U.S. patent application number 15/972012 was filed with the patent office on 2019-04-11 for remote loading of sparingly water-soluble drugs into lipid vesicles.
The applicant listed for this patent is ZONEONE PHARMA, INC.. Invention is credited to Mark E. HAYES, Charles O. NOBLE, Francis C. SZOKA.
Application Number | 20190105339 15/972012 |
Document ID | / |
Family ID | 55264423 |
Filed Date | 2019-04-11 |
View All Diagrams
United States Patent
Application |
20190105339 |
Kind Code |
A1 |
HAYES; Mark E. ; et
al. |
April 11, 2019 |
REMOTE LOADING OF SPARINGLY WATER-SOLUBLE DRUGS INTO LIPID
VESICLES
Abstract
The present invention provides liposome compositions containing
sparingly soluble drugs that are used to treat life-threatening
diseases. A preferred method of encapsulating a drug inside a
liposome is by remote or active loading. Remote loading of a drug
into liposomes containing a transmembrane electrochemical gradient
is initiated by co-mixing a liposome suspension with a solution of
drug, whereby the neutral form of the compound freely enters the
liposome and becomes electrostatically charged thereby preventing
the reverse transfer out of the liposome. There is a continuous
build-up of compound within the liposome interior until the
electrochemical gradient is dissipated or all the drug is
encapsulated in the liposome. However, this process as described in
the literature has been limited to drugs that are freely soluble in
aqueous solution or solubilized as a water-soluble complex. This
invention describes compositions and methods for remote loading
drugs with low water solubility (<2 mg/mL). In the preferred
embodiment the drug in the solubilizing agent is mixed with the
liposomes in aqueous suspension so that the concentration of
solubilizing agent is lowered to below its capacity to completely
solubilize the drug. This results in the drug precipitating but
remote loading is capability retained. The process is scalable and
the resulting drug-loaded liposomes are characterized by a high
drug-to-lipid ratios and predictable drug retention when the
liposome encapsulated drug is administered in patients.
Inventors: |
HAYES; Mark E.; (San
Francisco, CA) ; NOBLE; Charles O.; (San Francisco,
CA) ; SZOKA; Francis C.; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ZONEONE PHARMA, INC. |
San Francisco |
CA |
US |
|
|
Family ID: |
55264423 |
Appl. No.: |
15/972012 |
Filed: |
May 4, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15502092 |
Feb 6, 2017 |
10004759 |
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PCT/US2015/043594 |
Aug 4, 2015 |
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15972012 |
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62033073 |
Aug 4, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/496 20130101;
A61K 9/127 20130101; A61K 31/7048 20130101; A61K 9/19 20130101;
A61K 31/4196 20130101; A61K 9/1277 20130101; A61K 9/1271 20130101;
A61K 31/337 20130101; A61K 38/07 20130101; A61K 31/704 20130101;
A61K 47/02 20130101; A61K 31/395 20130101 |
International
Class: |
A61K 31/7048 20060101
A61K031/7048; A61K 31/704 20060101 A61K031/704; A61K 31/395
20060101 A61K031/395; A61K 9/127 20060101 A61K009/127; A61K 31/337
20060101 A61K031/337; A61K 31/4196 20060101 A61K031/4196; A61K 9/19
20060101 A61K009/19; A61K 38/07 20060101 A61K038/07; A61K 47/02
20060101 A61K047/02; A61K 31/496 20060101 A61K031/496 |
Claims
1. A pharmaceutical formulation comprising a sparingly
water-soluble agent encapsulated within a liposome, said
formulation manufactured by a method comprising: contacting an
aqueous suspension of said liposome with said aqueous suspension of
said agent under conditions appropriate to encapsulate said
sparingly water-soluble agent in said liposome, wherein said
liposome has an internal aqueous environment encapsulated by a
lipid membrane and said aqueous suspension of said liposome
comprises a gradient selected from a proton gradient, an ion
gradient and a combination thereof across said membrane, and
wherein said conditions are appropriate for said sparingly
water-soluble agent to traverse said membrane and concentrate in
said internal aqueous environment, thereby forming said
pharmaceutical formulation.
2. The pharmaceutical formulation according to claim 1, wherein
said aqueous suspension of said sparingly water-soluble agent is
prepared by a method comprising: essentially completely dissolving
the agent in an aprotic solvent, forming an agent solution and
subsequently diluting said agent solution with an aqueous medium
beyond the point of drug solubility.
3. The pharmaceutical formulation of claim 1 where said liposome is
selected from multilamellar vesicles (MLV), large unilamellar
vesicles (LUV) and small unilamellar vesicles (SUV), oligolamellar
vesicles (OLV), paucilamellar vesicles (PLV) or reverse phase
evaporation vesicles (REV).
4. The pharmaceutical formulation according to claim 1, wherein
said agent is a low molecular weight therapeutic agent having a
water solubility of less than or equal to about 2 mg/mL.
5. The pharmaceutical formulation of claim 1, wherein, the agent is
selected from an anthracycline compound, a camptothecin compound, a
vinca alkaloid, an ellipticine compound, a taxane compound, a
wortmannin compound, a geldanamycin compound, a polyene antibiotic,
a pyrazolopyrimidine compound, a steroid compound, a peptide-based
compound, a taxane, a derivative of any of the foregoing, a
pro-drug of any of the foregoing, and an analog of any of the
foregoing.
6. The pharmaceutical formulation according to claim 1, wherein
said agent is selected from carfilzomib, voriconazole, amiodarone,
ziprasidone, aripiprazole, imatinib, lapatinib, oprozomib,
cyclopamine, CUR-61414, PF-05212384, PF-4691502, toceranib,
PF-477736, PF-337210, sunitinib, SU14813, axitinib, AG014699,
veliparib, MK-4827, ABT-263, SU11274, PHA665752, Crizotinib, XL880,
PF-04217903, XR5000, AG14361, veliparib, bosutunib, PD-0332991,
PF-01367338, AG14361, NVP-ADW742, NVP-AUY922, NVP-LAQ824,
NVP-TAE684, NVP-LBH589, erubulin, doxorubicin, daunorubicin,
mitomycin C, epirubicin, pirarubicin, rubidomycin, carcinomycin,
N-acetyladriamycin, rubidazone, 5-imido daunomycin, N-acetyl
daunomycin, daunoryline, mitoxanthrone, camptothecin,
9-aminocamptothecin, 7-ethylcamptothecin,
7-Ethyl-10-hydroxy-camptothecin, 10-hydroxycamptothecin,
9-nitrocamptothecin,10,11-methylenedioxycamptothecin,
9-amino-1O,11-methylenedioxycamptothecin, 9-chloro-1 0,
11-methylenedioxycamptothecin, irinotecan, lurtotecan, silatecan,
(7-(4-methylpiperazinomethylene)-10,ll-ethylenedioxy-20(S)-camptothecin,
7-(4-methylpiperazinomethylene)-10,
II-methylenedioxy-20(S)-camptothecin,
7-(2-N-isopropylamino)ethyl)-(20S)-camptothecin, CKD-602,
vincristine, vinblastine, vinorelbine, vinflunine, vinpocetine,
vindesine, 2'succinylpaclitaxel, 2' succinyldocetaxel, 2'
succinylcabazitaxel, 2'glutarylpaclitaxel, 2'glutaryldocetaxel,
2'glutarylcabazitaxel, 2'succinyldimethylaminopropylamide of
paclitaxel, 2'succinyldimethylaminopropylamide of docetaxel,
2'succinyldimethylaminopropylamide of cabazitaxel, 2'
morpholine-4-ylethyl amide of succinylcabazitaxel, 2'
morpholine-4-ylethyl amide of succinylpaclitaxel, 2'
morpholine-4-ylethyl amide of succinyldocetaxel, 2'
morpholinopropylamide paclitaxel, 2' morpholinopropylamide
docetaxel, 2' morpholinopropylcabazitaxel. ellipticine,
6-3-aminopropyl-ellipticine, 2-diethylaminoethyl-ellipticinium,
datelliptium, retelliptine, paclitaxel, docetaxel, cabazitaxel,
diclofenac, bupivacaine, 17-allylamino-geldanamycin,
17-dimethylaminoethylamino-17-demethoxygeldanamycin, cetirizine,
fexofenadine, Onx 0912, Onx 0914, PD0332991, Axitinib, Lenvatinib,
PHA665752, SU11274, PF-02341066, foretinib, XL880, PX-478,
GDC-0349, PD0332991, AZD4547, Golotimod, SCH900776, TG02, UNC0638,
ARRY-520, Elacridar hydrochloride, golvatinib, MK-1775,
PF-03758309, AT13387, BAY 80-6946, cobicistat, GDC-0068, INNO-206,
MLN0905, resminostat, tariquidar, primidone and other
catecholamines, epinephrine, salts, prodrugs and derivatives of
these medicinal compounds and mixtures thereof.
7. The pharmaceutical formulation according to claim 1, wherein
said agent is selected from an antihistamine ethylenediamine
derivative, bromphenifamine, diphenhydramine, an anti-protozoal
drug, quinolone, iodoquinol, an amidine compound, pentamidine, an
antihelmintic compound, pyrantel, an anti-schistosomal drug,
oxaminiquine, an antifungal compound amphotericin B,
2'deoxyamphotericin B, triazole derivative, fliconazole,
itraconazole, ketoconazole, miconazole, an antimicrobial
cephalosporin, chelating agents, deferoxamine, deferasirox,
deferiprone, FBS0701, cefazolin, cefonicid, cefotaxime,
ceftazimide, cefuoxime, an antimicrobial beta-lactam derivative,
aztreopam, cefmetazole, cefoxitin, an antimicrobial of erythromycin
group, erythromycin, azithromycin, clarithromycin, oleandomycin, a
penicillin compound, benzylpenicillin, phenoxymethylpenicillin,
cloxacillin, methicillin, nafcillin, oxacillin, carbenicillin, a
tetracycline compound, novobiocin, spectinomycin, vancomycin; an
antimycobacterial drug, aminosalicycic acid, capreomycin,
ethambutol, isoniazid, pyrazinamide, rifabutin, rifampin,
clofazimine, an antiviral adamantane compound, amantadine,
rimantadine, a quinidine compound, quinine, quinacrine,
chloroquine, hydroxychloroquine, primaquine, amodiaquine,
mefloquine, an antimicrobial, qionolone, ciprofloxacin, enoxacin,
lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, a
sulfonamide; a urinary tract antimicrobial, nitrofurantoin,
trimetoprim; anitroimidazoles derivative, metronidazole, a
cholinergic quaternary ammonium compound, ambethinium, neostigmine,
physostigmine, an anti-Alzheimer aminoacridine, tacrine, an
anti-parkinsonal drug, benztropine, biperiden, procyclidine,
trihexylhenidyl, an anti-muscarinic agent, atropine, hyoscyamine,
scopolamine, propantheline, an adrenergic compound, dopamine,
serotonin, a hedgehog inhibitor, albuterol, dobutamine, ephedrine,
epinephrine, norepinephrine, isoproterenol, metaproperenol,
salmetrol, terbutaline, a serotonin reuptake inhibitor, an
ergotamine derivative, a myorelaxant, a curare series, a central
action myorelaxant, baclophen, cyclobenzepine, dentrolene,
nicotine, a nicotine receptor antagonist, a beta-adrenoblocker,
acebutil, amiodarone, abenzodiazepine compound, ditiazem, an
antiarrhythmic drug, diisopyramide, encaidine, a local anesthetic
compound, procaine, procainamide, lidocaine, flecaimide, quinidine,
an ACE inhibitor, captopril, enelaprilat, Hsp90 inhibitor,
fosinoprol, quinapril, ramipril; an opiate derivative, codeine,
meperidine, methadone, morphine, an antilipidemic, fluvastatin,
gemfibrosil, an HMG-coA inhibitor, pravastatin, a hypotensive drug,
clonidine, guanabenz, prazocin, guanethidine, granadril,
hydralazine, a non-coronary vasodilator, dipyridamole, an
acetylcholine esterase inhibitor, pilocarpine, an alkaloid,
physostigmine, neostigmine, a derivative of any of the foregoing, a
pro-drug of any of the foregoing, and ananalog of any of the
foregoing.
8. The pharmaceutical formulation of claim 1, wherein the aprotic
solvent is selected from dimethylsulfoxide, dioxane,
tetrahydrofuran, dimethylformamide, acetonitrile,
dimethylacetamide, sulfolane, gamma butyrolactone, pyrrolidones,
1-methyl-2-pyrrolidinone, methylpyrroline, ethylene glycol
monomethyl ether, diethylene glycol monomethyl ether, polyethylene
glycol.
9. The pharmaceutical formulation of claim 1, wherein the liposome
is prepared from one or more lipids selected from egg
phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG),
dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM),
cholesterol (Chol), cholesterol sulfate and its salts (CS),
cholesterol hemisuccinate and its salts (Chems), cholesterol
phosphate and its salts (CP), cholesterol phthalate,
cholesterylphosphorylcholine,
3,6,9-trioxaoctan-ol-cholesteryl-3e-ol,
dimyristoylphosphatidylglycerol (DMPG),
dimyristoylphosphatidylglycerol (DMPG),
dimyristoylphosphatidylcholine (DMPC),
distearoylphosphatidylcholine (DSPC), hydrogenated soy
phosphatidylcholine (HSPC), distearoylphosphatidylglycerol (DSPG),
sterol modified lipids (SML), inverse-phosphocholine lipids,
cationic lipids and zwitterlipids.
10. The pharmaceutical formulation of claim 1, wherein the
percentage of agent from said aqueous suspension of said agent
encapsulated in said internal aqueous medium of said liposome
ranges from about 10% to about 100% of the total agent in said
aqueous suspension of said agent.
11. The pharmaceutical formulation of claim 1, wherein said ion
gradient is caused by a difference in concentrations across said
membrane of a member selected from an amine salt and a metal salt
of a member selected from a carboxylate, a sulfate, and a
phosphate.
12. The pharmaceutical formulation according to claim 11, wherein
said carboxylate is acetate.
13. The pharmaceutical formulation according to claim 11, wherein
said amine salt and said metal salt are selected from a salt of a
member selected from a monovalent carboxylate, a multivalent
carboxylate, a sulfate and a phosphate.
14. The pharmaceutical formulation according to claim 11, wherein
the cation in said salt is selected from sodium, calcium,
magnesium, zinc, copper, potassium, primary, secondary, tertiary
and quaternary ammonium species.
15. The pharmaceutical formulation according to claim 1, wherein
said formulation is lyophilized.
16. The pharmaceutical formulation according to claim 1, wherein
said agent is present in said internal aqueous medium as a unit
dosage format.
17. A method of treating a disease state in a subject in need of
said treatment, said method comprising administering to said
patient a therapeutically effective amount of said formulation of
claim.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 15/502,092 filed Feb. 6, 2017, which is a 371 U.S. National
Phase of PCT International Application No. PCT/US2015/043594 filed
Aug. 4, 2015, which claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Application No. 62/033,073 filed on Aug.
4, 2014, all of which are incorporated herein by reference in their
entireties for all purposes.
BACKGROUND OF THE INVENTION
[0002] This invention relates to the fields of pharmaceutical
compositions, methods for making them and the uses of the resulting
compositions in drug therapy. The pharmaceutical compositions
include the active therapeutic agent encapsulated within the
aqueous interior of a liposome vesicle.
DESCRIPTION OF THE RELATED ART
[0003] The pharmaceutical industry, in its quest for improved
drugs, has generated a large number of potent compounds that are
sparingly soluble in water, the ubiquitous solvent that makes life
possible. The low water solubility of these new drugs has made it
difficult to deliver them in animals including humans. This has
created the need for drug delivery systems that can solubilize
sparingly water-soluble drugs to enable to their delivery in the
body.
[0004] Liposomes are vesicle structures usually composed of a
bilayer membrane of amphipathic molecules such as, phospholipids,
entrapping an aqueous core. The diameters and morphology of various
types of liposomes are illustrated in FIG. 1. Drugs are either
encapsulated in the aqueous core or interdigitated in the bilayer
membrane. Drugs interdigitated in the membrane transfer out of the
liposome when it is diluted into the body. Importantly, drugs that
are encapsulated in the aqueous core or held in complexes in the
aqueous core are retained substantially longer than drugs in the
bilayer. The use of liposomes with drugs encapsulated in the
aqueous core for drug delivery is well established (D. Drummond et
al., J. Pharm. Sci., (2008) 97(11):4696-4740, PMID 10581328).
[0005] A variety of loading methods for encapsulating functional
compounds, particularly drugs, in liposomes is available.
Hydrophilic compounds for example can be encapsulated in liposomes
by hydrating a mixture of the functional compounds and
vesicle-forming lipids. This technique is called passive loading.
