may be
injected intravenously. The formulations may, where appropriate, be
conveniently presented in discrete unit dosage forms and may be
prepared by any of the methods well known to pharmacy. Such methods
may include the step of bringing into association the therapeutic
agent with liquid carriers, solid matrices, semi-solid carriers,
finely divided solid carriers or combinations thereof, and then, if
necessary, introducing or shaping the product into the desired
delivery system.
[0168] When the therapeutic agents of the invention are prepared
for administration, in certain embodiments, they are combined with
a pharmaceutically acceptable carrier, diluent or excipient to form
a pharmaceutical formulation, or unit dosage form. The total active
ingredients in such formulations include from 0.1 to 99.9% by
weight of the formulation. A "pharmaceutically acceptable" is a
carrier, diluent, excipient, and/or salt that is compatible with
the other ingredients of the formulation, and not deleterious to
the recipient thereof. The active ingredient for administration may
be present as a powder or as granules, as a solution, a suspension
or an emulsion.
[0169] Pharmaceutical formulations containing the therapeutic
agents of the invention can be prepared by procedures known in the
art using well known and readily available ingredients. The
therapeutic agents of the invention can also be formulated as
solutions appropriate for parenteral administration, for instance
by intramuscular, subcutaneous or intravenous routes.
[0170] The pharmaceutical formulations of the therapeutic agents of
the invention can also take the form of an aqueous or anhydrous
solution or dispersion, or alternatively the form of an emulsion or
suspension.
[0171] Thus, the therapeutic agent may be formulated for parenteral
administration (e.g., by injection, for example, bolus injection or
continuous infusion) and may be presented in unit dose form in
ampules, pre-filled syringes, small volume infusion containers or
in multi-dose containers with an added preservative. The active
ingredients may take such forms as suspensions, solutions, or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredients may be in powder form,
obtained by aseptic isolation of sterile solid or by lyophilization
from solution, for constitution with a suitable vehicle, e.g.,
sterile, pyrogen-free water, before use.
[0172] It will be appreciated that the unit content of active
ingredient or ingredients contained in an individual aerosol dose
of each dosage form need not in itself constitute an effective
amount for treating the particular indication or disease since the
necessary effective amount can be reached by administration of a
plurality of dosage units. Moreover, the effective amount may be
achieved using less than the dose in the dosage form, either
individually, or in a series of administrations.
[0173] The pharmaceutical formulations of the present invention may
include, as optional ingredients, pharmaceutically acceptable
carriers, diluents, solubilizing or emulsifying agents, and salts
of the type that are well-known in the art. Specific non-limiting
examples of the carriers and/or diluents that are useful in the
pharmaceutical formulations of the present invention include water
and physiologically acceptable buffered saline solutions such as
phosphate buffered saline solutions pH 7.0-8.0, saline solutions,
and water.
General Terminology
[0174] "Synthetic" aptamers are those prepared by chemical
synthesis. The aptamers may also be produced by recombinant nucleic
acid methods. "Recombinant nucleic molecule" is a combination of
nucleic sequences that are joined together using recombinant
nucleic technology and procedures used to join together nucleic
sequences known in the art.
[0175] As used herein, the term "nucleic acid" and "polynucleotide"
refers to deoxyribonucleotides or ribonucleotides and polymers
thereof in either single- or double-stranded form, composed of
monomers (nucleotides) containing a sugar, phosphate and a base
that is either a purine or pyrimidine. Unless specifically limited,
the term encompasses nucleic acids containing known analogs of
natural nucleotides which have similar binding properties as the
reference nucleic acid and are metabolized in a manner similar to
naturally occurring nucleotides. Unless otherwise indicated, a
particular nucleic acid sequence also implicitly encompasses
conservatively modified variants thereof (e.g., degenerate codon
substitutions) and complementary sequences as well as the sequence
explicitly indicated. Specifically, degenerate codon substitutions
may be achieved by generating sequences in which the third position
of one or more selected (or all) codons is substituted with
mixed-base and/or deoxyinosine residues.
[0176] Deoxyribonucleic acid (DNA) in the majority of organisms is
the genetic material while ribonucleic acid (RNA) is involved in
the transfer of information contained within DNA into proteins. The
term "nucleotide sequence" refers to a polymer of DNA or RNA which
can be single- or double-stranded, optionally containing synthetic,
non-natural or altered nucleotide bases capable of incorporation
into DNA or RNA polymers.
[0177] The terms "nucleic acid," "nucleic acid molecule," "nucleic
acid fragment," "nucleic acid sequence or segment," or
"polynucleotide" may also be used interchangeably with gene, cDNA,
DNA and RNA encoded by a gene, e.g., genomic DNA, and even
synthetic DNA sequences. The term also includes sequences that
include any of the known base analogs of DNA and RNA.
[0178] By "fragment" or "portion" is meant a full length or less
than full length of the nucleotide sequence.
[0179] A "variant" of a molecule is a sequence that is
substantially similar to the sequence of the native molecule. For
nucleotide sequences, variants include those sequences that,
because of the degeneracy of the genetic code, encode the identical
amino acid sequence of the native protein. Naturally occurring
allelic variants such as these can be identified with the use of
well-known molecular biology techniques, as, for example, with
polymerase chain reaction (PCR) and hybridization techniques.
Variant nucleotide sequences also include synthetically derived
nucleotide sequences, such as those generated, for example, by
using site-directed mutagenesis that encode the native protein, as
well as those that encode a polypeptide having amino acid
substitutions. Generally, nucleotide sequence variants of the
invention will have in at least one embodiment 40%, 50%, 60%, to
70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,
generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%,
sequence identity to the native (endogenous) nucleotide
sequence.
[0180] The term "gene" is used broadly to refer to any segment of
nucleic acid associated with a biological function. Genes include
coding sequences and/or the regulatory sequences required for their
expression. For example, gene refers to a nucleic acid fragment
that expresses mRNA, functional RNA, or a specific protein,
including its regulatory sequences. Genes also include nonexpressed
DNA segments that, for example, form recognition sequences for
other proteins. Genes can be obtained from a variety of sources,
including cloning from a source of interest or synthesizing from
known or predicted sequence information, and may include sequences
designed to have desired parameters. In addition, a "gene" or a
"recombinant gene" refers to a nucleic acid molecule comprising an
open reading frame and including at least one exon and (optionally)
an intron sequence. The term "intron" refers to a DNA sequence
present in a given gene which is not translated into protein and is
generally found between exons.
[0181] "Naturally occurring," "native" or "wild type" is used to
describe an object that can be found in nature as distinct from
being artificially produced. For example, a nucleotide sequence
present in an organism (including a virus), which can be isolated
from a source in nature and which has not been intentionally
modified in the laboratory, is naturally occurring. Furthermore,
"wild-type" refers to the normal gene, or organism found in nature
without any known mutation.
[0182] "Homology" refers to the percent identity between two
polynucleotides or two polypeptide sequences. Two DNA or
polypeptide sequences are "homologous" to each other when the
sequences exhibit at least about 75% to 85% (including 75%, 76%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, and 85%), at least about
90%, or at least about 95% to 99% (including 95%, 96%, 97%, 98%,
99%) contiguous sequence identity over a defined length of the
sequences.
[0183] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence," (b) "comparison window," (c) "sequence
identity," (d) "percentage of sequence identity," and (e)
"substantial identity."
[0184] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full length cDNA or gene sequence,
or the complete cDNA or gene sequence.
[0185] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0186] Methods of alignment of sequences for comparison are well
known in the art. Thus, the determination of percent identity
between any two sequences can be accomplished using a mathematical
algorithm.
[0187] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics
Software Package, Version 8 (available from Genetics Computer Group
(GCG), 575 Science Drive, Madison, Wis., USA). Alignments using
these programs can be performed, using the default parameters.
[0188] Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information (see the
World Wide Web at ncbi.nlm.nih.gov). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold. These initial neighborhood
word hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always>0) and N (penalty score for
mismatching residues; always<0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when the cumulative
alignment score falls off by the quantity X from its maximum
achieved value, the cumulative score goes to zero or below due to
the accumulation of one or more negative-scoring residue
alignments, or the end of either sequence is reached.
