U.S. patent application number 09/771151 was filed with the patent office on 2001-10-25 for liposomes containing an entrapped compound in supersaturated solution.
Invention is credited to Abra, Robert M., Barenholz, Yechezkel, Lasic, Danilo D., Peleg-Shulman, Tal.
Application Number | 20010033861 09/771151 |
Document ID | / |
Family ID | 22653355 |
Filed Date | 2001-10-25 |
United States Patent
Application |
20010033861 |
Kind Code |
A1 |
Lasic, Danilo D. ; et
al. |
October 25, 2001 |
Liposomes containing an entrapped compound in supersaturated
solution
Abstract
A liposome composition having a compound entrapped in a
supersaturated solution and method for preparing such a composition
are described.
Inventors: |
Lasic, Danilo D.; (Newark,
CA) ; Abra, Robert M.; (San Francisco, CA) ;
Barenholz, Yechezkel; (Jerusalem, IL) ;
Peleg-Shulman, Tal; (Givataim, IL) |
Correspondence
Address: |
ALZA Corporation
1900 Charleston Road, P.O. Box 7210
M10-3
Mountain View
CA
94039-7210
US
|
Family ID: |
22653355 |
Appl. No.: |
09/771151 |
Filed: |
January 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60178650 |
Jan 28, 2000 |
|
|
|
Current U.S.
Class: |
424/450 ;
264/4.1 |
Current CPC
Class: |
A61K 9/1271 20130101;
A61P 35/00 20180101; A61K 33/243 20190101; A61K 9/1277
20130101 |
Class at
Publication: |
424/450 ;
264/4.1 |
International
Class: |
A61K 009/127 |
Claims
It is claimed:
1. A method for preparing liposomes comprising: selecting a
compound having room temperature water solubility capable of
exhibiting at least a two-fold increase in response to a condition;
selecting liposomes of a size effective to inhibit precipitation of
the compound when entrapped in a liposome; and entrapping the
compound in the liposomes in a supersaturated state.
2. The method of claim 1, wherein selecting the compound includes
selecting a compound having an increased water solubility at room
temperature in response to a condition selected from the group
consisting of: (i) increasing solvent temperature, (ii) adding a
co-solvent, and (iii) changing solvent pH.
3. The method of claim 1, wherein selecting the liposomes includes
selecting liposomes that have a liposome size of between about 60
nm to about 1000 nm.
4. The method of claim 1, wherein selecting the liposomes includes
preparing liposomes having an entrapped compound at liposome size
intervals between about 60 nm to about 1000 nm and analyzing the
liposomes for the presence or absence of a precipitated
compound.
5. The method of claim 1, wherein selecting the liposomes includes
preparing liposomes having an entrapped compound at liposome size
intervals between about 60 nm to about 1000 nm and analyzing the
liposomes for the presence or absence of a precipitated
compound.
6. The method of claim 1, wherein the entrapping includes preparing
a solution of lipids.
7. The method of claim 6, wherein the preparing includes preparing
a solution of lipids that include a lipid derivatized with a
hydrophilic polymer.
8. The method of claim 6, wherein the preparing includes preparing
a solution of lipids effective to form a rigid lipid bilayer.
9. The method of claim 1, further including removing from an
external liposome suspension medium the condition selected to
maintain the drug above the room temperature solubility limit.
10. A composition according to claim 1, wherein the compound is
entrapped in a central compartment of the liposomes in a
supersaturated condition.
11. A liposome composition comprising: a suspension of liposomes
composed of a vesicle-forming lipid, and a compound entrapped in
the liposomes, wherein the compound prior to entrapment is
maintained in the liposomes in a supersaturated state.
12. The composition of claim 11, wherein the compound exhibits a
two-fold increase in aqueous solubility in response to a condition
selected from the group consisting of: (i) increasing solvent
temperature, (ii) adding a co-solvent, and (iii) changing solvent
pH.
13. The composition of claim 11, wherein the liposomes have a
liposome size of between about 60 nm to about 1000 nm.
14. The composition of claim 1, wherein the liposomes further
comprise a lipid derivatized with a hydrophilic polymer chain.
15. The composition of claim 1, wherein the liposomes comprise
saturated vesicle-forming phospholipids.
16. A method for preparing liposomes comprising: preparing an
aqueous concentrated solution of a compound suitable for entrapment
in an internal aqueous compartment of the liposomes; hydrating a
lipid film or lipid solution with a concentrated solution of the
compound to form liposomes; and sizing the liposomes to a size
effective to inhibit formation of precipitated compound, thereby
maintaining the entrapped compound in a supersaturated state.
17. The composition of claim 11, wherein selection of a liposome
size includes a liposome size effective to inhibit formation of
precipitated drug in an internal liposome compartment.
18. The composition of claim 14, wherein the hydrophilic polymer
chain is polyethylene glycol.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The complete disclosure set forth in the U.S. provisional
patent application entitled "Liposomes Containing an Entrapped
Compound in Supersaturated Solution", Ser. No. 60/178,650, filed
with the United States Patent and Trademark Office on Jan. 28,
2000, is incorporated herein. The applications are commonly
owned.
FIELD OF THE INVENTION
[0002] The present invention relates to a liposome composition and
method of preparing liposomes. The liposomes of the invention have
entrapped in the liposomes' aqueous core a drug in supersaturated
solution, which provides the liposomes a high drug-to-lipid
ratio.
BACKGROUND OF THE INVENTION
[0003] Liposomes are completely closed lipid bilayer membranes
containing an entrapped aqueous volume. Liposomes may be
unilamellar vesicles (possessing a single membrane bilayer) or
multilameller vesicles (onion-like structures characterized by
multiple membrane bilayers, each separated from the next by an
aqueous layer). The bilayer is composed of two lipid monolayers
having a hydrophobic "tail" region and a hydrophilic "head" region.
The structure of the membrane bilayer is such that the hydrophobic
(nonpolar) "tails" of the lipid monolayers orient toward the center
of the bilayer while the hydrophilic "heads" orient towards the
aqueous phase.
[0004] A primary use of liposomes is to serve as carriers for a
variety of materials, such as, drugs, cosmetics, diagnostic
reagents, bioactive compounds, and the like. Hydrophilic agents
when entrapped in liposomes associate with the aqueous spaces in
the liposome structure, primarily the internal central compartment
and the spaces between lipid bilayers. Lipophilic drugs are
typically associated with the lipid bilayer.
[0005] Liposome drug formulations can improve treatment efficiency,
provide prolonged drug release and therapeutic activity, increase
the therapeutic ratio and possibly reduce the overall amount of
drugs needed for treating a given kind of ailment or disorder. For
a review, see Liposomes as Drug Carriers by G. Gregoriadis, Wiley
& Sons, New-York (1988).
[0006] Many methods exist for preparing liposomes and loading
liposomes with therapeutic compounds. The simplest method of drug
loading is by passive entrapment, wherein a dried lipid film is
hydrated with an aqueous solution containing the water-soluble drug
to form liposomes. Other passive entrapment methods involve a
dehydration-rehydration method where preformed liposomes are added
to an aqueous solution of the drug and the mixture is dehydrated
either by lyophilization, evaporation, or by freeze-thaw processing
method involving repeated freezing and thawing of multilamellar
vesicles which improves the hydration and hence increases
loading.
[0007] In general, however, passive entrapment yields a low
efficiency of drug entrapment. A low drug-to-lipid ratio can be a
significant disadvantage to achieving efficaceous therapy with a
liposome formulation. For example, for treatment of tumor tissue,
liposomes with a size of approximately 80-140 nm are required for
extravasation into the tumor tissue. The small size imposes a
limitation on the drug loading, especially when the drug is
passively entrapped and/or when the drug has a limited solubility
and/or the drug has a low affinity for the lipids. A low
drug-to-lipid ratio requires administration of a large lipid dose
to achieve the required drug dose.
[0008] The drug entrapment efficiency can be improved in part by
using a high lipid concentration or by a specific combination of
lipid components. For example, an amphiphatic amine, such as
doxorubicin, may be encapsulated more efficiently into liposome
membranes containing negative charge (Cancer Res. 42:4734-4739,
(1982)). This surface electrostatic drug association with
liposomes, while very stable in the test tube, leads to very rapid,
almost immediate, release upon systemic administration.
[0009] Thus, loading of uncharged and non-lipophilic drugs, and in
particular drugs having low solubility in aqueous phase at a
sufficiently high drug-to-lipid ratio for efficaceous therapy
remains a problematic issue.
SUMMARY OF THE INVENTION
[0010] In one embodiment, the invention provides a liposome
containing composition having a hydrophilic compound entrapped in
the liposome at a high drug-to lipid ratio. The high drug-to lipid
ratio is achieved by virtue of the drug being entrapped in the
liposome at a supersaturated state.