The functional compound is encapsulated in the liposome as the
nanoparticle is formed. The available lipid vesicle (liposome)
production procedures are satisfactory for most applications where
water-soluble drugs are encapsulated (G. Gregoriadis, Ed., Liposome
Technology, (2006) Liposome Preparation and Related Techniques, 3rd
Ed.) However, the manufacture of lipid vesicles that encapsulate
drugs sparing water-soluble (e.g., with a water solubility less
than 2 mg/mL) in the aqueous inner compartment of the liposome is
exceedingly difficult (D. Zucker et al., Journal of Controlled
Release (2009) 139:73-80, PMID 19508880).
[0006] Passive loading of lipophilic and, to a lesser extent
amphiphilic functional compounds, is somewhat more efficient than
hydrophilic functional compounds because they partition in both the
lipid bilayer and the intraliposomal (internal) aqueous medium.
However, using passive loading, the final
functional-compound-to-lipid ratio as well as the encapsulation
efficiency are generally low. The concentration of drug in the
liposome equals that of the surrounding fluid and drug not
entrapped in the internal aqueous medium is washed away after
encapsulation.
[0007] Certain hydrophilic or amphiphilic compounds can be loaded
into preformed liposomes using transmembrane pH- or ion-gradients
(D. Zucker et al., Journal of Controlled Release (2009) 139:73-80).
This technique is called active or remote loading. Compounds
amenable to active loading should be able to change from an
uncharged form, which can diffuse across the liposomal membrane, to
a charged form that is not capable thereof. Typically, the
functional compound is loaded by adding it to a suspension of
liposomes prepared to have a lower outside/higher inside pH- or
ion-gradient. Via active loading, a high
functional-compound-to-lipid mass ratio and a high loading
efficiency (up to 100%) can be achieved. Examples are active
loading of anticancer drugs doxorubicin, daunorubicin, and
vincristine (P. R. Cullis et al., Biochimica et Biophysica Acta,
(1997) 1331:187-211, and references therein).
[0008] Hydrophobic drugs are only considered capable of loading
into liposomes through membrane intercalation via some passive
loading/assembly mechanism. Wasan et al. states "Agents that have
hydrophobic attributes can intercalate into the lipid bilayer and
this can be achieved by adding the agent to the preformed
liposomes." in a description of the use of micelles to transfer
sparingly soluble agents to a liposome bilayer (US
2009/0028931).
[0009] Remote loading of a sparingly soluble drug into a liposome
under conditions where the drug is above its solubility limit and
is in the form of a precipitate is an unexpected event. D. Zucker
et al., Journal of Controlled Release (2009) 139:73-80 states
"Hydrophobic molecules may aggregate, and these aggregates have low
permeability across the liposomal membrane. Thus, when the
non-polar/polar surface area ratio is >2.31 (FIG. 4), it is
necessary that the drug would have a reasonable solubility, >1.9
mM, in order to achieve high loading because only soluble uncharged
molecules can enter the liposome." (D. Zucker et al., Journal of
Controlled Release (2009) 139:73-80).
[0010] To date, a method has not been developed for the active
loading of the aqueous core of a liposome with a sparingly
water-soluble agent from a precipitate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 illustrates the diameters and morphology of various
types of liposomes.
[0012] FIG. 2. Liposome formulations composed of HSPC/Chol/Peg-DSPE
containing either sodium sulfate (light shade) or ammonium sulfate
(dark shade) were incubated with carfilzomib at two input
drug-to-lipid ratios using conditions described below. The
liposomes were purified from unencapsulated drug and the amount of
encapsulated carfilzomib within the liposomes is shown, expressed
as .mu.g of carfilzomib per .mu.mol lipid.
[0013] FIG. 3 is a bar graph showing a trapping agent effect on
liposome loading of carfilzomib.
[0014] FIG. 4 is a bar graph showing a method of drug introduction
effect on liposome loading of carfilzomib.
[0015] FIG. 5 is a line graph showing carfilzomib loading from
precipitate demonstrated by reduction of light scattering at 600
nm.
[0016] FIG. 6 is a HPLC Chromatogram of Carfilzomib before loading
into liposomes (upper) and after loading into liposomes from a
precipitate and being released from liposomes using a reverse
ammonium sulfate gradient back to a precipitate in the
extraliposomal solution (lower).
[0017] FIG. 7 is a line graph showing liposome encapsulation
efficiency as a function of [DMSO]. The input drug-to-lipid ratio
was 200 .mu.g/.mu.mol.
[0018] FIG. 8 is a line graph showing light scattering of
carfilzomib solution as a function of DMSO concentration. The
concentration of carfilzomib was 0.2 mg/mL.
[0019] FIG. 9 is a bar graph showing the effect of delay time
between the formation of drug precipitate and liposome loading of
the precipitate.
[0020] FIG. 10 is a line graph showing the effect of ammonium
sulfate trapping agent concentration on liposome drug payload of
carfilzomib Loaded from Precipitate.
[0021] FIG. 11 is a line graph showing effect of ammonium sulfate
trapping agent concentration on liposome loading efficiency of
carfilzomib from precipitate.
[0022] FIG. 12 is a line graph loading insoluble carfilzomib
precipitate into liposomes using a triethylammonium sulfate
gradient.
[0023] FIG. 13 is a line graph showing the transfer of insoluble
carfilzomib precipitate into liposomes by remote loading.
[0024] FIG. 14 is a bar graph showing the comparison of liposome
loading of aripiprazole when mixed with liposomes as a SBCD complex
(Abilify) or when diluted from a stock DMSO solution directly into
liposomes, creating a drug suspension.
[0025] FIG. 15 is a bar graph showing absorbance at 600 nm
(scattering) of drug solutions (dark bars) and liposome drug
mixtures (gray bars). The rectangle indicates the samples where a
substantial decrease in scattering was measured upon incubation
with liposomes indicating drug loading.
[0026] FIG. 16 is a bar graph showing the loading efficiency of DFX
in calcium acetate liposomes.
[0027] FIG. 17 is a plot showing DFX loading capacity in liposomes
containing calcium acetate as a trapping agent.
[0028] FIG. 18 is a plot showing DFX loading capacity in liposomes
containing different acetate trapping agents.
[0029] FIG. 19A-FIG. 19B are illustrations of the structures of
paclitaxel, docetaxel and cabazitaxel and modifications to them
that enable the taxanes to be loaded from a precipitate into
liposomes containing an ion gradient.
SUMMARY OF THE INVENTION
[0030] In utilizing liposomes for delivery of functional compounds,
it is generally desirable to load the liposomes to high
concentration, resulting in a high functional-compound-lipid mass
ratio, since this reduces the amount of liposomes to be
administered per treatment to attain the required therapeutic
effect, all the more since several lipids used in liposomes have a
dose-limiting toxicity by themselves. The loading percentage is
also of importance for cost efficiency, since poor loading results
in a great loss of the active compound.
[0031] In an exemplary embodiment, the invention provides a
liposome comprising a liposomal lipid membrane encapsulating an
internal aqueous medium. The internal aqueous medium comprises an
aqueous solution of a complex between a trapping agent and a
sparingly water-soluble therapeutic agent.
[0032] In a further exemplary embodiment, the invention provides
pharmaceutical formulations comprising a liposome of the invention.
The formulations include the liposome and a pharmaceutically
acceptable diluent or excipient. In various embodiments, the
pharmaceutical formulation is in a unit dosage format, providing a
unit dosage of the therapeutic agent encapsulated in the
liposome.
[0033] In another exemplary embodiment, the invention provides
methods of making the liposomes of the invention. In various
embodiments, there is provided a method of remotely loading a
liposome with an agent that is sparingly water-soluble. The method
comprises: a) incubating an aqueous mixture comprising: (i) a
liposome suspension having a proton and/or ion gradient that exists
across the liposomal membrane; (ii) with an aqueous suspension of a
sparingly soluble drug (iii) wherein the drug suspension is made by
completely dissolving the drug in an aprotic solvent or polyol and
diluting it into the aqueous solution beyond the point of drug
solubility where a precipitate is formed, wherein incubating the
combined liposome drug precipitate mixture for a period of time
results in the drug accumulating within the liposome interior in
response to the proton/ion gradient. The mixture used to load the
liposome with the agent is prepared such that a proton- and/or
ion-gradient exists across the liposomal membrane between the
internal aqueous membrane and the external aqueous medium. The
incubating can be for any useful period but is preferably for a
period of time sufficient to cause at least part of the insoluble
drug precipitate to accumulate in the internal aqueous medium under
the influence of the proton and/or ion gradient.
[0034] Other embodiments, objects and advantages are set forth in
the Detailed Description that follows.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Introduction
[0035] In utilizing liposomes for delivery of functional compounds,
it is generally desirable to load the liposomes to high
concentration, resulting in a high agent-lipid mass ratio, since
this reduces the amount of liposomes to be administered per
treatment to attain the required therapeutic effect of the agent,
all the more since several lipids used in liposomes have a
dose-limiting toxicity by themselves. The loading percentage is
also of importance for cost efficiency, since poor loading results
in an increase loss of agent during the loading of the agent into
the liposome.
[0036] The present invention provides liposomes encapsulating
agents, e.g., sparingly water-soluble, methods of making such
liposomes, formulations containing such liposomes and methods of
making the liposomes and formulations of the invention.
[0037] In an exemplary embodiment, the invention provides a
liposome having a membrane encapsulating an aqueous compartment.
The liposome is prepared such that a proton- and/or ion-gradient
exists across the liposomal membrane between the internal aqueous
compartment and the external aqueous medium. The agent is dissolved
in an aprotic solvent at a concentration that when diluted in the
liposome suspension its solubility in the suspension is exceeded
and the agent forms a precipitate. A portion of the agent
precipitate is loaded into the liposome aqueous compartment using a
proton- and/or ion-gradient exists across the liposomal membrane
between the internal aqueous compartment and the external aqueous
medium.
[0038] In some embodiments, essentially the entire amount of the
insoluble agent precipitate is loaded into the aqueous compartment
of the liposome. In an exemplary embodiment, at least about 95%, at
least about 90%, at least about 85%, at least about 80% or at least
about 70% of the insoluble drug precipitate is loaded into the
aqueous compartment of the liposome.
Liposomes
[0039] The term liposome is used herein in accordance with its
usual meaning, referring to microscopic lipid vesicles composed of
a bilayer of phospholipids or any similar amphipathic lipids
encapsulating an internal aqueous medium. The liposomes of the
present invention can be unilamellar vesicles such as small
unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs),
and multilamellar vesicles (MLV), typically varying in size from 30
nm to 200 nm. No particular limitation is imposed on the liposomal
membrane structure in the present invention. The term liposomal
membrane refers to the bilayer of phospholipids separating the
internal aqueous medium from the external aqueous medium.
[0040] Exemplary liposomal membranes useful in the current
invention may be formed from a variety of vesicle-forming lipids,
typically including dialiphatic chain lipids, such as
phospholipids, diglycerides, dialiphatic glycolipids, single lipids
such as sphingomyelin and glycosphingolipid, cholesterol and
derivates thereof, and combinations thereof. As defined herein,
phospholipids are amphiphilic agents having hydrophobic groups
formed of long-chain alkyl chains, and a hydrophilic group
containing a phosphate moiety. The group of phospholipids includes
phosphatidic acid, phosphatidyl glycerols, phosphatidylcholines,
phosphatidylethanolamines, phosphatidylinositols,
phosphatidylserines, and mixtures thereof. Preferably, the
phospholipids are chosen from
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
dimyristoyl-phosphatidylcholine (DMPC), hydrogenated soy
phosphatidylcholine (HSPC), soy phosphatidylcholine (SPC),
dimyristoylphosphatidylglycerol (DMPG),
disrearoylphosphatidylglycerol
(DSPG),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)distearoyl
phosphatidylcholine (DSPC), egg yolk phosphatidylcholine (EYPC) or
hydrogenated egg yolk phosphatidylcholine (HEPC), sterol modified
lipids (SML), cationic lipids and inverse-zwitterlipids.
[0041] Liposomal membranes according to the present invention may
further comprise ionophores like nigericin and A23187.
[0042] In the method according to the present invention, an
exemplary liposomal phase transition temperature is between
-25.degree. C. and 100.degree. C., e.g., between 4.degree. C. and
65.degree. C. The phase transition temperature is the temperature
required to induce a change in the physical state of the lipids
constituting the liposome, from the ordered gel phase, where the
hydrocarbon chains are fully extended and closely packed, to the
disordered liquid crystalline phase, where the hydrocarbon chains
are randomly oriented and fluid. Above the phase transition
temperature of the liposome, the permeability of the liposomal
membrane increases. Choosing a high transition temperature, where
the liposome would always be in the gel state, could provide a
non-leaking liposomal composition, i.e. the concentration of the
sparingly water-soluble agent in the internal aqueous medium is
maintained during exposure to the environment. Alternatively, a
liposome with a transition temperature between the starting and
ending temperature of the environment it is exposed to provides a
means to release the sparingly water-soluble agent when the
liposome passes through its transition temperature. Thus, the
process temperature for the active-loading technique typically is
above the liposomal phase transition temperature to facilitate the
active-loading process. As is generally known in the art, phase
transition temperatures of liposomes can, among other parameters,
be influenced by the choice of phospholipids and by the addition of
steroids like cholesterol, lanosterol, cholestanol, stigmasterol,
ergosterol, and the like. Hence, in an embodiment of the invention,
a method according to any of the foregoing is provided in which the
liposomes comprise one or more components selected from different
phospholipids and cholesterol in several molar ratios in order to
modify the transition, the required process temperature and the
liposome stability in plasma. Less cholesterol in the mixture will
result in less stable liposomes in plasma. An exemplary
phospholipid composition of use in the invention comprises between
about 10 and about 50 mol % of steroids, preferably
cholesterol.
[0043] In accordance with the invention, liposomes can be prepared
by any of the techniques now known or subsequently developed for
preparing liposomes. For example, the liposomes can be formed by
the conventional technique for preparing multilamellar lipid
vesicles (MLVs), that is, by depositing one or more selected lipids
on the inside walls of a suitable vessel by dissolving the lipids
in chloroform and then evaporating the chloroform, and by then
adding the aqueous solution which is to be encapsulated to the
vessel, allowing the aqueous solution to hydrate the lipid, and
swirling or vortexing the resulting lipid suspension. This process
engenders a mixture including the desired liposomes. Alternatively,
techniques used for producing large unilamellar lipid vesicles
(LUVs), such as reverse-phase evaporation, infusion procedures, and
detergent dilution, can be used to produce the liposomes. A review
of these and other methods for producing lipid vesicles can be
found in the text Liposome Technology, Volume I, Gregory
Gregoriadis Ed., CRC Press, Boca Raton, Fla., (1984), which is
incorporated herein by reference. For example, the lipid-containing
particles can be in the form of steroidal lipid vesicles, stable
plurilamellar lipid vesicles (SPLVs), monophasic vesicles (MPVs),
or lipid matrix carriers (LMCs). In the case of MLVs, if desired,
the liposomes can be subjected to multiple (five or more)
freeze-thaw cycles to enhance their trapped volumes and trapping
efficiencies and to provide a more uniform interlamellar
distribution of solute.
[0044] Following liposome preparation, the liposomes are optionally
sized to achieve a desired size range and relatively narrow
distribution of liposome sizes. A size range of about 20-200
nanometers allows the liposome suspension to be sterilized by
filtration through a conventional filter, typically a 0.22 or 0.4
micron filter. The filter sterilization method can be carried out
on a high through-put basis if the liposomes have been sized down
to about 20-200 nanometers. Several techniques are available for
sizing liposomes to a desired size. Sonicating a liposome
suspension either by bath or probe sonication produces a
progressive size reduction down to small unilamellar vesicles less
than about 50 nanometer in size. Homogenization is another method
which relies on shearing energy to fragment large liposomes into
smaller ones. In a typical homogenization procedure, multilamellar
vesicles are recirculated through a standard emulsion homogenizer
until selected liposome sizes, typically between about 50 and 500
nanometers, are observed. In both methods, the particle size
distribution can be monitored by conventional laser-beam particle
size determination. Extrusion of liposome through a small-pore
polycarbonate membrane or an asymmetric ceramic membrane is also an
effective method for reducing liposome sizes to a relatively
well-defined size distribution. Typically, the suspension is cycled
through the membrane one or more times until the desired liposome
size distribution is achieved. The liposomes may be extruded
through successively smaller-pore membranes, to achieve a gradual
reduction in liposome size. Alternatively controlled size liposomes
can be prepared using microfluidic techniques wherein the lipid in
an organic solvent such as ethanol or ethanol-aprotic solvent
mixtures is rapidly mixed with the aqueous medium, so that the
organic solvent/water ratio is less than 30%, in a microchannel
with dimensions less than 300 microns and preferable less than 150
microns in wide and 50 microns in height. The organic solvent is
then removed from the liposomes by dialysis. Other useful sizing
methods such as sonication, solvent vaporization or reverse phase
evaporation are known to those of skill in the art.
[0045] Exemplary liposomes for use in various embodiments of the
invention have a size of from about 30 nanometers to about 40
microns.
