U.S. patent application number 12/135759 was filed with the patent office on 2009-05-14 for liposome drug delivery.
Invention is credited to Guru V. Betageri, Milton B. Yatvin.
Application Number | 20090123530 12/135759 |
Document ID | / |
Family ID | 24245259 |
Filed Date | 2009-05-14 |
United States Patent
Application |
20090123530 |
Kind Code |
A1 |
Betageri; Guru V. ; et
al. |
May 14, 2009 |
Liposome Drug Delivery
Abstract
This invention comprises pharmaceutical compositions for
administering a biologically active compound to an animal.
Particularly provided are proliposomal compositions that are
advantageously used to deliver biologically active compounds to the
gastrointestinal tract after oral administration.
Inventors: |
Betageri; Guru V.; (Cino
Hills, CA) ; Yatvin; Milton B.; (Portland,
OR) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE, 32ND FLOOR
CHICAGO
IL
60606
US
|
Family ID: |
24245259 |
Appl. No.: |
12/135759 |
Filed: |
June 9, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10889969 |
Jul 13, 2004 |
7387791 |
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12135759 |
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09562207 |
May 2, 2000 |
6761901 |
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10889969 |
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Current U.S.
Class: |
424/450 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 35/00 20180101; A61P 31/04 20180101; A61K 9/1277 20130101;
A61K 9/2072 20130101; A61P 3/02 20180101; A61P 31/10 20180101; A61K
9/2866 20130101; A61P 5/00 20180101; A61P 29/00 20180101; A61K
9/2846 20130101 |
Class at
Publication: |
424/450 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61P 35/00 20060101 A61P035/00 |
Claims
1-13. (canceled)
14. A pharmacological composition comprising a proliposomal
preparation of a biologically active compound, where the
biologically active compound is a nutrient, hormone, nucleic acid,
antibiotic drug, enzyme, antigen, antiviral drug, antineoplastic,
antiproliferative, peptide or a protein, in a capsule or tablet
comprising an enteric coating, a particle lubricant, and a
protective coating in between the proliposomal preparation and the
enteric coating, wherein the proliposomal preparation comprises a
positively charged phospholipid and a neutral lipid, and wherein
the protective coating further comprises a plasticizer.
15. A pharmacological composition according to claim 14 wherein the
enteric coating is cellulose acetate phthalate or a poly(acrylate,
methacrylate) copolymer.
16. A pharmacological composition according to claim 14 wherein the
protective coating is hydroxypropyl methylcellulose, polyethylene
glycol or ethylcellulose.
17. A pharmacological composition according to claim 14 wherein the
neutral lipid is cholesterol.
18. A pharmacological composition according to claim 14 wherein the
positively charged phospholipid is a phosphatidylcholine,
sphingosine, ceramide, or stearylamine.
19. A pharmacological composition according to claim 14 wherein the
plasticizer is triethylcitrate or polyvinyl pyrrolidine.
20. A pharmacological composition according to claim 14 wherein the
particle lubricant is talc, lactose, corn starch, ethyl cellulose,
fatty acids or salts thereof, agar, pectin, gelatin or acacia.
21. A pharmacological composition according to claim 14 wherein the
biologically active compound is aspirin, ibuprofen, erythromycin,
vasopressin insulin, dideoxyinosine, cyclosporine, taxol, heparin,
halofantrine, ethopropazine, griseofulvin, propofol, furosemide,
carbamazepine, diazepam, candesartan or cilexetil.
22. A pharmacological composition according to claim 14 wherein the
neutral lipid is cholesterol and the positively charged
phospholipid is a phosphatidylcholine, a phosphatidylethanolamine,
sphingosine, ceramide, or stearylamine.
23. A pharmacological composition according to claim 22 wherein the
phosphatidylcholine is distearylphosphatidylcholine,
dimyristylphosphatidylcholine or a mixture thereof.
24. A method for increasing the bioavailability of a biologically
active compound, said method comprising orally administering to a
human in need thereof a pharmaceutical composition of claim 14.
25. A method according to claim 24 wherein the biologically active
compound is aspirin, ibuprofen, erythromycin, vasopressin insulin,
dideoxyinosine, cyclosporine, taxol, heparin, halofantrine,
ethopropazine, griseofulvin, propofol, furosemide, carbamazepine,
diazepam, candesartan or cilexetil; the enteric coating is
cellulose acetate phthalate or a poly(acrylate, methacrylate)
copolymer; the protective coating is hydroxypropyl methylcellulose,
polyethylene glycol or ethylcellulose; the plasticizer is
triethylcitrate or polyvinyl pyrrolidine; and wherein the particle
lubricant is talc, lactose, corn starch, ethyl cellulose, fatty
acids or salts thereof, agar, pectin, gelatin or acacia.
26. A method of treating Crohn's disease, irritable bowel syndrome,
celia sprue, diverticulitis, immunoproliferative small intestine
disease, liver disease, diseases and disorders of the gall bladder,
disorders that are consequent to the removal of the gall bladder,
pancreatitis, Schwachman's syndrome, steatorrhea, Whipple's
disease, parasitic infection, malabsorption as a consequence of
chronic laxative use or abuse, pancreatic enzyme deficiency,
disaccharidase deficiency, or defects in fat absorption consequent
to surgical gastrectomy or other surgical interventions in the
gastrointestinal tract, said method comprising administering a
composition of claim 14 to human in need thereof.