[0189] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences. One measure of similarity
provided by the BLAST algorithm is the smallest sum probability
(P(N)), which provides an indication of the probability by which a
match between two nucleotide or amino acid sequences would occur by
chance. For example, a test nucleic acid sequence is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid sequence to the reference
nucleic acid sequence is less than about 0.1, less than about 0.01,
or even less than about 0.001.
[0190] To obtain gapped alignments for comparison purposes, Gapped
BLAST (in BLAST 2.0) can be utilized. Alternatively, PSI-BLAST (in
BLAST 2.0) can be used to perform an iterated search that detects
distant relationships between molecules. When using BLAST, Gapped
BLAST, PSI-BLAST, the default parameters of the respective programs
(e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be
used. The BLASTN program (for nucleotide sequences) uses as
defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff
of 100, M=5, N=-4, and a comparison of both strands. For amino acid
sequences, the BLASTP program uses as defaults a wordlength (W) of
3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See
the World Wide Web at ncbi.nlm.nih.gov. Alignment may also be
performed manually by visual inspection.
[0191] For purposes of the present invention, comparison of
nucleotide sequences for determination of percent sequence identity
to the sequences disclosed herein is made using the BlastN program
(version 1.4.7 or later) with its default parameters or any
equivalent program. By "equivalent program" is intended any
sequence comparison program that, for any two sequences in
question, generates an alignment having identical nucleotide or
amino acid residue matches and an identical percent sequence
identity when compared to the corresponding alignment generated by
a BLAST program.
[0192] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid sequences makes reference to a
specified percentage of residues in the two sequences that are the
same when aligned for maximum correspondence over a specified
comparison window, as measured by sequence comparison algorithms or
by visual inspection. When percentage of sequence identity is used
in reference to proteins, it is recognized that residue positions
that are not identical often differ by conservative amino acid
substitutions, where amino acid residues are substituted for other
amino acid residues with similar chemical properties (e.g., charge
or hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity." Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0193] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base occurs
in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison, and multiplying the result
by 100 to yield the percentage of sequence identity.
[0194] (e)(i) The term "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%; at
least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%; at least
90%, 91%, 92%, 93%, or 94%; or even at least 95%, 96%, 97%, 98%, or
99% sequence identity, compared to a reference sequence using one
of the alignment programs described using standard parameters.
[0195] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions (see below). Generally, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength and pH. However, stringent conditions
encompass temperatures in the range of about 1.degree. C. to about
20.degree. C., depending upon the desired degree of stringency as
otherwise qualified herein. Nucleic acids that do not hybridize to
each other under stringent conditions are still substantially
identical if the polypeptides they encode are substantially
identical. This may occur, e.g., when a copy of a nucleic acid is
created using the maximum codon degeneracy permitted by the genetic
code.
[0196] (e)(ii) For sequence comparison, typically one sequence acts
as a reference sequence to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0197] As noted above, another indication that two nucleic acid
sequences are substantially identical is that the two molecules
hybridize to each other under stringent conditions. The phrase
"hybridizing specifically to" refers to the binding, duplexing, or
hybridizing of a molecule only to a particular nucleotide sequence
under stringent conditions when that sequence is present in a
complex mixture (e.g., total cellular) DNA or RNA. "Bind(s)
substantially" refers to complementary hybridization between a
probe nucleic acid and a target nucleic acid and embraces minor
mismatches that can be accommodated by reducing the stringency of
the hybridization media to achieve the desired detection of the
target nucleic acid sequence.
[0198] "Stringent hybridization conditions" and "stringent
hybridization wash conditions" in the context of nucleic acid
hybridization experiments such as Southern and Northern
hybridizations are sequence dependent, and are different under
different environmental parameters. Longer sequences hybridize
specifically at higher temperatures. The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched nucleic acid.
Specificity is typically the function of post-hybridization washes,
the critical factors being the ionic strength and temperature of
the final wash solution. For DNA-DNA hybrids, the T.sub.m can be
approximated from the equation of Meinkoth and Wahl: T.sub.m
81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L. M is
the molarity of monovalent cations, % GC is the percentage of
guanosine and cytosine nucleotides in the DNA, % form is the
percentage of formamide in the hybridization solution, and L is the
length of the hybrid in base pairs. T.sub.n, is reduced by about
1.degree. C. for each 1% of mismatching; thus, T.sub.m,
hybridization, and/or wash conditions can be adjusted to hybridize
to sequences of the desired identity. For example, if sequences
with >90% identity are sought, the T.sub.m can be decreased
10.degree. C. Generally, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point (T.sub.m)
for the specific sequence and its complement at a defined ionic
strength and pH. However, severely stringent conditions can utilize
a hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than
the thermal melting point (T.sub.m); moderately stringent
conditions can utilize a hybridization and/or wash at 6, 7, 8, 9,
or 10.degree. C. lower than the thermal melting point (T.sub.m);
low stringency conditions can utilize a hybridization and/or wash
at 11, 12, 13, 14, 15, or 20.degree. C. lower than the thermal
melting point (T.sub.m). Using the equation, hybridization and wash
compositions, and desired T, those of ordinary skill will
understand that variations in the stringency of hybridization
and/or wash solutions are inherently described. If the desired
degree of mismatching results in a T of less than 45.degree. C.
(aqueous solution) or 32.degree. C. (formamide solution), the SSC
concentration is increased so that a higher temperature can be
used. Generally, highly stringent hybridization and wash conditions
are selected to be about 5.degree. C. lower than the thermal
melting point (T.sub.m) for the specific sequence at a defined
ionic strength and pH.
[0199] An example of highly stringent wash conditions is 0.15 M
NaCl at 72.degree. C. for about 15 minutes. An example of stringent
wash conditions is a 0.2.times.SSC wash at 65.degree. C. for 15
minutes. Often, a high stringency wash is preceded by a low
stringency wash to remove background probe signal. An example
medium stringency wash for a duplex of, e.g., more than 100
nucleotides, is 1.times.SSC at 45.degree. C. for 15 minutes. An
example low stringency wash for a duplex of, e.g., more than 100
nucleotides, is 4-6.times.SSC at 40.degree. C. for 15 minutes. For
short probes (e.g., about 10 to 50 nucleotides), stringent
conditions typically involve salt concentrations of less than about
1.5 M, such as about 0.01 to 1.0 M, Na ion concentration (or other
salts) at pH 7.0 to 8.3, and the temperature is typically at least
about 30.degree. C. and at least about 60.degree. C. for long
probes (e.g., >50 nucleotides). Stringent conditions may also be
achieved with the addition of destabilizing agents such as
formamide. In general, a signal to noise ratio of 2.times. (or
higher) than that observed for an unrelated probe in the particular
hybridization assay indicates detection of a specific
hybridization.
[0200] Very stringent conditions are selected to be equal to the
T.sub.n, for a particular probe. An example of stringent conditions
for hybridization of complementary nucleic acids which have more
than 100 complementary residues on a filter in a Southern or
Northern blot is 50% formamide, e.g., hybridization in 50%
formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.1.times.SSC at 60 to 65.degree. C. Exemplary low stringency
conditions include hybridization with a buffer solution of 30 to
35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulphate) at
37.degree. C., and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to
60.degree. C.
[0201] "Operably-linked" molecules refers to the association of
moieties to form a single molecule such that the function of one is
not affected by the other, e.g., an arrangement of elements wherein
the components so described are configured so as to perform their
usual function. For example, an aptamer is said to be "operably
linked to" or "associated with" a metal chelating group if the
aptamer and metal chelating group are situated such that the
aptamer still recognizes its target and the metal chelating group
still operates as an imageable group. "Operably-linked" in
reference to an azido group or an alkynyl group means that the
group is affixed to a molecule or surface in such a way as to
permit the azido or alkynyl group to undergo a 1,3-dipolar
cycloaddition with an alkynyl or azido group, respectively, on a
different molecule or surface, as applicable.