[0011] In another embodiment of the invention a method for
preparing a liposome containing composition having a hydrophilic
compound entrapped in the liposome in a supersaturated state is
provided.
[0012] In another embodiment, the invention includes a method for
preparing liposomes having a compound entrapped in a supersaturated
state in the liposomes. The method includes: (i) selecting a
compound having a room temperature water solubility capable of at
least a two-fold increase in response to a selected condition; (ii)
selecting a liposome size effective to inhibit precipitation of the
compound when entrapped in a liposome; and (iii) encapsulating the
compound in the liposomes.
[0013] In yet another embodiment, a compound for use in the method
is capable of an increase in its water solubility at room
temperature. One increase in water solubility is in response to a
condition selected from the group consisting of (i) increasing
solvent temperature, (ii) adding a co-solvent and (iii) changing
solvent pH.
[0014] The liposomes of the invention are, in one embodiment,
between about 60 to about 1000 nm in diameter. In another
embodiment, liposome size is achieved by preparing liposomes having
an entrapped compound at selected size intervals of between about
60 to about 1000 nm. The prepared liposomes can be analyzed
microscopically, or in alternative methods, spectroscopically or
via scattering techniques, such as extended x-ray adsorption fine
structure, for the presence or absence of precipitated
compound.
[0015] In another embodiment, a selected compound is encapsulated
in liposomes by hydrating a solution of lipids with the drug
solution. In another embodiment, the selected compound is entrapped
by hydrating a dried lipid film with the concentrated drug
solution.
[0016] In additional embodiments the liposomes may further include
a lipid derivatized with a hydrophilic polymer, such as
polyethylene glycol (PEG). In another embodiment, the lipids
selected to form the lipid bilayer are effective to form a rigid
lipid bilayer.
[0017] In another embodiment the method of the invention includes
removing from an external liposome suspension medium the condition
selected to maintain the drug above the room temperature solubility
limit.
[0018] In another aspect, the invention includes a liposome
prepared according to the method described above, where the
compound is entrapped in the liposomes' central compartment in a
supersaturated condition. In this aspect, the liposome composition
contains a suspension of liposomes composed of a vesicle-forming
lipid and a therapeutic compound entrapped in the liposomes,
wherein one compound is maintained in the liposomes in a
supersatured state via selection of a liposome size effective to
inhibit formation of precipitated drug in the liposomes' central
compartment.
[0019] The compound selected for use in a liposome is in one
embodiment capable of at least a two fold increase in aqueous
solubility. This increase in aqueous solubility is in response to a
condition selected from the group consisting of: (i) increasing
solvent temperature, (ii) adding a co-solvent and (iii) changing
solvent pH.
[0020] In yet another embodiment of the invention, a method for
preparing liposomes having a compound entrapped in the internal
aqueous compartment in a supersaturated state is described. The
method includes: (i) preparing an aqueous solution of the compound
where the compound is present in the solution at a concentration
above its solubility in the solution at room temperature; (ii)
hydrating a lipid film or lipid solution with the concentrated drug
solution to form liposomes; and (iii) sizing the liposomes to
inhibit formation of precipitated compound, thereby maintaining the
entrapped compound in a supersaturated solution.
[0021] In another embodiment, a selected compound is prepared such
that the compound is present in an aqueous solution at a
concentration above its solubility in the solution at a temperature
of about 37.degree. C.
[0022] These and other embodiments of the invention will be more
fully appreciated in view of the following Detailed Description of
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1A-1B are .sup.195Pt NMR spectra of
cis-Pt(.sup.15NH.sub.3).s- ub.2Cl.sub.2 with broad-band decoupling
(FIG. 1A) and without broad-band decoupling (FIG. 1B);
[0024] FIG. 2 is a .sup.195Pt NMR spectrum of cisplatin
encapsulated in liposomes;
[0025] FIGS. 3A-3B are .sup.31P NMR spectra of cisplatin-containing
liposomes (FIG. 3A) and of control, placebo liposomes (FIG.
3B);
[0026] FIGS. 4A-4B are spectrum obtained by heteronuclear single
quantum coherence spectroscopy of
cis-Pt(.sup.15NH.sub.3).sub.2Cl.sub.2 in water (FIG. 4A) and an
enlargement of region I(b( (FIG. 4B);
[0027] FIG. 5 is a spectrum obtained by heteronuclear single
quantum coherence spectroscopy of
cis-Pt(.sup.15NH.sub.3).sub.2Cl.sub.2 following encapsulation in
liposomes; the peak denoted by "x" is unidentified;
[0028] FIG. 6 is a spectrum obtained by heteronuclear single
quantum coherence spectroscopy of
cis-Pt(.sup.15NH.sub.3).sub.2Cl.sub.2 following encapsulation in
the presence of a histidine buffer where peaks 1-7 are attributed
to cis-Pt(.sup.15NH.sub.3).sub.2Cl.sub.2-histine species;
[0029] FIG. 7 is a spectrum obtained by heteronuclear single
quantum coherence spectroscopy of cisplatin following incubation
with N-acetyl-histidine;
[0030] FIG. 8 is a k.sup.2-weighted Pt L3-edge extended x-ray
absorption fine structure (EXAFS) spectra of crystalline cisplatin,
dissolved cisplatin and three liposome-encapsulated cisplatin
samples (liquid, frozen and freeze-dried). The spectra are shifted
vertically for clarity and the best of the data (represented by the
dots) is represented by the solid line;
[0031] FIG. 9 is the k.sup.2-weighted Fourier transform of the
EXAFS spectrum of crystalline cisplatin, where the solid line
represents the experimental data and the best fit model function is
represented by the dashed line;
[0032] FIG. 10 is the k.sup.2-weighted Fourier transform of the
EXAFS spectrum of cisplatin dissolved in water; with the same
notation as in FIG. 9;
[0033] FIG. 11 is the k.sup.2-weighted Fourier transform of the
EXAFS spectrum of liquid sample of liposome-entrapped cisplatin;
with the same notation as in FIG. 9;
[0034] FIG. 12 is the k.sup.2-weighted Fourier transform of the
EXAFS spectrum of frozen sample of liposome-entrapped cisplatin;
with the same notation as in FIG. 9; and
[0035] FIG. 13 is the k.sup.2-weighted Fourier transform of the
EXAFS spectrum of freeze-dried sample of liposome-entrapped
cisplatin; with the same notation as in FIG. 9.
DETAILED DESCRIPTION OF THE INVENTION
I. Preparation of Liposomes
[0036] In one aspect, the invention includes a method for preparing
a liposome composition having a compound entrapped in the liposomes
in a supersaturated condition. As used herein "supersaturated"
includes a condition wherein a solution contains more solute than
is normally necessary to achieve saturation under the same
conditions. That is, the solution holds more of a dissolved solute
than is required to produce equilibrium with its undissolved
solute.
[0037] In another aspect, liposomes are formed by selecting a
compound to be entrapped and preparing a solution having a
concentration of the compound that is above the saturation
concentration of the compound in water at room temperature. A
solution containing the compound and having a concentration above
the saturation concentration in water at room temperature can be
prepared by any one of a number of methods. For example, by heating
the solution to increase the solubility of the compound in the
solvent, by adding a co-solvent to the aqueous solvent or by
changing the pH of the aqueous solvent. This solution is referred
to herein as "the concentrated solution." The procedure used to
prepare the concentrated solution is, in a preferred embodiment,
effective to increase the solubility of the compound in the aqueous
solvent by about two-fold, preferably about three-fold, more
preferably about four-fold, over the solubility of the compound in
the aqueous solvent at room temperature.
[0038] Accordingly, compounds contemplated for use in the
compositions and methods of the invention include compounds capable
of at least about a two-fold, preferably at least about three-fold,
more preferably at least about a four-fold increase in room
temperature (20-25.degree. C.) aqueous solubility via: (i)
increasing solvent temperature, (ii) addition of a co-solvent, or
by (iii) changing solvent pH. Preferred compounds are those with
limited solubility in water at room temperature that undergo a
substantial increase in solubility with increase in temperature. In
the studies performed in support of the invention, cisplatin was
used as the model compound. Cisplatin has a solubility at room
temperature in water or saline of 1-2 mg/mL. At about 60-65.degree.
C., the solubility increases to about 8-10 mg/mL. Thus, temperature
was used as the means to prepare a concentrated solution of the
drug for preparation of liposomes.
[0039] Other compounds contemplated for use in the invention
include therapeutic drugs such as acyclovir, gancyclovir, taxol,
carboplatin and other platinum compounds and anthraquinones, such
as doxorubicin, epirubicin, daunorubicin and idarubicin, as well as
poorly water-soluble antibiotics, proteins and peptides.
[0040] Continuing the description of a method for preparing lipid
vesicles, in a separate container, lipids can be selected to
provide a desired lipid vesicle composition and dissolved in a
suitable solvent. Lipids suitable for formation of lipid vesicles
are known to those of skill in the art. See, for example, U.S. Pat.