27. A method according to claim 26 wherein the biologically active
compound is aspirin, ibuprofen, erythromycin, vasopressin insulin,
dideoxyinosine, cyclosporine, taxol, heparin, halofantrine,
ethopropazine, griseofulvin, propofol, furosemide, carbamazepine,
diazepam, candesartan or cilexetil.
28. A method according to claim 27, wherein the enteric coating is
cellulose acetate phthalate or a poly(acrylate, methacrylate)
copolymer; the protective coating is hydroxypropyl methylcellulose,
polyethylene glycol or ethylcellulose; the plasticizer is
triethylcitrate or polyvinyl pyrrolidine; and wherein the particle
lubricant is talc, lactose, corn starch, ethyl cellulose, fatty
acids or salts thereof, agar, pectin, gelatin or acacia.
29. A method according to claim 28 wherein the neutral lipid is
cholesterol and the positively charged phospholipid is a
phosphatidylcholine, a phosphatidylethanolamine, sphingosine,
ceramide, or stearylamine.
30. A method according to claim 29, wherein the phosphatidylcholine
is distearylphosphatidylcholine, dimyristylphosphatidylcholine or a
mixture thereof.
31. A method for delivering a biologically active compound to the
intestine or colon, said method comprising orally administering to
human in need thereof a composition of claim 14.
32. A method according to claim 31 wherein the biologically active
compound is aspirin, ibuprofen, erythromycin, vasopressin insulin,
dideoxyinosine, cyclosporine, taxol, heparin, halofantrine,
ethopropazine, griseofulvin, propofol, furosemide, carbamazepine,
diazepam, candesartan or cilexetil.
33. A method according to claim 32, wherein the enteric coating is
cellulose acetate phthalate or a poly(acrylate, methacrylate)
copolymer; the protective coating is hydroxypropyl methylcellulose,
polyethylene glycol or ethylcellulose; and the plasticizer is
triethylcitrate or polyvinyl pyrrolidine; and wherein the particle
lubricant is talc, lactose, corn starch, ethyl cellulose, fatty
acids or salts thereof, agar, pectin, gelatin or acacia.
34. A method according to claim 33 wherein the neutral lipid is
cholesterol and the positively charged phospholipid is a
phosphatidylcholine, a phosphatidylethanolamine, sphingosine,
ceramide, or stearylamine.
35. A method according to claim 34, wherein the phosphatidylcholine
is distearylphosphatidylcholine, dimyristylphosphatidylcholine or a
mixture thereof.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to delivery of drugs, nutrients and
other compounds to a biological organism. In particular, the
invention relates to liposomes and formulations of drugs, nutrients
and other compounds into liposomes to improve or effect delivery of
such beneficial compounds to cells and tissues in an organism.
Specifically, the invention provides such liposome compositions of
drugs, nutrients and other compounds in formulations advantageously
administered orally to an animal.
[0003] 2. Background of the Related Art
[0004] A major goal in the pharmacological arts has been the
development of reagents and methods that reduce the necessity of
administering therapeutic compounds, drugs and other agents
invasively (i.e., such as by injection). Most preferably, it has
been a consistent goal in the art to develop therapeutic compounds,
drugs and agents and formulations thereof that permit oral
administration (see, for example U.S. Pat. No. 4,963,526 to Ecanow
issued Oct. 16, 1990), although other reduced-invasiveness
formulations such as suppositories have also been developed. Among
the various routes of drug administration, the oral intake of drugs
is undoubtedly preferred because of its versatility, safety and
patient comfort.
[0005] In addition, it has been a goal in the nutritional arts to
develop preparations that increase transit of certain nutrients
through the gastrointestinal tract to increase uptake and delivery
of such nutrients into the bloodstream. In particular, such
preparations have been developed to permit chemically-labile
nutrients (such as vitamins and other sensitive compounds) to pass
through the chemically-hostile environment stomach for absorption
in the intestines (see, for example, U.S. Pat. No. 5,958,450 to
issued Sep. 28, 1999). Preparations having enhanced intestinal
uptake have also been deemed desirable.
[0006] One approach known in the prior art for improving efficiency
of delivery of therapeutic compounds, drugs and other agents has
been to envelop such compounds in a lipid structure termed a
liposome (see, for example, U.S. Pat. No. 4,744,989 to Payne et al.
issued May 17, 1988). Liposomes generically comprise an enclosed
lipid droplet having a core, typically an aqueous core, containing
the compound. In certain embodiments, the compound is chemically
conjugated to a lipid component of the liposome. In other
embodiments, the compound is simply contained within the aqueous
compartment inside the liposome.
[0007] Certain liposome formulations are known in the art.
[0008] U.S. Pat. No. 5,223,263, issued Jun. 29, 1993 to Hostetler
et al. discloses conjugates between antiviral nucleoside analogues
and polar lipids for inclusion in liposomes.
[0009] U.S. Pat. No. 5,466,468 to Schneider et al. issued Nov. 14,
1995 discloses parenterally administrable liposome formulation
comprising synthetic lipids.