[0046] The internal aqueous medium, as referred to herein,
typically is the original medium in which the liposomes were
prepared and which initially becomes encapsulated upon formation of
the liposome. In accordance with the present invention, freshly
prepared liposomes encapsulating the original aqueous medium can be
used directly for active loading. Embodiments are also envisaged
however wherein the liposomes, after preparation, are dehydrated,
e.g. for storage. In such embodiments the present process may
involve addition of the dehydrated liposomes directly to the
external aqueous medium used to create the transmembrane gradients.
However it is also possible to hydrate the liposomes in another
external medium first, as will be understood by those skilled in
the art. Liposomes are optionally dehydrated under reduced pressure
using standard freeze-drying equipment or equivalent apparatus. In
various embodiments, the liposomes and their surrounding medium are
frozen in liquid nitrogen before being dehydrated and placed under
reduced pressure. To ensure that the liposomes will survive the
dehydration process without losing a substantial portion of their
internal contents, one or more protective sugars are typically
employed to interact with the lipid vesicle membranes and keep them
intact as the water in the system is removed. A variety of sugars
can be used, including such sugars as trehalose, maltose, sucrose,
glucose, lactose, and dextran. In general, disaccharide sugars have
been found to work better than monosaccharide sugars, with the
disaccharide sugars trehalose and sucrose being most effective.
Other more complicated sugars can also be used. For example,
aminoglycosides, including streptomycin and dihydrostreptomycin,
have been found to protect liposomes during dehydration. Typically,
one or more sugars are included as part of either the internal or
external media of the lipid vesicles. Most preferably, the sugars
are included in both the internal and external media so that they
can interact with both the inside and outside surfaces of the
liposomes' membranes. Inclusion in the internal medium is
accomplished by adding the sugar or sugars to the buffer which
becomes encapsulated in the lipid vesicles during the liposome
formation process. In these embodiments the external medium used
during the active loading process should also preferably include
one or more of the protective sugars
[0047] As is generally known to those skilled in the art,
polyethylene glycol (PEG)-lipid conjugates have been used
extensively to improve circulation times for liposome-encapsulated
functional compounds, to avoid or reduce premature leakage of the
functional compound from the liposomal composition and to avoid
detection of liposomes by the body's immune system. Attachment of
PEG-derived lipids onto liposomes is called PEGylation. Hence, in
an exemplary embodiment of the invention, the liposomes are
PEGylated liposomes. PEGylation can be accomplished by incubating a
reactive derivative of PEG with the target liposomes. Suitable
PEG-derived lipids according to the invention, include conjugates
of DSPE-PEG, functionalized with one of carboxylic acids,
glutathione (GSH), maleimides (MAL), 3-(2-pyridyldithio) propionic
acid (PDP), cyanur, azides, amines, biotin or folate, in which the
molecular weight of PEG is between 2000 and 5000 g/mol. Other
suitable PEG-derived lipids are mPEGs conjugated with ceramide,
having either C8- or C16-tails, in which the molecular weight of
mPEG is between 750 and 5000 daltons. Still other appropriate
ligands are mPEGs or functionalized PEGs conjugated with
glycerophospholipds like
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and the
like. PEGylation of liposomes is a technique generally known by
those skilled in the art.
[0048] In various embodiments, the liposomes are PEGylated with
DSPE-PEG-GSH conjugates (up to 5 mol %) and/or DSPE-mPEG conjugates
(wherein the molecular weight of PEG is typically within the range
of 750-5000 daltons, e.g. 2000 daltons). The phospholipid
composition of an exemplary PEGylated liposome of the invention may
comprise up to 5-20 mol % of PEG-lipid conjugates.
[0049] Furthermore, in certain embodiments, one or more moieties
that specifically target the liposome to a particular cell type,
tissue or the like are incorporated into the membrane. Targeting of
liposomes using a variety of targeting moieties (e.g., ligands,
receptors and monoclonal antibodies) has been previously described.
Suitable examples of such targeting moieties include hyaluronic
acid, anti-ErbB family antibodies and antibody fragments,
lipoprotein lipase (LPL), [.alpha.]2-macroglobulin ([.alpha.]2M),
receptor associated protein (RAP), lactoferrin, desmoteplase,
tissue- and urokinase-type plasminogen activator (tPA/uPA),
plasminogen activator inhibitor (PAI-I), tPA/uPA:PAI-1 complexes,
melanotransferrin (or P97), thrombospondin 1 and 2, hepatic lipase,
factor Vila/tissue-factor pathway inhibitor (TFPI), factor Villa,
factor IXa, A[.beta.]1-40, amyloid-[.beta.] precursor protein
(APP), CI inhibitor, complement C3, apolipoproteinE (apoE),
pseudomonas exotoxin A, CRM66, HIV-I Tat protein, rhinovirus,
matrix metalloproteinase 9 (MMP-9), MMP-13 (collagenase-3),
spingolipid activator protein (SAP), pregnancy zone protein,
antithrombin III, heparin cofactor II, [.alpha.]1-antitrypsin, heat
shock protein 96 (HSP-96), platelet-derived growth factor (PDGF),
apolipoproteinJ (apoJ, or clusterin), A[.beta.] bound to apoJ and
apoE, aprotinin, angiopep-2 (TFFYGGSRGKRNNFKTEEY), very-low-density
lipoprotein (VLDL), transferrin, insulin, leptin, an insulin-like
growth factor, epidermal growth factors, lectins, peptidomimetic
and/or humanized monoclonal antibodies, dingle chain antibodies or
peptides specific for said receptors (e.g., sequences HAIYPRH and
THRPPMWSPVWP that bind to the human transferrin receptor, or
anti-human transferrin receptor (TfR) monoclonal antibody A24),
hemoglobin, non-toxic portion of a diphtheria toxin polypeptide
chain, all or a portion of the diphtheria toxin B chain, all or a
portion of a non-toxic mutant of diphtheria toxin CRM197,
apolipoprotein B, apolipoprotein E (e.g., after binding to
polysorb-80 coating), vitamin D-binding protein, vitamin
A/retinol-binding protein, vitamin B12/cobalamin plasma carrier
protein, glutathione and transcobalamin-B 12.
[0050] Targeting mechanisms generally require that the targeting
agents be positioned on the surface of the liposome in such a
manner that the target moieties are available for interaction with
the target, for example, a cell surface receptor. In an exemplary
embodiment, the liposome is manufactured to include a connector
portion incorporated into the membrane at the time of forming the
membrane. An exemplary connector portion has a lipophilic portion
which is firmly embedded and anchored in the membrane. An exemplary
connector portion also includes a hydrophilic portion which is
chemically available on the aqueous surface of the liposome. The
hydrophilic portion is selected so that it will be chemically
suitable to form a stable chemical bond with the targeting agent,
which is added later. Techniques for incorporating a targeting
moiety in the liposomal membrane are generally known in the
art.
Sparingly Water-Soluble Agent
[0051] As indicated above, the present invention provides liposomes
encapsulating a a sparingly water-soluble agent. In the context of
the present invention the term `sparingly water-soluble` means
being insoluble or having a very limited solubility in water, more
in particular having an aqueous solubility of less than 2 mg/mL,
e.g., less than 1.9 mg/mL, e.g., having an aqueous solubility of
less than 1 mg/mL. As used herein, water solubilities refer to
solubilities measured at ambient temperature, which is typically
about 20.degree. C., and pH=7.
[0052] According to an exemplary embodiment of the invention, the
sparingly water-soluble agent is a therapeutic agent selected from
the group of a therapeutic is selected from a group consisting of
an amphotericin B compound, an anthracycline compound, a
camptothecin compound, a vinca alkaloid, an ellipticine compound, a
taxane compound, a wortmannin compound, a geldanamycin compound, a
pyrazolopyrimidine compound, a peptide-based compound such as
carfilzomib, a steroid compound, a derivative of any of the
foregoing, a pro-drug of any of the foregoing, and an analog of any
of the foregoing.
[0053] Exemplary small molecule compounds having a water solubility
less than about 2 mg/mL include, but are not limited to,
amphotericin B, 2'deoxyamphotericin B, carfilzomib, voriconazole,
amiodarone, ziprasidone, aripiprazole, imatinib, lapatinib,
cyclopamine, oprozomib, CUR-61414, PF-05212384, PF-4691502,
toceranib, PF-477736, PF-337210, sunitinib, SU14813, axitinib,
AG014699, veliparib, MK-4827, ABT-263, SU11274, PHA665752,
Crizotinib, XL880, PF-04217903, XR5000, AG14361, veliparib,
bosutunib, PD-0332991, PF-01367338, AG14361, NVP-ADW742,
NVP-AUY922, NVP-LAQ824, NVP-TAE684, NVP-LBH589, erubulin,
doxorubicin, daunorubicin, mitomycin C, epirubicin, pirarubicin,
rubidomycin, carcinomycin, N-acetyladriamycin, rubidazone, 5-imido
daunomycin, N-acetyl daunomycin, daunory line, mitoxanthrone,
camptothecin, 9-aminocamptothecin, 7-ethylcamptothecin,
7-Ethyl-10-hydroxy-camptothecin, 10-hydroxycamptothecin,
9-nitrocamptothecin, 1O,11-methylenedioxycamptothecin,
9-amino-1O,11-methylenedioxycamptothecin, 9-chloro-1 0,
11-methylenedioxycamptothecin, ininotecan, lurtotecan, silatecan,
(7-(4-methylpiperazinomethylene)-10,ll-ethylenedioxy-20(S)-camptothecin,
7-(4-methylpiperazinomethylene)-10,
II-methylenedioxy-20(S)-camptothecin,
7-(2-N-isopropylamino)ethyl)-(20S)-camptothecin, CKD-602,
vincristine, vinblastine, vinorelbine, vinflunine, vinpocetine,
vindesine, ellipticine, 6-3-aminopropyl-ellipticine,
2-diethylaminoethyl-ellipticinium, datelliptium, retelliptine,
paclitaxel, docetaxel, diclofenac, bupivacaine,
17-dimethylaminoethylamino-17-demethoxygeldanamycin, cetirizine,
fexofenadine, primidone and other catecholamines, epinephrine,
(S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic
acid (deferitrin),
(S)-4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thiazolecarboxylic
acid (desferrithiocin),
(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9,12-tetraoxatridecyloxy)phenyl]-4-me-
thyl-4-thiazolecarboxylic acid,
(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6-dioxaheptyloxy)phenyl]-4-methyl-4-thi-
azolecarboxylic acid, ethyl
(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6-dioxaheptyloxy)phenyl]-4-methyl-4-thi-
azolecarboxylate,
(S)-4,5-dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)]-4-methyl-4-thiazol-
ecarboxylic acid, desazadesferrithiocin salts, 2'-hydroxyl modified
paclitaxel, 2'-hydroyxl modified docetaxel, 2'-hydroxy modified
carbazitaxel and prodrugs and derivatives of these medicinal
compounds and mixtures thereof.
[0054] An exemplary therapeutic agent is selected from: an
antihistamine ethylenediamine derivative, bromphenifamine,
diphenhydramine, an anti-protozoal drug, quinolone, iodoquinol, an
amidine compound, pentamidine, an antihelmintic compound, pyrantel,
an anti-schistosomal drug, oxaminiquine, an antifungal triazole
derivative, fliconazole, itraconazole, ketoconazole, miconazole, an
antimicrobial cephalosporin, chelating agents, deferoxamine,
deferasirox, deferiprone, FBS0701, cefazolin, cefonicid,
cefotaxime, ceftazimide, cefuoxime, an antimicrobial beta-lactam
derivative, aztreopam, cefmetazole, cefoxitin, an antimicrobial of
erythromycin group, erythromycin, azithromycin, clarithromycin,
oleandomycin, a penicillin compound, benzylpenicillin,
phenoxymethylpenicillin, cloxacillin, methicillin, nafcillin,
oxacillin, carbenicillin, a tetracycline compound, novobiocin,
spectinomycin, vancomycin; an antimycobacterial drug,
aminosalicycic acid, capreomycin, ethambutol, isoniazid,
pyrazinamide, rifabutin, rifampin, clofazimine, an antiviral
adamantane compound, amantadine, rimantadine, a quinidine compound,
quinine, quinacrine, chloroquine, hydroxychloroquine, primaquine,
amodiaquine, mefloquine, an antimicrobial, qionolone,
ciprofloxacin, enoxacin, lomefloxacin, nalidixic acid, norfloxacin,
ofloxacin, a sulfonamide; a urinary tract antimicrobial,
nitrofurantoin, trimetoprim; anitroimidazoles derivative,
metronidazole, a cholinergic quaternary ammonium compound,
ambethinium, neostigmine, physostigmine, an anti-Alzheimer
aminoacridine, tacrine, an anti-parkinsonal drug, benztropine,
biperiden, procyclidine, trihexylhenidyl, an anti-muscarinic agent,
atropine, hyoscyamine, scopolamine, propantheline, an adrenergic
compound, dopamine, serotonin, a hedgehog inhibitor, albuterol,
dobutamine, ephedrine, epinephrine, norepinephrine, isoproterenol,
metaproperenol, salmetrol, terbutaline, a serotonin reuptake
inhibitor, an ergotamine derivative, a myorelaxant, a curare
series, a central action myorelaxant, baclophen, cyclobenzepine,
dentrolene, nicotine, a nicotine receptor antagonist, a
beta-adrenoblocker, acebutil, amiodarone, abenzodiazepine compound,
ditiazem, an antiarrhythmic drug, diisopyramide, encaidine, a local
anesthetic compound, procaine, procainamide, lidocaine, flecaimide,
quinidine, an ACE inhibitor, captopril, enelaprilat, Hsp90
inhibitor, fosinoprol, quinapril, ramipril; an opiate derivative,
codeine, meperidine, methadone, morphine, an antilipidemic,
fluvastatin, gemfibrosil, an HMG-coA inhibitor, pravastatin, a
hypotensive drug, clonidine, guanabenz, prazocin, guanethidine,
granadril, hydralazine, a non-coronary vasodilator, dipyridamole,
an acetylcholine esterase inhibitor, pilocarpine, an alkaloid,
physostigmine, neostigmine, a derivative of any of the foregoing, a
pro-drug of any of the foregoing, and ananalog of any of the
foregoing.
[0055] This list of agents, however, is not intended to limit the
scope of the invention. In fact, the compound encapsulated within
the liposome can be any sparingly water-soluble amphipathic weak
base or amphipathic weak acid. As noted above, embodiments wherein
the sparingly water-soluble agent is not a pharmaceutical or
medicinal agent are also encompassed by the present invention.
[0056] Typically, within the context of the present invention,
sparingly water-soluble amphipathic weak bases have an
octanol-water distribution coefficient (log D) at pH 7 between -2.5
and 7.0 and pKa <11, while sparingly water-soluble amphipathic
weak acids have a log D at pH 7 between -2.5 and 7.0 and pKa >3.
The pKa is measured in water.
[0057] Typically, the terms weak base and weak acid, as used in the
foregoing, respectively refer to compounds that are only partially
protonated or deprotonated in water. Examples of protonable agents
include compounds having an amino group, which can be protonated in
acidic media, and compounds which are zwitterionic in neutral media
and which can also be protonated in acidic environments. Examples
of deprotonable agents include compounds having a carboxy group,
which can be deprotonated in alkaline media, and compounds which
are zwitterionic in neutral media and which can also be
deprotonated in alkaline environments.
[0058] The term zwitterionic refers to compounds that can
simultaneously carry a positive and a negative electrical charge on
different atoms. The term amphipathic, as used in the foregoing is
typically employed to refer to compounds having both lipophilic and
hydrophilic moieties. The foregoing implies that aqueous solutions
of compounds being weak amphipathic acids or bases simultaneously
comprise charged and uncharged forms of said compounds. Only the
uncharged forms may be able to cross the liposomal membrane.
[0059] When agents of use in the present invention contain
relatively basic or acidic functionalities, salts of such compounds
are included in the scope of the invention. Salts can be obtained
by contacting the neutral form of such compounds with a sufficient
amount of the desired acid or base, either neat or in a suitable
inert solvent. Examples of salts for relative acidic compounds of
the invention include sodium, potassium, calcium, ammonium, organic
amino, or magnesium salts, or a similar salts. When compounds of
the present invention contain relatively basic functionalities,
acid addition salts can be obtained by contacting the neutral form
of such compounds with a sufficient amount of the desired acid,
either neat or in a suitable inert solvent. Examples of acid
addition salts include those derived from inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from organic acids like acetic,
propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic,
fumaric, lactic, mandelic, phthalic, benzenesulfonic,
p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
Also included are salts of amino acids such as arginate and the
like, and salts of organic acids like glucuronic or galactunoric
acids and the like (see, for example, Berge et al., Journal of
Pharmaceutical Science 1977, 66: 1-19). Certain specific compounds
of the present invention contain both basic and acidic
functionalities that allow the compounds to be converted into
either base or acid addition salts.
[0060] The neutral forms of the compounds are preferably
regenerated by contacting the salt with a base or acid and
isolating the parent compound in the conventional manner. The
parent form of the compound differs from the various salt forms in
certain physical properties, such as solubility in polar solvents,
but otherwise the salts are equivalent to the parent form of the
compound for the purposes of the present invention.
