U.S. patent number 5,006,343 [Application Number 07/295,926] was granted by the patent office on 1991-04-09 for pulmonary administration of pharmaceutically active substances.
Invention is credited to Bradley J. Benson, JoRae Wright.
United States Patent |
5,006,343 |
Benson , et al. |
April 9, 1991 |
Pulmonary administration of pharmaceutically active substances
Abstract
Pulmonary administration of a pharmaceutically active substance
useful for local or systemic action which comprises, liposomes
containing an effective amount of a pharmaceutically active
substance, and an amount of alveolar surfactant protein effective
to enhance transport of the liposomes across a pulmonary
surface.
Inventors: |
Benson; Bradley J. (San
Francisco, CA), Wright; JoRae (San Francisco, CA) |
Family
ID: |
23139815 |
Appl.
No.: |
07/295,926 |
Filed: |
December 29, 1988 |
Current U.S.
Class: |
424/450; 252/181;
264/4.6; 514/1.2; 514/15.5 |
Current CPC
Class: |
A61K
9/0073 (20130101); A61K 9/0082 (20130101); A61K
9/127 (20130101) |
Current International
Class: |
A61K
9/00 (20060101); A61K 9/127 (20060101); A61K
037/22 () |
Field of
Search: |
;424/450 ;252/181
;264/4.6 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
|
|
|
8701586 |
|
Mar 1987 |
|
WO |
|
8804938 |
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Jul 1988 |
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WO |
|
Primary Examiner: Cashion, Jr.; Merrell C.
Assistant Examiner: Prater; P. L.
Attorney, Agent or Firm: Irell & Manella
Claims
We claim:
1. A composition for pulmonary administration of a pharmaceutically
active substance, said composition comprising 60-90% by weight
liposome forming compound, 5-20% by weight pharmacentically active
substance, and 5-20% by weight surfactant protein, based on the
total weight of liposome-forming substance, pharmacentically active
substance and alveolar surfactant protein, wherein said composition
comprises liposomes.
2. The composition of claim 1 wherein said alveolar surfactant
protein comprises at least one alveolar surfactant protein selected
from alveolar surfactant protein SP-A, alveolar surfactant protein
SP-B, and alveolar surfactant protein SP-C.
3. The composition of claim 1 wherein said pharmaceutically active
substance is water insoluble.
4. The composition of claim 1 wherein said pharmaceutically active
substance is water soluble.
5. The composition of claim 3 wherein said pharmaceutically active
substance is Vitamin E.
6. The composition of claim 1 wherein said liposome-forming
compound comprises 1-90% by weight dipalmitoyl phosphatidyl
choline, 1-90% by weight phosphatidyl choline, 1-30% by weight
phosphatidyl glycerol, and 1-30% by weight cholesterol.
7. A method of administering a pharmaceutically active substance
which comprises applying to pulmonary surfaces for transport across
said surfaces a composition comprising:
(a) liposomes formed from at least one liposome-forming compound,
said liposomes containing an effective amount of a pharmaceutically
active substance; and
(b) alveolar surfactant protein in an amount effective to enhance
transport of said liposomes across a pulmonary surface.
8. The method of claim 7 wherein said composition is applied to the
pulmonary surfaces in the form of an aerosol spray.
9. A method of preparing a composition for delivering a
pharmaceutically active substance comprising:
(a) forming liposomes containing at least one liposome-forming
compound and an effective amount of a pharmaceutically active
substance; and
(b) adding alveolar surfactant protein in an amount effective to
enhance transport of said liposomes across a pulmonary surface.
Description
TECHNICAL FIELD
This invention relates to the field of pharmaceutical
administration. More specifically it relates to pulmonary
administration of pharmaceutically active substances.
BACKGROUND ART
The mode of administration of a drug can affect its bioavailability
and pharmacokinetic profile, as well as patient compliance. Patient
compliance is best when the mode of administration is convenient
and does not involve patient discomfort. Oral administration is
often the preferred mode.
Despite the advantages of oral administration, it is still
unworkable for many drugs. One problem with oral administration
arises because there can be extensive metabolism of a drug during
transit from the gastrointestinal tract to the general circulation.
For example, the intestinal mucosa, through which an orally
administered drug passes before it enters the circulatory system,
is enzymatica))y very active and can thus metabolize a drug in many
ways. Therefore, bioavailability of orally administered drugs can
be very low.
