Field of the Invention
[0001] The present invention relates liposome encapsulated drugs and delivery systems, specifically
liposome encapsulated cisplatin. The drugs are useful to kill cancer cells in a variety
of human malignancies after intravenous injection.
Background of the Invention
[0002] Throughout this application various publications, patents and published patent specifications
are referenced by author and date or by an identifying patent number. Full bibliographic
citations for the publications are provided within this disclosure or immediately
preceding the claims. These publications, patents and published patent specifications
are included in order to more fully describe the state of the art to which this invention
pertains.
[0003] Cis-diamminedichloroplatinum(II), cis-[Pt(NH
3)
2Cl
2]
2+, abbreviated cisplatin or cis-DDP, is one of the most widely used antineoplastic
drugs for the treatment of testicular, ovarian carcinomas and against carcinomas of
the head and neck. More than 90% of testicular cancers are cured by cisplatin. The
most severe side effects are nephrotoxicity, bone marrow toxicity and gastrointestinal
irritation (Oliver and Mead, 1993). Bilateral optic neuropathy was observed in a patient
affected by ovarian carcinoma treated with 160 mg/m
2 cisplatin and 640 mg/m
2 carboplatin (Caraceni et al., 1997). Oral hexamethylmelamine treatment in a group
of 61 patients with epithelial ovarian carcinoma with cis- or carboplatin resistance
(relapse within 6 months after the end of that therapy) showed a 14% objective response
rate (Vergote et al., 1992).
[0004] Cationic cholesterol derivatives have been used to deliver therapeutic agents. For
example, they have been mixed with phosphatidylethanolamine and sonicated to form
small unilamellar vesicles which can complex with DNA and mediate the entry into the
cytosol from the endosome compartment. One of the liposome formulations, DC-Chol liposomes,
has been used in a gene therapy clinical trial against melanoma. Human immunodeficiency
virus-1 transactivating protein gene was-codelivered with a reporter gene under the
control of HIV-1 long terminal repeat. Human tumor cells selected for cisplatin resistance
or isolated from patients who had failed cisplatin therapy were highly transfectable
with cationic liposomes._ These results suggested a serial therapy protocol with cisplatin
and gene therapy for malignancy (Farhood et al., 1997).
[0005] Various platinum complexes prepared from 2-amino-methylpyrrolidine derivatives as
carrier ligands were tested for their antitumor activity against Colon 26 carcinoma
and P388 leukemia using subcutaneous and/or intraperitoneal injections in mice. 2-aminomethylpymolidine
proved to be the most effective carrier ligand in its amine derivatives (Morikawa
et al., 1990).
[0006] An optimum procedure was established by orthogonal test for preparing cisplatin albumin
microspheres (Cis-DDP-AMS) with emulsion-heating stabilization method (mean size was
148 µm). The distribution and elimination half times of platinum were prolonged 3.36
times and 1.23 times after hepatic arterial chemoembolization with Cis-DDP-AMS versus
Cis-DDP, respectively (Zhang et al., 1995).
[0007] The search for platinum (II)-based compounds with improved therapeutic properties
was prompted to design and synthesize a new family of water-soluble, third generation
cis-diaminedichloroplatinum (II) complexes linked to uracil and uridine. However,
none of the synthesized compounds showed any significant cytotoxic activity against
three cell lines that were treated (Kim et al., 1998).
[0008] The recently developed bioreductive agent 4-[3-(2-nitroimidazolyl)-propylamino]-7-chloroquinoline
hydrochloride (NLCQ-1) was found to potentiate the antitumor effect of the chemotherapeutic
agents melphalan (L-PAM), cisplatin (cisDDP) and cyclophosphamide (CPM) without concurrent
enhancement in bone marrow toxicity. Potentiation was strictly schedule dependent
and the optimum effect (1.5 to 2 logs killing beyond additivity) was observed when
NLCQ-1 was given 45-min before cisDDP. These results support the classification ofNLCQ-1,
based on clinical studies, as a chemosensitizer (Papadopoulou et al., 1998).
[0009] A combination ofpaciltaxel with cisplatin as second-line treatment in patients with
non-small cell lung cancer (NSCLC) who had previously undergone first-line therapy
with cisplatin achieved partial response (40%) in 14 patients (Stathopoulos et al.,
1999).
[0010] Abra et al. (U.S. Patent No. 5,945,122, issued August 31, 1999) describes a liposome
composition containing entrapped non-charged cisplatin in mostly neutral lipids. However,
the process of Abra et al. uses neutral lipids compared with the anionic lipid DPPG
disclosed in the present patent for cisplatin entrapment.
[0011] US Patent No. 5,567,434 (Szoka, Jr.) describes liposome and lipidic particle formulations
prepared by dissolving compounds (e.g., cisplatin) in a solution of liposome-forming
lipids in an aprotic solvent such as DMSO, optionally containing a lipid-solubilizing
amount of a lower alkanol, and either injecting the resulting solution into an aqueous
solution, or an aqueous solution into the resulting solution. The cisplatin is not
in its aqua form.
[0012] Speelmans et al., Biochimica et Biophysica Acta, 1996, Vol. 1283, pp. 60-66, describes
studies of the interaction between cisplatin and phospholipids of model membranes.
[0013] US Patent No. 5,945,122 (Abra et al.) describes a liposome composition containing
an entrapped cisplatin compound, as well as a method of forming such compositions
by heating an aqueous solution of cisplatin to increase its solubility (e.g, by 2
to 8 times) and then adding vesicle forming lipids.
[0014] US Patent No. 5,795,589 (Mayer et al.) describes methods for encapsulation of antineoplatic
agents in liposomes which exploits an active mechanism using a transmembrane ion gradient,
preferably a transmembrane pH gradient.
[0015] US Patent No. 5,908,777 (Lee et al.) describes methods of preparing a lipidic vector,
for delivery of nucleic acid and other molecules of therapeutic value which involve
bringing the molecule into contact with a polycation (e.g., polylysine hydrobromide
salt), thereby forming a complex, and then mixing the complex with an anionic lipidic
preparation.
[0016] US Patent No. 5,747,469 (Roth et al.) describes the use of tumor suppressor genes
(e.g., p53) in combination with a DNA damaging agent (e.g., cisplatin) for use in
killing cells and in particular cancerous cells.
[0017] US Patent No. 5,882,679 (Needham) describes liposomes which contain active agents
(e.g., paclitaxel) that are aggregated with a lipid surfactant and the liposome membrane
is formulated to resist the disruptive effects of the aggregate contained therein.
The liposome membrane contains polyethylene glycol in an amount sufficient to inhibit
fusion of the membrane with the active agent/surfactant aggregate entrapped therein.
[0018] Thus, while the prior reports indicate that liposome mediated delivery of cisplatin
and other therapeutic drugs is possible, therapeutic efficiency has been limited by
the low aqueous solubility and low stability of cisplatin. Therapeutic efficacy also
is limited by the high toxicity of the drug. Thus, a need exists to reduce the difficulties
involved in processing of cisplatin containing drugs and high toxicity of cisplatin
when used therapeutically. This invention satisfies this need and provides related
advantages as well.
SUMMARY OF THE INVENTION
[0019] One aspect of the present invention pertains to a method for producing cisplatin
micelles, comprising: a) combining: (i) cisplatin; and (ii) a phosphatidyl glycerol
lipid derivative in a cisplatin to lipid derivative molar ratio range of 1:1 to 1:2
to form a cisplatin mixture; and, b) combining the cisplatin mixture of step a) with
an effective amount of at least a 30% ethanol solution to form cisplatin micelles,
in which cisplatin is in its aqua form.
[0020] Another aspect of the present invention pertains to a method for producing cisplatin
micelles, comprising: a) combining: (i) cisplatin; and (ii) an effective amount of
at least a 30% ethanol solution; to form a cisplatin/ethanol solution; and, b) combining
the cisplatin/ethanol solution of step a) with a phosphatidyl glycerol lipid derivative
in a cisplatin to lipid derivative molar ratio range of 1:1 to 1:2 to form cisplatin
micelles, in which cisplatin is in its aqua form.
[0021] In one embodiment, the phosphatidyl glycerol lipid derivative is selected from: dipalmitoyl
phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl glycerol (DMPG), dicaproyl
phosphatidyl glycerol (DCPG), distearoyl phosphatidyl glycerol (DSPG), and dioleyl
phosphatidyl glycerol (DOPG).
[0022] In one embodiment, the molar ratio is 1:1.
[0023] In one embodiment, the method further comprises the step of combining with the mixture
or solution of step a) an effective amount of: a free fusogenic peptide, a fusogenic
peptide-lipid conjugate, or a fusogenic peptide-PEG-hydrogenated soy phosphatidylcholine
(HSPC) conjugate; wherein the fusogenic peptide is derivatized with a stretch of 1-6
negatively-charged amino acids at the N- or C-terminus and is thus able to bind electrostatically
to cisplatin in its aqua form .
[0024] In one embodiment, the free fusogenic peptide or fusogenic peptide lipid conjugate
comprises dioleyl phosphatidyl ethanolamine (DOPE) or DOPE/cationic lipid.
[0025] In one embodiment, the method further comprises the step of removal of the ethanol
from the cisplatin micelles.
[0026] In one embodiment, removal of the ethanol is by dialysis of the micelles through
permeable membranes to remove the ethanol.
[0027] Another aspect of the present invention pertains to a cisplatin micelle obtainable
by a method described above.
[0028] Another aspect of the present invention pertains to a method for encapsulating cisplatin
micelles, comprising mixing an effective amount of a vesicle-forming lipid with the
cisplatin micelles obtained by a method described above.
[0029] In one embodiment, the lipid is selected from premade neutral liposomes, composed
of cholesterol 10-60%, hydrogenated soy phosphatidylcholine (HSPC) 40-90%, and polyethyleneeglycol
hydrogenated soy phosphatidylcholine (PEG-HSPC) 1-7% or lipids in solution, lipids
in powder and polyethyleneglycol distearoyl phosphatidylethanolamine (PEG-DSPE).
[0030] In one embodiment, the lipid comprises 10-60% cholesterol.
[0031] Another aspect of the present invention pertains to encapsulated cisplatin obtainable
by the methods described above.
[0032] Another aspect of the present invention pertains to a method for obtaining a cisplatin/lipid
complex capable of evading macrophages and cells of the immune system when administered
to a subject, the method comprising mixing an effective amount of the cisplatin micelles
described above with an effective amount of polyethyleneglycol distearoyl phosphatidylethanolamine
(PEG-DSPE), polyethyleneglycol distearoyl phosphatidylcholine (PEG-DSPC) or hyaluronic
acid-distearoyl phosphatidylethanolamine.
[0033] Another aspect of the present invention pertains to encapsulated cisplatin obtainable
by the method described above.
[0034] Another aspect of the present invention pertains to a composition comprising the
encapsulated cisplatin described above and encapsulated oligonucleotides, ribozymes,
or polynucletotides.
[0035] Another aspect of the present invention pertains to a composition comprising the
encapsulated cisplatin as described above and a drug selected from: doxorubicin, fluorodeoxyuridine,
bleomycin, adriamycin, vinblastin, prednisone, vincristine, and taxol.
[0036] Another aspect of the present invention pertains to a method for delivering cisplatin
to a cell, in vitro, comprising contacting the cell with encapsulated cisplatin described
above.
[0037] Another aspect of the present invention pertains to encapsulated cisplatin described
above for use in a method of treatment of the human or animal body.
[0038] Another aspect of the present invention pertains to encapsulated cisplatin described
above for use in a method of treatment to inhibit the growth of a tumor in the human
or animal body.
[0039] Another aspect of the present invention pertains to encapsulated cisplatin described
above for use in a method of treatment to target solid tumors and metastases by intravenous
administration in the human or animal body.
[0040] Another aspect of the present invention pertains to encapsulated cisplatin described
above, and a gene selected from the group consisting of p53, pax5 and HSV-tk genes,
for use in a method of treatment to inhibit the growth of a tumor in the human or
animal body.
