U.S. patent application number 13/795955 was filed with the patent office on 2013-07-04 for methods and compositions for delivering active agents with enhanced pharmacological properties.
This patent application is currently assigned to DUKE UNIVERSITY. The applicant listed for this patent is DUKE UNIVERSITY. Invention is credited to Ashutosh Chilkoti.
Application Number | 20130172274 13/795955 |
Document ID | / |
Family ID | 48695305 |
Filed Date | 2013-07-04 |
United States Patent
Application |
20130172274 |
Kind Code |
A1 |
Chilkoti; Ashutosh |
July 4, 2013 |
METHODS AND COMPOSITIONS FOR DELIVERING ACTIVE AGENTS WITH ENHANCED
PHARMACOLOGICAL PROPERTIES
Abstract
Provided herein are methods of enhancing in vivo efficacy of an
active agent, comprising: administering to a subject an active
agent that is coupled to a bioelastic polymer or elastin-like
peptide, wherein the in vivo efficacy of the active agent is
enhanced as compared to the same active agent when administered to
the subject not coupled to (or not associated with) a bioelastic
polymer or ELP.
Inventors: |
Chilkoti; Ashutosh; (Durham,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DUKE UNIVERSITY; |
Durham |
NC |
US |
|
|
Assignee: |
DUKE UNIVERSITY
Durham
NC
|
Family ID: |
48695305 |
Appl. No.: |
13/795955 |
Filed: |
March 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13674285 |
Nov 12, 2012 |
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13795955 |
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12158190 |
Sep 2, 2008 |
8334257 |
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PCT/US2006/048572 |
Dec 20, 2006 |
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13674285 |
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60751896 |
Dec 20, 2005 |
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Current U.S.
Class: |
514/21.2 |
Current CPC
Class: |
C07K 2319/31 20130101;
A61K 49/0056 20130101; A61K 38/16 20130101; A61K 49/14 20130101;
C12Y 203/01028 20130101; A61K 47/6435 20170801; A61K 31/704
20130101; C12Y 203/02013 20130101; C12Y 113/12007 20130101; A61K
38/44 20130101; A61K 38/2271 20130101; A61K 38/45 20130101; A61K
38/17 20130101; A61K 38/2264 20130101; A61K 38/23 20130101; A61K
38/10 20130101; A61K 47/64 20170801; A61K 38/08 20130101; A61K
2039/505 20130101; A61K 38/095 20190101; A61K 38/22 20130101; A61K
38/1709 20130101; A61K 39/44 20130101 |
Class at
Publication: |
514/21.2 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with Government support under grant
number EB00188 and GM-061232 from the National Institutes of
Health. The US Government has certain rights to this invention.
Claims
1. A method of enhancing in vivo efficacy of a peptide agent,
comprising: administering to a subject the peptide agent as a
fusion with an amino acid sequence having a pattern of
proline-containing beta-turns forming an extended, non-globular
structure, the peptide fusion protein being administered about once
per week, about once every two weeks, or about monthly.
2. The method of claim 1, wherein the amino acid sequence forms a
spiral conformation.
3. The method of claim 1, wherein the amino acid sequence comprises
repeat amino acid motifs.
4. The method of claim 1, wherein administration is systemic.
5. The method of claim 1, wherein the peptide agent is administered
by one or more of subcutaneous injection, intraperitoneal
injection, intravenous injection, intramuscular injection,
intratumoral, oral administration, inhalation administration, and
transdermal administration.
6. The method of claim 1, wherein the administration is about once
per week.
7. The method of claim 1, wherein the administration is about every
two weeks.
8. The method of claim 1, wherein the administration is about
monthly.
Description
PRIORITY
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 13/674,285, filed Nov. 12, 2012, which is a
continuation of Ser. No. 12/158,190, filed Sep. 2, 2008 (now U.S.
Pat. No. 8,334,257), which is a national phase application of PCT
Application No. PCT/US2006/048572, filed Dec. 20, 2006, and
published in English on Jun. 28, 2007, as International Publication
No. WO 2007/073486, and which claims the benefit of U.S.
Provisional Patent Application Ser. No. 60/751,896, filed Dec. 20,
2005, the disclosure of each of which is incorporated by reference
herein in its entirety.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0003] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing (filename:
PHAS.sub.--003.sub.--03US_SeqList_ST25.txt, date recorded: Mar. 11,
2013, file size 4 kilobytes).
FIELD OF THE INVENTION
[0004] The present invention concerns methods and formulations for
improving pharmacological properties of active agents to be
delivered to a subject.
BACKGROUND OF THE INVENTION
[0005] A significant problem with many candidate drugs, or even
drugs in clinical use, is insufficient or unsatisfactory in vivo
efficacy. Insufficient in vivo efficacy can be manifested in a
variety of ways, such as (i) low bioavailability of the active
compound; (ii) undesirably short half-life of the active compound,
(iii) and/or undesirably high systemic toxicity of the active
compound. To avoid eliminating otherwise promising drugs from
clinical use, there remains a need for new approaches to enhancing
the in vivo efficacy of active compounds in their delivery to human
and animal subjects.
[0006] U.S. Pat. No. 6,004,782 to Danielle et al. describes
bioelastic polypeptides and the expression thereof in host cells.
The use thereof as fusion proteins containing therapeutics is
described in a cursory fashion at column 15, lines 43-53 therein.
Enhancing the in vivo efficacy of an active agent is neither
suggested nor described.
[0007] U.S. Pat. No. 6,582,926 to Chilkoti describes, among other
things, methods of targeting compounds to regions of interest in a
subject by administering the compound to be delivered as a
conjugate with a polymer that undergoes an inverse temperature
transition (such as an ELP). Compounds to be delivered include
certain radionuclides, chemotherapeutic agents, cytotoxic agents,
and imaging agents as set forth at column 11, lines 6-21. Enhancing
the in vivo efficacy of an active agent is neither suggested nor
described.
[0008] U.S. Pat. No. 6,852,834 to Chilkoti describes, among other
things, fusion proteins that are isolatable by phase transition,
primarily to improve the yield thereof during manufacturing. Fusion
proteins of therapeutic proteins are generally described at column
11, lines 10-24. Enhancing the in vivo efficacy of an active agent
is neither suggested nor described.
SUMMARY OF THE INVENTION
[0009] The present invention provides a method of enhancing in vivo
efficacy of an active agent, comprising: administering to a subject
an active agent that is coupled to a bioelastic polymer or
elastin-like peptide, wherein the in vivo efficacy of the active
agent is enhanced as compared to the same active agent when
administered to the subject not coupled to (or not associated with)
a bioelastic polymer or ELP. In vivo efficacy may be enhanced in
one, or more, of the following ways: solubility, bioavailability,
effective therapeutic dose, formulation compatibility, resistance
to proteolysis, half-life of the administered peptide active
therapeutic agent, persistence in the body subsequent to
administration, and rate of clearance from the body subsequent to
administration.