With the advent of recombinant DNA technology, many more peptides
are available for pharmaceutical use than ever before. However,
oral administration of peptides is particularly problematic because
peptide bonds are cleaved by proteases secreted into the gut, as
part of the digestive process. The only broadly applicable means of
administration of peptides at this time is parenteral
administration. This is not always a practical or desirable route,
especially if the drug is required to be administered chronically,
such as in the case of insulin treatment for diabetes. Many
individuals are reluctant or unable to self-inject a
parenterally-formulated drug on a routine basis.
Some of the shortcomings of oral and parenteral administration can
be circumvented by administering the drug by a route which avoids
digestive and gut-wall metabolism, and also eliminates the need for
injection. Examples of such alternative routes include transdermal
(Rajadgyaksha, V. et al., PCT publication WO 88/04938), nasal
(Carey, M. C., et al., U.S. Pat. No. 4,746,508), and pulmonary
delivery.
Transdermal administration is not workable for many drugs which
cannot penetrate the dermis unless they are formulated with
permeation enhancers, such as DMSO, which can cause undesirable
side effects. Nasal administration using low-toxicity permeation
enhancers of the fusidic acid derivative family has been effective
in many instances (Carey, M. C., supra), however, the surface area
of nose is relatively small.
Pulmonary delivery offers several potential advantages,
particularly in the case of administration of drugs intended to
treat conditions affecting the lungs themselves, because some drugs
have difficulty reaching the lungs by any route of administration.
For instance, Pseudomonas infections in patients with cystic
fibrosis can be difficult or impossible to treat, since antibiotics
delivered by conventional modes of administration do not easily
reach the lungs and therefore cannot stop the spread of
infection.
Currently, pulmonary drug delivery methods include mechanical means
such as aerosols and inhalers, which have been employed with or
without the addition of liposomes to the drug formulation. These
methods are effective at getting the drug to the lung, however,
they do not ensure efficient transport across the pulmonary
surface.
Hayward, J. A. (PCT patent publication #WO 87/01586) discloses
using liposomes which contain a drug or diagnostic agent in an
aerosol composition for inhalation. Although the
liposome-containing drug is more effective than the drug in
solution, the drug is still not efficiently administered.
A number of studies have been done which involved the pulmonary
administration of lipids for the treatment of respiratory distress.
Most of these studies administer liposomes only, without addition
of pharmaceutically active substances (Yoshida, T., et al., U.S.
Pat. No. 4,571,334). A few groups have investigated the
administration of peptides with liposomes for the treatment of
respiratory distress (Schilling, et al., U.S. Pat. No. 4,659,805
and Whitsett, J. A., PCT patent publication WO 87/01586). In these
cases, however, the lipids were not used as a drug delivery
vehicle. Rather the lipid/protein complex was administered to
compensate for a deficiency of a normally present lipid/protein
complex, which reduces surface tension along the alveolar
surfaces.
The compositions and methods of the present invention are widely
applicable to a variety of pharmaceutically active substances which
for the first time can be efficiently delivered across pulmonary
surfaces.
BRIEF DESCRIPTION OF THE INVENTION
The invention provides compositions and methods useful for
transporting a pharmaceutically active substance across pulmonary
surfaces which avoids some of the problems associated with other
modes of delivery. The compositions employed are admixtures
comprising: (a) liposomes formed from at least one liposome-forming
compound, said liposomes containing an effective amount of a
pharmaceutically active substance; and (b) an amount of alveolar
surfactant protein effective to enhance transport of the liposomes
across a pulmonary surface. The compositions can be administered to
the pulmonary surface through a variety of methods such as
endotracheal administration. The pharmaceutically active substances
contained in the compositions of the invention can be delivered
locally for pulmonary action or via the pulmonary surface into the
general circulation for systemic action.
The pharmaceutically active substance may be either water soluble
or water insoluble. While not wishing to be bound by any particular
theory or mechanism of action, it is believed in the case of water
soluble substances, the substance is contained in the liposome
through encapsulation. Also, it is believed in the case of water
insoluble substances, the substance is contained in the liposome
through interaction with the hydrophobic portions of the lipids
comprising the liposomes.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the protein sequence of human alveolar surfactant
protein SP-A.