[0010] U.S. Pat. No. 5,484,809, issued Jan. 16, 1996 to Hostetler
et al. discloses taxol and taxol derivatives conjugated to
phospholipids.
[0011] U.S. Pat. No. 5,580,571, issued Dec. 3, 1996 to Hostetler et
al. discloses nucleoside analogues conjugated to phospholipids.
[0012] U.S. Pat. No. 5,626,869 to Nyqvist et al. issued May 6, 1997
discloses pharmaceutical compositions wherein the pharmaceutically
active compound is heparin or a fragment thereof contained in a
defined lipid system comprising at least one amphiphatic and polar
lipid component and at least one nonpolar lipid component.
[0013] U.S. Pat. No. 5,744,461, issued Apr. 28, 1998 to Hostetler
et al. discloses nucleoside analogues conjugated to phosphonoacetic
acid lipid derivatives.
[0014] U.S. Pat. No. 5,744,592, issued Apr. 28, 1998 to Hostetler
et al. discloses nucleoside analogues conjugated to
phospholipids.
[0015] U.S. Pat. No. 5,756,116, issued May 26, 1998 to Hostetler et
al. discloses nucleoside analogues conjugated to phospholipids.
[0016] U.S. Pat. No. 5,843,509 to Calvo Salve et al. issued Dec. 1,
1998 discloses stabilization of colloidal systems through the
formation of lipid-polysaccharide complexes comprising a water
soluble and positively charged polysaccharide and a negatively
charged phospholipid.
[0017] International Patent Application Publication Number
WO89/02733, published April 1989 to Vical discloses conjugates
between antiviral nucleoside analogues and polar lipids.
[0018] European Patent Application Publication Number 0350287A2 to
Vical discloses conjugates between antiviral nucleoside analogues
and polar lipids.
[0019] International Patent Application Publication Number
WO93/00910 to Vical discloses conjugates between antiviral
nucleoside analogues and polar lipids.
[0020] Rahman et al., 1982, Life Sci. 31: 2061-71 found that
liposomes which contained galactolipid as part of the lipid
appeared to have a higher affinity for parenchymal cells than
liposomes which lacked galactolipid.
[0021] Gregoriadis, 1995, Trends in Biotechnology 13: 527-537
reviews the progress and problems associated with using liposomes
for targeted drug delivery.
[0022] Ledley, 1995, Human Gene Therapy 6: 1129-1144 reviews the
use of liposomes for gene therapy.
[0023] Mickisch, 1995, World J. Urology 13: 178-185 reviews the use
of liposomes for gene therapy of renal cell carcinoma.
[0024] Yang et al. 1997, J. Neurotrauma 14: 281-297 review the use
of cationic liposomes for gene therapy directed to the central
nervous system.
[0025] Storm & Crommelin, 1997, Hybridoma 16: 119-125 review
the preliminary use of liposomes for targeting chemotherapeutic
drugs to tumor sites.
[0026] Manusama et al., 1998, Semin. Surg. Oncol. 14: 232-237
reported on preclinical and clinical trials of
liposome-encapsulated tumor necrosis factor for cancer
treatments.
[0027] Although liposomes have conventionally been administered
parenterally (see, for example, U.S. Pat. No. 5,466,468), reports
of oral administration of liposome-related formulations have
appeared in the art.
[0028] U.S. Pat. No. 4,921,757 to Wheatley et al. issued May 1,
1990 discloses controlled release of biologically active
substances, such as drugs and hormones entrapped in liposomes which
are protected from the biological environment by encapsulation
within semi-permeable microcapsules or a permeable polymeric
matrix.
[0029] U.S. Pat. No. 5,043,165 to Radhakrishnan to Aug. 27, 1991
disclosed a liposome composition for sustained release of steroidal
drugs.
[0030] U.S. Pat. No. 5,762,904 to Okada et al. issued Jun. 9, 1998
discloses oral delivery of vaccines using polymerized
liposomes.
[0031] U.S. Pat. No. 5,955,451 to Lichtenberger et al. issued Sep.
21, 1999 discloses compositions comprising non-steroid
anti-inflammatory drugs (NSAID's) complexed with either
zwitterionic or neutral phospholipids, or both, having reduced
gastrointestinal irritating effects and enhanced antipyretic,
analgesic, and anti-inflammatory activity.
[0032] Proliposomes are an alternative to conventional liposomal
formulations. Proliposomes are dry, free-flowing granular products,
which, on addition of water, disperse to form a multi-lamellar
liposomal suspension. The stability problems associated with
conventional liposomes such as aggregation, susceptibility to
hydrolysis and/or oxidation are avoided by using proliposomes.
[0033] U.S. Pat. No. 5,635,206 to Ganter et al. issued Jun. 3, 1997
discloses a process for preparing liposomes or proliposomes.
[0034] Proliposomes of indomethacin were prepared using
effervescent granules, which upon hydration yielded liposomes of
high encapsulation efficiency and increased anti-inflammatory
activity with decreased ulcerogenic index (see, for example, Katare
et al., 1991, J. Microencapsulation 81: 1-7).
[0035] The proliposomal concept has been extended to administer
drugs through various routes and also to the food industry wherein
enzyme immobilization is essential for various food processing
regimes. A typical example is the immobilization of the enzyme,
chymotrypsin, in liposomes obtained from proliposomes.