Active Loading
[0061] The process of active loading, involves the use of
transmembrane potentials. The principle of active loading, in
general, has been described extensively in the art. The terms
active-loading and remote-loading are synonymous and will be used
interchangeably.
[0062] During active loading, the precipitate of the sparingly
water-soluble agent is transferred from the external aqueous medium
across the liposomal membrane to the internal aqueous medium by a
transmembrane proton- or ion-gradient. The term gradient of a
particular compound as used herein refers to a discontinuous
increase of the concentration of said compound across the liposomal
membrane from outside (external aqueous medium) to inside the
liposome (internal aqueous medium).
[0063] To create the concentration gradient, the liposomes are
typically formed in a first liquid, typically aqueous, phase,
followed by replacing or diluting said first liquid phase. The
diluted or new external medium has a different concentration of the
charged species or a totally different charged species, thereby
establishing the ion- or proton-gradient.
[0064] The replacement of the external medium can be accomplished
by various techniques, such as, by passing the lipid vesicle
preparation through a gel filtration column, e.g., a Sephadex or
Sepharose column, which has been equilibrated with the new medium,
or by centrifugation, dialysis, or related techniques.
[0065] The efficiency of active-loading into liposomes depends,
among other aspects, on the chemical properties of the complex to
be loaded and the type and magnitude of the gradient applied. In an
embodiment of the invention, a method as defined in any of the
foregoing is provided employing a gradient across the liposomal
membrane, in which the gradient is chosen from a pH-gradient, a
sulfate-, phosphate-, citrate-, or acetate-salt gradient, an
EDTA-ion gradient, an ammonium-salt gradient, an alkylated, e.g
methyl-, ethyl-, propyl- and amyl, ammonium-salt gradient, a
Ca.sup.2+, Cu.sup.2+, Fe.sup.+2, Mg.sup.2+, Mn.sup.2+, Zn.sup.2+,
Na.sup.+-, K.sup.+-gradient, with or without using ionophores, or a
combination thereof. These loading techniques have been extensively
described in the art.
[0066] Preferably, the internal aqueous medium of pre-formed, i.e.,
unloaded, liposomes comprises a so-called active-loading buffer
which contains water and, dependent on the type of gradient
employed during active loading, may further comprise a sulfate-,
phosphate-, citrate-, or acetate-salt, an ammonium-salt, an
alkylated, e.g., methyl-, ethyl-, propyl- and amyl, ammonium-salt,
an Ca.sup.2+, Cu.sup.2+, Fe.sup.+2, Mg.sup.2+, Mn.sup.2+,
Zn.sup.2+, Na.sup.+, and/or K.sup.+-salt, an EDTA-ion salt, and
optionally a pH-buffer to maintain a pH-gradient. The salts may be
polymeric such as dextran sulfate, polyethyleneimine,
polyamidoamine dendrimers, the 1.5 carboxylate terminal version of
polyamidoamines, polyphosphates, low molecular weight heparin, or
hyaluronic acid. In an exemplary embodiment, the concentration of
salts in the internal aqueous medium of unloaded liposomes is
between 1 and 1000 mM.
[0067] The external aqueous medium, used to establish the
transmembrane gradient for active loading, comprises water, the
precipitate of the sparingly water-soluble agent(s) to be loaded,
and optionally sucrose, saline or some other agent to adjust the
osmolarity and/or a chelator like EDTA to aid ionophore activity,
more preferably sucrose and/or EDTA.
[0068] In an exemplary embodiment the gradient is chosen from an
amine or a metal salt of a member selected from a carboxylate,
sulfate or phosphate or an acetate. As is generally known by those
skilled in the art, transmembrane pH- (lower inside, higher outside
pH) or cation acetate-gradients can be used to actively load
amphiphilic weak acids. Amphipathic weak bases can also be actively
loaded into liposomes using an ammonium sulfate- or triethylamine
sulfate, triethylamine dextran sulfate or ammonium
chloride-gradient.
[0069] Carboxylates of use in the invention include, without
limitation, carboxylate, citrate, diethylenetriaminepentaaceetate,
melletic acetate, 1,2,3,4-butanetetracarboxylate, benzoate,
isophalate, phthalate,
3,4-bis(carboxymethyl)cyclopentanecarboxylate, the carboxylate
generation of polyamidoamine dendrimers, benzenetricarboxylates,
benzenetetracarboxylates, ascorbate, ascorbate phosphate,
glucuronate, and ulosonate.
[0070] Sulfates of use in the invention include, but are not
limited to, sulfate, 1,5-naphthalenedisulfonate, dextran sulfate,
sulfobutlyether beta cyclodextrin, sucrose octasulfate benzene
sulfonate, poly(4-styrenesulfonate) trans
resveratrol-trisulfate.
[0071] Phosphates of use in the invention include, but are not
limited to, phosphate, ascorbate phosphate, hexametaphosphate,
phosphate glasses, polyphosphates, triphosphate, trimetaphosphate,
bisphosphonates, ethanehydroxy bisphosphonate, inositol
hexaphosphate
[0072] Exemplary salts of use in the invention include a mixture of
carboxylate, sulfate or phosphate including but not limited to:
2-carboxybenensulfonate, creatine phosphate, phosphocholine,
carnitine phosphate, the carboxyl generation of
polyamidoamines.
[0073] Amines of use in the invention include, but are not limited
to, monoamines, polyamines, trimethylammonium, triethylammonium,
tributyl ammonium, diethylmethylammonium, diisopropylethyl
ammonium, triisopropylammonium, N-methylmorpholinium,
N-ethylmorpholinium, N-hydroxyethylpiperidinium,
N-methylpyrrolidinium, N,N-dimethylpiperazinium,
isopropylethylammonium, isopropylmethylammonium,
diisopropylammonium, tert-butylethylammonium, dicychohexylammonium,
protonized forms of morpholine, pyridine, piperidine, pyrrolidine,
piperazine, imidazole, tert-butylamine, 2-amino-2-methylpropanol,
2-amino-2-methyl-propandiol, tris-(hydroxyethyl)-aminomethane,
diethyl-(2-hydroxyethyl)amine, tris-(hydroxymethyl)-aminomethane
tetramethylammonium, tetraethylammonium, N-methylglucamine and
tetrabutylammonium, polyethyleneimine, and polyamidoamine
dendrimers.
[0074] Depending upon the permeability of the lipid vesicle
membranes, the full transmembrane potential corresponding to the
concentration gradient will either form spontaneously or a
permeability enhancing agent, e.g., a proton ionophore can be added
to the medium. If desired, the permeability enhancing agent can be
removed from the liposome preparation after loading with the
complex is complete using chromatography or other techniques.
[0075] Typically the temperature of the medium during active
loading is between about -25.degree. C. and about 100.degree. C.,
e.g., between about 0.degree. C. and about 70.degree. C., e.g.,
between about 4.degree. C. and 65.degree. C.
[0076] The encapsulation or loading efficiency, defined as
encapsulated amount (e.g., as measured in grams of agent/moles of
phospholipid or g of drug/g total lipid) of the sparingly
water-soluble agent in the internal aqueous phase divided by the
initial amount in the external aqueous phase multiplied by 100%, is
at least 10%, preferably at least 50%, at least 90%.
[0077] In an exemplary embodiment, the invention provides a method
of loading a sparingly water-soluble agent into a liposome. An
exemplary method comprises, contacting an aqueous suspension of
said liposome with said aqueous suspension of said agent under
conditions appropriate to encapsulate said sparingly water-soluble
agent in said liposome, wherein said liposome has an internal
aqueous environment encapsulated by a lipid membrane and said
aqueous suspension of said liposome comprises a gradient selected
from a proton gradient, an ion gradient and a combination thereof
across said membrane, and wherein said conditions are appropriate
for said sparingly water-soluble agent to traverse said membrane
and concentrate in said internal aqueous environment, thereby
forming said pharmaceutical formulation.
[0078] In various embodiments, the reaction mixture above is
incubated for a selected period of time and the pH gradient,
sulfate gradient, phosphate gradient, carboxylate gradient (citrate
gradient, acetate gradient), EDTA ion gradient, ammonium salt
gradient, alkylated ammonium salt gradient, Ca.sup.2+, Cu.sup.2+,
Fe.sup.+2, Mg.sup.2+, Mn.sup.2+, Zn.sup.2+, Na.sup.+ gradient,
K.sup.+ gradient or a combination thereof, exists across the
liposomal membrane during the incubating.
[0079] In exemplary embodiments of the invention, the sparingly
water-soluble therapeutic agent is not covalently attached to a
component of the liposome, nor is it covalently attached to any
component of the pH or salt gradient used to form the liposomal
preparation of the invention.
Aprotic Solvent
[0080] In an exemplary embodiment, the sparingly water-soluble
agent is completely dissolved in an aprotic solvent that is
miscible with water. The agent solution is added to the aqueous
liposome suspension at a concentration that is greater than the
solubility of the drug agent in either the liposome suspension or
the liposome suspension/aprotic solvent mixture, thus a precipitate
is formed. Exemplary aprotic solvents include dimethylsulfoxide,
dioxane, tetrahydrofuran, dimethylformamide, acetonitrile,
dimethylacetamide, sulfolane, gamma butyrolactone, pyrrolidones,
1-methyl-2-pyrrolidinone, methylpyrroline, ethylene glycol
monomethyl ether, diethylene glycol monomethyl ether, PEG400 and
polyethylene glycols.
Sparingly Water-Soluble Agent Precipitate
[0081] The invention describes loading of an insoluble precipitate.
An exemplary precipitate is conceptualized as some insoluble
portion of the agent in suspension. The insoluble portion is
defined as a portion of the agent that is not solvated as indicated
by any of the following: any appearance of cloudiness greater than
that of the liposome suspension in the absence of the agent, any
degree of increased light scattering at a wavelength where the
contents do not absorb light, such at 600 nm greater than the
liposome suspension alone, any portion of the drug than can be
isolated (pelleted) through centrifugation at a rate below 12,000
RPM for 15 min, any portion of the drug agent than can be isolated
by filtration through 0.2 um filter.
Kits
[0082] In an exemplary embodiment, the invention provides a kit
containing one or more components of the liposomes or formulations
of the invention and instructions on how to combine and use the
components and the formulation resulting from the combination. In
various embodiments, the kit includes a the sparingly water-soluble
agent in one vessel and a liposome preparation in another vessel.
An exemplary liposome preparation includes a distribution of salt
on the outside and inside of the lipid membrane to establish and/or
maintain an ion gradient, such as that described herein. Also
included are instructions for combining the contents of the vessels
to produce a liposome or a formulation thereof of the invention. In
various embodiments, the amount of complex and liposome are
sufficient to formulate a unit dosage formulation of the complexed
agent.
[0083] In an exemplary embodiment, one vessel includes a liposome
or liposome solution, which is used to convert at least part of the
contents of a vessel of a sparingly water-soluble therapeutic agent
formulation (e.g., of an approved therapeutic agent) into a liquid
formulation of the liposome encapsulated therapeutic agent at the
point of care for administration to a subject. In an exemplary
embodiment, the contents of the vessels are sufficient to formulate
a unit dosage formulation of the therapeutic agent.
[0084] In the embodiment in which a unit dosage format is formed,
the vessel includes from about 1 mg to about 500 mg of the
therapeutic agent, e.g, from about 1 mg to about 200 mg, e.g., from
about 5 mg to about 100 mg, e.g., from about 10 mg to about 60
mg.
[0085] In an exemplary embodiment, the approved therapeutic agent
is carfilzomib and it is present in the vessel in an amount of from
about 40 mg to about 80 mg, e.g., from about 50 mg to about 70 mg.
In an exemplary embodiment, the carfilzomib is present in about 60
mg.
Methods of Treatment
[0086] In one aspect, the invention provides a method of treating a
proliferative disorder, e.g., a cancer, in a subject, e.g., a
human, the method comprising administering a composition that
comprises a pharmaceutical formulation of the invention to a
subject in an amount effective to treat the disorder, thereby
treating the proliferative disorder.
[0087] In one embodiment, the pharmaceutical formulation is
administered in combination with one or more additional anticancer
agent, e.g., chemotherapeutic agent, e.g., a chemotherapeutic agent
or combination of chemotherapeutic agents described herein, and
radiation.
[0088] In one embodiment, the cancer is a cancer described herein.
For example, the cancer can be a cancer of the bladder (including
accelerated and metastatic bladder cancer), breast (e.g., estrogen
receptor positive breast cancer; estrogen receptor negative breast
cancer; HER-2 positive breast cancer; HER-2 negative breast cancer;
progesterone receptor positive breast cancer; progesterone receptor
negative breast cancer; estrogen receptor negative, HER-2 negative
and progesterone receptor negative breast cancer (i.e., triple
negative breast cancer); inflammatory breast cancer), colon
(including colorectal cancer), kidney (e.g., transitional cell
carcinoma), liver, lung (including small and non-small cell lung
cancer, lung adenocarcinoma and squamous cell cancer),
genitourinary tract, e.g., ovary (including fallopian tube and
peritoneal cancers), cervix, prostate, testes, kidney, and ureter,
lymphatic system, rectum, larynx, pancreas (including exocrine
pancreatic carcinoma), esophagus, stomach, gall bladder, thyroid,
skin (including squamous cell carcinoma), brain (including
glioblastoma multiforme), head and neck (e.g., occult primary), and
soft tissue (e.g., Kaposi's sarcoma (e.g., AIDS related Kaposi's
sarcoma), leiomyosarcoma, angiosarcoma, and histiocytoma).
[0089] In an exemplary embodiment, the cancer is multiple myeloma.
In one embodiment, the pharmaceutical formulation of the invention
includes carfilzomib as the sparingly water-soluble therapeutic
agent.
[0090] In one aspect, the disclosure features a method of treating
a disease or disorder associated with inflammation, e.g., an
allergic reaction or an autoimmune disease, in a subject, e.g., a
human, the method comprises: administering a composition that
comprises a Pharmaceutical formulation of the invention to a
subject in an amount effective to treat the disorder, to thereby
treat the disease or disorder associated with inflammation.
[0091] In one embodiment, the disease or disorder associated with
iron overload such as occurs when a patient receives multiple units
of blood transfusion such as occurs in thalassemia, sickle anemia,
traumatic injury or after a bone marrow transplant. The iron
overload may be local, such as can occur in endometriosis due to
the extravasation of red blood cells into the local tissue where
the provoke an inflammatory immune response.
[0092] In one embodiment, the disease or disorder associated with
inflammation is a disease or disorder described herein. For
example, the disease or disorder associated with inflammation can
be for example, multiple sclerosis, rheumatoid arthritis, psoriatic
arthritis, degenerative joint disease, spondouloarthropathies,
gouty arthritis, systemic lupus erythematosus, juvenile arthritis,
rheumatoid arthritis, osteoarthritis, osteoporosis, diabetes (e.g.,
insulin dependent diabetes mellitus or juvenile onset diabetes),
menstrual cramps, cystic fibrosis, inflammatory bowel disease,
irritable bowel syndrome, Crohn's disease, mucous colitis,
ulcerative colitis, gastritis, esophagitis, pancreatitis,
peritonitis, Alzheimer's disease, shock, ankylosing spondylitis,
gastritis, conjunctivitis, pancreatitis (acute or chronic),
multiple organ injury syndrome (e.g., secondary to septicemia or
trauma), myocardial infarction, atherosclerosis, stroke,
reperfusion injury (e.g., due to cardiopulmonary bypass or kidney
dialysis), acute glomerulonephritis, vasculitis, thermal injury
(i.e., sunburn), necrotizing enterocolitis, granulocyte transfusion
associated syndrome, and/or Sjogren's syndrome. Exemplary
inflammatory conditions of the skin include, for example, eczema,
atopic dermatitis, contact dermatitis, urticaria, scleroderma,
psoriasis, and dermatosis with acute inflammatory components. In
some embodiments, the autoimmune disease is an organ-tissue
autoimmune diseases (e.g., Raynaud's syndrome), scleroderma,
myasthenia gravis, transplant rejection, endotoxin shock, sepsis,
psoriasis, eczema, dermatitis, multiple sclerosis, autoimmune
thyroiditis, uveitis, systemic lupus erythematosis, Addison's
disease, autoimmune polyglandular disease (also known as autoimmune
polyglandular syndrome), or Grave's disease.
[0093] In another embodiment, a pharmaceutical formulation of the
invention or method described herein may be used to treat or
prevent allergies and respiratory conditions, including asthma,
bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity,
emphysema, chronic bronchitis, acute respiratory distress syndrome,
and any chronic obstructive pulmonary disease (COPD). The
pharmaceutical formulation of the invention, particle or
composition may be used to treat chronic hepatitis infection,
including hepatitis B and hepatitis C.
[0094] In one aspect, the disclosure features a method of treating
cardiovascular disease, e.g., heart disease, in a subject, e.g., a
human, the method comprising administering a a pharmaceutical
formulation of the invention to a subject in an amount effective to
treat the disorder, thereby treating the cardiovascular
disease.