[0202] The terms "isolated and/or purified" refer to in vitro
isolation of a nucleic acid, e.g., a DNA or RNA molecule from its
natural cellular environment, and from association with other
components of the cell, such as nucleic acid or polypeptide, so
that it can be sequenced, replicated, and/or expressed. Thus, the
RNA or DNA is "isolated" in that it is free from at least one
contaminating nucleic acid with which it is normally associated in
the natural source of the RNA or DNA and in certain embodiments, is
substantially free of any other mammalian RNA or DNA. The phrase
"free from at least one contaminating source nucleic acid with
which it is normally associated" includes the case where the
nucleic acid is reintroduced into the source or natural cell but is
in a different chromosomal location or is otherwise flanked by
nucleic acid sequences not normally found in the source cell, e.g.,
in a vector or plasmid.
[0203] The nucleic acid molecules of the invention include
double-stranded interfering RNA molecules, which are also useful to
inhibit expression of a target gene.
[0204] As used herein, the term "recombinant nucleic acid," e.g.,
"recombinant DNA sequence or segment" refers to a nucleic acid,
e.g., to RNA, that has been derived or isolated from any
appropriate cellular source, that may be subsequently chemically
altered in vitro, so that its sequence is not naturally occurring,
or corresponds to naturally occurring sequences that are not
positioned as they would be positioned in a genome that has not
been transformed with exogenous DNA. An example of preselected RNA
"derived" from a source would be a RNA sequence that is identified
as a useful fragment within a given organism, and which is then
chemically synthesized in essentially pure form. An example of such
RNA "isolated" from a source would be a useful RNA sequence that is
excised or removed from said source by chemical means, e.g., by the
use of restriction endonucleases, so that it can be further
manipulated, e.g., amplified, for use in the invention, by the
methodology of genetic engineering.
[0205] Nucleic acid molecules having base substitutions (i.e.,
variants) are prepared by a variety of methods known in the art.
These methods include, but are not limited to, isolation from a
natural source (in the case of naturally occurring sequence
variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the nucleic acid molecule.
[0206] A "control" cell, tissue, sample, or subject is a cell,
tissue, sample, or subject of the same type as a test cell, tissue,
sample, or subject. The control may, for example, be examined at
precisely or nearly the same time the test cell, tissue, sample, or
subject is examined. The control may also, for example, be examined
at a time distant from the time at which the test cell, tissue,
sample, or subject is examined, and the results of the examination
of the control may be recorded so that the recorded results may be
compared with results obtained by examination of a test cell,
tissue, sample, or subject. The control may also be obtained from
another source or similar source other than the test group or a
test subject, where the test sample is obtained from a subject
suspected of having a disease or disorder for which the test is
being performed.
[0207] A "test" cell, tissue, sample, or subject is one being
examined.
[0208] The term "affected cell" refers to a cell of a subject
afflicted with a disease or disorder, which affected cell has an
altered phenotype relative to a subject not afflicted with a
disease or disorder. Cells or tissue are "affected" by a disease or
disorder if the cells or tissue have an altered phenotype relative
to the same cells or tissue in a subject not afflicted with a
disease or disorder. A disease or disorder is "alleviated" if the
severity of a symptom of the disease or disorder, the frequency
with which such a symptom is experienced by a patient, or both, is
reduced.
[0209] A "pathoindicative" cell, tissue, or sample is one which,
when present, is an indication that the animal in which the cell,
tissue, or sample is located (or from which the tissue was
obtained) is afflicted with a disease or disorder. By way of
example, the presence of one or more breast cells in a lung tissue
of an animal is an indication that the animal is afflicted with
metastatic breast cancer.
[0210] The terms "cell," "cell line," and "cell culture" may be
used interchangeably.
[0211] As used herein, a "derivative" of a compound refers to a
chemical compound that may be produced from another compound of
similar structure in one or more steps, as in replacement of H by
an alkyl, acyl, amino, or other chemically synthesized group.
[0212] The use of the word "detect" and its grammatical variants is
meant to refer to measurement of the species without
quantification, whereas use of the word "determine" or "measure"
with their grammatical variants are meant to refer to measurement
of the species with quantification. The terms "detect" and
"identify" are used interchangeably herein.
[0213] As used herein, a "detectable marker" or is an atom or a
molecule that permits the specific detection of a compound
comprising the marker in the presence of similar compounds without
a marker. Detectable markers include, but are not limited to,
radioactive isotopes, antigenic determinants, enzymes, nucleic
acids available for hybridization, chromophores, fluorophores,
chemiluminescent molecules, electrochemically detectable molecules,
and molecules that provide for altered fluorescence-polarization or
altered light-scattering.
[0214] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to
deteriorate.
[0215] A "disorder" in an animal is a state of health in which the
animal is able to maintain homeostasis, but in which the animal's
state of health is less favorable than it would be in the absence
of the disorder. Left untreated, a disorder does not necessarily
cause a further decrease in the animal's state of health.
[0216] A "therapeutic" treatment is a treatment administered to a
subject who exhibits signs of pathology for the purpose of
diminishing or eliminating those signs.
[0217] An "effective amount" or "therapeutically effective amount"
of a compound is that amount of compound which is sufficient to
provide a beneficial effect to the subject to which the compound is
administered. For example, an effective amount of an SIP receptor
antagonist is an amount that decreases the cell signaling activity
of the SIP receptor.
[0218] As used herein, an "instructional material" or
"instructions" includes a publication, a recording, a diagram, or
any other medium of expression which can be used to communicate the
usefulness of the composition of the invention for its designated
use. The instructional material of the kit of the invention may,
for example, be affixed to a container which contains the
composition or be shipped together with a container which contains
the composition. Alternatively, the instructional material may be
shipped separately from the container with the intention that the
instructional material and the composition be used cooperatively by
the recipient.
[0219] As used herein, the term "purified" and like terms relate to
an enrichment of a molecule or compound relative to other
components normally associated with the molecule or compound in a
native environment. The term "purified" does not necessarily
indicate that complete purity of the particular molecule has been
achieved during the process. A "highly purified" compound as used
herein refers to a compound that is greater than 90% pure.
[0220] As used herein, the term "pharmaceutically acceptable
carrier" includes any of the standard pharmaceutical carriers, such
as a phosphate buffered saline solution, water, emulsions such as
an oil/water or water/oil emulsion, and various types of wetting
agents. The term also encompasses any of the agents approved by a
regulatory agency of the US Federal government or listed in the US
Pharmacopeia for use in animals, including humans.
[0221] A "sample," as used herein, refers to a biological sample
from a subject, including, but not limited to, normal tissue
samples, diseased tissue samples, biopsies, blood, saliva, feces,
semen, tears, and urine. A sample can also be any other source of
material obtained from a subject who contains cells, tissues, or
fluid of interest. A sample can also be obtained from cell or
tissue culture.
[0222] The term "standard," as used herein, refers to something
used for comparison. For example, it can be a known standard agent
or compound which is administered or added to a control sample and
used for comparing results when measuring said compound in a test
sample. Standard can also refer to an "internal standard," such as
an agent or compound which is added at known amounts to a sample
and is useful in determining such things as purification or
recovery rates when a sample is processed or subjected to
purification or extraction procedures before a marker of interest
is measured.
[0223] A "subject" of analysis, diagnosis, or treatment is an
animal. Such animals include mammals, such as a human.
[0224] The term "treating" includes prophylaxis of the specific
disorder or condition, or alleviation of the symptoms associated
with a specific disorder or condition or preventing or eliminating
said symptoms. A "prophylactic" treatment is a treatment
administered to a subject who does not exhibit signs of a disease
or exhibits only early signs of the disease for the purpose of
decreasing the risk of developing pathology associated with the
disease.
[0225] A "functional" molecule is a molecule in a form in which it
exhibits a property by which it is characterized. By way of
example, a functional enzyme is one which exhibits the
characteristic catalytic activity by which the enzyme is
characterized.