[0010] Stated otherwise, the present invention provides a method of
delivering an active agent to a subject, comprising: administering
to said subject a conjugate of said active agent and an
elastin-like peptide; wherein the in vivo efficacy of said active
agent is enhanced in said subject when said active agent is
administered to said subject in conjugated form as said conjugate
as compared to the same amount of said active agent administered to
said subject in unconjugated form. In some embodiments, at least
one of: (i) the bioavailability of said active agent is greater;
(ii) the half-life of said active agent is greater, (iii) the
systemic toxicity of said active agent is less, in said subject
when said active agent is administered to said subject in
conjugated form as said conjugate as compared to the same amount of
said active agent administered to said subject in the same way
(e.g., the same dosage of active agent, administered in the same
vehicle or carrier composition, by the same route of
administration) in unconjugated form.
[0011] The active agent may be a diagnostic agent, a therapeutic
agent, an imaging agent, or a chemotherapeutical agent. In some
embodiments the active agent is a (i) small molecule, (ii)
radionuclide, (iii) peptide (iv) peptidomimetic, (v) protein, (vi)
antisense oligonucleotide, (vii) peptide nucleic acid, (viii)
siRNA, (ix) metal chelate, or (x) carbohydrate. In some embodiments
the active agent is a protein or peptide. In some embodiments the
active agent is an antibody such as a therapeutic or diagnostic
antibody.
[0012] The conjugate is generally to the subject in a
treatment-effective amount by any suitable route, such as
parenteral injection.
[0013] A further aspect of the present invention is a conjugate as
described herein in a pharmaceutically acceptable carrier.
[0014] A further aspect of the present invention is the use of an
active agent as described herein, in conjugated form as described
herein, for carrying out a method as described herein.
[0015] The foregoing and other objects and aspects of the invention
are explained in greater detail in the drawings herein and the
specification set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1. SDS-PAGE of a library of ELPs that are polymerized
at the gene level, expressed in E. coli, and purified by exploiting
the phase transition of the ELPs.
[0017] FIG. 2. SDS-PAGE analysis of (A) .sup.14C-ELP visualized by
copper staining, (B) .sup.14C-ELP autoradiography after SDS-PAGE.
(C) Pharmacokinetic analysis of .sup.14C-ELP in mice (Balb/c nu/nu)
exhibits a characteristic distribution and elimination response
with a terminal half-life of 8.4 hr.
[0018] FIG. 3. Uptake and localization of an ELP. All images are of
squamous cell carcinoma (FaDu) cells taken with a LSM-510 laser
scanning confocal fluorescence microscope. The cells are incubated
with ELP-Alexa488 (green) for 1 hour prior to co-staining (A) Cells
are stained with DiI-CM (red) to label the cell membrane. (B) Cells
are co-stained with lysotracker red (red) which selectively stains
the lysosomes. The ELP colocalizes with the lysotracker red dye
(note the yellow fluorescence).
[0019] FIG. 4. (A) Synthesis of a derivative with a terminal
maleimide: It shows that a derivative with a terminal maleimide is
prepared by attaching a pH sensitive hydrazone linker to
Doxorubicin (hereinafter as Dox), a cancer chemotherapeutic agent
at the 13-keto position. Then, the terminal maleimide of the
derivative is conjugated to an ELP, which presents one or more
Cysteine residues. (B) It is an example of cytotoxicity of
Doxorubicin conjugated to ELP2-160JM2 conjugate (hereinafter as
ELP-Dox) in a MTT cell viability assay. The cytotoxicity of
ELP-Doxorubicin and unconjugated Dox is a function of the
equivalent Doxorubicin concentration. Compared to the free drug,
ELP-Doxorubicin demonstrates almost equivalent cytotoxicity of the
free drugs. (C) ELP-Dox and Dox are injected at the same
concentration into mice via tail vein injections. After 1 h, no Dox
can be detected from the blood samples of the mice. However,
.about.20 injected gram/g serum (% ID/g) is detected from the mice
injected with ELP-Dox. The result of this experiment demonstrates
that the conjugated form has a greater plasma half-life of the
drug. (D) It demonstrates the biodistribution of Dox and ELP-Dox
injected nude mice with human tumor xenografts. Upon conjugation of
Dox to ELP, a different pattern of distribution is obtained. The
concentrations of Dox in the heart; liver and lung are greater than
those of ELP-OPDX, however, the concentration of ELP-Dox in tumor
is greater than that of Dox.
[0020] FIG. 5. Accumulation of .sup.14C-labeled ELPs in tumors. The
two ELPs reported are a thermally sensitive ELP1 and a thermally
insensitive ELP2 in tumors that are either heated to 41.5.degree.
C. or not heated.
[0021] FIG. 6. Expression of different ELP fusion proteins as
examples of recombinant ELP-protein conjugates. All ELP-protein
conjugates are prepared by fusion of the gene of the protein, ELP
and expression in a heterogeneous expression system (e.g., E.
coli). The left panel shows examples of blue fluorescent protein
(BFP), chloramphenicol acetyl transferase (CAT) and Kringle1-3
domains (K1-3: angiostatin). The right panel shows other examples
of purified ELP-protein conjugates.
[0022] FIG. 7. SDS-PAGE of purification of ELP fusion protein in
the following orientation: The preparation of the protein-ELP and
ELP-protein shows that protein conjugates of ELPs can be
synthesized in either orientation for CAT, BFP, and Trx. (A) Thin
layer chromatography showing activity of CAT, (B) Fluorescence of
BFP-ELP n ELP-BFP showing functionality of BDFP in the fusion.
[0023] FIG. 8. (I) SDS-PAGE characterization of inverse transition
purifications: It shows each stage of purification for the
thioredoxin/90-mer ELP fusion (49.9 kDa, lanes 1 through 5) Lane A:
soluble lysate; lane B: discarded supernatant containing
contaminating E. coli proteins; lane 3: resolubilized pellet
fraction containing purified fusion protein, lane 4, second round
supernatant; lane 5: second round pellet; lane 6: molecular weight
markers (kDa). (II) Total protein (green) and thioredoxin (Trx)
activity (red) for each stage of purification of the
thioredoxin/90-mer ELP. Values are normalized to those determined
for the soluble lysate.
[0024] FIG. 9. Examples of synthesis of ELP-peptide conjugates. All
conjugates are prepared recombinantly as fusions with ELP. The two
lanes in each SDS-PAGE gels from A-F show the fusion (conjugate) on
left, and the peptide on right. Mass spectrometry results for each
purified peptide are shown below the SDS-PAGE gels. (A) Morphine
modulating neuropeptide (MMN), (B) Neuropeptide Y (NPY) (2.7 kDa)
(note: although gel was overloaded, Commassie does not stain NPY)
(ELP4-60-NPY 222 mg/L fusion (conjugate) on left, NPY 20 mg/L
peptide on right), (C) Orexin B (3.0 kDa) (ELP4-60-Orexin B 320
mg/L fusion (conjugate) on left, Orexin B 19 mg/L peptide on
right), (D) Leptin (4.0 kDa) (ELP4-60-Leptin 415 mg/L fusion
(conjugate) on left, Leptin 19.5 mg/L peptide on right), (E) ACTH
(4.6 kDa) (ACTH-ELP1-90 133 mg/L fusion (conjugate) on left, ACTH
19 mg/L peptide on right), (F) Pro-calcitonin (6.2 kDa)
(ELP1-90-pro-CT 260 mg/L fusion (conjugate) on left, pro-CT 23 mg/L
peptide on right).