[0095] In one embodiment, cardiovascular disease is a disease or
disorder described herein. For example, the cardiovascular disease
may be cardiomyopathy or myocarditis; such as idiopathic
cardiomyopathy, metabolic cardiomyopathy, alcoholic cardiomyopathy,
drug-induced cardiomyopathy, ischemic cardiomyopathy, and
hypertensive cardiomyopathy. Also treatable or preventable using a
pharmaceutical formulation of the inventions, particles,
compositions and methods described herein are atheromatous
disorders of the major blood vessels (macrovascular disease) such
as the aorta, the coronary arteries, the carotid arteries, the
cerebrovascular arteries, the renal arteries, the iliac arteries,
the femoral arteries, and the popliteal arteries. Other vascular
diseases that can be treated or prevented include those related to
platelet aggregation, the retinal arterioles, the glomerular
arterioles, the vasa nervorum, cardiac arterioles, and associated
capillary beds of the eye, the kidney, the heart, and the central
and peripheral nervous systems. Yet other disorders that may be
treated with pharmaceutical formulation of the invention, include
restenosis, e.g., following coronary intervention, and disorders
relating to an abnormal level of high density and low density
cholesterol.
[0096] In one embodiment, the pharmaceutical formulation of the
invention can be administered to a subject undergoing or who has
undergone angioplasty. In one embodiment, the Pharmaceutical
formulation of the invention, particle or composition is
administered to a subject undergoing or who has undergone
angioplasty with a stent placement. In some embodiments, the
pharmaceutical formulation of the invention, particle or
composition can be used as a strut of a stent or a coating for a
stent.
[0097] In one aspect, the invention provides a method of treating a
disease or disorder associated with the kidney, e.g., renal
disorders, in a subject, e.g., a human, the method comprises:
administering a pharmaceutical formulation of the invention to a
subject in an amount effective to treat the disorder, thereby
treating the disease or disorder associated with kidney
disease.
[0098] In one embodiment, the disease or disorder associated with
the kidney is a disease or disorder described herein. For example,
the disease or disorder associated with the kidney can be for
example, acute kidney failure, acute nephritic syndrome, analgesic
nephropathy, atheroembolic renal disease, chronic kidney failure,
chronic nephritis, congenital nephrotic syndrome, end-stage renal
disease, good pasture syndrome, interstitial nephritis, kidney
damage, kidney infection, kidney injury, kidney stones, lupus
nephritis, membranoproliferative GN I, membranoproliferative GN II,
membranous nephropathy, minimal change disease, necrotizing
glomerulonephritis, nephroblastoma, nephrocalcinosis, nephrogenic
diabetes insipidus, nephrosis (nephrotic syndrome), polycystic
kidney disease, post-streptococcal GN, reflux nephropathy, renal
artery embolism, renal artery stenosis, renal papillary necrosis,
renal tubular acidosis type I, renal tubular acidosis type II,
renal underperfusion, renal vein thrombosis.
[0099] In one embodiment, the disease or disorder is caused by a
microbe or virus. These infectious agents can be viruses such as
HIV, fungi such as aspergillosis, bacteria such as staphylococcus,
protist, such as malaria or multicellular infectious agents, such
as schistosomyosis.
[0100] An "effective amount" or "an amount effective" refers to an
amount of the pharmaceutical formulation of the invention which is
effective, upon single or multiple dose administrations to a
subject, in treating a cell, or curing, alleviating, relieving or
improving a symptom of a disorder. An effective amount of the
composition may vary according to factors such as the disease
state, age, sex, and weight of the individual, and the ability of
the compound to elicit a desired response in the individual. An
effective amount is also one in which any toxic or detrimental
effects of the composition are outweighed by the therapeutically
beneficial effects.
[0101] As used herein, the term "prevent" or "preventing" as used
in the context of the administration of an agent to a subject,
refers to subjecting the subject to a regimen, e.g., the
administration of a pharmaceutical formulation of the invention
such that the onset of at least one symptom of the disorder is
delayed as compared to what would be seen in the absence of the
regimen.
[0102] As used herein, the term "subject" is intended to include
human and non-human animals. Exemplary human subjects include a
human patient having a disorder, e.g., a disorder described herein,
or a normal subject. The term "non-human animals" includes all
vertebrates, e.g., non-mammals (such as chickens, amphibians,
reptiles) and mammals, such as non-human primates, domesticated
and/or agriculturally useful animals, e.g., sheep, dog, cat, cow,
pig, etc.
[0103] As used herein, the term "treat" or "treating" a subject
having a disorder refers to subjecting the subject to a regimen,
e.g., the administration of a pharmaceutical formulation of the
invention such that at least one symptom of the disorder is cured,
healed, alleviated, relieved, altered, remedied, ameliorated, or
improved. Treating includes administering an amount effective to
alleviate, relieve, alter, remedy, ameliorate, improve or affect
the disorder or the symptoms of the disorder. The treatment may
inhibit deterioration or worsening of a symptom of a disorder.
[0104] The following examples are provided to illustrate exemplary
embodiments of the invention and are not to be construed as
limiting the scope of the invention.
EXAMPLES
Example 1
Carfilzomib Liposome Entrapment by Remote Loading
Materials and Method
[0105] Ammonium sulfate solution was prepared by dissolving
ammonium sulfate solid to a final concentration of 250 mM (500
mequivilents of anion/L) no pH adjustment was made to yield a final
pH of 5.6. Sodium sulfate solution (250 mM) was prepared by adding
0.35 g sodium sulfate to 10 mL deionized water.
[0106] The liposomes were formed by extrusion. Lipids were
dissolved in ethanol at a concentration of 500 mM HSPC (591 mg/mL
total lipid) at 65.degree. C. and the 9 volumes of the trapping
agent solution heated to 65.degree. C. was added to the
ethanol/lipid solution also at 65.degree. C. The mixture was
vortexed and transferred to a 10 mL thermostatically controlled
(65.degree. C.) Lipex Extruder. The liposomes were formed by
extruding 10 times through polycarbonate membranes having 0.1 um
pores. After extrusion the liposomes were cooled on ice. The
transmembrane electrochemical gradient was formed by purification
of the liposomes by dialysis in dialysis tubing having a molecular
weight cut off of 12,000-14,000. The samples are dialyzed against 5
mM HEPES, 10% sucrose pH 6.5 (stirring at 4.degree. C.) at volume
that is 100 fold greater than the sample volume. The dialysate was
changed after 2 h then 4 more times after 12 h each. The
conductivity of the liposome solution was measured and was
indistinguishable from the dialysis medium .about.40 .mu.S/cm.
[0107] The lipid concentration is determined by measuring the
cholesterol by HPLC using an Agilent 1100 HPLC with and Agilent
Zorbax 5 um, 4.6.times.150 mM, Eclipse XDB-C8 column and a mobile
phase of A=0.1% TFA, B=0.1% TFA/MeOH with an isocratic elution of
99% B. The flow rate is 1.0 mL/min, column temperature is
50.degree. C., 10 .mu.L injection and detection by absorbance at
205 nm. The retention time of cholesterol is 4.5 min. The liposome
size is measured by dynamic light scattering.
[0108] Carfilzomib (Selleck Chemicals) was dissolved in DMSO at a
concentration of 10 mg/mL. The carfilzomib was introduced to the
liposomes at a carfilzomib to HSPC ratio of 100 g drug/mol HSPC
(drug to total lipid ratio (wt/wt) of 0.12). The liposomes were
diluted with 50 mM citrate, 10% sucrose pH 4.0 to increase the
volume to a point where after addition of the drug the final DMSO
concentration is 2%. The carfilzomib/DMSO was added to the diluted
liposomes, which were mixed at room temperature then transferred to
a 65.degree. C. bath and swirled every 30 s for the first 3 min and
then swirled every 5 min over a total heating time of 30 min. All
samples were very cloudy when the drug was added and all became
clear (same as liposomes with no drug added) after 15 min. After
heating for 30 min all samples were placed on ice for 15 min. The
loaded liposomes were vortexed and 100 .mu.L of sample was kept as
the "before column" and the rest transferred to microcentrifuge
tubes and spun at 10,000 RPM for 5 min. The supernatants were
purified on a Sephadex G25 column collected and analyzed by HPLC.
The HPLC analysis of carfilzomib was performed on the same system
as described for analysis of cholesterol. The mobile phase consists
of A=0.1% TFA, B=0.1% TFA/MeOH with a gradient elution starting at
50% B and increasing to 83% B in 13 min with 7 min equilibration
back to 50% B. The flow rate is 1.0 mL/min, column temperature is
30 C, 10 .mu.l injection and detection by absorbance at 205 nm. The
retention time of carfilzomib is 12.2 min. The lipid concentration
is determined by analysis of the cholesterol by HPLC.
Results
[0109] The loading of liposomes containing 250 mM ammonium sulfate
resulted in a final drug to lipid ratio of 95.26.+-.3.47 g drug/mol
of HSPC liposomes when the drug was added at 100 g drug/mol of HSPC
lipid (95.26.+-.3.47% efficient) and a final drug to lipid ratio of
136.9.+-.7.35 g drug/mol of HSPC liposomes when the drug was added
at 200 g drug/mol of HSPC lipid (67.94.+-.3.67% efficient) (FIG.
2). This demonstrates that the loading capacity of these particular
liposomes is between 100 and 200 g drug/mol phospholipid. The
control liposomes containing 250 mM sodium sulfate which have no
electrochemical gradient for remote loading resulted in a final
drug load of 33.28.+-.0.79 and 29.01.+-.0.79 g drug/mol of HSPC
when the drug was added at a ratio of 100 and 200 g drug/mol of
HSPC respectively. This demonstrates that the capacity for loading
these liposomes was saturated below 100 g drug/mol of HSPC and at
this drug input ratio the remote loaded liposomes exhibit >3
fold higher loading capacity. Saturation of the drug loading
capacity for sodium sulfate liposomes at a ratio at least 3 fold
lower than the ammonium sulfate liposomes indicates that when no
electrochemical gradient is present for remote loading the drug
partitions into the lipid bilayer but does not form a salt with the
interior trapping agent. FIG. 5 illustrates the precipitate is
still present after the loading process with sodium sulfate
liposomes but not with ammonium sulfate liposomes.
Conclusion
[0110] Liposomes of identical lipid matrix composition and size but
varying in the composition of the sulfate salt internally trapped
had very different capabilities to load carfilzomib. The liposome
capable of generating an electrochemical gradient (ammonium
sulfate) was able to load close to 100% of the drug at optimal
conditions and the one incapable of creating a gradient had poor
loading efficiency suggesting that remote or active loading was the
primary mechanism for carfilizomib incorporation into the
liposome.
Example 2
Comparison of Liposome Trapping Agents
Introduction
[0111] Liposomes to be used for remote loading are formed in an
ionic solution that is intended to complex the loaded drug as a
salt. Trapping agents can form complexes with loaded drugs and the
stability of this complex is one factor that dictates liposome drug
loading ability, stability and drug release rates. Comparison of
different liposome trapping agents was made by evaluating the
efficiency of carfilzomib loading.
Methods
[0112] Three liposome formulations were used, all at a molar ratio
3 HSPC/2 Chol/0.15 PEG-DSPE each with a different trapping agent:
1. mellitic acid; 2. ammonium sulfate; and 3. napthelene
disulfononic acid.
[0113] Mellitic acid (MA) was dissolved in water and titrated with
diethylamine to a final pH of 5.5 and concentration of 83 mM (500
mequivilents of anion/L). Ammonium sulfate was prepared by
dissolving ammonium sulfate solid to a final concentration of 250
mM (500 mequivilents of anion/L) no pH adjustment was made to yield
a final pH of 5.6.
[0114] Napthelenedisulfonic acid (NDS) was dissolved in water and
titrated with diethylamine to a final pH of 8.0 and concentration
of 250 mM (500 mequivilents of anion/L).
[0115] See Example 1, Carfilzomib liposome entrapment by remote
loading for details on how the liposomes were made, purified and
characterized.
TABLE-US-00001 TABLE 1 Sizes of Liposomes Loaded with Carfilzomib.
before lyophilizing Z-ave Trapping agent (nm) PDI
(NH4).sub.2SO.sub.4 108 0.062 (Drug added quickly)
(NH4).sub.2SO.sub.4 109.2 0.035 (Drug added slowly)
Napthelenedisulfonic acid 111.9 0.039 Mellitic Acid 105.5 0.08
[0116] To ensure complete removal of the DMSO added with
carfilzomib, the liposomal carfilzomib samples were dialyzed in
dialysis tubing having a molecular weight cut off of 12,000-14,000.
The samples are dialyzed against 5 mM HEPES, 10% sucrose pH 6.5
(stirring at 4.degree. C.) at volume that is 100 fold greater than
the sample volume. The dialysate was changed after 2 h then 2 more
times after 12 h each. The carfilzomib liposomes were again
analyzed for drug and lipid concentration as described above.
Results
[0117] The efficiency of carfilzomib remote loading into liposomes
at 100 g drug/mol of HSPC lipid for liposomes with the trapping
agents mellitic acid, ammonium sulfate, and napthelenedisulfonic
acid were 37.4%.+-.2.01%, 97.0%.+-.2.38%, and 95.1%.+-.1.76%
respectively (FIG. 3).
Conclusion
[0118] The invention described here enables remote loading of
carfilzomib from an insoluble precipitate into liposomes can be
accomplished with various using the electrochemical gradient
generated by various trapping agents including mellitic acid,
ammonium sulfate and napthelene disulfononic acid.
Example 3
Comparison of Method for Introducing Drug
Method
[0119] A comparison of the method used for addition of the drug to
the liposomes during the loading procedure. The loading procedure
was the same as described above in Example 1 with the exception of
the drug being added to the liposome loading solution as a solid
powder, as a 10 mg/mL DMSO solution quickly and as 10 mg/mL DMSO
solution slowly.
Results
[0120] The efficiency of carfilzomib remote loading into liposomes
at 100 g drug/mol of HSPC lipid for the drug which was added as the
solid powder was 3.88%.+-.0.053% and 3.47%.+-.0.030% when heated to
65.degree. C. for 30 and 120 min respectively. The efficiency of
loading the drug as a 10 mg/mL DMSO solution was 97.0%.+-.2.38%
when the drug/DMSO was added quickly and 96.3%.+-.1.09% when the
drug/DMSO was added in 5 increments over 1 min to a liposome
solution while vortexing. The drug/liposome mixture that results
from the slow drug addition is clearer than the drug/liposome
mixture that results from rapid addition of the drug. However, both
solutions have no visible precipitate (or centrifugal precipitate
at 10,000 rpm for 5 min) after heating to 65.degree. C. for 30 min,
which is a result of all of the drug being loaded into the
liposomes regardless of the precipitate formed upon addition of the
drug (FIG. 4).
Example 4
[0121] Carfilzomib Loading into Liposomes at Room Temperature
Introduction
[0122] The ability to load a drug into liposomes at room
temperature is beneficial to reduce heat-induced drug degradation,
simplify manufacturing and allow for bedside liposome loading.
Efficient transport across the liposome membrane requires the
membrane to be in a fluid phase. This is accomplished with
saturated phospholipids having a high phase transition temperature
(T.sub.m) such as HSPC (T.sub.m=55.degree. C.) by heating the
liposomes above the T.sub.m during the loading process. An
alternative to heating is to use lipids that are fluid phase at
room temperature. The disadvantage of these lipids is that they are
unstable in circulation and result in rapid drug release. Sterol
modified lipids incorporate a novel lipid construction where
cholesterol (sterol) is covalently attached to the phosphate
headgroup. Sterol modified lipids have proven to render the sterol
non-exchangable from the lipid bilayer in circulation. Sterol
modified lipids are also fluid phase at room temperature, making
them ideal for room temperature loading of drugs into liposomes
that are to be used for in vivo delivery of therapeutics.
Method
[0123] The loading of carfilzomib into liposomes at room
temperature was performed by using two liposome formulations
composed of a molar ratio of 95 PChemsPC/5 PEG-DSPE and another
with a molar ratio of 3 POPC/2 Chol/0.15 PEG-DSPE each containing
250 mM ammonium sulfate as the trapping agent. The liposomes were
prepared using the procedure, drug/liposome ratio, buffers ad pH as
described in Example 1. The liposomes were stirred at room
temperature (20.degree. C.) and the carfilzomib was added as a 10
mg/mL DMSO solution in 5 increments over 1 min to result in a
cloudy solution. The liposome/drug mixture was stirred at room
temperature for a total of 30 min to yield a clear solution with
the same appearance as the liposome solution before the drug was
added.