Nos. 5,013,556 at Col. 9, lines 34-62, and 5,891,468 Col. 7 line 5
bridging to Col. 8, line 48. Lipid components used in forming the
liposomes are selected from a variety of vesicle-forming lipids,
and are typically phospholipids and sterols. The lipids can be
selected to achieve a more fluidic lipid bilayer via selection of
lipids with acyl chains that are relatively unsaturated, thereby to
achieve a lipid bilayer having a relatively higher permeability to
entrapped compound than a more rigid bilayer. More rigid lipid
bilayers are formed using highly saturated phospholipids in order
to form lipid bilayers with a lower permeability of entrapped
compound. In another embodiment of the invention, liposomes that
contain a lipid derivatized with a hydrophilic polymer chain, such
as polyethylene glycol or other polymers, are described, for
example, in U.S. Pat. Nos. 5,013,556 and 5,631,018.
[0041] The composition of the lipid bilayer is selectively varied
according to the drug selected for entrapment. Drugs having a
higher permeability in lipid bilayers are better retained in the
liposomes by forming a lipid bilayer with saturated or lipid
rigidifying components. Drugs that are not readily permeable
through lipid components in lipid bilayers can be entrapped in
liposomes formed with fluidic lipid components.
[0042] The selected lipid components, as described above, can be
dissolved in a suitable solvent. The lipids are hydrated with the
concentrated solution of drug to form liposomes. In detailed
studies described throughout the specification, a concentrated
solution of cisplatin was used to hydrate an ethanolic solution of
lipids, (Example 1). It will be appreciated by those of skill in
the art that the concentrated drug solution can also be used to
hydrate a dried lipid film, where the solvent solubilizing the
lipids is removed to leave a dried lipid film for hydration (see
for example, U.S. Pat. No. 5,013,556 Col. 10, lines 60-65. After
hydration, the lipid-drug mixture is mixed for a time sufficient
for liposome formation.
[0043] The liposomes are sized to a specific size via one of
several methods known in the art, such as sonication,
homogenization or extrusion. Typically, a liposome size is selected
to inhibit formation of precipitate in the liposomes' central
compartments when conditions of the liposome composition are
altered to reduce the solubility of the drug in the concentrated
solution. Such a size is determined by preparing several liposome
compositions having different sizes, altering a condition that
maintains the increased drug concentration and analyzing the
liposomes for the presence of drug precipitate. As used herein,
"precipitate" or "precipitation" is intended to include a drug in
any form, such as a crystalline form, semi-solid form or an
amorphous form drug. Liposomes having a size such that no drug
precipitate is present are within the scope of the invention.
[0044] Typically, liposomes having a size of between about 60-1,000
nm are suitable, preferably a size of between about 70-500 nm.
Analysis of the liposomes for the presence of drug precipitate can
be performed, for example, by visualization via microscopy
(electron microscopy or high resolution cryo-electron microscopy)
or by various spectroscopic techniques, including for example, IR,
Raman, NMR or by scattering techniques such as Extended x-ray
absorption fine structure (EXAFS).
[0045] The liposomes are typically sized to a size selected to
inhibit formation of precipitated drug. This typically occurs when
the condition which maintains the drug at a concentration above the
solubility of the drug in water at room temperature is altered in
the exterior phase of the liposome suspension. For example, if an
increase in temperature was used to increase the solubility of the
drug for preparation of the concentrated drug solution, the
temperature of the liposome suspension is lowered. If an increase
or decrease in pH was used to increase the drug solubility, the pH
is adjusted to the final desired pH. If a co-solvent was added to
the concentrated drug solution to increase the drug solubility, the
co-solvent is removed from the external liposome phase via a
selected method, such as diafiltration, density gradient
centrifugation, dialysis, distillation, vacuum removal, and the
like.
II. Characterization of the Liposomes
[0046] As described above, the invention provides efficient and
stable encapsulation of drugs having limited water solubility at
room temperature. Such compounds are known to be difficult to
entrap in liposomes in a drug-to-lipid ratio useful for therapy. In
the studies performed in support of the invention, the anti-cancer
agent cisplatin was used as an exemplary drug. It should be
appreciated, however, that the composition and method of the
invention are contemplated for use with a variety of compounds,
which have limited water solubility at room temperature, and other
exemplary drugs are described above.
[0047] Cisplatin (cis-diamminedichloroplatinum(II)), is a heavy
metal complex containing a central atom of platinum surrounded by
two chloride atoms and two ammonia molecules in the cis position.
Cisplatin is widely used for treating a variety of solid tumors,
including testicular, head and neck, and lung tumors. Like other
cancer chemotherapeutic agents, cisplatin is a highly toxic drug.
The main disadvantages of cisplatin are its extreme nephrotoxicity,
which is the main dose-limiting factor, its rapid excretion via the
kidneys, with a circulation half life of only a few minutes, and
its strong affinity to plasma proteins (Freise, J., et al., Arch.
Int. Pharmacodyn., 258:180-192 (1982)). Encapsulation of cisplatin
in liposomes as an approach to overcoming toxicity has been
described for example in Abra, et al., U.S. Pat. No. 5,945,122;
Gondal, J. A., et al., Eur. J. Cancer, 29A(11):1536-1542 (1993);
Sur, B., et al., Oncology, 40:372-376 (1983); Weiss, R. B., et al.,
Drugs, 46(3):360-377 (1993). Cisplatin is typically difficult
however, to efficiently entrap in liposomes because of the drug's
low aqueous solubility, approximately 1-2 mg/mL at room
temperature, and low lipophilicity, both of which contribute to a
low drug/lipid ratio.
[0048] In accordance with the invention, liposome-containing
cisplatin in a supersaturated state was prepared as described in
Example 1. Briefly, an aqueous solution of the drug was prepared
with the drug at a concentration above its solubility limit in the
solution at room temperature, about 20-25.degree. C., at pH=7. In
this case, the solubility of the drug was increased by heating the
temperature of the solution. Other methods to increase the
solubility of a drug in an aqueous solution at room temperature, as
discussed above, are also suitable. The prepared solution is
subsequently used to hydrate lipids selected for formation of the
liposome lipid bilayer, thereby forming liposomes containing the
supersaturated drug solution in the central core compartment of the
liposomes. More specifically, and in the particular case using
cisplatin, the liposomes contained cisplatin entrapped at a
concentration of about 8 mg/ml, about four times above cisplatin
solubility at room temperature (1-2 mg/mL). The liposomes were
sized by extrusion to an average mean particle diameter of 106
nm.
[0049] The liposomes containing cisplatin at 8 mg/ml were
characterized by NMR and by extended X-ray absorption fine
structure (EXAFS), as will now be described.
[0050] A. Characterization by NMR
[0051] NMR is a powerful tool for evaluation of liposomal
formulations since the anisotropic nature of molecular motion
within the lipid bilayers and the anisotropy of the membrane
structure can be evaluated by this technique [Fenske, P. B., Chem.
Phys. Lipids 64:143, (1993); Tilcock, C. P. S., Chem. Phys. Lipids
40:109, (1986)]. For cisplatin-loaded liposomes, NMR provides
information on both the liposome phospholipids (physical state,
rate of motion) and on the platinum complex (oxidation state and
coordination sphere). The combination of NMR and atomic absorption
spectrometry can be used to quantify the amount of soluble platinum
in the liposome formulations.
[0052] Liposomes containing .sup.15N-labeled cisplatin were
prepared as described in Example 2. The drug loading was performed
at a temperature of between 60-65.degree. C. to increase the
cisplatin solubility to about 8 mg/mL. After formation of large
unilamellar vesicles, the temperature of the liposome suspension
was lowered to 4.degree. C. at which the drug solubility is about
1-2 mg/mL.
[0053] 1. Analysis by .sup.195Pt NMR
[0054] .sup.195Pt NMR spectroscopy provides information on the
oxidation state of the metal and on the nature of the four ligands
in the platinum's first coordination sphere. This is due to the
fact that the chemical shift range of Pt complexes spans several
thousand ppm and that the chemical shift is sensitive to both the
oxidation state of the Pt (II and IV) and to the nature of the atom
which is bound to the Pt.
[0055] The cis-Pt(.sup.15NH.sub.3).sub.2Cl.sub.2, prepared as
described in Example 2A, was characterized by .sup.195Pt NMR
spectroscopy and the spectrum is shown in FIGS. 1A-1B. As seen in
FIG. 1A, the broad-band decoupled spectrum shows the expected
triplet due to the coupling of two equivalent .sup.15N atoms. When
the broad-band decoupling was turned off, each of the three
.sup.195Pt resonances was split to a septet by the six equivalent
hydrogens, as seen in FIG. 1B.