[0025] FIG. 10. Examples of ELP-peptide conjugate. Recombinant
fusion of antimicrobial peptide MSI-78 with ELP (ELP-peptide
conjugate). Sequence of MSI-78: Sequence=GIGKFLKKAKKFGKAFVKILKK
(SEQ ID NO.: 2). (A) Purification of ELP 1-90-MSI-78 and MSI-78.
SDS-Page gel shows both high purity of the conjugate and the
peptide produced recombinantly. (B) Purity of EP-MSI-78 conjugate
determined by liquid chromatography combined with mass
spectrometry. One compound was detected with a molecular weight of
2476.6 and purity is >99% by LC-ELSD (C) Bactericidal activity
of MSI-78.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The disclosures of all United States patent references cited
herein are to be incorporated by reference herein in their
entirety.
[0027] "Active agent" as used herein may be any suitable active
agent, including therapeutic and diagnostic or imaging agents.
[0028] Examples of imaging agents include, but are not limited to,
the following: radioisotopes (e.g., .sup.3H, .sup.14C, .sup.35S,
.sup.125I, .sup.131I) fluorescent labels (e.g., FITC, rhodamine,
lanthanide phosphors), MRI contrast agents (e.g., Gadolinum
chelates (Gd)) luminescent labels such as luminol; enzymatic labels
(e.g., horseradish peroxidase, beta-galactosidase, luciferase,
alkaline phosphatase, acetylcholinesterase), biotinyl groups (which
can be detected by marked avidin e.g., streptavidin containing a
fluorescent marker or enzymatic activity that can be detected by
optical or calorimetric methods), predetermined polypeptide
epitopes recognized by a secondary reporter (e.g., leucine zipper
pair sequences, binding sites for secondary antibodies, metal
binding domains, epitope tags). Indirect methods may also be
employed in which the primary antigen-antibody reaction is
amplified by the introduction of a second antibody.
[0029] "Therapeutic agent" as used herein may be any suitable
therapeutic agent, including but not limited to radionuclides,
chemotherapeutic agents; cytototoxic agents, parathyroid hormone
related protein (parathyroid hormone related protein), growth
hormone (GH) particularly human and bovine growth hormone, growth
hormone-releasing hormones; interferon including .alpha.-, .beta.-,
or .gamma.-interferons, etc, interleukin-I; interleukin-II;
erythropoietin including .alpha.- and .beta.-erythropoietin (EPO),
granulocyte colony stimulating factor (GCSF), granulocyte
macrophage colony stimulating factor (GM-CSF), anti-angiogenic
proteins (e.g., angiostatin, endostatin) PACAP polypeptide
(pituitary adenylate cyclase activating polypeptide), vasoactive
intestinal peptide (VIP), thyrotrophin releasing hormone (TRH),
corticotrophin releasing hormone (CRH), vasopressin, arginine
vasopressin (AVP), angiotensin, calcitonin, atrial naturetic
factor, somatostatin, adrenocorticotropin, gonadotropin releasing
hormone, oxytocin, insulin, somatotropin, HBS antigen of hepatitis
B virus, plasminogen tissue activator, coagulation factors
including coagulation factors VIII and IX, glucosylceramidase,
sargramostim, lenograstin, filgrastin, interleukin-2,
dornase-.alpha., molgramostim, PEG-L-asparaginase, PEG-adenosine
deaminase, hirudin, eptacog-.alpha. (human blood coagulation factor
VIIa) nerve growth factors, transforming growth factor, epidermal
growth factor, basic fibroblast growth factor, VEGF; heparin
including low molecular weight heparin, calcitonin; atrial
naturetic factor; antigens; monoclonal antibodies; somatostatin;
adrenocorticotropin, gonadotropin releasing hormone; oxytocin;
vasopressin; cromolyn sodium; vancomycin; desferrioxamine (DFO);
parathyroid hormone, anti-microbials, antifungals, an immunogen or
antigen, an antibody such as a monoclonal antibody, or any
combination thereof. See, e.g., U.S. Pat. Nos. 6,967,028;
6,930,090; and 6,972,300.
[0030] Example therapeutic agents include all of the therapeutic
agents set forth in paragraphs 0065 through 0388 of W. Hunter, D.
Gravett, et al., US Patent Application Publication No. 20050181977
(Published Aug. 18, 2005) (assigned to Angiotech International AG)
the disclosure of which is incorporated by reference herein in its
entirety.
[0031] "Radionuclide" as described herein may be any radionuclide
suitable for delivering a therapeutic dosage of radiation to a
tumor or cancer cell, including but not limited to .sup.227Ac,
.sup.211At, .sup.131Ba, .sup.77Br, .sup.109Cd, .sup.51Cr,
.sup.67Cu, .sup.165Dy, .sup.155Eu, .sup.153Gd, .sup.198Au,
.sup.166Ho, .sup.113mIn, .sup.115mIn, .sup.123I, .sup.125I,
.sup.131I, .sup.189Ir, .sup.191Ir, .sup.192Ir, .sup.194Ir,
.sup.52Fe, .sup.55Fe, .sup.59Fe, .sup.177Lu, .sup.109Pd, .sup.32P,
.sup.226Ra, .sup.186Re, .sup.188Re, .sup.153Sm, .sup.46Sc,
.sup.47Sc, .sup.72Se, .sup.75Se, .sup.105Ag, .sup.89Sr, .sup.35S,
.sup.177Ta, .sup.117mSn, .sup.121Sn, .sup.166Yb, .sup.169Yb,
.sup.90Y, .sup.212Bi, .sup.119Sb, .sup.197Hg, .sup.100Pd,
.sup.101mRh, and .sup.212Pb. Radionuclides may also be those useful
for delivering a detectable dosage for imaging or diagnostic
purposes, even where those compounds are not useful for therapeutic
purposes.
[0032] "Chemotherapeutic agent" as used herein includes but is not
limited to methotrexate, daunomycin, mitomycin, cisplatin
(cisplatinum or cis-dianminedichloroplatinum(II) (CCDP)),
vincristine, epirubicin, fluorouracil, verapamil, cyclophosphamide,
cytosine arabinoside, aminopterin, bleomycin, mitomycin C,
democolcine, etoposide, mithramycin, chlorambucil, melphalan,
daunorubicin, doxorubicin, tamoxifen, paclitaxel, vincristine,
vinblastine, camptothecin, actinomycin D, and cytarabine,
combrestatin and its derivatives.
[0033] "Cytotoxic agent" as used herein includes but is not limited
to ricin (or more particularly the ricin A chain), aclacinomycin,
diphtheria toxin, Monensin, Verrucarin A, Abrin, Vinca alkaloids,
Tricothecenes, and Pseudomonas exotoxin A.
