U.S. patent application number 14/274377 was filed with the patent office on 2014-12-11 for novel liposome compositions.
This patent application is currently assigned to Mebiopharm Co., Ltd.. The applicant listed for this patent is Mebiopharm Co., Ltd.. Invention is credited to Tadashi FUJISAWA, Tadayuki IBUKI, Donghyun KIM, Kazushi OKADA.
Application Number | 20140363491 14/274377 |
Document ID | / |
Family ID | 36992280 |
Filed Date | 2014-12-11 |
United States Patent
Application |
20140363491 |
Kind Code |
A1 |
OKADA; Kazushi ; et
al. |
December 11, 2014 |
NOVEL LIPOSOME COMPOSITIONS
Abstract
The present disclosure provides lipid-containing compositions,
including targeted liposomes encapsulating drug, and pharmaceutical
formulations thereof, as well as methods for the making and using
the lipid-containing compositions, including the use of the
targeted liposomes in the treatment of cancer and other
diseases.
Inventors: |
OKADA; Kazushi;
(Yokohama-Shi, JP) ; IBUKI; Tadayuki;
(Funabashi-Shi, JP) ; KIM; Donghyun; (Ota-Ku,
JP) ; FUJISAWA; Tadashi; (Minato-Ku, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mebiopharm Co., Ltd. |
Tokyo |
|
JP |
|
|
Assignee: |
Mebiopharm Co., Ltd.
Tokyo
JP
|
Family ID: |
36992280 |
Appl. No.: |
14/274377 |
Filed: |
May 9, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12891568 |
Sep 27, 2010 |
8758810 |
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14274377 |
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11371586 |
Mar 8, 2006 |
7829113 |
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12891568 |
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Current U.S.
Class: |
424/450 ;
514/281; 514/283; 514/44A; 514/449; 514/492 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 35/00 20180101; A61K 31/282 20130101; A61K 47/42 20130101;
A61K 9/1271 20130101 |
Class at
Publication: |
424/450 ;
514/283; 514/281; 514/44.A; 514/492; 514/449 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 47/42 20060101 A61K047/42; A61K 31/282 20060101
A61K031/282 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 10, 2005 |
JP |
2005-67469 |
Claims
1. A targeted liposome comprising one or more phosphatidylcholines
is DOPC or DSPC, an N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine, a targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, an encapsulated drug and, at least one additional
lipid, which is cholesterol; wherein the mol % of the one or more
phosphatidylcholines is 30-70 mol %; wherein the targeting
factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine comprises a transferrin linked to a
second N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine; and wherein the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 1, ##STR00028## and the second N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 3, ##STR00029## wherein R.sup.1, R.sup.2, R.sup.5 and
R.sup.6 are each an acyl group, wherein the acyl groups are acyl
groups from saturated or unsaturated aliphatic carboxylic acids
having 16-18 carbon atoms, wherein R.sup.1, R.sup.2, R.sup.5 and
R.sup.6 are the same; and m and p are equal and are an integer from
2 to 4; and; wherein the targeted liposome contains from about 10
.mu.g transferrin/mg lipid to about 50 .mu.g transferrin/mg lipid;
and, wherein the liposome does not comprise a non-derivatized
phosphatidyl ethanolamine, egg phosphatidylcholine or a hydrophilic
polymer.
2-15. (canceled)
16. The targeted liposome according to claim 1, wherein the
transferrin is in a holo-form but not in an apo-form.
17. The targeted liposome according to claim 1, wherein the mean
diameter of the liposome is from about 50 nm to about 250 nm.
18. The liposome of claim 1, wherein R.sup.1, R.sup.2, R.sup.5 and
R.sup.6 are oleoyl or stearoyl, and m and p are 3.
19. The targeted liposome of claim 18, wherein the one or more
phosphatidylcholine is DOPC.
20-21. (canceled)
22. The targeted liposome of claim 1, wherein m and p are 3.
23. The targeted liposome of claim 1, wherein R.sup.1, R.sup.2,
R.sup.5 and R.sup.6 are oleoyl, stearoyl, or palmitoyl.
24-30. (canceled)
31. The targeted liposome according to claim 1, wherein the drug is
an anticancer agent.
32. The targeted liposome according to claim 1, wherein the drug is
a cytotoxic drug.
33. The targeted liposome according to claim 1, wherein the drug is
a topoisomerase I inhibitor.
34. The targeted liposome according to claim 33, wherein the
topoisomerase I inhibitor is topotecan or irinotecan.
35. The targeted liposome according to claim 1, wherein the drug is
a vinca alkaloid.
36. The targeted liposome according to claim 35, wherein the vinca
alkaloid is vincristine, vinblastine, vinleurosine, vinrodisine,
vinorelbine or vindesine.
37. The targeted liposome according to claim 1, wherein the drug is
a nucleic acid.
38. The targeted liposome according to claim 37, wherein the
nucleic acid is an antisense oligonucleotide or a ribozyme.
39. The targeted liposome according to claim 1, wherein the drug is
a platinum compound.
40. The targeted liposome according to claim 39, wherein the
platinum compound is biplatin, cisplatin, carboplatin, ormaplatin,
oxaliplatin, zeniplatin, enloplatin, lobaplatin or spiroplatin.
41. The targeted liposome according to claim 40, wherein the
platinum compound is oxaliplatin.
42. (canceled)
43. The targeted liposome according to claim 1, wherein the drug is
an alkylating agent.
44. The targeted liposome according to claim 1, wherein the drug is
a taxane.
45. The targeted liposome according to claim 1, wherein the drug is
a metabolic antagonist.
46. The targeted liposome according to claim 1, wherein the drug is
an antitumour antibiotic.
47. The targeted liposome according to claim 1, wherein the drug is
a hormone therapy drug.
48. The targeted liposome according to claim 1, wherein the drug is
a molecular target drug.
49. The targeted liposome according to claim 41, wherein the
oxaliplatin is dissolved in an aqueous solution of a sugar selected
from the group consisting of trehalose, maltose, sucrose, mannose,
lactose, mannitol, glycerol and dextrose.
50. The targeted liposome of claim 49, wherein the sugar is at a
concentration of from about 1 to about 20% percent sugar (v/v).
51. The targeted liposome of claim 41, wherein the concentration of
oxaliplatin is from about 0.1 mg/ml to about 25 mg/ml within the
liposome.
52. (canceled)
53. The targeted liposome claim 1, wherein the liposome does not
comprise a cationic lipid.
54. The targeted liposome of claim 1, wherein the liposome does not
comprise an anionic lipid.
55-254. (canceled)
255. The targeted liposome of claim 1, wherein the mol % of the
second N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine is 0.5-2.5 mol %.
256. The targeted liposome of claim 1, wherein the mol % of the one
or more phosphatidylcholines is 35-55 mol %.
257. The targeted liposome of claim 1, wherein R.sup.1, R.sup.2,
R.sup.5 and R.sup.6 are oleoyl or stearoyl.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/891,568, filed Sep. 27, 2010, which is a
continuation of U.S. patent application Ser. No. 11/371,586 (now
U.S. Pat. No. 7,829,113 B2), filed Mar. 8, 2006, which claims the
benefit of Japanese Patent Application No. 2005-67469, filed Mar.
10, 2005, the disclosures of which are incorporated herein by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] The efficacy of treatments for many diseases, including
cancer, has improved dramatically in the past few decades, however,
many treatment regimens require the use of drugs with deleterious
side effects, including, for example, alopecia, nausea, vomiting,
tiredness, etc. Some treatment regimens may also entail the use of
drugs that are not stable under physiological conditions, for
example, bio-therapeutics (e.g., genes or gene products) and/or
other drugs that are easily degraded or otherwise altered upon
administration and thereby loose their effectiveness before
achieving the desired therapeutic result. Such instability also
makes the drugs more difficult and costly to store and prepare for
administration.
[0003] There are a number of classes of anticancer agents,
encompassing nearly 100 individual drugs, as well as numerous drug
combination therapies, methods of delivery and treatment regimens.
Anticancer agents may be classified according to several criteria,
such as class of compound and disease state treated. Certain agents
have been developed to take advantage of the rapid division of
cancer cells and target specific phases in the cell cycle,
providing another method of classification. Agents can also be
grouped according to the type and severity of their side effects or
method of delivery. However, the most common classification of
non-biotherapeutic based anticancer agents is by class of chemical
compound, which broadly encompasses the mechanism of action of
these compounds.
[0004] Depending on the reference source consulted, there are
slight differences in the classification of anticancer agents. The
classes of compounds are described in the Physician's Desk
Reference as follows: alkaloids; alkylating agents; anti-tumor
antibiotics; antimetabolites; hormones and hormone analogs;
immunomodulators; photosensitizing agents; and miscellaneous other
agents.
[0005] The alkaloid class of compounds may also be referred to as
mitotic inhibitors, as they are cell cycle phase specific and serve
to inhibit mitosis or inhibit the enzymes required for mitosis.
They are derived generally from plant alkaloids and other natural
products and work during the M-phase of the cell cycle. This class
of compounds is often used to treat neoplasias such as acute
lymphoblastic leukemia, Hodgkin's and non-Hodgkin's lymphoma;
neuroblastomas and cancers of the lung, breast and testes.
[0006] Alkylating agents make up a large class of chemotherapeutic
agents, including of the following sub-classes, which each
represent a number of individual drugs: alkyl sulfonates;
aziridines; ethylenimines and methylmelamines; nitrogen mustards;
nitrosoureas; and others, including platinum compounds. Alkylating
agents attack neoplastic cells by directly alkylating the DNA of
cells and therefore causing the DNA to be replication incompetent.
This class of compounds is commonly used to treat a variety of
diseases, including chronic leukemias, non-Hodgkin's lymphoma,
Hodgkin's lymphoma, multiple myeloma and certain lung, breast and
ovarian cancers.
[0007] Nitrosoureas are often categorized as alkylating agents, and
have a similar mechanism of action, but instead of directly
alkylating DNA, they inhibit DNA repair enzymes causing replication
failure. These compounds have the advantage of being able to cross
the blood-brain barrier and therefore can be used to treat brain
tumors.
[0008] Antitumor antibiotics have antimicrobial and cytotoxic
activity and also interfere with DNA by chemically inhibiting
enzymes and mitosis or by altering cell membranes. They are not
cell cycle phase specific and are widely used to treat a variety of
cancers.
[0009] The antimetabolite class of anticancer agents interfere with
the growth of DNA and RNA and are specific to the S-phase of the
cell-cycle. They can be broken down further by type of compound,
which include folic acid analogs, purine analogs, and pyrimidine
analogs. They are often employed in the treatment of chronic
leukemia, breast, ovary, and gastrointestinal tumors.
[0010] There are two classes of hormones or hormone analogs used as
anticancer agents, the corticosteroid hormones and sex hormones.
While some corticosteroid hormones can both kill cancer cells and
slow the growth of tumors, and are used in the treatment of
lymphoma, leukemias, etc., sex hormones function primarily to slow
the growth of breast, prostate and endometrial cancers. There are
numerous subclasses of hormones and hormone analogs, including,
androgens, antiadrenals, antiandrogens, antiestrogens, aromatase
inhibitors, estrogens, leutenizing hormone releasing hormone (LHRH)
analogs and progestins.
[0011] An additional smaller class of anticancer agents is
classified as immunotherapy. These are agents that are intended to
stimulate the immune system to more effectively attack the
neoplastic (cancerous) cells. This therapy is often used in
combination with other therapies.
[0012] There are also a number of compounds, such as campothectins,
which are generally listed as `other` anticancer agents and can be
used to treat a variety of neoplasias.
[0013] Combinations of anticancer agents are also utilized in the
treatment of a number of cancers. For example, Sanofi Syntholabo
markets ELOXATIN.TM. (oxaliplatin for injection) for the treatment
of colorectal cancer for use in combination with 5-fluorouracil and
leuvocorin. This combination of drugs is often used adjunctively
with surgery in the treatment of colorectal cancer. Oxaliplatin is
an alkylating agent that is believed to act by inhibiting both DNA
replication and transcription. Unlike other platinum agents,
oxaliplatin has demonstrated a decreased likelihood of resistance
development. Oxaliplatin is further described in U.S. Pat. Nos.
4,169,846; 5,338,874; 5,298,642; 5,959,133; 5,420,319; 5,716,988;
5,290,961; and in Wilkes G M. "New therapeutic options in colon
cancer: focus on oxaliplatin" Clin J Oncol Nurs. (2002)
6:131-137.
[0014] While there are a plethora of anticancer agents, the benefit
of these compounds is often outweighed by the severity of the side
effects produced by the agent. This comparison is often referred to
as the therapeutic index, which describes the balance between the
required dose to accomplish the destruction of the cancer cells
compared to the dose at which the substance is unacceptably toxic
to the individual. The drawback to most anticancer agents is the
relatively small range of the therapeutic index, (i.e., the narrow
dosage range in which cancer cells are destroyed without
unacceptable toxicity to the individual). This characteristic
limits the frequency and dosage where an agent is useful, and often
the side effects become intolerable before the cancer can be fully
eradicated.
[0015] The severe side effects experienced with the majority of
cancer chemotherapeutics are a result of the non-specific nature of
these drugs, which do not distinguish between healthy and cancerous
cells, and instead destroy both. Certain cell cycle specific drugs
attempt to lessen these effects, targeting phases of the cell cycle
involved in cell replication and division. These drugs do not,
however, distinguish between cancerous cells and healthy cells that
are undergoing normal cell division. The cells most at risk from
these types of chemotherapy are those which undergo cell division
often, including blood cells, hair follicle cells, and cells of the
reproductive and digestive tracts.
[0016] The most common side effects of anticancer agents are nausea
and vomiting. A large proportion of individuals also suffer from
myelosuppression, or suppression of the bone marrow, which produces
red blood cells, white blood cells and platelets. These and other
side effects are also exacerbated by the suppression of the immune
system concomitant with the destruction and lack of production of
white blood cells, and associated risk of opportunistic
infection.
[0017] Other side effects common to a wide range of anticancer
agents include: hair loss (alopecia); appetite loss; weight loss;
taste changes; stomatitis and esophagitis (inflammation and sores);
constipation; diarrhea; fatigue; heart damage; nervous system
changes; lung damage; reproductive tissue damage; liver damage;
kidney and urinary system damage.
[0018] The wide range of the side effects associated with most
anticancer agents and their severity in individuals who are already
debilitated with disease and possibly immune compromised has led
researchers to search for mechanisms by which they can alleviate
some of the side effects while maintaining the efficacy of the
treatment. Several approaches to this problem have been taken. They
include combination chemotherapy, where multiple anticancer agents
are administered together; adjuvant therapies, where additional
agents are prescribed along with the anticancer agent to fight the
side effects of the anticancer agent; combined modality treatments,
where chemotherapy is combined with radiation and/or surgery; and
alternative delivery vehicles for the administration of anticancer
agents, such as the encapsulation of anticancer agents in
liposomes.
[0019] Liposomes are formed when phospholipids and their
derivatives are dispersed in water. Upon dispersion in water the
phospholipids form closed vesicles called "liposomes", which are
characterized by lipid bilayers encapsulating an aqueous core.
Various liposomes have been used as carriers for entrapped
therapeutic agents, such as drugs, enzymes and genetic sequences
for use in medical science, in pharmaceutical science and in
biochemistry.
[0020] Examples of liposome compositions include U.S. Pat. Nos.
4,983,397; 6,476,068; 5,834,012; 5,756,069; 6,387,397; 5,534,241;
4,789,633; 4,925,661; 6,153,596; 6,057,299; 5,648,478; 6,723,338;
6,627218; U.S. Pat. App. Publication Nos: 2003/0224037;
2004/0022842; 2001/0033860; 2003/0072794; 2003/0082228;
2003/0212031; 2003/0203865; 2004/0142025; 2004/0071768;
International Patent Applications WO 00/74646; WO 96/13250; WO
98/33481; Papahadjopolulos D, Allen T M, Gbizon A, et al.
"Sterically stabilized liposomes: Improvements in pharmacokinetics
and antitumor therapeutic efficacy" Proc Natl Acad Sci U.S.A.
(1991) 88: 11460-11464; Allen T M, Martin F J. "Advantages of
liposomal delivery systems for anthracyclines" Semin Oncol (2004)
31: 5-15 (suppl 13). Weissig et al. Pharm. Res. (1998) 15:
1552-1556.
[0021] In earlier stages of developing liposomes, naturally
occurring phospholipids of the cell membrane such as egg-yolk
phospholipids and soybean phospholipids were used. In the case of
being intravenously administered, however, liposomes utilizing
these phospholipids are likely to be incorporated into the
reticuloendothelial system of liver or spleen, causing a problem of
low blood retention time and thereby reducing the efficacy of the
drug. Thereafter, as a means for solving this problem, synthetic
phospholipids whose lipid portion contains only saturated bonds
were used as a constituent of the liposome membrane in order to
harden the liposome membrane.
[0022] In an effort to prolong the circulatory half-life of
liposomes and avoid uptake by the reticuloendothelial system,
researchers developed liposomes that were modified by the
incorporation of polyethylene glycol or other hydrophilic polymers
(e.g., a PEG liposome where one or more of the constituent lipids
was modified by attachment of PEG). PEG-modified liposomes were
also often referred to as "shielded" liposomes. Doxil.TM.
(doxorubicin HCl liposome injection) is a liposome-enclosed
doxorubicin, with adjunct polyethylene glycol (PEG) utilized to
avoid the reticuloendothelial system (RES) and prolong drug
circulation time. See Vail D M, Amantea M A, Colbern G T, et al.,
"Pegylated Liposomal Doxorubicin: Proof of Principle Using
Preclinical Animal Models and Pharmacokinetic Studies." Semin
Oncol. (2004) 31 (Suppl 13): 16-35. However, adverse effects were
also caused by prolonged blood retention (e.g., hand-foot syndrome,
an adverse effect of Doxil.RTM. on the peripheral system, etc.)
became recognized as a problem.
[0023] Examples of liposomes include U.S. Pat. Nos. 4,983,397;
5,013,556; 6,316,024; 6,056,973; 5,945,122; 5,891,468; 6,126,966;
5,593,622, 5,676,971; 6,586,559; and 5,846,458 U.S. Pat. App.
Publication. Nos. 2003/0224037; 2004/0022842; 2003/0113262;
2002/0136707; International Patent Applications WO 99/30686; WO
02/41870 Aliminana et al., Prep. Biochem. Biotech. (2004) 34(1):
77-96. Liposomes are also described in U.S. Pat. Nos. 6,228,391;
6,197,333; 6,046,225; 5,292,524; and U.S. Pat. App. Pub. Nos.
20050271588; 20040213833; 20040029210; 20030175205; 20030162748;
20030130190; 20030059461; and 20020034537.
[0024] In addition to PEG-modified liposomes, researchers developed
a variety of other derivatized lipids. These derivatized lipids
could also be incorporated into liposomes. See, for example:
International Patent Application WO 93/01828; Park Y S, Maruyama K,
Huang L. "Some negatively charged phospholipids derivatives prolong
the liposome circulation in vivo." Biochimica et Biophysica Acta
(1992) 1108: 257-260; Ahl et al., Biochimica Biophys. Acta (1997)
1329: 370-382.
[0025] Additional lipid compositions are described in U.S. Pat.
Nos. 6,936,272; 6,897,196; 6,077,834; and U.S. Pat. App. Pub. Nos.
20050136064; 20040234588; 20030215490; 20030166601; and
20010038851.
[0026] In addition to modification of liposomes with PEG and other
hydrophilic polymers, researchers also developed liposomes that
aimed to specifically target particular cell types by incorporating
targeting factors (also referred to as targeting ligands) for
particular cell types. Examples of targeting factors/ligands
include asialoglycoprotein, folate, transferrin, antibodies, etc.
In some cases one or more of the constituent lipids could be
modified by the attachment of a targeting factor.
[0027] Examples of lipid compositions including targeting factors
include U.S. Pat. Nos. 5,049,390; 5,780,052; 5,786,214; 6,316,024;
6,056,973; 6,245,427; 6,524,613; 6,749,863; 6,177,059; 6,530,944;
U.S. Pat. App. Publication. Nos. 2004/0022842; 2003/0224037;
2003/143742; 2003/0228285; 2002/0198164; 2003/0220284;
2003/0165934; 2003/0027779; International Patent Application Nos.
WO 95/33841; WO 95/19434; WO 2001037807; WO 96/33698; WO
2001/49266; WO 9940789; WO 9925320; WO 9104014; WO 92/07959; EP
1369132; JP 2001002592; Iinuma H, Maruyama K, et al.,
"Intracellular targeting therapy of cisplatin-encapsulated
transferrin polyethylene glycol liposome on peritoneal
dissemination of gastric cancer" Int J Cancer (2002) 99 130-137;
Ishida 0, Maruyama K, Tanahashi H, Iwatsuru M, Sasaki K, et al.,
"Liposomes bearing polyethylene glycol-coupled transferrin with
intracellular targeting property to the solid tumors in vivo."
Pharmaceutical Research (2001) 18: 1042-1048; Holmberg et al.,
Biochem. Biophys. Res. Comm. (1989) 165(3):1272-1278; Nam et al.,
J. Biochem. Mol. Biol. (1998) 31(1): 95-100; Nag et al., J. Drug
Target. (1999) 6(6): 427-438.
[0028] In particular, Iinuma et al. developed a Tf-PEG-liposome,
with transferrin (Tf) attached at the surface of the liposome.
Iinuma et al., showed that a greater number of liposomes were bound
to the surface of the tumor cells, and there was a greater uptake
of liposomes by the tumor cells for Tf-PEG-liposome as compared to
PEG-liposome (Inuma et al., ibid; Ishida et al., ibid).
[0029] However, despite recent advances made in the drug and
labeled compound delivery field, including the use of liposome
compositions, there is still a need for improved liposome
compositions for the delivery of drugs and labeled compounds to
specific cells and/or tissues that achieve a therapeutic or
diagnostic effect. In particular in the cancer field, drug
formulations with improved specificity and reduced toxicity are
need to ensure therapeutic benefit without adversely effecting
healthy cells and which also do not result in deleterious side
effects for the individual being treated. Similarly, labeled
compounds that can be used to detect conditions, particularly
life-threatening conditions at an early stage (e.g., with high
specificity and/or high sensitivity) and also accurately monitor
the severity/extent of the condition (e.g., progression and/or
regression with or without treatment) would also significantly
improve the quality and success of therapy.
BRIEF SUMMARY OF THE INVENTION
[0030] The present invention relates to novel lipid-containing
compositions (including liposomes (e.g., targeted liposomes, blank
liposomes), lipid mixtures and liposome-containing compositions)
that may optionally incorporate a drug or labeled compound, or may
be used in the preparation of formulations that incorporate a drug
or labeled compound, where the lipid-containing composition confers
the benefit of reducing side effects from the drug or labeled
compound and/or also prevents degradation and/or loss of efficacy
of the drug or labeled compound. The invention also includes
methods of making and using the lipid-containing compositions
described herein. In certain aspects of the invention, the
lipid-containing compositions may be used to treat or diagnose
cancer (e.g., breast cancer, gastric cancer, colorectal cancer,
colon cancer, cancer of the pancreas, non small cell lung cancer,
small cell lung cancer, brain cancer, liver cancer, renal cancer,
prostate cancer, bladder cancer, ovarian cancer, or hematological
malignancies (e.g., leukemia, lymphoma, multiple myeloma,
etc.).
[0031] In certain embodiments are provided targeted liposomes
comprising one or more phospholipids, an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine, a targeting
factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine, an encapsulated drug or labeled compound
and, optionally, at least one additional lipid, wherein the
targeting factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine comprises a targeting ligand linked to a
second N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine; and wherein the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 1,
##STR00001##
and
[0032] the second N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is represented by Formula 3,
##STR00002## [0033] wherein R.sup.1, R.sup.2, R.sup.5 and R.sup.6
are each, independently, an acyl group, and m and p are,
independently, an integer from 1 to 10; and, wherein the liposome
does not comprise a non-derivatized phosphatidyl ethanolamine or
polyethylene glycol, and wherein the targeting ligand is not an
intact antibody.
[0034] In other embodiments are provided blank liposomes comprising
one or more phospholipids, an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine, a targeting
factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine, and, optionally, at least one additional
lipid, wherein the targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
comprises a targeting ligand linked to a second
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine; and wherein the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 1,
##STR00003##
and
[0035] the second N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine, where present, is represented by Formula
3,
##STR00004## [0036] wherein R.sup.1, R.sup.2, R.sup.5 and R.sup.6
are each, independently, an acyl group, and m and p are,
independently, an integer from 1 to 10; and, wherein the liposome
does not comprise a non-derivatized phosphatidyl ethanolamine,
polyethylene glycol, drug or labeled compound, and wherein the
targeting ligand is not an intact antibody.
[0037] In certain embodiments of the targeted liposomes and blank
liposomes, R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are oleoyl or
stearoyl, and m and p are 3. In certain embodiments, the targeting
ligand is transferrin. In particular embodiments, the one or more
phospholipids is DMPC or DSPC, and the at least one additional
lipid is present and is cholesterol. In certain embodiments of the
targeted liposomes and blank liposomes, R.sup.1, R.sup.2, R.sup.5
and R.sup.6 are oleoyl, m and p are 3, the one or more phospholipid
is DMPC and the additional lipid is cholesterol.
[0038] In certain embodiments of the targeted liposomes and blank
liposomes, m and p are each independently, an integer from 2 to 4.
In some embodiments, m and p are equal and are an integer from 2 to
4. In particular embodiments, m and p are equal and are 3. In
certain embodiments, R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are
each, independently, oleoyl, stearoyl, palmitoyl or myristoyl. In
some embodiments, R.sup.1 and are R.sup.2 are the same, and R.sup.5
and R.sup.6 are the same. In other embodiments, R.sup.1, R.sup.2,
R.sup.5 and R.sup.6 are the same. In particular embodiments,
R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are oleoyl or stearoyl. In
certain embodiments, R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are
oleoyl.
[0039] In further embodiments are provided lipid mixtures
comprising a mixture of one or more phospholipids, an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, a succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine, and, optionally, at
least one additional lipid, wherein the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 1,
##STR00005##
and
[0040] the succinimidyl ester of the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 2,
##STR00006## [0041] wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4
are each, independently, an acyl group, m and n are, independently,
an integer from 1 to 10; and, wherein the mixture does not comprise
a non-derivatized phosphatidyl ethanolamine or polyethylene
glycol.
[0042] In certain embodiments of the lipid mixtures, m and n are
each independently, an integer from 2 to 4. In some embodiments, m
and n are equal and are an integer from 2 to 4. In particular
embodiments, m and n are equal and are 3.
[0043] In certain embodiments of the lipid mixtures, R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are each, independently, oleoyl,
stearoyl, palmitoyl or myristoyl. In some embodiments, R.sup.1 and
are R.sup.2 are the same, and R.sup.3 and R.sup.4 are the same. In
particular embodiments, R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are
the same. In some embodiments, R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are oleoyl or stearoyl. In certain embodiments, m and n are
3, where the one or more phospholipids is DMPC or DSPC and the at
least one additional lipid is present and is cholesterol. In
certain embodiments of the lipid mixtures, R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are oleoyl, m and n are 3, the one or more
phospholipid is DMPC and the additional lipid is cholesterol.
[0044] In further embodiments are provided lipid mixtures
comprising a mixture of one or more phospholipids, an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, a targeting factor-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine, and, optionally, at
least one additional lipid, wherein the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 1,
##STR00007##
and
[0045] the targeting factor-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine comprises a targeting
ligand linked to a second N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine; and wherein the second
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is represented by Formula 3,
##STR00008## [0046] wherein R.sup.1, R.sup.2, R.sup.5 and R.sup.6
are each, independently, an acyl group, m and p are, independently,
an integer from 1 to 10; and, wherein the mixture does not comprise
a non-derivatized phosphatidyl ethanolamine or polyethylene glycol,
and wherein the targeting ligand is not an intact antibody.
[0047] In particular embodiments of the lipid mixtures, where a
targeting factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is present, m and p are each
independently, an integer from 2 to 4. In certain embodiments, m
and p are equal and are an integer from 2 to 4. In some
embodiments, m and p are equal and are 3.
[0048] In particular embodiments of the lipid mixtures, where a
targeting factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is present, R.sup.1, R.sup.2, R.sup.5 and
R.sup.6 are each, independently, oleoyl, stearoyl, palmitoyl or
myristoyl. In some embodiments, wherein R.sup.1 and are R.sup.2 are
the same, and R.sup.5 and R.sup.6 are the same. In further
embodiments, R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are the same. In
some embodiments, R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are oleoyl
or stearoyl. In certain embodiments, R.sup.1, R.sup.2, R.sup.5 and
R.sup.6 are oleoyl or stearoyl, m and p are 3, the one or more
phospholipids is DMPC or DSPC, the at least one additional lipid is
cholesterol, and the targeting ligand is transferrin. In certain
embodiments of the lipid mixtures, R.sup.1, R.sup.2, R.sup.5 and
R.sup.6 are oleoyl, m and p are 3, the one or more phospholipid is
DMPC, the additional lipid is cholesterol and the targeting ligand
is transferrin.
[0049] In certain embodiments are provided liposome-containing
compositions comprising liposomes comprising one or more
phospholipids, an N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine, a succinimidyl ester of an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, and, optionally, at least one additional lipid,
wherein the N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine is represented by Formula 1,
##STR00009##
and
[0050] the succinimidyl ester of the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 2,
##STR00010## [0051] wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4
are each, independently, an acyl group, m and n are, independently,
an integer from 1 to 10; and, wherein the composition does not
comprise a non-derivatized phosphatidyl ethanolamine or
polyethylene glycol.
[0052] In some embodiments of the liposome-containing compositions
m and n are each independently, an integer from 2 to 4. In certain
embodiments, m and n are equal and are an integer from 2 to 4. In
particular embodiments, m and n are equal are 3.
[0053] In some embodiments of the liposome-containing compositions,
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each, independently,
oleoyl, stearoyl, palmitoyl or myristoyl. In particular
embodiments, R.sup.1 and are R.sup.2 are the same, and R.sup.3 and
R.sup.4 are the same. In some embodiments, R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are the same. In certain embodiments, R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are oleoyl or stearoyl. In some
embodiments, R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are oleoyl or
stearoyl, and m and n are 3 and the one or more phospholipids is
DMPC, DSPC, POPC or DPPC. In certain embodiments, R.sup.1, R.sup.2,
R.sup.3 and R4.sup.6 are oleoyl, m and n are 3, the one or more
phospholipid is DMPC and the additional lipid is cholesterol.
[0054] In further embodiments of the liposome-containing
compositions are provided liposome-containing compositions
comprising liposomes comprising one or more phospholipids, an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, a targeting factor-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine, and, optionally, at
least one additional lipid, wherein the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 1,
##STR00011##
and
[0055] the targeting factor-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine comprises a targeting
ligand linked to a second N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine; and wherein the second
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is represented by Formula 3,
##STR00012## [0056] wherein R.sup.1, R.sup.2, R.sup.5 and R.sup.6
are each, independently, an acyl group, m and p are, independently,
an integer from 1 to 10; and, wherein the composition does not
comprise a non-derivatized phosphatidyl ethanolamine or
polyethylene glycol, and wherein the targeting ligand is not an
intact antibody.
[0057] In certain of the liposome-containing compositions, where a
targeting-factor modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is present, m and p are each
independently, an integer from 2 to 4. In particular embodiments, m
and p are equal and are an integer from 2 to 4. In some
embodiments, m and p are equal are 3.
[0058] In certain of the liposome-containing compositions, where a
targeting-factor modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is present, R.sup.1, R.sup.2, R.sup.5 and
R.sup.6 are each, independently, oleoyl, stearoyl, palmitoyl or
myristoyl. In particular embodiments, R.sup.1 and are R.sup.2 are
the same, and R.sup.5 and R.sup.6 are the same. In certain
embodiments, wherein R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are the
same. In some embodiments, R.sup.1, R.sup.2, R.sup.5 and R.sup.6
are oleoyl or stearoyl. In particular embodiments, R.sup.1,
R.sup.2, R.sup.5 and R.sup.6 are oleoyl or stearoyl, the one or
more phospholipids is DMPC or DSPC, the at least one additional
lipid cholesterol, and the targeting ligand is transferrin. In
certain embodiments, R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are
oleoyl, m and p are 3, the one or more phospholipid is DMPC, the
additional lipid is cholesterol and the targeting ligand is
transferrin.
[0059] In further embodiments of the liposome-containing
compositions a drug is included. In certain embodiments, the one or
more phospholipid is DMPC or DSPC, R.sup.1, R.sup.2 and, where
present, R.sup.5 and R.sup.6 are oleoyl or stearoyl, m and, where
present, p are 3, the at least one additional lipid, where present,
is cholesterol, the drug is oxaliplatin, and the targeting ligand,
where present, is transferrin. In certain embodiments, the
composition further includes a sugar at a concentration of from
about 1 to about 20% percent sugar (v/v). In certain embodiments,
the one or more phospholipid is DMPC or DSPC, R.sup.1, R.sup.2 and,
where present, R.sup.5 and R.sup.6 are oleoyl, m and, where
present, p are 3, the at least one additional lipid, where present,
is cholesterol, the drug is oxaliplatin, and the targeting ligand,
where present, is transferrin.
[0060] In further embodiments of the liposome-containing
compositions a labeled compound is included. In certain
embodiments, the labeled compound comprises a radioisotopic
moiety.
[0061] In certain embodiments of the targeted liposomes, blank
liposomes, lipid mixtures and liposome-containing compositions, the
at least one additional lipid is present. In particular
embodiments, the at least one additional lipid is cholesterol or a
cholesterol derivative.
[0062] In particular embodiments of the targeted liposomes, blank
liposomes, lipid mixtures and liposome-containing compositions, the
one or more phospholipids is a phosphatidylcholine, phosphatidic
acid, phosphatidylserine or phosphatidylglycerol. In particular
embodiments, the one or more phospholipids is a neutral
phospholipid. In some embodiments, the one or more phospholipids is
a phosphatidylcholine. In particular embodiments, the phosphatidyl
choline includes a moiety of a saturated fatty acid. In certain
embodiments, the one or more phospholipids is DMPC, DSPC, POPC or
DPPC. In particular of these embodiments, the at least one
additional lipid is present. And in certain of these, the at least
one additional lipid is cholesterol or a cholesterol derivative. In
particular embodiments, DMPC and cholesterol, DSPC and cholesterol,
POPC and cholesterol, or DPPC and cholesterol are incorporated. In
certain embodiments, DMPC and cholesterol.