[0056] .sup.15Pt NMR measurements of cisplatin containing liposomes
clearly show a single chemical shift (range -1350 to -2700 ppm) at
-2139.7 ppm, as seen in FIG. 2. This indicates that the cisplatin
is mainly (>90%) one species of Pt(I I), which is the intact
cisplatin. No additional platinum peaks were detected, indicating
that no other platinum species, i.e., platinum tri- or tetra-amine,
were formed, though adducts of Pt(II) with large molecules would
not be observed by .sup.195Pt NMR. Pt(II) can undergo a
two-electron disproportionation to give solid Pt(O) and soluble
Pt(IV) complexes, especially when they are heated in non-acidic
solutions. No black precipitate was detected in the samples,
suggesting that no Pt(0) was present, and no peaks were observed in
the .sup.195Pt NMR in the region between +1000 to -500 ppm,
indicating that no Pt(IV) complexes were present in the
solution.
[0057] 2. Analysis by .sup.31P NMR
[0058] .sup.31P NMR was used to verify the state of phospholipids
in the lipid vesicles, as well as to verify the homogeneity of
liposomal preparations with respect to size distribution and the
number of lamellae. The .sup.31P NMR spectra of the liposome
formulations for which samples were also measured by .sup.195Pt NMR
are described in Table 1 were measured; they displayed the slightly
asymmetric peaks typical of phospholipid vesicles. The same samples
were measured by .sup.195Pt NMR to verify their platinum content.
At room temperature, the .sup.31P peak observed was very broad.
This is in agreement with previous data of vesicles composed of
saturated phospholipids and cholesterol below the gel-to-liquid
crystalline phase transition temperature.TM. of the matrix lipids
(Tm=52.5.degree. C. for hydrogenated phosphatidylcholine)
(Lichtenberg D. et al., Methods of Biochemical Analysis, D. Glick,
Ed., Vol. 23, Wiley, New York, p. 337 (1998)). As seen in FIGS.
3A-3B, at 60.degree. C., above the Tm, sharp .sup.31P spectra were
observed for both the platinum containing (FIG. 3A) and control
(FIG. 3B) liposomes. At 60.degree. C., approximately 10 scans were
sufficient to produce reliable data with a high signal-to-noise
ratio. Peak analysis revealed somewhat asymmetric peaks for both
(skewness to the right), as is expected for phospholipid vesicles.
Measurement of the linewidths at half the height can serve as a
criterion for the interaction of the platinum with the
phospholipids in the formulation. Linewidths at half the height
(.DELTA..nu..sub.1/2) were determined at 60.degree. C., and values
of 6.3 ppm and 4.2 ppm were obtained for the control and for
cisplatin-liposomes, respectively. Comparison between the
Pt-containing liposomes and the control liposomes indicated minor
interactions between the platinum and the phospholipids. A weak
interaction may occur, as .DELTA..nu..sub.1/2
Pt-liposomes<.DELTA..nu..sub.1/2 control, although both vesicles
are of similar size. The data are in good agreement with .sup.31P
measurements of 100-200 nm diameter LUV (i.e. .about.5 ppm for egg
PC/DSPG 85/15 .DELTA..nu..sub.1/2) [Tilcock, C. P. S., Chem. Phys.
Lipids 40:109 (1986)]. The linewidth suggests no major
contamination with MLV. Thus, .sup.31P NMR has shown by the shape
of the peaks that there are no phospholipids in a hexagonal type II
state.
1TABLE 1 Liposome Formulations for .sup.31P and .sup.195Pt
Spectroscopy P Phospholipid total lipids Pt size Formulation
(mg/mL) (mM) (mg/mL) (mg/mL) (nm) placebo-lipo 2.65 86 109 -- 104
cisplatin-lipo 9.75 315 403 1.8 116
[0059] 3. Analysis by Heteronuclear Single Quantum Coherence
(HSQC)
[0060] While .sup.195Pt NMR spectroscopy provides information on
the oxidation state of the Pt complex and on the nature of atoms in
the first coordination sphere, it cannot distinguish between a
water ligand, a carboxy ligand or a phosphate ligand, since they
all bind the Pt through an oxygen atom. .sup.15N NMR spectroscopy
has been used in conjunction with .sup.195Pt NMR spectroscopy to
provide the missing information Barnham, K. J., Platinum and Other
Metal Coordination Compounds in Cancer Chemotherapy 2, Pinedo, H.
M., et al. (Eds.), Plenum Press, New York, p. 1-16 (1996);
Berners-Price, S. J., J. Am. Chem. Soc. 115:8649 (1993). The
.sup.15N chemical shift and the .sup.15N-.sup.195Pt coupling
constants are sensitive to the ligand that is trans to the .sup.15N
[Appleton, T. G., et al., Inorg. Chem. 24:4685 (1985)]. The lack of
.sup.14N causes the .sup.195Pt resonance to be sharp, and the
coupling constants (.sup.195Pt-.sup.15N, .sup.195Pt-.sup.1H) can be
obtained. .sup.15N-labeled cisplatin
[cis-Pt(.sup.15NH.sub.3).sub.2Cl.sub.2] is readily prepared as
described in Example 2, however, the low sensitivity of .sup.15N
NMR prevents its use for monitoring the Pt complexes in these
experiments.
[0061] Inverse detection experiments detect the protons that are
coupled to .sup.15N and thus provide information about the chemical
shifts and coupling constants of the .sup.15N which depend on the
ligands in the first coordination sphere of the Pt and on its
oxidation state. The main advantage of using .sup.15N-labeled
cisplatin is the ability to apply heteronuclear single or multiple
quantum coherence (HSQC or HMQC, respectively) NMR techniques. HSQC
and HMQC are two-dimensional inverse detection experiments that
have the proton chemical shifts on one axis and the .sup.15N
chemical shifts on the second axis. HSQC and HMQC of
.sup.15N-labeled Pt complexes have been used successfully, to
detect micromolar concentrations of Pt complexes. In the studies
performed in support of the invention, HSQC was used, since it
gives narrower lines and better resolution than HMQC. In a typical
HSQC experiment micromolar concentrations of cisplatin in
approximately 20 minutes were observed. In addition to the
tremendous gain in sensitivity, HSQC selectively detects only the
protons that are coupled to the .sup.15N, thus ignoring all the
other protons in the solution (from the liposomes) and simplifying
the interpretation of the data.
[0062] The HSQC of cis-Pt(.sup.15NH.sub.3).sub.2Cl.sub.2 in water
is depicted in FIGS. 4A-4B. The correlation peaks are located in
region I, which is characteristic of Pt(II) complexes. There are no
peaks in region II, where the Pt(IV) peaks are expected (Barnham,
K. J., Platinum and Other Metal Coordination Compounds in Cancer
Chemotherapy 2, Pinedo H. M., et al. (Eds.), Plenum Press, New
York, p. 1-16 (1996)). An enlargement of region I is shown in FIG.
4B which shows that there are three peaks (A, B, and C) and each
peak has additional satellites (A1', A1", A2' and A2"). Peak A
belongs to cis-Pt(.sup.15NH.sub.3).sub.2Cl.sub.- 2, where the two
equivalent nitrogens give rise to a single peak. Peaks B and C
belong to a single complex of
cis-[Pt(.sup.15NH.sub.3).sub.2Cl(H.su- b.2O)].sup.+ where peak B is
ascribed to the nitrogen that is trans to the aqua ligand, and peak
C, to the nitrogen that is trans to the chloride ligand. The
.sup.195Pt-.sup.15N coupling constants can be measured from the
distance between A1' and A1", and the .sup.195Pt-.sup.1H coupling
constants can be measured from the distance between A1" and A2".
The hydrolysis reaction of cisplatin, which is known to occur in
aqueous solutions, is not easily observed by .sup.195Pt NMR but is
easily observed by HSQC.
[0063] HSQC spectra of cis-Pt(.sup.15NH.sub.3).sub.2Cl.sub.2
passively encapsulated in the aqueous phase of approximately 100 nm
LUV appear almost identical to the spectrum of the native drug.
However, there is one small difference: the distance between the
satellite peaks is somewhat smaller than in the parent compound.
This may be related to the interaction of the
cis-Pt(.sup.15NH.sub.3).sub.2Cl.sub.2 with the lipids, or to the
rather low "activity" of the water inside the liposomes. The latter
is supported by the lack of peaks attributed to the aqua species,
which are clearly visible in the spectrum of the native drug.
Furthermore, as seen in FIG. 5, the spectrum of the
cisplatin-encapsulated liposomes does not have any peaks in the
Pt(IV) region, indicating that the process of the encapsulation of
cis-Pt(.sup.15NH.sub.3).sub.2Cl.sub.2 into the liposomes did not
cause disproportionation of the cisplatin. In addition, the lack of
.sup.15NH.sub.4.sup.+ peak in the spectrum suggests that no
deamination (loss of the .sup.15N ligand) occurred and that all the
Pt(II) complexes can be accounted for by the HSQC experiment. Thus,
it is safe to conclude that the only Pt species in the encapsulated
liposomes are cisplatin and some
cis-[Pt(.sup.15NH.sub.3).sub.2Cl(H.sub.2O)].sup.+. An unidentified
peak (marked as `X`) is visible in the spectrum, and HPLC
chromatograms of these samples detected only very minor impurities
(<5%) (results not shown).