[0034] "Immunogen" and "antigen" are used interchangeably and mean
any compound to which a cellular or humoral immune response is to
be directed against, and include bacterial antigens, viral
antigens, and tumor antigens. Non-living immunogens (e.g., killed
immunogens, subunit vaccines, recombinant proteins or peptides or
the like) are currently preferred. Examples of suitable immunogens
include those derived from bacterial surface polysaccharides which
can be used in carbohydrate-based vaccines. Bacteria typically
express carbohydrates on their cell surface as part of
glycoproteins, glycolipids, O-specific side chains of
lipopolysaccharides, capsular polysaccharides and the like.
Exemplary bacterial strains include Streptococcus pneumonia,
Neisseria meningitidis, Haemophilus influenza, Klebsiella spp.,
Pseudomonas spp., Salmonella spp., Shigella spp., and Group B
streptococci. A number of suitable bacterial carbohydrate epitopes
which may be used as the immunogen in the present invention are
described in the art (e.g., Sanders, et al. Pediatr. Res.
37:812-819 (1995); Bartoloni, et al. Vaccine 13:463-470 (1995);
Pirofski, et al., Infect. Immun. 63:2906-2911 (1995) and
International Publication No. WO 93/21948) and are further
described in U.S. Pat. No. 6,413,935. Exemplary viral antigen or
immunogen includes those derived from HIV (e.g., gp120, nef, tat,
pol). Exemplary fungal antigens include those derived from Candida
albicans, Cryptococcus neoformans, Coccidoides spp., Histoplasma
spp., and Aspergillus spp. Parasitic antigens include those derived
from Plasmodium spp., Trypanosoma spp., Schistosoma spp.,
Leishmania spp. and the like. Exemplary carbohydrate epitopes that
may be utilized as antigens or immunogens in the present invention
include but are not limited to the following:
Gal.alpha.1,4Gal.beta.-(for bacterial vaccines); GalNAc.alpha.-(for
cancer vaccines); Man.beta.1,2(Man.beta.).sub.nMan.beta.-(for
fungal vaccines useful against, for example, Candida albicans),
where n=O.fwdarw..infin.;
GalNAc.beta.1,4(NeuAc.alpha.-2,3)Gal.beta.1,4Glc.beta.-O-ceramide
(for cancer vaccines);
Gal.alpha.-1,2(Tyv.alpha.1,3)Man.alpha.1,4Rha.alpha.1,3Gal.alpha.1,2(Ty.a-
lpha.1,3)Man.alpha.4Rha- and
Gal.alpha.1,2(Abe.alpha.1,3)Man.alpha.1,4Rha.alpha.1,3Gal.alpha.1,2(Abe.a-
lpha.1,3)Man.alpha.1,4Rha.alpha.1,3Gal.alpha.1,2
(Abe.alpha.1,3)Man.alpha.1,4Rha-(both of which are useful against,
for example, Salmonella spp.). Carbohydrate epitopes as antigens or
immunogens and the synthesis thereof are described further in U.S.
Pat. No. 6,413,935. In one embodiment the immunogen may be an
anthrax immunogen; i.e. an immunogen that produces protective
immunity to Bacillus anthracis, such as anthrax vaccine, A,
(Michigan Department of Health, Lansing, Mich.; described in U.S.
Pat. No. 5,728,385). Other examples of immunogens or antigens
include but are not limited to those that produce an immune
response or antigenic response to the following diseases and
disease-causing agents: adenoviruses; Bordetella pertussus;
Botulism; bovine rhinotracheitis; Branhamella catarrhalis; canine
hepatitis; canine distemper; Chlamydiae; Cholera; coccidiomycosis;
cowpox; cytomegalovirus; cytomegalovirus; Dengue fever; dengue
toxoplasmosis; Diphtheria; encephalitis; Enterotoxigenic
Escherichia coli; Epstein Barr virus; equine encephalitis; equine
infectious anemia; equine influenza; equine pneumonia; equine
rhinovirus; feline leukemia; flavivirus; Globulin; haemophilus
influenza type b; Haemophilus influenzae; Haemophilus pertussis;
Helicobacter pylori; Hemophilus; hepatitis; hepatitis A; hepatitis
B; Hepatitis C; herpes viruses; HIV; HIV-1 viruses; HIV-2 viruses;
HTLV; Influenza; Japanese encephalitis; Klebsiellae species;
Legionella pneumophila; leishmania; leprosy; lyme disease; malaria
immunogen; measles; meningitis; meningococcal; Meningococcal
Polysaccharide Group A; Meningococcal Polysaccharide Group C;
mumps; Mumps Virus; mycobacteria and; Mycobacterium tuberculosis;
Neisseria; Neisseria gonorrhoeae; Neisseria meningitidis; ovine
blue tongue; ovine encephalitis; papilloma; parainfluenza;
paramyxovirus; paramyxoviruses; Pertussis; Plague; Pneumococcus;
Pneumocystis carinii; Pneumonia; Poliovirus; Proteus species;
Pseudomonas aeruginosa; rabies; respiratory syncytial virus;
rotavirus; Rubella; Salmonellae; schistosomiasis; Shigellae; simian
immunodeficiency virus; Smallpox; Staphylococcus aureus;
Staphylococcus species; Streptococcus pneumoniae; Streptococcus
pyogenes; Streptococcus species; swine influenza; tetanus;
Treponema pallidum; Typhoid; Vaccinia; varicella-zoster virus; and
Vibrio cholerae. The antigens or immunogens may, include various
toxoids, viral antigens and/or bacterial antigens such as antigens
commonly employed in the following vaccines: chickenpox vaccine;
diphtheria, tetanus, and pertussis vaccines; haemophilus influenzae
type b vaccine (Hib); hepatitis A vaccine; hepatitis B vaccine;
influenza vaccine; measles, mumps, and rubella vaccines (MMR);
pneumococcal vaccine; polio vaccines; rotavirus vaccine; anthrax
vaccines; and tetanus and diphtheria vaccine (Td). See, e.g., U.S.
Pat. No. 6,309,633. Antigens or immunogens that are used to carry
out the present invention include those that are derivatized or
modified in some way, such as by conjugating or coupling one or
more additional groups thereto to enhance function or achieve
additional functions such as targeting or enhanced delivery
thereof, including but not limited to those techniques described in
U.S. Pat. No. 6,493,402 to Pizzo et al. (.alpha.-2 macroglobulin
complexes); U.S. Pat. No. 6,309,633; U.S. Pat. No. 6,207,157; U.S.
Pat. No. 5,908,629, etc.