FIG. 2 shows the protein sequence of human alveolar surfactant
protein SP-B.
FIG. 3 shows the protein sequence of human alveolar surfactant
protein SP-C.
FIG. 4 is a schematic cross-sectional representation of a liposome,
which is depicted to contain both water soluble and water insoluble
pharmaceutically active substances.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
As used herein, "alveolar surfactant protein" refers to the
apoproteins associated with alveolar surfactant described herein
below. The human lung is composed of a large number of small sacs
or alveoli in which gases are exchanged between the blood and the
air spaces of the lung. The exchange is mediated by the presence of
alveolar surfactant which is a complex substance that lines the
epithelial surface of the lung. The surfactant is composed of
phospholipid components and protein components. The
surfactant-associated protein comprises both serum protein and
surfactant-specific apoproteins. There are three major
surfactant-specific proteins. One is a water-soluble protein having
a molecular weight of the order of 32,000 daltons (alveolar
surfactant protein SP-A) and the other two are very hydrophobic
proteins having molecular weights of the order of about 10,000
daltons (alveolar surfactant protein SP-B and alveolar surfactant
protein SP-C) (King, R. J., et al, Am. J. Physiol. (1973)
224:788-795). Granular pneumocytes (type II alveolar cells) secrete
the surfactant material (Goerke, J., Biochim. Biophys. Acta (1974)
344:241-261 and King, R. J., Fed. Proc. (1974) 33:2238-2247).
"Alveolar surfactant protein SP-A" refers to the relatively high
molecular weight (of the order of 32 kD) apoprotein associated with
the lung surfactant complex (Schilling, et al., U.S. Pat. No.
4,659,805). The alveolar surfactant protein SP-A of the invention
has an amino acid sequence substantially as shown in FIG. 1.
"Alveolar surfactant protein SP-B" refers to the larger of the two
hydrophobic proteins which has a molecular weight of about 18 kD on
gels under non-reducing conditions, but which shows a molecular
weight of about 10 kD on gels under reducing conditions and has an
amino acid sequence substantially as shown in FIG. 2.
"Alveolar surfactant protein SP-C" refers to the smaller of the two
hydrophobic proteins which has a molecular weight of about 8 kD or
5 kD on gels and has an amino acid sequence substantially as shown
in FIG. 3.
Minor modifications of the above three sequences which do not
destroy activity also fall within the definition of alveolar
surfactant protein as further set forth below. As is the case for
all proteins, the alveolar surfactant proteins can occur in neutral
form or in the form of basic or acid addition salts depending on
their mode of preparation, or, if in solution, upon its
environment. It is well understood that proteins in general, and,
therefore alveolar surfactant proteins, in particular, may be found
in the form of their acid addition salts involving the free amino
groups, or basic salts formed with free carboxyls. Pharmaceutically
acceptable salts may, indeed, enhance the functionality of the
protein. Suitable pharmaceutically acceptable acid acid addition
salts include those formed from inorganic acids such as, for
example, hydrochloric or sulfuric acids, or from organic acids such
as acetic or glycolic acid. Pharmaceutically acceptable bases
include the alkali hydroxides such as potassium or sodium
hydroxides, or such organic bases as piperidine, glucosamine,
trimethylamine, choline, or caffeine. In addition, the protein may
be modified by combination with other biological materials such as
lipids and saccharides, or by side chain modification, such as
acetylation of amino groups, phosphorylation of hydroxyl side
chains, or oxidation of sulfhydryl groups or other modification of
the encoded primary sequence. Indeed, in its native form, the
alveolar surfactant protein SP-A is a glycosylated protein, and
certain of the encoded proline residues have been converted to
hydroxyproline. Included within the definition of alveolar
surfactant protein SP-A are glycosylated and unglycosylated forms,
hydroxylated and non-hydroxylated forms, and any composition of an
amino acid sequence substantially similar to that of the native
sequences which retains activity. Also included within the
definition are fragments of the entire sequence of FIGS. 1, 2, and
3 which retain activity.
It is further understood that minor modifications of primary amino
acid sequence may result in proteins which have substantially
equivalent or enhanced activity as compared to the native
sequences. These modifications may be deliberate, as through
site-directed mutagenesis, or may be accidental, such as through
mutation of hosts which are alveolar surfactant protein producing
organisms. All of these modifications are included as long as
activity is retained.