[0226] The term "inhibit" refers to the ability of a disclosed
compound to reduce or impede a described function. In certain
embodiments, inhibition is by at least 10%, by at least 25%, by at
least 50%, or even by at least 75%.
[0227] The term "purified" and like terms relate to an enrichment
of a molecule or compound relative to other components normally
associated with the molecule or compound in a native environment.
The term "purified" does not necessarily indicate that complete
purity of the particular molecule has been achieved during the
process. A "highly purified" compound as used herein refers to a
compound that is greater than 90% pure.
[0228] The term "parenteral" means not through the alimentary canal
but by some other route such as subcutaneous, intramuscular,
intraspinal, or intravenous.
[0229] "Halo" or "halogen" refers to fluoro, chloro, bromo, or
iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both straight
and branched radical or linking groups; but reference to an
individual radical such as "propyl" embraces only the straight
chain radical or linking group, a branched chain isomer such as
"isopropyl" being specifically referred to. Aryl denotes a phenyl
radical or linking group, or an ortho-fused bicyclic carbocyclic
radical or linking group having about nine to ten ring atoms in
which at least one ring is aromatic. Heteroaryl encompasses a
radical or linking group, attached via a ring carbon of a
monocyclic aromatic ring containing five or six ring atoms
consisting of carbon and one to four heteroatoms each selected from
non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H,
O, (C.sub.1-C.sub.4)alkyl, phenyl or benzyl, as well as a radical
of an ortho-fused bicyclic heterocycle of about eight to ten ring
atoms derived therefrom, particularly a benz-derivative or one
derived by fusing a propylene, trimethylene, or tetramethylene
diradical thereto.
[0230] The term "Aryl" denotes a phenyl radical or linking group,
or an ortho-fused bicyclic carbocyclic radical or linking group,
having about nine to ten ring atoms in which at least one ring is
aromatic. "Aralkyl or arylalkyl" denotes an alkyl group substituted
with an aryl group such as benzyl.
[0231] "Linker" or "link" refers to a chemical moiety comprising a
covalent bond or a chain or group of atoms that covalently attaches
the azide group to the chelation portion of the molecule. Linkers
include moieties such as: repeating units of alkylene
(--(CH.sub.2).sub.n--), aryl (--(C.sub.6H.sub.4)--), alkyloxy
(e.g., polyethylenoxy, PEG, polymethyleneoxy) and alkylamino; and
diacid esters including succinate, succinamide, diglycolate,
malonate, and caproamide.
[0232] The term "Het" is a 4-16 membered saturated or unsaturated
monocyclic, bicyclic, or tricyclic ring system having 1, 2, 3, or 4
heteroatoms, such as oxygen (--O--), sulfur (--S--), oxygenated
sulfur such as sulfinyl (S.dbd.O) and sulfonyl (S(.dbd.O).sub.2),
or nitrogen, or an N-oxide thereof. Het includes "heteroaryl",
which encompasses a radical attached via a ring carbon of a
monocyclic aromatic ring containing five or six ring atoms
consisting of carbon and 1, 2, 3, or 4 heteroatoms, such as
non-peroxide oxygen (--O--), sulfur (--S--), oxygenated sulfur such
as sulfinyl (S.dbd.O) and sulfonyl (S(.dbd.O).sub.2), or nitrogen
N(X) wherein X is absent or is H, O, (C.sub.1-4) alkyl, phenyl or
benzyl, as well as a radical or linking group, of an ortho-fused
bicyclic heterocycle of about eight to ten ring atoms derived there
from, particularly a benz-derivative or one derived by fusing a
propylene, trimethylene, or tetramethylene diradical thereto. When
heteroaryl is an ortho-fused benz-derivative it can be attached via
any atom in an aromatic ring (e.g. an atom of the benz-ring).
[0233] The term "partially unsaturated", for example, a
C.sub.1-7alkylene which is optionally partially unsaturated, means
the named substituent or linking group has one or more
unsaturations, such as one or more double bonds, one or more triple
bonds, or both.
[0234] The term "optional" or "optionally" mean that the
subsequently described term, event or condition may be but need not
be present or occur, and that the description includes instances
where the term, event or condition is present or occurs and
instances in which it does not. For example, "optionally
substituted" means that the named substituent may be present, but
need not be present, and the description includes situations where
the named substituent is included and situations where the named
substituent is not included.
[0235] The invention is now described with reference to the
following examples. These examples are provided for the purpose of
illustration only and the invention should in no way be construed
as being limited to these examples, but rather should be construed
to encompass any and all variations which become evident as a
result of the teachings provided herein.
Example 1
PSMA Aptamers Target PSMA-Expressing Prostate Cancers
[0236] The ability of the PSMA aptamers to bind the surface of
prostate cancer cells expressing PSMA (LNCaP and 22Rv1 clone 1.7)
was tested. A PSMA-negative prostate cancer cell line (PC-3) was
used as a control for specificity. The surface expression of PSMA
was verified using flow cytometry. To determine whether the PSMA
aptamer can bind the surface of cells expressing PSMA,
.sup.32P-labeled PSMA aptamers were incubated with either LNCaP or
PC-3 cells. Binding of the PSMA aptamers was specific for cells
expressing PSMA.
[0237] Unless otherwise noted, all chemicals were purchased from
Sigma-Aldrich Co., all restriction enzymes were obtained from New
England BioLabs, Inc. (NEB), and all cell culture products were
purchased from GIBCO BRL/Life Technologies, a division of
Invitrogen.TM. Corp. Antibodies were purchased from the following
manufacturers: Plk1 (cat #33-1700; Zymed.RTM./Invitrogen.TM.,
Carlsbad, Calif.); Erkl K-23 (sc-94; Santa Cruz, Calif.); PSMA
(cat# M20454M; Biodesign.RTM., Saco, Me.); .beta.-actin_(cat#
A5441; Sigma-Aldrich Inc.); HRP-labeled rabbit anti-mouse IgG
secondary antibody (cat# 61-6420 Zymed.RTM./Invitrogen.TM.,
Carlsbad, Calif.).
[0238] Cell culture: Normal human foreskin fibroblasts cells were
maintained at 37.degree. C. and 5% CO.sub.2 in DMEM supplemented
with 10% FBS. Prostate carcinoma cell lines LNCaP (ATCC no.
CRL-1740) were maintained in Ham's F12-K medium supplemented with
10% FBS. PC-3 and 22Rv1(1.7) luciferase expressing cells (obtained
from Dr. Michael Henry, U Iowa) were grown in RPMI 1640 medium
(GIBCO.RTM.) supplemented with 10% FBS (Hyclone), 1 mM
non-essential amino acids (GIBCO.RTM.), and 100 .mu.g/mL G-418.
[0239] .sup.32P Binding Assays: PC-3 PSMA-negative or LNCaP and
22Rv1(1.7) PSMA-positive prostate cancer cell lines were used for
these experiments. 50,000 PC-3 or LNCaP cells (500 cells/A) in DPBS
(including calcium and magnesium) were blocked with 100 .mu.g/mL
tRNA and poly (I:C) for 15 min. Blocked cells were then incubated
at 37.degree. C. for 30 min with 500,000 cpms of .gamma.-.sup.32P
end-labeled PSMA aptamers or in block solution. Cells were then
washed profusely with DPBS (including calcium and magnesium) and
bound/internalized RNAs measured by scintillation counter. %
Aptamer Bound was calculated based on input counts. This experiment
was performed in triplicate. For determining the relative affinity
of the PSMA aptamers, LNCaP cells were fixed in 1% formaldehyde in
PBS for 20 min at RT. Fixed cells were washed several times after
which cells were diluted and blocked as mentioned above. Cells were
then incubated with serial dilutions of .gamma.-.sup.32P
end-labeled RNAs ranging from 2 nM to 0 nM at 37.degree. C. for 10
min. Bound RNAs were determined by filter binding assay as
described in McNamara et al., 2008.