[0035] Interferon (IFNs) are used herein refers to natural proteins
produced by the cells of the immune system of most vertebrates in
response to challenges by foreign agents such as viruses, bacteria,
parasites and tumor cells, and its function is to inhibit viral
replication within other cells. Interferons belong to the large
class of glycoproteins known as cytokines. Three major classes of
interferons for human have been discovered as type I, type II and
type III, classified according to the type of receptor through
which they signal. Human type I IFNs comprise a vast and growing
group of IFN proteins, designated IFN-.alpha., IFN-.beta.,
IFN-.kappa., IFN-.delta., IFN-.epsilon., IFN-.tau., IFN-.omega. and
IFN-.zeta.. [See Interferon-.zeta./limitin: Novel type I Interferon
that displays a narrow range of biological activity, Oritani Kenji
and Tomiyama Yoshiaki, International Journal of hematology, 2004,
80, 325-331; Characterization of the type I interferon locus and
identification of novel genes, Hardy et al., Genomics, 2004, 84,
331-345.] Homologous molecules to type I IFNs are found in many
species, including most mammals, and some have been identified in
birds, reptiles, amphibians and fish species. [See The interferon
system of non-mammalian vertebrates, Schultz et al., Developmental
and Comparative Immunology, 28, 499-508.] All type IIFNs bind to a
specific cell surface receptor complex known as the IFN-.alpha.
receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains. The
type II IFNs only has one member called IFN-.gamma.. Mature
IFN-.gamma. is an anti-parallel homodimer, which binds to the
IFN-.gamma. receptor (IFNGR) complex to elicit a signal within its
target cell. The type III IFN group consists of three IFN-.lamda.
molecules called IFN-.lamda.1, IFN-.lamda.2 and IFN-.lamda.3 (also
called IL29, IL28A and IL28B respectively). [See Novel interferons,
Jan Vilcek, Nature Immunology, 2003, 4, 8-9.] The IFN-.lamda.
molecules signal through a receptor complex consisting of IL10R2
(also called CRF2-4) and IFNLR1 (also called CRF2-12). [See Murine
interferon lambdas (type III interferons) exhibit potent antiviral
activity in vivo in a poxvirus infection model, Bartlett et al.,
Journal of General Virology, 2005, 86, 1589-1596.]
[0036] "Antibody" or "antibodies" as used herein refers to all
types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE.
The term "immunoglobulin" includes the subtypes of these
immunoglobulins, such as IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, etc. Of these immunoglobulins, IgM and IgG are
preferred, and IgG is particularly preferred. The antibodies may be
of any species of origin, including (for example) mouse, rat,
rabbit, horse, or human, or may be humanized or chimeric
antibodies. The term "antibody" as used herein includes antibody
fragments which retain the capability of binding to a target
antigen, for example, Fab, F(ab').sub.2, and Fv fragments, and the
corresponding fragments obtained from antibodies other than IgG.
Such fragments are also produced by known techniques. Antibodies
may be for diagnostic purposes or for therapeutic purposes.
Examples of therapeutic antibodies include but are not limited to
herceptin, rituxan, campath (Mellinium pharma Inc.), gemtuzumab
(Cell tech.), herceptin (Genentech), panorex (Centocor GSK),
rituximab (Genentech), bexxar (Coraxia GSK), edrecolomab
(Glaxo-wellcome), alemtuzumab (ILEX Pharmaceuticals), mylotrag
(Whety-Ayerst), IMC-C225, smartin 195, and mitomomab (Imclone
systems). Therapeutic antibodies include those coupled to a
therapeutic compound and "cold dose" antibodies, such as for
reducing non-specific binding. See, e.g., Abrams et al., U.S. Pat.
No. RE38,008.
[0037] "Treat" as used herein refers to any type of treatment or
prevention that imparts a benefit to a subject afflicted with a
disease or at risk of developing the disease, including improvement
in the condition of the subject (e.g., in one or more symptoms),
delay in the progression of the disease, delay the onset of
symptoms or slow the progression of symptoms, etc. As such, the
term "treatment" also includes prophylactic treatment of the
subject to prevent the onset of symptoms. As used herein,
"treatment" and "prevention" are not necessarily meant to imply
cure or complete abolition of symptoms." to any type of treatment
that imparts a benefit to a patient afflicted with a disease,
including improvement in the condition of the patient (e.g., in one
or more symptoms), delay in the progression of the disease,
etc.
[0038] "Treatment effective amount" as used herein means an amount
of the antibody sufficient to produce a desirable effect upon a
patient inflicted with a condition such as cancer, diabetes,
bacterial or viral infection, etc., including improvement in the
condition of the patient (e.g., in one or more symptoms), delay in
the progression of the disease, etc. With an immunogen a "treatment
effective amount" may be an amount effective to produce an immune
response or protective immunity (in whole or in part) against
subsequent infection by a bacterial, viral, fungal, protozoal, or
other microbial agent.
[0039] "Conjugate" as used herein refers to two or more moieties or
functional groups that are covalently or noncovalently joined to
one another, such that the two or more groups function together as
a single structure under the conditions of the methods described
herein. In one embodiment, the conjugate is a fusion protein. In
some embodiments, the conjugate refers to the two moieties that are
chemically or enzymatically attached to each other.
[0040] "Fusion protein" as used herein refers to a protein or
peptide, produced by recombinant means (i.e., expression from a
nucleic acid) that is comprised of a first protein or peptide
covalently joined on expression to a second protein or peptide.
[0041] A "polymer that undergoes an inverse temperature transition"
herein refers to a polymer that is soluble in an aqueous solution
at a lower temperature, and is insoluble in an aqueous solution at
a higher temperature.
[0042] "Transition temperature" or "T.sub.t" as used herein, refers
to the temperature above which a polymer that undergoes an inverse
temperature transition is insoluble in an aqueous system (e.g.,
water, physiological saline solution), and below which such a
polymer is soluble in an aqueous system.
[0043] A "bioelastic polymer" is, in general, a polypeptide that
exhibits an inverse temperature transition. Bioelastic polymers are
discussed in greater detail below. Such bioelastic polymers are
typically elastin-like peptides.
[0044] While the present invention is concerned primarily with the
treatment of human subjects, the invention may also be used for the
treatment of animal subjects, particularly mammalian subjects such
as dogs, cats, horses, cows, pigs, etc., for veterinary
purposes.
[0045] Subjects in need of treatment by the methods described
herein include subjects afflicted with any disorder conventionally
or currently treated or diagnosed by the active agents described
herein, including but not limited to subjects afflicted with solid
tumors or cancers such as lung, colon, breast, brain, liver,
prostate, spleen, muscle, ovary, pancreas, skin (including
melanoma) etc; subjects afflicted with or at risk of developing a
viral, bacterial, protozoal, or other microbial infection; etc.
[0046] Bioelastic Polymers.
[0047] Bioelastic polymers are known and described in, for example,
U.S. Pat. No. 5,520,672 to Urry et al. In general, bioelastic
polymers are polypeptides comprising elastomeric units of
bioelastic pentapeptides, tetrapeptides, and/or nonapeptides (that
is, "elastin-like peptides"). Thus in some embodiments the
elastomeric unit is a pentapeptide, in other embodiments the
elastomeric unit is a tetrapeptide, and in still other embodiments
the elastomeric unit is a nonapeptide. Bioelastic polymers that may
be used to carry out the present invention are set forth in U.S.
Pat. No. 4,474,851, which describes a number of tetrapeptide and
pentapeptide repeating units that can be used to form a bioelastic
polymer. Specific bioelastic polymers that can be used to carry out
the present invention are also described in U.S. Pat. Nos.