Studies have provided evidence that the phospholipids in the
alveolar surfactant are recycled through the lung as part of normal
surfactant turnover (Ozarzun, M. J., et al, Am. Rev. Resp. Disease
(1980) 121:709-721, Hallman, M., et al, J. Clin. Invest. (1981)
68:742-751, and Jacobs, H., et al, J. Dio. Chem. (1982)
257:1085-1810).
The present invention takes advantage of the knowledge that
surfactant phospholipid and protein components are taken up by type
II cells of the lung. Alveolar surfactant protein SP-A is therefore
used to enhance delivery of pharmaceutically active substances
which are contained in liposomes.
The mechanism for surfactant phospholipid uptake is currently not
understood, but the seeming specificity of the metabolism by type
II cells has prompted investigators to consider protein-mediated or
protein-directed uptake. In the first studies, the low molecular
weight hydrophobic surfactant proteins (alveolar surfactant protein
SP-B and alveolar surfactant protein SP-C) were mixed with
exogenous phospholipids and formed into liposomes by sonic
irradiation (Claypool, W. D., et al, J. Clin. Invest. (1984)
74:677-684 and Claypool, W. D., et al, Exp. Lung Res. (1984)
6:215-222). The phospholipids used were the major types found in
surfactant from mammalian lungs. The hydrophobic proteincontaining
liposomes were incubated with isolated type II cells from rat
lungs. The amount of lipid transferred was 20-70% above control.
This small amount could be attributed to non-specific protein
transfer of lipids between cell membranes and liposomes, fusion of
the liposome with a cell membrane or merely "sticking" of the
liposome to the cells. There also was no cell specificity.
In more recent studies, alveolar surfactant protein SP-A was added
to liposomes composed of synthetic phospholipids which were in the
appropriate proportions as found in normal alveolar surfactant
(Wright, J. R., et al, J. Biol. Chem. (1987) 262:2888-2894). The
results of incubation of this formulation with type II cells were
dramatic. The alveolar surfactant protein SP-A enhanced lipid
uptake about 10-20 fold (1000%) over control. Furthermore,
antibodies to alveolar surfactant protein SP-A blocked the lipid
uptake by :he cells almost completely (95%). In addition the
effects seem specific to type II cells and macrophages.
None of the previous studies gave any indication that a
pharmaceutically active substance could be included in the
liposome-surfactant protein compositions, nor certainly did they
show that such a composition would effectively transport the drug
across the surface of the lung. The present invention is based on
the discovery that delivery of pharmaceutically active ingredients
contained in liposomes is rendered more efficient by the addition
of alveolar surfactant protein to the delivery vehicle.
The present invention also takes advantage of the knowledge that
alveolar surfactant proteins SP-A, SP-B and SP-C have an activity
("ASP activity") which enhances the formation of a lipid film at an
air/aqueous interface and causes spreading across the pulmonary
surfaces. The spreading activity allows for a greater surface area
through which a pharmaceutically active substance can be
administered.
"ASP activity" for a protein is defined as the ability, when
combined with lipids either alone or in combination with other
proteins, to exhibit activity in the in vivo assay of Robertson, B.
Lung (1980) 158:57-68. In this assay, the sample to be assessed is
administered through an endotracheal tube to fetal rabbits or lambs
delivered prematurely by Caesarian section. (These "preemies" lack
their own alveolar surfactant, and are supported on a ventilator.)
Measurements of lung Compliance, blood gases and ventilator
pressure provide indices of activity.
Preliminary assessment of activity may also be made by an in vitro
assay, for example that of Kihg, R. J., et al, Am J Physiol (1972)
223:715-726, or that illustrated below of Hawgood, et al, which
utilizes a straightforward measurement of surface tension at an
air-water interface when the protein is mixed with a phospholipid
vesicle preparation. The alveolar surfactant proteins SP-A, SP-B
and SP-C described herein show ASP activity in combination as well
as independently. The ASP activity can be used to enhance pulmonary
administration of pharmaceutically active substances.
"Pulmonary administration" refers to any mode of administration
which delivers a pharmaceutically active substance to any surface
of the lung. The modes of delivery can include, but are not limited
to those suitable for endotracheal administration, i.e., generally
as a liquid suspension, as a dry powder "dust" or as an aerosol.