[0240] PSMA Cell-Surface Expression: PSMA cell-surface expression
was determined by Flow cytometry and/or immunoblotting using
antibodies specific to human PSMA. Flow cytometry: HeLa, PC-3, and
LNCaP cells were trypsinized, washed three times in phosphate
buffered saline (PBS), and counted using a hemocytometer. 200,000
cells (1.times.10.sup.6 cells/mL) were resuspended in 500 .mu.l of
PBS+4% fetal bovine serum (FBS) and incubated at room temperature
(RT) for 20 min. Cells were then pelleted and resuspended in 100
.mu.L of PBS+4% FBS containing 20 .mu.g/mL of primary antibody
against PSMA (anti-PSMA 3C6: Northwest Biotherapeutics) or 20
.mu.g/mL of isotype-specific control antibody. After a 40 min
incubation at RT cells were washed three times with 500 .mu.L of
PBS+4% FBS and incubated with a 1:500 dilution of secondary
antibody (anti-mouse IgG-APC) in PBS+4% FBS for 30 min at RT. Cells
were washed as detailed above, fixed with 400 .mu.L of PBS+1%
formaldehyde, and analyzed by Flow cytometry. Immunoblots: HeLa,
PC-3, and LNCaP cells were collected as described above. Cell
pellets were resuspended in 1.times.RIPA buffer (150 mM NaCl, 50 mM
Tris-HCl pH 8.0, 1 mM EDTA, 1% NP-40) containing 1.times. protease
and phosphatase inhibitor cocktails (Sigma) and incubated on ice
for 20 min. Cells were then pelleted and 25 .mu.g of total protein
from the supernatants were resolved on a 7.5% SDS-PAGE gel. PSMA
was detected using an antibody specific to human PSMA (anti-PSMA
3C6; Northwest Biotherapeutics).
[0241] Cell-Surface Binding of Aptamers: PC-3 or LNCaP cells were
trypsinized, washed twice with 500 .mu.L PBS, and fixed in 400
.mu.L of FIX solution (PBS+1% formaldehyde) for 20 min at RT. After
washing cells to remove any residual trace of formaldehyde, cell
pellets were resuspended in 1.times. Binding Buffer (IXBB) (20 mM
HEPES pH 7.4, 150 mM NaCl, 2 mM CaCJ.sub.2, 0.01% BSA) and
incubated at 37.degree. C. for 20 min. Cells were then pelleted and
resuspended in 50 .mu.L of 1xBB (pre-warmed at 37.degree. C.)
containing either 400 nM FAM-G labeled PSMA aptamer. Concentrations
of FAM-G labeled PSMA aptamer for the relative affinity
measurements varied from 0 to 4 .mu.M. Cells were incubated with
the RNA for 40 min at 37.degree. C., washed three times with 500
.mu.L of 1xBB pre-warmed at 37.degree. C., and finally resuspended
in 400 .mu.L of FIX solution pre-warmed at 37.degree. C. Cells were
then assayed using Flow cytometry as detailed above and the
relative cell surface binding affinities of the PSMA aptamer was
determined.
Example 2
Azides for Molecular Targeting
[0242] Chemical reagents for reactions presented in this example
were ACS grade or better. DOTAzide precursor
1,4,7,10-Tetraazacyclododecane-1,4,7-tris(t-butyl
acetate)-10-succinimidyl acetate (DOTA-NHS) of
1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) was
purchased from Macrocyclics, Inc. (Dallas, Tex., USA, catalog
numbers: M140 and B270). 1-amino-3-bromopropane of sufficient
purity was purchased from Alfa Aesar (Ward Hill, Mass. USA, stock
number: B23254 or L12962). Custom oligonucleotides were purchased
from Integrated DNA Technologies, Inc. (IDT, Coralville, Iowa,
USA). Alkyne-modified 5'-/5-hexynyl-phosphoramidite
(6-hexyn-1-yl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) was
obtained from Glen Research (Sterling, Va. USA; catalog number
10-1908-xx). Copper catalyst was prepared using 99.99+%
metals-basis anhydrous copper sulfate (CuSO.sub.4) obtained from
Sigma-Aldrich, Inc. (Milwaukee, Wis. USA, catalog number:
451657-50G). Water soluble Cu(I) stabilizing ligand
tris-hydroxypropyltriazolyl amine (TriL) was employed as described
by Graham et al. (Graham, D., et al., J. Am. Chem. Soc., Perk Trans
1, 1998, Volume 6, 1132-1138.). Other chemicals and reagents were
obtained from either Fisher Scientific (Pittsburg, Pa. USA) or
Sigma-Aldrich Chemicals, Inc. (Milwaukee, Wis., USA) and used
without further modification. All aqueous experiments and reagents
were conducted and prepared using double-distilled deionized 18 Mf
water obtained using a model Synthesis Milli-Q Quantam.RTM. EX
ultra-water purification system, equipped with Ultrapurex Organex
(cat. QTUOOEX) and Q-gard.RTM. 2 (cat. QGARDOOD2) purification
columns (Millipore, Billerica, Mass. USA).
[0243] Chemical reactions were conducted under high-purity argon or
ultra-pure nitrogen gas unless otherwise stated. Synthesis of
DOTAzide and 1-amino-3-azidopropane precursor was conducted in
glass under argon. Glass reaction vessels were cleaned by washing
with dilute hydrochloric acid (HCl), followed by water, 10 M sodium
hydroxide (NaOH), a second water rinse, and final cleansing with
100% ethyl alcohol (EtOH) and finally acetone. After cleaning,
glass reaction vessels were oven dried for at least 30 minutes at
80.degree. C. Click chemical reactions were conducted in plastic
0.5 and 1.5 mL snap-cap v-vials, used without further treatments
(USA Scientific, Ocala, Fla. USA) under ultra-high-purity nitrogen.
Gravimetric measurements were performed using a model AT261
Deltarange digital balance with a readability of 10 .mu.g
(Mettler-Toledo, Toledo, Ohio USA). Mass analysis of low-molecular
weight precursors and high-molecular weight oligonucleotides and
final bioconjugates were conducted by electrospray ionization (ESI)
and MALDI-TOF mass spectrometry. Presence of azide functional
groups was confirmed infrared spectrometry. Chemical structure ID
NMR data were collected using a Unity Inova 600 MHz NMR
spectrometer (Varian, Palo Alto, Calif. USA). NMR analysis was
carried out using 7 in, 5 mm ID thin-wall glass NMR tubes (Wilmad
Lab Glass, Buena, N.J. USA) in 99.99+% chloroform-d
(Sigma-Aldrich). Spectra were collected using a 6 kHz sweep width,
centered at 4.5 ppm, with a 2 s acquisition time and 1 s delay. The
number of scans was scaled to reasonably achieve a signal to noise
ratio of approximately 64:1. Spectra were evaluated qualitatively
using the software program NUTS (Acorn NMR, Inc., Livermore, Calif.
USA). Retention-time identification and purification of
bioconjugates was performed using high performance liquid
chromatography using model Agilent 1100 series (Agilent, Santa
Clara, Calif. USA) or UltiMate 3000 HPLCs (Dionex, Sunnyvale,
Calif. USA). Final purification of desired bioconjugates was
carried out by HPLC using a 50.times.4.6 mm, column Clarity 5.mu.
Oligo RP (cat. 00B-4442-E0, Phenomenex, Torrance, Calif. USA).
Desalting of bioconjugate solutions was carried out using
Illustra.TM. G-25 "NAPS" single-use size exclusion columns
(Sephadex G-25, in gravity flow mode, using purified water as the
mobile phase buffer (cat. 17-0853-02, maximum capacity 1 mg
mL.sup.-1 DNA, GE Healthcare Bio-Sciences Corp, Piscataway, N.J.
USA).