[0041] Another aspect of the present invention pertains to encapsulated cisplatin described
above; a gene selected from the group consisting of p53, pax5 and HSV-tk genes; and
an effective amount of encapsulated ganciclovir; for use in a method of treatment
to inhibit the growth of a tumor in the human or animal body
[0042] Another aspect of the present invention pertains to encapsulated cisplatin described
above, and a gene selected from the group consisting of p53, pax5 and HSV-tk genes,
for use in a method of treatment to inhibit the growth of a tumor in the human or
animal body, wherein the genes to be combined with cisplatin are any of, or combinations
of encapsulated IL-2, IL-4, IL-7, IL-12, GM-CSF, IFN-gamma, TNF-alpha, RB, BRCA1,
E1A, cytosine deaminase in combination with encapsulated 5-fluorcytosine, bcl-2, MDR-1,
p21, p16, bax, bcl-xs, E2F, IGFI, VEGF, and TGF-beta.
[0043] Another aspect of the present invention pertains to use of encapsulated eisplatin
described above for the manufacture of a medicament for use in the treatment to inhibit
the growth of a tumor in the human or animal body.
[0044] Another aspect of the present invention pertains to use of encapsulated cisplatin
described above for the manufacture of a medicament for use in the treatment by intravenous
administration to target solid tumors and metastases in the human or animal body.
[0045] Another aspect of the present invention pertains to use of encapsulated cisplatin
described above, and a gene selected from the group consisting of p53, pax5 and HSV-tk
genes, for the manufacture of a medicament for use in the treatment to inhibit the
growth of a tumor in the human or animal body.
[0046] Another aspect of the present invention pertains to use of encapsulated cisplatin
described above; a gene selected from the group consisting of p53, pax5 and HSV-tk
genes; and an effective amount of encapsulated ganciclovir; for the manufacture of
a medicament for use in the treatment to inhibit the growth of a tumor in the human
or animal body.
[0047] Another aspect of the present invention pertains to use of encapsulated cisplatin
described above, and a gene selected from the group consisting of p53, pax5 and HSV-tk
genes, for the manufacture of a medicament for use in the treatment to inhibit the
growth of a tumor in the human or animal body, wherein the genes to be combined with
cisplatin are any of, or combinations of encapsulated IL-2, IL-4, IL-7, IL-12, GM-CSF,
IFN-gamma, TNF-alpha, RB, BRCA1, E1A, cytosine deaminase in combination with encapsulated
5-fluorcytosine, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGFI, VEGF, and TGF-beta.
[0048] This invention provides a method for encapsulating cisplatin and other positively-charged
drugs into liposomes having a different lipid composition between their inner and
outer membrane bilayers and able to reach primary tumors and their metastases after
intravenous injection to animals and humans. In one aspect, the method includes complex
formation between cisplatin with DPPG (dipalmitoyl phosphatidyl glycerol) or other
lipid molecules to convert cisplatin to its aqua form by hydrolysis which is positively-charged
and is the active form of cisplatin endowed with the antineoplastic activity. At this
stage membrane fusion peptides and other molecules with fusogenic properties may be
added to improve entrance across the cell membrane of the complex. The aqua cisplatin-DPPG
micelles are converted into liposomes by mixing with vesicle forming lipids such as
pre-made liposomes or lipids followed by dialysis and extrusion through membranes,
entrapping and encapsulating cisplatin to a very high yield. Doxorubicin or other
positively-charged compounds can be substituted for cisplatin in these formulations.
The encapsulated cisplatin has a high therapeutic efficacy in eradicating a variety
of solid human tumors including but not limited to breast carcinoma and prostate carcinoma.
Combination of the encapsulated cisplatin with encapsulated doxorubicin or with other
antineoplastic drugs are claimed to be of therapeutic value. Also of therapeutic value
in cancer eradication are claimed to be combinations of encapsulated cisplatin with
a number of anticancer genes including but not limited to p53, IL-2, IL-12, angiostatin,
and oncostatin encapsulated into liposomes as well as combinations of encapsulated
cisplatin with HSV-tk plus encapsulated ganciclovir.
Brief Description of the Figures
[0049]
Figure 1 depicts cisplatin encapsulation.
Figure 2 shows MCF-7 tumor regression in SCID mice after 3-4 injections of encapsulated
cisplatin.
Figure 3 shows histology of tumors in SCID mice with or without treatment with cisplatin.
Figure 3A shows untreated MCF-7 tumors grown in SCID mice. 40X magnification. Notice
the homogeneous pattern of structures characteristic of tumor tissue. Figure 3B shows
cisplatin-treated mice (4 injections). Cells are apoptotic, there are groups of cells
into structures and nuclei stain bigger and darker, characteristic of apoptotic cells.
Figure 3C shows tumors from untreated animals showing invasion to muscle. 20X. Figure
3D shows cisplatin-treated mice. Invasion is not evident. 20X magnification.
Figure 4 shows macroscopic (visual) difference in tumor size between an animal treated
with encapsulated cisplatin (A) and an untreated animal (B)).
Modes for Carrying Out the Invention
[0050] The practice of the present invention will employ, unless otherwise indicated, conventional
techniques of immunology, molecular biology, microbiology, cell biology and recombinant
DNA. These methods are described in the following publications. See,
e.g., Sambrook, et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS
IN ENZYMOLOGY (Academic Press, Inc.); "PCR: A PRACTICAL APPROACH" (M. MacPherson,
et al., IRL Press at Oxford University Press (1991)); PCR 2: A PRACTICAL APPROACH
(M.J. MacPherson, B.D. Hames and G.R Taylor eds. (1995)); ANTIBODIES, A LABORATORY
MANUAL (Harlow and Lane, eds. (1988)); and ANIMAL CELL CULTURE (R.I. Freshney, ed.
(1987)).
[0051] As used in the specification and claims, the singular form "a," "an" and "the" include
plural references unless the context clearly dictates otherwise. For example, the
term "a cell" includes a plurality of cells, including mixtures thereof
[0052] The term "comprising" is intended to mean that the compositions and methods include
the recited elements, but not excluding others. "Consisting essentially of" when used
to define compositions and methods, shall mean excluding other elements of any essential
significance to the combination. Thus, a composition consisting essentially of the
elements as defined herein would not exclude trace contaminants from the isolation
and purificationmethod and pharmaceutically acceptable carriers, such as phosphate
buffered saline, preservatives, and the like. "Consisting of shall mean excluding
more than trace elements of other ingredients and substantial method steps for administering
the compositions of this invention. Embodiments defined by each of these transition
terms are within the scope of this invention.
[0053] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to
refer to polymeric forms of nucleotides of any length. The polynucleotides may contain
deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides may have
any three-dimensional structure, and may perform any function, known or unknown. The
term "polynucleotide" includes, for example, single-, double-stranded and triple helical
molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,
recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated
DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
A nucleic acid molecule may also comprise modified nucleic acid molecules.
[0054] A "gene" refers to a polynucleotide containing at least one open reading frame that
is capable of encoding a particular polypeptide or protein after being transcribed
and translated.
[0055] A "composition" is intended to mean a combination of active agent and another compound
or composition, inert (for example, a detectable agent or label or a pharmaceutically
acceptable carrier) or active, such as an adjuvant.
[0056] A "pharmaceutical composition" is intended to include the combination of an active
agent with a carrier, inert or active, making the composition suitable for diagnostic
or therapeutic use
in vitro, in vivo or
ex vivo.
[0057] As used herein, the term "pharmaceutically acceptable carrier" encompasses any of
the standard pharmaceutical carriers, such as a phosphate buffered saline solution,
water, and emulsions, such as an oil/water or water/oil emulsion, and various types
of wetting agents. The compositions also can include stabilizers and preservatives.
For examples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'S PHARM.
SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).
[0058] An "effective amount" is an amount sufficient to effect beneficial or desired results.
An effective amount can be administered in one or more administrations, applications
or dosages.
[0059] A "subject," "individual" or "patient" is used interchangeably herein, which refers
to a vertebrate, preferably a mammal., more preferably a human. Mammals include, but
are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
[0060] A "control" is an alternative subject or sample used in an experiment for comparison
purpose. A control can be "positive" or "negative." For example, where the purpose
of the experiment is to determine a correlation of an altered expression level of
a gene with a particular type of cancer, it is generally preferable to use a positive
control (a subject or a sample from a subject, carrying such alteration and exhibiting
syndromes characteristic of that disease), and a negative control (a subject or a
sample from a subject lacking the altered expression and clinical syndrome of that
disease).
[0061] A "gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotides
into a host cell. Examples of gene delivery vehicles are liposomes, cationic liposomes,
viruses, such as baculovirus, adenovirus, adeno-associated virus, and retrovirus,
bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically
used in the art which have been described for expression in a variety of eukaryotic
and prokaryotic hosts, and may be used for gene therapy as well as for simple protein
expression.
[0062] A "viral vector" is defined as a recombinantly produced virus or viral particle that
comprises a polynucleotide to be delivered into a host cell, either
in vivo, ex vivo or
in vitro. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated
virus vectors and the like. In aspects where gene transfer is mediated by a retroviral
vector, a vector construct refers to the polynucleotide comprising the retroviral
genome or part thereof, and the inserted polynucleotide. As used herein, "retroviral
mediated gene transfer" or "retroviral transduction" carries the same meaning and
refers to the process by which a gene or nucleic acid sequences are stably transferred
into the host cell by virtue of the virus entering the cell and integrating its genome
into the host cell genome. The virus can enter the host cell via its normal mechanism
of infection or be modified such that it binds to a different host cell surface receptor
or ligand to enter the cell. As used herein, retroviral vector refers to a viral particle
capable of introducing exogenous nucleic acid into a cell through a viral or viral-like
entry mechanism.
[0063] Retroviruses carry their genetic information in the form ofRNA; however, once the
virus infects a cell, the RNA is reverse-transcribed into the DNA form, which integrates
into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.
In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus
(Ad) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide
comprising the viral genome or part thereof, and a polynucleotide to be inserted.
Adenoviruses (Ads) are a relatively well characterized, homogenous group of viruses,
including over 50 serotypes. (see,
e.g., WO 95/27071). Ads are easy to grow and do not require integration into the host
cell genome. Recombinant Ad-derived vectors, particularly those that reduce the potential
for recombination and generation of wild-type virus, have also been constructed. (see,
WO 95/00655; WO 95/11984). Wild-type AAV has high infectivity and specificity integrating
into the host cells genome. (Hermonat and Muzyczka (1984) PNAS USA 81:6466-6470; Lebkowski,
et al. (1988) Mol. Cell. Biol. 8:3988-3996).
[0064] Vectors that contain both a promoter and a cloning site into which a polynucleotide
can be operatively linked are well known in the art. Such vectors are capable of transcribing
RNA
in vitro or
in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and
Promega Biotech (Madison, WI). In order to optimize expression and/or
in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated
portions of the clones to eliminate extra, potential inappropriate alternative translation
initiation codons or other sequences that may interfere with or reduce expression,
either at the level of transcription or translation. Alternatively, consensus ribosome
binding sites can be inserted immediately 5' of the start codon to enhance expression.
[0065] Gene delivery vehicles also include several non-viral vectors, including DNA/liposome
complexes, and targeted viral protein DNA complexes. Liposomes that also comprise
a targeting antibody or fragment thereof can be used in the methods of this invention.
To enhance delivery to a cell, the nucleic acid or proteins of this invention can
be conjugated to antibodies or binding fragments thereof which bind cell surface antigens,
e.g., TCR, CD3 or CD4.
[0066] Polynucleotides are inserted into vector genomes using methods well known in the
art. For example, insert and vector DNA can be contacted, under suitable conditions,
with a restriction enzyme to create complementary ends on each molecule that can pair
with each other and be joined together with a ligase. Alternatively, synthetic nucleic
acid linkers can be ligated to the termini of restricted polynucleotide. These synthetic
linkers contain nucleic acid sequences that correspond to a particular restriction
site in the vector DNA. Additionally, an oligonucleotide containing a termination
codon and an appropriate restriction site can be ligated for insertion into a vector
containing, for example, some or all of the following : a selectable marker gene,
such as the neomycin gene for selection of stable or transient transfectants in mammalian
cells; enhancer/promoter sequences from the immediate early gene of human CMV for
high levels of transcription; transcription termination and RNA processing signals
from SV40 for mRNA stability; SV40 polyoma origins of replication and ColE1 for proper
episomal replication; versatile multiple cloning sites; stabilizing elements 3' to
the inserted polynucleotide, and T7 and SP6 RNA promoters for
in vitro transcription of sense and antisense RNA. Other means are well known and available
in the art.