[0036] There remains a need in the art for a general, inexpensive
and effective means for delivering biologically-active compounds,
including drugs, hormones, enzymes, genetic material, antigens
including vaccines, and nutrients, to an animal by oral
administration. Advantageous embodiments of such delivery means are
formulated to efficiently deliver biologically-active compounds to
the appropriate portion of the gastrointestinal tract for efficient
absorption.
SUMMARY OF THE INVENTION
[0037] The present invention is directed to an improved method for
delivering biologically-active compounds, particularly drugs,
hormones, enzymes, genetic material, antigens including vaccines,
and nutrients, to an animal by oral administration. This delivery
system achieves specific delivery of such biologically-active
compounds through associating the compounds with liposomes and
proliposome components.
[0038] In preferred embodiments, the biologically active compound
is formulated as a proliposomal composition that can be
reconstituted in vivo to provide a liposomal preparation.
Preferably, the invention provides pharmaceutical compositions
comprising the biologically active compound and a lipid formulated
as a proliposomal preparation. In more preferred embodiments, the
pharmaceutical compositions of the invention are formulated for
oral administration. Most preferably, the pharmaceutical
compositions of the invention formulated for oral administration
comprise an enteric coating sufficient to prevent dissolution of
the composition in the stomach of an animal. In alternative
embodiments, the pharmaceutical compositions also comprise a
protective coating between the enteric coating and the core of the
composition comprising the proliposomal components thereof.
Additional advantageous components of said orally-administrable
pharmaceutical compositions further comprise the pharmaceutical
compositions as will be understood by those with skill in the
art.
[0039] Specific preferred embodiments of the present invention will
become evident from the following more detailed description of
certain preferred embodiments and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIGS. 1A through 1C depict thermograms produced by
differential scanning calorimetry as set forth in Example 1.
[0041] FIGS. 2 and 3 depict transfer rates of glyburide through a
Caco-2 cellular monolayer using the liposomal compositions of the
invention, as set forth in Example 2.
[0042] FIGS. 4 and 5 depict total accumulation of glyburide in the
receiving chamber of a transwell comprising a Caco-2 cellular
monolayer using the liposomal compositions of the invention, as set
forth in Example 2.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0043] The present invention provides compositions of matter and
methods for facilitating the delivery of biologically-active
compounds to the tissues of an animal after oral administration.
For the purposes of this invention, the term "biologically-active
compound" is intended to encompass all naturally-occurring or
synthetic compounds capable of eliciting a biological response or
having an effect, either beneficial or cytotoxic, on biological
systems, particularly cells and cellular organelles. These
compounds are intended to include but are not limited to all
varieties of drugs, including but not limited to antibiotic,
antibacterial, antiviral, antimycotic, antiproliferative and
antineoplastic drugs; hormones, including peptide hormones and
steroid hormones, and most particularly including endocrine and
exocrine gland hormones; genes, recombinant nucleic acids,
oligonucleotides or other nucleic acids encoding all or a portion
of a mammalian gene, a viral gene or a gene from a microorganism;
antigens, particularly in the form of vaccines; enzymes,
particularly digestive enzymes and most particularly enzymes
involved in processing, modifying, converting or degrading a
nutrient into a form more easily absorbed by an animal's
gastrointestinal tract; nutrients, and most preferably vitamins and
minerals; and most particularly any biologically active compound,
including particularly nutrients, that inefficiently transits the
gastrointestinal tract or is unstable in a compartment thereof.
[0044] Pharmaceutical compositions comprising the biologically
active compounds of the invention are preferably provided as
proliposomal compositions that can be reconstituted, most
preferably in vivo, to produce liposomal compositions of the
biologically active compounds. As used herein, the term
"proliposome" and "proliposomal" are intended to encompass dry,
free-flowing granular products, which, on addition of water,
disperse to form multi-lamellar liposomal suspensions comprising
the biologically active compounds of the invention. Advantageously,
the stability problems associated with the conventional liposomes
(such as aggregation, susceptibility to hydrolysis and oxidation)
are avoided by using proliposomes
[0045] The proliposomal compositions provided by the invention are
reconstituted, particularly in vivo, to provide liposomal
compositions wherein the biologically active compounds of the
invention are encapsulated in said liposomes. In preparing the
proliposomal compositions of the invention, lipid components
including neutral lipids, positively-charged lipids,
negatively-charged lipids, amphoteric lipids such as phospholipids,
and cholesterol are advantageously used. As defined herein, the
"lipid component" of the proliposomal compositions of the invention
are intended to encompass a single species of lipid (such as a
particular phospholipid) or combinations of such lipids, either of
one type such as combinations of phospholipids (for example,
phosphatidylcholine plus phosphatidyl ethanolamine) or of different
types (such as a phospholipid plus a charged lipid or a neutral
lipid). Combinations comprising a multiplicity of different lipid
types are also advantageously encompassed by the proliposomal
compositions of the invention (see, Lehninger, 1975, Biochemistry,
2d ed., Chapters 11 & 24, Worth Publishers: New York; and
Small, 1986, "From alkanes to phospholipids," Handbook of Lipid
Research: Physical Chemistry of Lipids, Volume 4, Chapters 4 and
12, Plenum Press: New York).