Results
[0124] The efficiency of carfilzomib remote loading into liposomes
at 100 g drug/mol of PChemsPC was 95.5%.+-.1.23% The efficiency of
carfilzomib remote loading into liposomes at 187 g drug/mol of 3
POPC/2 Chol/0.15 PEG-DSPE was 100.52%.+-.1.01%
Conclusion
[0125] The invention described here was not able to load
carfilzomib into liposomes by adding the crystal form of the drug
directly to the loading solution. The drug requires solubilization
in some solvent prior to addition to the loading solution at a
concentration above the solubility of the drug. Liposome loading
efficiency of the precipitate that is formed upon addition of the
drug to the loading solution is not dependent upon the rate of
addition in this case using carfilzomib.
Example 5
[0126] Drug Precipitate Loading into Liposomes as Determined by
Light Scattering at 600 nm.
Introduction
[0127] Liposome loading of drug from a precipitate into liposomes
is evidenced by the resulting drug to lipid ratio and clarifying of
the solution as the drug precipitate transfers into the liposome.
To get a quantitative measure liposome loading from a drug
precipitate the light scattering was measured at 600 nm during the
loading process.
Method
[0128] Liposomes containing 250 mM ammonium sulfate as trapping
agent and 250 mM sodium sulfate as control liposomes which would
not remote load drug. The liposomes were prepared and loaded using
the procedure described in Example 1 except a disposable
polystyrene cuvette was used as the reaction vessel. The scattering
of light at 600 nm was measured with a UV/vis spectrophotometer
during the loading process.
Results/Conclusion
[0129] The sodium sulfate liposomes do not show any clarification
of the precipitate during the loading procedure indication that the
drug is not remote loading into the liposomes. (see FIG. 5). The
ammonium sulfate liposomes efficiently load the drug resulting in
clarification of the solution within 15 min.
Example 6
[0130] Confirmation of Drug Release from Remote Loaded
Liposomes
Introduction
[0131] A reverse gradient was used to attempt to release the active
drug from within the liposome. The theory is that if a drug can be
released from within a liposome with a reverse gradient there is a
good chance the that drug release will be possible in vivo.
Method
[0132] Liposomes were loaded with carfilzomib as described in
Example 1 and were purified into deionized water. The sample was
divided into two aliquots. To the first aliquot, concentrated Hepes
pH 7.4 and NaCl was added so that the final concentration was 5 mM
Hepes, 145 mM NaCl (HBS). To the second aliquot, concentrated
ammonium sulfate was added so that the final concentration was 250
mM. No obvious physical changes were initially observed. The
samples were then heated at 65.degree. C. for 30 min. The samples
were transferred to clean eppendorf tubes and centrifuged for
10,000 rpm for 5 min after which the supernatants and precipitates
were separated and tested for carfilzomib content by HPLC assay.
Released drug precipitated, liposome encapsulated drug remained in
the supernatant. The % carfilzomib released was calculated by
% Release = amt . of drug in precipitate amt . of total drug
##EQU00001##
Results
TABLE-US-00002 [0133] TABLE 2 The reverse gradient-directed drug
release from liposomes Solution Composition % Carfilzomib Released
Hepes buffered saline 10.6 .+-. 0.28 Ammonium sulfate 68.5 .+-.
1.82
[0134] The drug released using the reverse gradient is 6.5-fold
greater than the drug released from the control with no reverse
gradient (Table 2). HPLC chromatogram of the released drug was
identical to the starting material indicating that no degradation
of carfilzomib had taken place (FIG. 7). HPLC retention time for
the stock solution of carfilzomib was 12.15 min and the retention
time for the carfilzomib that was released from the remote loaded
liposome was 12.27 min, as shown in FIG. 6.
Conclusion
[0135] Carfilzomib was released from the liposome using a reverse
gradient to yield the original molecule as indicated by HPLC
analysis.
Example 7
Carfilzomib Loading as a Function of DMSO Content
Introduction
[0136] The physical form of the drug when added to liposomes is
important for loading efficiency, i.e., when added as a dry powder
almost no loading is observed but addition using a predissolved
solution in an aprotic solvent can lead to high entrapment
efficiency. This study looks at the effect of aprotic solvent
concentration on drug loading efficiency of carfilzomib.
Method
[0137] Ammonium sulfate containing liposomes were diluted in 50 mM
citric acid sucrose (10% wt/wt) buffer pH 4.0 to 1 mM phospholipid.
Various amounts of DMSO were added so that when 200 .mu.g drug was
added from a 10 mg/mL carfilzomib solution in DMSO the final DMSO
concentration ranged from 1-10% v/v.
Results
[0138] DMSO had a dramatic effect on the ability of carfilzomib to
remote load into liposomes. When absent, there is practically no
loading. At concentrations 1% and above the loading efficiency
ranges from 74-94%, with higher efficiencies observed at higher
DMSO concentrations (FIG. 7). It should be noted that drug
precipitates were observed in all samples before loading commenced,
suggesting that the concentrations of DMSO used here are below the
minimum concentration required to effectively solubilize
carfilzomib at the drug concentration used (0.2 mg/mL).
Conclusions
[0139] The introduction of pre-solubilized carfilzomib is necessary
for efficient remote loading. However, above 1% DMSO there is a
relatively small change in loading efficiency, up as far as
10%.
Example 8
[0140] Carfilzomib Solubility as a Function of DMSO Content
Introduction
[0141] Under the conditions described above, carfilzomib is
solubilized in DMSO before diluting in liposome buffer solution
prior to loading. It then immediately precipitates before remote
loading. This study is designed to determine the DMSO concentration
that is required to effectively solubilize carfilzomib at room
temperature and at the temperature required for liposome loading
into liposomes composed of high T.sub.m lipids (65.degree. C.).
Method
[0142] Carfilzomib was added from a stock 10 mg/mL solution in DMSO
to 1 mL of a citric acid/DMSO mixture so that the composition of
DMSO was 2%, 25%, 50%, 75% and 100%. The final drug concentration
was 0.2 mg/mL. The solutions were prepared and measured for optical
density at 600 nm. The optical density at 600 nm is a good measure
of how turbid or how much scattering material (such as drug
precipitates) are in a solution, generally, the more precipitates
the higher the absorbance. From FIG. 8 is apparent that at DMSO
concentrations below 50% vol/vol (25.degree. C.) and 25% vol/vol
(65.degree. C.) the drug remains in a precipitated form. Only when
the concentration of DMSO is increased does it become effectively
solubilized at this concentration of 0.2 mg/mL.
[0143] To test the integrity of the liposomes in 25% DMSO we
attempted to remote load the water-soluble weak base drugs
doxorubicin and 17-dimethylaminoethylamino-17-demethoxygeldanamycin
(17-DMAG) and compared to the same loading without DMSO. We found
that the loading efficiency was adversely affected (Table 3).
Results
[0144] At 0.2 mg/mL carfilzomib the drug precipitates and the
aggregates are large enough to cause a light scattering signal at
600 nm. As the % DMSO is increased the signal is reduced and
indicates solubilization of the drug. We observed that >25%
vol/vol DMSO is required to completely dissolve the drug at 0.2
mg/mL at a temperature of 65.degree. C.
TABLE-US-00003 TABLE 3 Comparison of remote loading doxorubicin and
17-DMAG into ammonium sulfate containing liposomes in the presence
and absence of 25% DMSO. % Efficiency compared to Drug % DMSO
control of no DMSO doxorubicin 25 92.7 .+-. 0.43 17-DMAG 25 73.1
.+-. 1.4
Conclusions
[0145] Previous studies loading carfilzomib were done using 10% v/v
DMSO or less and the light scattering results above show that the
vast majority of the drug under these conditions is in a
precipitated form at the concentrations used. Adding enough aprotic
solvent to completely solubilize the drug (i.e., greater than 25%
DMSO at 65.degree. C.) has a negative impact of the liposome
loading of amphipathic weak base drugs indicating liposome
instability caused by contents leakage or electrochemical gradient
dissipation for example. Under conditions that maintain good
liposome stability, we have not found a DMSO concentration that
will solubilize carfilzomib completely or alternatively we have not
found conditions using DMSO where simultaneously the drug is
completely solubilized and the liposomes are not adversely
destabilized.
Example 9
[0146] Effect of Delay on Liposome Loading of Carfilzomib after the
Drug Precipitate is Formed
Introduction
[0147] The invention described in this application allows for
loading of an insoluble drug precipitate into liposomes. Example 9
evaluates the effect of the time between the formation of the drug
precipitate and the time it is loaded into liposomes.
Procedure
[0148] Liposomes were prepared from the same composition and
methods as described in Example 1.
[0149] Carfilzomib was dissolved in DMSO at a concentration of 10
mg/mL and we added to a final concentration of 2% (v/v) to 50 mM
citrate, 10% sucrose at pH 3.5 containing no liposomes. Upon
addition of the drug to the citrate buffer a precipitate was
formed. The liposomes for loading were added to the solution
containing drug precipitate either immediately after formation,
after a 1 h delay or after a 12 h delay and then the precipitate
was loaded into the liposomes using the loading conditions
described in Example 1.
Results/Conclusion
[0150] The time between the formation of the drug precipitate and
the loading of the precipitate does not have a significant impact
on the efficiency of the liposome loading procedure for carfilzomib
even if the delay time is up to 12 h.
Example 10
[0151] Effect of Liposome Drug Payload on Efficiency of Carfilzomib
Loaded from Precipitate Procedure
[0152] Liposomes were prepared from the same composition and
methods as described in Comparison of Trapping Agents except the
concentration of ammonium sulfate internal trapping agent was
either 250 mM or 500 mM.
[0153] Carfilzomib was dissolved in DMSO at a concentration of 10
mg/mL. The carfilzomib was introduced to the liposomes at
carfilzomib to HSPC ratios of 91.8, 167, 251, 338 and 433, g
drug/mol HSPC for the liposomes having 250 mM ammonium sulfate as
the trapping agent and 451, 546, 639, and 759 g drug/mol HSPC for
the liposomes having 500 mM ammonium sulfate as the trapping agent.
The liposomes were diluted with 50 mM citrate, 10% sucrose pH 4.0
to increase the volume to a point where after addition of the drug
the final DMSO concentration is 10%. The carfilzomib/DMSO was added
to the diluted liposomes, which were mixed at room temperature then
transferred to a 65.degree. C. bath and swirled every 30 s for the
first 3 min and then swirled every 5 min over a total heating time
of 30 min. All samples were very cloudy when the drug was added and
all became clear (same as liposomes with no drug added) after 15
min. After heating for 30 min all samples were placed on ice for 15
min. The loaded liposomes were purified and analyzed as described
in Example 1.
Results/Conclusion
TABLE-US-00004 [0154] TABLE 4 Effect of Ammonium Sulfate Trapping
Agent Concentration on Liposome Drug Payload of Carfilzomib Loaded
from Precipitate Input Drug trapping payload/carrier agent Output
weight ratio drug/HSPC SD [(NH.sub.4).sub.2SO.sub.4] drug/HSPC SD
SD (g drug/g total (g/mol) (g/mol) (mM) (g/mol) (g/mol) efficiency
% (g/mol) lipid) 91.8 0.3 250 83.5 2.7 90.9 3.0 0.07 167.3 4.2 250
127.2 1.5 76.0 2.1 0.11 251.8 5.8 250 174.1 3.9 69.1 2.2 0.15 338.1
4.1 250 210.7 2.5 62.3 1.1 0.18 432.8 14.4 250 240.5 4.4 55.6 2.1
0.20 450.6 9.6 500 345.2 7.1 76.6 2.3 0.29 545.9 17.1 500 380.9
21.4 69.8 4.5 0.32 639.2 42.5 500 438.9 10.2 68.7 4.8 0.37 758.7
12.4 500 468.2 4.9 61.7 1.2 0.40
[0155] The resulting drug payload increases as the drug to liposome
input lipid ratios is increased in the loading solution. The
efficiency is greatest at the lowest input ratio used for each
different concentration of ammonium sulfate trapping agent. Using
the conditions described in this example, carfilzomib can be loaded
into liposomes from an insoluble precipitate up to a final drug
payload of 469.+-.4.9 g drug/mol HSPC (drug/carrier total lipid
weight ratio of 0.4) at an efficiency of 61.7.+-.1.2%.
Example 11
[0156] Loading of Insoluble Carfilzomib into Liposomes Using a
Triethylammonium Sulfate Gradient
Introduction
[0157] Remote loading of drugs into liposomes is commonly
accomplished using an ammonium sulfate gradient. Some drug
molecules including the example carfilzomib used here have an
epoxide group which is required for activity. The epoxide of these
drugs is potentially susceptible to aminolysis from any remaining
ammonia that is used in the ammonium sulfate remote loading. In
this, Example 11, the ammonium sulfate is replaced with a
triethylammonium sulfate remote loading agent to eliminate
potential ammonia/epoxide reactions by replacement with nonreactive
triethylamine.
Methods
[0158] The liposomes were prepared by using the same compositions
and procedure as described in Carfilzomib Liposome Entrapment by
Remote Loading with the following exception that 50 mM
triethylammonium sulfate was used as the trapping agent.
Triethylammonium Sulfate was prepared by titrating 1 M sulfuric
acid with triethylamine to a final pH of 7.3 and sulfate
concentration of 500 mM.
[0159] Carfilzomib was dissolved in DMSO at a concentration of 10
mg/mL. The carfilzomib was introduced to the liposomes at
carfilzomib to HSPC ratios of 650 g drug/mol HSPC. The liposomes
were diluted with 50 mM citrate, 10% sucrose pH 4.0 to increase the
volume to a point where after addition of the drug the final DMSO
concentration is 10%. The carfilzomib/DMSO was added to the diluted
liposomes, which were mixed at room temperature then transferred to
a 65.degree. C. bath and swirled every 30 s for the first 3 min and
then swirled every 5 min. A sample of the loading mixture was
removed at 10, 20, 30 and 40 min during the loading procedure and
placed on ice for 15 min. The loaded liposomes were vortexed and
100 .mu.L of sample was kept as the "before column" and the rest
transferred to microcentrifuge tubes and spun at 10,000 RPM for 5
min. The supernatants were purified on a Sephadex G25 column
collected and analyzed by HPLC. The drug precipitate pellets were
dissolved in DMSO/MeOH (10:1) and analyzed by HPLC.
Results/Conclusion
[0160] Loading an insoluble carfilzomib precipitate into liposomes
using a triethylammonium sulfate gradient results in similar
liposomes to those produced using an ammonium sulfate gradient
(Example 1). FIG. 12 illustrates the time dependence on the
liposome loading, which begins quickly by 10 min. The greatest
payload achieved was 440.+-.12.6 g drug/mol HSPC (efficiency of
65.9.+-.1.98%) was achieved at 30 min. This result using 500 mM
triethylamine as a trapping agent at drug to HSPC ratios of 650 g
drug/mol HSPC is very similar to that using 500 mM ammonium sulfate
as the trapping agent drug to HSPC ratios of 639 g drug/mol HSPC
which resulted in a final drug to lipid ratio of 440.+-.10.2 g
drug/mol HSPC (efficiency of 68.7.+-.4.80%).
[0161] The insoluble drug precipitate on the liposome exterior is
transferred (remote loaded) to the liposome interior as indicated a
reduction in the amount of precipitate in the mixture over the
course of the loading process. FIG. 12 shows the greatest reduction
in the extraliposomal precipitate happens between 0-10 min which
corresponds to the loading of precipitate into liposomes as seen in
FIG. 13.
Example 12
[0162] Loading Another Sparingly Soluble Drug from a
Precipitate
Introduction
[0163] Another drug, aripiprazole is formulated with sulfobutyl
cyclodextran (SBCD) and is used to treat bipolar disorders and
schizophrenia (Abilify, Pfizer). The drug is very insoluble in
water and when added to a liposome suspension, fine precipitates
are immediately observed.
[0164] Whether aripiprazole would remote load under similar
conditions outlined above for carfilzomib was tested.
Method
[0165] Liposomes (HSPC/Chol/PEG-DSPE 3/2/0.15 mol/mol/mol)
containing 250 mM ammonium sulfate or 250 mM sodium sulfate were
diluted in 1 mL of 50 mM citric acid, 10% (wt/wt) sucrose, pH 4.0
to a concentration of 6 mM phospholipid. 0.3 mg of aripiprazole was
added from a stock solution of 15 mg/mL in DMSO, so that the final
DMSO concentration was 2% (v/v). Fine precipitates were immediately
observed after the drug was added to both liposome samples. The
samples were heated at 65.degree. C. for 30 min, the cooled on ice.
The samples were filtered through a 0.2 um polyethersulfone syringe
filter to remove any drug precipitates, followed by purification on
a Sephadex G25 column equilibrated with HBS, pH 6.5 to remove any
soluble extraliposome drug. The turbid fraction was collected and
analyzed for lipid and drug as described above.
Results
TABLE-US-00005 [0166] TABLE 5 Results of loading aripiprazole into
liposomes containing ammonium and sodium sulfate. Input %
Efficiency D/L Output D/L % NH4SO4/ Loading Agent ug/umol ug/umol
Efficiency NaSO4 (NH.sub.4).sub.2SO.sub.4 50 42.28 .+-. 0.49 84.56
.+-. 0.99 49.3 (Na).sub.2SO.sub.4 50 0.86 .+-. 0.51 1.71 .+-.