[0067] "Host cell" is intended to include any individual cell or cell culture which can
be or have been recipients for vectors or the incorporation of exogenous polynucleotides,
polypeptides and/or proteins. It also is intended to include progeny of a single cell,
and the progeny may not necessarily be completely identical (in morphology or in genomic
or total DNA complement) to the original parent cell due to natural., accidental.,
or deliberate mutation. The cells may be prokaryotic or eukaryotic, and include but
are not limited to bacterial cells, yeast cells, plant cells, insect cells, animal
cells, and mammalian cells, e.g., murine, rat, simian or human.
[0068] As used herein, the terms "neoplastic cells," "neoplasia," "tumor," "tumor cells,"
"cancer" and "cancer cells," (used interchangeably) refer to cells which exhibit relatively
autonomous growth, so that they exhibit an aberrant growth phenotype characterized
by a significant loss of control of cell proliferation (i.e., de-regulated cell division).
Neoplastic cells can be malignant or benign.
[0069] "Suppressing" tumor growth indicates a growth state that is curtailed when compared
to control cells. Tumor cell growth can be assessed by any means known in the art,
including, but not limited to, measuring tumor size, determining whether tumor cells
are proliferating using a
3H-thymidine incorporation assay, or counting tumor cells. "Suppressing" tumor cell
growth means any or all of the following states: slowing, delaying, and stopping tumor
growth, as well as tumor shrinkage.
Embodiments of the Invention
Micelles, Liposomes and Processes for Obtaining Them
[0070] Described herein is a new method for entrapping cisplatin into lipids which enhances
the content of cisplatin per volume unit, reduces its toxicity, is able to target
primary tumors and their metastases after intravenous injection, and shows shrinkage
of tumors and complete therapy of SCID mice bearing human tumors.
[0071] Cisplatin is a heavy metal complex containing two chloride atoms and two amino groups
in the cis position attached to one atom of the transitory heavy metal platinum in
its divalent form. It is a bifunctional alkylating agent as well as DNA intercalator
inhibiting DNA synthesis. In one form, cisplatin is a yellow powder of a molecular
weight of 300.1 and of limited solubility of 1 mg/ml in water. It is widely used for
the treatment of cancer patients, especially those of testicular, lymphomas, endometrial,
bladder, ovarian, head and neck squamous cell carcinomas, breast carcinomas, and many
other malignancies, often in combination with adriamycin, vinblastin, bleomycin, prednisone,
vincristine, taxol, and others antineoplastic drugs as well as radiation therapy.
The methods described herein facilitate a reduction in total cisplatin volume required
for patient treatment because of an increase in its solubility in its lipid entrapment
form.
[0072] The volume used for intravenous injection is usually large (about 180 ml per adult
patient or about 20-120 mg/m
2) administered as a 24-hour infusion. It is cleared from the plasma in a rapid phase
of 25-80 min followed by a slower secondary phase of 58-73 h; it is bound by plasma
proteins and excreted by the kidneys (explaining the severe kidney toxicity in treated
patients). Dose related nephrotoxicity can be partially overcome with vigorous hydration,
mannitol, furosemide and other drugs. Other toxicities incurred by cisplatin include
ototoxicity, nausea and vomiting, anemia, and mild myelosuppression (The Merck Manual
of Diagnosis and Therapy). The present invention overcomes the limitations of prior
art processes and compositions.
[0073] Described herein are methods for producing cisplatin micelles, by combining cisplatin
and a phosphatidyl glycerol lipid derivative (PGL derivative) in a range of 1:1 to
1:2.1 to form a cisplatin mixture. In alternative embodiments, the range of cisplatin
to PGL derivative is in the ranges 1:1.2; or 1:1.4; or 1:1.5; or 1:1.6; or 1:1.8 or
1:1.9 or 1:2.0 or 1:2.1. The mixture is then combined with an effective amount of
at least a 20% organic solvent such as an ethanol solution to form cisplatin micelles.
[0074] As used herein, the term "phosphatidyl glycerol lipid derivative (PGL derivative)"
is any lipid derivative having the ability to form micelles and have a net negatively
charged head group. This includes but is not limited to dipalmitoyl phosphatidyl glycerol
(DPPG), dimyristoyl phosphatidyl glycerol, and dicapryl phosphatidyl glycerol. In
one aspect, phosphatidyl derivatives include those with a carbon chain of 10 to 28
carbons and having unsaturated side aliphatic side chain. The complexation of cisplatin
with negatively-charged phosphatidyl glycerol lipids having variations in the molar
ratio giving the particles a net positive (1:1) neutral (1:2) or slightely negative
(1:2.1) charge will allow targeting of different tissues in the body after administration.
However, complexing of cisplatin with negatively changed PGL has been shown to enhance
the solubility of cisplatin, thus reducing the volume of the drug required for effective
antineoplastic therapy. In addition, the complexation of cisplatin and negatively
charged PGL proceeds to very high encapsulation efficiency minimizing drug loss during
the manufacturing process. These complexes are stable, do not form precipitates and
retain therapeutic efficacy after storage at 4°C for at least 4 months.
[0075] As used herein, the term "cisplatin" included analogs. Examples include carboplatin,
ormaplatin, oxaplatin, 2-aminomethylpyrrolidine (1,1-cyclobutane dicarboxylato)platinum,
lobaplatin, 1-cyclobutane-dicarboxylato(2-)-(2-methyl-1,4-butanediamine-N,N')platinum,
zeniplatin, enloplatin, 254-S nedaplatin and JM-216 (bis-acetato-amine-dichlorocyclohexylamine-platinum(IV)).
[0076] It is to be understood, although not always explicitly stated, that other positively
charged drugs, including but not limited to the antineoplastic drug doxorubicin can
be substituted for cisplatin. Alternatively, other types of drugs that are neutral
can be used upon their conversion into positively charged drugs by derivation with
positively charged groups. Modification of a neutral or negatively-charged anticancer
or other type of drug to a positively-charged molecule can be accomplished by a number
of methods well established in organic synthesis. This can be achieved for example
by replacing a hydroxyl group in the drug with an amino group or by a trimethylamino
group thus introducing a positive charge to the compound. Replacement of a ring hydroxyl
group with an amino group is discussed in United States Patent 5,837,868, Wang, et
at. isssued November 17,1998.
[0077] The above method does not require that the steps be performed in the order indicated
above. For example, the method can be practiced by combining cisplatin with an effective
amount of at least a 20% organic solvent solution to form a solution. The solution
is combined with a phosphatidyl glycerol lipid (PGL) derivative in a range of 1:1
to 1:2.1 to form a cisplatin micelle. As above, the range of cisplatin to PGL derivative
is in the ranges 1:1.2; or 1:1.4; or 1:1.5; or 1:1.6; or 1:1.8 or 1:1.9 or 1:2.0 or
1:2.1.
[0078] Any organic solvent or formulation of ethanol, or any other alcohol that does not
form a two phase system, or other organic solvent (i.e., choroform), that is miscible
in 20% alcohol, is useful in the methods described herein. For example, the alcohol
solution can be any of at least 30%, 35%, 40%, 45% up to 90%, including any increment
in between. Preferably, the alcohol solution is 30% ethanol for DPPG, and for other
lipids the optimal percentage may be different.
[0079] In one embodiment, partial replacement of DPPG molecules by peptides with a net negative
charge gives to cisplatin complexes having fusogenic properties able to cross the
cell membrane of the target. Fusogenic peptides may also be covalently attached at
the free and of PEG for their better exposure. Addition of a small amount of cationic
lipids replacing positive charges of aqua cisplatin at the final cisplatin/DPPG complex
also endows the complex with fusogenic properties; the percentage of positive charges
to be substituted by cationic lipids (
e.g., DDAB, dimethyldioctadecyl ammonium bromide; DMRIE: N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)
ammonium bromide; DMTAP: 1,2-dimyristoyl-3-trimethylammonium propane; DOGS: Dioctadecylamidoglycylspennine;
DOTAP: N-(1-(2,3-dioleoyloxy)propyl)-N,N,N trimethylammonium chloride; DPTAP: 1,2-dipalmitoyl-3-trimethylammonium
propane; DSTAP: 1,2-disteroyl-3-trimethylammonium propane) is small because of the
toxicity of cationic lipids. Some formulations contain an amount of the fusogenic
amphiphilic lipid DOPE in the micelles.
[0080] In a further aspect, the cisplatin micelles are encapsulated into vesicle forming
lipids, for example, for use in drug delivery.
[0081] The lipid encapsulated cisplatin has a high therapeutic efficacy in eradicating a
variety of solid human tumors including but not limited to breast carcinoma, prostate
carcinoma, glioblastoma multiform, non-small lung-cell-carcinoma, pancreatic carcinoma,
head and neck squamous cell carcinoma and T-cell lymphomas. Described herein is a
method for the treatment of a variety of human malignancies using an encapsulated
cisplatin or alternatively, other positively-charged antineoplastic drugs into liposomes
having a different lipid composition between their inner and outer membrane bilayers.
The liposome encapsulated drugs are able to reach primary tumors and their metastases
after intravenous injection to animals and humans.
[0082] A combination of the encapsulated cisplatin with doxorubicin or with other antineoplastic
drugs have a higher therapeutic efficacy than cisplatin alone. Also of higher therapeutic
efficacy in cancer eradication are combinations of encapsulated cisplatin with a number
of anticancer genes including but not limited to p53, IL-2, IL-12, angiostatin, and
oncostatin encapsulated into similar type of liposomes as well as combinations of
encapsulated cisplatin with HSV-tk plus encapsulated ganciclovir.
[0083] Also described herein is a combination of the encapsulated cisplatin with tfie following
genes:
(i) A wild-type (wt) p53 cDNA expression vector under control of the CMV, beta-actin,
or other promoters, and human origins of replication able to sustain long term expression
of the p53 gene; viral origins of replication which require viral replication initiator
proteins such as T antigen for their activation are nor suitable for the transfer
of the p53 gene because p53 protein interacts strongly with T antigen.
(ii) A PAX5 cDNA expression vector, the only suppressor of the p53 gene known (both
of the wt and mutant p53 genes) interacting with a short (10 nucleotide) regulatory
region within intron 1 of the p53 gene. A major drawback in p53 gene therapy is the
inactivation of the wt p53 protein by the endogenous mutated forms of p53 which are
overexpressed in tumors and which are able to tetramerize with wt p53 protein; the
endogenous p53 genes will be suppressed by expression ofPax5, a potent transcriptional
repressor of the p53 gene. The wt p53 cDNA vector lacks intron 1 and by consequence
the suppressive PAX5 binding region. It is important to suppress the endogenous mutant
p53 gene expression and eliminate mutant p53 from the cancer cells to potentiate induction
of apoptosis and tumor suppression.
(iii) The herpes simplex virus thymidine kinase (HSV-tk) gene. The herpes simplex
virus thymidine kinase (HSV-tk) suicide gene will be also included in combinations
of p53 and Pax5 genes causing interruption in DNA synthesis after ganciclovir (GCV)
treatment of the animal model and human patient; this is expected to increase the
strand-breaks in the cancer cells and to potentiate the tumor suppressor functions
of p53 known to bind to strand breaks and to damaged DNA sites. In a further embodiment,
ganciclovir is combined and encapsulated into liposomes.
[0084] Gene therapy is a new era of biomedical research aimed at introducing therapeutic
genes into somatic cells of patients (reviewed by Boulikas, 1998a; Martin and Boulikas,
1998). Two major obstacles prohibit successful application of somatic gene transfer.
(1) the small percentage of transduced cells and (2) the loss of the transcription
signal of the therapeutic gene after about 3-7 days from injection in vivo.
[0085] The first problem arises (i) from inability of delivery vehicles carrying the gene
to reach the target cell surface (the vast majority of liposome-DNA complexes are
eliminated from blood circulation rapidly); (ii) from difficulty to penetrate the
cell membrane and (iii) to release the DNA from endosomes after internalization by
cells; (iv) from inefficient import into nuclei. Others have used stealth liposomes
(Martin and Boulikas, 1998a), which persist___ in circulation for days and concentrate
in tumors. However, classical stealth liposomes are not taken up by cancer cells.
Disclosed herein are strategies that are designed to enhance liposome internalization
(fusogenic-peptides).