[0244] 1-amino-3-azidopropane: Precursor 1-amino-3-azidopropane was
prepared by reaction of 10% equivalent excess sodium azide
(NaN.sub.3, 5.39 g dissolved in 10 mL water) with
1-amino-3-bromopropane (8.39 g dissolved in 10 mL water). Reactants
were combined by slowly adding NaN.sub.3 solution for a total
volume of 20 mL and mixed continuously under Ar at 80.degree. C.
for 48 hours (under argon) to apparent completion as revealed by
iodine TLC staining, employing a mobile phase of 10% methyl alcohol
(MeOH) in chloroform (CHCl.sub.3). Following the reaction period,
the solution was cooled to <10.degree. C. in an ice bath,
transferred to a separatory funnel and purification of the desired
1-amino-3-azidopropane product was accomplished by solvent
extraction in 30 mL diethyl ether (prepared with dissolution of 0.7
g, 5 pellets NaOH immediately prior to extraction). The extraction
was conducted a total of three times (cooling the solution to
10.degree. C. each time), followed by drying over magnesium
sulfate, glass wool filtration and careful in vacuo removal of
solvents at 40.degree. C. Remaining diethyl ether solvent was
removed by final evaporation using a gentle stream of high purity
argon to yield 1.5 g of pure product. .sup.1H NMR observed .delta.
1.19 (s, 2.32H); 1.73 (quintet, 2.0H); 2.81 (triplet, 2.0H); 3.37
(triplet, 2.0H). IR (neat, cm.sup.-1) observed 2102; theoretical
2100 (N.sub.3). LC-MS observed mass M+H 101.2; theoretical 100.12.
Although perhaps not necessary for subsequent steps, the final
purification step to remove remaining diethyl ether makes
interpretation of .sup.1H NMR straightforward (FIG. 1A-1C).
[0245] DOTAzide:
2,2',2''-(10-(2-(3-azidopropylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclodo-
decane-1,4,7,-triyl)triacetate was prepared as shown in (FIG. 2):
To a clean 100-mL triple-port glass reaction vessel was added 446
mg (0.67 mmol) DOTA-NHS. The DOTA-NHS reactant was dissolved in
approximately 50 mL tetrahydrofuran (THF) to visually-apparent
dissolution at room temperature (a few minutes). In a separate 2-mL
glass vial, 79 mg (0.79 mmol, 1.2 eq. relative to DOTA-NHS)
1-amino-3-azidopropane was added to approximately 1 mL THF. The
solution of 1-amino-3-azidopropane was then added dropwise to the
DOTA-NHS solution (while stirring) over a period of about 1 minute.
The reaction mixture was then placed in a -20.degree. C. freezer
and allowed to react overnight (15 hours). Following the reaction
period, the solvent was removed in vacuo and used without further
purification for initial click chemistry experiments. Confirmatory
mass analysis LC-MS=655.36; ESI-MS=654.35; theoretical 654.84. No
trace of DOTA-NHS starting material observed. Minor peaks
associated with ionization of t-butyl protecting groups could be
identified. Deprotection was carried out by dissolution of dried
product in 10 mL DMSO and addition of 5 mL 2 M HCl. The solution
was heated at 80.degree. C. overnight, cooled and analyzed without
further purification yielding a single-observable mass peak (LC-MS)
485.5 M+H; theoretical 484. (See FIGS. 3A-3B). Function of the
prepared DOTAzide for click chemical conjugation to alkyne modified
molecular structures was confirmed by performing the click chemical
reaction using the prepared DOTAzide and phenyl acetylene (FIG.
4).
[0246] Conjugation by Click Chemistry: A stock solution of a 20mer
DNA alkyne-functionalized (via 5' conjugation of
5'-/5-hexynyl-phosphoramidite
(6-hexyn-1-yl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,
FIG. 5) oligonucleotide (2.5 nmol .mu.L.sup.-1 in water) was
prepared from purified-dried oligonucleotide obtained from the
manufacturer. For experimental preparations, 10 .mu.L aliquots were
transferred to 500 .mu.L plastic snap-cap v-vials and stored at
-20.degree. C. A solution of deprotected DOTAzide was prepared in a
concentration of 25 nmol .mu.L.sup.-1 in water. To achieve the best
results, the reactants were combined in the following way: A frozen
aliquot of 25 nmol alkyne-modified oligo was removed from the
-20.degree. C. freezer and allowed to thaw slowly. To a solution of
100 .mu.L 0.2 M sodium chloride (NaCl) was added 25 nmol (1 .mu.L
stock solution) DOTAzide, followed by 1 .mu.L of a 1 .mu.L calcium
chloride (CaCl.sub.2) solution (1 .mu.mol Ca.sup.2+). The Ca.sup.2+
is added to interfere with Cu.sup.1+ complexation by the DOTAzide
ring, potentially inhibiting Cu.sup.1+ catalytical activity
required for the click cycloaddition reaction. The
DOTAzide/Ca.sup.2+ mixture was incubated for 1 hour at 80.degree.
C. in a hot water bath, removed and allowed to cool for 15 minutes.
During the cooling period, a reagent solution was prepared: To a
solution of 100 .mu.L 0.2 M NaCl was added in order, 16 .mu.L TriL
solution (30 nmol .mu.L.sup.-1 in 0.2 M NaCl); 1 .mu.L sodium
ascorbate (NaAsc, 2.5 .mu.mmol .mu.L.sup.-1 in 0.2 M NaCl); 2.5
.mu.L CuSO.sub.4 solution (0.1 M in 0.2 M NaCl). The reagent
solution was degassed by application of a gentle stream of
ultra-high-purity N2 for 5 min. and capped tightly. The cooled
solution of DOTAzide/Ca.sup.2+ was combined with the thawed oligo
aliquot and degassed five minutes. The degassed solutions were then
combined and allowed to react for 30 minutes at room temperature
with continuous application of a gentle stream of N.sub.2.
Preparation was confirmed by ESI mass spectrometry and included a
slight impurity of known composition that can be removed easily by
preparatory HPLC, size exclusion, or other chromatographic methods
(FIG. 6). FIG. 7 shows the structure of difluoromethylene
cyclooctyne modified phosphoramidite and Cu-catalyst-free click
chemical reaction with DOTAzide.
Example 3
Conjugation an Amine Modified RNA Aptamer to a Reactive NOTA
Derivative
(2,2',2''-(2-(4-isothiocyanatobenzyl)-1,4,7-triazonane-1,4,7-triyl)triace-
tic acid)
[0247] Chemical reagents for reactions presented in this example
were ACS grade or better. DOTAzide precursor
1,4,7,10-Tetraazacyclododecane-1,4,7-tris(t-butyl
acetate)-10-succinimidyl acetate (DOTA-NHS) of
1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) was
purchased from Macrocyclics, Inc. (Dallas, Tex., USA, catalog
numbers: M140 and B270). 1-amino-3-bromopropane of sufficient
purity was purchased from Alfa Aesar (Ward Hill, Mass. USA, stock
number: B23254 or L12962). Custom oligonucleotides were purchased
from Integrated DNA Technologies, Inc. (IDT, Coralville, Iowa,
USA). Alkyne-modified 5'-/5-hexynyl-PSMA-A10-3.2 RNA aptamer was
custom ordered from Integrated DNA Technologies, Inc. (Iowa City,
Iowa). Copper catalyst was prepared using 99.99+% metals-basis
anhydrous copper sulfate (CuSO.sub.4) obtained from Sigma-Aldrich,
Inc. (Milwaukee, Wis. USA, catalog number: 451657-50G). Water
soluble Cu(I) stabilizing ligand tris-hydroxypropyltriazolyl amine
(TriL) was employed as described by Graham et al. (Graham, D., et
al., J. Am. Chem. Soc., Perk Trans 1, 1998, Volume 6, 1132-1138.).
Other chemicals and reagents were obtained from either Fisher
Scientific (Pittsburg, Pa. USA) or Sigma-Aldrich Chemicals, Inc.
(Milwaukee, Wis., USA) and used without further modification. All
aqueous experiments and reagents were conducted and prepared using
double-distilled deionized 18 M.OMEGA. water obtained using a model
Synthesis Milli-Q Quantam.RTM. EX ultra-water purification system,
equipped with Ultrapurex Organex (cat. QTUOOEX) and Q-gard.RTM. 2
(cat. QGARDOOD2) purification columns (Millipore, Billerica, Mass.