[0046] Biologically active compounds that are unstable in the
stomach, such as proteins and peptides, vitamins and other small
molecule nutrients, or biologically active compounds that irritate
the stomach, such as various analgesics like aspirin, and those
compounds that are preferentially absorbed in the small intestine
are preferred biological compounds useful with the liposomal
formulations of the invention. In preferred embodiments, said
compounds include but are not limited to aspirin, ibuprofen,
erythromycin, vasopressin, insulin, dideoxyinosine (ddI),
cyclosporine, taxol, heparin, halofantrine, ethopropazine,
griseofulvin, propofol, furosemide, carbamazepine, diazepam,
candesartan and cilexetil.
[0047] The proliposomal preparations comprising the biologically
active compounds of the invention are preferably provided in a form
that can be orally administered, including but not limited to
syrups, elixirs, capsules, tablets, and emulsions. Preferred forms
are tablets or capsules, most preferably comprising an enteric
coating to prevent premature dissolution under the chemically harsh
environment of the stomach. Enteric coatings are prepared as will
be understood by one having skill in the art, and preferably
include coatings including but not limited to eudragit and
cellulose acetate phthalate.
[0048] In alternative embodiments, the tablets or capsules of the
invention comprise a protective coating between the enteric coating
and the core of the capsule or tablet comprising the proliposomal
preparations of the invention. In such embodiments, the protective
coating is prepared as will be understood by one having skill in
the art, and preferably include coatings including but not limited
to hydroxypropyl methylcellulose, polyethylene glycol and
ethylcellulose. In additional embodiments, the protective coating
further comprises a plasticizing agent, including but not limited
to triethylcitrate and polyvinyl pyrrolidone.
[0049] The tablets, capsules and other like embodiments of the
proliposomal preparations and pharmaceutical compositions of the
invention further advantageously comprise particle lubricants that
minimize the tendency of the granular proliposomal compositions to
agglomerate. By "particle lubricant" as used herein is meant the
class of materials used in the manufacturing of pharmaceutical
tablets as lubricants to improve the flowability and prevent
agglomeration of an active agent during the tableting process.
Examples of particle lubricants include talc, lactose, corn starch,
ethyl cellulose, fatty acid salts such as magnesium stearate, agar
pectin, fatty acids such as stearic acid, gelatin and acacia.
[0050] The invention specifically provides methods for preparing
and administering the proliposomal compositions of the invention as
disclosed in the Examples below, and pharmaceutical compositions
comprising the proliposomal preparations of biologically active
compounds.
[0051] Animals to be treated with the proliposomal preparations and
pharmaceutical compositions of the invention are intended to
include all vertebrate animals, preferably domesticated animals,
such as cattle, horses, goats, sheep, fowl, fish, household pets,
and others, as well as wild animals, and most preferably
humans.
[0052] One advantage of orally-administered liposomal formulations
over parenterally-administered formulations is that oral
administration reduces uptake of liposomes by the liver, thus
reducing liver toxicity (which is a particular liability of
parenterally-administered liposomal formulations). Oral
formulations are targeted to deliver biologically active compounds
to the intestine, which is a large surface for absorption and
results in slow release of the administered compound. Finally, oral
administration avoids transport-mediated saturation of drugs like
dideoxyinosine.
[0053] The formulations of the invention are advantageously used
for treating diseases that cause or result in malabsorption,
including but not limited to Crohn's disease, irritable bowel
syndrome, celiac sprue, diverticulitis, immunoproliferative small
intestine disease, liver disease, diseases and disorders of the
gall bladder (including those disorders that are consequent to
surgical removal of the gall bladder), pancreatitis, Schwachman's
syndrome, steatorrhea, Whipple's disease, parasitic infection,
malabsorption as a consequence of chronic laxative use or abuse,
pancreatic enzyme deficiency, disaccharidase deficiency, or defects
in fat absorption consequent to surgical gastrectomy or other
surgical interventions in the gastrointestinal tract.
[0054] The following Examples illustrate certain aspects of the
above-described method and advantageous results. The following
examples are shown by way of illustration and not by way of
limitation.
EXAMPLE 1
[0055] Proliposomal formulations useful for oral administration
were developed using an in vitro model system. Human Caco-2 cells
(colon adenocarcinoma cells), grown on semipermeable filters,
provide a simple and reliable in vitro model for studying drug
transport across the intestinal mucosa. Caco-2 cells are recognized
in the art for yielding useful predictions on oral absorption of
new drug formulations.
[0056] In order to assay the proliposomal tablets of the invention,
glyburide (glybenclamide), an oral blood-glucose-lowering drug of
the sulfonylurea class, was used as model drug, because uptake in
the CaCo-2 system can be monitored by measuring transport across
monolayers formed by this cell line.
[0057] Proliposomal tablets were prepared as follows. The
identities and amounts of each of the reagents used to prepare the
tablets of the invention are shown in Table I. Phospholipids DMPC
and DSPC were obtained from Avanti Polar Lipids (Alabaster, Ala.);
glyburide, cholesterol, stearylamine, dicetylphosphate and all
tissue culture reagents were obtained from Sigma Chemical Co. (St.