[0086] The second problem results from the loss of the plasmids in the nucleus by nuclease
degradation and failure to replicate autonomously leading to their dilution during
cell proliferation among progeny cells or by inactivation of the foreign DNA after
integration into the chromosomes of the host cell. However, the use of human sequences
able to sustain extrachromosomal replication of plasmids for prolonged periods (see
U.S. Patent No. 5,894,060 on "Cloning method for trapping human origins of replication"
by Teni Boulikas will overcome this limitation.
[0087] Also described herein are tumor regression and reduction in tumor mass volume of
breast, prostate and other cancers in animal models and in humans after delivery of
encapsulated cisplatin (termed Lipoplatin™) or encapsulated doxorubicin, and combinations
of these drugs with genes including but not limited to the p53, PAX5, and HSV-tk/encapsulated
ganciclovir, IL-2, IL-12, GM-CSF, angiostatin, IL-4, IL-7, IFN-gamma, TNF-alpha, RB,
BRCA1, E1A, cytosine deaminiase in combination with encapsulated 5-fluorocytosine,
bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF, TGF-beta genes and the like.
[0088] Also described herein is a method for delivering cisplatin or other therapeutic agent
to a cell comprising contacting the cell with the encapsulated drugs or other intermediate,
obtainable by the methods ofthis invention. Also described herein is a method for
inhibiting the growth of a tumor in a subject, comprising administering to the subject
an effective amount of the encapsulated drugs obtainable by the methods of described
herein. The methods can be practiced
in vitro, ex vivo or
in vivo.
[0089] Also described herein is combination therapy using encapsulated drugs and polynucleotides.
As used herein, a polynucleotide includes but is not limited to genes encoding proteins
and polypeptides as well as sequences encoding ribozymes and antisense. The combination
therapy is more effective in eradicating cancer than either treatment alone because
the two mechanisms are different and can achieve a synergism. For example, the property
of p53 protein to bind to damaged DNA regions and free ends of DNA is known and also
to trigger the mechanism of apoptosis in severely-damaged cells (reviewed by Boulikas,
1998a). Free ends of DNA in cancer cells are expected to be produced after damage
by cisplatin enhancing the induction of an apoptotic pathway in these cells by the
expression of the transferred wt p53 (many tumors have mutated p53 and might be unable
to induce effectively this pathway). The therapeutic polynucleotide also can be inserted
into a gene transfer vector prior to incorporation into the micelle.
[0090] Transfer of the Wild-type p53 gene has been successfully used to slow-down tumor
cell proliferation in vivo and in cell culture in numerous studies. Intratumoral injection
using, adenoviral/p53 vectors has been shown to be effective against lung tumors in
recent clinical trials (Roth et al., 1996) and against prostate tumors on animal models
(reviewed by Boulikas, 1998a). The intratumoral injection method, however, may not
be applicable to metastases often associated with late stages of cancer. Systemic
delivery of the p53 gene with encapsulated cisplatin and targeting of tumors in any
region of the body is an effective treatment for cancer cure. Described herein are
strategies for ameliorating or partially overcoming the four main obstacles for successful
somatic gene transfer using liposomal delivery of the wt p53, pax5, HSV-tk, GM-CSF,
IL-12., IL-2, lL-4, IL-7, IFN-gamma, TNF-alpha, RB, BRCA1, E1A, cytosine deaminiase
in combination with encapsulated 5-fluorocytosine, bcl-2, MDR-1, p21, p16, bax, bcl-xs,
E2F, IGF-I VEGF, TGF-beta, angiostatin and other genes in combination with encapsulated
cisplatin and other drugs to a variety of human cancers in animal models and in patients
to be tested in clinical trials. These include: (i) concentration and encapsulation
of the drug and gene bullets into liposomes reducing their toxicity; (ii) targeting
of solid tumors and metastases by coating of the surface of the complexes with polyethylene
glycol (PEG), hyaluronic acid or other polymers; (iii) enhancement in uptake of drugs
and plasmids by cancer cells because of the fusogenic peptides or small percentage
of cationic lipids, and (iv) sustained expression of the genes using human origins
of replication (ORIs) able to sustain episomal replication or long term expression
of therapeutic genes and high levels of expression.
[0091] In a further embodiment, the polynucleotides or genes further comprise regulatory
DNA sequences that sustain expression of the genes for months rather than days. This
translates into fewer treatments and less suffering of the cancer patient. It can
also exert a strong therapeutic effect because of higher levels of expression of the
anticancer gene; the same gene placed under control of weak regulatory DNA will be
ineffective.
[0092] Several experimental strategies for cancer treatment have been designed using p53
gene delivery; novelty exists in that the endogenous mutant p53 forms, which are overexpressed
in over half of human prostate malignancies especially those from advanced prostate
cancer are suppressed using the PAX5 expression vector. Mutated forms of p53 have
amino acid substitutions mainly in their DNA binding domain but are still able to
tetramerize with the wt p53 form; p53 acts as a tetramer and the presence of high
levels of endogenous mutant p53 in human cancers cells interferes with the tumor suppressor
functions of the wt p53 to be delivered.
[0093] PAX5 is an homeodomain protein which determines body structures during development;
PAX5 is expressed at early stages of mammalian development and in the adult during
differentiation in hematopoietic stem cells; p53 gene expression is eliminated by
the PAX5 suppressor protein at early stages of development allowing cells to multiply
fast in the developing embryo. PAX5 is switched off at later stages throughout adulthood
allowing p53 to be expressed and exert its tumor suppressive functions and to regulate
apoptosis especially in the hematopoietic cell lineage.
[0094] A number of delivery systems are being used in somatic gene transfer, each associated
with advantages and drawbacks. Recombinant adenoviruses do not replicate efficiently,
recombinant murine retroviruses integrate randomly and are inactivated by chromatin
surroundings; recombinant AAV integrates randomly and cannot achieve high titers for
clinical utility. All have a maximum capacity of 3.5-7.5 kb of foreign DNA because
of packaging limitations. Naked DNA is rapidly degraded (half-life 5 minutes) after
systemic delivery. Cationic liposomes are toxic do not survive in circulation beyond
a heart beat and target mainly the endothelium of the lung, liver, and heart So far,
only "stealth" liposomes have been proven capable of concentrating in tumor sites
(also in liver and spleen) and to survive for prolonged periods in blood circulation
(eg., one day compared with minutes for non-stealth neutral liposomes and a few seconds
for cationic liposomes). However, stealth liposomes are not taken readily by tumor
cells remaining in the extracellular space where they release their load over days
after lysis (reviewed by Martin and Boulikas, 1998); however, the method described
below modifies stealth liposomes with fusogenic peptides or by providing a partially
cationic lipid composition or DOPE at their inner bilayer which would endow them to
enter the tumor cell membrane by causing disturbance of the lipid bilayer.
[0095] Having attained concentration and uptake of the drug and gene_bullets in solid tumors
in animals with stealth liposomes, the second step is efficacy of the drug and gene
targeting approach. A human clinical trial at M.D. Anderson Cancer Center uses transfer
of the wild-type p53 gene in patients suffering with non-small cell lung cancer and
shown to have p53 mutations in their tumors using local injection of an Ad5/CMV/p53
recombinant adenovirus at the site of tumor in combination with cisplatin. The first
results of this clinical trial are encouraging after intratumor injection of p53 (Roth
et al., 1996; reviewed by Boulikas, 1998a). However, local injection is not applicable
to metastases often associated with advanced stages of malignancies; in particular,
prostate cancer gives metastases to bones by a mechanism involving stimulation in
prostate tumor proliferation by insulin-like growth factor I(IGF-I) which is especially
secreted by bone cells. Therefore, the delivery system proposed here, able to concentrate
into the tumor cell mass after systemic injection, is likely to treat not only the
primary tumor but also its metastases.
[0096] The proposed cisplatin liposomes will primarily target tumors because of the nature
of our delivery system. The genes in the combination therapy will primarily target
dividing cells because of the use of HSV-tk and ganciclovir that incorporates into
replicating DNA, and primarily vascularizing tumors because of the use of stealth
liposomes. Thus, liver and spleen cells that are also reached by stealth liposomes
will not be killed.
[0097] In a further embodiment, the liposome encapsulated drugs described herein further
comprise an effective amount of a fusogenic peptide. Fusogenic peptides belong to
a class of helical amphipathic peptides characterized by a hydrophobicity gradient
along the long helical axis. This hydrophobicity gradient causes the tilted insertion
of the peptides in membranes, thus destabilizing the lipid core and, thereby, enhancing
membrane fusion (Decout et al., 1999).
[0098] Hemagglutinin (HA) is a homotrimeric surface glycoprotein of the influenza virus.
In infection, it induces membrane fusion between viral and endosomal membranes at
low pH. Each monomer consists of the receptor-binding HA1 domain and the membrane-interacting
HA2 domain. The NH2-terminal region of the HA2 domain (amino acids 1 to 127), the
so-called "fusion peptide," inserts into the target membrane and plays a crucial role
in triggering fusion between the viral and endosomal membranes. Based on substitution
of eight amino acids in the region 5-14 with cysteines and spin-labeling electron
paramagnetic resonance it was concluded that the peptide forms an alpha-helix tilted
approximately 25 degrees from the horizontal plane of the membrane with a maximum
depth of 15 angstroms (A) from the phosphate group (Macosko et al., 1997). Use of
fusogenic peptides from influenza virus hemagglutinin HA-2 enhanced greatly the efficiency
of transferrin-polylysine-DNA complex uptake by cells; in this case the peptide was
linked to polylysine and the complex was delivered by the transferrin receptor-mediated
endocytosis (reviewed by Boulikas, 1998a). This peptide had the sequence: GLFEAIAGFIENGWEGMIDGGGYC
(SEQ ID NO:1) and was able to induce the release of the fluorescent dye calcein from
liposomes prepared with egg yolk phosphatidylcholine which was higher at acidic pH;
this peptide was also able to increase up to 10-fold the anti-HIV potency of antisense
oligonucleotides, at a concentration of 0.1-1 mM, using CEM-SS lymphocytes in culture.
This peptide changes conformation at the slightly more acidic environment of the endosome
destabilizing and breaking the endosomal membrane (reviewed by Boulikas, 1998a).
[0099] The presence of negatively charged lipids in the membrane is important for the manifestation
of the fusogenic properties of some peptides but not of others; whereas the fusogenic
action of a peptide, representing a putative fusion domain of fertilin, a sperm surface
protein involved in sperm-egg fusion, was dependent upon the presence of negatively
charged lipids. However, that of the HIV2 peptide was not (Martin and Ruysschaert,
1997).
[0100] For example, to analyze the two domains on the fusogenic peptides of influenza virus
hemagglutinin HA, HA-chimeras were designed in which the cytoplasmic tail and/or transmembrane
domain of HA was replaced with the corresponding domains of the fusogenic glycoprotein
F of Sendai virus. Constructs of HA were made in which the cytoplasmic tail was replaced
by peptides of human neurofibromin. type 1 (NF1) (residues 1441 to 1518) or c-Raf-1,
(residues 51 to 131). The constructs were expressed in CV-1 cells by using the vaccinia
virus-T7 polymerase transient-expression system. Membrane fusion between CV-1 cells
and bound human erythrocytes (RBCs) mediated by parental or chimeric HA proteins showed
that, after the pH was lowered, a flow of the aqueous fluorophore calcein from preloaded
RBCs into the cytoplasm of the protein-expressing CV-1 cells took place. This indicated
that membrane fusion involves both leaflets of the lipid bilayers and leads to formation
of an aqueous fusion pore (Schroth-Diez et al., 1998).
[0101] A remarkable discovery was that the TAT protein of HIV is able to cross cell membranes
(Green and Loewenstein, 1988) and that a 36-amino acid domain of TAT, when chemically
crosslinked to heterologous proteins, conferred the ability to transduce into cells.
It is worth mentioning that the 11-amino acid fusogenic peptide of TAT CYGRKKRRQRRR
(SEQ ID NO:2)) is a nucleolar localization signal (see Boulikas, 1998b).
[0102] Another protein of HIV, the glycoprotein gp41, contains fusogenic peptides. Linear
peptides derived from the membrane proximal region of the gp41 ectodomain have potential
applications as anti-HIV agents and inhibit infectivity by adopting a helical conformation
(Judice et al., 1997). The 23 amino acid residues N-terminal peptide of HIV-1 gp41
has the capacity to destabilize negatively charged large unilamellar vesicles. In
the absence of cations the main structure was a pore-forming alpha-helix, whereas
in the presence of Ca2
+ the conformation switched to a fusogenic, predominantly extended beta-type structure.