[0063] In particular embodiments of the targeted liposomes, blank
liposomes, lipid mixtures and liposome-containing compositions,
where present, targeting ligand is directed to a target cell. In
some embodiments, the targeting ligand is directed to a cell
surface receptor of a target cell. In particular embodiments, the
targeting ligand is transferrin, folic acid, hyaluronic acid, a
sugar chain or a fragment of a monoclonal antibody. In certain
embodiments, the targeting ligand is transferrin, folic acid,
hyaluronic acid or a sugar chain. In other embodiments, the
targeting ligand is transferrin, folic acid, hyaluronic acid or a
sugar chain. In some embodiments, the targeting ligand is
transferrin. In particular embodiments, the transferrin is in a
holo-form but not in an apo-form. In some embodiments, the
transferrin is in a holo-form.
[0064] In certain embodiments of the targeted liposomes and blank
liposomes, the mean diameter of the liposome is from about 50 nm to
about 250 nm. In others, the mean diameter of the liposome is from
about 90 nm to about 200 nm.
[0065] In particular embodiments of the targeted liposomes and
blank liposomes, the zeta potential of the liposome is negative. In
certain embodiments, the zeta potential is from about -75 mV to
about -90 mV. In others, the zeta potential is from about -80 mV to
about -85 mV.
[0066] In certain embodiments of the liposome-containing
compositions, targeted liposomes and blank liposomes, the
formulations further include a solution.
[0067] In particular embodiments of the targeted liposomes and
liposome-containing compositions, the drug is present.
[0068] In particular embodiments of the targeted liposomes and
liposome-containing compositions, the drug is oxaliplatin. In
certain embodiments where the drug is oxaliplatin, the targeting
ligand is transferrin. In certain embodiments, the at least one
additional lipid is present and is cholesterol.
[0069] In certain embodiments, the drug is an anticancer agent. In
particular embodiments, the drug is a cytotoxic drug. In some
embodiments, the drug is a topoisomerase I inhibitor. In particular
embodiments, the topoisomerase I inhibitor is topotecan or
irinotecan. In other embodiments, the drug is a vinca alkaloid. In
particular embodiments, the vinca alkaloid is vincristine,
vinblastine, vinleurosine, vinrodisine, vinorelbine or vindesine.
In some embodiments, wherein the drug is a nucleic acid. In certain
embodiments, the nucleic acid is an antisense oligonucleotide or a
ribozyme. In some embodiments, the drug is an alkylating agent. In
particular embodiments, the drug is a taxanes. In other
embodiments, the drug is a metabolic antagonist. In certain
embodiments, the drug is an antitumour antibiotic. In some
embodiments, the drug is a hormone therapy drug. In some
embodiments, the drug is a molecular target drug.
[0070] In some embodiments of the targeted liposomes and
liposome-containing compositions, the drug is a platinum compound.
In particular embodiments, the platinum compound is biplatin,
cisplatin, carboplatin, ormaplatin, oxaliplatin, zeniplatin,
enloplatin, lobaplatin or spiroplatin. In some embodiments, the
platinum compound is oxaliplatin.
[0071] In some embodiments where the drug is oxaliplatin, R.sup.1,
R.sup.2, R.sup.5 and R.sup.6 are oleoyl or stearoyl, m and p are 3,
the targeting ligand is transferrin, the one or more phospholipids
is DMPC or DSPC, and the at least one additional lipid is present
and is cholesterol. In certain embodiments where the drug is
oxaliplatin, R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are oleoyl, m
and p are 3, the targeting ligand is transferrin, the one or more
phospholipids is DMPC, and the at least one additional lipid is
present and is cholesterol. In particular embodiments, the targeted
liposomes and liposome-containing compositions are free of other
lipid components.
[0072] In some embodiments where the drug is oxaliplatin, the
oxaliplatin is dissolved in an aqueous solution of a sugar selected
from the group consisting of trehalose, maltose, sucrose, mannose,
lactose, mannitol, glycerol and dextrose. In certain embodiments,
the sugar is at a concentration of from about 1 to about 20%
percent sugar (v/v). In particular embodiments, the concentration
of oxaliplatin is from about 0.1 mg/ml to about 25 mg/ml within the
liposome. In other embodiments, the concentration of oxaliplatin is
from about 0.5 mg/ml to about 10 mg/ml within the liposome. In
still other embodiments, the concentration of oxaliplatin is from
about 0.5 mg/ml to about 3 mg/ml.
[0073] In particular embodiments of the of the targeted liposomes
and liposome-containing compositions, the labeled compound is
present. In certain embodiments, the labeled compound includes a
radioisotopic moiety. In particular embodiments, the radioisotopic
moiety incorporates .sup.125I.
[0074] In particular embodiments of the targeted liposomes and the
liposome-containing compositions, the concentration of targeting
ligand incorporated in the liposome is from about 1.0 mg/ml to
about 3.0 mg/ml. In others, the concentration of targeting ligand
incorporated in the liposome is from about 1.0 mg/ml to about 2.5
mg/ml.
[0075] In particular embodiments of the targeted liposomes and the
liposome-containing compositions where drug is present and is
oxaliplatin, the targeting ligand is transferrin. In particular
embodiments, the transferrin is in a holo-form. In some
embodiments, ferric iron is in a concentration of from about 0.4 to
about 3.0 .mu.g/ml. In other embodiments, ferric iron is in a
concentration of from about 0.4 to about 1.5 .mu.g/ml.
[0076] In particular embodiments of the targeted liposomes, blank
liposomes, lipid mixtures and liposome-containing compositions, the
liposomes, lipid mixtures or liposome-containing composition does
not comprise a cationic lipid. In particular embodiments, the
liposomes, lipid mixtures or liposome-containing composition do not
comprise an anionic lipid. In some embodiments, the liposomes,
lipid liposome, lipid mixtures or liposome-containing composition
do not comprise either an anionic lipid or a cationic lipid.
[0077] In particular embodiments of the targeted liposomes, blank
liposomes, lipid mixtures and liposome-containing compositions, the
formulations further include a solution. In certain embodiments,
the lipid mixtures are free of solution. In particular embodiments,
the solution is an aqueous solution or a mixture of an aqueous
solution and a water-miscible solvent.
[0078] In particular embodiments of the targeted liposomes, blank
liposomes, lipid mixtures and liposome-containing compositions, the
formulations further include sucrose.
[0079] In a further aspect of the invention are provided
pharmaceutical formulations of the lipid-containing compositions
described herein. Particular embodiments of the liposome-containing
compositions, targeted liposomes and blank liposomes include the
liposome-containing compositions, targeted liposomes or blank
liposomes as described herein and one or more pharmaceutically
acceptable carriers, excipients, diluents, stabilizers, or
preservatives.
[0080] In yet another aspect of the invention are provided kits
including the lipid-containing compositions described herein.
Certain embodiments of the liposome-containing compositions,
targeted liposomes and blank liposomes include the
liposome-containing compositions, targeted liposomes or blank
liposomes as described herein, packaging and instructions for
use.
[0081] In certain embodiments of the kits, the liposome-containing
compositions, targeted liposomes or blank liposomes as described
herein is contained in a first container and one or more
pharmaceutically acceptable carriers, excipients, diluents,
stabilizers, or preservatives are contained in a second
container.
[0082] In particular embodiments are provided kits incorporating
the pharmaceutical formulations described herein, packaging and
instructions for use.
[0083] In another aspect of the invention are provided methods of
making the lipid-containing compositions described herein.
[0084] In particular embodiments are provided methods of making
targeted liposomes as described herein, comprising the steps
of:
[0085] a) mixing the one or more phospholipids, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, the targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, and, optionally, the at least one additional lipid,
to form a lipid mixture;
[0086] b) adding a drug or labeled compound to the lipid mixture
formed in step (a);
[0087] c) forming a liposome.
[0088] In a further embodiments of the method is provided a step
(d), purifying the liposome of step (c). In particular embodiments,
the drug in step (b) is in aqueous solution prior to mixing. In
certain embodiments, step (c) comprises sonication or stirring. In
some embodiments, step (c) comprises extrusion.
[0089] In other embodiments are provided methods of making targeted
liposomes as described herein, comprising the steps of
[0090] a) mixing the one or more phospholipids, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, a succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine, and, optionally, the at
least one additional lipid, to form a lipid mixture
[0091] wherein the succinimidyl ester of the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is represented by Formula 2,
##STR00013## [0092] wherein R.sup.3 and R.sup.4 are each,
independently, an acyl group, n is, independently, an integer from
1 to 10;
[0093] b) adding drug or labeled compound to the lipid mixture
formed in step (a);
[0094] c) forming a liposome; and,
[0095] d) linking a targeting ligand to the succinimidyl ester of
an N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine.
[0096] In certain embodiments of the above-described method, the
method also includes a step (e), purifying the liposome of step
(d).
[0097] In particular embodiments, the drug in step (b) is in
aqueous solution prior to mixing. In some embodiments, step (c)
comprises sonication or stirring. In certain embodiments, step (c)
comprises extrusion. In particular embodiments, step (c) comprises
stirring.
[0098] In certain embodiments are provided methods of making blank
liposomes as described herein, comprising the steps of
[0099] a) mixing the one or more phospholipids, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, the targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, and, optionally, the at least one additional lipid,
to form a lipid mixture; and
[0100] b) forming a liposome.
[0101] In certain embodiments of the methods of preparing the blank
liposomes, the method further comprises a step (c), purifying the
liposome of step (b).
[0102] In particular embodiments, step (b) comprises sonication or
stirring. In some embodiments, step (b) comprises extrusion.
[0103] In other embodiments are provided methods of making blank
liposomes as described herein, comprising the steps of
[0104] a) mixing the one or more phospholipids, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, and a succinimidyl ester of an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, and, optionally, the at least one additional lipid,
to form a lipid mixture,
[0105] wherein the succinimidyl ester of the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is represented by Formula 2,
##STR00014## [0106] wherein R.sup.3 and R.sup.4 are each,
independently, an acyl group, n is, independently, an integer from
1 to 10;
[0107] b) forming a liposome; and,
[0108] c) linking a targeting ligand to the succinimidyl ester of
an N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine to form a targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine.
[0109] In certain of the methods of preparing the blank liposomes,
the methods further include a step (d), purifying the liposome of
step (c).
[0110] In particular embodiments, step (b) comprises sonication or
stirring. In some embodiments, step (b) comprises extrusion.
[0111] In other embodiments are provided methods of making the
lipid-containing compositions as described herein, comprising the
step of mixing the one or more phospholipids, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
and the succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine.
[0112] In further embodiments are provided methods of making the
lipid-containing compositions where the at least one additional
lipid is present, as described herein, comprising the step of
mixing the one or more phospholipids, the at least one additional
lipid, the N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine and the succinimidyl ester of an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine.
[0113] In another embodiments are provided methods of making the
lipid-containing compositions as described herein, comprising the
step of mixing the one or more phospholipids, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
and the targeting factor-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine.
[0114] In further embodiments are provided methods of making the
lipid-containing compositions, where the at least one additional
lipid is present, as described herein, comprising the step of
mixing the one or more phospholipids, the at least one additional
lipid, the N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine and the targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine.
[0115] Also provided are methods of making the liposome-containing
compositions described herein, comprising the steps of
[0116] a) mixing the one or more phospholipid lipids, and the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
and, where present, the succinimidyl ester of an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, or targeting-factor modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine, and, optionally, where
present, at least one additional lipid, to form a lipid mixture;
and
[0117] b) adding a drug to the lipid mixture formed in step (a);
and,
[0118] c) forming a liposome.
[0119] In certain embodiments of the methods of making the various
lipid-containing compositions (targeted liposomes, blank liposomes,
liposome-containing compositions), where a drug is present, the
drug is in an aqueous solution. In certain embodiments, step a) is
performed in the presence of organic solvent. In some embodiments,
aqueous solution further comprises a sugar. In certain embodiments
the aqueous solution may also include a water-miscible organic
solvent.
[0120] In other embodiments are provided methods of making the
liposome-containing compositions, comprising the steps of
[0121] a) mixing the one or more phospholipid lipids, and the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
and,
[0122] where present, the succinimidyl ester of an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, or the targeting-factor modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, and, optionally, where present, the at least one
additional lipid, to form a lipid mixture; and
[0123] b) adding a labeled compound to the lipid mixture formed in
step (a).
[0124] c) forming a liposome
[0125] In certain embodiments of the methods of making the various
lipid-containing compositions (targeted liposomes, blank liposomes,
liposome-containing compositions), where a labeled compound is
present, the labeled compound is in an aqueous solution. In certain
embodiments, step a) is performed in the presence of organic
solvent. In certain embodiments the aqueous solution may also
include a water-miscible organic solvent.
[0126] In certain embodiments are also provided methods of making
the liposome-containing compositions as described herein, wherein
the liposome-containing composition includes a targeting
factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine, comprising the steps of
[0127] a) mixing the one or more phospholipids, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
and the targeting factor-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine and, optionally, at
least one additional lipid, to form a lipid mixture; and
[0128] b) adding solvent to the mixture formed in step (a) to form
a liposome-containing composition.
[0129] In particular embodiments, the mixing step (a) is performed
in the presence of an organic solvent. In particular embodiments,
the solvent in step (b) is an aqueous solution or a mixture of
aqueous solution and a water-miscible solvent.
[0130] In certain embodiments, step (b) comprises sonication or
stirring. In some embodiments, step (b) comprises extrusion.
[0131] In particular embodiments of the methods of making the
lipid-containing compositions, in step (a) the at least one
additional lipid is present.
[0132] In some embodiments of the methods of making the
lipid-containing compositions, in step (a) the succinimidyl ester
of an N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine is present.
[0133] In certain embodiments of the methods of making the
lipid-containing compositions in step (a) the targeting
factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is present.
[0134] In an additional aspect of the invention are provided
methods of treatment or diagnosis using the lipid-containing
compositions described herein.
[0135] In particular embodiments are provided methods for treating
cancer comprising, a) administering a targeted liposome as
described herein to an individual in need thereof in an amount
effective to treat cancer, wherein the targeted liposome comprises
a drug, and the drug is an anticancer agent.
[0136] In certain embodiments of the method of treatment or
diagnosis, the individual is a mammal. In particular embodiments,
the individual is a human.
[0137] In certain embodiments of the methods of treatment, the
cancer is breast, gastric, colon, colorectal cancer, cancer of the
pancreas, non small cell lung cancer, small cell lung cancer, brain
cancer, liver cancer, renal cancer, prostate cancer, bladder
cancer, ovary cancer, or a hematological malignancies.
[0138] In some embodiments of the methods of treatment, step (a) is
performed prior to, concurrently with or after combined modality
cancer therapy. In particular embodiments, the combined modality
cancer therapy comprises chemotherapy, radiation therapy, or
surgery.
[0139] In particular embodiments of the methods of treatment, step
(a) is performed prior to, concurrently with or after adjunctive
cancer therapy. In particular embodiments, the adjunctive cancer
therapy comprises administration of one or more agents to reduce
hair loss, vomiting, immune suppression, nausea, diarrhea, rash,
sensory disturbance, anemia, fatigue, stomatitis, or hand foot
syndrome. In some embodiments, step (a) is performed prior to,
concurrently with or after administration of one or more additional
anticancer agents. In certain embodiments, the one or more
additional anticancer agents comprise 5-fluorouracil, leucovorin,
capecitabine, UFT/LV (tegafur-uracil and leucovorin), irinotecan,
an-anti EGFR antibody, an anti-VEGF antibody, a tyrosine kinase
inhibitor, or combinations thereof.
[0140] In some embodiments of the methods of treatment, the
targeted liposome is administered via parenteral administration. In
particular embodiments, the parenteral administration is via
injection or intravenous infusion.
[0141] Also provided are methods of diagnosis comprising the steps
of
[0142] a) administering a targeted liposome as described herein to
an individual in need thereof in an amount effective for detection,
wherein the targeted liposome comprises a labeled compound;
and,
[0143] b) detecting the labeled compound.
[0144] In additional embodiments of the methods of diagnosis, the
methods further comprise a step (c), comparing a level of labeled
compound detected with the amount of labeled compound detected at a
previous point in time.
[0145] In further embodiments of the methods of diagnosis, step (b)
comprises detection via a gamma counter.
[0146] In a further aspect of the invention are provided
transferrin-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamines, where the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 3,
##STR00015## [0147] wherein R.sup.5 and R.sup.6 are each,
independently, an acyl group and p is an integer from 1 to 10, and
transferrin is liked linked to the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine.
[0148] In certain embodiments of the transferrin-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, p is an integer from 2 to 4. In particular
embodiments, p is 3.
[0149] In some embodiments of the transferrin-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, R.sup.5 and R.sup.6 are each, independently, oleoyl,
stearoyl, palmitoyl or myristoyl. In particular embodiments,
R.sup.5 and R.sup.6 are the same. In some embodiments, R.sup.5 and
R.sup.6 are oleoyl or stearoyl. In certain embodiments, R.sup.5 and
R.sup.6 are oleoyl and p is 3.
[0150] Also provided in an additional aspect are a pharmaceutical
formulations comprising the transferrin-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines as described herein and one or more pharmaceutically
acceptable carriers, excipients, diluents, stabilizers, or
preservatives.
[0151] In certain embodiments are provided lipid mixtures
comprising a mixture of at least two different neutral lipids, an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
and a succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine, wherein the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is represented by Formula 1,
##STR00016##
and the succinimidyl ester of the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 2,
##STR00017##
wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each,
independently, an acyl group, m and n are, independently, an
integer from 1 to 10; and, wherein the mixture does not comprise a
non-derivatized phosphatidyl ethanolamine or polyethylene
glycol.
[0152] In certain embodiments are provided liposome-containing
compositions comprising at least two different neutral lipids, an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, a succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine and an encapsulated drug
or labeled compound, wherein the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 1,
##STR00018##
and the succinimidyl ester of the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 2,
##STR00019##
wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each,
independently, an acyl group; m and n are, independently, an
integer from 1 to 10; and, wherein the mixture does not comprise a
non-derivatized phosphatidyl ethanolamine or polyethylene
glycol.
[0153] In particular embodiments of the liposome-containing
compositions and the lipid mixtures, m and n are each
independently, an integer from 2 to 4. In certain embodiments, m
and n are equal and are an integer from 2 to 4. In other
embodiments, m and n are equal and are 3.
[0154] In some embodiments of the liposome-containing compositions
and the lipid mixtures, R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are
each, independently, oleoyl, stearoyl, palmitoyl or myristoyl. In
some embodiments, R.sup.1 and are R.sup.2 are the same, and R.sup.3
and R.sup.4 are the same. In particular embodiments R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are the same. In some embodiments,
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are oleoyl
[0155] In particular embodiments of the liposome-containing
compositions and the lipid mixtures, the molar ratio of neutral
lipids:N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine:succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is about 95:4:1.
[0156] In some embodiments of the liposome-containing compositions
and the lipid mixtures, where the neutral lipids are DMPC and
cholesterol, the molar ratio of
DMPC:cholesterol:(N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine+succinimidyl ester of an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine) is 50:45:5. In certain of these embodiments, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is NG-DOPE and the succinimidyl ester of an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is NHS-NG-DOPE.
[0157] In certain embodiments are provided targeted liposomes
comprising at least two different neutral lipids, a
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, a targeting factor-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine and an encapsulated drug
or labeled compound, wherein the targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
comprises a targeting ligand linked to a second
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine; and, wherein the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 1,
##STR00020##
and the second N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is represented by Formula 3,
##STR00021##
wherein R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are each,
independently, an acyl group, and m and p are, independently, an
integer from 1 to 10; and, wherein the liposome does not comprise a
non-derivatized phosphatidyl ethanolamine or polyethylene glycol,
and wherein the targeting ligand is not an intact antibody.
[0158] In particular embodiments are provided targeted liposome
comprising a neutral phosphatidyl choline, cholesterol or a
cholesterol derivative, an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine, a transferrin-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
and encapsulated oxaliplatin, wherein the transferrin-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
comprises transferrin linked by a carboxylic acid amide bond to a
second N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine; and wherein the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 1,
##STR00022##
and
[0159] the succinimidyl ester of the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is represented by
Formula 2, and the second N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is represented by Formula 3,
##STR00023##
[0160] wherein R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are each,
independently, an acyl group, and m and p are, independently, an
integer from 1 to 10; and, wherein the liposome does not comprise a
non-derivatized phosphatidyl ethanolamine or polyethylene glycol.
In certain embodiments, the targeted liposome is substantially free
of EDC and/or DCC.
[0161] In certain embodiments of the targeted liposomes, m and p
are each independently, an integer from 2 to 4. In some
embodiments, m and p are equal and are an integer from 2 to 4. In
particular embodiments, m and p are equal are 3.
[0162] In some embodiments of the targeted liposomes, R.sup.1,
R.sup.2, R.sup.5 and R.sup.6 are each, independently, oleoyl,
stearoyl, palmitoyl or myristoyl. In certain embodiments, R.sup.1
and are R.sup.2 are the same, and R.sup.5 and R.sup.6 are the same.
In particular embodiments, R.sup.1, R.sup.2, R.sup.5 and R.sup.6
are the same. In some embodiments, R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are oleoyl or stearoyl. In certain embodiments, R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are oleoyl.
[0163] In certain embodiment of the targeted liposomes, the
targeting ligand is directed to a target cell. In particular
embodiments, the targeting ligand is directed to a cell surface
receptor of a target cell. In some embodiments, the targeting
ligand is transferrin, folic acid, hyaluronic acid, a sugar chain
or a fragment of a monoclonal antibody. In certain embodiments, the
targeting ligand is transferrin, folic acid, hyaluronic acid or a
sugar chain. In particular embodiments, the targeting ligand is
transferrin. In some of these embodiments, the transferrin is in a
holo-form but not in an apo-form. In other embodiments, the
transferring is in an apo-form.
[0164] In certain embodiments of the lipid mixtures,
liposome-containing compositions and the targeted liposomes, the
formulations do not comprise an anionic lipid. In some embodiments,
the formulations do not comprise a cationic lipid. In some
embodiments, the formulations do not comprise a cationic lipid or
an anionic lipid. In certain embodiments, the formulation do not
comprise a phosphatidyl glycerol or derivative thereof. In
particular embodiments, the formulations do not comprise egg
phosphatidylcholine.
[0165] In some embodiments of the lipid mixtures,
liposome-containing compositions and the targeted liposomes, the at
least two different neutral lipids are one or more phospholipids
and cholesterol or a cholesterol derivatives. In some embodiments,
at least one of the at least two different neutral lipids is a
phospholipid. In certain embodiments of the lipid mixtures,
liposome-containing compositions and the targeted liposomes, the at
least two different neutral lipids are a phosphatidyl choline and
cholesterol. In particular embodiments, one of the at least two
different neutral lipids is DMPC, DSPC or DPPC. In some of the
embodiments, one of the at least two different neutral lipids is
cholesterol or a cholesterol derivative. In particular embodiments,
the at least two different neutral lipids are DMPC and cholesterol,
DSPC and cholesterol, or DPPC and cholesterol. In particular
embodiments, the at least two different neutral lipids are DMPC and
cholesterol.
[0166] In some embodiments of the targeted liposomes the mean
diameter of the liposome is from about 50 nm to about 250 nm. In
certain embodiments, the mean diameter of the liposome is from
about 90 nm to about 200 nm. In particular embodiments, the mean
diameter of the liposome is from about 100 nm to about 140 nm.
[0167] In certain embodiments of the targeted liposomes the zeta
potential of the liposome is negative. In particular embodiments,
the zeta potential is from about -75 mV to about -90 mV. In some
embodiments, the zeta potential is from about -80 mV to about -85
mV.
[0168] In some embodiments of the lipid mixtures,
liposome-containing compositions and the targeted liposomes, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is NG-DOPE (where NG-DOPE is equivalent to R.sup.1 and R.sup.2
being oleoyl and m being 3) and, where present, the succinimidyl
ester of an N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine is NHS-NG-DOPE (where NHS-NG-DOPE is equivalent to
R.sup.3 and R.sup.4 being oleoyl and n being 3) or, where present
the targeting-factor modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is TF-NG-DOPE (where
TF-NG-DOPE is equivalent to R.sup.5 and R.sup.6 being oleoyl and p
being 3).
[0169] In some embodiments of the liposome-containing compositions
and the targeted liposomes, the formulations further include a
solution.
[0170] In certain embodiments of the targeted liposomes, the molar
ratio of neutral lipids:N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine:targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is about 95:4:1.
[0171] In certain embodiments of the targeted liposomes, the molar
ratio of DMPC:cholesterol:(N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine+targeting-factor
modified N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine) is 50:45:5.
[0172] In particular embodiments of the liposome-containing
compositions and the targeted liposomes, a labeled compound is
present. In certain embodiments, the labeled compound includes a
radioisotopic moiety. In particular embodiments, the labeled
compound includes .sup.125I.
[0173] In some embodiments of the liposome-containing compositions
and the targeted liposomes, a drug is present. In particular
embodiments, the drug is an anticancer agent. In some embodiments,
the drug is a cytotoxic drug. In certain embodiments, the drug is a
topoisomerase I inhibitor. In particular embodiments, the
topoisomerase I inhibitor is topotecan or irinotecan. In other
embodiments, the drug is a vinca alkaloid. In some embodiments, the
vinca alkaloid is vincristine, vinblastine, vinleurosine,
vinrodisine, vinorelbine or vindesine. In other embodiments, drug
is a nucleic acid. In some of these embodiments, the nucleic acid
is an antisense oligonucleotide or a ribozyme. In particular
embodiments, the drug is a platinum compound. In certain
embodiments, the platinum compound is biplatin, cisplatin,
carboplatin, ormaplatin, oxaliplatin, zeniplatin, enloplatin,
lobaplatin or spiroplatin. In particular embodiments, the platinum
compound is oxaliplatin. In some embodiments drug is an alkylating
agent. In particular embodiments, the drug is a taxanes. In other
embodiments, the drug is a metabolic antagonist. In certain
embodiments, the drug is an antitumour antibiotic. In some
embodiments, the drug is a hormone therapy drug. In particular
embodiments, the drug is a molecular target drug.
[0174] In particular embodiments, where oxaliplatin is present, the
oxaliplatin is dissolved in an aqueous solution of a sugar selected
from the group consisting of trehalose, maltose, sucrose, lactose,
mannose, mannitol, glycerol and dextrose. In certain embodiments,
the sugar is at a concentration of from about 1 to about 20%
percent sugar (v/v). In some embodiments, the concentration of
oxaliplatin is from about 0.1 mg/ml to about 25 mg/ml within the
liposome. In other embodiments, the concentration of oxaliplatin is
from about 0.5 mg/ml to about 10 mg/ml within the liposome. In
still other embodiments, the concentration of oxaliplatin is from
about 0.5 mg/ml to about 3 mg/ml.
[0175] In particular embodiments where targeting-factor modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is present, the concentration of targeting ligand incorporated in
the liposome is from about 1.0 mg/ml to about 3.0 mg/ml. In certain
embodiments, the concentration of targeting ligand incorporated in
the liposome is from about 1.0 mg/ml to about 2.5 mg/ml.
[0176] In certain embodiments, where a targeting-factor modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is present, the targeting ligand is transferrin. In some
embodiments, the transferrin is in a holo-form but not in an
apo-form. In certain embodiments, the transferrin is in a
holo-form. In some embodiments, the ferric iron is in a
concentration of from about 0.4 to about 3.0 .mu.g/ml. In other
embodiments, the ferric iron is in a concentration of from about
0.4 to about 1.5 .mu.g/ml.
[0177] In some embodiments of the lipid mixtures,
liposome-containing compositions and the targeted liposomes,
formulations are free of lipid components other than two different
neutral lipids, the N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine and the targeting-factor modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine. In particular embodiments, the formulations are free
of lipid components other than phosphatidyl choline, cholesterol or
a cholesterol derivative, the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine and the
transferrin-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine.
[0178] In another aspect are provided pharmaceutical formulations
comprising a targeted liposome or liposome-containing composition
as described herein and one or more pharmaceutically acceptable
carriers, excipients, diluents, stabilizers, or preservatives.
[0179] In yet another aspect are provided kits containing one or
more of the lipid mixtures, liposome-containing compositions or
targeted liposomes described herein, packaging and instructions for
use.
[0180] In certain embodiments, the kit includes targeted liposomes.
In particular embodiments, the targeted liposome is contained in a
first container and one or more pharmaceutically acceptable
carriers, excipients, diluents, stabilizers, or preservatives are
contained in a second container.
[0181] Unless otherwise noted, the lipid-containing compositions as
described herein are intended for use in the methods of treatment
and diagnosis as described herein and may be incorporated in the
pharmaceutical formulations and kits described herein. The
lipid-containing compositions described herein (including lipid
mixtures, liposome-containing compositions), liposomes (including
targeted liposomes, blank liposomes, etc.)) may, unless otherwise
noted, be made by the methods of production as described
herein.
[0182] In another aspect are provided methods for making the
lipid-containing compositions described herein, comprising the step
of mixing the at least two different neutral lipids, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
and the succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine.
[0183] In certain embodiments are provided methods for making the
liposome-containing compositions described herein, comprising the
steps of
[0184] a) mixing the at least two different neutral lipids, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
and the succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine to form a lipid
mixture;
[0185] b) adding a drug to the lipid mixture formed in step (a);
and,
[0186] c) forming a liposome.
[0187] In certain embodiments, the mixing step (a) is performed in
the presence of an organic solvent.
[0188] In some embodiments, the drug in step (b) is in aqueous
solution prior to mixing.
[0189] In certain embodiments, step (c) comprises sonication or
stirring. In particular embodiments, step (c) comprises
extrusion.
[0190] In further embodiments are provided methods of making a
targeted liposome as described herein, comprising the steps of
[0191] a) mixing the at least two different neutral lipids, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
and the succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine to form a lipid
mixture;
[0192] b) adding drug or labeled compound to the lipid mixture
formed in step (a);
[0193] c) forming a liposome; and,
[0194] d) linking a targeting ligand to the succinimidyl ester of
an N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine.
[0195] In certain embodiments of the above method, the method
further comprising a step (e), purifying the liposome of step (d).
In particular embodiments, the drug in step (b) is in aqueous
solution prior to mixing. In certain embodiments step (c) comprises
sonication or stirring. In some embodiments, step (c) comprises
extrusion.
[0196] Also provided are additional methods of making a targeted
liposome comprising the steps of
[0197] a) mixing the phosphatidyl choline, cholesterol or a
cholesterol derivative, and N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine to form a lipid
mixture;
[0198] b) adding oxaliplatin to the lipid mixture formed in step
(a);
[0199] c) forming a liposome; and,
[0200] d) functionalizing a portion of the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine to form a succinimidyl
ester of the N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine; and
[0201] e) linking transferrin to the succinimidyl ester of an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine.
[0202] In certain embodiments, the method further comprises a step
(f) of purifying the liposome of step (e).
[0203] In particular embodiments of the method the drug in step (b)
is in aqueous solution prior to mixing.
[0204] In some embodiments of the method step (c) comprises
sonication or stirring.
[0205] In a further aspect of the invention is provided use of the
lipid-containing compositions (including targeted liposomes) and
formulations thereof as described herein in the manufacture of a
medicament. Particularly, the manufacture of a medicament for use
in the treatment or diagnosis of conditions as described herein.
Further, the pharmaceutical formulations thereof, variously
described herein, are also intended for use in the manufacture of a
medicament for use in treatment and diagnosis of the conditions
and, in accordance with the methods, described herein, unless
otherwise noted.
[0206] In a further aspect of the invention is provide methods for
treating cancer comprising the step of a) administering a targeted
liposome as described herein to an individual in need thereof in an
amount effective to treat cancer, wherein the drug is an anticancer
agent.
[0207] In some embodiments, the individual is a mammal. In
particular embodiments, the individual is a human.
[0208] In certain embodiments, the cancer is breast, gastric,
colon, colorectal cancer, cancer of the pancreas, non small cell
lung cancer, small cell lung cancer, brain cancer, liver cancer,
renal cancer, prostate cancer, bladder cancer, ovary cancer, or a
hematological malignancies.
[0209] In some embodiments of the methods of treatment, step (a) is
performed prior to, concurrently with or after combined modality
cancer therapy. In particular embodiments, the combined modality
cancer therapy comprises chemotherapy, radiation therapy, or
surgery.
[0210] In some embodiments of the methods of treatment, step (a) is
performed prior to, concurrently with or after adjunctive cancer
therapy. In particular embodiments, the adjunctive cancer therapy
comprises administration of one or more agents to reduce hair loss,
vomiting, immune suppression, nausea, diarrhea, rash, sensory
disturbance, anemia, fatigue, stomatitis, or hand foot syndrome. In
certain embodiments, step (a) is performed prior to, concurrently
with or after administration of one or more additional anticancer
agents. In particular embodiments, the one or more additional
anticancer agents include 5-fluorouracil, leucovorin, capecitabine,
UFT/LV (tegafur-uracil and leucovorin), irinotecan, an-anti EGFR
antibody, an anti-VEGF antibody, a tyrosine kinase inhibitor, or
combinations thereof.
[0211] In certain embodiments of the methods of treatment, the
targeted liposome is administered via parenteral administration. In
particular embodiments, the parenteral administration is via
injection or intravenous infusion.
BRIEF DESCRIPTION OF THE DRAWINGS
[0212] FIG. 1 shows a schematic representation of a targeted
liposome.
[0213] FIG. 2 shows a schematic representation of the active drug
targeting of tumor cells using targeted liposomes.
[0214] FIG. 3 shows a schematic representation of the proposed mode
of action of targeted liposomes containing oxaliplatin.
[0215] FIG. 4 shows a schematic depiction of production process A
for targeted liposomes.
[0216] FIG. 5 shows a schematic of production process B for
targeted liposomes.
[0217] FIG. 6 shows the cytotoxicity of oxaliplatin on AsPC-1 cells
at various oxaliplatin concentrations.
[0218] FIG. 7 shows the number of transferrin receptors present on
the cell surface of normal leukocytes and tumor-derived cell
lines.
[0219] FIG. 8 shows the results of size distribution for the
liposome-containing mixtures prepared in Example 6 and obtained by
QELS; A) Entry 1, B) Entry 2, C) Entry 3, D) Entry 4, E) Entry 5,
F) Entry 6.
[0220] FIG. 9 shows the concentrations of liposomes in the blood,
where (.diamond-solid.) indicates the Tf-PEG-liposomes prepared in
Example 9, (.box-solid.) indicates the Tf-NG-DSPE:NG-DSPE:DSPC:CH
liposomes prepared in Example 8, and () indicates the
Tf/PEG-NG-DSPE liposomes prepared in Example 9.