0.10
[0167] The liposomes containing ammonium sulfate were found to load
approximately 85% of the drug, while the loading into sodium
sulfate liposomes was less than 2%, with about a 50-fold increase
in loading attributable to the ability of ammonium sulfate
liposomes to facilitate remote loading (Table 5).
[0168] Ariprazole, when introduced to the liposome solution in the
form of a SBCD complex (from the pharmaceutical product Abilify)
gave a loading efficiency of 68% under the same concentration and
loading conditions (FIG. 14).
Conclusion
[0169] This is another example of a poorly soluble drug, that can
be remote loaded into liposomes using the approach described above,
and gives slightly better loading than if the drug was introduced
as a SBDC complex.
Example 13
[0170] Loading Sparingly Soluble Drug from Precipitates Made by
Diluting Various Drug Solvent Solutions into Liposome Solution
[0171] This Example describes a technique for remote loading poorly
soluble drugs into liposomes that begins with dissolving the drug
in a solubilizing agent that initially forms drug precipitates when
added to an aqueous solution of liposomes. After some incubation
time the drug enters the liposome in response to an electrochemical
gradient, accumulating in the liposome core. Solvents that may be
used include but not limited to dimethylsulfoxide, dioxane,
tetrahydrofuran, dimethylformamide, acetonitrile,
dimethylacetamide, sulfolane, gamma butyrolactone, pyrrolidones,
1-methyl-2-pyrrolidinone, methylpyrroline, ethylene glycol
monomethyl ether, diethylene glycol monomethyl ether, polyethylene
glycol.
Method
[0172] Aripiprazole was dissolved in a range of solvents indicated
below at 4 mg/mL. Liposomes composed of HSPC/Chol/PEG-DSPE
(3/2/0.15 mol/mol/mol) that were prepared in 250 mM ammonium
sulfate were used and diluted to 6 mM in Hepes buffered sucrose 10%
(wt/wt) (HBSuc pH 6.5). 0.3 mg of drug was introduced by slow
addition of each solvent while vortexing. The final solvent
concentration was 7.5% for all samples. As controls, the drug was
added from each solvent to the same volume of HBSuc pH 6.5 without
the liposomes. The samples were heated at 65.degree. C. for 30 min
then cooled on ice. After reaching room temperature again, the
samples were measured for absorbance at 600 nm (Cary 100 Bio UV-Vis
spectrometer) and the values are displayed below (FIG. 15).
Results
[0173] All the solutions without liposomes became extremely turbid
or there was gross precipitation and settling (especially in the
case of methanol and 1-butanol). Some of the liposome samples were
also very turbid, but some clarified the drug precipitate
consistent with earlier results indicating drug loading of the drug
precipitate had taken place (namely in the cases where the drug was
initially dissolved in DMSO, 1-4-methylpyrrolidone,
diethylenemonoethylether or polyethyleneglycol (MW400), see FIG.
15.
Example 14
[0174] Remote Loading of an Insoluble Precipitate of Deferasirox
into Liposomes Using an Acetate Gradient
[0175] Remote loading of deferasirox into liposomes containing
calcium acetate demonstrates the use of an acetate gradient for
loading an iron chelating agent. Calcium acetate gradient remote
loading differs from ammonium sulfate remote loading in that the
drug molecule being loaded must have a carboxylate (or hydroxamate)
rather than an amine. Deferasirox is known to have significant
kidney toxicity and liposome delivery is a technique for reducing
kidney toxicity.
Method
[0176] The remote loading of a deferasirox insoluble precipitate
into iposomes using an acetate gradient is performed in the same
manner as acetate loading of soluble carboxyfluoroscein and
nalidixic acid by Clerc and Barenholtz 1995 (PMID 8541297).
Liposomes are prepared as described in Example 1 but in this case
the liposomes are extruded in a solution of 120 mM calcium acetate
at pH 8. The acetate gradient is formed by exchanging the external
media for 120 mM sodium sulfate at pH 6.0. Deferasirox is dissolved
in DMSO at a concentration of 10 mg/ml and added to the liposome
suspension where it forms a precipitate. The precipitate is loaded
into the liposomes by heating to 65.degree. C. for 1 h and
purification and analysis is performed as described in Example
1.
Results
[0177] Deferasirox forms a precipitate when diluted from a 10 mg/ml
DMSO stock to a concentration of 1 mg/ml in the liposome loading
suspension due to its poor water solubility (.about.0.038 mg/mL).
The insoluble deferasirox precipitate is loaded into the liposomes
using a calcium acetate gradient at an efficiency at least 5-fold
greater than it is loaded into control liposomes which contain
sodium sulfate and no acetate gradient.
[0178] Remote loading an insoluble precipitate of deferasirox into
the liposome provides an example of the use of an acetate gradient
to remote load a carboxylate drug from a precipitate. In this
example the drug being loaded is a chelating agent, in particular
an iron chelating agent. The 5-fold greater loading into the
liposomes having an acetate gradient over control liposomes
indicates that the majority of the deferasirox is remote loaded
rather than intercalated in the lipid bilayer.
Example 15
Introduction
[0179] One goal of liposomal delivery of carfilzomib is to protect
the drug from degradation and elimination which required the drug
to be retained within the liposome. One technique for evaluating
the drug retention within the liposome, and thus the benefits
obtained from liposome delivery, is to measure the pharmacokinetics
of the drug in mice. Stable formulations with greater drug
retention within the liposome will result in a higher concentration
of non-metabolized drug in mouse plasma compared to less stable
formulations or unencapsulated drug.
Materials and Methods
[0180] 100 nm liposomes comprised of HSPC/Cholesterol/PEG-DSPE
(60/40/5 mol/mol/mol) and sphingomyelin/cholesterol/PEG-DSPE
(55/45/2.8, mol/mol/mol) were formed, purified and drug loaded with
carfilzomib using the methods described in Example 1. The trapping
agents used to remote load carfilzomib were triethylammonium
dextran sulfate (1.0 M SO.sub.4) or triethylammonium
sucroseoctasulfate (1.0 M SO.sub.4). The drug loaded liposomes were
purified by tangential flow filtration with buffer exchange into
HBS, pH 6.5. The liposomes were sterile filtered through 0.2 um
polyethersulfone filters and assayed for carfilzomib and lipid
content as described in Example 1. The drug-to-lipid ratio, drug
concentration and loading efficiency were calculated and results
shown in Table 6.
Results.
TABLE-US-00006 [0181] TABLE 6 Carfilzomib concentration in mouse
plasma after IV. administration of liposome formulations. Drug
Lipid Formulation Loading CFZ/PL # (mol/mol/mol) Trapping Agent
Efficiency (.mu.g/.mu.mol) % ID @ 4 h 1 HSPC/Chol/PEG- Ammonium
Sucrose 94.1 .+-. 0.43 329.2 .+-. 2.26 0.65 .+-. 0.29 DSPE
(60/40/5) Octasulfate (1.0M SO.sub.4) 2 HSPC/Chol/PEG-
Triethylammonium 94.6 .+-. 7.55 381 .+-. 12.1 4.48 .+-. 1.10 DSPE
(60/40/5) Dextran Sulfate (1.0M SO.sub.4) 3 HSPC/Chol/PEG-
Triethylammonium 94.6 .+-. 7.55 381 .+-. 12.1 5.53 .+-.
1.69.dagger. DSPE (60/40/5) Dextran Sulfate (1.0M SO.sub.4) 4
SM/Chol/PEG-DSPE Triethylammonium 80.4 .+-. 0.71 321.8 .+-. 7.98
66.3 .+-. 20.3 (55/45/2.8) Dextran Sulfate (1.0M SO.sub.4)
.dagger.formulation #3 is the same as #2 except it was stored at
4.degree. C. for 30 days before PK analysis
[0182] In addition, we examined the pharmacokinetics of carfilzomib
encapsulated in the liposome formulations in male CD1 mice. The
mice were dosed by IV bolus injection through the tail vein at 5
mg/kg carfilzomib using 3 mice per formulation. At 4 h, the mice
were sacrificed and plasma harvested by centrifugation of the
blood. 0.1 mL of plasma was mixed with 0.2 mL methanol, mixed well
and carfilzomib concentration measured by HPLC as described in
Example 1. The results are shown in Table 6. While no effort was
made to distinguish between non-liposome entrapped and liposome
entrapped drug in the plasma as our analysis measures total drug
content we presume that the majority of the measured carfilzomib is
liposome entrapped because the drug is very rapidly eliminated in
the blood stream (t.sub.1/2<20 min) (Yang et al 2011, Drug Metab
Dispos. 2011 October; 39(10):1873-82). We observed a 100-fold range
of drug retention from 0.65% to 66.3% ID depending on the liposome
formulation composition. The most stable liposome tested was
sphingomyelin based and contained an internal ammonium dextran
sulfate solution. The liposomes described above increased the
plasma retention of carfilzomib 46-to-4735 fold more than a SBCD
formulation, or 5-to-510 fold higher than published liposome
formulations at 4 h post administration. (Chu et al 2012 AAPS
Meeting, Poster T2082).
Example 16
[0183] Remote Loading of an Insoluble Precipitate of Deferasirox
into Liposomes Using an Acetate Gradient
Introduction
[0184] Remote loading of deferasirox (DFX) into liposomes
containing calcium acetate demonstrates the use of an acetate
gradient for loading an iron chelating agent. Calcium acetate
gradient remote loading differs from ammonium sulfate remote
loading in that the drug molecule being loaded must have a
carboxylate (or hydroxamate) rather than an amine. Deferasirox is
known to have significant kidney toxicity and liposome delivery is
a technique for reducing kidney toxicity.
Method
[0185] Liposomes were prepared using the extrusion and purification
method described in Example 1. The lipid composition was
HSPC/Cholesterol (3/0.5, mol/mol) or POPC/cholesterol (3/0.5,
mol/mol). The trapping agent consisted of calcium acetate or sodium
sulfate each at a concentration of 120 mM. A solution of DFX in
DMSO at 20 mg/mL was added to the liposome solution slowly over 30
seconds while vortexing to produce a drug precipitate in the
liposome solution. The target drug to phospholipid ratio was 100 g
DFX/mol phospholipid. The solution was heated for 30 min (at
45.degree. C. for POPC liposomes and 65.degree. C. for HSPC
liposomes) and then cooled on ice. A sample was removed to
determine the input drug to lipid ratio and the remaining solution
was spun in a centrifuge at 12,000 RPM for 5 minutes to pellet any
unloaded drug. The supernatant was further purified from unloaded
drug using a Sephadex G25 size exclusion column eluted with 5 mM
HEPES, 145 mM NaCl at pH 6.5. The purified liposomes are analyzed
for drug and lipid content by HPLC using the system described in
Example 1 and a program consisting of gradient elution of 65% B to
98% B in 6 min with 5 min equilibration back to 65% B (A=0.1% TFA,
B=0.1% TFA/MeOH, 1.0 mL/min), column temperature held constant at
30.degree. C., 10 ul injection, and detection by absorbance at 254
nm.
Results
[0186] Upon addition of the drug in DMSO to the liposomes
containing calcium acetate as the trapping agent, the solution of
POPC liposomes were less cloudy than the solution of HSPC
liposomes, both contained precipitated drug before loading. After
heating, the solutions clarified and appeared like liposomes with
no drug precipitate. Liposomes containing sodium sulfate as the
control trapping agent never clarified during the heating process
and a drug precipitate pellet was formed upon centrifugation. The
loading of liposomes containing calcium acetate made from POPC and
HSPC was very efficient. Both liposomes containing the calcium
acetate trapping resulted in >90% loading efficiency. The
liposomes containing sodium sulfate resulted in 3.3% loading
efficiency, which indicates that the loading of DFX into calcium
acetate liposomes is not passive but can be described as remote
loading. The DFX loading results are shown in Table 7 (FIG.
16).
TABLE-US-00007 TABLE 7 Loading Efficiency of DFX in Calcium Acetate
Liposomes 2 DFX loading Lipid composition Interior buffer
efficiency 3 mol POPC/0.5 mol Chol 120 mM calcium acetate 94.8 .+-.
1.46% 3 mol HSPC/0.5 mol Chol 120 mM calcium acetate 92.5 .+-.
0.33% 3 mol HSPC/0.5 mol Chol 120 mM sodium sulfate 3.3 .+-.
0.14%
Conclusion
[0187] Remote loading an insoluble precipitate of deferasirox into
the liposome provides an example of the use of an acetate gradient
to remote load a carboxylate drug from a precipitate. In this
example the drug loaded was a chelating agent, in particular an
iron chelating agent. The 28-fold greater loading into the
liposomes having an acetate gradient over control liposomes
indicates that the majority of the deferasirox is remote loaded
rather than intercalated in the lipid bilayer.
Example 17
[0188] Remote Loading of an Insoluble Precipitate of Deferasirox
into Liposomes. Evaluation of Drug to Lipid Ratio and Calcium
Acetate Trapping Agent Concentration.
Introduction
[0189] The remote loading capacity of DFX in liposomes containing
calcium acetate was evaluated by using different concentrations
calcium acetate on the liposome interior and loading a range of
different DFX-to-lipid ratios.
Method
[0190] Liposomes were prepared using the extrusion and purification
method described in Example 1. The lipid composition was
POPC/cholesterol (3/0.5, mol/mol). The trapping agent consisted of
calcium acetate 120 mM, 250 mM or 500 mM. A solution of DFX in DMSO
at 20 mg/mL was added to the liposome solution slowly over 30
seconds while vortexing to produce a drug precipitate in the
liposome solution. The target drug to phospholipid ratio was 100,
200 or 300 g DFX/mol phospholipid. The solution was heated for 30
min at 45.degree. C. and then cooled on ice. A sample was removed
to determine the input drug to lipid ratio and the remaining
solution was spun in a centrifuge at 12,000 RPM for 5 minutes to
pellet any unloaded drug. The supernatant was further purified from
unloaded drug using a Sephadex G25 size exclusion column eluted
with 5 mM HEPES, 145 mM NaCl at pH 6.5. The purified liposomes are
analyzed for drug and lipid content by HPLC as described in Example
16.
Results
[0191] Upon addition of the drug in DMSO to the liposomes
containing calcium acetate as the trapping agent the DFX forms a
precipitate before loading. After heating, the solutions clarify
and look like liposomes with no drug precipitate. The maximum drug
load was higher for liposomes containing 250 and 500 mM calcium
acetate compared to 120 mM calcium acetate. The maximum drug load
and efficiency was achieved at an input of 200 g DFX/mol
phospholipid for liposomes containing either 250 mM calcium acetate
or 500 mM calcium acetate. The efficiency of loading for a target
of 100 g DFX/mol phospholipid ranged from 99.2 to 103% for all
three concentrations of internal calcium acetate. When the target
drug load was increased to 200 g DFX/mol phospholipid the
efficiency of liposomes having 250 or 500 mM internal calcium
acetate was at least two-fold greater than liposomes having an
internal calcium acetate concentration of 120 mM. The capacity of
all three liposomes was exceeded at input of 300 g DFX/mol
phospholipid resulting in n efficiency <24%. The results are
shown in FIG. 17.
Conclusion
[0192] The drug payload capacity of DFX when remote loaded into
liposomes can be substantially increased by increasing the
concentration of the trapping agent concentration inside the
liposome. This example demonstrates the dependence of loading
capacity on calcium acetate trapping agent concentration. This
example also demonstrates DFX liposome loading can have an optimum
drug to lipid ratio where the efficiency and drug load are both
greatest. The achieved drug to lipid ratio allows for the DFX to be
administered to an animal using a tolerated dose of lipid.
Example 18
[0193] Remote Loading of an Insoluble Precipitate of Deferasirox
into Liposomes. Evaluation of Trapping Agent.
[0194] The remote loading capacity of DFX in liposomes containing
calcium acetate, magnesium acetate and zinc acetate was evaluated
by preparing liposomes with different trapping agents on the
interior and loading a range of different DFX-to-lipid ratios.
Method
[0195] Liposomes were prepared using the extrusion and purification
method described in Example 1. The lipid composition was
POPC/cholesterol (3/0.5, mol/mol). The trapping agent consisted of
calcium acetate, magnesium acetate or zinc acetate at 120 mM. A
solution of DFX in DMSO at 20 mg/mL was added to the liposome
solution slowly over 30 seconds while vortexing to produce a drug
precipitate in the liposome solution. The target drug to
phospholipid ratio was 100, 150 or 200 g DFX/mol phospholipid. The
solution was heated for 30 min at 45.degree. C. and then cooled on
ice. A sample was removed to determine the input drug to lipid
ratio and the remaining solution was spun in a centrifuge at 12,000
RPM for 5 minutes to pellet any unloaded drug. The supernatant was
further purified from unloaded drug using a Sephadex G25 size
exclusion column eluted with 5 mM HEPES, 145 mM NaCl at pH 6.5. The
purified liposomes are analyzed for drug and lipid content by HPLC
as described in Example 16.