The fusion activity of HIV(ala) (bearing the R22(A substitution) was reduced by 70%
whereas fusogenicity was completely abolished when a second substitution (V2(E) was
included arguing that it is not an alpha-helical but an extended structure adopted
by the HIV-1 fusion peptide that actively destabilizes cholesterol-containing, electrically
neutral membranes (Pereira et al., 1997).
[0103] The prion protein (PrP) is a glycoprotein of unknown function normally found at the
surface of neurons and of glial cells. It is involved in diseases such as bovine spongiform
encephalopathy, and Creutzfeldt-Jakob disease in the human, where PrP is converted
into an altered form (termed PrPSc). According to computer modeling calculations,
the 120 to 133 and 118 to 135 domains of PrP are tilted lipid-associating peptides
inserting in a oblique way into a lipid bilayer and able to interact with liposomes
to induce leakage of encapsulated calcein (Pillot et al., 1997b).
[0104] The C-terminal fragments of the Alzheimer amyloid peptide (amino acids 29-40 and
29-42) have properties related to those of the fusion peptides of viral proteins inducing
fusion of liposomes in vitro. These properties could mediate a direct interaction
of the amyloid peptide with cell membranes and account for part of the cytotoxicity
of the amyloid peptide. In view of the epidemiologic and biochemical linkages between
the pathology of Alzheimer's disease and apolipoprotein E (apoE) polymorphism, examination
of the potential interaction between the three common apoE isoforms and the C-terminal
fragments of the amyloid peptide showed that only apoE2 and apoE3, not apoE4, are
potent inhibitors of the amyloid peptide fusogenic and aggregational properties. The
protective effect of apoE against the formation of amyloid aggregates was thought
to be mediated by the formation of stable apoE/amyloid peptide complexes (Pillot et
al., 1997a; Lins et al., 1999). The fusogenic properties of an amphipathic net-negative
peptide (WAE 11), consisting of 11 amino acid residues were strongly promoted when
the peptide was anchored to a liposomal membrane; the fusion activity of the peptide
appeared to be independent of pH and membrane merging and the target membranes required
a positive charge which was provided by incorporating lysine-coupled phosphatidylethanolamine
(PE-K). Whereas the coupled peptide could cause vesicle aggregation via nonspecific
electrostatic interaction with PE-K, the free peptide failed to induce aggregation
of PE-K vesicles (Pecheur et al., 1997).
[0105] A number of studies suggest that stabilization of an alpha-helical secondary structure
of the peptide after insertion in lipid bilayers in membranes of cells or liposomes
is responsible for the membrane fusion properties of peptides; Zn
2+, enhances the fusogenic activity of peptides because it stabilizes the alpha-helical
structure. For example, the HEXXH domain of the salivary antimicrobial peptide, located
in the C-terminal functional domain of histatin-5, a recognized zinc-binding motif
is in a helicoidal conformation (Martin et al., 1999; Melino et al., 1999; Curtain
et al., 1999).
[0106] Fusion peptides have been formulated with DNA plasmids to create peptide-based gene
delivery systems. A combination of the YKAKnWK peptide, used to condense plasmids
into 40 to 200 nm nanoparticles, with the GLFEALLELLESLWELLLEA (SEQ ID NO:3) amphipathic
peptide, which is a pH-sensitive lytic agent designed to facilitate release of the
plasmid from endosomes enhanced expression systems containing the beta-galactosidase
reporter gene (Duguid et al., 1998).
[0107] DOPE (dioleyl phosphatidyl ethanolamine) is a fusogenic lipid; elastase cleavage
of N-methoxy-succinyl-Ala-Ala-Pro-Val-DOPE converted this derivative to DOPE (overall
positive charge) to deliver an encapsulated fluorescent probe, calcein, into the cell
cytoplasm (Pak et al., 1999). An oligodeoxynucleic sequence of 30 bases complementary
to a region of beta-endorphin mRNA elicited a concentration-dependent inhibition of
beta-endorphin production in cell culture after it was encapsulated within small unilamellar
vesicles (50 nm) containing dipalmitoyl-DL-alpha-phosphatidyl-L-serine endowed with
fusogenic properties (Fresta et al., 1998).
[0108] Additional fusogenic peptides useful in the methods of this invention are described
in Table 1, below.
Fusogenic peptide |
Source Protein |
Properties |
Reference |
GLFEAIAGFIENG WEGMIDGGGYC |
Influenza virus hemagglutinin HA-2 |
|
Bongartz et al, 1994; |
YGRKKRRQRRR |
TAT of HIV |
|
Green and Loewenstein, 1988; |
the 23-residue fusogenic N-terminal peptide |
HIV-1 transmembrane glycoprotein gp41 |
Was able of inserting as an alpha-helix into neutral phospholipid bilayers |
Curtain et al, 1999 |
120 to 133 and 118 to 135 domains |
prion protein |
tilted lipid-associating peptide; interact with liposomes to induce leakage of encapsulated
calcein |
Pillot et al, 1997b |
29-42-residue fragment |
Alzheimer's beta-amyloid peptide |
Endowed with capacities resembling those of the tilted fragment of viral fusion proteins |
Lins et al, 1999 |
nonaggregated amyloid beta-peptide (1-40) |
Alzheimer's beta-amyloid peptide |
induces apoptotic neuronal cell death |
Pillot et at, 1999 |
LCAT 56-68 helical segment |
lecithin cholesterol acyltransferase (LCAT) |
forms stable beta-sheets in lipids |
Peelman et at, 1999; Decout et at, 1999 |
70 residue peptide (SV-117) |
Fusion peptide and N-terminal heptad repeat of Sendai virus |
Induced lipid mixing of egg phosphatidylcholine/phosphatid yiglycerol (PC/PG) large
unilamellar vesicles (LUVs) |
Ghosh and Shai, 1999 |
MSGTFGGILAGL IGLL |
N-terminal region of the S protein of duck hepatitis B Virus (DHBV) |
Was inserted into the hydrophobic core of the lipid bilayer and induced leakage of
internal aqueous contents from both neutral and negatively charged liposomes |
Rodriguez-Crespo et al, 1999 |
MSPSSLLGLLAG LQVV |
S protein of woodchuck hepatitis B virus (WHV) |
Was inserted into the hydrophobic core of the lipid bilayer and induced leakage of
internal aqueous contents from both neutral and negatively charged liposomes |
Rodriguez-Crespo et al, 1999 |
peptide sequence B18 sequence |
membrane-associated sea urchin sperm protein bindin |
Triggers fusion between lipid vesicles; a histidine-rich motif for binding zinc, is
required for the fusogenic function |
Ulrich et al, 1999 |
|
histatin-5 (salivary antimicrobial peptide) |
Aggregates and fuses negatively charged small unilamellar vesicles in the presence
of Zn2+ |
Melino et al, 1999 |
amphipathic negatively charged peptide consisting of 11 residues (WAE) |
|
Forms an alpha-helix inserted and anchored into the membrane (favored at 37°C) oriented
almost parallel to the lipid acyl chains; promotes fusion of large unilamellar liposomes
(LUV) |
Martin et al, 1999 |
A polymer of polylysine (average 190) partially substituted with histidyl residues |
|
histidyl residues become cationic upon protonation of the imidazole groups at pH below
6.0.; disrupt endosomal membranes |
Midoux and Monsigny, 1999 |
GLFEALLELLESL WELLLEA |
|
amphipathic peptide; a pH-sensitive lytic agent to facilitate release of the plasmid
from endosomes |
Duguid et al, 1998 |
(LKKL)4 |
|
amphiphilic fusogenic peptide, able to interact with four molecules of DMPC |
Gupta and Kothekar, 1997 |
residues 53-70 (C-terminal helix) |
apolipoprotein (apo) AII |
induces fusion of unilamellar lipid vesicles and displaces apo AI from HDL and r-HDL |
Lambert et al, 1998 |
residues 90-111 |
PH-30 alpha (a protein functioning in sperm-egg fusion) |
membrane-fusogenic activity to acidic phospholipid bilayers |
Niidome et al, 1997 |
N-terminus of Nef |
Nef protein of human immunodeficien cy type 1 (HIV-1) |
membrane-perturbing and fusogenic activities in artificial membranes; causes cell
killing in E. coli and yeast |
Macreadie et al, 1997 |
casein signal peptides |
alpha s2- and beta-casein |
Interact with dimyristoylphosphatidyl-glycerol and -choline liposomes; show both lytic
and fusogenic activities |
Creuzenet et al, 1997 |
amino-terminal sequence F1 polypeptide |
F1 polypeptide of measles virus (MV) |
Can be used as a carrier system for CTL epitopes |
Partidos et al, 1996 |
23 hydrophobic amino acids in the amino-terminal region |
S protein of hepatitis B virus (HBV) |
A high degree of similarity with known fusogenic peptides from other viruses. |
Rodriguez-Crespo et al, 1994 |
19-27 amino acid segment |
glycoprotein gp51 of bovine leukemia virus |
Adopts an amphiphilic structure and plays a key role in the fusion events induced
by bovine leukemia virus. |
Voneche et al, 1992 |
Ac-(Leu-Ala-Arg-Leu)3-NHCH3 |
basic amphipathic peptides |
caused a leakage of contents from small unilamellar vesicles composed of egg yolk
phosphatidylcholine and egg yolk phosphatidic acid (3:1) |
Suenaga et al, 1989; Lee et al, 1992 |
amphiphilic anionic peptides E5 and E5L |
|
can mimic the fusogenic activity of influenza hemagglutinin(HA) |
Murata et al, 1991 |
30-amino acid peptide with the major repeat unit Glu-Ala-Leu-Ala (GALA)7 |
designed to mimic the behavior of the fusogenic sequences of viral fusion proteins |
becomes an amphipathic alpha-helix as the pH is lowered to 5.0 ; fusion of phosphatidylcholine
small unilamellar vesicles induced by GALA requires a peptide length greater than
16 amino acids |
Parente et al, 1988 Parente et al, 1988 |
pardaxin |
amphipathic polypeptide, purified from the gland secretion of the Red Sea Moses sole
flatfish Pardachirus marmoratus |
forms voltage-gated, cation-selective pores; mediated the aggregation of liposomes
composed of phosphatidylserine but not of phosphatidylcholine phosphatidylcholine |
Lelkes and Lazarovici, 1988 |
Gramicidin (linear hydrophobic polypeptide) |
|
Antibiotic; induces aggregation and fusion of vesicles |
Massari and Colonna, 1986; Tournois et al, 1990 |
poly(Glu-Aib-Leu-Aib) (Aib represents 2-aminoisobutyric acid), |
|
Amphiphilic structure upon the formation of alpha-helix; caused fusion of EYPC liposomes
and dipalmitoylphosphatidylcholine liposomes more strongly with decreasing pH |
Kono et al, 1993 |
[0109] After the micelles have been formed, they are mixed with an effective amount of a
vesicle forming lipid to form drug containing liposomes. Useful lipids for this invention
include premade neutral liposomes, lipids in powder, PEG-DSPE or hydrogenated soy
phosphatidylchline (HSPC). Vesicle-forming lipids are selected to achieve a specified
degree of fluidity or rigidity of the final complex providing the lipid composition
of the outer layer. These can be composed of 10-60% cholesterol and the remaining
amounts include bipolar phospholipids, such as the phosphatidylcholine (PC) or phosphatidylethanolamine
(PE), with a hydrocarbon chain length in the range of 14-22, and saturated with one
or more double C=C bonds. A preferred lipid for use in the present invention is cholesterol
(10-60%), hydrogenated soy phosphatidylcholine (HSPC) at 40-90%, and the derivatized
vesicle-forming lipid PEG-DSPE at 1-7%. The liposomes provide the outer lipid bilayer
surfaces that are coated with the hydrophilic polymer, PEG. The PEG chains have a
molecular weight between 1,000-5,000 Dalton. Other hydrophilic polymers include hyaluronic
acid, polyvinylpyrrolidone, DSPE, hydroxyethylcellulose, and polyaspartamide. PEG-DSPC
and PEG-HSPC are commercially available from Syngena.
[0110] Prior to mixture with the vesicle forming lipid, the ethanol or other organic solvent
can be removed by any method known in the art, e.g., dialysis of the micelles through
permeable membranes.