USA).
[0248] Chemical reactions were conducted under high-purity argon or
ultra-pure nitrogen gas unless otherwise stated. Synthesis of
1-amino-3-azidopropane precursor was conducted in as described in
Example 2 above. Click chemical reactions were conducted in plastic
0.5 and 1.5 mL snap-cap v-vials, used without further treatments
(USA Scientific, Ocala, Fla. USA) under ultra-high-purity nitrogen.
Gravimetric measurements were performed using a model AT261
Deltarange digital balance with a readability of 10 .mu.g
(Mettler-Toledo, Toledo, Ohio USA). Mass analysis of low-molecular
weight precursors and high-molecular weight oligonucleotides and
final bioconjugates were conducted by electrospray ionization (ESI)
and MALDI-TOF mass spectrometry. Presence of azide functional
groups was confirmed infrared spectrometry. Chemical structure
.sup.1H 1D NMR data were collected using a Unity Inova 600 MHz NMR
spectrometer (Varian, Palo Alto, Calif. USA). NMR analysis was
carried out using 7 in, 5 mm ID thin-wall glass NMR tubes (Wilmad
Lab Glass, Buena, N.J. USA) in 99.99+% chloroform-d
(Sigma-Aldrich). Spectra were collected using a 6 kHz sweep width,
centered at 4.5 ppm, with a 2 s acquisition time and 1 s delay. The
number of scans was scaled to reasonably achieve a signal to noise
ratio of approximately 64:1. Spectra were evaluated qualitatively
using the software program NUTS (Acorn NMR, Inc., Livermore, Calif.
USA). Retention-time identification and purification of
bioconjugates was performed using high performance liquid
chromatography using model Agilent 1100 series (Agilent, Santa
Clara, Calif. USA) or UltiMate 3000 HPLC's (Dionex, Sunnyvale,
Calif. USA). Final purification of desired bioconjugates was
carried out by HPLC using a 50.times.4.6 mm, column Clarity 5.mu.
Oligo RP (cat. 00B-4442-E0, Phenomenex, Torrance, Calif. USA).
Desalting of bioconjugate solutions was carried out using
Illustra.TM. G-25 "NAPS" single-use size exclusion columns
(Sephadex G-25, in gravity flow mode, using purified water as the
mobile phase buffer (cat. 17-0853-02, maximum capacity 1 mg
mL.sup.-1 DNA, GE Healthcare Bio-Sciences Corp, Piscataway, N.J.
USA).
[0249] Amine-modified RNA aptamer: 100 nmoles of
5'-/5-hexynyl-PSMA-A10-3.2 RNA was dissolved in 100 .mu.L of
deionized-distilled water. To this solution was added 2000 nmoles
1-amino-3-azidopropane (described above) and the solution was
allowed to mix gently several minutes. Meanwhile, in a separate
reaction vessel, a reagent solution was prepared containing 100
.mu.L DMSO, 200 .mu.L 0.2 M NaCl, 100 .mu.moles sodium ascorbate,
14 .mu.moles triazolyl ligand Cu(I) stabilizer, and 4 .mu.moles
CuSO.sub.4. The solution was bubbled gently for several minutes
with high purity argon at which time reagent solution was added to
the solution containing the RNA and 1-amino-3-azidopropane and the
combined solutions were allowed to react under argon for 35 minutes
at room temperature. To this solution was added 100 .mu.L of 620
nmole .mu.L-1 DOTA as a scavenger of free Cu in solution. The
solution was heated to 65.degree. C. and allowed to incubate with
gentle mixing for several minutes and slowly cooled to room
temperature. The solutions were transferred to 10,000 MWCO Ambicon
spin filters and diluted to 3.5 mL, followed by centrifugation at
3000 rpm until the unfiltered fraction containing the desired
product was approximately 250 .mu.L. To this solution was added a
second 100 .mu.L of 620 nmole .mu.L-1 DOTA as a scavenger of free
Cu in solution and the mixture was allowed to stand at room
temperature for 10 minutes, followed by repeated centrifugation
using the Ambicon spin filter to a final volume of <500 .mu.L
which was subsequently desalted on a NAPS size-exclusion column as
described earlier. The final 1 mL eluate of the desalting step was
analyzed for RNA concentration by spectrophotometry (Nanodrop,
Nanodrop Technologies, Wilmington, Del.). The final solution was
then lyophilized overnight and dissolved readily in 75 .mu.L of 0.2
M NaCl. To this solution was added 10 .mu.L 62 nmole .mu.L.sup.-1
DOTA and the solution was heated to 65.degree. C. and allowed to
cool slowly. These solutions were purified by RP-HPLC as described
above to obtain 5 mL solutions that were subsequently concentrated
using 10,000 MWCO Ambicon spin filters as described above to a
final volume of <500 mL followed by a final desalting step by
NAPS size exclusion chromatography as described above. The solution
was freeze dried overnight and removed from the lyophilizer and
stored at -20 for five days. A NOTA conjugate of the obtained
amine-modified RNA was afforded by addition of the 1460 nmoles of
2,2',2''-(2-(4-isothiocyanatobenzyl)-1,4,7-triazonane-1,4,7-triyl)triacet-
ic acid (NOTA-NCS) in a solution of 50 .mu.L 1 M NaHCO3 at pH 9.5,
40.degree. C., for 5 hours. The solution was then allowed to cool
and desalted as described above (NAPS). Samples were lyophilized
and stored at -20.degree. C. for three days, redissolved in 75 mL
of purified water and purified by RP-HPLC (4%-20% CH3CN (B); 100 mM
triethylamine acetate/5% CH3CN (A), 45 minutes, retention time of
conjugate 28.1 minutes, collected manually. Final fractions were
lyophilized and dissolved in 25 .mu.L pure water. Nanodrop
measurements revealed a overall synthetic yield of 22% and overall
purity of 90% with no trace of Cu or Fe contaminants that could
interfere with radiolabeling.
[0250] Schema of the preparations of DOTA- and NOTA-conjugated RNA
aptamer A10-3.2 are provided in FIGS. 8A-8C. Illustration of
synthesis and confirmation of the preparation of NOTA and DOTA
modified PSMA RNA aptamer by mass spectrometry is shown in FIGS.
9A-9C.
[0251] FIGS. 10A-10C show the radiochemical purity achievable by
reacting imagable radiometals .sup.64Cu (FIG. 10B), .sup.68Ga (FIG.
10A) with a NOTA conjugated RNA aptamer and .sup.111In (FIG. 10C)
with a DOTA conjugated RNA aptamer.
[0252] Results of representative cell binding assays of the RNA
aptamer radiolabeled with .sup.111In and .sup.64Cu when contacted
with cells that express PSMA and cells that do not express PSMA are
provided in FIGS. 11A-11C. These assays demonstrated that the RNA
aptamer binding affinity to PSMA expressing cells was preserved
when modified to include a chelator, linker, and radionuclide for
imaging or therapy. Chemical conjugation of the NOTA or DOTA
chelator to the A 10-3.2 aptamer had minimal effect on binding
affinity, and .sup.111In-DOTA- and .sup.64Cu-NOTA-labeled A10-3.2
bound with comparable affinity to PSMA-expressing prostate cancer
cells.
Example 4
In Vivo Imaging of NOTA-Conjugated PSMA-Targeted RNA Aptamer
A10-3.2 by Positron Emission Tomography
[0253] To evaluate the effectiveness of the
[.sup.68Ga]-NOTA-PSMA-Aptamer described above for imaging prostate
cancer by PET, imaging studies were carried out to examine the
characteristics of the .sup.68Ga image quality, and test the
ability of the radiolabeled aptamer to accumulate in a flank
xenograft model of prostate cancer in the mouse. For these
experiments, Athymic nude male mice (nu/nu) 6-10 weeks old were
obtained from Harlan Sprague Dawley, Inc. and maintained in a
sterile environment according to guidelines established by the US
Department of Agriculture and the American Association for
Accreditation of Laboratory Animal Care (AAALAC). Xenograft tumors
were induced by subcutaneous injection of 22Rv1(1.7) cells as
described above and tumor growth was monitored by BLI as described
above. For imaging, animals were fasted for 12 hours with ready
access to water and preinjected with 100 .mu.L of isotonic saline
30 minutes prior to radiopharmaceutical injection.