Results
[0196] Upon addition of the drug in DMSO to the liposomes, the DFX
forms a precipitate before loading. After heating, the solutions
containing liposomes with calcium acetate and magnesium acetate
became much less turbid than the liposomes containing zinc acetate
as the trapping agent. The maximum drug load was highest for the
liposomes containing magnesium the second highest for the liposomes
containing calcium acetate and the liposomes containing zinc
acetate resulted in the lowest drug payload. The efficiency of
loading for a target of 100 g DFX/mol phospholipid was 5.3.+-.0.07%
but the efficiency using calcium acetate and magnesium acetate were
97.6.+-.0.41% and 99.2.+-.2.42% respectively. The results are shown
in FIG. 18.
Conclusion
[0197] The drug payload capacity of DFX when remote loaded into
liposomes can be dependent on the particular metal salt of acetate
used for remote loading. This example demonstrates that magnesium
acetate is a better trapping agent for DFX than calcium acetate or
zinc acetate.
Example 19
[0198] Remote Loading of an Insoluble Precipitate of Amphotericin B
into Liposomes Method
[0199] Liposomes were prepared with a lipid composition of
HSPC/DSPG/Chol/PEG-DSPE in the ratio 2/0.6/2/0.3 containing 1.0 M
(SO.sub.4) TEA dextran sulfate. The liposomes were separated form
the non-entrapped TEA dextran sulfate by anion exchange and then by
dialysis against 5 mM Hepes buffered 10% (wt/wt) sucrose pH 6.5.
The liposomes were exchanged into 0.01 N HCL, 10% sucrose pH 2.0
before drug loading. Amphotericin B was dissolved in DMSO at 10
mg/ml.
[0200] In a typical preparation, the DMSO amphotericin B solution
was added dropwise to the liposomes at room temperature while the
liposomes suspension was rapidly mixed on a vortex mixer. The
concentration of the liposomal lipid was 5 umol (phospholipid)/mL
and 0.1 mL of the amphotericin B solution was added per mL of
liposomes so that the final amphotericin B concentration was about
1.0 mg/ml AmB and the final DMSO concentration was about 10% (V/V).
By adjusting the amount of liposomes in the preparation various
AmB/PL ratios were tested, e.g., 200, 400, 800 g/mol.
[0201] The samples were heated at 65.degree. C. for 15 min. Then
cooled on ice for 30 minutes. The preparation was then passed
through a PD10 (Sephadex G25) gel filtration column to remove any
precipitated amphotericin B that was not incorporated into the
liposome. The buffer was also exchanged into 5 mM Hepes, 144 mM
NaCL pH 6.5. to remove the DMSO from the liposomal amphotericin B
preparation. The diameter of the drug loaded liposomes was not
changed at the 200 and 400 g drug/mole lipid input ratio and were
slighted larger circa 10% in the 800 g drug lipid ratio. The
purified liposomes were analyzed for drug and lipid content by the
HPLC method as described in Example 1.
Results
[0202] Amphotericin B was remote loaded into liposomes at 96%
efficiency at a 200 g drug/mole lipid, at the input ratio of 400 g
drug/mole of lipid the drug was about 90% encapsulated to provide a
purified preparation of 360 g amphotericin B/mole lipid. At 800 g
drug/mole lipid, amphotericin B was about 70% encapsulated, to
provide 560 g amphotericin B/mole lipid. All of these values are
substantially greater than the approximate value of 120 g
amphotericin B/mole lipid that is contained in the drug product
Ambisome.RTM.. The final liposome preparation was readily
concentrated to a 10 mg/mL amphotericin B concentration in the 5 mM
Hepes, 144 mM NaCl, pH 7.4 buffer.
Conclusion
[0203] The drug payload capacity of liposomes for amphotericin B
when amphotericin B is remote loaded into pre-formed liposomes in
containing TEA dextran sulfate from the precipitated amphotericin B
greatly exceeds the amount of amphotericin B that can be loaded
into the liposome membrane by the classical mixing of the drug with
the lipid components and then forming the liposomes. This example
demonstrates that the exceptionally insoluble compound amphotericin
B can be remote loaded from the precipitate to provide a high
concentration of liposome encapsulated amphotericin B, something
that has not been previously possible with amphotericin B lipid
formulations.
Example 20
[0204] Remote Loading of an Insoluble Precipitate of a Taxane
Derivative, 2' Succinyl Cabazitaxel, into Liposome Method for the
Synthesis of Sparingly Soluble Taxane Derivatives with pH Dependent
Solubility
[0205] In order to take full advantage of the precipitate loading
method, it is advantageous to prepare drugs with a pH adjustable
solubility so one can optimize both the encapsulation and release
of the encapsulated drug in the correct place in the body using the
various remote-loading agents described in this application. To
create sparingly soluble taxane derivatives with properties that
enable them to be loaded into liposomes, we modified paclitaxel,
docetaxel and carbazitaxel with either carboxylic, dimethylamino or
morpolino containing moieties at the 2' position (FIG. 19) by
standard chemical reactions, well known to those skilled in the
art. The parent compounds are illustrated on the right hand side of
FIG. 19 with the 2' position indicated by an R group. In the parent
compound the R group is a hydrogen. R can be a succinate,
glutarate, morpholino or a succinyl dimethylaminopropylamide,
glutaryl dimethylamino propylamide, a
succinyl-2-(morpholin-4-yl)ethanamide or
glutaryl-2-(morpholin-4-yl)ethanamide.
[0206] The reaction of carbazitaxel with either succinic anhydride
or glutaric anhydride in pyridine solution at room temperature will
give the crystalline mono 2'-adducts succinyl and glutaryl,
respectively. The contaminating diesters (about 5% or less) are
easily removed by crystallization. This site selectivity is in
agreement with published results, which indicates much higher
reactivity of the 2'-hydroxyl as compared to the 7-hydroxyl group
for acylation reactions. 2'-succinylcarbazitaxel is synthesized by
reacting a solution of 0.050 g (0.060 mmol) of carbazitaxel and
0.090 g (0.076 mmol) of succinyl anhydride for three hours at room
temperature in 3 mL of pyridine. The reaction mixture is evaporated
to dryness in vacuo. The residue is treated with 10 mL of water,
stirred for 20 min, and filtered. The precipitate is dissolved in
acetone, water is slowly added, and the tiny crystals are
collected. The crystals are recrystalled from chloroform/benzene to
yield the product, 2'succinylcarbazitaxel.
[0207] The 2' succinyl ester of carbazitaxel is converted into a
carbazitaxel with a basic group attached to the carboxylate of the
2' glutarylic acid by coupling with 3-(dimethylamino)-1-propylamine
in the presence of CDI with acetonitrile as a solvent. To a
well-stirred solution of 4 g (4.1 mmol) of 2'succinylcarbazitaxel
in 40 mL of acetonitrile is added 0.88 g (5.43 mmol) of CDI, and
the mixture is heated to 45.degree. C. for 5 min. After the mixture
is cooled to room temperature, a solution of 0.47 g (4.61 mmol) of
3-(dimethylamino)-1-propylamine in 3 mL of acetonitrile is added
over a period of 20 min. After 30 min, the solvent is evaporated,
and the residue is treated with 150 mL of water and 40 mL
chloroform. The organic layer is washed five times with 150 mL of
water, dried with K.sub.2CO.sub.3, and evaporated to yield 3.6 g
(83%) an oil. Recrystallization from methylene chloride/ethyl
acetate will yield circa 3.6 g (83%) of the title compound. The
hydrochloride salt is prepared by the addition of 1 equiv of
hydrochloric acid, followed by freeze-drying of the aqueous
solution.
[0208] The 2' succinyl ester of carbazitaxel was converted into a
morpholin-4-ylethyl amide by reacting the 2' glutaryl ester of
carbazitaxel in the presence of CDI with acetonitrile as a solvent
with 2-(morpholin-4-yl)ethanamine. To a well-stirred solution of 4
g (4.1 mmol) of 2'-succinylcarbazitaxel in 40 mL of acetonitrile is
added 0.88 g (5.43 mmol) of CDI, and the mixture is heated to
45.degree. C. for 5 min. After the mixture is cooled to room
temperature, a solution of 0.6 g (4.61 mmol) of
2-(morpholin-4-yl)ethanamine in 3 mL of acetonitrile is added over
a period of 20 min. After 30 min, the solvent is evaporated, and
the residue is treated with 150 mL of water and 40 mL chloroform.
The organic layer is washed five times with 150 mL of water, dried
with K.sub.2CO.sub.3, and evaporated to yield circa 3.6 g of an oil
of the title compound. The hydrochloride salt was prepared by the
addition of 1 equiv of hydrochloric acid. The HCl salt of 2'
Morpholine-4-ylethyl amide of succinylcabazitaxel was isolated in
good yield by crystallization from a mixture of methylene chloride
and ethyl acetate.
[0209] An alternative approach to the preparation of a basic
derivative of carbazitaxel was to react (4-morpholinyl)propanoic
acid with carbazitaxel via the activation of the carboxylate on the
morpholinyl propanoic acid. A stirred solution of
4-morpholinylpropanic acid in pyridine (circa 0.1 g, 0.5 mM, 1.2
equivalents) in 4 mL pyridine containing DBU (1.4 mL, circa 3
equivalents in a 50 mL roundbottom flask was cooled on in a water
ice bath and then 2.0 mL of acetonitrile was added. Then
carbazitaxel (0.3 grams, 0.35 mmoles) was added. Then 2.5
equivalents (circa 1.8 grams) of
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide was added in
portions over 30 minutes. The suspension was stirred and the ice
cold solution was allowed to come to room temperature over the
course of 24 hours. Analysis of the reaction mixture on TLC
(ethylacetate/hexane/methanol/ammonium hydroxide: 60/30/6/4: V/V)
indicated the cabazitaxel is substantially reacted over the course
of the 24 hours. Ethanol, circa 4 mL was added to the reaction
mixture and the material was concentrated to an oil by removal of
the solvent under reduced pressure on a rotary evaporator. The oil
was dissolved in circa 8 mL of ethanol and concentrated again. The
dried residue was dissolved in about 6 mL of methylchloride and
loaded on to a silica flash column that had been pre-equilibrated
with ethylacetate/hexane/methanol/ammonium hydroxide: 60/30/2/4:
V/V and was eluted with an increasing polar solvent from 2-10%
methanol in ethylacetate/hexanes/methanol/ammonium hydroxide
(60/30/2-10/4. The fractions containing the desired material were
pooled and concentrated. Derivatives of paclitaxel and docetaxel
are prepared in a similar manner to the above synthesis to provide
sparingly soluble taxane derivatives whose solubility can be
adjusted by altering the pH of the liposome solution in which the
aprotic solution of the derivatives are added.
[0210] Liposomes were prepared using the extrusion and purification
method described in Example 1. The lipid composition was
POPC/cholesterol (3/0.5, mol/mol). The trapping agent consisted of
zinc acetate or magnesium acetate at 120 mM. A solution of either
unmodify carbazitaxel or 2'succinylcarbazitaxel in DMSO at 20 mg/mL
was added to the liposome solution slowly over 30 seconds while
vortexing to produce a drug precipitate in the liposome solution.
The target drug to phospholipid ratio was 100 g/mole lipid for
unmodified carbazitaxel and 100, 150 or 200 g for
succinylcarbazitaxel/mol phospholipid. The solutions are heated for
30 min at 45.degree. C. and then cooled on ice. A sample was
removed to determine the input drug to lipid ratio and the
remaining solution was spun in a centrifuge at 12,000 RPM for 5
minutes to pellet any unloaded drug. The supernatant was further
purified from unloaded drug using a Sephadex G25 size exclusion
column eluted with 5 mM HEPES, 145 mM NaCl at pH 6.5. The purified
liposomes were analyzed for drug and lipid content by HPLC as
described in Example 16.
Results
[0211] Upon addition of the drug in DMSO to the liposomes
containing the divalent acetate buffer, both the carbazitaxel and
2'succinylcarbazitaxel formed a white precipitate before loading.
After heating, the white precipitate of the unmodified carbazitaxel
solutions containing liposomes was unchanged. However, after
heating the 2'-succinylcabazitaxel solution with zinc acetate or
magnesium acetate liposomes, the turbidity was substantially less.
The maximum 2'succinylcarbazitaxel load was highest for the
liposomes containing magnesium acetate and second highest for the
liposomes containing zinc acetate. The loading efficacy was greater
than 85% in loading the 2'succinylcabazitaxel. The efficiency of
loading unmodified carbazitaxel added at 100 g carbazitaxel/mol
phospholipid was very low in both the calcium acetate and magnesium
acetate liposomes.
[0212] To encapsulate the 2' morpholine-4-ylethyl amide of
succinylcabazitaxel, liposomes were prepared with a lipid
composition of HSPC/DSPG/Chol/PEG-DSPE in the ratio 2/0.6/2/0.3
containing 1.0 M (SO.sub.4) TEA dextran sulfate. The liposomes were
separated form the non-entrapped TEA dextran sulfate by anion
exchange and then by dialysis against 5 mM Hepes buffered 10%
(wt/wt) sucrose, pH 6.5. 2' morpholine-4-ylethyl amide of
succinylcabazitaxel was dissolved in DMSO at 10 mg/mL.
[0213] In a typical preparation, the DMSO 2' morpholine-4-ylethyl
amide of succinylcabazitaxel solution was added dropwise to the
liposomes at room temperature while the liposomes suspension was
rapidly mixed on a vortex mixer. The concentration of the liposomal
lipid was 5 umol (phospholipid)/mL and 0.1 mL of the 2'
morpholine-4-ylethyl amide of succinylcabazitaxel solution was
added per mL of liposomes so that the final 2' morpholine-4-ylethyl
amide of succinylcabazitaxel concentration was about 1.0 mg/ml and
the final DMSO concentration was about 10% (V/V). By adjusting the
amount of liposomes in the preparation various 2'
morpholine-4-ylethyl amide of succinylcabazitaxel/PL ratios were
tested, e.g., 200, 400, 800 g/mol. To determine if the unmodified
cabazitaxel was loaded into the TEA-dextran sulfate liposomes,
cabazitaxel was dissolved in DMSO and added to the liposomes as
described for the 2' morpholine-4-ylethyl amide of
succinylcabazitaxel compound. The solutions were heated for 30 min
at 45.degree. C. and then cooled on ice. A sample was removed to
determine the input drug to lipid ratio and the remaining solution
is spun in a centrifuge at 12,000 RPM for 5 minutes to pellet any
unloaded drug. The supernatant was further purified from unloaded
drug using a Sephadex G25 size exclusion column eluted with 5 mM
HEPES, 145 mM NaCl at pH 6.5. The purified liposomes were analyzed
for drug and lipid content by HPLC as described in Example 16.
Using a similar method for precipitate loading the 2'
morpholine-propyl ester cabazitaxel (FIG. 19) can be precipitate
loaded into liposomes containing the TEA-dextran sulfate
gradient.
Results
[0214] The unmodified carbazitaxel formed a white turbid
precipitate when added to the liposome suspension in a DMSO
solution but the precipitate did not clear up upon heating. The
encapsulation of cabazitaxel was less then 5% of the added drug.
The 2' morpholine-4-ylethyl amide of succinylcabazitaxel and 2'
morpholinopropyl derivatives of cabazitaxel (FIG. 19) formed a
white turbid precipitate when added to the liposomes that cleared
upon heating. The precipitate was loaded into TEA-dextran sulfate
liposomes at greater than 90% of the added drug at the 200 and 400
g drug/mole lipid ratios and greater than 70% at the 800 g
drug/mole lipid.
Conclusion
[0215] pH titratable taxane derivatives can be remote loaded into
liposomes from a precipitate formed when the taxane derivative is
added in an aprotic solvent to a preformed liposome containing
remote loading agent where the concentration of the mobile ion
species is greater on the inside than on the outside of the
liposome. Taxanes modified to contain a carboxylate on the 2'OH
position can be loaded into liposomes that contain a divalent
cation with a mobile anionic salt such as acetate. Taxanes modified
at the 2'hydroxyl group to contain a titratable amine can be remote
loaded in liposomes containing a mobile cation such as ammonium or
triethylamine and an impermeable anion such as sulfate or dextran
sulfate. Thus converting an uncharged taxane into a titratable
taxane allows the taxane to be loaded into a liposome from a
precipitate with high efficiency and retained in the liposome when
the liposome is injected into an animal.
[0216] The foregoing descriptions of specific embodiments of the
present invention have been presented for purposes of illustration
and description. They are not intended to be exhaustive or to limit
the invention to the precise forms disclosed, and obviously many
modifications and variations are possible in light of the above
teaching. The embodiments were chosen and described in order to
best explain the principles of the invention and its practical
application, to thereby enable others skilled in the art to best
utilize the invention and various embodiments with various
modifications as are suited to the particular use contemplated. It
is intended that the scope of the invention be defined by the
claims appended hereto and their equivalents.
[0217] All publications, patents, and patent applications cited
herein are hereby incorporated by reference in their entirety for
all purposes.
* * * * *