[0064] The final formulations with the cisplatin included a
histidine buffer. .sup.1H-.sup.15N-HSQC NMR is sensitive enough to
detect even the slightest impurities in the sample. Such impurities
were observed when histidine buffer was present during a process
identical to the liposome preparation but in the absence of lipids
(no encapsulation) or when the histidine buffer that was present in
the process of platinum encapsulation in the liposomes. As seen in
FIG. 6, the .sup.1H-.sup.15N-HSQC spectra measured revealed peaks
additional to those of the .sup.15N-labeled compound. Any
cisplatin-histidine species detected are attributed to aqueous
cisplatin during encapsulation in the liposomes.
[0065] Further experiments with known quantities of the amino acid
histidine indicated that these foreign peaks could be attributed to
binding of the drug to the histidine in the buffer during the
encapsulation process. These associations are irreversible, thus
making the drug unavailable, once bound. The reactivity of the
.sup.15N-labeled cisplatin towards biological buffers was examined
using carboxy groups of the N-acetyl-histidine, in order to assess
the contribution of the histidine amino group. Following 24-hour
incubation of a 1:1 mole ratio of .sup.15N-cisplatin and N-acetyl
histidine, various new peaks appeared, probably belonging to
cisplatin-histidine species, as seen in FIG. 7. These results were
further verified by means of HPLC; the chromatograms obtained
indeed verified the presence of a cisplatin-histidine species
(approximately 20-30% of the platinum detected) (results not
shown).
[0066] A similar study was carried out with N-BOC-methionine. This
study suggested that in cisplatin-encapsulated liposomes in which
the encapsulation was performed the absence of histidine buffer and
the histidine is added after the encapsulation process is finished,
as described in Example 1 (FIG. 5) there is no contact between the
histidine buffer and .sup.15N-cisplatin, resulting in liposomes
which are non-leaky and very stable. The study further demonstrated
that there was no leakage of the .sup.15N-labeled cisplatin from
the liposomes, as peaks belonging to cisplatin-histidine species
were not observed.
[0067] 4. Quantification of Platinum Content by .sup.195Pt NMR
[0068] Other parameters to be determined regarding the platinum
encapsulated in the liposomes include the drug-to-lipid ratio, and
how much of the liposomal drug is behaving like water-soluble
cisplatin. In the case of cisplatin, it is expected that only the
soluble portion of the platinum within the liposome will be
discharged and able to act upon cellular DNA; any platinum
precipitate may be considered as unavailable drug. Thus, a
comparative study carried out using an internal reference of
K.sub.2PtCl.sub.4 indicated that, unlike atomic absorption
spectroscopy, it was possible to use .sup.195Pt NMR measurements to
quantify selectively only the soluble cisplatin and thus obtain a
clear indication as to the physical state of the platinum contained
in the liposomes. Any platinum in the solid state (i.e., in a
precipitate or even in a suspension of very fine particles) would
be undetected; its T.sub.2 would be very short, broadening the
lines to such an extent that they would disappear into the
baseline.
[0069] The liposome samples were measured at both 37.degree. C. and
60.degree. C. in order to observe the effect of the rise in
temperature on the solubility of the platinum in the liposomes. The
rise in temperature did not increase the observed peak areas;
however, very small peaks of the aqua species were detected at
60.degree. C. This agrees with the data obtained in the HSQC NMR
measurements discussed above.
[0070] The four liposome samples used in this part of the study are
described in Table 2 and are of somewhat varying size and lipid
concentration. These factors may influence the viscosity of the
sample and therefore contribute to broader line widths in the NMR
spectrum. A significant difference between samples of soluble
cisplatin in water and the liposome cisplatin samples was not
observed. When measured at 60.degree. C., lines were broader, which
is consistent with a shorter T.sub.2 and a rapidly decaying signal.
A direct correlation between the liposome size and the line width
of the cisplatin peak was found; as the liposome size increased, so
did the width of the cisplatin peak. An inverse correlation existed
between the lipid concentration and the width of the cisplatin
peak: the line width increased as the lipid concentration
decreased.
2TABLE 2 Liposomal Formulations For [.sup.1H, .sup.15N] HSQC NMR
Spectroscopy Parameter Sample 1 Sample 2 Sample 3 Sample 4 Size
(nm) 112 125 100 107 cis-Pt (mg/mL) 1.0 1.0 0.9 0.9 Lipid (mM) 104
89 110 110 External Phase 10% sucrose 0.9 NaCl 3% sucrose 3%
sucrose 1 mM NaCl 10 mM histidine 3.3% NaCl 3.3% NaCl 10 mM
histidine pH 6.5 10 mM histidine 10 mM histidine pH 6.5 pH 6.5 pH
6.5 Comment never frozen frozen and thawed
[0071] The liposomes for use in this study were prepared in the
presence of ethanol during the first stages of liposome formulation
and a study was performed to determine the effect of ethanol on the
solubility of cisplatin. Hence, the solubility of cisplatin at 1
and 8 mg/ml at room temperature and at 65.degree. C. in 0.9% NaCl
and in 20% ethanol in 0.9% NaCl was examined. At 1 mg/ml, cisplatin
was soluble under all conditions, while at 8 mg/ml, most of the
cisplatin precipitated at room temperature, yet was mostly soluble
at 65.degree. C. Lowering the temperature back to room temperature
led to the precipitation of most of the 8 mg/ml of the cisplatin,
in both the absence and presence of 20% ethanol. Thus, it can be
concluded that the presence of 20% ethanol did not improve the
solubility of cisplatin. NMR measurements indicate that the
solubility of free cisplatin in the aqueous phase is limited to
.about.2 mg/ml, and is increased upon a rise in temperature to
60.degree. C. The NMR experiments show detection of a peak whose
integration is proportional to .about.2 mg/ml, whereas the
insoluble platinum precipitate is in fact undetected. In the case
of the liposomes, nearly all the cisplatin accounted for by atomic
absorption is soluble in the intraliposomal aqueous phase, which
suggests that the intraliposomal concentration is higher than 2
mg/ml, which is the solubility at room temperature. It was found
that in spite of the fact that the concentration of cisplatin
during liposome preparation was above the solubility at room
temperature (or 4.degree. C.), nearly all the cisplatin in the
liposomes behaved as if soluble in the intraliposomal aqueous
phase. From the solubility studies it is clear that ethanol is not
responsible for the higher than expected drug-to-lipid ratio.
[0072] The NMR studies have shown that cisplatin remains intact
during loading into the liposomes and that the encapsulated
cisplatin resemble, with minor differences, the parent compound
dissolved in the aqueous phase. All the cisplatin present in the
liposomes behaves like a water-soluble drug, and there is no
suggestion of an insoluble drug. The drug within the liposomes does
not precipitate. This fact is further confirmed by the EXAFS study
described below.
[0073] B. Characterization by Extended X-ray Absorption Fine
Structure (EXAFS)
[0074] Extended X-ray Absorption Fine Structure (EXAFS) provides
information on local structure of selected atoms in a sample. In
the method, the scattering of a photoelectron, emitted in the
process of x-ray photoeffect, is exploited to scan the immediate
atomic neighborhood. The interference pattern, resulting from the
scattering, is resolved as a small oscillation of the x-ray
absorption coefficient on the energy or photoelectron wavenumber
scale. The effect is resolved only for slow photoelectrons, i.e.,
immediately above any of the x-ray absorption edges. Hence, the
selectivity of the method with regard to the target atom. From the
amplitude, phase and period of the oscillations, most readily
retrieved by Fourier transform of the data, the number, species and
distance of the neighbor atoms can be deduced. Further analysis can
also provide the degree of thermal and/or structural disorder, so
that materials in crystalline as well as various amorphous states
can be studied.
[0075] For platinum, the L3 absorption edge falls into the energy
region where best resolution of monochromators for synchrotron
radiation can be expected. Thus, Pt L3-edge EXAFS spectra were
measured on the liposome encapsulated cisplatin to determine the
local structure around Pt atoms in the sample. The system was
studies in the liquid, frozen and freeze dried state. For
comparison, Pt L3-edge EXAFS of free drug in solid and dissolved
form were also measured.
[0076] Standard k2-weighted EXAFS spectra .sub..chi.(k) at the Pt
L3 edge for crystalline and dissolved cisplatin and for three
liposome-encapsulated cisplatin samples (liquid, frozen and
freeze-dried), prepared as described in Example 3, are shown in
FIG. 8.