[0221] FIG. 10 shows the concentrations of liposomes in cancer
tissues; where (.diamond-solid.) indicates the Tf-PEG-liposomes
prepared in Example 9, (.box-solid.) indicates the
Tf-NG-DSPE:NG-DSPE:DSPC:CH liposomes prepared in Example 8, and ()
indicates the Tf/PEG-NG-DSPE liposomes prepared in Example 9.
[0222] FIG. 11 shows the accumulation in tumor tissue of NG-PE
liposomes, prepared as described in Example 13, after intravenous
injection, where NGPE liposomes encapsulated tyraminyl inulin
labeled with .sup.125I were injected into Colon 26 tumor-bearing
mice. Data shown as mean. Data shown as mean.+-.SD (n=5).
(.quadrature.) 0 mol % (-); (.box-solid.) 1 mol % (+) Tf-NG-DOPE. *
Significant difference from 0 mol % (-).
[0223] FIG. 12 shows the inhibitory effects of liposomes on tumor
growth by plotting the tumor growth ratio vs. days after initial
treatment, where (.diamond-solid.) indicates the Tf-PEG-liposomes
prepared in Example 9; (.box-solid.) indicates the PEG-liposomes
prepared in Example 9 without Tf; () indicates the
Tf-NG-DSPE:NG-DSPE:DSPC:CH liposomes prepared in Example 8;
(.largecircle.) indicates the NG-DSPE:NG-DSPE:DSPC:CH liposomes
prepared in Example 8, without Tf; (*) indicates the Tf/PEG-NG-DSPE
liposomes prepared in Example 9; ( ) indicates the PEG-NG-DSPE
liposomes prepared in Example 9, without Tf; (+) indicates l-OHP
solution; and (-) indicates no treatment.
[0224] FIG. 13 shows the effect of varying concentration of NG-PE
(NG-DSPE) on the percentage of drug dose detected in blood, where
concentration (% of total lipid content) of NG-DSPE is as follows:
(.diamond-solid.) 0%, (.box-solid.) 1%; () 3%; (x) 6%;
(.largecircle.) 12%; and with the following lipids: ( ) MPB 6%; (+)
PDP 6%.
[0225] FIG. 14 shows the blood retention of liposomes with various
dicarboxylic acid linkers, with and without Tf; where
(.diamond-solid.) Tf-NGPE, (.box-solid.) Tf-NSPE; () TF-MPB; (x)
NGPE (no Tf), (*) MPB (no Tf).
[0226] FIG. 15 shows exemplary analysis of liposomes by
electrophoresis: lane 6 (transferrin-N-glutaryl-distearoyl
phosphatidyl ethanolamine-liposome (Tf-NG-DSPE liposome)); lane 5
(transferrin-polyethyleneglycol-distearoyl phosphatidyl
ethanolamine-liposome (Tf-PEG-DSPE liposome)) are shown. Lanes 1-4,
contain h-apo-Tf (240 ng), h-apo-Tf (120 ng), h-apo-Tf (60 ng), and
h-apo-Tf (30 ng), respectively.
[0227] FIG. 16 shows the amount of transferrin binding to
Tf-NG-DSPE liposomes with (lane 5) and without (lane 4) non-NG-DSPE
incorporated into the liposome. Lanes 1-3, contain h-apo-Tf (400
ng), h-apo-Tf (200 ng), and h-apo-Tf (50 ng), respectively.
[0228] FIG. 17 shows the accumulation oxaliplatin in blood after
administration of (.box-solid.) NG-DOPE:Tf-NG-DOPE:DMPC:CH
liposomes at 5 mg/kg and ( ) Tf-PEG liposomes at 5 mg/kg.
[0229] FIG. 18 shows the accumulation oxaliplatin in colon 26
tumors after administration of (.zeta.) NG-DOPE:Tf-NG-DOPE:DMPC:CH
liposomes at 5 mg/kg and ( ) Tf-PEG liposomes at 5 mg/kg in mouse
colon 26 tumors.
[0230] FIG. 19 shows the antitumor effect in colon 26 tumor-bearing
mice of NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes, where ( ) indicates
vehicle control (9% sucrose); () indicates l-OHP solution at 5
mg/kg, (.diamond-solid.) indicates NG-DOPE:Tf-NG-DOPE:DMPC:CH
liposomes at 5 mg/kg and (.box-solid.) indicates Tf-PEG liposomes
at 5 mg/kg.
[0231] FIG. 20 shows the antitumor effect in HCT-116 human colon
tumor xenograft-bearing mice of NG-DOPE:Tf-NG-DOPE:DMPC:CH
liposomes, where ( ) indicates vehicle control (300 mM (10.27%)
sucrose); (.largecircle.) indicates blank (no drug) liposomes, ()
indicates l-OHP solution at 15 mg/kg, (.diamond-solid.) indicates
NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes at 10 mg/kg and (.box-solid.)
indicates NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes at 15 mg/kg. All
liposomes were administered by exact body weight with an injection
volume of 0.103 mL/10 g body weight.
[0232] FIG. 21 shows the antitumor effect in HT-29 human colon
tumor xenograft-bearing mice of NG-DOPE:Tf-NG-DOPE:DMPC:CH
liposomes, where ( ) indicates vehicle control (300 mM (10.27%)
sucrose), () indicates NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes at 15
mg/kg, (.box-solid.) indicates NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes
at 10 mg/kg, and (.diamond-solid.) indicates
NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes at 6.7 mg/kg.
[0233] FIG. 22 shows the antitumor effect in MKN45 gastric human
tumor xenograft-bearing mice of NG-DOPE:Tf-NG-DOPE:DMPC:CH
liposomes, where ( ) indicates vehicle control (300 mM (10.27%)
sucrose), () indicates NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes at 15
mg/kg, (.box-solid.) indicates NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes
at 10 mg/kg, and (.diamond-solid.) indicates
NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes at 6.7 mg/kg.
[0234] FIG. 23 shows the antitumor effect in COLO 205 human tumor
xenograft-bearing mice of NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes,
where ( ) indicates vehicle control (9% sucrose); () indicates
l-OHP solution at 5 mg/kg q4d.times.3 (day 16), 10 mg/kg
q2d.times.2 (day 47), 2 mg/kg q2d.times.6 (day 51);
(.diamond-solid.) indicates NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes at
5 mg/kg q4d.times.3 (day 16), 10 mg/kg q2d.times.2 (day 47), 2
mg/kg q2d.times.6 (day 51); and (.box-solid.) indicates
NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes at 10 mg/kg q4d.times.3 (day
16), 15 mg/kg q2d.times.2 (day 47), 4 mg/kg q2d.times.6 (day
51).
[0235] FIG. 24 shows the pattern of SDS-PAGE after reduction with
2-mercaptoethanol, where lane 1 is the molecular weight markers,
lane 2 is holo-transferrin, lanes 3-5 are
NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes, and lane 6 is Tf-NG-DOPE.
[0236] FIG. 25 shows an exemplary HPLC chromatogram of system
suitability.
DETAILED DESCRIPTION OF THE INVENTION
[0237] Provided herein are lipid-containing compositions (including
targeted liposomes, blank liposomes, liposome-containing
compositions, lipid mixtures, etc.), and methods of making and
using the lipid-containing compositions described herein. The
lipid-containing compositions, and the liposomes in particular that
are provide herein, are suitable for the preparation of
pharmaceutical formulations and for use in the treatment or
diagnosis of a variety of conditions, including cancer. The
compositions, including the pharmaceutical formulations, provide
for more effective treatment and diagnosis regimens with reduced
adverse effects associated with the drug or labeled compound being
delivered to the individual. The increased efficacy and reduced
adverse effects should increase the therapeutic index of the drug
formulation and provide an opportunity for successful treatment of
a variety of conditions, including cancer and should also increase
the efficacy and reduce the adverse effects associated with
diagnosis. The increased specificity of the drug formulations with
the concomitant reduction in adverse effects should ensure
therapeutic benefit to a greater number and range of individuals
being treated, thus, saving or prolonging lives and improving the
quality of life of individuals in need of treatment. The increased
specificity of the labeled compound formulations with the
concomitant reduction in adverse effects should increase the number
of individuals who can be successfully diagnosed, for example, able
to tolerate the diagnosis formulation, and also increase the
accuracy (e.g., sensitivity, etc.) of diagnosis, including allowing
for earlier diagnosis of conditions and more effective monitoring
of the severity of the disease (e.g., progression or regression
with or without therapy).
[0238] Included in the compositions presently described are
pharmaceutical formulations of the lipid-containing compositions.
The lipid-containing compositions described herein include, but are
not limited to, liposomes that encapsulate drugs and labeled
compounds and can be used in the treatment or diagnosis of disease
or other conditions requiring treatment or diagnosis, including,
for example, cancer (e.g., breast, gastric, colorectal or colon
cancer).
[0239] When conventional anticancer (including cytotoxic) agents
are administered intravenously, the entire body is exposed and
affected by the drug non-selectively. As a result, a number of
adverse reactions may occur, the cancer is not targeted, and/or the
drug effect may be lost during the circulation process.
Encapsulation of a drug in a liposome composition prior to
administration may result in a one or more benefits, including
reducing the adverse effect(s) of the drug on a normal cell,
protecting the drug until it arrives at a target pathological cell
in the case where the drug may be unstable, prolonging the presence
of the drug in the circulatory system to enable delivery to
pathological cells, and/or in facilitating delivery of the drug to
a particular target pathological cell. More specific targeting of
the drug and the reduction of loss of drug by uptake in the RES
also includes the benefit of reducing the amount of drug that needs
to be administered and thereby also reduces the cost of therapy, as
well as the other benefits described herein.
[0240] Similarly, many labeled compounds have adverse effects
and/or may be degraded in the time between administration and
diagnosis (e.g., the time at which the diagnostic technique is
performed--for example, radioisotope detection, magnetic resonance
imaging, ultrasound, etc.). Incorporation of the labeled compounds
in the lipid-containing compositions described herein should
increase the efficacy of the labeled compound, such as, for
example, the threshold of detection may be achieved at lower doses
of labeled compound, reducing the adverse effects of the agent,
and/or extending the window of time in which the diagnosis can be
performed.
[0241] The lipid-containing compositions also include lipids
modified with targeting ligands (e.g., targeted liposomes, blank
liposomes, liposome-containing compositions, lipid mixtures) or
other derivatization. For example, liposomal compositions
incorporating targeting factor (e.g., transferrin, folate, etc.)
and derivatized lipids (e.g., N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines) were developed to
improve the safety and efficacy of anticancer agents (e.g.,
oxaliplatin, etc.) through the prolongation of drug circulation
time in plasma (compared to drug administered in solution alone)
and by targeting factor-specific receptors on tumor cells. This
improved bioavailability and tumor-targeting, should result in
improved safety and increased antitumor activity, and therefore a
greater likelihood of effective treatment for individuals in need
thereof, while also reducing the adverse effects associated with
many drugs, particularly the severe adverse effects associated with
most anticancer agents. Similarly, such derivatization and
targeting factors may also be employed to efficiently target
particular sites (e.g., tumor types, organs, tissues, etc.) for
delivery of labeled compounds.
[0242] The invention also provides transferrin-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines, which can be used in the lipid-containing
compositions and formulations thereof described herein.
[0243] The lipid-containing compositions, including the liposomes,
described herein, may be made by the methods herein described, as
well as methods for liposome manufacture known to the skilled
artisan and appropriate in view of the teaching provide in the
present specification. Unless otherwise noted, the liposomes and
liposome-containing compositions described herein can be
incorporated without limitation in pharmaceutical formulations
and/or kits, including pharmaceutical formulations and/or kits as
described herein and, additionally, those that would be apparent to
a skilled artisan in view of the teaching provided in the present
specification. Similarly the liposomes and liposome-containing
compositions and pharmaceutical formulations incorporating the
liposomes and liposome-containing compositions may be used without
limitation, unless otherwise noted, in the methods of treatment or
diagnosis consistent with the description provided throughout the
present specification and in accordance with the practice of skill
artisans in view of the teaching provided herein.
[0244] An exemplary targeted liposome incorporating a drug
(oxaliplatin) is represented schematically in FIG. 1. Proposed
mechanisms of the uptake and mode of action of targeted liposomes
are provided in FIGS. 2 and 3. As used herein, the term "targeted
liposome" refers generally to a liposome with components including
at least one or more phospholipid(s), N.omega.PE, TF-N.omega.PE and
which also incorporates a drug or labeled compound as described
herein. Each of these components as described throughout the
specification, without limitation, can be incorporated into the
targeted liposomes of the present invention in keeping with the
teaching provided herein. It is noted that blank liposomes,
described in greater detail herein, can also be "targeted," in that
they can incorporate TF-modified N.omega.PEs, but generally do not
incorporate a drug or labeled compound.
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines
[0245] The lipid-containing compositions described herein
incorporate at least one N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine according to Formula 1, below:
##STR00024##
[0246] wherein R.sup.1 and R.sup.2 are, independently, an acyl
group, and m is and integer from 1 to 10.
[0247] As used herein, the term "N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine," and its cognates,
refer to the N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines encompassed by Formula 1 as provided herein.
Similarly the abbreviation N.omega.PE is used to refer to the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines encompassed by Formula 1 (e.g., N.omega.-DOPE,
N.omega.-DSPE, NG-DOPE, etc.), and, for example NG-PE refers to
N-glutaryl phosphatidyl ethanolamine(s) of Formula 1, unless
otherwise noted.
[0248] It is intended that the only phosphatidyl ethanolamine(s)
incorporated in the lipid-containing compositions (including
targeted liposomes) described herein are N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines of Formula 1 or
succinimidyl esters of N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamines of Formula 2 or the targeting
factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamines of Formula 3, as described in greater
detail below. As used herein, the term "non-derivatized
phosphatidyl ethanolamine," and cognates thereof, refer to
phosphatidyl ethanolamine, semi-synthetic phosphatidyl
ethanolamine(s), synthetic phosphatidyl ethanolamine(s) and/or
derivatives thereof that are not encompassed by Formula 1, Formula
2 or Formula 3.
[0249] A wide variety of acyl groups, represented by R.sup.1 and
R.sup.2, may be used in Formula 1, as is well understood by those
of ordinary skill in the field.
[0250] In some embodiments, the acyl group is derived from
saturated or unsaturated aliphatic carboxylic acids having 12-22
carbon atoms. Exemplary acyl groups include, but are not limited
to, acyl groups derived from lauric acid, tridecanoic acid,
myristic acid, pentadecanoic acid, palmitic acid, margaric acid,
stearic acid, nonadecanoic acid, arachic acid, heneicosanic acid,
behenic acid, 2-lauroleic acid, 5-lauroleic acid, 11-lauroreic
acid, 5-myristoleic acid, myristoleic acid, 2-palmitoleic acid,
7-palmitoleic acid, cis-9-palmitoleic acid, trans-9-palmitoleic
acid, petroselinic acid, petroselidinic acid, oleic acid, elaidic
acid, vaccenic acid, gondoic acid, trans-gondoic acid, erucic acid,
linoleic acid, linoelaidic acid, .alpha.-eleostearic acid,
.beta.-eleostearic acid, linolenic acid, pseudoeleostearic acid,
arachidonic acid, eicosapentaenic acid, or docosahexaenic acid.
[0251] In certain embodiments, the acyl group is derived from
saturated or unsaturated aliphatic carboxylic acids having 14-18
carbon atoms. Exemplary acyl groups of this type include, but are
not limited to those derived from oleic acid (18 carbons), palmitic
acid (16 carbons), stearic acid (18 carbons), or myristic acid (14
carbons). As is recognized by those of skill, the corresponding
acyl groups are oleoyl, palmitoyl, stearoyl, and myristoyl,
respectively.
[0252] In other embodiments, the acyl groups are derived from
saturated or unsaturated aliphatic carboxylic acids having 14-18,
14-20, 14-22, 16-18, 16-20, 16-22, 18-20, 18-22, 12, 14, 16, 18, 20
or 22 carbons. In certain embodiments, the acyl groups are derived
from saturated or unsaturated aliphatic carboxylic acids having an
even number of carbons.
[0253] In some embodiments, the acyl groups are derived from oleic
acid (oleoyl), stearic acid (stearoyl), palmitic acid (palmitoyl),
linoleic acid (linoleoyl, 18 carbons), or myristic acid
(myristoyl). In other embodiments, the acyl groups are derived from
oleic acid (oleoyl). In certain embodiments, the acyl groups are
derived from stearic acid (stearoyl). In still other embodiments,
the acyl groups are derived from palmitic acid (palmitoyl). In
other embodiments, the acyl groups are derived from myristic acid
(myristoyl).
[0254] In some embodiments, the acyl group is derived from a
saturated aliphatic carboxylic acid, such as, but not limited to
palmitic acid (16 carbons), stearic acid (18 carbons), or myristic
acid (14 carbons).
[0255] In other embodiments, the acyl group is derived from an
unsaturated aliphatic carboxylic acid, such as, but not limited to
oleic acid (oleoyl, 18 carbons), linoleic acid (linoleoyl, 18
carbons) or linolenic acid (linolenoyl, 18 carbons). In some
embodiments, the acyl group is derived from linoleic acid.
[0256] In certain embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10. In other embodiments, m is an integer from 1-8, 1-6, 1-5, 1-7,
1-3, 1-2, 2-8, 2-6, 2-5, 2-4, 2-3, 3-4, 3-5, or 3-6. In some
embodiments, m is an integer from 2-4. In other embodiments, m is
1, 2 or 3.
[0257] In certain embodiments, m is an integer from 1 to 4. As is
recognized by those of ordinary skill, m=1 corresponds to a malonic
acid derivative of the phosphatidyl ethanolamine (PE), while m=2,
3, or 4, represent succinic acid, glutaric acid, and adipic acid
derivatives of the PE, respectively. In some embodiments, m is 3
(glutaric acid).
[0258] In certain embodiments, R.sup.1 and R.sup.2 are the same
acyl group. In other embodiments, R.sup.1 and R.sup.2 are different
acyl groups. In certain embodiments, R.sup.1 and R.sup.2 are
oleoyl, stearoyl, palmitoyl or myristoyl. In some embodiments,
R.sup.1 and R.sup.2 are oleoyl. In other embodiments, R.sup.1 and
R.sup.2 are stearoyl. In particular embodiments, R.sup.1 and
R.sup.2 are palmitoyl. In other embodiments, R.sup.1 and R.sup.2
are myristoyl.
[0259] In some embodiments, the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine of Formula 1 is
N-glutaryl-dioleoyl phosphatidyl ethanolamine (NG-DOPE (i.e., where
R.sup.1 and R.sup.2 are oleoyl and m is 3)). In other embodiments,
it is N-glutaryl-distearoyl phosphatidyl ethanolamine (NG-DSPE
(i.e., where R.sup.1 and R.sup.2 are stearoyl and m is 3). In other
embodiments, it is N-glutaryl-dimyristoyl phosphatidyl ethanolamine
(NG-DMPE (i.e., where R.sup.1 and R.sup.2 are myristoyl and m is
3)). In other embodiments, it is N-glutaryl-dipalmitoyl
phosphatidyl ethanolamine (NG-DPPE (i.e., where R.sup.1 and R.sup.2
are palmitoyl and m is 3)). In other embodiments, it is
N-succinyl-distearoyl phosphatidyl ethanolamine (NS-DSPE (i.e.,
where R.sup.1 and R.sup.2 are stearoyl and m is 2)). In other
embodiments, it is N-adipinyl-distearoyl phosphatidyl ethanolamine
(NA-DSPE (i.e., where R.sup.1 and R.sup.2 are stearoyl and m is
4)). In certain embodiments the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine of Formula 1 is NG-DOPE
or NG-DSPE.
Preparation of N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamines
[0260] The N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines described herein can be obtained by binding a
dicarboxylic acid to the amino group of phosphatidyl
ethanolamine.
[0261] Phospholipids, including phosphatidyl ethanolamines and
their derivatives utilized for the purposes described herein, must
be of high purity and should ideally be homogeneous. Known methods
for the preparation of high purity phospholipids include extraction
of the lipid from a buffer solution and purification using column
chromatography. For example, production methods for N-succinyl
dipalmitoylphosphatidylethanolamine are described in International
Patent Application Publication WO93/01828 (JPAH7-501316) and U.S.
Pat. Nos. 5,804,552 and 5,554,728, the contents of which are hereby
incorporated in their entirety. These production methods include
the purification of phospholipid derivative from the reaction
mixture by silica-gel 60 column chromatography of the reaction
mixture. Dipalmitoyl phosphatidyl ethanolamine (DPPE) is reacted
with succinic acid anhydride with triethylamine catalyst at room
temperature under nitrogen gas for 16 hours.
[0262] Other production methods of N-(.omega.-carboxy)
acylamido-phosphatidyl ethanolamine phospholipid derivatives are
described in Japanese published patent application JPA2001-261688,
which includes purification by separating a liquid layer after
addition of pH3.5-7.5 buffer solution to the reaction mixture, and
is herein incorporated by reference in its entirety. In this case,
the PE was reacted with dicarboxylic acid anhydride with
triethylamine alkali catalyst at 4.degree. C. for 1 hr. This method
may not work well for all phosphatidyl ethanolamine
derivatives.
[0263] DOPE (dioleoyl-phosphatidyl ethanolamine) can also be
commercially obtained or prepared by methods known to the skilled
artisan. For example, briefly, lecithin (API Starting Material) can
be chemically hydrolyzed to generate glycerol-phospho-choline that
is isolated by precipitation. The lipid is then acylated using
activated oleic acid, and DOPC (dioleoyl-phosphatidyl choline) is
isolated by normal phase column chromatography and passed through
ion exchange column to purify. DOPE can be prepared from DOPC by
reacting with ethanolamine using phospholipase D.
[0264] The N-(.omega.-carboxy) acylamido-phosphatidylethanolamine
phospholipid derivatives can also be prepared in such a manner as
described in, for example, U.S. Pat. No. 4,534,899, which is hereby
incorporated in its entirety. Briefly, a dicarboxylic anhydride is
reacted with a phospholipid such as phosphatidylethanolamine to
obtain a dicarboxylic acid-derivatized
phosphatidylethanolamine.
Succinimidyl esters of N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamines
[0265] Succinimidyl esters of N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines as described herein are
represented by the following Formula 2:
##STR00025##
[0266] wherein R.sup.3 and R.sup.4 are, independently, an acyl
group, and n is and integer from 1 to 10.
[0267] As used herein, the term "succinimidyl esters of
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine," and its cognates, refer to the succinimidyl esters
of N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines encompassed by Formula 2 as provided herein.
Similarly the abbreviation SuccN.omega.PE can be used to refer to
the succinimidyl esters of N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines encompassed by Formula
2 (e.g., SuccN.omega.-DOPE, SuccN.omega.-DSPE, SuccNG-DOPE, etc.),
and, for example NHS-NG-PE refers to a the succinimidyl ester of
N-glutaryl phosphatidyl ethanolamine(s) of Formula 2 formed by
reaction with NHS, unless otherwise noted.
[0268] A wide variety of acyl groups that are represented by
R.sup.3 and R.sup.4 may be used, as is well understood by those of
ordinary skill in the field and as described above for R.sup.1 and
R.sup.2. Unless otherwise noted herein, it is expressly intended
that the description provided herein of the acyl groups with
respect to Formula 1 (e.g., R.sup.1 and R.sup.2) is equally
applicable to the acyl groups with respect to Formula 2 (e.g.,
R.sup.3 and R.sup.4). Including, in particular, the description
above in the section entitled "N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines."
[0269] In certain embodiments, R.sup.3 and R.sup.4 are the same
acyl group. In other embodiments, R.sup.3 and R.sup.4 are different
acyl groups. In certain embodiments, R.sup.3 and R.sup.4 are
oleoyl, stearoyl, palmitoyl or myristoyl. In some embodiments,
R.sup.3 and R.sup.4 are oleoyl. In other embodiments, R.sup.3 and
R.sup.4 are stearoyl. In certain embodiments, R.sup.3 and R.sup.4
are palmitoyl. In other embodiments, R.sup.3 and R.sup.4 are
myristoyl.
[0270] In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10. In other embodiments, n is an integer from 1-8, 1-6, 1-5, 1-7,
1-3, 1-2, 2-8, 2-6, 2-5, 2-4, 2-3, 3-4, 3-5, or 3-6. In some
embodiments, n is an integer from 2-4. In other embodiments, n is
1, 2 or 3.
[0271] In certain embodiments, n is an integer from 1 to 4. As is
recognized by those of ordinary skill, n=1 corresponds to a malonic
acid derivative of the phosphatidyl ethanolamine (PE), while n=2,
3, or 4, represent succinic acid, glutaric acid, and adipic acid
derivatives of the PE, respectively. In some embodiments, n is 3
(glutaric acid).
[0272] In some embodiments, the succinimidyl ester of the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
of Formula 2 is a succinimidyl ester of N-glutaryl-dioleoyl
phosphatidyl ethanolamine (NG-DOPE). In other embodiments, it is a
succinimidyl ester of N-glutaryl-distearoyl phosphatidyl
ethanolamine (NG-DSPE). In certain embodiments the succinimidyl
ester of the N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine of Formula 2 is a succinimidyl ester of NG-DOPE or
NG-DSPE.
[0273] In some embodiments, the succinimidyl ester of the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
of Formula 2 is the succinimidyl ester of the N-glutaryl-dioleoyl
phosphatidyl ethanolamine (SuccNG-DOPE (i.e., where R.sup.3 and
R.sup.4 are oleoyl and n is 3)). In other embodiments, it is the
succinimidyl ester of N-glutaryl-distearoyl phosphatidyl
ethanolamine (SuccNG-DSPE (i.e., where R.sup.3 and R.sup.4 are
stearoyl and n is 3). In other embodiments, it is the succinimidyl
ester of N-glutaryl-dimyristoyl phosphatidyl ethanolamine
(SuccNG-DMPE (i.e., where R.sup.3 and R.sup.4 are myristoyl and n
is 3)). In other embodiments, it is the succinimidyl ester of
N-glutaryl-dipalmitoyl phosphatidyl ethanolamine (SuccNG-DPPE
(i.e., where R.sup.3 and R.sup.4 are palmitoyl and n is 3)). In
other embodiments, it is the succinimidyl ester of
N-succinyl-distearoyl phosphatidyl ethanolamine (SuccNS-DSPE (i.e.,
where R.sup.3 and R.sup.4 are stearoyl and n is 2)). In other
embodiments, it is the succinimidyl ester of N-adipinyl-distearoyl
phosphatidyl ethanolamine (SuccNA-DSPE (i.e., where R.sup.3 and
R.sup.4 are stearoyl and n is 4)). In certain embodiments the
succinimidyl ester of the N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine of Formula 2 is SuccNG-DOPE or
SuccNG-DSPE.
[0274] In some embodiments, the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is N.omega.-DOPE and the
succinimidyl ester of the N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is SuccN.omega.-DOPE. In other
embodiments, the N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is N.omega.-DSPE and the succinimidyl
ester of the N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine is SuccN.omega.-DSPE. In still other embodiments, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is N.omega.-DOPE and the succinimidyl ester of the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is SuccN.omega.-DSPE. In certain other embodiments, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is N.omega.-DSPE and the succinimidyl ester of the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is SuccN.omega.-DOPE. In certain of these embodiments, the
succinimidyl ester may be NHS (e.g., NHS-N.omega.-DOPE,
NHS-N.omega.-DSPE, etc.).
[0275] In some embodiments, the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is NG-DOPE and the
succinimidyl ester of the N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is SuccNG-DOPE. In other embodiments, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is NG-DSPE and the succinimidyl ester of the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is SuccNG-DSPE. In still other embodiments, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is NG-DOPE and the succinimidyl ester of the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is SuccNG-DSPE. In certain other embodiments, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is NG-DSPE and the succinimidyl ester of the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is SuccNG-DOPE. In certain of these embodiments, the succinimidyl
ester may be NHS (e.g., NHS-NG-DOPE, NHS-NG-DSPE, etc.).
Preparation of Succinimidyl esters of N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines
[0276] The succinimidyl esters of N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines described herein can be
obtained by derivatization of the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines described herein,
prepared as known in the art and described herein. Preparation of
the succinimidyl esters of N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines is also described in
greater detail below, including in the Examples. In view of the
teaching provided in the present specification, the skilled artisan
will also be able to modify the methods described herein.
[0277] A method for the production of the succinimidyl esters of
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines of the present inventions is as follows: [0278] To 1
equivalent of N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamines represented by Formula 2 as described
herein is added about 0.7-1.3 equivalents of NHS, which are
dissolved in an organic solvent that does not have an active
hydrogen. The mixture is then reacted with about 0.7-1.3
equivalents of a carbodiimide compound at 0-50 C..degree., for
about 1-7 days.
[0279] Exemplary, organic solvents that do not have an active
hydrogen include, but are not limited to, esters (e.g., ethyl
acetate, butyl acetate, etc.), aliphatic hydrocarbons (e.g.,
hexane, heptanes, etc.), aromatic hydrocarbons (e.g., toluene,
xylene, etc.), halogenated hydrocarbone (e.g., chloroform,
dichloromethane, dichloroethane, etc.), ethers (e.g., THF, dioxane,
diethyl ether, etc.), cyclic hydrocarbons (e.g., cyclohexane,
etc.), DMF and DMSO. The organic solvent may also be
dehydrated.
[0280] A wide variety of carbodiimide compounds can be used, so
long as the compounds posses a carbodiimide group
(--N.dbd.C.dbd.N--). For example, carbodiimide compounds that made
be used include, but are not limited to, carbodiimide groups such
as N,N'-dicyclohexyl-carbodiimide (DCC),
N,N'-diisopropyl-carbodiimide,
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride
(EDC), etc. In certain embodiments, DCC used. In others EDC is
used.
[0281] The above-described reaction may also be performed under
conditions that minimize or eliminate production of by-products.
Unwanted by-products include urea compounds (e.g.,
N,N'-dicyclohexylurea, N-ethyl-N'-(3-dimethylaminopropyl)urea,
etc.), N-acylated urea compounds, carboxyanhydride compounds and
5-oxazolone compounds. Conditions and materials which disfavor or
minimize the formation of by-products include, 1) slowly dissolving
the carbodiimide compounds in organic solvent, 2) performing the
reaction below 0 C..degree. to prevent heat evolution by the
reaction, etc. Other means for optimizing the reaction and
minimizing the production of by-products will be understood by
those of ordinary skill in the field, particularly in view of the
teaching provided herein.
[0282] The organic solvent that is capable of dissolving the
carbodiimide compounds is the same as the organic solvent, which
does not have an active hydrogen as described above. The solvent
used to dissolve the carbodiimide and the organic solvent without
active hydrogens may be the same or different.
[0283] The progress of the reaction can monitored by a thin-layer
chromatography (TLC) systems, high-performance liquid
chromatography (HPLC), and/or evaporated light scattering
detectors. Other methods for monitoring the progress of the
reaction will also be known by those of skill in the art.
[0284] Purification can be performed by silica gel column
chromatography using a mixture of chloroform and methanol.
Additional purification methods will be known to those of skill in
the art.
[0285] In fully dehydrated organic solvents and in the absence of
strong acidity or strong alkali, the succinimidyl esters of
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines as described herein are usually stable.
Targeting Factor-Modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamines
[0286] Targeting factor-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines include a targeting
ligand linked to a second N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine, where the second
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is represented by Formula 3,
##STR00026##
[0287] wherein R.sup.5 and R.sup.6 are, independently, an acyl
group, and p is and integer from 1 to 10.
[0288] As used herein, the term "targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine," and its cognates, refer to the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines encompassed by Formula 3 and modified with a
targeting factor as provided herein. Similarly the abbreviation
TF-N.omega.PE can be used to refer to the targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines (e.g., TF-N.omega.-DOPE, TF-N.omega.-DSPE,
TF-NG-DOPE, etc.), and, for example TF-NG-PE refers to a targeting
ligand linked to N-glutaryl phosphatidyl ethanolamine(s) of Formula
3.
[0289] A wide variety of acyl groups that are represented by
R.sup.5 and R.sup.6 may be used, as is well understood by those of
ordinary skill in the field and as described above for R.sup.1 and
R.sup.2. Unless otherwise noted herein, it is expressly intended
that the description provided herein of the acyl groups with
respect to Formula 1 (e.g., R.sup.1 and R.sup.2) is equally
applicable to the acyl groups with respect to Formula 3 (e.g.,
R.sup.5 and R.sup.6). Including, in particular, the description
above in the section entitled "N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines."
[0290] In certain embodiments, R.sup.5 and R.sup.6 are the same
acyl group. In other embodiments, R.sup.5 and R.sup.6 are different
acyl groups. In certain embodiments, R.sup.5 and R.sup.6 are
oleoyl, stearoyl, palmitoyl or myristoyl. In some embodiments,
R.sup.5 and R.sup.6 are oleoyl. In other embodiments, R.sup.5 and
R.sup.6 are stearoyl. In certain embodiments, R.sup.5 and R.sup.6
are palmitoyl. In other embodiments, R.sup.5 and R.sup.6 are
myristoyl.
[0291] In certain embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10. In other embodiments, p is an integer from 1-8, 1-6, 1-5, 1-7,
1-3, 1-2, 2-8, 2-6, 2-5, 2-4, 2-3, 3-4, 3-5, or 3-6. In some
embodiments, p is an integer from 2-4. In other embodiments, p is
1, 2 or 3.
[0292] In certain embodiments, p is an integer from 1 to 4. As is
recognized by those of ordinary skill, p=1 corresponds to a malonic
acid derivative of the phosphatidyl ethanolamine (PE), while p=2,
3, or 4, represent succinic acid, glutaric acid, and adipic acid
derivatives of the PE, respectively. In some embodiments, p is 3
(glutaric acid).
[0293] In some embodiments, the targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
of Formula 3 is targeting factor-modified N-glutaryl-dioleoyl
phosphatidyl ethanolamine (TF-NG-DOPE). In other embodiments, the
targeting factor-modified N-glutaryl-distearoyl phosphatidyl
ethanolamine is (TF-NG-DSPE). In certain embodiments the targeting
factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine of Formula 3 is TF-NG-DOPE or
TF-NG-DSPE.