4,132,746; 4,187,852; 4,500,700; 4,589,882; and 4,870,055. Still
other examples of bioelastic polymers are set forth in U.S. Pat.
No. 6,699,294 to Urry, U.S. Pat. No. 6,753,311 to Fertala and Ko;
and U.S. Pat. No. 6,063,061 to Wallace.
[0048] As disclosed in U.S. Pat. No. 4,474,851, elastomeric
peptides may have a sequence of regularly appearing 13-turns,
forming an overall spiral conformation (e.g., a .beta.-spiral,
which is a series of regularly repeating .beta.-turns). The spiral
structures are more open than the more common .alpha.-helix. As a
result, the atoms in the peptide backbone have a high freedom of
movement (e.g., as compared to the freedom of movement for an
.alpha.-helix). This is particularly true of librational motions
involving peptide moieties. A libration is a torsional oscillation
involving simultaneous rotational motions of the two single bonds
on each side of a librating moiety. The moiety involved in a
libration may be a single peptide bond or several peptide residues.
For adequate freedom of motion to exist, it is important, however,
that the carbonyl oxygen and the amino hydrogen of the peptide bond
not be involved in hydrogen bonding to other parts of the molecule
or to other molecules. Otherwise a greater energy barrier to the
libration exists and motion will be restricted. Since
non-hydrogen-bonded segments having freedom of motion exist in the
.beta.-spiral between the points of hydrogen bonding for the
.beta.-turns, these segments may be said to be librationally
suspended. Librationally suspended segments therefore are a
structural feature that exists in certain elastic peptides because
of the repeating .beta.-turns with relative infrequent hydrogen
bonding. Librationally suspended segments resulting from the
.beta.-spiral structure are thought to give rise to elasticity, as
will be further discussed.
[0049] Another factor leading to the high librational freedom of
such molecules is the absence of significant polar interactions
between the amino acid residues, either intrachain or interchain,
other than a hydrogen bond within the .beta.-turn. The amino acid
residues present are mostly hydrophobic or glycine and accordingly
do not exert significant forces on one another through space. If a
significant number of charged or polar groups were present,
electrostatic interactions might limit librational freedom and
restrict the number of available states in the relaxed
(non-extended) form of the molecules. Polar and charged amino acid
residues are not strictly prohibited, however, if their presence
does not destroy the elasticity of the elastic peptide component as
a whole. For example, an occasional serine residue is present in
naturally occurring tropoelastin without destroying elasticity.
Accordingly, hydrophobic amino acid residues and glycines are
preferred in forming elastomeric polypeptides of the present type
although other amino acids may be present to a some extent.
[0050] Although not intending to be bound by theory, the elasticity
of polypeptides of the .beta.-turn structure may be caused by
thermodynamic drive toward greater entropy. The relaxed state of
the .beta.-spiral has a large degree of librational freedom and
thus the atoms of the peptide chain can exist in a large number of
positions. When the molecules are stretched, the degree of freedom
is reduced, particularly for librational motions, and when the
tension is released, a thermodynamic driving force toward higher
entropy results in reformation of the contracted .beta.-spiral.
[0051] In one embodiment, the bioelastic polymers used to carry out
the present invention are polypeptides of the general formula
(VPGXG).sub.m (SEQ ID NO.: 1), where X is any amino acid (e.g.,
Ala, Leu, Phe) and m is any suitable number such as 2, 3 or 4 up to
60, 80 or 100 or more. The frequency of the various amino acids as
the fourth amino acid can be changed, as well as the frequency of
X. For example, the bioelastic polymers used to carry out the
present invention may be polypeptides of the general formula:
[(VPGXG).sub.m(VPGKG).sub.n].sub.o (SEQ ID NO.: 3), where m is 2, 3
or 4 to 20 or 30, n is 1, 2 or 3, o is at least 2, 3 or 4 up to 30,
40 or 50 or more. Any ratios of X/K can be possible, which means
where m is 1, 2, or 3 up to 100, 150, or 300 or more, n is 1, 2 or
3 up to 100 or 150 or 300 or more, or is at least 1, 2, or 3 up to
100, 150 or 300 or more.
[0052] For example, bioelastic polymers used to carry out the
present invention may comprise repeating elastomeric units selected
from the group consisting of bioelastic pentapeptides and
tetrapeptides, where the repeating units comprise amino acid
residues selected from the group consisting of hydrophobic amino
acid and glycine residues and where the repeating units exist in a
conformation having a beta-turn of the formula:
##STR00001##
wherein R.sub.1-R.sub.5 represent side chains of amino acid
residues 1-5, and m is 0 when the repeating unit is a tetrapeptide
or 1 when the repeating unit is a pentapeptide. Nonapeptide
repeating units generally consist of sequential tetra- and
pentapeptides. Preferred hydrophobic amino acid residues are
selected from the group consisting of alanine, valine, leucine,
isoleucine, proline, phenylalanine, tryptophan, and methionine. In
many cases, the first amino acid residue of the repeating unit is a
residue of valine, leucine, isoleucine or phenylalanine; the second
amino acid residue is a residue of proline; the third amino acid
residue is a residue of glycine; and the fourth amino acid residue
is glycine or a very hydrophobic residue such as tryptophan,
phenylalanine or tyrosine. Particular examples include the
tetrapeptide Val-Pro-Gly-Gly (SEQ ID NO.: 4), the tetrapeptide GGVP
(SEQ ID NO.: 5), the tetrapeptide GGFP (SEQ ID NO.: 6), the
tetrapeptide GGAP (SEQ ID NO.: 7), the pentapeptide is
Val-Pro-Gly-Val-Gly (SEQ ID NO.: 8), the pentapeptide GVGVP (SEQ ID
NO.: 9), the pentapeptide GKGVP (SEQ ID NO.: 10), the pentapeptide
GVGFP (SEQ ID NO.: 11), the pentapeptide GFGFP (SEQ ID NO.: 12),
the pentapeptide GEGVP (SEQ ID NO.: 13), the pentapeptide GFGVP
(SEQ ID NO.: 14), and the pentapeptide GVGIP (SEQ ID NO.: 15). See,
e.g., U.S. Pat. No. 6,699,294 to Urry.
[0053] Coupling of conjugates may be carried out by any suitable
means, including chemical and recombinant means. Chemical or
enzymatic coupling may be carried out by procedures known in the
art. (See, e.g., U.S. Pat. Nos. 6,930,090; 6,913,903; 6,897,196;
and 6,664,043). Coupling of conjugates by recombinant means (e.g.,
where elastin is joined to a protein or peptide such as an
interleukin, by recombinant means such as by expression of a fusion
protein) may also be carried out by procedures known in the art
(See, e.g., U.S. Pat. Nos. 6,974,572; 6,972,322; 6,962,978; and
6,956,112).
[0054] Formulations and Administration.