[0077] FIGS. 8-13 are Fourier-transformed k.sup.2-weighted EXAFS
spectra, calculated in the k interval 3 .ANG..sup.-1 to 12
.ANG..sup.-1 for the individual samples shown in FIG. 8. The peaks
in the spectra correspond to consecutive shells of atoms, neighbors
to the Pt atom. Structural parameters are quantitatively resolved
from these spectra by comparing the measured signals with model
signals, constructed ab initio from the set of scattering paths of
the photoelectron in a tentative spatial distribution of neighbor
atoms with the FEFF6 programme code [Rehr, J. J., et al., Phys.
Rev. Lett. 69:3397 (1992)]. In the same way, the atomic species of
a neighbor is recognized by its specific scattering phase
shift.
[0078] In the initial step, the signal of the crystalline cisplatin
sample is analyzed. Although it is not directly relevant for the
study of the encapsulation effects, its high-quality signal serves
as a benchmark of the achievable resolution. Most importantly,
crystallographic data can be used to build a model of the
neighborhood of the Pt atom (Milburn, G. H. W., et al., J. Chem.
Soc. A. JCSIA, p. 1609 (1966)). Indeed, a perfect agreement is
found for the signal of the first-shell N and C1 neighbors at 2.05
.ANG. and 2.33 .ANG. respectively. Both species are identified
unambiguously together with their occupation number of 2.
[0079] Further structure in the FT spectrum between 2.4 and 4.0
.ANG. can be completely explained by the contribution of higher
order scattering on the first shell neighbors, and of the
scattering on the second shell of neighbors, comprising two Pt at
3.37 .ANG., as suggested by the crystallographic data (Milburn, G.
H. W., et al., J. Chem. Soc. A. JCSIA, p. 1609 (1966)). However,
the contributions of further C1 and N neighbors at about the same
distance, predicted by the crystallographic model cannot be
accommodated. Apparently, the measurement and the model refer to
different crystal modifications. In this way, the closest
neighbors, the N and C1 atoms of the cisplatin molecule itself and
the closest Pt atoms of adjacent molecules would be found in their
predicted positions, while further neighbors, brought into the
vicinity of the Pt atom by the particular stacking of molecules
within the crystal, would not. For the neighborhood thus
constructed and refined with the best fit of the data in the k
region of 3 .ANG..sup.-1 -12 .ANG..sup.-1 and the R region of 1.1
.ANG.-4.1 .ANG. the parameters are given in Table 3.
3TABLE 3 Parameters of the nearest coordination shells around Pt in
crystalline cisplatin, saturated water solution of cisplatin and in
liquid, frozen and freeze dried samples of liposome-encapsulated
cisplatin. Crystalline cisplatin Pt neigh..sup.a N.sup.a R(X).sup.a
.sigma..sup.2(x.sup.2).sup.a Fit r-factor.sup.c N 2.0(2).sup.b
2.052(4) 0.0022(9) 0.007 Cl 2.0(2) 2.330(1) 0.0025(3) Pt 2.0(5)
3.37(2) 0.016(3) Cisplatin dissolved in water N 2.3(4) 2.02(1)
0.002(1) 0.006 Cl 1.9(2) 2.29(1) 0.003(1) Liposome-encapsulated
cisplatin: liquid N 2.2(4) 2.01(1) 0.002(1) 0.017 Cl 2.3(3) 2.29(1)
0.003(1) Liposome-encapsulated cisplatin: frozen N 2.4(4) 2.00(1)
0.002(1) 0.022 Cl 2.2(3) 2.29(1) 0.003(1) Liposome-encapsulated
cisplatin: freeze-dried N 1.9(3) 2.03(1) 0.002(1) 0.005 Cl 2.2(3)
2.32(1) 0.003(1) .sup.aAbbreviations: type of neighbor atom, N =
number, R = distance, .sigma..sup.2 = Debye-Waller factors
.sup.bUncertainty of the last digit is given in parentheses.
.sup.cFit r-factor as a measure of goodness of fit is given in the
last column (Stern, E. A., et al., Physica B 208, 209:117,
(1995)).
[0080] The crystalline sample, with its well-defined nearest
neighbors, can also be used to determine another parameter, the
amplitude reduction factor S.sub.0.sup.2 of the EXAFS signal for Pt
atom. This number is transferable between different samples with
the central atom in a similar chemical (valence, coordination)
state. The result (S.sub.0.sup.2=0.77.+-.0.03) is in good agreement
with theoretical estimates [Roy, M., et al., J. Phys. IV France
7:C2-151-C2-152 (1997)] and is used in the analysis of subsequent
cisplatin samples.
[0081] The FT spectrum of the saturated aqueous solution of
cisplatin shown in FIG. 10 is another, possibly closer, template
for identification of the Pt atom neighborhood in the encapsulated
samples. The comparison with the crystalline cisplatin shows a
remarkable similarity in all details up to the distance of 3.5
.ANG.. The model of the Pt neighborhood is thus based on the
closest shell of 2 N and 2 Cl atoms of the molecule, as in the
crystalline sample. A very good fit (Table 3) is obtained for the
interval from 1.1 to 2.5 .ANG., with model parameters identical to
those of the crystalline sample within error interval. The
higher-order scattering contributions from the same shell extend
the validity of the model up to 4 .ANG., providing the explanation
of the small double peak within the region. The presence of Pt atom
neighbors at a larger distance, such as confirmed in the
crystalline sample, is completely excluded by the fit. The opposite
finding would point to aggregation of the cisplatin molecules in
the solution which, if true, should certainly have been known from
physicochemical data (osmotic pressure, freezing-point depression).
Among conceivable expansions of the basic closest-neighbor model,
the only one supported by the data is a large diffuse shell of O
atoms. Apparently, it describes the hydration shell of the
molecule. Its parameters, subject to strong intercorrelation,
cannot be determined with satisfactory precision. The important
point, however, is that it does not extend inwards to the immediate
vicinity of the central Pt atom.
[0082] The spectra of the three samples of encapsulated cisplatin
shown in FIGS. 11-13 (liquid, frozen and freeze-dried,
respectively), although considerably more noisy, agree perfectly
with that of the cisplatin solution in k- as well as in r-space.
The quantitative analysis in the R interval from 1.1 .ANG. to 2.5
.ANG. confirms this observation (Table 3). The presence of the
second-shell Pt atom neighbors is again excluded, so that
aggregation of encapsulated cisplatin molecules is not indicated.
The vestigial contribution of the hydration shell is occluded by
the noise.
[0083] The determination of the crystal structures in the
neighborhood of the Pt atom does not only help to determine the
aggregation state but can also shed light on its chemical structure
and consequently, chemical stability. The first neighbors of the Pt
atom, the directly bonded C1 and N atoms are found at the unchanged
distance and coordination number in all samples which show that the
cisplatin molecule is not appreciably affected by the physical
state of the system.
[0084] The observation of two N and two C1 atoms in the first
coordination shell demonstrates that the encapsulated cisplatin
molecules are chemically stable and do not hydrolyze, i.e. exchange
one or two C1 atoms with water. This is an important observation
because it can be obtained directly from the unperturbed sample
while other analyses of such complex and subtle systems may lead to
chemical and physical changes during sample preparation.
[0085] The absence of Pt neighbors at 3.37 .ANG. of the Pt atom
clearly demonstrates that the cisplatin solution inside the
liposomes does not crystallize. To be more specific, it does not
crystallize in the modification of the crystalline sample [Milburn,
G. H. W., et al., J. Chem. Soc. A. JCSIA, p. 1609 (1966); Moeller,
M., et al., Curr. Op. Cooo. Interf. Sci. 2:177-187 (1997)].
However, another crystal modification of cisplatin is known
[Milburn, G. H. W., et al., J. Chem. Soc. A. JCSIA, p. 1609
(1966)]. It is hydrated cisplatin in which the closest Pt neighbors
are removed to a distance beyond 4 .ANG., on behalf of interposed
water molecules which contribute two O neighbors to each Pt atom at
the distance of 2.03 .ANG. [Kuroda, R., et al., Inorganic Chemistry
22:3620 (1983); Faggiani, R., et al., Canadian Journal of Chemistry
60:529 (1982)]. Neither the encapsulated samples nor even the plain
solution show O atoms so close to the central Pt. Therefore
formation of hydrated crystal structure liposomes is also
excluded.
[0086] EXAFS results are supported by extensive absorption studies
of the drug to lipid bilayers, which have shown that cisplatin does
not absorb onto phosphatidylcholine bilayers [Speelmans, G., et
al., Biochim. Biophys. Acta 1283:60 (1996)]. Additionally,
.sup.195Pt NMR studies of the same system did not show any line
broadening and all the Pt signal could be attributed to the narrow
isotropic signal of rapidly rotating drug also characterized in
free drug solution.