Pulmonary administration can be utilized for both local and
systemic delivery of pharmaceutically active substances.
"Transport across a pulmonary surface" refers to any mode of
passage which penetrates or permeates the interior surface of the
lung. This includes passage through any lung surface, including
alveolar surfaces, bronchiolar surfaces and passage between any of
these surfaces. Preferably the passage is through alveolar
surfaces. Most preferably, the passage is through the type II cells
on the alveolar surfaces. Passage can be either directly to the
pulmonary tissues for local action or via the pulmonary tissues
into the circulatory system for systemic action.
"Liposomes" refers to completely closed vesicular bilayer
structures wherein the bilayers have hydrophilic surfaces on either
side and a hydrophobic middle as illustrated in FIG. 4. The
liposomes are composed of known liposome-forming compounds as
described hereinbelow (see, also, Szoka, F. Jr., et al Ann. Rev.
Biophys. Bioeng. (1980) 9:467-508).
"Liposome-forming compound" refers to any compound capable of
forming a liposome either alone or in combination with any other
liposome-forming compound(s). Liposomeforming compounds are usually
lipids. Liposome-forming compounds generally have polar heads and
nonpolar tails as shown in FIG. 4. The polar heads are hydrophilic,
while the nonpolar tails are hydrophobic. The polar heads line both
the interior and exterior surfaces of the liposome. The nonpolar
tails are embedded in the interior of the liposome. Preferred
liposome-forming compounds are phospholipids, alone or in
combination with simple lipids.
"Phospholipids" refer to amphipathic lipids which are composed of a
nonpolar hydrophobic tail, a glycerol or sphingosine moiety, and a
polar head. The nonpolar hydrophobic tail is usually a saturated or
unsaturated fatty acid group. The polar head is usually a phosphate
group attached to a nitrogen-containing base. Exemplary of useful
phospholipids are synthetic phospholipids such as phosphatidyl
choline, distearoyl phosphatidyl choline, dipalmitoyl phosphatidyl
choline, dipalmitoyl phosphatidylethanolamine, etc. and natural
phospholipids such as soybean lecithin, egg yolk lecithin,
sphingomyelin, phosphatidyl serine, phosphatidyl glycerol,
phosphatidyl inositol, diphosphatidyl glycerol,
phosphatidylethanolamine, etc. Preferred phospholipids are
dipalmitoyl phosphatidyl choline, phosphatidyl choline, and
phosphatidyl glycerol.
"Simple lipids" refers to those lipids which do not contain fatty
acids. All simple lipids are derived from two major parent classes;
the steroids and the terpenes. The preferred simple lipid is
cholesterol. Cholesterol is a suitable simple lipid since it is
believed to close up the "pores" in the bilayer by occupying spaces
between the lipid side chains at the interior, which results in
tighter packing and a decrease in permeability. Simple lipids alone
do not form liposomes. However, as described above, they are
effective liposome-forming substances in combination with
phospholipids.
"Pharmaceutically active substance" refers to an agent which is a
biologically-active synthetic or natural substance, other than
alveolar surfactant proteins themselves, that is useful for
treating a medical or veterinary disorder or trauma, preventing a
medical or veterinary disorder, or regulating the physiology of a
human being or animal. Any pharmaceutically active substance can be
delivered using the methods of this invention. Whether the
pharmaceutically active substance will reside in the aqueous phase
or in the lipid phase of the liposome will determine how the
composition is prepared.
Preferred pharmaceutically active substances are those which are
useful in treating disorders localized in or near the lungs or
respiratory tract, such as infant or adult respiratory distress
syndrome, oxygen toxicity associated with respirator therapy,
pneumonia, bronchitis, asthma, emphysema, tuberculosis, chronic
obstructive pulmonary disorders, and lung cancer. The
pharmaceutically active substances which can be used to treat the
above disorders include known antivirals, antibacterials,
antifungals, antibiotics, antimicrobials, protease inhibitors,
anti-oxidants, anti-inflammatory agents, anti-allergenics,
xanthines, sympathomimetic amines, mucolytics, corticosteroids,
antihistamines, and chemotherapeutic agents.