[0294] In some embodiments, the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine of Formula 3 is
N-glutaryl-dioleoyl phosphatidyl ethanolamine (NG-DOPE (i.e., where
R.sup.5 and R.sup.6 are oleoyl and p is 3)). In other embodiments,
it is N-glutaryl-distearoyl phosphatidyl ethanolamine (NG-DSPE
(i.e., where R.sup.5 and R.sup.6 are stearoyl and p is 3). In other
embodiments, it is N-glutaryl-dimyristoyl phosphatidyl ethanolamine
(NG-DMPE (i.e., where R.sup.5 and R.sup.6 are myristoyl and p is
3)). In other embodiments, it is N-glutaryl-dipalmitoyl
phosphatidyl ethanolamine (NG-DPPE (i.e., where R.sup.5 and R.sup.6
are palmitoyl and p is 3)). In other embodiments, it is
N-succinyl-distearoyl phosphatidyl ethanolamine (NS-DSPE (i.e.,
where R.sup.5 and R.sup.6 are stearoyl and p is 2)). In other
embodiments, it is N-adipinyl-distearoyl phosphatidyl ethanolamine
(NA-DSPE (i.e., where R.sup.5 and R.sup.6 are stearoyl and p is
4)). In certain embodiments the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine of Formula 3 is NG-DOPE
or NG-DSPE.
[0295] In certain embodiments the targeting ligand is transferrin
(Tf), which is described in greater detail below, and the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
incorporated in the transferrin-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is of Formula 3, as
described herein.
[0296] In some embodiments, the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is N.omega.-DOPE and the
targeting ligand is transferrin (Tf), and the targeting
factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is Tf-N.omega.-DOPE. In other
embodiments, the N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is N.omega.-DSPE and the targeting ligand
is transferrin (Tf), and the targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is Tf-N.omega.-DSPE. In still other embodiments, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is N.omega.-DOPE and the targeting ligand is transferrin (Tf), and
the targeting factor-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is Tf-N.omega.-DSPE. In
certain other embodiments, the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is N.omega.-DSPE and the
targeting ligand is transferrin (Tf), and the targeting
factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is Tf-N.omega.-DOPE. In certain of these
embodiments, m may be 3 and the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is a NG-PE according to
Formula 1.
[0297] In some embodiments, the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine is NG-DOPE and the
targeting factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine is TF-NG-DOPE. In other embodiments, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is NG-DSPE and the targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is TF-NG-DSPE. In still other embodiments, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is NG-DOPE and the targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is TF-NG-DSPE. In certain other embodiments, the
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is NG-DSPE and the targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
is TF-NG-DOPE.
[0298] The targeting-factor modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines may be prepared from
the SuccN.omega.PE by reaction with the targeting ligand and other
reactants as described herein in greater detail. The TF-N.omega.PE
may either be prepared prior to mixture with other lipid components
of the lipid-containing compositions described herein (and
optionally purified) or may be prepared in situ from the reaction
of the pre-prepared SuccN.omega.PE that has been incorporated in a
lipid-containing composition.
Additional Lipid Components
[0299] The lipid-containing compositions as described herein also
contain one or more additional lipid components in addition to the
N.omega.PEs, SuccN.omega.PEs and/or TF-N.omega.PEs described
herein. A variety of additional lipid components can be used,
however, the term "additional lipid component(s)" is not intended
to include non-derivatized phosphatidyl ethanolamines (PEs) or
derivatives of PEs as in Formulae 1, 2 or 3. In some embodiments,
the one or more additional lipid component(s) may be a phospholipid
or one or more phospholipids. In certain embodiments, the one or
more additional lipid components may include at least two neutral
lipids. In other embodiments, one or more phospholipids may be
present and, optionally, an "additional lipid" (which is not a
phospholipid). A variety of neutral lipids can be used, however,
the at least two neutral lipids are not intended to include
non-derivatized phosphatidyl ethanolamines (PEs) or derivatives of
PEs as in Formulae 1, 2 or 3. It is not intended that the term
"phospholipid," as used herein, should include PEs or derivatives
thereof, as in Formulae 1, 2 or 3. Similarly, the term "additional
lipid" does not include PEs or derivatives thereof, as in Formulae
1, 2 or 3, nor does it include other phospholipids.
[0300] In particular embodiments, phospholipid(s) may be used in
the lipid-containing compositions and formulations described
herein. For example, one or more, at least two, at least three, at
least four phospholipids; or, two, three, or four phospholipids. In
particular embodiments there is one phospholipid. In certain
embodiments the lipid components of the compositions are limited to
one phospholipid, the N.omega.PE, and the SuccN.omega.PE (or
TF-modified N.omega.PE, where the reaction with the targeting
factor has been performed).
[0301] In particular embodiments, two or more neutral lipids may be
used in the lipid-containing compositions and formulations
described herein. For example, at least two, at least three, at
least four neutral lipids; or, two, three, or four neutral lipids.
In particular embodiments there are two neutral lipids. In certain
embodiments the lipid components of the compositions are limited to
two neutral lipids, the N.omega.PE, and the SuccN.omega.PE (or
TF-modified N.omega.PE, where the reaction with the targeting
factor has been performed).
[0302] In some embodiments, where the additional lipid
components(s) includes a phospholipid, the phospholipid may be a
phosphatidylcholine, including naturally occurring, semi-synthetic
or synthetic phosphatidylcholines (e.g., DSPC, DMPC, etc.). In some
embodiments, the phosphatidylcholine is a non-naturally occurring
phosphatidylcholine (e.g., not egg phosphatidylcholine). In
particular embodiments, the phosphatidylcholine is an acyl
phosphatidylcholine (e.g., DMPC, DPPC, POPC, DSPC, etc.). In some
embodiments, the phospholipid is cationic. In other embodiments the
phospholipid is anionic. In still other embodiments, the
phospholipid is neutral. In particular embodiments, the one or more
phospholipid(s) is not anionic. In other embodiments, the one or
more phospholipid(s) is not cationic. In certain embodiments where
more than one phospholipid is present, an anionic and neutral lipid
may be included. Exemplary phospholipids include, but are not
limited to, phosphatidylcholines (PCs), phosphatidic acid,
phosphatidylserine, phosphatidylglycerol, etc. In some embodiments,
the lipid-containing compositions do not include phosphatidylserine
or phosphatidylglycerol.
[0303] In certain embodiments, at least one of the at least two
neutral lipids may be a phospholipid. In some embodiments, the
phospholipid may be a phosphatidylcholine, including naturally
occurring, semi-synthetic or synthetic phosphatidylcholines (e.g.,
DSPC, DMPC, etc.). In some embodiments, the phosphatidyl choline is
a non-naturally occurring phosphatidyl choline (e.g., not egg
phosphatidyl choline). In particular embodiments, the phosphatidyl
choline is an acyl phosphatidyl choline (e.g., DMPC, DPPC, POPC,
DSPC, etc.).
[0304] In some embodiments, at least one of the at least two
neutral lipids may be cholesterol or a cholesterol derivative
(e.g., cholesterol pullulan, positively-charged cholesterol (e.g.,
DC-Chol)), incorporating a moiety of a radioisotope (e.g., .sup.3H,
.sup.14C, .sup.125I, .sup.131I, etc.), having a functional moiety
(e.g., a fluorescent moiety, etc.).
[0305] In certain embodiments, where the lipid-containing
compositions include one or more phospholipid, the compositions may
additionally comprise an additional neutral, non-phospholipid as
the additional lipid. For example, cholesterol or a cholesterol
derivative as described above.
[0306] Phospholipids (non-PEs) for use in the lipid-containing
compositions described herein include synthetic, semi-synthetic and
naturally occurring phospholipids. Exemplary phospholipids include,
but are not limited to, phosphatidylcholine (PC), phosphatidic
acid, phosphatidylserine, phosphatidylglycerol, etc. In other
embodiments, the one or more phospholipids include
phosphatidylcholine (PC) or phosphatidic acid and do not include
phosphatidylserine or phosphatidylglycerol.
[0307] In some embodiments, the phospholipid is a
phosphatidylcholine. In certain embodiments, the
phosphatidylcholine may be, e.g., distearoyl phosphatidyl choline
(DSPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl
phosphatidylcholine (DPPC), palmitoyl oleoyl phosphatidylcholine
(POPC), egg phosphatidylcholine (EPC), hydrogenated soya
phosphatidylcholine (HSPC), etc. In particular embodiments, at
least one phospholipid is a phosphatidylcholine. In certain of
these embodiments, the phosphatidylcholine is DMPC. In other
embodiments, the phosphatidylcholine is DSPC. In other embodiments,
the phosphatidylcholine is DPPC. In other embodiments, the
phosphatidylcholine is POPC. In other embodiments, the
phosphatidylcholine is EPC. In other embodiments, the
phosphatidylcholine is HSPC. In some embodiments, one phospholipid
is included and is DMPC, DSPC, DPPC, POPC, EPC or HSPC. In
particular embodiments where there are lipid-containing composition
includes a single phospholipid (non-PE phospholipid), the
phospholipid is DMPC. In other embodiments where the
lipid-containing composition includes a single phospholipid (non-PE
phospholipid), the phospholipid is DSPC. In other embodiments where
the lipid-containing composition includes a single phospholipid
(non-PE phospholipid), the phospholipid is DPPC. In other
embodiments where the lipid-containing composition includes a
single phospholipid (non-PE phospholipid), the phospholipid is
POPC. In other embodiments where the lipid-containing composition
includes a single phospholipid (non-PE phospholipid), the
phospholipid is EPC. In other embodiments where the
lipid-containing composition includes a single phospholipid (non-PE
phospholipid), the phospholipid is HSPC.
[0308] In certain embodiments, the additional lipid components(s)
may include at least one phospholipid and, optionally, an
additional lipid such as cholesterol or a cholesterol derivative.
In particular embodiments, the additional lipid component(s) are a
single phospholipid and cholesterol. In certain embodiments, the
additional lipid component(s) include at least one
phosphatidylcholine and cholesterol. In particular embodiments, the
additional lipid component(s) include a single phosphatidylcholine
and cholesterol. In certain embodiments where cholesterol is
included, the phospholipid is DSPC, DMPC, DPPC, POPC, EPC or HSPC.
In some embodiments, the additional lipid component(s) include
cholesterol and DMPC. In other embodiments, the additional lipid
component(s) include cholesterol and DSPC. In certain embodiments,
the additional lipid component(s) are cholesterol and one of DMPC
or DSPC. In certain embodiments, the additional lipid component(s)
are cholesterol and DMPC. In other embodiments, the additional
lipid component(s) are cholesterol and DSPC. In other embodiments,
the additional lipid component(s) include cholesterol and DPPC. In
other embodiments, the additional lipid component(s) include
cholesterol and POPC. In other embodiments, the additional lipid
component(s) include cholesterol and EPC. In other embodiments, the
additional lipid component(s) include cholesterol and HSPC. In
certain embodiments, the additional lipid component(s) are
cholesterol and one of DMPC, DSPC, DPPC, POPC, EPC or HSPC. In
certain embodiments, the additional lipid component(s) are
cholesterol and DMPC. In other embodiments, the additional lipid
component(s) are cholesterol and DSPC. In other embodiments, the
additional lipid component(s) are cholesterol and DPPC. In other
embodiments, the additional lipid component(s) are cholesterol and
POPC. In other embodiments, the additional lipid component(s) are
cholesterol and EPC. In other embodiments, the additional lipid
component(s) are cholesterol and HSPC.
[0309] In particular embodiments, there is one phospholipid and the
phospholipid is not HSPC or EPC.
[0310] In particular embodiments, there are one or more
phospholipids. In certain embodiments, the one or more phospholipid
includes a phosphatidyl choline. In particular embodiments, are
included one or more phospholipids and cholesterol (or a
cholesterol derivative). In certain embodiments, the phospholipid
is a phosphatidyl choline and the composition additionally includes
a cholesterol (or a cholesterol derivative). In particular
embodiments, the phosphatidylcholine is a phosphatidylcholine which
includes a moiety of saturated fatty acid (e.g., DMPC, DSPC or
DPPC). In certain embodiments the phosphatidylcholine is not egg
phosphatidylcholine. In particular embodiments, the
phosphatidylcholine is not HSPC.
[0311] In particular embodiments, there are two neutral lipids. In
some embodiments, the two neutral lipids are cholesterol (or a
cholesterol derivative) and a phosphatidyl choline. In particular
embodiments, the phosphatidylcholine is a phosphatidylcholine which
includes a moiety of saturated fatty acid (e.g., DMPC, DSPC or
DPPC). In certain embodiments the phosphatidylcholine is not egg
phosphatidylcholine.
[0312] The additional lipid component(s) as described herein, and
those known to the skilled artisan, are commercially available from
a number of suppliers, including, for example Avanti Polar Lipids,
Inc. (Alabaster, Ak.), Northern Lipid Inc. (Canada), Lipoid GmbH
(Germany), NOF Corporation (Japan), Nippon Fine Chemical Co., Ltd
(Japan).
Drugs
[0313] A variety of drugs may be included in the lipid-containing
compositions of the present invention, for example, a compound or a
gene. In certain embodiments, the drug may be an anticancer agent,
for example, an anticancer agent suitable for encapsulation in a
liposome. The amount of drug to be included in the lipid-containing
compositions, and formulations thereof, as described herein can be
readily determined by the skilled artisan in view of the teaching
herein provided and depending on the drug selected and the use
intended for the composition or formulation, taking into account
factors specific to both the drug and the individual to be treated,
as described further herein.
[0314] In certain embodiments, the drug may be a nucleic acid, for
example, nucleic acid encoding for sequences with anticancer
properties. For example, but not limited to, antisense
oligonucleotides, ribozymes, etc.
[0315] In some embodiments, the anticancer agent can be a cytotoxic
drug, including those known by skill in the art and medical
practitioners. Exemplary anticancer agents include topoisomerase I
inhibitors, vinca alkaloids, alkylating agents (including platinum
compounds), taxanes and others known to those of skill in the
art.
[0316] In some embodiments, the anticancer drug may be a
topoisomerase I inhibitor, for example, but not limited to,
topotecan, irinotecan, etc.
[0317] The anticancer drug may also be a vinca alkaloid, for
example, vincristine, vinblastine, vinleurosine, vinrodisine,
vinorelbine, vindesine, etc.
[0318] Further, the anticancer drug may also be a platinum
compound. Non-limiting examples of platinum compounds include
biplatin, cisplatin, carboplatin, ormaplatin, oxaliplatin,
zeniplatin, enloplatin, lobaplatin, spiroplatin, etc.
[0319] Oxaliplatin (platinum (II) cis-oxalato complex of
trans-1-1,2-diaminocyclohexane) is a platinum, more specifically,
an oragnoplatinum, complex having a structure represented by the
following formula shown below. Oxaliplatin is also known as the
following: diaminocyclohexane platinum, DACH-platinum, and
cis-[(1R,2R)-1,2-cyclohexanediamine-N,N][oxalato(2)-O,O']platinum
(C.sub.8H.sub.14N.sub.2O.sub.4Pt; MW 397.4 g/mol). As mentioned
previously, oxaliplatin is the active pharmaceutical ingredient in
Eloxatin.TM..
##STR00027##
[0320] Oxaliplatin is useful as an antitumor agent, since it has a
therapeutic activity similar to that of cisplatin and relatively
low nephrotoxicity and emetogenicity (vomiting). Production
processes for oxaliplatin is well known in the art (e.g.,
JP-A-9-40685; U.S. Pat. Nos. 4,169,846, 5,338,874; 5,959,133;
5,298,642; and 5,290,961 (the disclosures of which are hereby
incorporated in their entirety). Oxaliplatin is further described
in Chaney S G et al. "Recognition and processing of cisplatin-and
oxaliplatin-DNA adducts." Crit Rev Oncol Hematol. (2005) 53: 3-11
(herein incorporated by reference in its entirety).
[0321] In certain embodiments, the oxaliplatin concentration
encapsulated within the liposome is about 1 mg/ml, for example
about 0.8 mg/ml.
[0322] Generally, the liposome composition of the present invention
contains from about 1 to about 50 .mu.g oxaliplatin/mg lipid and
from about 1 to about 150 .mu.g TF/mg lipid. For example, from
about 10 to about 50 .mu.g oxaliplatin/mg lipid and from 10 to
about 150 .mu.g TF/mg lipid.
[0323] In certain embodiments, the compositions contain from about
1 to about 45 .mu.g oxaliplatin/mg lipid, from about 1 to about 40
.mu.g oxaliplatin/mg lipid, from about 1 to about 35 .mu.g
oxaliplatin/mg lipid, from about 1 to about 30 .mu.g oxaliplatin/mg
lipid, from about 1 to about 25 .mu.g oxaliplatin/mg lipid, from
about 1 to about 20 .mu.g oxaliplatin/mg lipid, from about 1 to
about 15 .mu.g oxaliplatin/mg lipid, from about 1 to about 10 .mu.g
oxaliplatin/mg lipid, from about 1 to about 5 .mu.g oxaliplatin/mg
lipid, from about 5 to about 50 .mu.g oxaliplatin/mg lipid, from
about 5 to about 45 .mu.g oxaliplatin/mg lipid, from about 5 to
about 35 .mu.g oxaliplatin/mg lipid, from about 5 to about 25 .mu.g
oxaliplatin/mg lipid, from about 5 to about 20 .mu.g oxaliplatin/mg
lipid, from about 5 to about 15 .mu.g oxaliplatin/mg lipid, from
about 5 to about 10 .mu.g oxaliplatin/mg lipid, about 1 .mu.g
oxaliplatin/mg lipid, about 2 .mu.g oxaliplatin/mg lipid, about 4
.mu.g oxaliplatin/mg lipid, about 5 .mu.g oxaliplatin/mg lipid,
about 10 .mu.g oxaliplatin/mg lipid, about 15 .mu.g oxaliplatin/mg
lipid, about 20 .mu.g oxaliplatin/mg lipid, about 30 .mu.g
oxaliplatin/mg lipid, about 40 .mu.g oxaliplatin/mg lipid, or about
50 .mu.g oxaliplatin/mg lipid.
[0324] In certain embodiments, the compositions contain from about
1 to about 145 .mu.g TF/mg lipid, from about 1 to about 120 .mu.g
TF/mg lipid, from about 1 to about 115 .mu.g TF/mg lipid, from
about 1 to about 100 .mu.g TF/mg lipid, from about 1 to about 90
.mu.g TF/mg lipid, from about 1 to about 70 .mu.g TF/mg lipid, from
about 1 to about 60 .mu.g TF/mg lipid, from about 1 to about 50
.mu.g TF/mg lipid, from about 1 to about 25 .mu.g TF/mg lipid, from
about 10 to about 150 .mu.g TF/mg lipid, from about 10 to about 140
.mu.g TF/mg lipid, from about 10 to about 125 .mu.g TF/mg lipid,
from about 10 to about 100 .mu.g TF/mg lipid, from about 10 to
about 80 .mu.g TF/mg lipid, from about 10 to about 50 .mu.g TF/mg
lipid, from about 10 to about 25 .mu.g TF/mg lipid, about 1 .mu.g
TF/mg lipid, about 5 .mu.g TF/mg lipid, about 10 .mu.g TF/mg lipid,
about 25 .mu.g TF/mg lipid, about 40 .mu.g TF/mg lipid, about 50
.mu.g TF/mg lipid, about 70 .mu.g TF/mg lipid, about 100 .mu.g
TF/mg lipid, about 120 .mu.g TF/mg lipid, about 140 .mu.g TF/mg
lipid, or about 150 .mu.g TF/mg lipid.
[0325] In some embodiments, from about 0.5 to about 50 .mu.g
oxaliplatin/mg lipid and from about 1 to about 150 .mu.g TF/mg
lipid. In some embodiments, from about 5 to about 50 .mu.g
oxaliplatin/mg lipid and from about 10 to about 100 .mu.g/mg. In
certain embodiments, from about 2 to about 50 .mu.g oxaliplatin/mg
lipid and from about 5 to about 150 .mu.g TF/mg lipid; from about 3
to about 50 .mu.g oxaliplatin/mg lipid and from about 5 to about
150 .mu.g TF/mg lipid; from about 4 to about 50 .mu.g
oxaliplatin/mg lipid and from about 5 to about 150 .mu.g TF/mg
lipid; from about 2 to about 40 .mu.g oxaliplatin/mg lipid and from
about 5 to about 150 .mu.g TF/mg lipid; from about 3 to about 40
.mu.g oxaliplatin/mg lipid and from about 5 to about 150 .mu.g
TF/mg lipid; from about 4 to about 40 .mu.g oxaliplatin/mg lipid
and from about 5 to about 150 .mu.g TF/mg lipid; from about 2 to
about 50 .mu.g oxaliplatin/mg lipid and from about 10 to about 150
.mu.g TF/mg lipid; from about 3 to about 50 .mu.g oxaliplatin/mg
lipid and from about 10 to about 150 .mu.g TF/mg lipid; from about
4 to about 50 .mu.g oxaliplatin/mg lipid and from about 10 to about
150 .mu.g TF/mg lipid; from about 5 to about 50 .mu.g
oxaliplatin/mg lipid and from about 5 to about 100 .mu.g TF/mg
lipid; from about 5 to about 50 .mu.g oxaliplatin/mg lipid and from
about 5 to about 100 .mu.g TF/mg lipid; or from about 0.5 to about
50 .mu.g oxaliplatin/mg lipid and from about 5 to about 100 .mu.g
TF/mg lipid.
[0326] In certain embodiments, the oxaliplatin concentration in the
liposome formulation is 0.8+/-10% mg/ml.
[0327] In certain embodiments, where the drug is oxaliplatin, the
oxaliplatin may be dissolved in a solution (e.g., an aqueous
solution). In some embodiments, the solution includes a sugar
(e.g., trehalose, maltose, sucrose, lactose, mannose, mannitol,
glycerol, dextrose, fructose, etc.) The concentration of the sugar
may be of several percent. For example sugar concentrations (v/v)
of about 0.1-12%; 0.5-12%, 1%-12%, 2%-8%, 2%-6%, 2%-5%, 2%-4%,
2%-5%, 2%-6%, 2%-8%, 2%-9%, 2%-10%, 4%-10%, 4%-9%, 4%-8%, 4%-6%,
3%-4%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,
about 8%, about 9% or about 10%. In certain embodiments the
solution includes a sugar and is aqueous. It is intended that the
solution in which oxaliplatin is dissolved can also contain
additional components, including those known to the skilled
artisan.
[0328] In certain embodiments, the sugar concentration is about 5%,
about 7%, about 8%, about 9% or about 10%. In other embodiments,
the sugar concentration is about 5% to about 10%. In some
embodiments, the sugar is dextrose and the concentration of
dextrose in the oxaliplatin solution is about 5%. In some
embodiments, the sugar is dextrose and the concentration of
dextrose in the oxaliplatin solution is about 9%. In certain
embodiments, the sugar is sucrose and the concentration of sucrose
in the oxaliplatin solution is about 9%. In certain embodiments,
the sugar is sucrose and the concentration of sucrose in the
oxaliplatin solution is about 10%.
[0329] In some embodiments, the concentration of sugar in solution
may be, for example about 50 mg/ml to about 150 mg/ml, about 50
mg/ml to about 130 mg/ml, about 50 mg/ml to about 120 mg/ml, about
50 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml,
about 90 mg/ml to about 150 mg/ml, about 90 mg/ml to about 130
mg/ml, about 60 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100
mg/ml, about 110 mg/ml, about 105 mg/ml, about 120 mg/ml, or about
140 mg/ml.
[0330] The solution may also contain other ingredients known to
those of skill in the art, such as, but not limited to, salts,
buffers, sugar alcohol, etc. In certain embodiments, the solution
in which oxaliplatin is dissolved contains sodium phosphate (e.g.,
monobasic and/or dibasic sodium phosphate).
[0331] In certain embodiments, the concentration of sodium
phosphate may be about 5 to about 15 mM. For example, from about 5
to about 12 mM, from about 5 to about 10 mM, from about 5 to about
7 mM, from about 7 to about 12 mM, from about 7 to about 15 mM,
from about 9 to about 12 mM, about 5 mM, about 7 mM, about 10 mM,
about 12 mM or about 15 mM.
[0332] In certain embodiments, the sugar solution may additionally
include about 1.0 to about 1.5 mg/ml sodium phosphate. For example,
about 1.2 to about 1.5 mg/ml, 1.0 to about 1.7 mg/ml, 1.0 to about
2 mg/ml, 1.0 to about 2.5 mg/ml, 1.0 to about 3 mg/ml, 0.5 to about
3.5 mg/ml sodium phosphate.
[0333] In some embodiments, the solution pH will be about 6.5 to
about 7.5, be about 6.7 to about 7.5, be about 7 to about 7.5,
about 7, about 7.5, about 6.8, or about 6.5.
[0334] In some embodiments, the drug is oxaliplatin, and is
contained in a solution of about 9% sucrose. In some embodiments,
the drug is oxaliplatin, and is contained in a solution of about 9%
sucrose in an oxaliplatin concentration of about 1 mg/ml. In some
embodiments, the drug is oxaliplatin, and is contained in a
solution of about 105 mg/ml sucrose. In some embodiments, the drug
is oxaliplatin, and is contained in a solution of about 105 mg/ml
sucrose in an oxaliplatin concentration in liposome solution of
about 1 mg/ml. In certain of these embodiments, the solution
further contains sodium phosphate. In certain embodiments,
oxaliplatin is in a concentration of about 0.8+/-10% mg/ml of
liposome solution.
Labeled Compounds
[0335] A variety of labeled compounds may also be included in the
lipid-containing compositions of the present invention. Generally,
the labeled compound may be an agent useful in carrying out in vivo
diagnostic procedures.
[0336] As with the incorporation and use of drugs as described
herein, the amount of labeled compound to be included in the
lipid-containing compositions, and formulations thereof, as
described herein can be readily determined by the skilled artisan
in view of the teaching herein provided and depending on the
labeled compound selected and the use intended for the composition
or formulation, taking into account factors specific to both the
labeled compound and the individual to be diagnosed, as described
further herein.
[0337] Exemplary labeled compounds include, for example, materials
comprising radioisotopes (e.g., .sup.3H, .sup.14C, .sup.67Ga,
.sup.111In, .sup.125I, .sup.131I, .sup.133Xe, etc.), material
comprising fluorescent moieties (e.g., fluorescein, fluorescein
isothiocyanate, etc.), material comprising enzyme (e.g.,
peroxidase, alkaline phosohatase, etc.), as well as additional
labeled compounds known to those of skill in the art.
[0338] As will be appreciated by the skilled artisan, the selection
of the labeled compound and methods used in diagnosis will depend
upon the organ (e.g., liver, pancreas, prostate, etc.), tissue
(e.g., malignant or non-malignant or tissue type (e.g., breast,
etc.)) to be investigated. For example, lipid-containing
compositions (e.g., targeted liposomes, liposome-containing
compositions, etc.) incorporating .sup.125I are particularly useful
for identifying the presence and determining the severity (e.g.,
initially, during a course of treatment, after treatment) of
various cancers (e.g., breast cancer, gastric cancer, colorectal
cancer, colon cancer, etc.) by gamma-counter.
Targeting Factors
[0339] Unless otherwise noted, the terms "targeting factor" and
"targeting ligand" may be used interchangeably herein.
[0340] The lipid-containing compositions described herein are
characterized by incorporating an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine modified with a
targeting factor (i.e., TF-N.omega.PE) directed to a particular
target cell. The term "targeting factor" refers to a moiety that
can bind to a receptor or a surface antigen present on the surface
of a target cell. In certain embodiments, the targeting factors are
directed to cell surface receptors on a particular target cell. The
targeting factor is often a protein or a peptide that can be
attached to a lipid component of the lipid-containing
composition.
[0341] Most effectively, targeting factors are selected such that
the targeted receptor or antigen is present only on cells that are
targeted for the delivery of the drug or labeled compound (e.g.,
pathogenic cells) and not present on healthy cells. Alternatively,
a greater number of receptors or antigens are expressed on the
target cells (e.g., pathogenic or diseased cells) compared to
non-targeted (e.g., healthy) cells. Preferably, the receptor or
antigen that binds the targeting factor is either not present or
present in low numbers on healthy cells such that binding with the
targeting factor does not occur with frequency. In other words,
targeting factors need to selectively deliver the liposomes as
described herein (including encapsulated drug) to the targeted
cells (e.g., pathogenic, unhealthy, etc.). Selective delivery of
the encapsulated drug to the targeted cells thus reduces the
occurrence of adverse effects due to the effect of encapsulated
drug or labeled compound on non-targeted (e.g., healthy) cells,
thereby also reducing the adverse effects experienced by the
individual to whom the composition, or formulation thereof, is
administered.
[0342] Exemplary targeting factors include, but are not limited to,
transferrin, folic acid, folate, hyaluronic acid, sugar chains
(e.g., galactose, mannose, etc.), fragments of monoclonal
antibodies, asialoglycoprotein, etc., as well as other targeting
factors known to the skilled artisan.
[0343] In particular embodiments, the targeting factor is a protein
or peptide directed to a cell surface receptor (e.g., transferrin,
folate, folic acid, asialoglycoprotein, etc.).
[0344] In other embodiments, the targeting factor is directed to an
antigen (e.g., fragments of monoclonal antibodies (e.g., Fab, Fab',
F(ab').sub.2, Fc, etc.)). It is not intended that targeting factors
include intact or whole monoclonal antibodies. The term "whole
antibody" or "intact antibody," and cognates thereof, as used
herein generally refer to antibody IgG of immune globulin. A
fragment of a monoclonal antibody generally refers to a
decomposition product of the monoclonal antibody, for example, a
fragment obtained by using protease digestion, such as pepsin,
etc.
[0345] In certain embodiments, the targeting factor is not directed
to an antigen (e.g., is not a fragment of a monoclonal antibody,
e.g., Fab, Fab', F(ab').sub.2, Fc, etc).
[0346] In a certain embodiments, the targeting factor is
transferrin.
[0347] Transferrin (Tf) is an iron binding protein with a molecular
weight of 80,000, which is synthesized in hepatocytes and found in
the blood. Transferrin supplies iron (Fe) to the cells through Tf
receptors on the surface of each cell. The transferrin receptor is
generally expressed in tumor tissues in a larger amount compared
with normal tissues regardless of the types of the tumors. Tumor
cell membranes are known to over-express transferrin receptors to
maintain cell proliferation. See Shindelman J E, Ortmeyer A E,
Sussman H H. "Demonstration of the transferrin receptor in human
breast cancer tissue. Potential marker for identifying dividing
cells." Int J Cancer. (1981) 27(3):329-34; Lloyd J M, O'Dowd T,
Driver M, Tee D E. "Demonstration of an epitope of the transferrin
receptor in human cervical epithelium--a potentially useful cell
marker." J Clin Pathol. (1984) 37(2):131-5; and Habeshaw J A,
Lister T A, Stansfeld A G, Greaves M F. "Correlation of transferrin
receptor expression with histological class and outcome in
non-Hodgkin lymphoma." Lancet. (1983) 1(8323): 498-501. Binding of
the therapeutic agents to transferrin will therefore enhance the
uptake of the drug into tumor cells through the transferrin
receptor. While not being limited to a mechanism of action, the
likely route of uptake of transferrin liposomes as described herein
is represented schematically in FIGS. 2 and 3. Transferrin is
commercially available, or can be produced recombinantly as
described in, for example, U.S. Pat. No. 5,026,651, which is hereby
incorporated by reference in its entirety.
[0348] While not being bound by theory, it is believed that the
conjugation of transferrin (Tf) to the MADE occurs by the reaction
of a primary amine with the N.omega.PE which results in the
formation of a carboxylic acid amide bond between the lipid anchor
and the protein.
[0349] In certain embodiments, the molar ratio of Tf to total lipid
present in the targeted liposome product is approximately 0.00014:1
mol/mol (Tf:total lipid) (0.015, wt/wt). In other embodiments, the
molar ratio of Tf:total lipid is from about 0.016 to about
0.029:about 126 about 158 mM/mM.
Lipid-Containing Compositions
[0350] The lipid-containing compositions described herein include
targeted liposomes incorporating derivatized lipids, additional
lipids and encapsulated drug or labeled compound, as well as the
intermediates used to prepare the targeted liposomes, including
lipid mixtures and liposome-containing compositions, as described
herein, where the lipid-containing compositions (including targeted
liposomes) are free of non-derivatized phosphatidyl ethanolamine
and hydrophilic polymers, such as, but not limited to, polyethylene
glycol. The lipid-containing compositions also include liposomes
which incorporate a TF, but do not include a drug or labeled
compound (e.g., blank liposomes).
[0351] As used herein, the term "hydrophilic polymer" and cognates
thereof, refers to polymers such as polyethylene glycol (PEG) and
other polyethoxylated polymers that are used in the liposome field
to shield liposomes in an attempt to enhance the circulatory
half-life of the liposome. It is intended that this term
encompasses free hydrophilic polymers associated non-covalently
with the liposomes as well as hydrophilic polymers that are in some
way conjugated or covalently linked to a particular component of
the liposome (e.g. PEG-modified lipids, etc.). Such hydrophilic
polymers are also alternatively referred to in the field as
"water-soluble" polymers. Additional exemplary hydrophilic polymers
include, but are not limited to, polyvinyl alcohol, polylactic
acid, polyglycolic acid, polyvinylpyrrolidone, polyacrylamide,
polyglycerol, polyaxozlines, etc.
[0352] As used herein, the term "lipid mixture," and cognates
thereof, refer to mixtures of lipid components as described herein,
where the lipid mixture does not incorporate solution, for example,
aqueous solution (e.g., water, buffer or a mixture of water and a
water-miscible solvent (e.g., sugar (e.g., trehalose, sucrose,
lactose, mannose, dextrose, fructose, etc.), sugar alcohol (e.g.,
sorbitol, maltitol, lactitol, glycerol, mannitol, etc), alcohol
(e.g., ethanol, t-butanol, etc.), etc.)) or organic solvent.
[0353] The term "liposome-containing composition," and cognates
thereof, refers to mixtures of lipids and, optionally, drug(s) or
labeled compound(s), in which aqueous solution (e.g., water, buffer
(e.g., acetate buffer, phosphate buffer, citrate buffer, borate
buffer, tartrate buffer, etc.) or mixture of water and a
water-miscible solvent) has been incorporated by mixing (e.g., one
or more of, stirring, shaking, etc.). The aqueous solution may also
include additional components such as one or more sugars (e.g.,
trehalose, maltose, sucrose, lactose, mannose, dextrose, fructose,
etc.), sugar alcohol (e.g., sorbitol, maltitol, lactitol, mannitol,
glycerol, etc.), alcohol (e.g., ethanol, t-butanol, etc.), etc. And
the aqueous solution may also include organic solvent ((e.g.,
esters (e.g., ethyl acetate, butyl acetate, etc.), aliphatic
hydrocarbons (e.g., hexane, heptane, etc.), aromatic hydrocarbons
(e.g., toluene, xylene, etc.), halogenated hydrocarbons (e.g.,
chloroform, dichloromethane, dichloroethane, etc.), ethers (e.g.,
THF, dioxane, diethyl ether, isopropyl ether, etc.), cyclic
hydrocarbons (e.g., cyclohexane, etc.), DMF, DMSO, etc.) or
mixtures thereof). The liposome-containing composition will
generally contain a non-homogenous mixture of lipids, aqueous
solution, and liposomes having a broad distribution around
100-10,000 nm and a mean diameter of 500-2,000 nm. Characterization
of exemplary liposome-containing compositions is further provided
in the Examples.