[0087] Frozen and freeze dried liposomal sample are similar to the
liquid one. The data in Table 3 show that neither freezing nor
freeze drying induce drug crystallization. This is consistent with
the fact that water in pores (<200 nm) of comparable size to the
liposome interior does not behave like bulk water. Properties such
as freezing and melting are affected by the large surface to volume
ratio for encapsulate water molecules and it may simply freeze into
a glassy state without crystallizing out solutes [Holly, R., et
al., J. Chem. Phys. 108:4183 (1998)].
[0088] The data shown above, as well as NMR observations, suggest
that cisplatin in the liposome interior forms a supersaturated
solution. Depending on the liposome size, typically between
1000-3000 drug molecules are entrapped. In this study, from the
liposome size, and lipid and drug concentrations it is reasonable
to estimate that each liposome contains on average around 3000
cisplatin molecules. Assuming a molecular volume of 60 .ANG..sup.3
for each cisplatin molecule, the size of a hypothetical crystal
these molecules would form upon precipitation can be calculated. If
the molecules would associate into a solid cube, its side would be
55-60 .ANG..
[0089] While not intending to be bound to any particular theory,
one explanation for the above observations is that the number of
compartmentalized molecules may be simply too small to permit
crossing the energy barrier from crystal embryo to real crystal.
So, the encapsulated cisplatin molecules are constantly associating
into crystallization nuclei and because there are not enough
molecules to overcome the barrier to start crystallization, the
aggregates constantly disassociate. This would be an example of a
"frustrated system" because the compartmentalization of molecules
and their small number and (small) size prevent them from achieving
a stable thermodynamic equilibrium.
[0090] It will be appreciated that the phenomena of a
supersaturated compound in a liposome will find use in fields other
than drug delivery. For example, applications where a high
concentration of an agent is required on demand, such as reaction
kinetics, would benefit from a method to maintain a high
concentration of a compound ready for use. Other areas of use
include diagnostic kits or storage means for proteins and
peptides.
III. EXAMPLES
[0091] The following examples further illustrate the invention
described herein and are in no way intended to limit the scope of
the invention.
Example 1
Preparation of Liposomes Containing Cisplatin
[0092] A. Step 1: Drug Solution Preparation
[0093] Sterile water was heated to 63-67.degree. C. in a
TEFLON-lined pressure vessel and sodium chloride (0.9%) was added.
Cisplatin was added at a concentration of 8.5 mg/ml and mixed until
dissolved, approximately 15-25 minutes.
[0094] B. Step 2: Lipid Dissolution
[0095] 257.0 g PEG-DSPE, 719.4 g HSPC and 308.4 g cholesterol
(molar ratio of 50.6/44.3/5.1) were added to 900 ml dehydrated
ethanol at 60-65.degree. C. and mixed until dissolved,
approximately 2 hours. The dissolved lipids were added to 7670 g of
drug solution to give a total lipid concentration of approximately
150 mg/ml.
[0096] C. Step 3: Lipid Hydration/Drug Loading
[0097] The warm lipid solution was rapidly added to the warm
(63-67.degree. C.) drug solution, with mixing, to form a suspension
of liposomes having heterogeneous sizes. The suspension was mixed
for one hour at 63-67.degree. C. The cisplatin concentration in the
hydration mixture was 7.2 mg/ml and, at this stage, approximately
30% of the drug was encapsulated in the liposomes. 10% of the total
solution volume was ethanol and the total lipid concentration was
150 mg lipid/ml.
[0098] D. Step 4: Extrusion
[0099] The liposomes were sized to the desired mean particle
diameter by controlled extrusion through polycarbonate filter
cartridges housed in Teflon-lined stainless steel vessels. The
liposome suspension was maintained at 63-65.degree. C. throughout
the extrusion process, a period of 6-8 hours.
[0100] E. Step 5: Low-Grade Filtering
[0101] After sizing, the liposome suspension was cooled to room
temperature (20-25.degree. C.) and filtered through a 1.2 .mu.m
142-mm Gelman Versapor filter (acrylic copolymer on a Nylon 66
support) to remove precipitated drug. At this stage approximately
50% of the drug was encapsulated.
[0102] F. Step 6: Diafiltration
[0103] A sucrose/sodium chloride solution was prepared by
dissolving sucrose (100 mg/ml) and sodium chloride (0.058 mg/ml) in
sterile water. The pH of the solution was adjusted to approximately
5.5 with 2 N HCl or NaOH. The solution was filtered through a 0.22
.mu.m Durapore filter.
[0104] The liposome suspension was diluted in approximately a 1:1
(v/v) ratio with the sucrose/sodium chloride solution and
diafiltered through a polysulfone hollow-fiber ultrafilter. Eight
volume exchanges were performed against the sucrose/sodium chloride
solution to remove the ethanol and unencapsulated drug. The process
fluid temperature was maintained at about 20-30.degree. C. Total
diafiltration time was approximately 4.5 hours.
[0105] The liposome suspension was then concentrated to
approximately 1.2 mg cisplatin/ml by ultrafiltration. The post
diafiltration process fluid was analyzed for cisplatin content by
HPLC. The liposomes had an internal phase of 8.5 mg/ml cisplatin in
0.9% sodium chloride and an external phase of sucrose/sodium
chloride solution.
[0106] G. Step 7: Dilution
[0107] A diluent was prepared by dissolving histidine (10 mM) in a
sucrose/sodium chloride (10% sucrose/1 mM NaCl) solution to a
target histidine concentration of 1.55 mg/ml in the final mixture.
The liposome suspension was diluted to a target cisplatin
concentration of 1.05 mg/ml with the histidine diluent. The pH of
the suspension was adjusted to 6.5 with 2N NaOH or HCl.
[0108] H. Step 8: Sterile Filtration
[0109] The liposome suspension was heated to 33-38.degree. C. and
filtered through a 0.2 .mu.m Gelman Supor polyethersulfone filter.
Total filtration time was approximately 10 minutes.
Example 2
NMR Analysis
[0110] A. Preparation and characterization of .sup.15N-labeled
cisplatin [cis-Pt(.sup.15NH.sub.3).sub.2Cl.sub.2]
[0111] cis-.sup.15N-diamminedichloroplatinum(II) (cisplatin) was
synthesized according to the procedure described by Boreham, et
al., Aust. J. Chem. 34:659 (1981). The product was characterized by
both .sup.195Pt NMR spectroscopy and by the thiourea test. Thiourea
was added to an aqueous solution of the .sup.15N-labeled cisplatin,
and the solution was heated to 40-50.degree. C. The appearance of a
yellow color was indicative that the product was in the cis
configuration, whereas the appearance of a colorless solution
indicated the presence of the trans isomer. The cis-.sup.15N-DDP
was dissolved in DMF and analyzed by .sup.195PtNMR.
[0112] B. Preparation of liposomes containing cisplatin
[0113] Initial .sup.195PtNMR measurements were carried out using
.sup.2000PEG-DSPE [polyethylene-glycol (M.W. approximately
2000)-derivatized distearoyl-phosphatidyl-ethanolamine],
cholesterol, large unilamellar liposomes, with 99 mg/ml total
lipids, 78 mM=2.40 mg/ml P, containing 1.64 mg/ml Pt.
[0114] 1. Preparation of .sup.15N-cisplatin encapsulated in
sterically-stabilized liposomes
[0115] 8.5 mg/ml .sup.15N-labeled cisplatin was dissolved in 0.9%
NaCl at 65.degree. C. and left at this temperature for 1 hour.
Lipids (HSPC/cholesterol/.sup.2000PEG-DSPE 51:44:5) were dissolved
in ethanol. The lipids were hydrated by adding this ethanolic
solution to the drug mixture. Final lipid concentration was 150
mg/ml (15%) in 10% ethanol, at 65.degree. C. The mixture was kept
stirring for 1 hour at 60.degree. C., then extruded at 65.degree.
C. through 100 nm and 200 nm pore size polycarbonate filters using
the Lipofast syringe extruder. Sized liposomes (.about.100 nm) were
allowed to cool to room temperature. During the cooling, a heavy
yellow precipitate formed. The supernatant was collected and
allowed more standing-time at room temperature. More precipitation
occurred, and the supernatant was collected again. The sample was
diluted twofold and dialyzed 5 times against 100 volumes of 10%
sucrose containing 1 mM NaCl at room temperature. Under these
conditions, a complete equilibration with 10% sucrose containing 1
mM NaCl should occur. Finally, histidine buffer (pH 6.5) was added
to a final concentration of 10 mM. The final liposome dispersion
was translucent, white. Aliquots of the liposomes and of both
precipitates were analyzed by heteronuclear single quantum
coherence (HSQC) at 30.degree. C.