Diagnostic and Therapeutic Methods
[0111] Described herein is the therapy of a subject,
e.g., mammals such as mice, rats, simians, and human patients, with human cancers including,
but not limited to breast, prostate, colon, non-small lung, pancreatic, testicular,
ovarian, cervical carcinomas, head and neck squamous cell carcinomas. Described herein
is intravenous injection of cisplatin encapsulated into liposomes as well as by combinations
of encapsulated cisplatin with encapsulated doxorubicin, fluorodeoxyuridine, bleomycin,
adriamycin, vinblastin, prednisone, vincristine, taxol or radiation therapy, encapsulated
oligonucleotides, ribozymes endowed with anticancer properties and a number of anticancer
genes including but not limited to p53/Pax5/HSV-tk genes. The approach consists of
two major parts: (i) the ability to target cancer cells (ii) effectiveness of our
approach to kill cancer cells.
[0112] Accordingly, also described herein is a method for delivering cisplatin or other
therapeutic agent to a cell comprising contacting the cell with the encapsulated drugs
obtainable by the methods described herein. Also described herein is a method for
inhibiting the growth of a tumor in a subject, comprising administering to the subject
an effective amount of the encapsulated drugs obtainable by the methods described
herein. Depending on the composition of the lipid/micelle formulation, also described
herein are methods for targeting solid tumors and metastases in a subject by intravenous
administration of an effective amount of the encapsulated drug and methods for penetrating
the cell membrane of a tumor in a subject by administration of an effective amount
of the encapsulated drug, wherein the micelle contains a free fusogenic peptide or
a fusogenic peptide-lipid conjugate.
[0113] The methods can be practiced in vitro, ex vivo or in vitro.
[0114] In vitro practice of the method involves removal of a tumor biopsy or culturing of
a cell sample containing tumor cells. The final liposome complex or any intermediate
product arising during cisplatin encapsulation (e.g., micelles shown in Figure 1A)
are contacted with the cell culture under conditions suitable for incorporation of
the drug intracellularly. The in vitro method is useful as a screen to determine the
best drug therapy for each individual patient. Inhibition of cell growth or proliferation
indicates that the cell or tumor is suitably treated by this therapy. Effective amount
of drug for each therapy varies with the tumor being treated and the subject being
treated. Effective amounts can be empirically determined by those of skill in the
art.
[0115] When delivered to an animal, the method is useful to further confirm efficacy of
the drug or therapy for each tumor type. As an example of suitable animal models,
groups of SCID mice or nude mice (Balb/c NCR nu/nu female, Simonsen, Gilroy, CA) may
be subcutaneously inoculated with about 10
5 to about 10
9 cancer or target cells as defined herein. When the tumor is established, the liposome
is administered.
[0116] As used herein, "administration, delivered or administered" is intended to include
any method which ultimately provides the drug/liposome complex to the tumor mass.
Examples include, but are not limited to, topical application, intravenous administration,
parenteral administration or by subcutaneous injection around the tumor. Tumor measurements
to determine reduction of tumor size are made in two dimensions using venier calipers
twice a week.
[0117] For in vivo administration, the pharmaceutical compositions are preferably administered
parenterally, i.e., intravenously, intraperitoneally, subcutaneously, intrathecally,
injection to the spinal cord, intramuscularly, intraarticularly, portal vein injection,
or intratumorally. More preferably, the pharmaceutical compositions are administered
intravenously or intratumorally by a bolus injection. In other methods, the pharmaceutical
preparations may be contacted with the target tissue by direct application of the
preparation to the tissue. The application may be made by topical, "open" or "closed"
procedures. By "topical", it is meant the direct application of the pharmaceutical
preparation to a tissue exposed to the environment, such as the skin, nasopharynx,
external auditory canal, eye, inhalation to the lung, genital mucosa and the like.
"Open" procedures are those procedures which include incising the skin of a patient
and directly visualizing the underlying tissue to which the pharmaceutical preparations
are applied. This is generally accomplished by a surgical procedure, such as a thoracotomy
to access the lungs, abdominal laparotomy to access abdominal viscera, or other direct
surgical approach to the target tissue. "Closed" procedures are invasive procedures
in which the internal target tissues are not directly visualized, but accessed via
inserting instruments through small wounds in the skin. For example, the preparations
may be administered to the peritoneum by needle lavage. Likewise, the pharmaceutical
preparations may be administered to the meninges or spinal cord by infusion during
a lumbar puncture followed by appropriate positioning of the patient as commonly practiced
or spinal anesthesia or metrazamide imaging of the spinal cord. Alternatively, the
preparations may be administered through endoscopic devices.
[0118] Administration
in vivo can be effected in one dose, continuously or intermittently throughout the course
of treatment. Methods of determining the most effective means and dosage of administration
are well known to those of skill in the art and will vary with the composition used
for therapy, the purpose of the therapy, the target cell being treated, and the subject
being treated. Single or multiple administrations can be carried out with the dose
level and pattem being selected by the treating physician. Suitable dosage formulations
and methods of administering the agents can be empirically determined by those of
skill in the art.
[0119] The agents and compositions described herein can be used in the manufacture of medicaments
and for the treatment of humans and other animals by administration in accordance
with conventional procedures, such as an active ingredient in pharmaceutical compositions.
[0120] Ideally, the drug/lipid formulation should be administered to achieve peak concentrations
of the active compound at sites of disease. This may be achieved, for example, by
the intravenous injection of the drug/lipid formula. Desirable blood levels of the
drug may be maintained by a continuous infusion to provide a therapeutic amount of
the active ingredient within disease tissue. The use of operative combinations is
contemplated to provide therapeutic combinations requiring a lower total dosage of
each component drugs than may be required when each individual therapeutic compound
or drug is used alone, thereby reducing adverse effects.
[0121] While it is possible for the drug/lipid formula to be administered alone, it is preferable
to present it as a pharmaceutical formulation comprising at least one active ingredient,
as defined above, together with one or more pharmaceutically acceptable carriers therefor
and optionally other therapeutic agents. Each carrier must be "acceptable" in the
sense of being compatible with the other ingredients of the formulation and not injurious
to the patient.
[0122] Designing the third generation of vehicles for the delivery of anticancer drugs and
genes to solid tumors at as described herein was the result of five major improvements
over existing technologies:
- 1.) Encapsulation of antineoplastic drugs into sterically stabilized liposomes has
reduced manifold their toxicity. This is anticipated to bring to an end the nightmare
of cancer patients subject to chemotherapy. Most antineoplastic drugs under current
use have severe - side effects such as hair loss, vomiting, weight loss and cause
infarction as well as damage to kidney, brain, liver and all other vital tissue. The
antineoplastic drugs described herein are hidden inside the lumen of the lipid bilayer,
are not visible to most tissues and concentrate into their tumor targets, not in every
tissue in the body. Upon their uptake by the solid tumor they exert a specific cytotoxic
effect to cancer cells without damaging normal cells.
- 2.) Targeting of solid tumors and their metastases all over the body. Over 95% of
cancer patients succumb from complications connected to metastases, not from the primary
tumor. The gene and drug delivery system described herein has been designed to evade
the immune system after intravenous administration of the gene and drug bullet reaching
not only the primary tumor but also every metastasis in the animal and human body
regardless of the size of the tumor. It is based on the long circulation time of our
drug and gene carrying vehicles and their extravasation through the vascular endothelium
of tumors because of its imperfections and leakiness at its initial stage of formation
(neoangiogenesis in growing tumors) as well as because of differences in hydrostatic
pressure between the growing solid tumor and normal body tissues. The liposomes of
this invention have a different composition between their inner and outer lipid layers
permitting efficient encapsulation and tumor targeting.
- 3.) Uptake of the liposome bullet by the cancer cell. The liposome bullets are able
to promote fusion with the cell membrane. Similar "stealth" bullets developed elsewhere
are unable to cross the membrane barrier of the cancer cell.
- 4.) Reaching nearly 100% liposome encapsulation efficiency for anticancer drugs, oligonucleotides
and genes is a major advancement. This means minimal loss and cost effective use of
drugs and genes. It also translated into simpler steps in manufacturing the anticancer
bullet.
- 5.) The unique technology described herein can identify regulatory DNA sequences that
sustain expression of the genes in the anticancer bullet for months rather than days.
This translates into fewer treatments and less suffering of the cancer patient. It
can also exert a strong therapeutic effect because of higher levels of expression
of the anticancer gene; the same gene placed under control of weak regulatory DNA
will be ineffective.
[0123] The following examples are intended to illustrate, and not limit the invention, as
defined by the appended claims.
Examples
Preparation of Micelles and Lipid-Encapsulated Cisplatin
[0124] One formula for encapsulation includes the steps of: (A) mixing cisplatin (in powder
or other form) with DPPG (dipalmitoyl phosphatidyl glycerol) or other negatively-charged
lipid molecules at a 1:1 to 1:2 molar ratio in at least a 30% ethanol, 0.1 M Tris
HCl, pH 7.5 to achieve about 5 mg/ml final cisplatin concentration. Variations in
the molar ratio between cisplatin and DPPG are also of therapeutic value targeting
different tissues. (B) Heating at 50° C. During steps A and B the initial powder suspension,
which tends to give a precipitate of the yellow cisplatin powder, is converted into
a gel (colloidal) form; during steps A and B there is conversion of cisplatin to its
aqua form (by hydrolysis of the chloride atoms and their replacement by water molecules
bound to the platin) which is positively-charged and is the active form of cisplatin
endowed with the antineoplastic activity; the aqua cisplatin is simultaneously complexed
with the negatively-charged lipid into micelles in 30% ethanol. This cisplatin-DPPG
electrostatic complex has already improved properties over free cisplatin in tumor
eradication. (C) The properties of the complex (and of the final formulation after
step D, see below) in passing through the tumor cell membrane after reaching its target
are improved by addition of peptides and other molecules that give to the complex
this property. (D) The cisplatin-DPPG micelle complex is converted into liposomes
encapsulating the cisplatin-DPPG monolayer (Figure 1 top) or to other type of complexes
by direct addition of premade liposomes followed by dialysis against saline and extrusion
through membranes to downsize these to 100-160 nm in diameter (Figure 1 bottom). It
is the lipid composition of added liposomes that determines the composition of the
outer surface of our final cisplatin formulation.
[0125] Variations in step (A) permit encapsulation of doxorubicin and other positively charged
antineoplastic compounds. Addition of positively charged groups to neutral or negatively-charged
compounds allows their encapsulation similarly into liposomes.
Therapeutic Application
[0126] Ninety (90) day-release estrogen pellets were implanted subcutaneously into SCID
female mice. The mice were subcutaneously injected at mammary fat pad with 7.5 million
MCF-7 (a human breast carcinoma available from the ATCC) cells in 0.1 ml PBS. After
establishment of tumors, the mice were injected intravenously at tail vein with 0.1
ml of cisplatin liposomes. Results are shown in Figures 2 to 4.
[0127] While the invention has been described in detail and with reference to specific embodiments
thereof, it will be apparent to one skilled in the art that various changes and modifications
can be made therein without departing from the scope thereof, as defined by the appended
claims.
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1. A method for producing cisplatin-micelles; comprising:
a) combining:
(i) cisplatin; and
(ii) a phosphatidyl glycerol lipid derivative in a cisplatin to lipid derivative molar
ratio range of 1:1 to 1:2 to form a cisplatin mixture; and,
b) combining the cisplatin mixture of step a) with an effective amount of at least
a 30% ethanol solution to form cisplatin micelles, in which cisplatin is in its aqua
form.
2. A method for producing cisplatin micelles, comprising:
a) combining:
(i) cisplatin; and
(ii) an effective amount of at least a 30% ethanol solution; to form a cisplatin/ethanol
solution; and,
b) combining the cisplatin/ethanol solution of step a) with a phosphatidyl glycerol
lipid derivative in a cisplatin to lipid derivative molar ratio range of 1:1 to 1:2
to form cisplatin micelles, in which cisplatin is in its aqua form.
3. A method according to claim 1 or 2, wherein the phosphatidyl glycerol lipid derivative
is selected from:
dipalmitoyl phosphatidyl glycerol (DPPG),
dimyristoyl phosphatidyl glycerol (DMPG),
dicaproyl phosphatidyl glycerol (DCPG),
distearoyl phosphatidyl glycerol (DSPG), and
dioleyl phosphatidyl glycerol (DOPG).