Louis, Mo.); purified talc and anhydrous lactose were obtained from
J.T. Baker (Phillipsburg, N.J.) and Quest, Int'l. (Hoffman Estates,
Ill.); chloroform, methanol and ethanol were obtained from Fisher
Scientific (Fairlawn, N.J.); Caco-2 cells were obtained from the
American Type Culture Collection (Manassas, Va.; Accession No. HTB
37); and transwell culture chambers were obtained from Costar
(Cambridge, Mass.).
[0058] Glyburide, lipid and cholesterol were dissolved at room
temperature in 10 mL chloroform. Lactose (25 mg/tablet) was
suspended in the organic mixture and the suspension evaporated to
dryness at 60.degree. C. in a conventional coating pan. The solid
residue was collected and sifted through a #60 mesh screen. The
sifted residue was then mixed with Explotab.RTM. (3 mg/tablet),
lactose (50 mg/tablet) and talc (2 mg/tablet) and compressed into
tablets using a Manesty B3B 16 station press. The tablets were then
coated with a solution of hydroxypropyl methylcellulose in ethyl
alcohol (3% w/v) containing triethyl citrate (15% of polymer
weight) as a plasticizer. Eudragit L30 D-55 (7% w/w) was then
applied on the coated tablets.
TABLE-US-00001 TABLE I Formulary for Preparing Proliposome Tablets
Quantity of each ingredient used (mg/tablet) Formulation Glyburide
DSPC DMPC CHO STA DCP DSCP/Neu 5.0 10.0 -- -- -- -- DSPC.Neu.Cho
5.0 10.0 -- 2.45 -- -- DSPC/Pos 5.0 10.0 -- -- 0.35 -- DSPC/Pos/Cho
5.0 10.0 -- 2.45 0.35 -- DSPC/Neg 5.0 10.0 -- -- -- 0.69
DSPC/Neg/Cho 5.0 10.0 -- 2.45 -- 0.69 DMPC/Neu 5.0 -- 10.0 -- -- --
DMPC/Neu/Cho 5.0 -- 10.0 2.85 -- -- DMPC/Pos/Cho 5.0 -- 10.0 2.85
0.40 -- DSPC = distearylphosphatidylcholine DMPC =
dimyristylphosphatidylcholine STA = stearylamine (Pos: positively
charged lipid) CHO = cholesterol (Neu: neutral lipid) DCP =
dicetylphosphate (Neg: negatively charged lipid)
[0059] The purity of the reagents used to make the proliposome
tablets of the invention described herein was tested using
differential scanning calorimetry. Samples were prepared by
dissolving lipid with glyburide and cholesterol separately at a
ratio of 1:1 (w/w) in an excess of chloroform. The organic layer
was removed and thermograms obtained using a differential scanning
calorimeter (TA Instruments, New Castle, Del., Model 2910). Each
component was scanned both individually and using a mixture
comprising glyburide, lipid and cholesterol at a ratio of 1:1:1
(w:w:w). 2-5 mg of sample was scanned at a rate of 20.degree. C.
per minute over a suitable temperature range (25-225.degree. C.) in
a hermetically-sealed aluminum pan. The peak transition
temperatures of the dispersion were compared with the pure
compounds. The results of these experiments are shown in FIGS. 1A
through 1C.
[0060] FIG. 1A shows a thermogram of DMPC alone compared with
mixtures of DMPC and cholesterol (DMPC/CHOL), DMPC and glyburide
(DMPC/GLYB) and DMPC, cholesterol and glyburide (DMPC/CHOL/GLYB).
Peak transition temperatures are shown in the Figure. In contrast
to the simple and easily-recognizable peak transition temperature
obtained for DMPC, the mixtures are heterogeneous, having more than
one localized peak region where a thermal transition occurs.
[0061] FIG. 1B shows a thermogram of DSPC alone compared with
mixtures of DSPC and cholesterol (DSPC/CHOL), DSPC and glyburide
(DMPC/GLYB) and DSPC, cholesterol and glyburide (DSPC/CHOL/GLYB).
Peak transition temperatures are shown in the Figure. A similar
pattern is observed herein, where there is a simple and
easily-recognizable peak transition temperature obtained for DSPC,
but the mixtures are heterogeneous, having more than one localized
peak region where a thermal transition occurs.
[0062] Thermograms were also obtained individually and in mixtures
for glyburide and cholesterol, and these results are shown in FIG.
1C. From these thermograms, it is evident that the presence of
cholesterol acts as an "impurity" in the drug, lowering its melting
point. The same effect is observed in mixtures of the drug and
lipid. In the presence of both cholesterol and lipid, the melting
point of glyburide is further decreased, demonstrating a
synergistic effect. These results also indicate that the amount of
heat required to melt the drug in a pure state is far higher than
the amount needed when the drug is combined with cholesterol or
lipid. This explains the increased solubility of the drug when
prepared in a solid dispersion of lipid and/or cholesterol.
[0063] Liposomes were reconstituted from proliposomal tablets by
adding one tablet to 1 mL phosphate buffered saline in a sterile
glass vial. The tablet was allowed to stand at 37.degree. C. for 1
hour with shaking, which was sufficient to dissolve the tablet and
reconstitute the liposomal preparation.