[0116] 2. Sample preparation for HSQC experiments
[0117] The .sup.15N-cisplatin was dissolved in either 0.9% NaCl or
in 10% sucrose (8.5 mg/ml), at 65.degree. C. Control samples,
without cisplatin, were prepared and processed according to the
same procedures. After cooling, histidine buffer (10 mM, pH=6.5)
was added. Treated samples were stored at 4.degree. C. or at
(-20).degree. C.
[0118] C. NMR measurements
[0119] 1. .sup.195PtNMR
[0120] .sup.195PtNMR measurements were carried out on a Varian
VXR-300S spectrometer with a 7.05 T magnet equipped with a 5 mm
computer switchable probe. The platinum chemical shifts were
measured relative to the external reference signal of
K.sub.2PtCl.sub.4 set at -1624 ppm. Data were collected with and
without broad-band decoupling of the protons, using 100.0 kHz
spectral width, acquisition time of 0.010 s. Usually, 350,000
pulses or more were acquired and a line broadening of 300 Hz was
applied. The samples were measured, without spinning, at 37.degree.
C. and 60.degree. C. A capillary containing 2 mg/ml
(4.82.times.10.sup.-3M) K.sub.2PtCl.sub.4 was added to the NMR
tubes as an internal reference of a known concentration. The data
were apodized using a line broadening equal to the natural line
width (in Hz) prior to processing and integration.
[0121] 2. .sup.31P NMR
[0122] .sup.31P measurements were carried out at room temperature
and at 60.degree. C. using a 5 mm computer-switchable probe. The
.sup.31P chemical shifts were measured relative to phosphoric acid
set at 0 ppm. Data were collected with broad-band decoupling of the
protons, using 10000 Hz spectral width, and an acquisition time of
1.6 s. Usually, 250 pulses or more were acquired and a line
broadening of 20.0 Hz was applied. Placebo (control) and
cisplatin-loaded liposomes were studied by .sup.31P NMR. The
liposomes contained large unilamellar vesicles (LUV) and were
composed of hydrogenated phosphatidylcholine (HPC), cholesterol,
and .sup.2000PEG-DSPE at a mole ratio of 55:40:5. Samples were
prepared by mixing 700 .mu.l of the original liposome sample with
70 .mu.l of D.sub.2O.
[0123] 3. HSQC NMR measurements
[0124] All [.sup.1H, .sup.15N] HSQC data were obtained using a
Bruker DXR 400 MHz NMR spectrometer, equipped with a 5 mm
multinuclear inverse detection probe. The 2D data were recorded
using Bruker sequence of INVIGSTP (inverse detection 2D .sup.1H-X
correlation via double INEPT transfer, phase sensitive using TPPI
with decoupling during acquistion). .sup.15N spins were irradiated
during the acquisition time using the GARP-1 sequence. The .sup.15N
chemical shifts were externally referenced to 1.5 M NH.sub.4Cl in
1M HCl: .sup.1H chemical shifts were externally referenced to TSP
(Me.sub.3Si(CD.sub.2).sub.2CO.sub.2Na). 2-8 transients were
acquired, using an acquisition time of, 0.251 s. spectral widths of
2 KHz in both f.sub.2 and f.sub.1 dimensions, and 256 increments of
t.sub.1. All spectra were acquired at 300.degree. K. (27.degree.
C.). Spectra were collected in approximately 20 minutes and the
data were processed using Bruker software, with no line
broadening.
[0125] 4. Quantification of platinum content by .sup.195PtNMR
[0126] K.sub.2PtCl.sub.4 was chosen as an internal reference since
it is a stable compound whose chemical shift (-1624 ppm) is in
close proximity to that of cisplatin (-2100 ppm), yet is distant
enough to avoid overlapping. The K.sub.2PtCl.sub.4 (2 mg/mL, 4.82
mM), was sealed in a capillary, and concentrated hydrochloric acid
was added to prevent its hydrolysis. The capillary was inserted
into the 5 mm NMR tube containing the sample, and the .sup.195Pt
NMR spectrum was acquired as described above.
[0127] To test the method's accuracy, three cisplatin samples of
varying concentrations (1.54, 2.0, and 2.31 mg/ml) were measured in
the presence of the capillary at 37.degree. C. The areas under the
curves were integrated relative to the area under the reference
peak, and the elemental platinum concentration was calculated. To
verify the calculated figures, atomic absorption (AA) measurements
were performed.
[0128] 5. Atomic Absorption Spectroscopy
[0129] Atomic absorption measurements were performed on a Varian
SpectrAA Zeeman 300 spectrometer. The platinum concentration was
calculated according to a calibration curve of a known
concentration of a K.sub.2PtCl.sub.4 stock solution (250 ng/ml,
6.02.times.10.sup.-7M).
Example 3
EXAFS Analysis
[0130] A. Liposome Preparation
[0131] 1. Materials
[0132] Cholesterol (CH) from Croda, Fullerton, Calif.; Hydrogenated
Soy Phosphatidylcholine (HSPC) from Lipoid, Ludwigshafen, Germany;
N-Carbonyl-methoxypolyethylene glycol
2000)-1,2-distearoyl-sn-glycerophos- phoethanolamine sodium salt
(MPEG-DSPE)from Sygena, Liestal, Switzerland. Cisplatin (USP grade,
not less than 98% pure) from Heraeus GmbH, Hanau, Germany.
[0133] 2. Liposome preparation
[0134] Liposome-encapsulated cisplatin was prepared as described in
Example 1, where multilamellar vesicles were formed by injection of
ethanolic lipid solution into drug solution at 65.degree. C.,
followed by cooling and dialysis. The liposomal lipid composition
was HSPC/CH/MPEG-DSPE in a molar ratio of 51:44:5. The cisplatin
concentration was 1.1 mg/ml and the drug encapsulation, as
determined by gel-exclusion chromatography, was 98%. Total lipid
concentration was 118 mM and mean particle diameter was 106 nm,
determined by dynamic laser-light scattering (Coulter model N4MB,
Miami, Fla.). The encapsulated cisplatin was dissolved in 0.9%
(w/v) NaCl solution and the liposomes suspended in an aqueous phase
of 0.9% NaCl solution, 10 mM histidine, pH 6.5.
[0135] Placebo liposomes were made identically to cisplatin
liposomes, but contained no drug. The placebo liposomes had a mean
particle diameter of 101 nm and a total lipid concentration 73
mM.
[0136] Freeze-dried cisplatin liposomes were prepared as follows. A
sample of cisplatin-containing liposomes was dialysed using
molecular weight cut-off 14,000 dialysis tubing and four,
twenty-volume exchanges of 10% (w/v) sucrose solution over twelve
hours at room temperature. The resulting liposome suspension in 10%
sucrose solution was then rapidly frozen using a
dry-ice/isopropanol mixture and lyophilized overnight at high
vacuum (100 mTorr).
[0137] B. EXAFS Measurements
[0138] Platinum L.sub.3-edge EXAFS spectra of the samples were
measured in a transmission mode at the x-ray beamline ROEMO2 (X1.1)
in Hamburger Synchrotronstrahlungslabor HASYLAB at Deutschen
Elektronen-Synchrotron DESY (Hamburg, Germany). A Si(311)
fixed-exit double-crystal monochromator was used with 2 eV
resolution at 12 keV. Harmonics were effectively eliminated by
detuning the monochromator crystal using a stabilization feedback
control. Ionization cells filled with argon at 1 bar were used to
detect incident and transmitted flux of the monochromatic x-ray
beam through the sample. The absorption spectra were recorded as a
function of the x-ray photon energy with an integration time of 1
s/point. Standard stepping progression within a 1000 eV region
above the edge was adopted.
[0139] Liquid, frozen and freeze dried liposome-encapsulated
cisplatin samples and a water solution of cisplatin were prepared
in a variable-length liquid absorption cell with KAPTON.TM.
windows. Due to the very low concentration of Pt in the samples,
the optimum absorption thickness was found with a 5 mm thick layer
of the liquid samples and a 2 mm layer of the freeze dried sample.
The obtained PtL.sub.3 edge jump was only 0.04 and 0.1 for liposome
encapsulated samples and for aqueous solution respectively at the
total absorption thickness of 2. Ten experimental runs were
superimposed to improve the signal to noise ratio. A reference
spectrum was taken on a 5 mm thick layer of an aqueous solution of
empty liposomes. Powdered crystalline sample was prepared on
multiple layers of adhesive tape, with the edge jump of about 1. A
reference spectrum was measured on empty tapes.
[0140] The complete disclosures of the patents, patent documents,
and publications cited herein are incorporated by reference in
their entirety as if each were individually incorporated. Various
modifications and alterations to this invention will become
apparent to those skilled in the art without departing from the
scope of this invention. It should be understood that this
invention is not intended to be unduly limited by the illustrative
embodiments and examples set forth herein and that such examples
and embodiments are presented by way of example only with the scope
of the invention intended to be limited only by the claims set
forth herein as follows.
* * * * *