4. A method according to claim 1 or 2, wherein the molar ratio is 1:1.
5. A method according to claim 1 or 2, further comprising the step of combining with
the mixture or solution of step a) an effective amount of:
a free fusogenic peptide,
a fusogenic peptide-lipid conjugate, or
a fusogenic peptide-PEG-hydrogenated soy phosphatidylcholine (HSPC) conjugate;
wherein the fusogenic peptide is derivatized with a stretch of 1-6 negatively-charged
amino acids at the N- or C-terminus and is thus able to bind electrostatically to
cisplatin in its aqua form.
6. A method according to claim 5, wherein the free fusogenic peptide or fusogenic peptide
lipid conjugate comprises dioleyl phosphatidyl ethanolamine (DOPE) or DOPE/cationic
lipid.
7. A method according to claim 1 or 2, further comprising the step of removal of the
ethanol from the cisplatin micelles.
8. A method according to claim 7, wherein removal of the ethanol is by dialysis of the
micelles through permeable membranes to remove the ethanol.
9. A cisplatin micelle obtainable by a method according to any one of claims 1 to 8.
10. A cisplatin micelle obtainable by a method according to claim 5.
11. A method for encapsulating cisplatin micelles, comprising mixing an effective amount
of a vesicle-forming lipid with the cisplatin micelles obtained by a method according
to any one of claims 1 to 8.
12. A method according to claim 11, wherein the lipid is selected from premade neutral
liposomes, composed of cholesterol 10-60%, hydrogenated soy phosphatidylcholine (HSPC)
40-90%, and polyethyleneeglycol hydrogenated soy phosphatidylcholine (PEG-HSPC) 1-7%
or lipids in solution, lipids in powder and polyethyleneglycol distearoyl phosphatidylethanolamine
(PEG-DSPE).
13. A method according to claim 11, wherein the lipid comprises 10-60% cholesterol.
14. Encapsulated cisplatin obtainable by a method according to any one of claims 11 to
13.
15. A method for obtaining a cisplatin/lipid complex capable of evading macrophages and
cells of the immune system when administered to a subject, the method comprising mixing
an effective amount of the cisplatin micelles according to claim 7 or 8 with an effective
amount of polyethyleneglycol distearoyl phosphatidylethanolamine (PEG-DSPE), polyethyleneglycol
distearoyl phosphatidylcholine (PEG-DSPC) or hyaluronic acid-distearoyl phosphatidylethanolamine.
16. Encapsulated cisplatin obtainable by a method according to claim 15.
17. A composition comprising the encapsulated cisplatin according to claim 14 and encapsulated
oligonucleotides, ribozymes, or polynucletotides.
18. A composition comprising the encapsulated cisplatin according to claim 14 and a drug
selected from: doxorubicin, fluorodeoxyuridine, bleomycin, adriamycin, vinblastin,
prednisone, vincristine, and taxol.
19. A method for delivering cisplatin to a cell, in vitro, comprising contacting the cell
with encapsulated cisplatin according to claim 14 or 16.
20. Encapsulated cisplatin according to claim 14 or 16 for use in a method of treatment
of the human or animal body.
21. Encapsulated cisplatin according to claim 14 or 16 for use in a method of treatment
to inhibit the growth of a tumor in the human or animal body.
22. Encapsulated cisplatin according to claim 14 or 16 for use in a method of treatment
to target solid tumors and metastases by intravenous administration in the human or
animal body.
23. Encapsulated cisplatin according to claim 14 or 16, and a gene selected from the group
consisting of p53, pax5 and HSV-tk genes, for use in a method of treatment to inhibit
the growth of a tumor in the human or animal body.
24. Encapsulated cisplatin according to claim 14 or 16; a gene selected from the group
consisting of p53, pax5 and HSV-tk genes; and an effective amount of encapsulated
ganciclovir; for use in a method of treatment to inhibit the growth of a tumor in
the human or animal body.
25. Encapsulated cisplatin according to claim 14 or 16, and a gene selected from the group
consisting of p53, pax5 and HSV-tk genes, for use in a method of treatment to inhibit
the growth of a tumor in the human or animal body, wherein the genes to be combined
with cisplatin are any of, or combinations of encapsulated IL-2, IL-4, IL-7, IL-12,
GM-CSF, IFN-gamma, TNF-alpha, RB, BRCA1, E1A, cytosine deaminase in combination with
encapsulated 5-fluorcytosine, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGFI, VEGF,
and TGF-beta.
26. Use of encapsulated cisplatin according to claim 14 or 16 for the manufacture of a
medicament for use in the treatment to inhibit the growth of a tumor in the human
or animal body.
27. Use of encapsulated cisplatin according to claim 14 or 16 for the manufacture of a
medicament for use in the treatment by intravenous administration to target solid
tumors and metastases in the human or animal body.
28. Use of encapsulated cisplatin according to claim 14 or 16, and a gene selected from
the group consisting of p53, pax5 and HSV-tk genes, for the manufacture of a medicament
for use in the treatment to inhibit the growth of a tumor in the human or animal body.
29. Use of encapsulated cisplatin according to claim 14 or 10, a gene selected from the
group consisting of p53, pax5 and HSV-tk genes; and an effective amount of encapsulated
ganciclovir, for the manufacture of a medicament for use in the treatment to inhibit
the growth of a tumor in the human or animal body.
30. Use of encapsulated cisplatin according to claim 14 or 16, and a gene selected from
the group consisting of p53, pax5 and HSV-tk genes, for the manufacture of a medicament
for use in the treatment to inhibit the growth of a tumor in the human or animal body,
wherein the genes to be combined with cisplatin are any of, or combinations of encapsulated
IL-2, IL-4, IL-7, IL-12, GM-CSF, IFN-gamma, TNF-alpha, RB, BRCA1, E1A, cytosine deaminase
in combination with encapsulated 5-fiuorcytosine, bcl-2, MDR-1, p21, p16, bax, bcl-xs,
E-2F, IGFI, VEGF, and TGF-beta.
1. Méthode de production de micelles de cisplatine, comprenant:
a) la combinaison:
(i) cisplatine; et
(ii) un dérivé lipidique de phosphatidyl glycérol à une gamme de rapport molaire de
cisplatine au dérivé lipidique de 1:1 à 1:2 pour former un mélange de cisplatine;
et,
b) la combinaison du mélange de cisplatine de l'étape a) avec une quantité efficace
d'au moins une solution à 30% d'éthanol pour former des micelles de cisplatine, où
le cisplatine est sous sa forme aqua.
2. Méthode de production de micelles de cisplatine, comprenant:
a) la combinaison:
(i) cisplatine; et
(ii) une quantité efficace d'au moins une solution à 30% d'éthanol; pour former une
solution cisplatine/éthanol; et
b) la combinaison de la solution cisplatine/éthanol de l'étape a) avec un dérivé lipidique
de phosphatidyl glycérol dans une gamme de rapport molaire cisplatine à dérivé lipidique
de 1:1 à 1:2 pour former des micelles de cisplatine dans lesquelles le cisplatine
est sous sa forme aqua.
3. Méthode selon la revendication 1 ou 2, où le dérivé lipidique de phosphatidyl glycérol
est sélectionné parmi:
dipalmitoyl phosphatidyl glycérol (DPPG),
dimyristoyl phosphatidyl glycérol (DMPG),
dicaproyl phosphatidyl glycérol (DCPG),
distéaroyl phosphatidyl glycérol (DSPG), et
dioléyl phosphatidyl glycérol (DOPG).
4. Méthode selon la revendication 1 ou 2, où le rapport molaire est de 1:1.
5. Méthode selon la revendication 1 ou 2, comprenant de plus l'étape de combiner au mélange
ou à la solution de l'étape a) une quantité efficace de:
un peptide fusogène libre,
un conjugué peptide fusogène-lipide, ou
un conjugué peptide fusogène-PEG-phosphatidylcholine du soja hydrogéné (HSPC);
où le peptide fusogène est dérivatisé avec un tronçon de 1 -6 acides aminés négativement
chargés au N- ou C - terminal et est ainsi capable de se lier électrostatiquement
au cisplatine sous sa forme aqua.
6. Méthode selon la revendicatiobn 5, où le peptide fusogène libre ou conjugué lipide
peptide fusogène comprend dioléyl phosphatidyl éthanolamine (DOPE) ou DOPE/lipide
cationique.
7. Méthode selon la revendication 1 ou 2, comprenant de plus l'étape d'éliminer l'éthanol
des micelles de cisplatine.
8. Méthode selon la revendication 7, où l'élimination de l'éthanol est par dialyse des
micelles à travers des membranes perméables pour éliminer l'éthanol.
9. Micelle de cisplatine pouvant être obtenue par une méthode selon l'une quelconque
des revendications 1 à 8.
10. Micelle de cisplatine pouvant être obtenue par une méthode selon la revendication
5.
11. Méthode pour encapsuler des micelles de cisplatine, comprenant le mélange d'une quantité
efficace d'un liquide formant des vésicules avec les micelles de cisplatine obtenues
par une méthode selon l'une quelconque des revendications 1 à 8.
12. Méthode selon la revendication 11, où le lipide est sélectionné parmi des liposomes
neutres préfabriqués composés de 10-60% de cholestérol, 40-90% de phosphatidylcholine
de soja hydrogéné (HSPC) et 1-7% de polyéthylèneglycol phosphatidylcholine de soja
hydrogéné (PEG-HSPC) ou des lipides en solution, des lipides en poudre et polyéthylèneglycol
distéaroyl phosphatidyléthanolamine (PEG-DSPE).
13. Méthode selon la revendication 11, où le lipide comprend 10-60% cholestérol.
14. Cisplatine encapsulé pouvant être obtenu par une méthode selon l'une quelconque des
revendications 11 à 13.
15. Méthode pour obtenir un complexe cisplatine/lipide capable d'évacuer les macrophages
et les cellules du système immun lors d'une administration à un sujet, la méthode
comprenant le mélange d'une quantité efficace de micelles de cisplatine selon la revendication
7 ou 8 avec une quantité efficace de polyéthylèneglycol distéaroyl phosphatidyléthanolamine
(PEG-DSPE), polyéthylèneglycol distéaroyl phosphatidylcholine (PEG-DSPC) ou acide
hyaluronique-distéaroyl phosphatidyléthanolamine.
16. Cisplatine encapsulé pouvant être obtenu par une méthode selon la revendication 15.
17. Composition comprenant le cisplatine encapsulé selon la revendication 1 4 et des oligonucléotides
ribozymes, ou polynucléotides encapsulés.
18. Composition comprenant le cisplatine encapsulé selon la revendication 14 et un produit
pharmaceutique sélectionné parmi: doxorubicine, fluorodésoxyuridine, bléomycine, adriamycine,
vinblastine, prednisone, vincristine, et taxol.
19. Méthode pour délivrer du cisplatine à une cellule, in vitro, comprenant la mise en
contact de la cellule avec du cisplatine encapsulé selon la revendication 14 ou 16.
20. Cisplatine encapsulé selon la revendication 14 ou 16 à utiliser dans une méthode de
traitement du corps humain ou animal.
21. Cisplatine encapsulé selon la revendication 14 ou 16 à utiliser dans une méthode de
traitement pour inhiber la croissance d'une tumeur dans le corps humain ou animal.
22. Cisplatine encapsulé selon la revendication 14 ou 16 à utiliser dans une méthode de
traitement pour cibler des tumeurs solides et des métastases par administration intraveineuse
au corps humain ou animal.
23. Cisplatine encapsulé selon la revendication 14 ou 16, et un gène sélectionné dans
le groupe consistant en gènes p53, pax5 et HSV -tk, à utiliser dans une méthode de
traitement pour inhiber la croissance d'une tumeur dans le corps humain ou animal.
24. Cisplatine encapsulé selon la revendication 14 ou 16; un gène sélectionné dans le
groupe consistant en gènes p53, pax5 et HSV -tk; et une quantité efficace de ganciclovir
encapsulé; à utiliser dans une méthode de traitement pour inhiber la croissance d'une
tumeur dans le corps humain ou animal.