[0354] In certain embodiments, the lipid-containing compositions do
not incorporate hydrophilic polymers. In particular embodiments,
the lipid-containing compositions do not incorporate PEG.
[0355] In some embodiments, the intermediate lipid mixtures include
at least two different neutral lipids or one or more phospholipids,
and an N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, wherein the lipid components are described in greater
detail herein and wherein the mixture is free of non-derivatized
phosphatidyl ethanolamine and hydrophilic polymers, such as
polyethylene glycol. Optionally, an aqueous solution as herein
described may be mixed with the lipid components to form a
liposome-containing composition. In certain embodiments, the lipid
mixture does not include a drug or labeled compound. In particular
embodiments, the lipid-mixture may be treated to form a
liposome-containing composition or a liposome formulation.
[0356] In some embodiments, the intermediate lipid mixtures include
at least two different neutral lipids or one or more phospholipids,
an N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine and a drug or labeled compound, wherein the lipid
components and drug/labeled compound are described in greater
detail herein and wherein the mixture is free of non-derivatized
phosphatidyl ethanolamine and hydrophilic polymers, such as
polyethylene glycol. Optionally, an aqueous solution as herein
described may be mixed with the lipid components to form a
liposome-containing composition, for example, as where the drug or
labeled compound is added as an aqueous solution of drug/labeled
compound. In certain embodiments, the liposome-containing
composition can be treated (e.g., by one or more of extrusion, size
exclusion chromatography, etc. or methods known in the art) to form
a liposome.
[0357] In some embodiments, the lipid mixtures include one or more
phospholipids or at least two different neutral lipids, an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
and a succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine, wherein the lipid
components are described in greater detail herein and wherein the
mixture is free of non-derivatized phosphatidyl ethanolamine and
hydrophilic polymers, such as polyethylene glycol. The mixtures may
also be substantially free of non-NHS starting material, byproduct
and/or decomposition product associated with synthesis of the
succinimidyl ester of an N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine (e.g., carbodiimmides (e.g., DCC, EDC,
etc.), acylated urea compounds, etc.). Optionally, an aqueous
solution as herein described may be mixed with the lipid components
to form a liposome-containing composition.
[0358] In some embodiments, the intermediate lipid mixtures include
one or more phospholipids or at least two different neutral lipids,
an N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine and a targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, wherein the lipid components are described in greater
detail herein and wherein the mixture is free of non-derivatized
phosphatidyl ethanolamine and hydrophilic polymers, such as
polyethylene glycol. The mixtures may also be substantially free of
non-NHS starting material, byproduct and/or decomposition product
associated with synthesis of the succinimidyl ester of an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
(e.g., carbodiimmides (e.g., DCC, EDC, etc.), acylated urea
compounds, etc.). Optionally, an aqueous solution as herein
described may be mixed with the lipid components to form a
liposome-containing composition. In some embodiments, the lipid
mixture does not include a drug or labeled compound. The lipid
mixture may be treated to form a liposome-containing composition or
a liposome formulation.
[0359] In some embodiments, the intermediate lipid mixtures include
one or more phospholipids or at least two different neutral lipids,
an N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine and a succinimidyl ester of an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
and a drug or labeled compound, wherein the lipid and drug/labeled
compound components are described in greater detail herein and
wherein the composition is free of non-derivatized phosphatidyl
ethanolamine and hydrophilic polymers, such as polyethylene glycol.
The mixtures may also be substantially free of non-NHS starting
material, byproduct and/or decomposition product associated with
synthesis of the succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine (e.g., carbodiimmides
(e.g., DCC, EDC, etc.), acylated urea compounds, etc.). Optionally,
an aqueous solution as herein described may be mixed with the lipid
components to form a liposome-containing composition.
[0360] In certain embodiments, the intermediate lipid-containing
compositions include a liposome containing one or more
phospholipids or at least two different neutral lipids, an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
and a succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine and an encapsulated
drug, wherein the lipid and drug components are described in
greater detail herein and wherein the liposome is free of
non-derivatized phosphatidyl ethanolamine and hydrophilic polymers,
such as polyethylene glycol. The liposomes may also be
substantially free of non-NHS starting material, byproduct and/or
decomposition product associated with synthesis of the succinimidyl
ester of an N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine (e.g., carbodiimides (e.g., DCC, EDC, etc.), acylated
urea compounds, etc.). In some embodiments, the lipid mixture does
not include a drug or labeled compound. The lipid mixture may be
treated to form a liposome-containing composition or a liposome
formulation.
[0361] In certain embodiments, the lipid mixtures include one or
more phospholipids or at least two different neutral lipids, an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, a targeting-factor-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine and an encapsulated drug
or labeled compound, wherein the lipid, targeting factor and
drug/labeled compound components are described in greater detail
herein and wherein the liposome is free of non-derivatized
phosphatidyl ethanolamine and hydrophilic polymers, such as
polyethylene glycol. In some embodiments of the lipid mixtures, the
liposomes are substantially free of non-NHS starting material,
byproduct or decomposition products associated with synthesis of
the succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine (e.g., carbodiimmides
(e.g., DCC, EDC, etc.), acylated urea compounds, etc.). In
particular embodiments, the lipid mixture is a liposome-containing
composition (e.g., where drug or labeled compound is added as an
aqueous solution). Optionally, an aqueous solution as herein
described may be mixed with the lipid components to form a
liposome-containing composition.
[0362] In certain embodiments, the targeted liposomes include
liposomes containing one or more phospholipids or at least two
different neutral lipids, an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine and a targeting-factor
modified N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine and an encapsulated drug or labeled compound, wherein
the lipid, targeting factor and drug or labeled compound components
are described in greater detail herein and wherein the liposome is
free of non-derivatized phosphatidyl ethanolamine and hydrophilic
polymers, such as polyethylene glycol. In some embodiments of the
targeted liposomes, the liposomes are substantially free of non-NHS
starting material, byproduct or decomposition products associated
with synthesis of the succinimidyl ester of an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
(e.g., carbodiimmides (e.g., DCC, EDC, etc.), acylated urea
compounds, etc.). However, in some embodiments, the succinimidyl
ester of an N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine may be present in the initial stages in the
preparation of the targeted liposomes (e.g., NHS-NG-PEs (e.g.,
NHS-NG-DOPE, NHS-NG-DSPE, etc.), for example, prior to hydrolysis
of the succinimidyl ester which may yield, for example NHS and
NG-DOPE in the final formulation. In certain embodiments, where
SuccN.omega.PE is not incorporated into the interior of the
liposome, the liposome or liposome-containing composition can also
be free or substantially free of NHS, as in when TF-N.omega.PE is
pre-formed and used as starting material. In particular
embodiments, the targeted liposomes are substantially free of DCC
and EDC. In certain embodiments, the targeted liposomes are
substantially free of DCC.
[0363] Additionally, each of the liposome-containing compositions
as described herein can be treated to produce liposomes. The
production of liposomes is well known in the art and, additionally,
can be accomplished according to the methods described herein, for
examples as described for production methods A and B, described in
greater detail below. Production methods for liposomes from
liposome-containing compositions include, but are not limited to,
extrusion, sonication, reverse phase vesicle, freeze-thaw, size
exclusion chromatography, ultrafiltration, etc. and combinations
thereof. The liposomes formed from the liposome-containing
compositions herein described may incorporate drug or labeled
compound or may be free of drug or labeled compound (e.g., for
liposomes also referred to herein also as "blank liposomes"). In
particular embodiments, the liposome-containing compositions,
liposomes (including blank liposomes) and targeted liposomes maybe
formulated as pharmaceutical formulations and, additionally, may be
used in the methods of treatment or diagnosis and/or kits described
herein.
[0364] The term "substantially free" refers to levels of materials
that are undetectable or minimally detectable by routine analytical
methods used in the field. For example, HPLC (see e.g., European
Phmaracopoeia 5.sup.th Ed.), TLC, gas chromatography, etc., as well
as other analytical methods known to the skilled artisan.
[0365] For example, the lipid-containing compositions may contain
less than about 0.1%, less than about 0.5%, less than about 1%,
less than about 2%, less than about 3%, less than about 4%, less
than about 5%, or less than about 6% by weight of a particular
starting material, byproduct or decomposition product associated
with synthesis of the succinimidyl ester of an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
to the total lipid content. In particular embodiments, the
compositions will contain less than about 10%, less than about 7%,
less than about 5%, less than about 3%, less than about 2%, or less
than about 1% total impurities (e.g., % sum of starting material,
byproduct and decomposition product associated with synthesis of
the succinimidyl ester of an N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine).
[0366] In some embodiments, the targeted liposomes include
liposomes containing a phosphatidylcholine (e.g., neutral, anionic
or cationic), cholesterol or a cholesterol derivative, an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, a targeting factor-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine and an encapsulated drug
or labeled compound, wherein the lipid, targeting factor and drug
components are described in greater detail herein and wherein the
liposome is free of non-derivatized phosphatidyl ethanolamine and
hydrophilic polymers, such as polyethylene glycol. In particular
embodiments, the phosphatidyl choline is a neutral phosphatidyl
choline.
[0367] In particular embodiments, the targeted liposomes include
liposomes containing a phosphatidyl choline (e.g., neutral, anionic
or cationic), cholesterol or a cholesterol derivative, an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, a transferrin-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine and encapsulated drug or
labeled compound, wherein the lipid components and drug or labeled
compound are described in greater detail herein and wherein the
liposome is free of non-derivatized phosphatidyl ethanolamine and
hydrophilic polymers, such as polyethylene glycol. In particular
embodiments, the phosphatidyl choline is a neutral phosphatidyl
choline.
[0368] In particular embodiments, the targeted liposomes include
liposomes containing a phosphatidyl choline (e.g., neutral, anionic
or cationic), cholesterol or a cholesterol derivative, an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, a transferrin-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine and encapsulated
oxaliplatin, wherein the lipid components are described in greater
detail herein and wherein the liposome is free of non-derivatized
phosphatidyl ethanolamine and hydrophilic polymers, such as
polyethylene glycol. In particular embodiments, the phosphatidyl
choline is a neutral phosphatidyl choline.
[0369] In certain embodiments, the lipid-containing compositions
(including targeted liposomes and blank liposomes), and
formulations thereof, described herein may further contain lipids
obtained by derivatizing phosphatidylglycerol, sphingosine,
ceramide, a cholesterol derivative or the like with a dicarboxylic
acid. These dicarboxylic acid-derivatives may be prepared as
described herein for the N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamines and according to preparation methods
known to the skilled artisan.
[0370] In some embodiments, the lipid-containing compositions
(including targeted liposomes and blank liposomes), and
formulations thereof, described herein do not include anionic
lipids (e.g., phosphatidylserines, phosphatidylinsitosols,
phosphatidylglycerols, etc.) or cationic lipids (e.g., sphingosine,
DOTAP, DOTMA, DC-CHOL, etc.). In particular embodiments, the
compositions are free of anionic lipids. In other embodiments, the
compositions are free of cationic lipids. In certain embodiments,
the compositions are free of cationic and anionic lipids.
[0371] In some embodiments, the lipid-containing compositions, the
composition comprises a drug. In other embodiments, the
lipid-containing compositions comprise a labeled compound.
[0372] In certain embodiments of the lipid-containing compositions,
the drug is oxaliplatin, the targeting factor (TF) is transferrin
(Tf) and the lipid components include: DMPC or DSPC, and,
cholesterol or a cholesterol derivative, and an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamine, and a transferrin-modified N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine, wherein the
transferrin-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine comprises a transferrin linked to an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
by a carboxylic acid amide bond.
[0373] In some embodiments, the N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamine and targeting
factor-modified N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamine are NG-DOPE or NG-DSPE.
[0374] In particular embodiments, the lipid components of the
lipid-containing compositions are DMPC, cholesterol, NG-DOPE and
TF-modified NG-DOPE. In other embodiments, the lipid components are
DSPC, cholesterol, NG-DOPE and TF-modified NG-DOPE. In still other
embodiments, the lipid components are DMPC, cholesterol, NG-DSPE,
and TF-modified NG-DSPE. In certain other embodiments, the lipid
components are DSPC, cholesterol, NG-DSPE, and TF-modified NG-DSPE.
In certain of these embodiments, the targeting factor (TF) is
transferrin and the drug is oxaliplatin.
[0375] In particular embodiments, the lipid components are DPPC,
cholesterol, NG-DOPE and TF-modified NG-DOPE. In other embodiments,
the lipid components are POPC, cholesterol, NG-DOPE and TF-modified
NG-DOPE. In still other embodiments, the lipid components are DPPC,
cholesterol, NG-DSPE, and TF-modified NG-DSPE. In certain other
embodiments, the lipid components are POPC, cholesterol, NG-DSPE,
and TF-modified NG-DSPE. In certain of these embodiments, the
targeting factor (TF) is transferrin and the drug is
oxaliplatin.
[0376] In particular embodiments, the lipid components are HSPC,
cholesterol, NG-DOPE and TF-modified NG-DOPE. In other embodiments,
the lipid components are EPC, cholesterol, NG-DOPE and TF-modified
NG-DOPE. In still other embodiments, the lipid components are HSPC,
cholesterol, NG-DSPE, and TF-modified NG-DSPE. In certain other
embodiments, the lipid components are EPC, cholesterol, NG-DSPE,
and TF-modified NG-DSPE. In certain of these embodiments, the
targeting factor (TF) is transferrin and the drug is
oxaliplatin.
[0377] Ratios of Lipid Components
[0378] Generally, the molar percent of starting materials NG-DOPE
will be between about 2.5 mole % to about 4.5 mol %, compared to
the total lipid content. Additionally, the molar percent of
starting materials NHS-NG-DOPE will be between about 0.5 mole % to
about 2.5 mol %, compared to the total lipid content. In some
embodiments, the relative mole ratio of NG-DOPE to NHS-NG-DOPE will
be about 3.4:1. In certain embodiments, the relative mole % ratio
of NG-DOPE to NHS-NG-DOPE may be about 4:1. In particular
embodiments, where a neutral phospholipid and a neutral lipid are
present, the molar ratio of (e g., DMPC:Chol:NG-DOPE:NHS-NG-DOPE)
may be 43.0:38.5:3.42:1, which can also be expressed as 50:45:4:1
by mol %.
[0379] In certain embodiments, the relative mol % of additional
lipid(s) to N.omega.PE to SuccN.omega.PE (e.g., at least two
neutral lipids:N.omega.PE:SuccN.omega.PE or one or more
phospholipids:N.omega.PE:SuccN.omega.PE or (one or more
phospholipids+neutral lipid(s)):N.omega.PE:SuccN.omega.PE) may be
from about 98 mol % to about 87 mol % additional lipids: from about
1 mol % to about 12 mol % N.omega.PE: about 0.5 mol % to about 1%
SuccN.omega.PE; where the total mol % of all components is 100 mol
%. For example, additional lipids:N.omega.PE:SuccN.omega.PE may be
approximately 95:4:1, 90:9:1, 92:7:1, 93:6:1, etc.
[0380] In particular embodiments, where the additional lipids
include a phospholipid and another lipid such as cholesterol,
cholesterol derivatives, etc., the range of mol % for each lipid
component is from about 30 mol % to about 64%, where the total of
additional lipids is from about 98 mol % to about 87 mol %.
[0381] In certain embodiments, where the additional lipids are two
different neutral lipids, the range of mol % for each neutral lipid
is from about 30 mol % to about 64%, where the total of neutral
lipids is from about 98 mol % to about 87 mol %.
[0382] In an exemplary embodiment, where one additional lipid is a
phosphatidyl choline and a second additional lipid is cholesterol
or a cholesterol derivative, the mol % of the phosphatidyl choline
is from about 30 to about 70 mol %, (e.g., from about 50 to about
64 mol %, from about 40 to about 65 mol %, from about 40 to about
60 mol %, from about 50 to about 62 mol %, from about 55 to about
60 mol %, from about 35 to about 55 mol %, about 30 mol %, about 40
mol %, about 45 mol %, about 50 mol %, about 55 mol %, about 60 mol
%, about 65 mol %, about 70 mol %) and the mol % of the cholesterol
or cholesterol derivative is from about 30 to about 60 mol % (e.g.,
from about 32 to about 45 mol %, from about 32 to about 42 mol %,
from about 32 to about 40 mol %, from about 40 to about 60 mol %,
from about 35 to about 55 mol %, from about 35 to about 60 mol %,
from about 45 to about 60 mol %, from about 35 to about 45 mol %,
about 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %,
about 50 mol %, about 55 mol % or about 60 mol %). In some
embodiments, the phosphatidyl choline is about 50 mol %, about 52
mol %, about 55 mol %, about 58 mol %, about 60 mol %, about 62 mol
% and the cholesterol or cholesterol derivatives is from about 30
mol %, about 32 mol %, about 34 mol %, about 35 mol %, about 37 mol
%, about 38 mol %, about 40 mol %, about 42 mol %, about 43 mol %,
about 45 mol %.
[0383] In particular embodiments, the mol % of N.omega.PE is about
1 to about 11 mol %, about 1 to about 10 mol %, about 1 to about 8
mol %, about 1 to about 6 mol %, about 1 to about 5 mol %, about 1
to about 4 mol %, about 1 to about 3 mol %, about 1 to about 2 mol
%, about 2 to about 10 mol %, about 2 to about 5 mol %, about 1 mol
%, about 2 mol %, about 3 mol %, about 4 mol %, about 5 mol %,
about 7 mol %, about 8 mol %, about 9 mol % about 10 mol %, about
11 mol %, or about 12 mol %.
[0384] In certain embodiments, the relative mol % of the first
additional lipid:second additional lipid:N.omega.PE:SuccN.omega.PE
is, for example 50:45:4:1. In some embodiments, the first
additional lipid is a phosphatidylcholine (e.g., DMPC, DOPC, DPPC,
DSPC, etc.) and the second additional lipid is cholesterol. In some
embodiments, the PE of the N.omega.PE is DOPE or DSPE. In
particular embodiments, the N.omega.PE is NG-DOPE or NG-DSPE. In
some embodiments, the lipids are DMPC:Chol:NG-DOPE:NHS-NG-DOPE and
their relative mol % is 50:45:4:1. In other embodiments, the lipids
are DSPC:Chol:NG-DSPE:NHS-NG-DSPE and their relative mol % is
50:45:4:1. In other embodiments, the lipids are
DSPC:Chol:NG-DSPE:NHS-NG-DSPE and their relative mol % is
62:33:4:1.
[0385] In some embodiments, the total mol % of N.omega.PE and
TF-N.omega.PE (N.omega.PE+TF-N.omega.PE) is about 2 to about 13 mol
% of total lipid content. For example, about 2 to about 12 mol %,
about 2 to about 10 mol %, about 2 to about 8 mol %, about 2 to
about 6 mol %, about 2 to about 4 mol %, about 2 mol %, about 3 mol
%, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %,
about 8 mol %, about 9 mol %, about 10 mol %, about 11 mol %, or
about 12 mol %.
[0386] Generally, the total mol % of TF-N.omega.PE is about 0.002
to about 0.2 mol % relative to total lipid content. For example, in
some embodiments, the total mol % of TF-N.omega.PE is about 0.002
to about 0.15 mol %, total mol % of TF-N.omega.PE is about 0.002 to
about 0.1 mol %, 0.002 to about 0.05 mol %, about 0.01 to about
0.03 mol %, about 0.005 to about 0.2 mol %, about 0.007 to about
0.2 mol %, about 0.007 to about 0.05 mol %, about 0.01 to about
0.025 mol %, about 0.015 to about 0.025 mol %, about 0.01 to about
0.2 mol %, about 0.02 to about 0.2 mol %, about 0.04 to about 0.2
mol %, about 0.06 to about 0.2 mol %, about 0.08 to about 0.2 mol
%, about 0.002 mol %, about 0.008 mol %, about 0.01 mol %, about
0.02 mol %, about 0.03 mol %, about 0.025 mol %, about 0.015 mol %,
about 0.06 mol %, about 0.08 mol %, about 0.1 mol %, about 0.15 mol
%, or about 0.2 mol %.
[0387] Characterization of Liposomes and Liposome-Containing
Compositions
[0388] In addition to characterizing the lipid-containing
compositions by the ratios of components (e.g., ratios of lipids,
ratio of drug/labeled compound to lipid, etc.), the
liposome-containing compositions as described herein may also be
characterized (e.g., physicochemical properties, etc.) using
standard analytical methods, as will be appreciated by the skilled
artisan. Such analytical methods include, but are not limited to
and where appropriate, determination of mean diameter, encapsulated
volume, net charge (zeta potential), amount of entrapped (i.e.,
encapsulated) drug, particle size, stability under various
conditions (e.g., in storage, as prepared for administration, in
vitro), osmotic properties, amount of conjugated targeting factor,
etc. Exemplary analytical methods for such characterization are set
forth below, as well as in the Examples, and additional methods
known to the skilled artisan may also be used to characterize the
compositions.
[0389] As will be appreciated by the skilled artisan, the drug
content of liposomes can be determined using established methods
for HPLC analysis, with appropriate controls, as routinely
practiced in the art and, additionally, as described in the
Examples. Using appropriate controls, the identity of the
encapsulated drug can also be determined by HPLC or, for certain
drugs (e.g., platinum containing drugs) by analytical methods such
as ICP-MS (Inductively Coupled Plasma-Mass Spectrometry) as is
practiced by those of skill in the field.
[0390] In certain embodiments, the amount of drug (e.g.,
oxaliplatin, etc.) or labeled compound encapsulated within the
liposome or liposome-containing composition may be from about 0.1
mg/ml to about 15 mg/ml within the liposome. For example, the drug
concentration may be from about 0.5 mg/ml to about 15 mg/ml, about
0.5 mg/ml to about 10 mg/ml, about 0.1 mg/ml to about 10 mg/ml,
about 0.5 mg/ml to about 5 mg/ml; about 0.5 mg/ml to about 3 mg/ml,
about 0.5 mg/ml to about 2 mg/ml, about 0.5 mg/ml to about 1.5
mg/ml, about 0.8 mg/ml to about 3 mg/ml, about 0.8 mg/ml to about 2
mg/ml, about 0.8 mg/ml to about 1.5 mg/ml, about 0.7 mg/ml to about
3 mg/ml, about 0.7 mg/ml to about 2 mg/ml, about 0.7 mg/ml to about
1.7 mg/ml, about 0.7 mg/ml to about 1.5 mg/ml, about 0.7 mg/ml to
about 1.4 mg/ml, about 0.7 mg/ml to about 1.3 mg/ml, about 0.5
mg/ml, about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1
mg/ml, about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4
mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 2 mg/ml, about 3
mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml,
about 8 mg/ml, about 9 mg/ml, about 10 mg/ml or about 15 mg/ml
within the liposome.
[0391] The electric potential at the shear plane is called the zeta
potential of a liposome. As is known the skilled artisan, the zeta
potential of liposomes can be experimentally determined using
appropriate instrumentation, for example as measured by an ELS-6000
(Otsuka Electronics, Japan) with the laser-Doppler
microelectrophoresis method or other instrumentation and protocols
available to the skilled artisan. For example, J. Colloid and
Interface Sci., 39, 670-675(1972),
nition.com/en/products/zeecom_s.htm, etc.
[0392] In certain embodiments, the liposomes (including the
targeted liposomes and the liposomes of the liposome-containing
composition) as described herein will exhibit an overall net
negative zeta potential. In some embodiments, the zeta potential is
from about -10 mV to about -200 mV. For example, from about -50 mV
to about -150 mV, from about -50 mV to about -130 mV, from about
-60 to about -120 mV, from about -50 to about -100 mV, from about
-75 mV to about -90 mV, from about -80 mV to about -90 mV, from
about -80 mV to about -85 mV, from about -85 mV to about -90 mV,
from about -75 mV to about -85 mV, from about -70 mV to about -90
mV, about -75 mV, about -80 mV, about -85 mV, about -83 mV, about
-90 mV, about -100 mV, about -120 mV.
[0393] After the intravenous injection of small liposomes, they are
thought to pass through the fenestrae of the liver sinusoids and
will rapidly come into contact with hepatocytes. Liposomes of
intermediate size are thought to be retained within the blood
compartment and can circulate for a considerable length of time.
However, large liposomes pass more slowly through the liver
sinusoids, and are rapidly taken up by Kupffer cells. Thus the size
of the liposome is very important to determine the behavior in
vivo. See, for example, Liu et al., Biochim. Biophys. Acta (1992)
1104(1):95-101; Harashima et al., J. Drug Target. (1995)
3(4):253-261; (which are hereby incorporated by reference in their
entirety) etc.
[0394] Liposome particle size can be obtained from the correlation
function by using various algorithms using Photon Correlation
Spectroscopy (PCS; Dynamic Light Scattering or Quasi-Elastic Light
Scattering (QELS)). The particle size obtained by these techniques
is comparable to the mean diameter determined by PCS. PCS uses
standard deviation and .chi.2 to describe the size distribution. In
PCS systems, .chi.2 determines whether the system is unimodal
(Gaussian distribution) or multimodal (Nicomp distribution). The
mean particle size can be determined using intensity weighted
measurements and is reported from the Gaussian distribution if
.chi.2.ltoreq.5. If .chi.2>5, then the mean of the principal
peak in a Nicomp distribution. Such analysis will be familiar to
the skilled artisan, as will suitable hardware, for example Nicomp
QELS Particle Sizer, PSS Model 380ZLS, S/N 0103301;
pssnicomp.com/zetaspec.htm.
[0395] In certain embodiments of the liposomes, particularly the
targeted liposomes, described herein the liposome mean diameter
will be from about 50 to about 275 nm. For example, the liposome
mean diameter may be from about 50 to about 200 nm, from about 50
to about 265 nm, from about 50 to about 250 nm, from about 50 to
about 225 nm, from about 50 to about 175 nm, from about 50 to about
150 nm, from about 50 to about 120 nm, from about 50 to about 100
nm, from about 75 to about 250 nm, from about 75 to about 200 nm,
from about 75 to about 175 nm, from about 75 to about 150 nm, from
about 75 to about 120 nm, from about 75 to about 100 nm, from about
90 to about 100 nm, from about 90 to about 120 nm, from about 90 to
about 150 nm, from about 90 to about 200 nm, from about 95 to about
100 nm, from about 95 to about 120 nm, from about 95 to about 125
nm, from about 95 to about 130 nm, from about 95 to about 150 nm,
from about 95 to about 175 nm, about 90 nm, about 95 nm, about 100
nm, about 120 nm, about 130 nm, or about 150 nm. For a particular
liposome composition, the targeted liposome will be approximately
about 15 to about 25 nm greater in diameter than the a liposome
formed of the same components but with the targeting factor not
incorporated.
[0396] The liposomes (e.g., targeted liposomes, blank liposomes,
liposomes in liposome-containing compositions) described herein can
also be characterized by the concentration of targeting ligand that
is incorporated into the liposome. Depending on the targeting
ligand selected, various means of quantifying the amount of
targeting ligand will be apparent to the skilled artisan. For
example, as described in the examples, the transferrin (Tf) content
of liposomes can be determined by use of electrophoretic migration
(e.g., as measured by SDS-PAGE) the liposome compared to
appropriate controls.
[0397] In brief, confirmation of transferrin content in liposomes
can be evaluated using two assays for the content and/or identity
of transferrin conjugated to liposomes. Firstly, the
electrophoretic migration of transferrin the liposome as analyzed
by SDS-PAGE can be compared to the migration pattern of purified
conjugated transferrin, e.g., TF-N.omega.PE. In addition, the
electrophoretic migration of conjugated transferrin in can also be
compared to free transferrin reference standard. Additional support
for the identity of transferrin in liposomes can be obtained using
ELISA, a research-grade formulation that demonstrates specific
binding of anti-transferrin antibody to the targeted liposomes. The
concentration of transferrin-targeted liposomes can be measured
using colorimetric protein quantification assays, such as a BCA, an
assay well known to the skilled artisan. The skilled artisan in
view of the teachings herein will also appreciate similar methods
and others known in the field for determining the amount of a
variety of targeting factors.
[0398] Briefly, the amount of transferrin in a liposome can be
analyzed using the bicinchoninic acid (BCA) assay reagent. Copper
(II) is reduced to copper (I) by protein under alkaline conditions.
The copper (I) ion generated forms a soluble, intensely colored
complex with BCA. The total microparticle-bound protein is measured
by the reaction of a known amount of microparticle suspension with
the BCA reagents. Once color formation occurs, the microparticles
are removed by filtration and the color is measured
spectrophotometrically.
[0399] In some embodiments, the concentration of targeting ligand
incorporated in the liposome will be from about 0.5 mg/ml to about
5.0 mg/ml, from about 0.5 mg/ml to about 2.0 mg/ml, from about 1.0
mg/ml to about 2.0 mg/ml, from about 1.0 mg/ml to about 3.0 mg/ml,
from about 1.0 mg/ml to about 2.5 mg/ml, from about 1.0 mg/ml to
about 2.0 mg/ml, or from about 1.3 mg/ml to about 2.5 mg/ml.
[0400] The role of ferric ion is very important in binding
transferrin to the surface of tumor cells. Therefore, the ferric
ion content of targeted liposomes incorporating Tf is another
meaningful way of characterizing the liposomes. While a number of
methods for determining ferric ion content will be known to those
of skill in the art, one method is ICP-MS.
[0401] Where the a liposome contains transferrin, the ferric ion
content of the liposome may be, for example, from about 0.25
.mu.g/mL to about 3 .mu.g/mL, 0.4 .mu.g/mL to about 3 .mu.g/mL,
0.25 .mu.g/mL to about 2 .mu.g/mL, 0.25 .mu.g/mL to about 1.5
.mu.g/mL, 0.25 .mu.g/mL to about 1 .mu.g/mL, 0.4 .mu.g/mL to about
2 .mu.g/mL, 0.4 .mu.g/mL to about 1.5 .mu.g/mL, 0.5 .mu.g/mL to
about 2 .mu.g/mL, about 0.5 .mu.g/mL to about 1.4 .mu.g/mL, or
about 0.5 .mu.g/mL to about 1.5 .mu.g/mL.
[0402] The liposomes, including targeted liposomes and blank
liposomes, may also be characterized by their osmotic pressure at a
given temperature. The osmotic pressure at a given temperature
depends upon the molar concentration of sugar (sucrose) solution.
And it also depends on the total ion density and the size of the
molecules within the solution. Normally osmotic pressure can be
measured using an instrument known as an osmometer, which measures
osmotic pressure in suitable pressure units, as will be appreciated
by the skilled artisan.
[0403] In certain embodiments the osmotic pressure of the
liposomes, particularly the targeted liposomes and blank liposomes,
at room temperature will be from about 310 to about 410 mOsm/kg.
For example, the osmotic pressure may be from about from about 310
to about 400 mOsm/kg, from about 310 to about 380 mOsm/kg, from
about 320 to about 360 mOsm/kg, from about 315 to about 375
mOsm/Kg, from about 320 to about 375 mOsm/Kg, from about 315 to
about 370 mOsm/Kg, from about 320 to about 370 mOsm/Kg, about 360
mOsm/Kg, about 350 mOsm/Kg, about 340 mOsm/Kg, about 370 mOsm/Kg or
about 380 mOsm/Kg at room temperature.
[0404] Under the various conditions described herein (e.g.,
conditions for storage, prepared for administration and/or in vitro
conditions) the osmotic pressure may vary less than about 25%, less
than about 20%, less than about 15% when monitored over a
particular time period associated with the various conditions as
described herein. For example, 360+/-50 mOsm/kg.
Production of Lipid-Containing Compositions
[0405] There are three main requirements for drugs and labeled
compounds intended for administration to individuals in the course
of therapy, namely, efficacy, safety, and assurance of quality.
Despite proven efficacy and safety, the ability to use a drug in
therapy is undermined if its quality (e.g., purity, homogeneity,
reproducibility of dosage, stability over time, etc.) cannot be
consistently guaranteed during manufacture and distribution.
Methods of production of drugs or labeled compound that do not
assure consistently high quality drug also increase the likelihood
of adverse reactions in the individuals to whom the drug is
administered. Throughout the lifetime of a drug or labeled
compound, it is important that the product that is manufactured and
distributed meet the same standards as the product that initially
received regulatory approval. Thus, the ability to consistently
produce drugs or labeled compounds of high quality is necessary for
producing safe drug or labeled compound products and drugs or
labeled compound that can be routinely and easily manufactured and
purified are advantageous from both a commercial and a safety
perspective. Where, for example, efficacy of a labeled compound
refers to its ability to be useful in the diagnosis of a particular
disease or conditions in conjunction with the particular diagnostic
methods (e.g., the activity of the labeled compound (for example,
ability to be visualized by gamma counter, etc.) is not impaired by
an unacceptable level batch to batch or during storage.
[0406] Described below are general methods for the production of
the compositions described herein that can be used to consistently
produce high quality (e.g., of high purity, homogeneity, etc.)
targeted liposomes (and intermediates thereof) and blank liposomes.
These methods are also represented schematically in FIGS. 4
(production method A) and 5 (production method B).
[0407] The succinimidyl esters of N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines and
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines as described herein are useful as components of
phospholipid complexes such as liposomes, polymer micelles, micro-
and nano-spheres, emulsions and water-soluble polymers. The
preparation of these PE derivatives is described herein and methods
for their production are also known in the art, as mentioned
previously.
[0408] In particular, the succinimidyl esters of
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines and N-(.omega.)-dicarboxylic acid-derivatized
phosphatidyl ethanolamines are also useful as components of the
lipid-containing compositions as described herein. The
lipid-containing compositions can be prepared according to the
methods described herein, though modifications of these methods
will also be apparent to the skilled artisan. For example, various
methods known to the skilled artisan may be used in liposome
formation from lipid components (e.g., sonication, stirring,
extrusion, dehydration, etc.) As, for example, as described in U.S.
Pat. App. Pub. No. 2004/0142025, the contents of which are hereby
incorporated by reference in its entirety.
[0409] Use of the general methods described herein, including in
the Examples, to produce the targeted liposomes also encompasses
methods for the production of the other lipid-containing
compositions (e.g., lipid mixtures, liposome-containing
compositions, blank liposomes, and intermediate liposomes), as
described herein.
Production Method A
[0410] Production method A is depicted schematically in FIG. 4.