[0254] Radiolabeling was conducted by elution of the Eckert-Zeigler
generator (740 MBq) in 10 mL 0.1 M HCl directly to a post-elution
purification cation exchange column. Metal impurities and minor
breakthrough of parent .sup.68Ge were removed by passing 1 mL 80%
acetone-0.5 M HCl over the column and discarding to waste. Purified
.sup.68Ga was eluted in 98% acetone-0.05 M HCl directly to a
solution (2.5 mL sodium acetate/2.5 mL acetic acid) containing 15
nmole NOTA-conjugated PSMA aptamer and reacted for 20 minutes at
60.degree. C. Final purification of the radiolabeled species was
conducted using a StrataX C18 RP column to remove unlabeled
.sup.68Ga. The animal was anesthetized according to approved
protocols at 4% isofluorane (maintained at 1.5% through the imaging
procedure). The anesthetized animal was fixed prone to the bed
holder, kept at a constant temperature of 23.degree. C. for the
entire procedure. The final purified [.sup.68Ga]-PSMA-A10-3.2-RNA
aptamer was injected by retroorbital injection of 422 .mu.Ci (16
MBq) [.sup.68Ga]-NOTA-PSMA-Aptamer, with a specific activity of 11
MBq nmole.sup.-1 NOTA-RNA Aptamer A10-3.2 in 50 .mu.L of isotonic
saline, followed by a 120 minute accumulation period.
[0255] At 120 minutes post-injection, a 15 min. static scan was
undertaken and the animal was revived and recaged according to
approved protocols. Bladder accumulation was rapid and obvious,
based on examination a preliminary 15 minute scan performed at 60
minutes post-injection and previous studies using .sup.32P-labeled
A10-3.2 PSMA-aptamers (data not shown). Examination of the 120
minute scan demonstrates excellent conspicuity of the xenograft
tumor (maximum intensity coronal slice of the right flank shown
here, FIG. 12A-12B). These images demonstrate excellent
[.sup.68Ga]-NOTA-PSMA-Aptamer accumulation in the xenograft tumor
in sufficient quantity to enable PET imaging of the location of the
PSMA expressing tumor.
Example 5
In Vivo Imaging of DOTA-Conjugated PSMA-Targeted RNA Aptamer
A10-3.2 by Single Photon Emission Computed Tomography
[0256] To evaluate the effectiveness of the
[.sup.111In]-DOTA-PSMA-Aptamer described above for imaging prostate
cancer by PET, imaging studies were carried out to examine the
characteristics of the .sup.111In image quality, and test the
ability of the radiolabeled aptamer to accumulate in a xenograft
model of prostate cancer in the mouse. For these experiments,
Athymic nude male mice (nu/nu) 6-10 weeks old were obtained from
Harlan Sprague Dawley, Inc. and maintained in a sterile environment
according to guidelines established by the US Department of
Agriculture and the American Association for Accreditation of
Laboratory Animal Care (AAALAC). Xenograft tumors were induced by
subcutaneous injection of 22Rv1(1.7) cells as described above and
tumor growth was monitored by BLI as described above. For imaging,
animals were fasted for 12 hours with ready access to water and
preinjected with 100 .mu.L of isotonic saline 30 minutes prior to
radiopharmaceutical injection.
[0257] Radiolabeling was conducted by adding .sup.111In dissolved
in acetate buffer directly to a solution (2.5 mL sodium acetate/2.5
mL acetic acid) containing 3.8 nmole DOTA-conjugated PSMA aptamer
and reacted for 30 minutes at 100.degree. C. Final purification of
the radiolabeled species was conducted by spin-filter dialysis to
remove free .sup.111In. Radiochemical purity of >98% was
achieved for these experiments. The animal was anesthetized
according to approved protocols at 4% isofluorane (maintained at
1.5% through the imaging procedure). The anesthetized animal was
fixed prone to the bed holder, kept at a constant temperature of
23.degree. C. for the entire procedure. The final purified
[.sup.111In]-PSMA-A10-3.2-RNA aptamer was injected by tail vein
injection of 1000 .mu.Cui (37 MBq) [.sup.111In]-DOTA-PSMA-Aptamer,
with a specific activity of 37 MBq nmol.sup.-1 DOTA-RNA Aptamer A
10-3.2 in 50 .mu.L of isotonic saline, followed by a four hour
accumulation period.
[0258] At four hours post-injection, a 60 min. static scan was
undertaken and the animal was revived and recaged according to
approved protocols. A second scan was performed at 24 hours post
injection. Examination of the 24 hour scan demonstrates excellent
tumor targeting affinity and exceptional specificity for the PSMA
positive xenograft tumor, while no apparent accumulation was
observed in a PSMA negative tumor present on the opposite shoulder
of the animal. FIG. 13A-13C show views of a SPECT/CT imaging study
of the biodistribution of [.sup.111In]-DOTA-PSMA-Aptamer in a
xenograft (subcutaneous, 22RV1 right flank) nude mouse model of
prostate cancer (injected dose: 622 .mu.Ci, 17 MBq) in 100 .mu.L
phophate buffered saline (approximately 1 nmole, specific activity
17 MBq nmole.sup.-1). Image was taken at 24 hours post injection.
No apparent accumulation is observed in a large PSMA negative tumor
injected in the left shoulder area. The PSMA negative tumors were
approximately 40 times the mass of the PSMA positive tumor, which
demonstrates excellent accumulation and specificity of the
radiolabeled PSMA-Aptamer for a prostate cancer tumor. These images
demonstrate excellent [.sup.111In]-DOTA-PSMA-Aptamer accumulation
in the xenograft tumor in sufficient quantity to enable SPECT or
SPECT/CT imaging of the location of the PSMA expressing tumor.
[0259] Flow cytometry was used to confirm expression of PSMA in the
22Rv1(1.7) cells used for in vitro binding experiments and in vivo
xenograft experiments (FIG. 14). The population of cells labeled
with a fluorescent PE-conjugated primary antibody against PSMA is
shown in gray, compared with the population of unlabeled cells
(unshaded).
[0260] Although the foregoing specification and examples fully
disclose and enable the present invention, they are not intended to
limit the scope of the invention, which is defined by the claims
appended hereto.
[0261] All publications, patents and patent applications are
incorporated herein by reference. While in the foregoing
specification this invention has been described in relation to
certain embodiments thereof, and many details have been set forth
for purposes of illustration, it will be apparent to those skilled
in the art that the invention is susceptible to additional
embodiments and that certain of the details described herein may be
varied considerably without departing from the basic principles of
the invention.
[0262] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to") unless otherwise noted. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0263] Embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those embodiments may become apparent to
those of ordinary skill in the art upon reading the foregoing
description. The inventors expect skilled artisans to employ such
variations as appropriate, and the inventors intend for the
invention to be practiced otherwise than as specifically described
herein. Accordingly, this invention includes all modifications and
equivalents of the subject matter recited in the claims appended
hereto as permitted by applicable law. Moreover, any combination of
the above-described elements in all possible variations thereof is
encompassed by the invention unless otherwise indicated herein or
otherwise clearly contradicted by context.
Sequence CWU 1
1
7120RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1nnncggauca gcnnnguuua 20220RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2augcggauca gccauguuua 20328RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 3nnnnnnncgg aucagcnnng uuuannnn 28428RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4gacgaugcgg aucagccaug uuuacguc 28539RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 5gggaggacga ugcggaucag ccauguuuac gucacuccu
39640RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 6nnnnnnnnnn nncggaucag cnnnguuuan
nnnnnnnnnn 40719DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 7gcacttggca aagccgccc 19
* * * * *