25. Cisplatine encapsulé selon la revendication 14 ou 16, et un gène sélectionné dans
le groupe consistant en gènes p53, pax5 et HSV-tk, à utiliser dans une méthode de
traitement pour inhiber la croissance d'une tumeur dans le corps humain ou animal,
où les gènes à combiner au cisplatine sont l'un de ou une combinaison de IL -2, IL-4,
IL-7, IL-12, GM-CSF, IFN-gamma, TNF-alpha, RB, BRCA1, E1A, cytosine désaminase encapsulés
en combinaison avec 5-fluorocytosine, bcl-2, MDR-1, p21, p16, bax, bcl -xs, E2F, IGFI,
VEGF, et TGF-béta encapsulés.
26. Utilisation de cisplatine encapsulé selon la revendication 14 ou 16 pour la fabrication
d'un médicament à utiliser dans le traitement pour inhiber la croissance d'une tumeur
dans le corps humain ou animal.
27. Utilisation de cisplatine encapsulé selon la revendication 14 ou 16 pour la fabrication
d'un médicament à utiliser dans le traitement par administration intraveineuse pour
cibler des tumeurs solides et des métastases dans le corps humain ou animal.
28. Utilisation de cisplatine encapsulé selon la revendication 14 ou 16 et un gène sélectionné
dans le groupe consistant en gènes p53, pax5 et HSV -tk, pour la fabrication d'un
médicament à utiliser dans le traitement pour inhibe r la croissance d'une tumeur
dans le corps humain ou animal.
29. Utilisation de cisplatine encapsulé selon la revendication 14 ou 16; un gène sélectionné
dans le groupe consistant en gènes p53, pax5 et HSV -tk; et une quantité efficace
de ganciclovir encapsulé, pour la fabrication d'un médicament à utiliser dans le traitement
pour inhiber la croissance d'une tumeur dans le corps humain ou animal.
30. Utilisation de cisplatine encapsulé selon la revendication 14 ou 16, et d'un gène
sélectionné dans le groupe consistant en gènes p53, pax5 et HSV -tk, pour la fabrication
d'un médicament à utiliser dans le traitement pour inhiber la croissance d'une tumeur
dans le corps humain ou animal, où les gènes à combiner avec le cisplatine sont l'un
de ou une combinai son de IL-2, IL-4, IL-7, IL-12, GM-CSF, IFN-gamma, TNF-alpha, RB,
BRCA1, E1A, cytosine désaminase encapsulés en combinaison avec 5-fluorocytosine, bcl-2,
MDR-1, p21, p16, bax, bcl -xs, E2F, IGFI, VEGF, et TGF-béta encapsulés.
1. Verfahren zur Herstellung von Cisplatinmicellen, umfassend:
a) das Kombinieren von:
(i) Cisplatin; und
(ii) einem Phosphatidylglycerinlipidderivat in einem Molverhältnis zwischen Cisplatin
und Lipidderivat von 1:1 bis 1:2, um ein Cisplatingemisch herzustellen; und
b) das Kombinieren des Cisplatingemischs aus Schritt a) mit einer wirksamen Menge
einer zumindest 30%igen Ethanollösung, um Cisplatinmicellen zu bilden, worin Cisplatin
in seiner Aquaform vorliegt.
2. Verfahren zur Herstellung von Cisplatinmicellen, umfassend:
a) das Kombinieren von:
(i) Cisplatin; und
(ii) einer wirksamen Menge einer zumindest 30%igen Ethanollösung, um eine Cisplatin/Ethanol-Lösung
zu bilden; und
b) das Kombinieren der Cisplatin/Ethanol-Lösung aus Schritt a) mit einem Phosphatidylglycerinlipidderivat
in einem Molverhältnis zwischen Cisplatin und Lipidderivat von 1:1 bis 1:2, um Cisplatinmicellen
herzustellen, worin das Cisplatin in seiner Aquaform vorliegt.
3. Verfahren nach Anspruch 1 oder 2, worin das Phosphatidylglycerinlipidderivat ausgewählt
ist aus:
Dipalmitoylphosphatidylglycerin (DPPG),
Dimyristoylphosphatidylglycerin (DMPG),
Dicaproylphosphatidylglycerin (DCPG),
Distearoylphosphatidylglycerin (DSPG) und
Dioleylphosphatidylglycerin (DOPG).
4. Verfahren nach Anspruch 1 oder 2, worin das Molverhältnis 1:1 beträgt.
5. Verfahren nach Anspruch 1 oder 2, ferner den Schritt des Kombinierens des Gemischs
oder der Lösung aus Schritt a) mit einer wirksamen Menge von:
einem freien fusogenen Peptid,
einem fusogenen Peptid-Lipid-Konjugat oder
einem fusogenen Peptid-PEG-hydriertes-Soja-Phosphatidylcholin- (-HSPC-) Konjugat;
worin das fusogene Peptid mit einer Folge von 1-6 negativ geladenen Aminosäuren am
N- oder C-Terminus derivatisiert ist und deshalb in der Lage ist, elektrostatisch
an Cisplatin in seiner Aquaform zu binden.
6. Verfahren nach Anspruch 5, worin das freie fusogene Peptid oder fusogene Peptid-Lipid-Konjugat
Dioleylphosphatidylethanolamin (DOPE) oder DOPE/kationisches Lipid umfasst.
7. Verfahren nach Anspruch 1 oder 2, ferner den Schritt des Entfernens von Ethanol von
den Cisplatinmicellen umfassend.
8. Verfahren nach Anspruch 7, worin das Entfernen von Ethanol durch Dialyse der Micellen
durch permeable Membranen stattfindet, um das Ethanol zu entfernen.
9. Cisplatinmicelle, erhältlich durch ein Verfahren nach einem der Ansprüche 1 bis 8.
10. Cisplatinmicelle, erhältlich durch ein Verfahren nach Anspruch 5.
11. Verfahren zum Einkapseln von Cisplatinmicellen, umfassend das Vermischen einer wirksamen
Menge eines vesikelbildenden Lipids mit den durch ein Verfahren nach einem der Ansprüche
1 bis 8 erhaltenen Cisplatinmicellen.
12. Verfahren nach Anspruch 11, worin das Lipid aus vorgefertigten neutralen Liposomen,
bestehend aus 10-60 % Cholesterin, 40-90 % hydriertem Soja-Phosphatidylcholin (HSPC)
und 1-7 % Polyethylenglykol-hydriertes-Sojaphosphatidylcholin (PEG-HSPC), oder Lipiden
in Lösung, Lipiden in Pulverform und Polyethylenglykol-Distearoylphosphatidylethanolamin
(PEG-DSPE) ausgewählt ist.
13. Verfahren nach Anspruch 11, worin das Lipid 10-60 % Cholesterin umfasst.
14. Eingekapseltes Cisplatin, erhältlich durch ein Verfahren nach einem der Ansprüche
11 bis 13.
15. Verfahren zum Erhalten eines Cisplatin/Lipid-Komplexes, der in der Lage ist, Makrophagen
und Zellen des Immunsystems zu meiden, wenn er einem Patienten verabreicht wird, wobei
das Verfahren das Vermischen einer wirksamen Menge Cisplatinmicellen nach Anspruch
7 oder 8 mit einer wirksamen Menge Polyethylenglykol-Distearoylphosphatidylethanolamin
(PEG-DSPE), Polyethylenglykol-Distearoylphosphatidylcholin (PEG-DSPC) oder Hyaluronsäure-Distearoylphosphatidylethanolamin
umfasst.
16. Eingekapseltes Cisplatin, erhältlich durch ein Verfahren nach Anspruch 15.
17. Zusammensetzung, umfassend das eingekapselte Cisplatin nach Anspruch 14 und eingekapselte
Oligonucleotide, Ribozyme oder Polynucleotide.
18. Zusammensetzung, umfassend eingekapseltes Cisplatin nach Anspruch 14 und ein Arzneimittel,
das ausgewählt ist aus: Doxorubicin, Fluordesoxyuridin, Bleomycin, Adriamycin, Vinblastin,
Prednison, Vincristin und Taxol.
19. Verfahren zur Zufuhr von Cisplatin zu einer Zelle, in vitro, umfassend das Kontaktieren
der Zelle mit eingekapseltem Cisplatin nach Anspruch 14 oder 16.
20. Eingekapseltes Cisplatin nach Anspruch 14 oder 16 zur Verwendung in einem Verfahren
zur Behandlung eines menschlichen oder tierischen Körpers.
21. Eingekapseltes Cisplatin nach Anspruch 14 oder 16 zur Verwendung in einem Behandlungsverfahren
zur Hemmung des Wachstums eines Tumors in einem menschlichen oder tierischen Körper.
22. Eingekapseltes Cisplatin nach Anspruch 14 oder 16 zur Verwendung in einem Behandlungsverfahren
zum Zielen auf feste Tumoren und Metastasen durch intravenöse Verabreichung in einen
menschlichen oder tierischen Körper.
23. Eingekapseltes Cisplatin nach Anspruch 14 oder 16 und ein Gen, ausgewählt aus der
aus p53-, pax5- und HSV-tk-Genen bestehenden Gruppe, zur Verwendung in einem Behandlungsverfahren
zur Hemmung des Wachstums eines Tumors in einem menschlichen oder tierischen Körper.
24. Eingekapseltes Cisplatin nach Anspruch 14 oder 16, ein Gen, ausgewählt aus der aus
p53-, pax5- und HSV-tk-Genen bestehenden Gruppe, und eine wirksame Menge eingekapseltes
Ganciclovir zur Verwendung in einem Behandlungsverfahren zur Hemmung des Wachstums
eines Tumors in einem menschlichen oder tierischen Körper.
25. Eingekapseltes Cisplatin nach Anspruch 14 oder 16 und ein Gen, ausgewählt aus der
aus p53-, pax5- und HSV-tk-Genen bestehenden Gruppe, zur Verwendung in einem Behandlungsverfahren
zur Hemmung des Wachstums eines Tumors in einem menschlichen oder tierischen Körper,
worin die mit Cisplatin zu kombinierenden Gene eingekapseltes IL-2, IL-4, IL-7, IL-12,
GM-CSF, IFN-γ, TNF-α, RB, BRCA1, E1A, Cytosindesaminase in Kombination mit eingekapseltem
5-Fluorcytosin, blc-2, MDR-1, p21, p16, bax, bcl-xs, E2F, lGFl, VEGF und TGF-β oder
Kombinationen daraus sind.
26. Verwendung von eingekapseltem Cisplatin nach Anspruch 14 oder 16 zur Herstellung eines
Medikaments zur Verwendung bei der Behandlung zur Hemmung des Wachstums eines Tumors
in einem menschlichen oder tierischen Körper.
27. Verwendung von eingekapseltem Cisplatin nach Anspruch 14 oder 16 zur Herstellung eines
Medikaments zur Verwendung bei der Behandlung von festen Tumoren und Metastasen in
einem menschlichen oder tierischen Körper durch intravenöse Verabreichung.
28. Verwendung von eingekapseltem Cisplatin nach Anspruch 14 oder 16 und eines Gens, ausgewählt
aus der aus p53-, pax5- und HSV-tk-Genen bestehenden Gruppe, zur Herstellung eines
Medikaments zur Verwendung bei der Behandlung zur Hemmung des Wachstums eines Tumors
in einem menschlichen oder tierischen Körper.
29. Verwendung von eingekapseltem Cisplatin nach Anspruch 14 oder 16, eines Gens, ausgewählt
aus der aus p53-, pax5- und HSV-tk-Genen bestehenden Gruppe, und einer wirksamen Menge
an eingekapseltem Ganciclovir zur Herstellung eines Medikaments zur Verwendung bei
der Behandlung zur Hemmung des Wachstums eines Tumors in einem menschlichen oder tierischen
Körper.
30. Verwendung von eingekapseltem Cisplatin nach Anspruch 14 oder 16 und eines Gens, ausgewählt
aus der aus p53-, pax5- und HSV-tk-Genen bestehenden Gruppe, zur Herstellung eines
Medikaments zur Verwendung bei der Behandlung zur Hemmung des Wachstums eines Tumors
in einem menschlichen oder tierischen Körper, worin die mit Cisplatin zu kombinierenden
Gene eingekapseltes IL-2, IL-4, IL-7, IL-12, GM-CSF, IFN-γ, TNF-α, RB, BRCA1, E1A,
Cytosindesaminase in Kombination mit eingekapseltem 5-Fluorcytosin, blc-2, MDR-1,
p21, p16, bax, bcl-xs, E2F, IGFI, VEGF und TGF-β oder Kombinationen daraus sind.