[0411] A: Production of N.omega.PE:SuccN.omega.PE:Additional Lipid
Mixture (Intermediate 1)
[0412] The succinimidyl esters of N-(.omega.)-dicarboxylic
acid-derivatized phosphatidyl ethanolamines (SuccN.omega.PE) and
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines (N.omega.PE) as described herein are mixed with
additional lipid(s) ((e.g., at least one phospholipid(s) (e.g. PC
(e.g., DMPC, DSPC, etc.), PI, sphingomyelin, phosphatidic acid,
etc.) and at least one other additional lipid (e.g., cholesterol)),
then dissolved in a suitable solvent (e.g., ethanol, t-BuOH,
chloroform, isopropylether, etc.). The amount of the solvent is
generally 1 to 100 v/w (vs. total lipid weight). In certain
embodiments, this is 2 to 20 v/w.
[0413] The SuccN.omega.PE and N.omega.PE used as described in the
preceding paragraph can be prepared and purified by methods
described herein or by methods known to the skilled artisan. The
SuccN.omega.PE and N.omega.PE, as well as other components
described herein for the production of the targeted liposomes and
intermediates thereof, should be of sufficient purity and
homogeneity to ultimately yield targeted liposomes of sufficient
purity and homogeneity to fall within regulatory guidelines for the
administration of the targeted liposomes to individuals and in
accordance with good laboratory practice (GLP) and good
manufacturing practice (GMP) guidelines.
[0414] Where an additional lipid(s) (in additional to one or more
phospholipids) is used in combination with the phospholipid, the
ratio of phospholipid(s) to other additional lipid(s) is about 2:1.
A mixing ratio of the SuccN.omega.PE derivatives to the
phospholipids is from about 1 to about 12% (1:99 to 12:88), or
about 3-6% (3:97 to 6:94) of the total concentration/ratio of
(NHS-NGPE+NGPE) to (CHOL+Phospholipid). For example, exemplary
ratio include 50:45:4:1 (e.g., PC:Chol:NG-PE:NHS-NG-PE), where, for
example 17.5 mg of NHS-NG-DOPE, 63.1 mg of NG-DOPE, 312 mg of Chol
and 607 mg of phospholipids in 1 g of mixture.
[0415] B: Production of Drug:N.omega.PE:SuccN.omega.PE:Additional
Lipid Mixture (Intermediate 2)
[0416] The N.omega.PE:SuccN.omega.PE:additional lipid mixture
prepared in step A is then mixed with aqueous solution (e.g.,
buffer, etc.) containing the drug or labeled compound to be
encapsulated (e.g., anticancer agent (e.g., oxaliplatin,
topoisomerase I inhibitor, vinca alkaloid, etc.)) to obtain the
drug:N.omega.PE:SuccN.omega.PE:neutral lipid mixture (Intermediate
2).
[0417] Where the drug is oxaliplatin (l-OHP), the concentration of
the oxaliplatin solution is about 8 mg/ml in an approximately 9%
sucrose solution. For example, the concentration of oxaliplatin in
the targeted liposome is about 0.8 mg/mL+/-10%.
[0418] C: Production of Drug:N.omega.PE:SuccN.omega.PE:Additional
Lipid Liposome (Intermediate 3)
[0419] The drug:N.omega.PE:SuccN.omega.PE:additional lipid mixture
(Intermediate 2) obtained in step B is then sonicated or stirred
followed by evaporation of the solvent to form the
drug:N.omega.PE:SuccN.omega.PE:additional lipid liposome
(Intermediate 3). Methods and conditions for performing sonication,
stirring and evaporation and means for accomplishing these steps
are well understood by the skilled artisan and are also further
described in the Examples. See for example, methods of production
by reverse phase vesicle (REV) methods, U.S. Pat. No. 4,235,871
(incorporated by reference in its entirety). General liposome
production methods such as simple hydration methods and ethanol
injection methods, known to the skilled artisan, can be also
used.
[0420] The drug:N.omega.PE:SuccN.omega.PE:additional lipid liposome
formed as described above is then extruded by size and the
drug:N.omega.PE:SuccN.omega.PE:additional lipid liposome is
isolated. Optionally, ultrafiltration can then be used to
concentrate the liposome solution.
[0421] Where the liposome contains l-OHP (drug), DMPC (additional
lipid/phospholipid (neutral)), cholesterol (CHOL, additional
lipid/neutral lipid), N-glutaryl-DOPE (NG-DOPE) and NHS-NG-DOPE, a
liposome with a mean diameter of about 0.2 micrometer (200 nm) can
be isolated. Similarly sized liposomes can also be obtained for
liposomes containing l-OHP (drug), DSPC (additional
lipid/phospholipid (neutral), cholesterol, N-glutaryl-DSPE
(NG-DSPE) and NHS-NG-DSPE. Exemplary target amounts of the lipid
components are, for example, about 40 mg/mL DMPC (an additional
lipid/phosphatidyl choline/phospholipid/neutral lipid), about 20
mg/mL CHOL (additional lipid/neutral lipid) and about 5 mg/mL
NG-DOPE (combined amount of NG-PE and NHS-NG-PE). An exemplary
ratio for the lipid components is 50:45:5 (additional lipid 1 (e.g.
a phosphatidyl choline)):additional lipid 2 (e.g., CHOL):NG-PE
(e.g., NG-DOPE+NHS-NG-DOPE).
[0422] D: Production of Drug:N.omega.PE:TF-N.omega.PE:Additional
Lipid Liposome (Targeted Liposome)
[0423] The drug:N.omega.PE:SuccN.omega.PE:additional lipid liposome
formed as described in step C can then be functionalized with the
targeting factor of choice to produce the
drug:N.omega.PE:TF-N.omega.PE:additional lipid liposome (also
referred to as the "targeted liposome").
[0424] Attachment of the targeting factor (TF) (e.g.,
functionalization of the intermediate liposome (Intermediate 3)
with targeting factor) is accomplished by covalently binding the
targeting factor to SuccN.omega.PE by reaction of the succinimidyl
moiety with the targeting factor. Through appropriate reaction
conditions, succinimidyl groups on the exposed surface of the
liposome (on the exterior of the lipid bilayer, where the drug or
labeled compound is encapsulated in the interior of the liposome)
can be covalently modified to form targeting factor-modified
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl
ethanolamines (TF-N.omega.PE). Attachment of the targeting factor
to the liposome results in the formation of the
drug:N.omega.PE:TF-N.omega.PE:additional lipid liposome (targeted
liposome).
[0425] More specifically, under appropriate conditions, the
succinyl carboxyl moiety of the SuccN.omega.PE as described herein
is functionalized. If the targeting factor has an amino group(s),
the amino group(s) on the targeting factor is reacted with the
succinyl carboxyl moiety and forms carboxylic acid amide bond.
Conditions appropriate for this reaction are further described
herein, including in the Examples, and will also be well understood
by the skilled artisan. The skilled artisan will also be able to
modify the reaction conditions to optimize conditions for
particular combinations of targeting factor and liposomes without
undue experimentation given the teaching herein.
[0426] Various targeting factors as described herein and known to
the skilled artisan can be obtained commercially or produced by
methods known to the artisan of ordinary skill
[0427] When, for example, transferrin is selected as the targeting
factor, transferrin is can be commercially obtained as purified
protein, for example, from Celliance Corp., GA, USA. Transferrin
can also be obtained using recombinant methods well understood in
the art (e.g., by using prokaryotic cell (E. coli, etc.), by using
eukaryotic cell (CHO, BHK, etc.), etc.). As is well understood,
transferrin can be obtained and used in the targeted liposomes in
either its apo or holo forms. Alternatively, targeted liposomes
incorporating apo-transferrin can be treated with ferric compounds,
such as ferric citrate, iron (III) chloride, etc., to produce
targeted liposomes incorporating holo-transferrin derivatized
liposome.
[0428] An exemplary amount of transferrin as targeting factor is,
for example, about 2 mg/mL transferrin. Such an amount would be
appropriate for an exemplary targeted liposome containing about 40
mg/mL DMPC, about 20 mg/mL CHOL and about 5 mg/mL NG-DOPE (combined
amount of NG-PE and NHS-NG-PE).
[0429] Upon functionalization of the liposome (intermediate 3) with
targeting factor as described above to obtain the targeted
liposomes, the resulting liposomes can optionally be further
purified using methods known to those of skill in the art,
including those purification methods described herein, particularly
those described in connection with step C, above.
[0430] Where the liposome contains l-OHP (drug), DMPC, cholesterol
(CHOL), N-glutaryl-DOPE (NG-DOPE) and Tf-NG-DOPE, a liposome with a
mean diameter of about 0.05 micrometer to about 0.2 micrometer
(about 50 nm to about 200 nm) can be isolated. Similarly sized
liposomes can also be obtained for liposomes containing l-OHP
(drug), DSPC, cholesterol, N-glutaryl-DSPE (NG-DSPE) and
NHS-NG-DSPE.
[0431] Production of the targeted liposomes by the above-described
method (referred to for convenience as production method A)
reproducibly produces targeted liposomes of high purity and
homogeneity. In particular, the targeted liposomes are
substantially free of the non-NHS starting materials (described
herein) and by-products (e.g., acylated urea compounds, etc.)
associated with the generation of SuccN.omega.PEs. In particular,
preparation and purification of Succ N.omega.PEs prior to liposome
formation yields liposomes (intermediate 3) and targeted liposomes
substantially free of carbodiimide starting materials (e.g., DCC,
EDC, etc.) used to functionalize N.omega.PEs to form
SuccN.omega.PEs. As mentioned previously, drugs and labeled
compounds, including targeted liposomes incorporating drugs or
labeled compounds, which are intended for administration to
individuals in the course of therapy or diagnosis, must necessarily
be of high quality.
[0432] Optionally, the lipid mixtures described in A (intermediate
1) can be treated with targeting factor to form a lipid mixture
containing TF-N.omega.PE. Further, this lipid mixture may be mixed
with aqueous solution to form a liposome-containing composition.
Finally, the liposome-containing composition may be treated to
produce a liposome formulation. Optionally, the aqueous solution
may include a drug or labeled compound.
[0433] Alternative Production Method (Method B)
[0434] Production method B is depicted schematically in FIG. 5.
[0435] A. Production of Drug: N.omega.PE:Additional Lipids
Liposome
[0436] The targeted liposomes as described herein can also be
produced by dissolving the additional lipids and N.omega.PE in a
suitable solvent (e.g., ethanol, t-BuOH, chloroform,
isopropylether, etc.), dispersing the resultant solution in an
aqueous solution optionally containing a drug or labeled compound,
and then performing ultrasonication or reverse phase vesicle of the
resultant dispersion to form a liposome (drug:N.omega.PE:neutral
lipids). The liposome solution may be concentrated by
ultrafiltration.
[0437] As a non-limiting example, the liposomes can be produced by
reverse phase vesicle (REV) method (U.S. Pat. No. 4,235,871,
incorporated by reference). Of course, general liposome composition
methods such as simple hydration methods and ethanol injection
methods can be also used.
[0438] In order to stably retain the N.omega.PE(s) in the lipid
bilayer, the N.omega.PE(s) can be prepared and purified, and then
N.omega.PE(s), together with the additional lipids (e.g.,
phospholipid(s), cholesterol, etc.) are used to prepare the
liposome according to methods known to the skilled artisan.
[0439] As a non-limiting example, additional lipids (e.g., one or
more phospholipid (e.g., DSPC, DMPC, etc.), and, optionally,
another additional lipid (e.g., cholesterol, etc.) and at least one
N.omega.PE are mixed together and dissolved in a suitable organic
solvent.
[0440] Where the additional lipids are a phospholipid and
cholesterol, the mixing ratio of the phospholipid and the
cholesterol may be, for example, about 1:1, for example, about
1.1:1, about 1.2:1, about 0.9:1 (e.g., DMPC and cholesterol, 50:45
(mol %). The content of the N.omega.PE(s) as a proportion of total
lipid content is, for example, 6% relative to the phospholipid.
Then, the resultant solution is mixed with a solution of
oxaliplatin in an aqueous buffer. The N.omega.PE can be about 0.8
mol % to about 12 mol % to total lipid content. For example, from
about 1 mol % to about 10 mol %, about 1 mol % to about 8 mol %,
about 1 mol % to about 6 mol %, about 1 mol % to about 5 mol %,
about 1 mol % to about 4 mol %, about 1 mol % to about 3 mol %,
about 1 mol % to about 2 mol %, about 2 mol % to about 12 mol %,
about 2 mol % to about 10 mol %, about 3 mol % to about 8 mol %,
about 1 mol %, about 2 mol %, about 3 mol %, about 4 mol %, about 5
mol %, about 6 mol %, about 8 mol %, about 10 mol %, or about 12
mol %.
[0441] The concentration of drug or labeled compound in solution
can be as described herein, and, in particular as above for
production method A. Similarly, the solution containing the drug or
labeled compound includes the solution components as described
herein.
[0442] Liposomes incorporating l-OHP (drug), DSPC, cholesterol and
N-glutaryl-DSPE (NG-DOPE) prepared by this method can be isolated
to provide an oxaliplatin-containing liposome (e.g., by
gel-filtration, by size exclusion chromatography, by
ultrafiltration, by ultracentrifugation, etc.) having a mean
diameter of about 0.2 .mu.m.
[0443] B. Production of Drug: SuccN.omega.PE:N.omega.PE:Additional
Lipids Liposome
[0444] Following step A, a portion of the N.omega.PE present in the
liposome prepared in step A (drug:N.omega.PE:additional lipids
liposome) is functionalized to yield a liposome incorporating
SuccN.omega.PE (i.e., drug: SuccN.omega.PE:N.omega.PE:additional
lipids liposome), which can later be modified to form
TF-N.omega.PE.
[0445] In order to form SuccN.omega.PE, the carboxyl group at a
terminus of the N.omega.PE is modified to form a succinimidyl
group. Such functionalization can be accomplished using the methods
described for the production of SuccN.omega.PE(s).
[0446] For example, a carbodiimmide (e.g., EDC, DCC, etc.) and
N-hydroxysulfosuccinimide (NHS) are reacted in the presence of the
liposome to yield drug:SuccN.omega.PE:N.omega.PE:additional lipids
liposome.
[0447] C. Production of Drug: TF-N.omega.PE:N.omega.PE:Additional
Lipids Liposome
[0448] After step B, the drug: SuccN.omega.PE:N.omega.PE:additional
lipids liposome prepared in step B is reacted with targeting factor
to form drug: TF-N.omega.PE:N.omega.PE:additional lipids liposome.
The methods and conditions for the reaction are as described for
production method A, step D.
[0449] The drug: TF-N.omega.PE:N.omega.PE:additional lipids
liposomes obtained by production method B can be purified and
concentrated using methods as described herein and known to the
skilled artisan.
[0450] Comparison of Production Methods
[0451] Production method A has several advantages over production
method B, though both can be used to obtain
drug:TF-N.omega.PE:N.omega.PE:additional lipids liposomes (targeted
liposomes, optionally containing either drug or labeled compound).
Most notably, the liposomes obtained by method A will be free or
substantially free of impurities (e.g., non-NHS starting materials
and/or by-products) related to the production of the
SuccN.omega.PEs. In particular, as noted previously, targeted
liposomes prepared by production method A will be free, or
substantially free, of e.g., carbodiimmides (e.g., EDC, DCC, etc.),
and acylated ureas. In certain embodiments, where SuccN.omega.PE is
not incorporated into the interior of the liposome, the liposome or
liposome-containing composition can also be free or substantially
free of NHS. Additionally, the larger a scale of reaction, the more
a preparation time. The time for production method A is
substantially shorter than production method B.
[0452] While purification of the drug:
TF-N.omega.PE:N.omega.PE:additional lipids liposomes prepared by
production method B will reduce the amount of such impurities, it
is more difficult to purify liposomes (e.g., the drug:
SuccN.omega.PE:N.omega.PE:additional lipids liposomes obtained in
step B of production method B) than lipids (e.g., SuccN.omega.PEs
prepared and purified prior to step A of production method A). As
some of the SuccN.omega.PE is likely oriented to the interior of
the liposomes (e.g., the succinimidyl ester functionality is on the
interior of the lipid bilayer and inaccessible to reaction with
targeting factor), it is likely that targeted liposomes prepared by
production method A might have some residual SuccN.omega.PE
incorporated therein.
[0453] An additional advantage of production method A is that the
relative content of TF-N.omega.PE vs. N.omega.PE in the final drug:
TF-N.omega.PE:N.omega.PE:additional lipids liposomes can be
controlled more accurately when production method A is used. The
relative amounts of these lipids are directly related to the
relative amounts of the SuccN.omega.PE and N.omega.PE used as
starting materials in step A of production method A. Thus, the
amount of TF-modified SuccN.omega.PE can also be controlled more
accurately.
[0454] When production method B is used, the relative amounts of
N.omega.PE vs. SuccN.omega.PE are dependent upon the reaction
efficiency of method B step B. This reaction is believed to go to
completion for about approximately 10% of the N.omega.PE present in
the liposome, but experimental variation would be expected from
batch to batch. The low reaction efficiency is likely due, in part,
to the steric hindrance of the pre-formed liposome. When
SuccN.omega.PE is formed from isolated N.omega.PE (lipid only),
there is far less steric hindrance and the reaction goes farther to
completion. Also, subsequent to formation of the SuccN.omega.PE (in
lipid only form), the resultant product can be purified from the
reaction mixture, thus removing unreacted N.omega.PE, carbodiimmide
and NHS, as well as other by-products that may form during the
reaction.
[0455] The liposomes formed by either method appear to be more
homogeneous (and therefore can be used to produce a more
reproducible drug/diagnostic product) than liposomes that
incorporate PEG or other hydrophilic polymers, such as those
described in the Background section of the present specification.
In general, when PEG or other hydrophilic polymers are used to
increase the circulation time of liposomes (e.g., to shield the
liposomes from uptake by the RES), their use results in liposomes
with a distribution of molecular size-due to the broad distribution
of the PEG or hydrophilic polymers themselves. This distribution
increases the difficulties associated with manufacture (e.g.,
reproducibility and/or purification) and may also increase the
variability in clinical efficacy. Targeted liposomes prepared by
either method A or B should be superior in these respects.
[0456] Additional Production Methods
[0457] Lipid mixtures and liposome-containing compositions (which
may be used to prepare liposomes) may also be prepared by
modification of production methods A and B. For example, in some
embodiments, lipid mixtures and liposome-containing compositions
incorporating additional lipid
component(s):N.omega.PE:TF-N.omega.PE or additional lipid
component(s):N.omega.PE:TF-N.omega.PE:drug/labeled compound (where
"additional lipid component(s)" refers to one or more phospholipids
(e.g., one or more neutral, one or more anionic, one or more
cationic phospholipids or combinations of two or more of the
foregoing), optionally additionally comprising one or more
additional lipids as described herein (e.g., cholesterol or a
derivative thereof); or at least two different neutral lipids as
described herein, (e.g., at least one phospholipid(s) (e.g. PCs
(e.g., DMPC, DSPC, etc.), PI, sphingomyelin, phosphatidic acid,
etc.) and at least one other neutral lipid (e.g., cholesterol)) may
be prepared by the production methods described below, as well as
other modifications of the methods envisioned by the skilled
artisan in view of the teaching of the present specification. The
additional lipids, N.omega.PE, TF-N.omega.PE and, where present,
drug or labeled compound components may be as described throughout
the present specification. Similarly, the relative amounts of the
components are also as described through the present
specification.
[0458] In certain embodiments, the lipid mixture produced in the
first step of production method B (the lipid mixture produced by
dissolving the additional lipids and N.omega.PE in a suitable
organic solvent) may be modified to incorporate NHS and then
modified with a TF to produce a additional lipid
component(s):N.omega.PE:TF-N.omega.PE lipid mixture. This lipid
mixture can then be mixed with aqueous solution (optionally
containing drug or labeled compound) to form a liposome-containing
composition. Alternatively, a drug or labeled compound can be
incorporated after the liposome-containing composition has been
prepared. In some embodiments, drug or labeled compound free of
aqueous solution can be incorporated in the lipid mixture, formed
after modification with NHS and TF, to form a additional lipid
component(s):N.omega.PE:TF-N.omega.PE:drug/labeled compound lipid
mixture. This lipid mixture can subsequently be mixed with an
aqueous solution to form a liposome-containing composition.
[0459] In some embodiments, the lipid mixture produced in the first
step of production method B (the lipid mixture produced by
dissolving the additional lipid component(s) and N.omega.PE in a
suitable solvent) may be mixed with aqueous solution to form a
liposome-containing composition (additional lipid
component(s):N.omega.PE). This liposome-containing composition may
then either be treated with NHS and TF and subsequently mixed with
drug or labeled compound to form a additional lipid
component(s):N.omega.PE:TF-N.omega.PE:drug/labeled compound
liposome-containing composition. Alternatively, the additional
lipid component(s):N.omega.PE liposome-containing composition may
be treated with drug or labeled compound and then modified with NHS
followed by TF.
[0460] In some embodiments, the lipid mixture produced in the first
step of production method B (the lipid mixture produced by
dissolving the additional lipid component(s) and N.omega.PE in a
suitable solvent) may then be mixed with drug or labeled compound
(optionally including aqueous solution) to form a lipid mixture
(where the drug or labeled compound does not include aqueous
solution) or liposome-containing composition. Where a lipid mixture
is formed, the lipid mixture can then be treated with NHS and TF to
form a additional lipid
component(s):N.omega.PE:TF-N.omega.PE:drug/labeled compound lipid
mixture, which can then be mixed with aqueous solution to form a
liposome-containing composition. Alternatively, where a
liposome-containing composition is formed (e.g., when the drug or
labeled compound is incorporated in aqueous solution), this
liposome-containing composition can subsequently be treated with
NHS and TF to also yield a additional lipid
component(s):N.omega.PE:TF-N.omega.PE:drug/labeled compound
liposome-containing composition.
[0461] In a further alternative production method, method C,
individual components are simultaneous mixed in organic solvent to
form a lipid mixture (C-1) (e.g., components: additional lipid
component(s); N.omega.PE; TF-N.omega.PE or components: additional
lipid component(s):N.omega.PE:TF-N.omega.PE:drug or labeled
compound), where the TF-N.omega.PE is prepared and optionally
purified prior to admixture. Lipid mixture C-1 can then be mixed
with aqueous solution to form a liposome-containing composition C-2
(additional lipid component(s):N.omega.PE:TF-N.omega.PE (optionally
containing drug or labeled compound). Where the liposome-containing
composition so formed does not contain drug or labeled compound,
the drug or labeled compound may be added after formation of the
liposome-containing composition (C2-A). Alternatively, where a drug
or labeled compound including an aqueous solution is used as an
initial starting component, the liposome-containing composition can
be formed simultaneously upon the mixing of all the starting
components.
[0462] C-2 (optionally containing drug or labeled compound) or
C2-A, may then be treated to form a liposome (C-3). Where C-3 does
not include a drug or labeled compound, the liposome will be an
blank liposome, as previously described (e.g., additional lipid
component(s):N.omega.PE:TF-N.omega.PE liposome). Where C-3 includes
a drug or labeled compound, the liposome will be a targeted
liposome as described herein. Where C-3 is an blank liposome, a
drug or labeled compound may, as previously described, be added to
the blank liposome in a subsequent step to form a targeted
liposome, which may be performed immediately after preparation of
C-3 or after a delay, which may include storage of the C-3 blank
liposome for a period of time.
[0463] In a further alternative production method, method D,
individual components are simultaneous mixed in organic solvent to
form a lipid mixture (D-1) (e.g., components: additional lipid
component(s); N.omega.PE; or components: additional lipid
component(s):N.omega.PE:drug or labeled compound). Lipid mixture
D-1 can then be mixed with aqueous solution to form a
liposome-containing composition D-2 (additional lipid
component(s):N.omega.PE:(optionally containing drug or labeled
compound). Liposome-containing composition D-2 can then be mixed
with TF-N.omega.PE to form a liposome-containing composition D-3
(additional lipid component(s):N.omega.PE:TF-N.omega.PE (optionally
containing drug or labeled compound)), where the TF-N.omega.PE is
prepared and optionally purified prior to admixture. Where the
liposome-containing composition so formed does not contain drug or
labeled compound, the drug or labeled compound may be added after
formation of the liposome-containing composition (D3-A).
Alternatively, where a drug or labeled compound including an
aqueous solution is used as an initial starting component, the
liposome-containing composition can be formed simultaneously upon
the mixing of all the components.
[0464] D-3 (optionally containing drug or labeled compound) or
D3-A, may then be treated to form a liposome (D-4). Where D-4 does
not include a drug or labeled compound, the liposome will be an
blank liposome, as previously described (e.g., additional lipid
component(s):N.omega.PE:TF-N.omega.PE liposome). Where D-4 includes
a drug or labeled compound, the liposome will be a targeted
liposome as described herein. Where D-4 is an blank liposome, a
drug or labeled compound may, as previously described, be added to
the blank liposome in a subsequent step to form a targeted
liposome, which may be performed immediately after preparation of
D-4 or after a delay, which may include storage of the D-4 blank
liposome for a period of time.
[0465] As with lipid mixtures, liposome-containing compositions,
and liposomes (including targeted liposomes, blank liposomes, etc.)
formed by production method A, the lipid-containing compositions
prepared by production method C or method D will be substantially
free of non-NHS starting material, byproduct and/or decomposition
product associated with synthesis of the succinimidyl ester of an
N-(.omega.)-dicarboxylic acid-derivatized phosphatidyl ethanolamine
(e.g., carbodiimmides (e.g., DCC, EDC, etc.), acylated urea
compounds, etc.), so long as the starting material (e.g., TF-NG-PE)
is substantially free of these substances prior to the initial step
of production method C or method D. Where SuccN.omega.PE is not
incorporated as a starting material (and therefore not incorporated
into the interior of the liposome), the liposome or
liposome-containing composition can also be free or substantially
free of NHS, as in when TF-N.omega.PE is pre-formed and used as
starting material. In particular embodiments, the lipid-containing
compositions prepared by production method C or method D are
substantially free of DCC and EDC. In certain embodiments, the
lipid-containing compositions prepared by production method C or
method D are substantially free of DCC.
[0466] In certain embodiments, the additional lipid component(s)
includes one or more phospholipid (e.g., a phosphatidyl choline,
etc.) and a cholesterol or cholesterol derivative. In particular
embodiments, the phosphatidyl choline is DMPC, POPC, DSPC, etc. as
herein described. In certain embodiments, the phosphatidyl choline
is DMPC or DSPC. In particular embodiments, the additional lipid(s)
are a phospholipid and cholesterol. In certain embodiments, the
phospholipid is a neutral phospholipid.
[0467] In certain embodiments, the additional lipid component(s)
include at least two different neutral lipids, which include a
phospholipid (e.g., a phosphatidyl choline, etc.) and a cholesterol
or cholesterol derivative. In particular embodiments, the
phosphatidyl choline is DMPC, POPC, DSPC, etc. as herein described.
In certain embodiments, the phosphatidyl choline is DMPC or DSPC.
In particular embodiments, the at least two different neutral
lipids are a phospholipid and cholesterol.
[0468] In some embodiments, the N.omega.PE is an NG-PE. In
particular embodiments, the N.omega.PE is N.omega.-DOPE or
N.omega.-DSPE. In certain embodiments, the N.omega.PE is NG-DOPE or
NG-DSPE.
[0469] In particular embodiments, the TF is for example,
asialoglycoprotein, folate, transferrin, etc. In certain
embodiments, the TF is transferrin (Tf). In some embodiments, the
TF-N.omega.PE is a Tf-N.omega.PE (e.g., Tf-NG-DOPE or
Tf-NG-DSPE).
[0470] In particular embodiments, the drug is, for example, an
anticancer agent (e.g., oxaliplatin, topoisomerase I inhibitor,
vinca alkaloid, etc.). In other embodiments, the lipid mixture or
liposome-containing composition includes a labeled compound. In
some embodiments, the lipid mixture or liposome-containing
composition does not include a labeled compound or drug.
[0471] As mentioned previously, each of the lipid mixtures may be
mixed with an aqueous solution to form liposome-containing
compositions and each of the liposome-containing compositions may
be treated to form the corresponding liposomes (e.g., targeted
liposomes (e.g., incorporating drug or labeled compound),
intermediate liposomes, blank liposomes, etc.), as described in
detail herein.
[0472] With respect to the variations of the production methods
described herein, it is intended that the modification of
N.omega.PE with NHS, the modification of NHS-N.omega.PE with TF,
the preparation of liposome-containing compositions from lipid
mixtures, and the preparation of liposomes from liposome-containing
compositions may be accomplished by the skilled artisan as
described herein without undue experimentation given the teaching
provided in the present specification, including, in particular,
the detailed description of production methods A and B and as
presented in the examples.
Pharmaceutical Formulations
[0473] In another aspect, the present invention provides
pharmaceutical formulations for treatment or diagnosis of
individuals in need thereof, comprising lipid-containing
compositions as described herein and one or more pharmaceutically
acceptable carriers, excipients, diluents, stabilizers,
preservatives, or other inactive ingredients, including
combinations of the foregoing, known to skilled artisans and
described further herein.
[0474] In certain embodiments of the pharmaceutical formulations,
the lipid-containing composition is a targeted liposome as
described herein. In other embodiments, the lipid-containing
composition is a liposome-containing composition. In some
embodiments, the lipid-containing composition is an blank liposome.
In certain embodiments, the composition comprises a drug. In other
embodiments, the composition comprises a labeled compound.
[0475] In certain embodiments, the carrier may include one or more
of sterile water, a buffer solution or saline, diluent, and
combinations thereof.
[0476] The pharmaceutical formulations may further comprise one or
more of different salts, sugars, proteins, starch, gelatin, plant
oils, polyethylene glycol and the like, including combinations of
two or more of the foregoing.
[0477] An additional aspect of the invention includes use of the
compositions and formulations thereof as described herein in the
manufacture of a medicament. Particularly, the manufacture of a
medicament for use in the treatment or diagnosis of conditions as
described herein. Further, the active compositions and formulations
thereof, variously described herein, are also intended for use in
the manufacture of a medicament for use in treatment or diagnosis
of the conditions and, in accordance with the methods, described
herein, unless otherwise noted.
Use of the Compositions
[0478] Administration
[0479] As noted previously, in one aspect is provided methods of
treatment or diagnosis of conditions as described herein using the
drug- or labeled compound-containing lipid-containing compositions
(e.g., targeted liposomes, drug-/labeled compound-containing
liposome-containing compositions) and pharmaceutical formulations
as described herein.
[0480] In one embodiment, the methods may be practiced as a
therapeutic approach towards the treatment of the conditions
described herein. Thus, in a specific embodiment, the
drug-containing lipid-containing compositions or pharmaceutical
formulations may be used to treat the conditions described herein
in individuals in need thereof, including humans. The methods
generally comprise administering to the individual an amount of a
composition, or formulation described herein, effective to treat
the condition.
[0481] In another embodiment, the methods may practiced as a
diagnostic approach towards the diagnosis of the conditions
described herein. Thus, in a specific embodiment, the labeled
compound-containing lipid-containing compositions or pharmaceutical
formulations may be used to diagnosis the conditions described
herein in individuals in need thereof, including humans. The
methods generally comprise administering to the individual an
amount of a composition, or formulation described herein, effective
to diagnosis the condition. Such administration is generally
undertaken in conjunction with methods to detect the condition.
[0482] In some embodiments, the individual is a mammal, including,
but not limited to, human, bovine, horse, feline, canine, rodent,
or primate. In other embodiments, the individual is a human.
[0483] The terms, "pharmaceutically effective amount" or
"therapeutically effective amount" refer to an amount of a
composition sufficient to treat a specified disorder, condition or
disease or one or more of its symptoms and/or to prevent the
occurrence of the disease or disorder. In reference to cancers, a
pharmaceutically or therapeutically effective amount comprises an
amount sufficient to, among other things, cause a tumor to shrink
or to decrease the growth rate of the tumor.
[0484] The terms "an amount effective to diagnose" or
"diagnostically effective amount" or "amounts effective for
diagnosis" cognates thereof, refer to an amount of a composition
sufficient to diagnose a specified disorder, condition or disease,
and/or one or more of its manifestations, where diagnosis includes
identification of the existence of the disease and/or detection of
the extent or severity of the disease. For example, in reference to
cancers, a "diagnostically effective amount" comprises an amount
sufficient to detect, for example, the presence and/or
concentration of one or more of malignant cells, tumor(s) or other
manifestation of the cancer. Often, diagnosis will be carried out
with reference to a baseline or background detection level observed
for individuals without the condition. Levels of detection above
background or baseline levels (elevated levels of detection) are
indicative of the presence and, in some cases, the severity of the
condition.
[0485] When used with respect to methods of treatment and the use
of drug-containing lipid-containing compositions, an individual "in
need thereof" may be an individual who has been diagnosed with or
previously treated for the condition to be treated. With respect to
methods of diagnosis and the use of labeled compound-containing
compositions, an individual "in need thereof" may be an individual
who is suspected to have a condition, is at risk for a condition
(e.g., a family history of the condition, life-style factors
indicative of risk for the condition (e.g., smoking as a risk
factor for lung cancer, etc.)) or has previously been diagnosed
with the condition (e.g., diagnosis can include monitoring of the
severity (e.g., progression/regression) of the disease over time
and/or in conjunction with therapy).
[0486] In certain embodiments, the condition to be treated or
diagnosed is cancer. In some embodiments the cancer may be a
gastric, colon, colorectal or breast cancer. In certain
embodiments, the cancer is a colon cancer. In other embodiments,
the cancer is a breast cancer. In still other embodiments, the
cancer is a gastric cancer. In some embodiments the cancer is
cancer of the pancreas, non small cell lung cancer, small cell lung
cancer, brain cancer, liver cancer, renal cancer, prostate cancer,
bladder cancer, ovarian cancer, or hematological malignancies
(e.g., leukemia, lymphoma, multiple myeloma, etc.).
[0487] The drug-containing compositions, including formulations
described herein, may be used alone or in conjunction with (e.g.,
prior to, concurrently with, or after) other modes of treatments
(e.g., adjunctive cancer therapy, combined modality treatments).