[0064] Reconstituted liposomes were characterized for size
distribution by large-angle dynamic light scattering using a
particle size analyzer (Brookhaven Instruments, Model BI-90). Each
preparation was diluted with filtered saline to an appropriate
concentration to achieve a medium viscosity of 0.089 centipoise and
a medium relative refractive index of 1.332 at room temperature.
Measurements obtained under these condition are shown in Table II.
These results indicated that the particle size of the resulting
liposomes varied both with the presence or absence of cholesterol
and with the identity of the phospholipid component. The mean
diameter of the liposomes was greater in neutral liposome
embodiments than in charged liposome embodiments, and can be
explained by the greater propensity of neutral liposomes to
aggregate or fuse with one another. Encapsulation efficiency,
defined as the percentage of the glyburide encapsulated in
liposomes, was determined using the protamine-induced aggregation
method as described in Kulkarni et al. (1995, Pharm. Sci. 1:
359-362). Briefly, each tablet was disintegrated in 1 mL of
phosphate-buffered saline (PBS, pH 7.4) to give a concentration of
10 mg/mL of lipid. To 100 .mu.L of the preparation, equal
quantities of a protamine solution (50 mg/mL) in PBS was added and
vortexed for about 1 min. The mixture was then incubated for about
12 hours at room temperature. After incubation, the mixture was
centrifuged at about 16,000.times.g for about 5 minutes. 100 .mu.L
of the supernatant was removed and the pellet was dissolved in
about 1 mL of reagent-grade alcohol (95% ethanol) and sonicated for
5 minutes.
TABLE-US-00002 TABLE II Liposome Particle Size (nm) of Different
Tablet Formulations Lipid Type Formulation/Charge DSPC DMPC Neutral
1413 1825 Neutral/Cholesterol 1035 748 Positive 1059 N.D.
Positive/Cholesterol 867 629 Negative 3633 N.D.
Negative/Cholesterol 800 N.D. N.D.: not determined
[0065] The quantity of glyburide in the pellet and the supernatant
was determined by HPLC analysis using the Star.RTM. 9010 solvent
system and Star 9095.RTM. variable-wavelength ultraviolet/visible
spectrum spectrophotometric detector (Varian Associates, Walnut
Creek, Calfi.) and the data analyzed by a Dynamax.RTM.
MacIntegrator (Rainin Instrument Co., Woburn, Mass.). HPLC analysis
was performed using a C18 column (Phenominex.RTM.) packed with 5
.mu.m particles and having dimensions of 250 mm in length and an
internal diameter of 4.6 mm. The mobile phase was a solution of
methanol in 0.1M phosphate buffer, pH 3.5 at a ratio of 75:25 by
volume. Column flow rate was 1.0 mL/min and the output was scanned
at a wavelength of 225 nm.
[0066] The results of these characterization experiments are shown
in Table III.
TABLE-US-00003 TABLE III Drug Encapsulation Efficiency (% .+-.
s.d.) Lipid Type Formulation DSPC DMPC Neutral 81.6 .+-. 0.4 86.7
.+-. 2.7 Neutral/Cholesterol 80.4 .+-. 0.6 88.8 .+-. 1.2 Positive
78.4 .+-. 0.7 N.D. Positive/Cholesterol 81.0 .+-. 1.2 87.6 .+-. 0.6
Negative 81.2 .+-. 0.1 N.D. Negative/Cholesterol 80.4 .+-. 0.4 N.D.
N.D.: Not determined
[0067] These results demonstrated that a slightly higher percentage
of the drug was encapsulated in DMPC. These results are consistent
with a slightly higher amount of the drug being encapsulated in
"fluid" liposomes (i.e., those comprising DMPC) than liposomes in a
gel state (i.e., those comprising DSPC) at 37.degree. C.
EXAMPLE 2
[0068] Caco-2 cell cultures were prepared as monolayers on
polycarbonate transwells having a membrane pore size of 4 nm.
Caco-2 cells were first grown in T-150 flasks (Falcon, Lincoln
Park, N.J.) at 37.degree. C. under an atmosphere of 5% CO.sub.2 and
95% air in Dulbecco's modified Eagle's medium (pH 7.2, Sigma
Chemical Co., St. Louis, Mo.), with conventional supplements. The
medium was changed every other day until the monolayers reached
about 90% confluency. Media was removed and the cells were washed
with Hank's balanced salt solution (HBSS, Sigma). The cells were
trypsinized by adding 0.5 mL of a 0.25% trypsin solution containing
1 mM EDTA to each flask and incubating the monolayers for 10 min at
37.degree. C. The separated cells were removed from the flasks and
collected into centrifuge tubes, centrifuged at 200.times.g for 10
min, the supernatant removed and the pellet resuspended in a
sufficient amount of Dulbecco's modified Eagle medium to yield a
suspension that would produce about 60,000 cells/cm.sup.2 on
plating. The Caco-2 cells were then seeded into Transwell
semipermeable membrane inserts having 4 .mu.m pore size. In the
transwells, media was changed every other day until the cells were
used for the transport studies described below.
[0069] Caco-2 cell cultures on transwell membranes prepared as
described above were used for transport studies about 17 days after
plating. Proliposome tablets were dissolved as described above by
incubation for 1 h with shaking at 37.degree. C. in 2 mL HBSS. As a
control, pure glyburide treated with chloroform was compressed into
tablet form with lactose and Explotab.RTM.; all controls were
treated exactly as experimental.