"Water soluble" pharmaceutically active substance refers to a
substance which dissolves in water or is equivalently termed
hydrophilic. Such a substance when dissolved in water forms a
homogeneous, single phase liquid system. A water soluble
pharmaceutically active substance when mixed with liposome-forming
compounds will be encapsulated within the liposomes of the
invention. Water soluble pharmaceutically active substances are
depicted by triangles in FIG. 4. Many substances fall into this
definition and include, for example, growth hormone, insulin,
tissue plasminogen activator, sodium oxide dismutase, catalase,
glucocorticoids, growth factors and antibiotics.
"Water insoluble" pharmaceutically active substance refers to a
substance which does not readily dissolve in water or is
equivalently termed hydrophobic. All pharmaceutically active
substances dissolve in water to a certain degree. However, water
insoluble pharmaceutically active substances are not capable of
dissolving enough to become a homogeneous, single phase liquid.
Water insoluble pharmaceutically active substances associate
preferably with the hydrophobic tails of the phospholipids of the
present invention. Such substances when mixed with liposome-forming
compounds are associated with the hydrophobic middles of the
liposomes employed in the invention. Water insoluble
pharmaceutically active substances are depicted by circles in FIG.
4. Many substances fall into this definition and include, for
example, Vitamin E. Vitamin A, Vitamin D, Vitamin K, and steroids
such as progesterone, estrogen, and androgen.
There is a continuum which allows pharmaceutically active
substances to be characterized as water soluble or water insoluble,
however, the localization of the pharmaceutically active substance
in the liposome (association with the hydrophobic tails vs.
entrapment amongst the polar heads) is the final determination of
classification.
B. Method for Making Compositions of the Invention
The compositions of the present invention are capable of effecting
the delivery of pharmaceutically active substances across pulmonary
surfaces. The compositions are prepared by mixing liposomes which
contain a pharmaceutically active substance with alveolar
surfactant protein. Alveolar surfactant proteins SP-A, SP-B and
SP-C are each intended for use in the compositions. Alveolar
surfactant protein SP-A is the preferred alveolar surfactant
protein.
The liposomes of the invention can be made by liposome-forming
procedures known in the art (see, e.g., Szoka, F. Jr., et al Ann.
Rev. Biophys. Bioeng. (1980) 9:467-508). The following methods are
illustrative of those that can be used. However, other known
liposome-forming procedures may also be employed.
Structurally, there are three principal types of liposomes:
multiple compartment liposomes, single compartment liposomes and
macrovesicles. Multiple compartment liposomes are multilamellar,
while single compartment liposomes and macrovesicles are
unilamellar. Multiple compartment liposomes are non-uniform
aggregates each containing several layers of phospholipid molecules
separated from each other by water molecules. Single compartment
liposomes are generally prepared by sonication of multiple
compartment liposomes. Macrovesicles are best formed by the slow
injection of ether solutions of the lipid into warm aqueous buffer
(Deamer, D., et al., Biochim. Biophys. Acta (1976)
443:629.varies.634). Principally, they are large unilamellar
liposomes which can encapsulate approximately ten times more
material than single compartment liposomes.
FIG. 4 is a cross-sectional view of a unilamellar liposome. A
liposome is a completely closed bilayer composed of one or more
liposome-forming compounds 16, one of which is encircled in the
FIGURE. The liposome-forming compounds are each made up of a polar
head 18 and a non-polar, hydrophobic tail 20. The hydrophobic
nature of the non-polar tails of the liposome-forming substances
causes the non-polar tails to associate with other non-polar tails,
to form a hydrophobic environment. There is thus formed a
hydrophobic phase 12 which is disposed within the bilayered
structure. The polar heads of the liposome-forming substances tend
to associate with the hydrophilic environment. Water insoluble
pharmaceutically active substances, 22 when added to a liposome
preparation, become associated with the hydrophobic phase 12. The
hydrophilic nature of the polar heads of the liposome-forming
substances causes them to be orientated away from the hydrophobic
interior, thereby forming an exterior hydrophilic surface 10 and an
interior hydrophilic surface 14. Water soluble pharmaceutically
active substances 24, when added to a liposome preparation, become
encapsulated in the hydrophilic interior of the liposome, within
the interior hydrophilic surface 14.
Multiple compartment or multilamellar liposomes are most commonly
prepared by admixing in a vessel liposomeforming compounds, such
as, phospholipid, cholesterol and other hydrophobic molecules in
the presence of an organic solution, preferably chloroform. The
organic solution is then evaporated, preferably by rotary
evaporation, leaving a thin film of lipid on the wall of the
vessel. Subsequently, the thin film of lipid is removed from the
wall of the vessel by shaking with aqueous buffer solutions. This
process yields multicompartment or multilamellar liposomes.