For example, in combination with other therapeutic agents (e.g.,
cancer chemotherapeutic agents as described herein and known to
those of skill in the art (e.g., alkylating agents, taxanes,
metabolic antagonist, antitumour antibiotic, plant alkaloids,
hormone therapy drug, molecular target drug, etc.)), surgery,
and/or radiation therapy. Where the condition being treated is
cancer, the compositions described herein can be administered in
conjunction with one or more of other anticancer agents or
cytotoxic compounds as described herein and as know in the art, one
or more additional agents to reduce the occurrence and/or severity
of adverse reactions and/or clinical manifestations thereof,
surgery (e.g., to remove a tumor or lymph nodes, etc.) or
radiation. Where one or more of surgery or radiation are part of
the treatment regimen, the compositions may be administered before,
concurrently, or after the radiation therapy or surgery. Likewise,
the compositions, and formulations thereof, as described herein may
be administered before, concurrently, or after the administration
of one or more anticancer agents. The targeted liposomes and
formulations thereof described herein may also be administered in
conjunction with (e.g., prior to, concurrently with, or after)
drugs to alleviate the symptoms associated with the condition or
the treatment regimen (e.g., drugs to reduce vomiting, hair loss,
immunosuppression, diarrhea, rash, sensory disturbance, anemia,
fatigue, stomatitis, hand foot syndrome, etc.). The targeted
liposomes may also be administered at more than one stage of
(including throughout) the treatment regimen (e.g., after surgery
and concurrently with and after radiation therapy, etc.).
[0488] The labeled compound-containing compositions, including
formulations described herein, may be used alone or in conjunction
with (e.g., prior to, concurrently with, or after) modes of
treatments (e.g., adjunctive cancer therapy, combined modality
treatments). For example, the compositions may be used to monitor
the progress of treatment. For example, to determined if the
condition being treated is detectable before, after or concurrently
with a treatment regimen (as described above with respect to
methods of treatment).
[0489] In certain embodiments, the compositions are administered
prior to or after surgery (e.g., removal of a tumor or lymph nodes,
etc.). In other embodiments, the compositions are administered
after surgery and prior to, concurrently with or after radiation
therapy. The optimal combination of one or more of surgery and/or
radiation therapy in conjunction with administration of the
compositions described herein, and, optionally, additional one or
more chemotherapeutic agents, can be determined by an attending
physician based on the individual and taking into consideration the
various factors effecting the particular individual, including
those described herein.
[0490] In particular embodiments, the drug-containing compositions
or pharmaceutical formulations may be administered in combination
with one or both of 5-fluorouracil and/or leucovorin. In other
embodiments, the drug-containing composition or pharmaceutical
formulations may be administered in combination with one or more
other anti cancer drugs such as capecitabine, UFT/LV
(tegafur-uracil and leucovorin), irinotecan, anti EGFR antibody
(e.g., cetuximab, etc.), anti VEGF antibody (e.g., avastin, etc.),
tyrosine kinase inhibitor (e.g., erlotinib), etc. Such
administration may also be combined with a treatment regimen
including radiation therapy and/or surgery. In certain embodiments,
the encapsulated drug in the targeted liposome is oxaliplatin.
[0491] In conjunction with the methods of use described herein, the
lipid-containing compositions or pharmaceutical formulations of the
present invention may be administered parenterally. Parenteral
administration may be accomplished via bolus injection (IV),
infusion (IV), intraperitoneal injection, or via local injection
(such as intracranial injection). In some embodiments, the
administration is via a bolus injection or continuous infusion.
[0492] Continuous intravenous infusion may be administered over a
period of minutes or hours. For example, but not limited to, from
about 10 minutes to about 5 hours, from about 15 minutes to about 4
hours; from about 30 minutes to about 4 hours; from about 45
minutes to about 4 hours, from about 60 minutes to about 4 hours,
from about 45 minutes to about 3 hours, from about 60 minutes to
about 2 hours, from about 90 minutes to about 3 hours, from about
90 minutes to about 2 hours, about 10 minutes, about 15 minutes,
about 20 minutes, about 30 minutes, about 45 minutes, about 50
minutes, about 60 minutes, 80 minutes, about 1.5 hours, about 2
hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4
hours, about 5 hours, about 12 hours, about 24 hours, about 36
hours, or about 48 hours.
[0493] Formulation and Dosage
[0494] As noted previously, the lipid-containing compositions and
pharmaceutical formulations as described herein may be administered
to individuals in need thereof for the treatment or diagnosis of
conditions as described herein in conjunction with the methods of
use described herein.
[0495] The lipid-containing compositions described herein, and, in
particular the targeted liposomes described herein, will generally
be used in an amount effective to achieve the intended result, for
example in an amount effective to treat or prevent the particular
condition being treated. The composition(s) may be administered
therapeutically to achieve therapeutic benefit. By therapeutic
benefit is meant eradication or amelioration of the underlying
disorder being treated and/or eradication or amelioration of one or
more of the symptoms associated with the underlying disorder such
that the patient reports an improvement in feeling or condition,
notwithstanding that the patient may still be afflicted with the
underlying disorder. Therapeutic benefit also includes halting or
slowing the progression of the disease, regardless of whether
improvement is realized.
[0496] In some embodiments, where the condition being treated is a
cancer, an effective amount is an amount sufficient to reduce tumor
growth (e.g., as measured by rate of increase of mean tumor volume
prior to and/or after treatment). In certain embodiments, an
effective amount is an amount sufficient to decrease mean tumor
volume (e.g., where mean tumor volume after treatment is reduced
compared to mean tumor volume prior to treatment).
[0497] The amount of compositions administered in order to
administer an effective amount of encapsulated drug (e.g.,
oxaliplatin) will depend upon a variety of factors, including, for
example, the particular condition being treated, the mode of
administration, the severity of the condition being treated and the
age and weight of the patient, the bioavailability of the
composition, the adverse effects experienced by the individual
being treated, etc. Determination of an effective dosage is well
within the capabilities of those skilled in the art in view of the
teachings provided herein.
[0498] In certain embodiments, the dose of encapsulated oxaliplatin
administered at a particular time point will be in the range from
about 1 to about 400 mg/m.sup.2/day. For example, in the range from
about 1 to about 350 mg/m.sup.2/day, 1 to about 300 mg/m.sup.2/day,
1 to about 250 mg/m.sup.2/day, 1 to about 200 mg/m.sup.2/day, 1 to
about 150 mg/m.sup.2/day, 1 to about 100 mg/m.sup.2/day, from about
5 to about 80 mg/m.sup.2/day, from about 5 to about 70
mg/m.sup.2/day, from about 5 to about 60 mg/m.sup.2/day, from about
5 to about 50 mg/m.sup.2/day, from about 5 to about 40
mg/m.sup.2/day, from about 5 to about 20 mg/m.sup.2/day, from about
10 to about 80 mg/m.sup.2/day, from about 10 to about 70
mg/m.sup.2/day, from about 10 to about 60 mg/m.sup.2/day, from
about 10 to about 50 mg/m.sup.2/day, from about 10 to about 40
mg/m.sup.2/day, from about 10 to about 20 mg/m.sup.2/day, from
about 20 to about 40 mg/m.sup.2/day, from about 20 to about 50
mg/m.sup.2/day, from about 20 to about 90 mg/m.sup.2/day, from
about 30 to about 80 mg/m.sup.2/day, from about 40 to about 90
mg/m.sup.2/day, from about 40 to about 100 mg/m.sup.2/day, from
about 80 to about 150 mg/m.sup.2/day, from about 80 to about 140
mg/m.sup.2/day, from about 80 to about 135 mg/m.sup.2/day, from
about 80 to about 130 mg/m.sup.2/day, from about 80 to about 120
mg/m.sup.2/day, from about 85 to about 140 mg/m.sup.2/day, from
about 85 to about 135 mg/m.sup.2/day, from about 85 to about 135
mg/m.sup.2/day, from about 85 to about 130 mg/m.sup.2/day, or from
about 85 to about 120 mg/m.sup.2/day. The does administered at a
particular time point may also be about 130 mg/m.sup.2/day, about
120 mg/m.sup.2/day, about 100 mg/m.sup.2/day, about 90
mg/m.sup.2/day, about 85 mg/m.sup.2/day, about 80 mg/m.sup.2/day,
about 70 mg/m.sup.2/day, about 60 mg/m.sup.2/day, about 50
mg/m.sup.2/day, about 40 mg/m.sup.2/day, about 30 mg/m.sup.2/day,
about 20 mg/m.sup.2/day, about 15 mg/m.sup.2/day, or about 10
mg/m.sup.2/day.
[0499] The dose administered may be higher or lower than the dose
ranges described herein, depending upon, among other factors, the
bioavailability of the composition, the tolerance of the individual
to adverse side effects, the mode of administration and various
factors discussed above. Dosage amount and interval may be adjusted
individually to provide plasma levels of the composition that are
sufficient to maintain therapeutic effect, according to the
judgment of the prescribing physician. Skilled artisans will be
able to optimize effective local dosages without undue
experimentation in view of the teaching provided herein.
[0500] Dosages may also be estimated using in vivo animal models,
as will be appreciated by those skill in the art.
[0501] Multiple doses (e.g., continuous or bolus) of the
compositions as described herein may also be administered to
individuals in need thereof of the course of hours, days, weeks, or
months. For example, but not limited to, daily, every other day,
every 10 days, weekly, monthly, twice weekly, three times a week,
twice monthly, three times a month, four times a month, five times
a month, every other month, every third month, every fourth month,
etc.
Kits
[0502] Also provided are kits for administration of the
compositions described herein, including pharmaceutical
formulations comprising the compositions.
[0503] In certain embodiments the kits may include a dosage amount
(e.g., as used for therapy or diagnosis) of at least one
lipid-containing composition, or pharmaceutical formulation
thereof, as disclosed herein. Kits may further comprise suitable
packaging and/or instructions for use of the composition. Kits may
also comprise a means for the delivery for the composition, or
pharmaceutical formulation thereof, such as a syringe for injection
or other device as described herein and known to those of skill in
the art.
[0504] In some embodiments the kits may include a dosage amount
(e.g., as used for therapy or diagnosis) of a blank liposome, or
pharmaceutical formulation thereof, as disclosed herein. Kits may
further comprise suitable packaging and/or instructions for use of
the composition. Kits may also comprise a means for the delivery
for the composition, or pharmaceutical formulation thereof, such as
a syringe for injection or other device as described herein and
known to those of skill in the art. Additionally, in certain
embodiments, the kit may contain a separate dosage amount of the
drug or labeled compound to be incorporated into the blank
liposome.
[0505] Additionally, the lipid-containing composition, or
pharmaceutical formulation thereof may be assembled in the form of
kits. The kit provides the lipid-containing composition, or
pharmaceutical formulation thereof and reagents to prepare a
composition for administration. The composition may be in a dry or
lyophilized form, or in a solution, particularly a sterile
solution. When the composition is in a dry form, the reagent may
comprise a pharmaceutically acceptable diluent for preparing a
liquid formulation. Such diluents include those known to those of
skill in the art, for example, sugar solutions, e.g., dextrose,
sucrose, etc. In certain embodiments the kits may includes sugar
solutions of about 1% to about 20%, about 1% to about 18%, about 1%
to about 15%, about 1% to about 10%, about 3% to about 10%, about
3% to about 6%, about 1%, about 2%, about 3%, about 4%, about 5%,
about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about
15%, about 18%, or about 20% sugar. In certain embodiments, the
solution may be a dextrose solution (e.g., about 1%, about 2%,
about 5% dextrose, etc.). In certain embodiments, the
lipid-containing composition may be, for example, a targeted
liposome, blank liposome, lipid mixture, or liposome-containing
composition (optionally containing drug or labeled compound).
[0506] The kit may also contain a device for administration or for
dispensing the compositions, including, but not limited to syringe,
pipette, or other device known to those of skill. When in a wet
form, the composition may be stored in an ampoule or other sterile
sealed container, including those known to persons of skill in the
art.
[0507] The kits may include other therapeutic compounds for use in
conjunction with the compounds described herein. In one embodiment,
the therapeutic agents are other anticancer agents. These agents
may be provided in a separate form, or mixed with the compounds of
the present invention, provided such mixing does not reduce the
effectiveness of either the additional therapeutic agent of the
compositions and formulations described herein. Similarly the kits
may include additional agents for adjunctive therapy. For example,
agents to reduce the adverse effects of the drug (e.g., anti-nausea
agents, anti-alopecia agents, immuno-enhancing agents, etc.).
[0508] The kits will include appropriate instructions for
preparation and administration of the composition, side effects of
the compositions, and any other relevant information. The
instructions may be in any suitable format, including, but not
limited to, printed matter, videotape, computer readable disk, or
optical disc.
[0509] In another aspect of the invention, kits for treating an
individual who suffers from or is susceptible to the conditions
described herein are provided, comprising a first container
comprising a dosage amount of a lipid-containing composition or
formulations thereof as disclosed herein, and instructions for use.
The container may be any of those known in the art and appropriate
for storage and delivery of intravenous formulations. In certain
embodiments the kit further comprises a second container comprising
a pharmaceutically acceptable carrier, diluent, adjuvant, etc. for
preparation of the composition to be administered to the
individual.
[0510] Kits may also be provided that contain sufficient dosages of
the compositions or formulations thereof as disclosed herein to
provide effective treatment for an individual for an extended
period, such as a week, 2 weeks, 3, weeks, 4 weeks, 6 weeks, 8
weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months,
9 months or more.
[0511] Kits may also include multiple doses of the lipid-containing
composition or formulations thereof and instructions for use and
packaged in quantities sufficient for storage and use in
pharmacies, for example, hospital pharmacies and compounding
pharmacies.
[0512] All patents, patent applications and publications referred
to herein are hereby incorporated herein by reference in their
entirety.
EXAMPLES
[0513] The present invention is further described with reference to
the following Examples; however, these Examples are not limiting
the scope of the present invention.
Example 1
Cytotoxicity Test for Oxaliplatin
[0514] An oxaliplatin (l-OHP) solution was prepared by dissolving
oxaliplatin in a 9% sucrose solution (sucrose/distilled water) at a
concentration of 8 mg/ml. The cell viability was determined using a
commercially available cytotoxicity assay kit (WST-1 kit, Wako Pure
Chemical Industries, Ltd., Japan).
[0515] AsPC-1 cells (provided by Dr. Hironobu Yanagie of Research
Center for Advanced Science and Technology, the University of
Tokyo, Japan) cultured in RPMI 1640 medium supplemented with 10%
FCS (fetal calf serum; SIGMA, USA) were treated with concentrations
of l-OHP solutions [200.times.(1/2).sup.(0-10) nM] at 37.degree. C.
in 5% CO.sub.2 for 48 hours. Then, the medium was removed and a
substrate (WST-1, Cell Counting Kit, Dojindo Laboratories, Japan)
was added to the cells which were incubated at 37.degree. C. in 5%
CO.sub.2 for 2 hours to develop the colored product. The developed
color was measured at an absorbance of 450 nm (reference
wavelength: 620 nm) on an Immuno Mini NJ-2300 (Cosmo Bio Co., Ltd.,
Japan).
[0516] The results are shown in FIG. 6. The cytotoxicity of l-OHP
was found to be LD.sub.50>8 .mu.g/ml.
Example 2
Determination of the Number of Transferrin Receptors on the Cell
Surface
[0517] Human normal leukocytes and human malignant tumor-derived
cell lines (K562, MKN45P and HL60) were used in the experiment and
obtained as follows: K562:TKG0210 (Cell Resource Center for
Biomedical Research, Institute of Department, Aging and Cancer,
Tohoku University, Japan); MKN45P: Dr. Hisae Iinuma, Teikyo
University School of Medicine, Japan; HL60:TKG0345 (Cell Resource
Center for Biomedical Research, Institute of Department, Aging and
Cancer, Tohoku University, Japan).
[0518] The number of Tf (transferrin) receptors on the cell surface
of each was determined by Scatchard analysis (Comp. Biochem.
Physiol., 116B, 137-160 (1949), using Microsoft Excel). A solution
of .sup.125I-labeled Tf (Na--.sup.125I (PerkinElmer Japan Co.,
Ltd., Japan) and h-Tf (T-4132, SIGMA, USA) were combined by iodogen
method (Biochem. Biophys. Res. Commun., 122, 319-325 (1984)) was
added to each cell culture at different concentrations ranging from
[300.times.(1/2).sup.(0-9) nM] at 4.degree. C. and incubated for 1
hour.
[0519] The concentration of .sup.125I-labeled Tf was determined by
protein quantification assay by the Lowry method (J. Biol. Chem.,
193, 265-270 (1951)) and the radioactivity was measured using a
gamma counter (Auto Well Gamma System ARC-300, Aloka Co., Ltd.,
Japan]. Briefly, the solution was centrifuged to precipitate the
cells, and the cell fraction was washed with an ice-cooled PBS
(180.times.g (gravity), for 3 min, which was repeated 3 times,
followed by the measurement of radioactivity with a gamma counter
to determine the concentration of Tf bound to the cell surface. The
number of cells was determined by protein quantification assay,
using the Lowry method (J. Biol. Chem., 193, 265-270 (1951)).
[0520] For each data point, the concentration of unbound Tf was
determined by subtracting the concentration of bound Tf from the
known concentration of Tf added. The Scatchard plot was drawn by
plotting the concentration of bound Tf on the horizontal axis and
the ratio of the concentration of bound Tf to the concentration of
unbound Tf on the vertical axis. The number of the bound Tf (i.e.,
the number of the receptors) was determined from the x intercept of
the graph, as described in Proc. Natl. Acad. Sci. USA, 80 2263-2266
(1983); J. Cell Physiol., 132, 492-500 (1987); Proc. Natl. Acad.
Sci. USA, 92 3318-3322 (1995); J. Pharm. Sci., 84, 216-220 (1995);
Eur. J. Biochem., 186, 367-373 (1989); J. Biol., Chem., 258,
4715-4724 (1983), which are hereby incorporated by reference in
their entirety.
[0521] The number of .sup.125I-Tf bound to the cell surface in the
different cell types are shown in FIG. 7. It was determined that
the number of transferrin (Tf) receptors on the cell surface of the
cell lines derived from human malignant tumors was significantly
higher than that in normal leukocytes.
Example 3
Preparation of NHS-NG-DOPE
[0522] A mass of 200 mg of NG-DOPE (Avanti Polar Lipids, Inc., USA)
(Cat. No. 870242, MW 880.13)) was weighed into a conical flask with
2 legs. To the flask was added 39.2 mg NHS (Sigma, USA, MW=115.09).
Next, 5 mL of chloroform/Ethyl acetate (1:1 (v/v), Wako Pure
Chemical Industries, Ltd., Japan) was added and swirled to begin
dissolving the NG-DOPE and NHS. Slight cloudiness observed.
[0523] Following initial mixing, a stir bar was added and the flask
set up (balloon filled with nitrogen gas) to blow nitrogen gas
gently into one leg of the flask and sealed with rubber stopper.
Stirring under nitrogen was accomplished using a stir bar and stir
plate. The second leg was sealed with a tube. The reaction was
performed at ambient temperature (20-23.degree. C.). The mixture
was stirred for 5-10 minutes. A 20 .mu.L sample of Lipid+NHS
reaction mixture was set aside for use as a TLC control.
[0524] In a separate flask, a solution of DCC (99%, Aldrich, USA,
MW: 206.33 g/mol) was prepared by dissolving 70 mg of DCC in 5 mL
ethyl acetate. The DCC dissolved quickly in the solvent to yield a
clear solution. The DCC solution so prepared (approximately 5 mL)
was then added dropwise to the lipid/NHS reaction mixture over
10-15 minute period. The reaction mixture became more cloudy upon
addition of DCC.
[0525] TLC was performed on the control (lipid/NHS) and on an
aliquot of lipid/NHS/DCC at time 0 for reference, as follows.
Sample was spotted 50 .mu.g (2.5 .mu.L of 20 mg/mL) on TLC plate
(aluminum sheet-silica gel 60F.sub.254 from EM Science (Gibbstown,
N.J., USA) Cat No. SP05554M), dried and then placed in the
developing chamber where solvent (70% chloroform, 28% methanol, 2%
water) was allowed to migrate. The solvent front was marked and
then the TLC plate was dipped in ammonium molybdate (5% ammonium
molybdate in 10% H.sub.2SO.sub.4) and dehydrated with dryer.
[0526] The lipid/NHS/DCC reaction mixture was stirred under
nitrogen flow and the formation of product was monitored (Rf
0.3-0.4) over time.
[0527] After 18 hours, conversion to NHS-NG-DOPE was not complete
conversion after 18 hours, and more NHS (26 mg in 2 mL ethyl
acetate) and DCC (47 mg in 1 mL ethyl acetate) were added. Reaction
progression was again tested by TLC at T=20 hr.
[0528] The reaction was allowed to proceed over the weekend at
ambient temperature with nitrogen flow and stirring (protected from
light). Some starting materials remained prior to purification.
[0529] Purification:
[0530] The reaction mixtures was chilled reaction on ice for
.about.30 minutes. The chilled reaction mixture was then filtered
through a Buckner funnel and then washed 3 times with 2.times.5 mL
chloroform. All of the liquid obtained was collected and dried by
rotary evaporation. A semi-solid paste was obtained after
evaporation. The paste was then resuspended in 2-3 mL
chloroform.
[0531] Silica gel for purification of the suspended paste was
prepared using silica (400 mesh) 4 g--hydrated in chloroform. The
silica gel was packed onto a 1 cm.times.28 cm column with stopcock.
The approximate size of the bed was 1 cm.times.14 cm. The column
was equilibrated with chloroform (gravity packed).
[0532] Sample was loaded onto the equilibrated (but not dried)
silica gel column. Added 10 mL chloroform to column (5.times.2 mL).
Collected 5.times.2 mL fractions. Flow rate was a function of
gravity, but 5.times.10 mL fractions collected in 10-20 min and
designated fractions 1-5.
[0533] Next, 50 mL chloroform/methanol (90/10, vol/vol) was added
to the column (5.times.10 mL). And 5.times.10 mL fractions were
collected and designated fractions 6-10.
[0534] After collection of fractions 6-10, a volume of 100 mL
chloroform/methanol (5/1 (v/v)) was added to the column
(10.times.10 mL). And additional 10.times.10 mL fractions were
collected and designated fractions 11-15.
[0535] Fractions 6-15 were assayed (5 .mu.L aliquot) by TLC as
described above.
[0536] Following TLC of fractions 6-15, fractions 7-11 were pooled
and dried to a thin film using rotary evaporation. The final
product obtained after evaporation was 130 mg (65% yield), as
determined by TLC against the unpurified reaction product and
comparison with a standard product (NHS-NG-DOPE) obtained from NOF
(Japan).
Example 4
Preparation of NHS-NG-DOPE
[0537] Pre-prepared and purified NG-DOPE (200 mg) (NOF Corporation
Japan) and NHS (N-hydroxysulfosuccinimide; 34 mg) were weighed and
placed in a 5 mL conical flask with 2 openings. One opening was
sealed with a rubber stopper and a stir bar was added through the
remaining opening.
[0538] The flask was then placed under vacuum and filled with
nitrogen gas flowing gently (repeat at three times). The flask was
then kept under nitrogen using a nitrogen balloon.
[0539] After placing under nitrogen, to the flask was added 2.5 mL
dry chloroform, was then stirred using the stir bar and stir plate.
The reaction was performed at ambient temperature for approximately
30 minutes and swirling was used to dissolve the starting
materials. A 20 .mu.L sample of Lipid+NHS was set aside for use in
the TLC control/monitoring of the reaction.
[0540] A solution of 61 mg of DCC (1,3-dicyclohexylcarbodiimide)
dissolved in 2.5 mL dry chloroform (clear, dissolved quickly) was
prepared then prepared. The DCC solution was added dropwise to the
lipid/NHS mixture over a 15 minute period. The solution turned
cloudy upon addition of DCC.
[0541] At time 0, a TLC (70% chloroform, 30% methanol, 5% water) on
lipid/NHS and lipid/NHS/DCC was performed to monitor the reaction
by spotting 50 mg of chloroform on the TLC plate, allowing to dry,
which was then placed in the reaction chamber (70% chloroform, 30%
methanol, 5% water) to migrate.
[0542] The reaction mixture continued with stirring under nitrogen
flow and the formation of product (Rf 0.3-0.4) was monitored over
time.
[0543] The reaction was allowed to proceed over a time period of
2-3 days at ambient temperature with nitrogen flow and
stirring.
[0544] The reaction mixture was then filtered through a Bruchner
funnel and washed twice with 2.times.5 mL chloroform. The entire
solution was collected and dried by rotary-evaporation. A
semi-solid paste was obtained.
[0545] The semi-solid paste was resuspended in 2.times.3 mL
chloroform, and then filtered and dried. This process was repeated
three times. Finally, after three times the product was resuspended
in 2.times.3 mL chloroform.
[0546] Column silica gel was prepared by mixing silica in
chloroform which was then packed on a 1 cm.times.28 cm column with
stopcock. The approximate size of the column bed was 1 cm.times.14
cm. The column was equilibrated with chloroform (gravity
packed).
[0547] Samples were loaded onto equilibrated (but not dried) silica
gel column. Then, was added 100 mL chloroform to column and
aliquots were collected in 100 mL fractions. The flow rate was a
function of gravity. (Fraction 1)
[0548] Fraction 1. Added 100 mL chloroform/methanol (90/10,
vol/vol) to column. Collected 100 mL fractions. (Fraction 2)
[0549] Added 200 mL chloroform/methanol (50/10, vol/vol) to column
(20.times.10 mL). Collected 20.times.10 mL fractions. (Fraction
3-23.)
[0550] Fractions 1 to 23 were assayed using 5 mL aliquots via
TLC.
[0551] Fractions 9 through 22 were pooled and dried to a thin film
using rotary-evaporation and lyophilization. The final weight of
NHS-NG-DOPE from these fractions was 61.9 mg (27.9% yield).
Example 5
Preparation of Lipid Mixture (NG-DOPE:Tf-NG-DOPE:DMPC:CH)
[0552] 583 mg of DMPC (NOF corporation, Japan), 299 mg of
cholesterol (Wako Pure Chemical Industries, Ltd., Japan) and 75.7
mg of NG-DOPE (NOF corporation, Japan) were mixed and dissolved in
t-BuOH (10 v/w vs. lipids (10 mL)) at 45-50.degree. C.
[0553] The resulting solution was poured into a vial and frozen for
about 8 hours on a shelf at -40.degree. C. It was depressurized to
about 0.1 mmHg and kept at reduced pressure for 2 days with rising
the temperature from -40.degree. C. to 25.degree. C. stepwise, from
which process a lyophilized lipid mixture was obtained.
[0554] A powder of the lyophilized lipid mixture as obtained above
was mixed with 20 mg of powdery Tf-NG-DOPE (as prepared in Example
29) and crashed. A homogeneous powder of lipid mixture was thus
obtained, with a lipid ratio of 50:45:5
(DMPC:Chol:NG-DOPE+Tf-NG-DOPE).
Example 6
Preparation of Liposome-Containing Compositions
[0555] Lipid mixtures were prepared according to the previous
examples with the components as detailed below:
[0556] Entry 1: DMPC/Chol/NG-DOPE (155 mg/79.4 mg/16.1 mg)
[0557] Entry 2: DMPC/Chol/NG-DOPE (155 mg/79.4 mg/16.1 mg)
[0558] Entry 3: DMPC/Chol/NG-DOPE/NHS-NG-DOPE (152 mg/77.9 mg/15.8
mg/4.38 mg)
[0559] Entry 4: DMPC/Chol/NG-DOPE/NHS-NG-DOPE (152 mg/77.9 mg/15.8
mg/4.38 mg)
[0560] Entry 5: DMPC/Chol/NG-DOPE/Tf-NG-DOPE (148 mg/76.0 mg/15.4
mg/4.8 mg)
[0561] Entry 6: DMPC/Chol/NG-DOPE/Tf-NG-DOPE (148 mg/76.0 mg/15.4
mg/4.8 mg)
[0562] Entries 1, 3 and 5 were each hydrated and stirred with 300
mM of aqueous sucrose solution (20 v/w vs. lipids (5 mL) (5 mL of
sucrose solution (20 v/w) was added to the dry lipid mixture and
stirred) for 30 min. at 40-45.degree. C. Entries 2, 4 and 6 were
each hydrated and stirred with an aqueous solution of l-OHP (8 mg
l-OHP/mL, 20 v/w vs. lipids (5 mL) in a 300 mM sucrose solution)
for 30 min. at 40-45.degree. C. A liposome-containing mixture was
thus obtained.
[0563] Liposome diameter was determined by QELS and the results are
shown in FIG. 8. The liposomes present in the liposome-containing
mixture have a mean diameter of 500-2,000 nm and have a broad
distribution of sizes around 100-10,000 nm.
Example 7
Preparation of Oxaliplatin-Containing Liposome
(NG-DOPE:Tf-NG-DOPE:DMPC:CH)
[0564] The composition of the liposome was as follows:
Dimyristoyl phosphatidylcholine
(1,2-dimyristoyl-sn-glycero-3-phosphocholine: DMPC) (NOF
Corporation, Japan)
Cholesterol (CH) (Solvay Pharmaceuticals B.V., Netherlands)
[0565] N-glutaryl-dioleoyl phosphatidyl ethanolamine
(N-glutaryl-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, sodium
salt: DOPE-CO--(CH.sub.2).sub.3--COOH; hereinafter represented by
NG-DOPE) (NOF Corporation, Japan) Succ-N-glutaryl-dioleoyl
phosphatidyl ethanolamine
(N-(succinimidyl-glutaryl)-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,
sodium salt: DOPE-CO--(CH.sub.2).sub.3--CO--OSu; hereinafter
represented by NHS-NG-DOPE) (NOF Corporation, Japan)
DMPC:CH:NG-DOPE:NHS-NG-DOPE=50:45:4:1 (m/m).
[0566] As the aqueous phase, an aqueous solution of l-OHP (8 mg/ml,
in a 300 mM sucrose solution) was used.
[0567] A mixture of DMPC, CH, NG-DOPE and NHS-NG-DOPE (at the mole
ratio of 50:45:4:1) was dissolved in 4 v/w (vs. total lipid weight)
of warm ethanol/t-butanol/water solvent. The lipid solution was
injected into 300 mM sucrose solution containing about 8 mg/ml
l-OHP at about 45.degree. C., so that the concentration of the
solvent became about 14% v/v.
[0568] The suspension was passed through an Extruder that was
lapped over five pieces of 100 nm filters (Cat. No. 112105, Whatman
plc, UK) under pressure of about 200-800 psi at about 45.degree. C.
The liposome was thus obtained, having an average diameter in the
vicinity of 100 nm. Liposome diameter was determined using
QELS.
[0569] 6 L of phosphate buffered saline (pH 7.9), 6 L of
transferrin (Cat. No. 4455, Selorogicals, GA, USA) solution (20
mg/ml), and 18 L of liposome suspension were mixed and stirred at
30.degree. C. for 15-60 min. This resulted in a reaction mixture
which contained 4 mg/ml of transferring and 20 mg/ml of lipids.
[0570] The quantitative analysis of transferrin was performed by
the bicinchoninic acid (BCA) assays according to instructions
provided by the vendor.
[0571] The increase of molecular weight after incorporation of
transferrin was investigated by SDS-PAGE (Sodium Dodecyl Sulfate
polyacrylamide gel electrophoresis). The analysis of NG-DOPE was
performed by high-performance liquid chromatography (HPLC) with
evaporative light scattering detector (ELSD2000, Alltech, MD, USA)
using a Silicagel column (YMC PVA Silica Column, 4.6.times.250 mm,
5 .mu.m).
Example 8
Preparation of Oxaliplatin-Containing Liposome
(NG-DSPE:Tf-NG-DSPE:DSPC:CH)
[0572] The composition of the liposome was as follows:
Distearoyl phosphatidylcholine
(1,2-distearoyl-sn-glycero-3-phosphocholine: DSPC) Cholesterol (CH)
N-glutaryl-distearoyl phosphatidyl ethanolamine
(N-glutaryl-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium
salt: DSPE-(CH.sub.2).sub.3--COOH; hereinafter represented by
NG-DSPE) DSPC:CH:NG-DSPE=2:1:0.2 (mol/mol).
[0573] As the aqueous phase, an aqueous solution of l-OHP (8 mg/ml,
in a 9% sucrose solution) was used, as described in Example 1.
[0574] A mixture of DSPC (MC8080, NOF, Japan), cholesterol
(038-03005, Wako Pure Chemical Industries, Ltd., Japan) and NG-DSPE
(Dr. Kazuo Maruyama, Teikyo University, Faculty of Pharmaceutical
Sciences, Japan) at the ratio of 2:1:0.2 (m/m) was dissolved in
chloroform and isopropyl ether.
[0575] To the resultant solution, a solution of l-OHP (in a 9%
sucrose solution) was added, and then the resulting mixture was
sonicated for about 15-30 minutes. The solution was then evaporated
by rotary evaporation at 60.degree. C. to remove the solvent and
the freeze/thawing was repeated five times. The suspension was
frozen (by being immersed in dry-ice/acetone bath) and thawed (by
being left to stand and immersed in warm water). This was repeated
five times.
[0576] Then, the resultant product was sized at 60.degree. C. using
EXTRUDER filters (twice at 400 nm and then five times at 100 nm),
(Lipex.TM. Extruder, Model No. T-001, Northern Lipids Inc., Canada)
and ultracentrifuged (200,000.times.g, 60 min, about 4.degree. C.).
The precipitate was resuspended in a 9% sucrose solution or MES
buffer (pH 5.5) (MES buffer. Cat. No. 345-01625, Dojindo
Laboratories, Japan) to obtain l-OHP-encapsulated NG-DSPE:DSPC:CH
liposome.
[0577] Subsequently, the l-OHP-encapsulated NG-DSPE:DSPC:CH
liposome was derivatized with transferrin (Tf). To the thus
obtained l-OHP-encapsulated NG-DSPE liposome,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC;
Cat. No. #22980, Pierce Biotechnology, Inc., USA) (in an amount of
2.7% relative to the weight of the lipid) and
N-hydroxysulfosuccineimide (S--NHS; 038-0432, Wako Pure Chemical
Industries, Ltd., Japan) (in an amount of 7.3% relative to the
weight of the lipid) were added, and the mixture was left at room
temperature for 10 minutes.
[0578] Then, to the resultant solution, transferrin (Tf) (in an
amount of 20% relative to the weight of the lipid; (Cat. No. T4132,
SIGMA, USA) was added and stirred at room temperature for 3 hours.
A 1 mM PBS (Phosphate buffered saline) solution of transferrin (Tf)
((in an amount of 20% relative to the volume of the total reaction
mixture) and 1 mM PBS were added (in an amount of 20% relative to
the volume of the total reaction mixture), and the resulting
solution stirred at room temperature for 1 hour.
[0579] To the thus obtained apo-form of Tf-NG-DSPE liposome, 10-40
eq. (vs. transferrin) of iron citrate-sodium citrate (Wako Pure
Chemical Industries, Ltd., Japan) was added to the suspension and
stirred at room temperature for 15 minutes. The resultant solution
was ultrafiltrated as above. The precipitate was then resuspended
in a 9% sucrose solution, whereby a holo-form of Tf-NG-DSPE
liposome was obtained. The solution was ultrafiltrated
(200,000.times.g, 60 min, about 4.degree. C.) and then the
precipitate was resuspended in a 9% sucrose solution.