[0070] The medium from the transwell plates was gently removed
using a micropipette. 0.5 mL of the reconstituted liposomal
suspension was gently added to the donor compartment of the
transwell and 1.5 mL of HBSS was added to the receiver compartment.
100 .mu.L of FITC-Dextran was then added to the donor compartment
to a final concentration of 10 .mu.g/mL of FITC-Dextran in the
donor side. FITC-Dextran was used as a marker to test for the
presence of leaks, if any, on the monolayers covering the
semipermeable transwell membranes. Samples (300 .mu.L) were
carefully withdrawn from the receiver side at 50, 120, 180, 240,
300 minutes after addition, and the receiver side was replenished
with 300 .mu.L of fresh HBSS each time the sample was taken. Cells
were incubated at 37.degree. C. in a 5% CO.sub.2/95% air atmosphere
at all times during these assays. Sampling was done under aseptic
conditions in a laminar air-flow hood.
[0071] The amount of glyburide transported during each sampling
interval was determined by injecting 90 .mu.L of the sample onto
the HPLC system described above in Example 1 and peak areas were
recorded. These experiments were performed in triplicate and the
average of the results was reported. The results of the experiments
are shown in FIGS. 2 through 5.
[0072] FIG. 2 shows the results of glyburide transit across Caco-2
cell monolayers in formulations containing
distearylphosphatidylcholine (DSPC). Control experiments performed
in the absence of DSPC had a flow rate of almost 1
.mu.g/hr.cndot.cm.sup.2. Formulations of glyburide with DSPC (a
"neutral" lipid at physiological pH) showed a similar level of flux
across the monolayer, although the addition of cholesterol to these
formulations increased the flux about two-fold. Formulations of
glyburide with negatively-charged lipid, on the other hand, in
either the presence or absence of cholesterol were transported
across the monolayer at an even lower rate. In contrast,
formulations of glyburide with positively-charged lipid were
transported across the membranes at a rate about fourfold higher
than control, and the addition of cholesterol increased this to a
rate of about fivefold higher than control.
[0073] FIG. 3 shows the results of parallel experiments using
dimyristylphosphatidylcholine as the lipid component. A similar
pattern of glyburide flux was seen in these experiments; however,
the degree of enhancement of transit across the Caco-2 cell
monolayer was much higher for formulations containing DMPC. For
example, glyburide formulations containing DMPC and
positively-charged lipid had a transit rate almost thirty-fold
higher than control. Formulations of neutral lipid were elevated to
a lesser degree; in the presence of cholesterol such formulations
had a transit rate about eightfold higher than control, and in the
absence of cholesterol this rate was about fivefold higher than
control.
[0074] FIGS. 4 and 5 show the cumulative amount of transported
glyburide using DSPC- and DMPC-containing formulations over a five
hour period. FIG. 4 shows DSPC-containing formulations, wherein the
highest accumulation levels were achieve with glyburide
formulations containing DSPC and positively-charged lipid (about 27
.mu.g). Similar formulations additionally containing cholesterol
had lower total amounts (about 13 .mu.g). DSPC formulations
containing neutral lipid and cholesterol showed slower kinetics but
achieved essentially the same total accumulation as DSPC/positive
lipid/cholesterol formulations. Formulations containing DSPC and
neutral lipids in the absence of cholesterol showed the same total
accumulation as control (about 2.5 .mu.g), while DSPC formulations
with negatively-charged lipid (in the presence or absence of
cholesterol) showed lower total accumulation amounts.
[0075] FIG. 5 shows the results of similar experiments performed
with DMPC formulations. Total accumulation levels were noticeably
higher than control only for formulations containing DMPC,
positively-charged lipid and cholesterol (about 34 .mu.g), while
DMPC formulations with neutral lipid (in the presence or absence of
cholesterol) resulted in total accumulation at levels equivalent to
control (about 2-5 .mu.g).
[0076] These results demonstrated that liposomes can be
successfully prepared for oral administration in the form of
enteric-coated proliposome tablets. The presence of cholesterol
reduces the particle size of the formulation. Proliposomes provide
a stable system of production of liposomes for oral administration.
Degradation of proliposome contents of the tablet in the stomach
can be effectively avoided by administering the proliposomes as
enteric-coated tablets. Enhanced transport of glyburide across
Caco-2 cells was observed with such liposomal formulations.
Although the transport of glyburide with DMPC formulations is
higher than transport in the DSPC formulation in vitro, DSPC
formulations are better suited for in vivo conditions because of
the rigidity and increased stability of the membrane against the
attack of bile salts and enzymes of the intestine. Since in vitro
transport across Caco-2 cells is an indication of bioavailability,
an increased transport with the liposome formulation suggests an
increased bioavailability of compounds that are poorly absorbed
otherwise. For example, using a suitable polymer coating for the
proliposomal tablets of the invention, colonic delivery of drugs,
especially peptides may be possible.
[0077] Proliposomes are ideally suited for lipophilic compounds,
since the majority of such a biologically active compound will
partition into the lipid phase. These results also have
implications for developing formulations that stabilize the
encapsulated drug.
[0078] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims.
* * * * *