[0055] Administering of the conjugate to the subject may be carried
out by any suitable means, such as subcutaneous injection,
intraperitoneal injection, intravenous injection, intramuscular
injection, intratumoral, oral administration, inhalation
administration, transdermal administration, etc. Preferred
administration techniques are typically "systemic" in that a
particular region of interest is not specifically targeted.
[0056] The conjugates (or "active compounds") described above may
be formulated for administration in a single pharmaceutical carrier
or in separate pharmaceutical carriers for the treatment of a
variety of conditions. In the manufacture of a pharmaceutical
formulation according to the invention, the active compounds
including the physiologically acceptable salts thereof, or the acid
derivatives of either thereof are typically admixed with, inter
alia, an acceptable carrier. The carrier must, of course, be
acceptable in the sense of being compatible with any other
ingredients in the formulation and must not be deleterious to the
patient. The carrier may be a solid or a liquid, or both, and is
preferably formulated with the compound as a unit-dose formulation,
for example, a tablet, which may contain from 0.5% to 95% by weight
of the active compound. One or more active compounds may be
incorporated in the formulations of the invention, which may be
prepared by any of the well-known techniques of pharmacy consisting
essentially of admixing the components, optionally including one or
more accessory ingredients.
[0057] The formulations of the invention include those suitable for
oral, rectal, topical, buccal (e.g., sub-lingual), parenteral
(e.g., subcutaneous, intramuscular, intradermal, or intravenous)
and transdermal administration, although the most suitable route in
any given case will depend on the nature and severity of the
condition being treated and on the nature of the particular active
compound which is being used.
[0058] Formulations suitable for oral administration may be
presented in discrete units, such as capsules, cachets, lozenges,
or tablets, each containing a predetermined amount of the active
compound; as a powder or granules; as a solution or a suspension in
an aqueous or non-aqueous liquid; or as an oil-in-water or
water-in-oil emulsion. Such formulations may be prepared by any
suitable method of pharmacy, which includes the step of bringing
into association the active compound and a suitable carrier (which
may contain one or more accessory ingredients as noted above). In
general, the formulations of the invention are prepared by
uniformly and intimately admixing the active compound with a liquid
or finely divided solid carrier, or both, and then, if necessary,
shaping the resulting mixture. For example, a tablet may be
prepared by compressing or molding a powder or granules containing
the active compound, optionally with one or more accessory
ingredients. Compressed tablets may be prepared by compressing, in
a suitable machine, the compound in a free-flowing form, such as a
powder or granules optionally mixed with a binder, lubricant, inert
diluent, and/or surface active/dispersing agent(s). Molded tablets
may be made by molding, in a suitable machine, the powdered
compound moistened with an inert liquid binder. Formulations of the
present invention suitable for parenteral administration
conveniently comprise sterile aqueous preparations of the active
compound, which preparations are preferably isotonic with the blood
of the intended recipient. These preparations may be administered
by means of subcutaneous, intravenous, intramuscular, or
intradermal injection. Such preparations may conveniently be
prepared by admixing the compound with water or a glycine buffer
and rendering the resulting solution sterile and isotonic with the
blood.
[0059] Formulations suitable for transdermal administration may be
presented as discrete patches adapted to remain in intimate contact
with the epidermis of the recipient for a prolonged period of time.
Formulations suitable for transdermal administration may also be
delivered by iontophoresis (see, for example, Pharmaceutical
Research 3 (6):318 (1986)) and typically take the form of an
optionally buffered aqueous solution of the active compound.
Suitable formulations comprise citrate or bis/tris buffer (pH 6) or
ethanol/water and contain from 0.1 to 0.2M active ingredient. The
therapeutically effective dosage of any one active agent, the use
of which is in the scope of present invention, will vary somewhat
from compound to compound, patient to patient, and will depend upon
factors such as the condition of the patient and the route of
delivery. Such dosages can be determined in accordance with routine
pharmacological procedures known to those skilled in the art,
particularly in light of the disclosure provided herein. In one
example, the dosage is from 1 to 10 micrograms of active compound
per Kilogram subject body weight.
[0060] In another example, where the therapeutic agent is
.sup.131I, the dosage to the patient is typically from 10 mCi to
100, 300 or even 500 mCi. Stated otherwise, where the therapeutic
agent is .sup.131I, the dosage to the patient is typically from
5,000 Rads to 100,000 Rads (preferably at least 13,000 Rads, or
even at least 50,000 Rads). Doses for other radionuclides are
typically selected so that the tumoricidal dosage is equivalent to
the foregoing range for .sup.131I.
[0061] In a preferred embodiment, the improved pharmacological
properties of the invention are utilized to improve the delivery
and/or dosage regime to the subject. For example, an improved half
live of the active agent is utilized to reduce the frequency of
dosages to the patient (e.g., one dosage or administration every
three or four days; more preferably one administration per week,
still more preferably one administration every two weeks; still
more preferably one administration per month); an improved
bioavailability is utilized to reduce the overall dosage of the
active agent administered to the patient, etc.
[0062] The present invention is explained in greater detail in the
following non-limiting examples.
EXAMPLES
[0063] The goal of this invention is to selectively deliver drugs
or imaging agents to diseased sites in order to improve therapeutic
efficacy and limit systemic toxicity.
[0064] The invention has four parts:
[0065] 1. Drug or Imaging Agent carriers: The carrier is a novel
macromolecular drug carrier, consisting of elastin-like
polypeptides (ELPs). ELPs belong to a unique class of biopolymers
that undergo an inverse temperature phase transition; they are
soluble at temperatures below their transition temperature
(T.sub.t) but become insoluble and aggregate at temperatures above
their T.sub.t[1-3].
[0066] (i) The ELP may be designed with a T.sub.t that is below the
local temperature at the diseased site so that it will aggregate at
the diseased site.
[0067] (ii) Alternatively the ELP may be designed to have a T.sub.t
that is above the diseased site so as to remain in soluble
form.
[0068] (iii) The ELP may contain sites for the covalent or
enzymatic attachment of drugs or imaging agents or targeting
moieties.
[0069] (iv) The ELP may also be designed to contain genetically
encodable targeting moieties (one or more) such as a peptide or
protein to specifically target the ELP to the diseased site or
organ.
[0070] 2. Definition of
[0071] (A) Drug: Any molecule that has therapeutic value against
any disease.
[0072] (B) Imaging agent: Any molecule that provides visualization
of the diseased site or organ
[0073] Example of the drug or imaging agent would include, though
not exclusively: (i) small molecule, (ii) radionuclide, (iii)
peptide, (iv) peptidomimetic, (v) protein, (vi) antisense
oligonucleotide, (vii) peptide nucleic acid, (viii) siRNA, (ix)
metal chelate, (x) carbohydrates.
[0074] 3. Attachment or association of drug or imaging agent. The
drug can be covalently linked to the ELP through a stable or labile
linkage scheme. The drug may be hydrophobically associated with the
ELP. The drug may be attached to the ELP through a chelation
method. The drug may be associated with the ELP through molecular
recognition through secondary bonds. The drug may also be attached
to the ELP through the action of an enzyme. In the case of
molecules such as peptides proteins that can be produced
recombinantly, the ELP and drug may be produced as a single entity
in suitable host (E. coli, Pichia pastoris, mammalian cells, or
baculovirus) from a synthetic or cloned gene. The "ELP-drug/imaging
agent conjugate" may be synthesized so that the link between the
conjugate may be stable so as to deliver the single entity as a
therapeutic or imaging agent or designed to be labile under the
action of pH or light, or the action of enzymes to liberate the
drug from the ELP.