[0580] The quantitative analysis of transferrin was performed by
the bicinchoninic acid (BCA) assays, performed in accordance with
the vendor's instruction (Cat. No. 23227, BCA.TM. Protein Assay
Kit, Pierce Biotechnology, Inc., USA).
[0581] The increase in molecular weight upon derivatization was
investigated by SDS-PAGE (Sodium Dodecyl Sulfate polyacrylamide gel
electrophoresis). The analysis of NG-DSPE was performed by
high-performance liquid chromatography (HPLC) with evaporative
light scattering detector (ELSD2000, Alltech, MD, USA) using a
Silicagel column (YMC PVA Silica Column, 4.6.times.250 mm, 5
.mu.m).
Example 9
Preparation of PEGylated Oxaliplatin-Containing Liposomes
[0582] Following the experimental protocol in Example 8,
DSPC:cholesterol:DSPE-PEG(2K)-OMe:DSPE-PEG(3.4K)-COOH liposomes
(Tf-PEG-liposomes) were prepared. In these liposomes the ratio of
components was as follows:
DSPC:cholesterol:DSPE-PEG(2K)-OMe:DSPE-PEG(3.4K)-COOH=2:1:0.16:0.03.
[0583] This liposome contained 6% by mole of PEG-lipid and 1% by
mole of PEG-COOH-lipid, and Tf is bound to the liposome through
PEG-COOH.
[0584] Also made by the method of Example 8 were Tf/PEG-DSPE
liposomes (Tf/PEG-NG-DSPE liposomes).
[0585] In these liposomes the ratio of components was as follows:
DSPC:cholesterol:DSPE-PEG(2K)-OMe:NG-DSPE=2:1:0.16:0.03.
[0586] This liposome is derivatized with PEG, and the Tf was bound
to the liposome through NG-DSPE. PEG-derivatized liposomes can also
be produced by the methods described in U.S. Pat. App. Pub. Nos.
2003/0224037 and 2004/0022842, the disclosures of which are hereby
incorporated in their entirety.
Example 10
Preparation of Blank Liposome
[0587] A mixture of DMPC, Chol (Wako Pure Chemical Industries,
Ltd., Japan), NG-DOPE (NOF Corporation, Japan) and NHS-NG-DOPE (NOF
Corporation, Japan) (at the mole ratio of 50:45:4:1; 410 g of DMPC,
211 g of Chol, 43 g of NG-DOPE and 12 g of NHS-NG-DOPE,
respectively) was dissolved in 4 v/w (vs. total lipid weight) of
warm ethanol/t-butanol/water solvent. The resulting suspension with
a volume of 20 L was incubated at 45.degree. C. with stirring and
passed through an Extruder (Stevested Machinery & Engineering
Ltd., Canada) that was lapped with five stacking of polycarbonate
100 nm filters (Cat. No. 112105, Whatman plc, UK) under pressure of
about 200-800 psi at about 45.degree. C. The liposomes were thus
obtained, having an average diameter in the vicinity of 100 nm.
Liposome diameter was determined by QELS.
[0588] The liposome suspension, PBS buffer (pH 7.9) and PBS
solution of transferrin (Cat. No. 4455, Selorogicals, GA, USA) (pH
7.0) were mixed at the ratio of 3:1:1 (v/v), then stirred for 15-60
min. at 30.degree. C. Approximately 6 L blank liposome was thus
obtained.
[0589] 20 g (about 19 mL) of liposome solution was poured into a
vial and frozen for about 8 hours on a shelf at -40.degree. C. It
was depressurized to about 0.1 mmHg and kept in the reduced
pressure for 2 days with rising temperature from -40.degree. C. to
25.degree. C. stepwise over the 2-day period. At the completion of
this process about 3.5 g of lyophilized blank liposome was thus
obtained. The liposomes were subsequently stored at 4.degree.
C.
Example 11
Encapsulation of Oxaliplatin in Pre-Prepared Blank Liposome
[0590] An aqueous solution of l-OHP (8 mg/mL, in a 300 mM sucrose
solution) was added to about 3.5 g of lyophilized blank liposome
and rehydrated by stirring for 2 hours at 40.degree. C. After
stirring, liposomal l-OHP was separated from free l-OHP by
fractionation using Sephadex G-25 (.phi.1.times.45 cm). The
liposome l-OHP and free l-OHP were monitored by VIS 600 nm and UV
210 nm respectively.
[0591] The amount of l-OHP and cholesterol was measured. The l-OHP
concentration was calculated for the case of condensing the
liposome fraction to the original cholesterol concentration
finally, the yield of l-OHP was measured by comparison of the l-OHP
concentration of liposome and a feeding concentration of l-OHP.
[0592] Total l-OHP concentration for the case of condensing the
liposome fraction to the original cholesterol concentration was 210
m/mL. And the yield of l-OHP was 2.6%.
[0593] This indicates 210 .mu.g/mL of l-OHP was encapsulated into
the lyophilized blank liposome.
Example 12
Comparison of Liposome Levels in Blood and Organs
[0594] A comparative study was carried out to evaluate the blood
retention and accumulation in organs of l-OHP-encapsulated
Tf-modified liposome compositions in tumor-bearing mice. Male
BALB/c mice aged 5 weeks were used as the animal models, and Colon
26 cells (derived from mouse colon cancer) were used as the tumor
cells. The cells were obtained from Laboratory of Biopharmaceutics,
Teikyo University School of Pharmaceutical Sciences, Japan.
[0595] Colon 26 cells (2.times.10.sup.6 cells) subcultured in vitro
were subcutaneously implanted into the dorsal region of the mice. A
mouse bearing a tumor with a diameter of about 8 to 10 mm (after 8
to 10 days growth on average) was used as the colon cancer-bearing
mouse. A solution of each of the liposomes prepared in Examples 8
and 9 or l-OHP (8 mg/ml in a 9% sucrose solution) was injected into
the tail vein. The concentration of oxaliplatin was adjusted at 5
mg l-OHP/kg body weight in each case. As the liposomes, Tf-NG-DSPE
liposome ((.box-solid.); Example 8), Tf/PEG-NG-DSPE liposome (();
Example 9) and Tf-PEG-DSPE liposome ((.diamond-solid.); Example 9)
were used.
[0596] Blood, plasma, liver, spleen, kidney, heart, lung and tumor
tissues were collected from 3 mice at each time point for each
group at 1, 3, 6, 24, 48 and 72 hours after the administration. The
Pt concentration in the blood, each organ and tumor tissues were
determined using atomic absorption (AA), and the l-OHP
concentration was calculated and reported as the ratio (%) to the
dose. The concentrations in the blood are shown in FIG. 9.
[0597] Tf-NG-DSPE liposome showed substantially the same blood
retention until 3 hours after the administration compared with
Tf-PEG-DSPE liposome and Tf/PEG-NG-DSPE liposome. However, after 6
hours, Tf-NG-DSPE liposome showed some blood retention, but it
disappeared more quickly from the blood compared with the PEG
liposomes. The concentrations in the tumor tissues are shown in
FIG. 10. Tf-NG-DSPE liposome showed substantially the same
accumulation to tumor tissues as Tf-PEG-DSPE liposome and
Tf/PEG-NG-DSPE liposome, despite being retained at a lower
concentration in the blood over time.
[0598] From the above results, it was found that about 6 hours of
retention time in the blood after administration is necessary and
sufficient deliver a sufficient concentration of a drug to tumor
tissue at a significant level or higher in mice. It is considered
that a retention time in the blood longer than this may increase
the possibility of causing an adverse effect on a normal
tissue.
Example 13
Preparation of Diagnostic Liposome and Accumulation of .sup.125I in
Tumor Tissue
[0599] Liposomes were prepared in the same manner as in Example 7
with the exception that [.sup.125I]-Tyraminyl inulin (in PBS
solution) replaced l-OHP and
DMPC/CH/NG-DOPE/Tf-NG-DOPE/[.sup.125I]-Tyraminyl inulin liposomes
were obtained. Lipid components were obtained also as described in
Example 7. Two liposome formulations were prepared, with the
components as shown below. The liposome lacking Tf-NG-DOPE served
as a control for non-targeted distribution of the liposome.
Targeted Liposome: DMPC/CH/NG-DOPE/Tf-NG-DOPE (63.3/31.7/4/1 (m/m))
Non-Targeted Liposome (control): DMPC/CH/NG-DOPE (63.3/31.7/5
(m/m))
[0600] .sup.125I was bound to tyraminyl inulin by combining
Na--.sup.125I (PerkinElmer Japan Co., Ltd., Japan) and tyraminyl
inulin (Dr. Kazuo Maruyama, Teikyo University, Faculty of
Pharmaceutical Sciences, Japan) using the iodogen method (Biochem.
Biophys. Res. Commun., 122, 319-325 (1984), incorporated by
reference in its entirety). .sup.125I-Tyraminyl inulin was thus
obtained. .sup.125I-Tyraminyl inulin/PBS(-) solution at a
concentration of about 1 mg/mL was then encapsulated into the
liposome as described in Example 7.
[0601] 100 .mu.l of each of the liposome solutions was injected
into the tail vein of murine colon cancer-bearing mice described in
Example 12. Tumor tissue and tail were collected from 5 mice at
each time point for each group at 1, 6, 24 and 48 hours after the
administration. The weight of the tumor tissue was measured and the
radioactivity (unit: cpm) in the tumor tissue and the tail was
measured using a gamma counter (Aloka Auto Gamma System ARC-300,
Japan). Results were evaluated as the distribution amount in the
tumor tissue (% of dose/g-tumor)=[(count value in the tumor
tissue)-(value of b.g.)].times.100/[(count value of Std.)-(count
value in the tail)]/(weight of tumor tissue (g)). The half-life
radioactive of .sup.125I is approximately 60 days.
[0602] The radioactivity of 100 .mu.l of the administered solution
(standard: Std.) was defined as 100% and the count value of an
empty test tube was defined as the value of the background (b.g.).
The results are shown in FIG. 11. As is apparent from FIG. 11
Tf-modified liposome shows high accumulation to a tumor tissue,
while non-targeted liposomes do not show a high accumulation. These
results demonstrate that a liposome encapsulating a radioactive
compound are useful for the detection of tumor tissue.
Example 14
Comparison of Antitumor Effects of Liposomes
[0603] A comparative study was carried out to evaluate the
antitumor effects on colon cancer Colon 26-bearing mice for
l-OHP-encapsulated Tf-modified liposome compositions
(Tf-PEG-liposomes prepared in Example 9, Tf-NG-DSPE:NG-DSPE:DSPC:CH
liposomes prepared in Example 8, Tf/PEG-NG-DSPE liposomes prepared
in Example 9; 9 mice in each group) and for each of the liposome
compositions to which transferrin is not bound ((-)TF; 6 mice in
each group).
[0604] The tumor-bearing mice were prepared in the same manner as
in Example 12. As the control, a solution of l-OHP (8 mg/ml in a 9%
sucrose solution) was used. The date when 1-OHP was administered at
doses of 5 mg/kg was defined as the start date, and on day 4, l-OHP
was administered at doses of 5 mg/kg again. The size of the tumor
on day 0 was defined as 1, and the size was shown as the ratio
based on this starting size. The size of the tumor was measured on
day 0, 2, 5, 7, 10, 13, 15, 18 and 21, and the survival days were
surveyed.
[0605] The results are shown in FIG. 12.
[0606] As can be seen in FIG. 12, the liposome compositions to
which transferrin is bound exhibited an inhibitory effect on tumor
growth. On the other hand, the liposome compositions to which
transferrin is not bound had a weaker inhibitory effect on tumor
growth compared with that of the liposome compositions to which
transferrin was bound. From the results depicted in FIGS. 9 and 10,
it was found that about 6 hours of retention time in the blood
after administration is necessary and sufficient for a liposome to
which transferrin is bound to have an inhibitory effect on tumor
growth and to make the concentration of a drug accumulating to a
tumor tissue be significant and substantially the same level. It is
considered that a retention time in the blood longer than this may
increase the possibility of causing an adverse effect on a normal
tissue.
Example 15
Optimization of NG-DSPE Content
[0607] In order to determine the optimal blending ratio of NG-DSPE
in a liposome, the blood retention of NG-DSPE in which an
anticancer agent was not encapsulated was investigated in normal
mice. Liposome compositions in which an anticancer agent was not
encapsulated were prepared in the same manner as in Example 8, but
using water instead of a solution of l-OHP as the aqueous phase,
with differing amounts of NG-DSPE.
[0608] The total molar amount of the total lipid components
constituting a liposome is defined as 100% and the contents of
NG-DSPE are shown as the ratio (% by mole) of NG-DSPE to the total
lipid components. In addition, a liposome containing 6% by mole of
MPB lipid (MPB-DSPE) or PDP lipid (PDP-DSPE) as the constituent
lipid was also prepared. MPB liposome is obtained by forming a
liposome by binding maleimide-phenylbutyrate (MPB) to the amino
group of the ethanolamine of the lipid, and binding Tf to the
liposome through MPB (870013(16:0), Avanti Polar Lipids, Inc, USA).
PDP (870205(16:0, Avanti Polar Lipids, Inc, USA) liposome is
obtained by forming a liposome by binding 2-pyridylthio propionate
(PDP) to the amino group of ethanolamine of the lipid, and binding
Tf to the liposome through PDP.
[0609] In the experiment, 105 mice (ICR male, 6 weeks of age)
(Tokyo Laboratory Animal Science Co., Ltd., Japan) were used. As
the tracer, .sup.125I was bound to tyraminyl-inulin, (prepared as
described in Example 13) and this inulin solution at a
concentration of about 1 mg/ml was encapsulated into the liposome.
The weight of the collected blood and organs for each case was
measured, and the radioactivity (unit: cpm) of the liposome marker
was measured using a gamma counter (Aloka Auto Gamma System
ARC-300, Japan). In addition, the radioactivity of each
administered solution (100 .mu.l) to the tail vein was measured.
The radioactivity of 100 .mu.l of the administered solution
(standard: Std.) was defined as 100%, and the value (% of dose) for
each organ was expressed as a percentage. The total blood amount
was estimated to be 7.3% of the body weight, and the liposome
amount in the blood was expressed as the amount in the total blood.
The count value of an empty test tube was defined as the value of
the background (b.g.), which was subtracted from the count value
for each sample.
Distribution amount in the blood (%)=[(count value of the
blood)-(value of b.g.)].times.(body weight of mouse
(g)).times.0.073.times.100/[(count value of Std.)-(count value of
tail).times.(weight of blood (g))].
[0610] The results are shown in FIG. 13. With regard to the
concentration in the blood after 6 hours, NG-DSPE liposome shows
high blood retention when the lipid content is 3% by mole or
higher. As for the maleimide-liposome (MPB 6%), the blood retention
was low.
Example 16
Effect of Tf and Dicarboxylic Acid on Blood Retention
[0611] In order to investigate the effects of the presence or
absence of the liposome-bound transferrin and the types of
dicarboxylic acids (e.g., glutaryl, succinyl, etc.), the blood
retention of a transferrin-bound liposome in which an anticancer
agent was not encapsulated was examined in normal mice. The
experimental method was the same as described in Example 15.
[0612] A liposome containing a phospholipid to which succinic acid
was bound instead of glutaric acid was prepared.
[0613] NG-DSPE (glutaric) was prepared as follows. In the dark
under a nitrogen gas stream, DSPE (ME-8080, NOF Corporation, Japan)
was suspended in dehydrated chloroform of 10 times the volume of
DSPE. Then, 1.3 equivalent amounts of triethylamine (208-02643,
Wako Pure Chemical Industries, Ltd., Japan) were added, and a
dehydrated chloroform solution of glutaric acid anhydrous (G0071,
Tokyo Chemical Industry, Japan) (dissolved in dehydrated chloroform
of the same volume as DSPE) was added dropwise at room temperature.
After completion, the solution was reacted at 30.degree. C. for 2
hours while stirring.
[0614] Then, the reaction solution was washed 3 times with an
acetate buffer (pH 4.5), and the organic layer was dehydrated with
magnesium sulfate and filtrated by suction filtration with a water
flow aspirator. Then, the filtrate was concentrated under reduced
pressure at 30.degree. C. When it became oily (about 2 times the
volume of DSPE), methanol was added to form crystals, and then
filtered. It was dissolved in chloroform again, and this procedure
was repeated twice. Then, the crystal was dried under reduced
pressure at room temperature, whereby a target product was obtained
as white crystal. NG-DSPE liposome was prepared by the same method
as in Example 8.
[0615] The results are shown in FIG. 14. The liposome to which
transferrin is bound through dicarboxylic acid (NG-DSPE:
N-glutaryl-distearoyl phosphatidyl ethanolamine, NS-DSPE:
N-succinyl-distearoyl phosphatidyl ethanolamine) shows high blood
retention. However, in the case of the liposome to which
transferrin is bound through an S--S bond by maleimide (MPB), the
blood retention was low even though the same ligand, transferrin,
was bound.
Example 17
Electrophoretic Analysis of Liposomes
[0616] As one example of analytical methods for characterizing the
liposomes, an example of electrophoresis is shown. Liposome was
dissolved and denatured at 95.degree. C. for 5 min in sample buffer
containing 2.5% of SDS and 5% of 2-mercaptoethanol. By using about
7.5% to 10% polyacrylamide gel (Funakoshi, Easy gel (II), precast
gel, Japan), 5 .mu.l of each sample was applied on the gel, and
electrophoresis was carried out under a constant current of 20 mA
for 1 to 2 hours.
[0617] After the electrophoresis, the gel was silver stained with a
silver staining kit (Wako Pure Chemical Industries, Silver Staining
II Kit Wako, Japan). The results are shown in FIG. 15 for the
following liposomes: lane 6 (transferrin-N-glutaryl-distearoyl
phosphatidyl ethanolamine-liposome (Tf-NG-DSPE liposome)); lane 5
(transferrin-polyethyleneglycol-distearoyl phosphatidyl
ethanolamine-liposome (Tf-PEG-DSPE liposome)) are shown. Lanes 1-4,
contain h-apo-Tf (240 ng), h-apo-Tf (120 ng), h-apo-Tf (60 ng), and
h-apo-Tf (30 ng), respectively.
[0618] In the case of the comparative example, Tf-PEG-DSPE
liposome, since polyethyleneglycol has some molecular weight
distribution, a complicated electrophoresis image with several
bands appeared. In the case of Tf-NG-DSPE liposome, a single band
appeared, which is much more easily analyzed and enhances the
ability to purify the liposome. These results indicate that, for
the liposome composition according to the present invention, an
analytical assay method is simpler than that for a PEG-derivatized
liposome composition.
Example 18
Effect of Free PE on Liposome Compositions
[0619] In order to investigate the effects of the presence of free
phosphatidyl ethanolamine (non-NG-PE) in a liposome, the binding
ability of Tf was measured for Tf-NG-DSPE liposome and a liposome
prepared by adding distearoyl phosphatidyl ethanolamine (DSPE) (no
NG present). Tf-NG-DSPE liposome was prepared from DSPC (64 parts),
CH (32 parts) and NG-DSPE (4 parts), and Tf-NG-DSPE+DSPE liposome
was prepared from DSPC (64 parts), CH (32 parts), NG-DSPE (4 parts)
and DSPE (10 parts) in the same manner as in Example 8.
[0620] Subsequently, Tf was bound to NG-DSPE by using 10 equivalent
amounts of NHS and ECD/HCl and 0.05 equivalent amounts of Tf. Then,
the liposome samples in an amount corresponding to 1 mg of lipid
were separated by SDS-PAGE, and the bands were visualized by silver
staining as described in Example 17.
[0621] The results are shown in FIG. 16. It was found that, in the
case of NG-DSPE+DSPE liposome to which 10% by mole of DSPE was
added, the bound amount of Tf was significantly low compared with
that of NG-DSPE liposome that contained no non-NG-DSPE. It is
likely that this is because the amino group of Tf and the amino
group of DSPE compete each other in the reaction where Tf is bound
to the carboxyl group of NG-DSPE.
Example 19
Comparison of Liposome Levels in Blood and Organs
[0622] Using the protocols described in Example 12, the levels of
NG-DOPE:Tf-NG-DOPE:DMPC:CH (Tf-NG-DOPE:NG-DOPE) liposomes (prepared
as in Example 7) and Tf-PEG-DSPE liposomes (prepared as in Example
9) in blood and tumor were compared. The results for the amount of
liposome retained in blood are depicted in FIG. 17 and the amount
of liposome detected in tumors is shown in FIG. 18.
[0623] The results in FIGS. 17 and 18 show that while the
Tf-NG-DOPE:NG-DOPE liposomes show a lower accumulation in the blood
(FIG. 17) than the Tf-PEG-DSPE liposomes, they were able to deliver
a greater amount of oxaliplatin to the tumor (FIG. 18). Lower
accumulation of liposomes in the blood is likely to reduce the
adverse systemic effects of the oxaliplatin.
Example 20
Comparison of Liposome Antitumor Effects in Colon 26 Tumor-Bearing
Mice
[0624] Using the protocols described in Example 14, the effect of
NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes (prepared as in Example 7) and
Tf-PEG-DSPE liposomes (prepared as in Example 9) on colon 26 tumors
in mice were compared. The results are depicted in FIG. 19.
[0625] As can be seen in FIG. 19, both liposomes show an inhibition
of tumor growth compared to oxaliplatin solution, however, as noted
in Example 19, the accumulation of less of the
NG-DOPE:Tf-NG-DOPE:DMPC:CH in blood (plasma) will likely mean that
these liposomes are better tolerated by the individuals to whom
they are administered.
Example 21
Liposome Antitumor Effects on Xenograft HCT-116 Colon Tumor
Model
[0626] The antitumor efficacy of the NG-DOPE:Tf-NG-DOPE:DMPC:CH
liposomes (prepared as in Example 7) when administered against
subcutaneously implanted HCT-116 human colon tumor xenografts. The
test was performed at Southern Research Institute, AL, USA] in male
athymic NCr-nu mice (02/A/08F17T9, Frederick Cancer Research and
Development Center, MD, USA; 50 mice) was studied, with oxaliplatin
in solution used as a reference compound. The antitumor activity of
NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes is summarized in FIG. 20.
[0627] NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes were administered
intravenously (i.v.) every four days for four injections
(q4d.times.4) as doses of 15 and 10 mg/kg/injection. Oxaliplatin
was administered on the same schedule at a dose of 15
mg/kg/injection. Vehicle (about 10.3% sucrose) and blank liposome
control groups were injected on the same schedule.
[0628] Mean tumor volume for the HCT-116 colon tumor model,
following treatment with NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes every
4 days was 28.9% of the control tumor volume for the 15 mg/kg group
and 35.9% of the control tumor volume for the 10 mg/kg group. The
antitumor activity of NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes was also
compared to non-liposomal oxaliplatin in the HCT-116 model, where
NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes showed greater efficacy in
terms of relative tumor volume, when administered at 15 mg/kg
(28.9% of the control tumor volume) every four days (4.times.).
Non-liposomal oxaliplatin delivered at 15 mg/kg every four days
(4.times.) yielded 39.3% of the control tumor volume.
Example 22
Liposome Antitumor Effects on Xenograft HT-29 Colon Tumor Model
[0629] The antitumor efficacy of NG-DOPE:Tf-NG-DOPE:DMPC:CH
liposomes (prepared as in Example 7) when administered against
subcutaneously implanted HT-29 human colon tumor xenografts. The
test was performed at Panapharm Laboratories Co., Ltd., Japan in
female athymic BALB/cA Jcl-nu mice (CLEA Japan, Inc., Japan; 50
mice) and the results are summarized in FIG. 21. Groups of 4 mice
were administered NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes at doses of
6.7, 10 or 15 mg/kg, or vehicle control. The vehicle and 6.7 and 10
mg/kg treated groups were injected on days 10, 14 and 19 and the 15
mg/kg treated group was injected on days 10 and 14.
[0630] The mean tumor volume for the HT-29 colon tumor model,
following treatment with NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes was
66.3% of the control tumor volume for the 6.7 mg/kg group and 39.5%
of the control tumor volume for the 10 mg/kg group (p value
0.01).
Example 23
Liposome Antitumor Effects on Xenograft MKN45 Gastric Tumor
Model
[0631] The antitumor efficacy of NG-DOPE:Tf-NG-DOPE:DMPC:CH
liposomes (prepared as in Example 7) when administered against
subcutaneously implanted MKN45 human gastric tumor xenografts. The
test was performed at Panapharm Laboratories Co., Ltd., Japan in
male athymic BALB/cA Jcl-nu mice (CLEA Japan, Inc., Japan; 50 mice)
was studied and is summarized FIG. 22.
[0632] Groups of 4 mice were administered
NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes at doses of 6.7, 10 or 15
mg/kg, or vehicle control. The vehicle and 6.7 and 10 mg/kg treated
groups were injected on days 7, 12 and 24 and the 15 mg/kg treated
group was injected on days 7 and 24. The mean tumor volume for the
MKN45 gastric tumor model following treatment with
NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes was 65.4% of the control tumor
volume for the 6.7 mg/kg group (p value .ltoreq.0.05), 49.6% of the
control tumor volume for the 10 mg/kg group (p value .ltoreq.0.01),
and 48.5% of the control tumor volume for the 15 mg/kg group (p
value .ltoreq.0.01; delivered every 17 days).
Example 24
Liposome Antitumor Effects on Xenograft COLO 205 Colon Tumor
Model
[0633] The antitumor efficacy of NG-DOPE:Tf-NG-DOPE:DMPC:CH
liposomes (prepared as in Example 7) when administered against
subcutaneously implanted COLO 205 human colon tumor xenografts The
test was performed at Southern Research Institute, AL, USA in male
athymic NCr-nu mice (01/A/09F3T8, Federic Cancer Research and
Development Center, MD, USA) was studied, with oxaliplatin in
solution used as a reference compound and is summarized in FIG.
23.
[0634] Into 40 mice NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes were
administered by i.v. injection every four days for three injections
(q4d.times.3) at doses of 10 and 5 mg/kg/injection. Oxaliplatin was
administered at a dose of 5 mg/kg/injection on the same schedule.
The control group was injected on the same schedule. Advanced-stage
tumors were retreated beginning on day 47 for all groups.
[0635] NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes were administered every
other day for two injections at doses of 15 and 10 mg/kg/injection
followed by treatment every other day for six injections at doses
of 4 and 2 mg/kg/injection, respectively. Oxaliplatin was
administered on the same schedule at doses of 10 and 2
mg/kg/injection. The control group was treated on the same
schedule.
[0636] The mean tumor volume for the COLO 205 colon tumor model
following treatment with NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes
administered initially at doses of 10 and 5 mg/kg, with subsequent
treatment of advanced stage, previously treated tumor with
NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes at doses of 15 and 10 mg/kg,
followed by NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes at doses of 4 and
2 mg/kg using various treatment schedules, was between 53.2% and
69.5% of the control tumor volume (p value 0.05 or 0.01).
Example 25
Encapsulation of Oxaliplatin in Targeted Liposomes
[0637] In order to measure the proportion of oxaliplatin
encapsulated in NG-DOPE:Tf-NG-DOPE:DMPC:CH (Tf-NG-DOPE:NG-DOPE)
liposomes prepared as in Example 7, the following procedure was
used.
[0638] The extent of encapsulation was determined by passing an
aliquot of the sample down a 3,000 MWCO (molecular weight cut-off)
spin column (30K MWCO cellulose ultrafilter membrane column, Cat.
No. 42410, Millipore Corp., USA) and measuring oxaliplatin
concentration in the eluent using HPLC with isocratic elution of 1%
acetonitrile in diluted phosphoric acid water solution (pH
3.0).
[0639] The level of oxaliplatin was determined following membrane
filtration using HPLC analysis to quantify the levels of
unencapsulated (free) drug. The trapping efficiencies of 3 batches,
prepared as in Example 7, were greater than 98% (see Table 1).
TABLE-US-00001 TABLE 1 The Ratio of Encapsulation of Oxaliplatin in
Lipsome Lot I II III % of encapsulation 98.8 99.6 99.1
Example 26
pH of Targeted Liposomes
[0640] The pH of targeted liposomes can be determined by place the
liposomes of the invention in distilled water and measuring with a
standard pH meter as described below.
[0641] The pH of NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes (prepared as
in Example 7) was determined by pH meter (VWR Model 8000), with an
Ag/AgCl gel-filled electrode. The pH values for 4 lots of liposome
ranged from pH 7.17-7.23, as shown in Table 2 below.
TABLE-US-00002 TABLE 2 pH of Liposome Lot 1 2 3 4 pH 7.17 7.17 7.23
7.20
[0642] The appearance of the liposomes at varying pHs are
summarized in Table 3. These results indicate that a low pH led to
aggregation, sedimentation, and precipitation, which may be due to
protonation of NG-DOPE and Tf followed by aggregation of the
bilayer and denaturation of transferrin.
TABLE-US-00003 TABLE 3 Condition of Liposome at Various pH Values
pH Observations 7.19 Liquid, translucent, light pink 6.98 Liquid,
translucent, no change as compared to no addition, light pink 6.83
Liquid, translucent, no change as compared to no addition, light
pink 6.37 Liquid, small white precipitates upon addition, clears
within a minute to as compared to no addition, light pink 5.53
Liquid, white precipitates upon addition, clears within a minute
but is slightly more cloudy, slightly white in color 5.07 Liquid,
small white precipitates, cloudy, white in color 4.33 Increased
viscosity, significant amount of white precipitates, very cloudy,
white in color 3.72 Very viscous, sample does not move when the
microcentrifuge tube is turned, white in color upside down, opaque,
white in color
Example 27
Identification of Conjugated Transferrin and SDS-PAGE Pattern of
Transferrin in Targeted Liposomes
[0643] This study was performed to confirm transferrin conjugation
to NG-DOPE in the NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes prepared as
in Example 7. When transferrin is conjugated to NG-DOPE, the
complex shows a higher molecular weight than non-conjugated
transferrin in this method.
[0644] Liposome was dissolved and denatured at 95.degree. C. for 5
min in sample buffer containing 2.5% of SDS and 5% of
2-mercaptoethanol. The samples were then applied to 5-10% gradient
polyacrylamide gel and then they were electrophoresed in the
presence of SDS. Migrated protein bands were visualized by using a
brilliant blue G-colloidal (B2025, SIGMA, USA).
[0645] The transferrin in the liposome was detected as transferrin
conjugated to NG-DOPE, which showed a higher molecular weight than
that of intact transferrin (see FIG. 24). A minor band with a lower
molecular weight was detected as free transferrin.
[0646] The ratio of free transferrin to transferrin conjugated to
NG-DOPE on the SDS-PAGE (FIG. 24) was calculated as the area of the
peak using Scion Image soft ware (freely available at
www.microsoft.com.DirectX). The ratio of free Tf in total Tf of
NG-DOPE:Tf-NG-DOPE:DMPC:CH liposome was approximately 4.7%.
Example 28
Analysis of Osmotic Pressure
[0647] The osmotic pressure at a given temperature depends on
sucrose and salts such as sodium chloride and phosphate buffer. It
does not depend on the solute, but on the total ion density and the
size of the molecules within the solution. Normally osmotic
pressure can be measured using an instrument known as an osmometer,
which measures osmotic pressure in suitable pressure units.
[0648] The osmotic pressure NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes
prepared as in Example 7 at room temperature was measured using an
osmometer (Vapro Vapor Pressure Osmometer Model 5520, Wescor, Inc.,
USA). The osmolarity values for 3 preparations of liposomes ranged
from 360-370 mOsm/kg, as reported in Table 4.
TABLE-US-00004 TABLE 4 Osmolarity Pressure Lot A B C Osmolarity 360
370 368 (mOsm/Kg)
Example 29
Isolation of Tf-NG-DOPE
[0649] 900 mL of EtOH was added to 100 mL of blank liposome
(DMPC/Chol/NG-DOPE/Tf-NG-DOPE) (as prepared in Example 10 prior to
lyophilization) and stirred fully. The mixture was then centrifuged
(9,000 rpm, 10 min, 20.degree. C.; CF16RX, Hitachi Koki Co., Ltd.,
Japan) and a pellet was obtained.
[0650] 100 mL of EtOH was then added to this pellet and stirred
fully. The mixture was centrifuged (9,000 rpm, 10 min, 20.degree.
C.; CF16RX, Hitachi Koki Co., Ltd., Japan) again and off-white
(light orange) pellet was obtained. This washing process was
repeated once more.
[0651] The obtained pellet above was dried with N.sub.2 gas for 30
min. The dried material was then dissolved in 10 mL of distilled
water and passed through a sterile filter (0.22 .mu.m) (Millipore
Corp., USA).
[0652] The filtrate was poured into a vial and frozen for about 8
hours on a shelf at -40.degree. C. The sample was depressurized to
about 0.1 mmHg and kept under reduced pressure for 2 days with
rising temperature from -40.degree. C. to 25.degree. C. stepwise.
Approximately 444 mg of Tf-NG-DOPE (about 45% of transferrin
content of the blank liposome was thus obtained.
Example 30
Preparation of Tf-NG-DSPE
[0653] 200 .mu.L of NHS (Wako Pure Chemical Industries, Ltd.,
Japan) aqueous solution (0.1 mol/L), 200 .mu.L of EDC
(N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride)
(Tokyo Chemical Industry Co., Ltd., Japan) aqueous solution (0.25
mol/L) and 1 mL of NG-DSPE solution (2 mmol/L) containing 2% (w/v)
of OG (n-octyl-D-glucopyranoside) (Wako Pure Chemical Industries,
Ltd., Japan) in 50 mmol/L MES buffer (pH 5.5) were mixed and
stirred for 10 minutes.
[0654] Surplus reagents were eliminated by Sephadex G-15 column
(1.5 cm.times.20 cm, 0.1% (w/v) of OG in 50 mmol/L HEPES buffer (pH
8.0), GE Healthcare Bio-Sciences Corp., USA) and fractioned to
about 1 mL/tube.
[0655] 5 mL of 1% transferrin (Sigma, USA) aqueous solution was
added dropwise to the fractions containing NG-DSPE and stirred
gently for 20 hours at 4.degree. C. Identification was by MS
determination of each fraction.
[0656] The reaction product was then fractioned to about 1.7
mL/tube by TOYOPEARL HW-55S column (1.5 cm.times.45 cm, 0.9% NaCl,
Tosoh Bioscience LLC, USA). Tf-NG-DSPE was estimated by mass
spectrometry (MALDI-TOF/MS) and SDS-PAGE with CBB (Coomasie
Brilliant Blue, Wako Pure Chemical Industries, Ltd., Japan)
staining.
* * * * *
References