[0075] 4. Administration: The ELP-drug conjugate or fusion protein
will be: (i) injected into the subject systemically (iv, ia, ip or
im) (ii) locally into the diseased site or organ, (iii) or
delivered orally, or (iv) parenterally.
[0076] The injected ELP-drug/imaging agent conjugate or fusion
protein will exhibit as compared to the free drug one or more of
the following: (1) enhanced solubility of the drug/imaging agent in
its conjugated form over free drug/imaging agent, increased
circulation half-life, exhibit reduced clearance from the body, or
increased bioavailability of the drug/imaging agent, resulting in
reduced dose and frequency of injection, an improved therapeutic
index or improved visualization of the diseased site or organ.
[0077] Synthesis and Characterization of ELPs.
[0078] ELPs are typically prepared by a recombinant synthesis in E.
coli. However, other hosts may be used for recombinant synthesis as
well. ELP may also be prepared by a chemical synthesis. In a
typical example of a recombinant synthesis, the polymerization
process is carried out at the gene level by a method called
recursive directional ligation (RDL), in which a synthetic gene for
a repeat sequence for the ELP (typically encoding .about.10
pentapeptides of VPGXG (SEQ ID NO.: 1)) are ligated in a
head-to-tail manner recursively. After n rounds of ligation into a
plasmid, this provides a library of n+1 ELP genes, all of which
encode the same peptide sequence, but with MWs that are multiples
of the drug.
[0079] ELP-Drug Conjugation.
[0080] An ELP containing a unique C-terminal cysteine residue is
synthesized and purified by inverse transition cycling (ITC) and
conjugated to Doxorubicin molecules through four different
pH-sensitive, maleimide-activated, hydrazone linkers. The linkers'
structures or length have little effect on the T.sub.t of the
ELP-Doxorubicin conjugates, since all conjugates' T.sub.ts are
similar to those of the native ELP (data not shown). However, the
ELP-Doxorubicin conjugates with longer linkers exhibits slower
transition kinetics than the ELP-Doxorubicin conjugates with
shorter linkers. At pH 4, the release of Doxorubicin from the
ELP-Doxorubicin conjugate with the shortest linker reached almost
80% over 72 h.
[0081] Cytotoxicity of ELP-Doxorubicin Conjugates.
[0082] An acid-labile ELP-Doxorubicin conjugate is tested for
cytotoxicity in an in vitro cell culture assay with FaDu cells. The
unconjugated ELP, the control conjugate, does not show any inherent
cytotoxicity, and thus it indicates that ELPs are non-toxic despite
of substantial internalization (FIG. 4). By contrast, the
ELP-Doxorubicin conjugate shows substantial cytotoxicity during
either 24 or 72 h, and the level of toxicity is similar to those of
an equivalent Doxorubicin concentration.
[0083] Accumulation of ELPs in Solid Tumors.
[0084] Biodistribution studies are carried out by systematically
injecting .sup.14C-labeled ELP into nude mice bearing a FaDu solid
carcinoma. The accumulation of the ELPs in implanted tumors is in
the range of 10-20% injected dose per gram (% ID/g). When an ELP
with a T.sub.t of .about.40.degree. C. is systematically injected
into a mice and implanted tumors are heated to 42.degree. C., the
accumulation is .about.20% ID/g. By contrast, when the same ELP is
injected without heating the tumors, the accumulation was
.about.10% ID/g. This data shows that a significant concentration
(% ID/g) of the radiolabeled ELP localized in the tumor even when
the tumor is not heated. By contrast, the injection of a small
radiolabeled molecule (molecular weight <500 Da) in unconjugated
form results in significantly lower accumulation in the tumor.
TABLE-US-00001 TABLE 1 List of ELP-protein conjugates synthesized
recombinantly (ELP fusion proteins), molecular weight (MW) of the
target proteins, and their yield from a 1 Liter shaker flask
culture of Escherichia coli. MW Yield Target Proteins (kDa) (mg/L)
Angiostatin (K1-3) 30.7 27 Blue fluorescent protein (BFP) 26.9 100
Calmodulin (CalM) 16.7 75 Chloramphenicol acetyltransferase (CAT)
25.7 80 Green fluorescent protein (GFP) 26.9 78-1600 Interleukin 1
receptor antagonist (IL1rRa) 17.0 50 Luciferase 60.8 10 Tissue
transglutaminase (tTg) 77.0 36 Tendamistat 7.9 22 Thioredoxin (Trx)
11.7 120
TABLE-US-00002 TABLE 2 Yield of peptide-ELP conjugates synthesized
recombinantly in E. coli. Both yield of the conjugate (fusion) and
the target peptide is shown, as well as purity as determined by
mass spectrometry. MW Yield Fusion Yield Peptide Peptide (kDa)
(mg/L culture) (mg/L culture) Purity Morphine Modulating 2.0 224 17
99% Neuropeptide (MMN) Neuropeptide Y (NPY) 2.7 222 20 98% Orexin B
3.0 320 19 91% Leptin 4.0 415 19 97% ACTH 4.6 133 19 99% Calcitonin
6.2 260 23 98%
[0085] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
Sequence CWU 1
1
1515PRTUnknownELP pentapeptide 1Val Pro Gly Xaa Gly 1 5
222PRTUnknownAnti-microbial peptide MSI-78 2Gly Ile Gly Lys Phe Leu
Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe 1 5 10 15 Val Lys Ile Leu
Lys Lys 20 310PRTUnknownELP decapeptide 3Val Pro Gly Xaa Gly Val
Pro Gly Lys Gly 1 5 10 44PRTUnknownELP tetrapeptide 4Val Pro Gly
Gly 1 54PRTUnknownELP tetrapeptide 5Gly Gly Val Pro 1
64PRTUnknownELP tetrapeptide 6Gly Gly Phe Pro 1 74PRTUnknownELP
tetrapeptide 7Gly Gly Ala Pro 1 85PRTUnknownELP pentapeptide 8Val
Pro Gly Val Gly 1 5 95PRTUnknownELP pentapeptide 9Gly Val Gly Val
Pro 1 5 105PRTUnknownELP pentapeptide 10Gly Lys Gly Val Pro 1 5
115PRTUnknownELP pentapeptide 11Gly Val Gly Phe Pro 1 5
125PRTUnknownELP pentapeptide 12Gly Phe Gly Phe Pro 1 5
135PRTUnknownELP pentapeptide 13Gly Glu Gly Val Pro 1 5
145PRTUnknownELP pentapeptide 14Gly Phe Gly Val Pro 1 5
155PRTUnknownELP pentapeptide 15Gly Val Gly Ile Pro 1 5
* * * * *