Sonication of multicompartment liposomes results in the formation
of fairly uniform single compartment vesicles. Gel filtration
and/or ultracentrifugation separate the single compartment
liposomes from the multilamellar ones (Fendler, J. H., et al., Life
Sciences (1977) 20:1109-1120).
Both water soluble and water insoluble pharmaceutically active
substances can be incorporated in liposomes. To prepare liposomes
containing water insoluble pharmaceutically active substances, the
latter are dissolved in an organic solvent, such as chloroform,
along with liposome-forming substances. The chloroform is then
evaporated, for example in a rotary evaporator, whereupon the water
insoluble pharmaceutically active substances become embedded in the
thin film of liposomes deposited on the wall of the vessel. The
liposomes are reconstituted by the addition of aqueous buffer. The
water insoluble pharmaceutically active substance is associated
with the hydrophobic phase of the liposome upon aqueous
reconstitution.
To prepare liposomes containing water soluble pharmaceutically
active substances, liposomes are first prepared, for example, by
the previously described method involving rotary evaporation of the
liposome-forming substances. The resultant thin film of evaporated
liposome-forming substances which is deposited on the wall of the
vessel is reconstituted by the addition of an aqueous buffer which
contains the water soluble pharmaceutically active substance,
thereby forming liposomes which contain entrapped water soluble
pharmaceutically active substances. The water soluble
pharmaceutically active substances have become incorporated in the
aqueous phase of the liposomes. The liposomes containing the
entrapped water soluble pharmaceutically active substance can be
readily separated from the excess free water soluble
pharmaceutically active substance by gel filtration using, for
example, a Sephadex G-50 column. Once the water soluble
pharmaceutically active substance is entrapped in the liposome, it
can only be liberated by leakage through the bilayer of the
liposome or by destruction of the vesicle.
In order to prepare the composition of the invention, the alveolar
surfactant protein SP-A is admixed with the reconstituted liposome
suspension containing the pharmaceutically active substance. This
may be accomplished by adding the alveolar surfactant protein
either during or after reconstitution of the liposomes. When
alveolar surfactant proteins SP-B or SP-C are included in the
composition of the invention, the alveolar surfactant protein SP-B
or SP-C is admixed in an organic solvent with the liposome-forming
substances and the preparation of the composition proceeds as
described above.
C. Formulation and Administration
The composition of the present invention is prepared by forming
liposomes which comprise at least one iposome-forming compound, an
effective amount of a pharmaceutically active substance, and
alveolar surfactant protein in an amount effective to transport the
composition across a pulmonary surface. Alveolar surfactant protein
can include alveolar surfactant proteins SP-A, SP-B, and SP-C.
Alveolar surfactant protein SP-A is preferred. The formulation of
the composition comprises about 20% to 99.9% by weight
liposome-forming compounds, 40% to less than 1% by weight
pharmaceutically active substance, and 40% to less than 1% alveolar
surfactant protein, based on the weight of the composition
exclusive of water. Preferably, the composition of the invention
comprises 60-90% by weight liposome-forming compounds, 5-20% by
weight pharmaceutically active substance and 5-20% by weight
alveolar surfactant protein, exclusive of water.
The liposome-forming portion of the composition preferably contains
one or more compounds selected from dipalmitoyl phosphatidyl
choline, phosphatidyl choline, phosphatidyl glycerol,
triacylglycerols, palmitic acid and cholesterol. The
liposome-forming portion of the composition preferably comprises
1-90% dipalmitoyl phosphatidyl choline, 1-90% phosphatidyl choline,
1-30% phosphatidyl glycerol and 1-30% cholesterol based on the
weight of the liposome-forming portion of the composition.
The compositions of the present invention are preferably
administered in a form suitable for endotracheal administration,
i.e., generally as a liquid suspension, as a dry powder "dust" or
as an aerosol. For direct endotracheal administration, the
composition is suspended in a liquid with suitable excipients such
as, for example, water, saline, dextrose, or glycerol and the like.
The compositions may also contain small amounts of non-toxic
auxiliary substances such as pH buffering agents, for example,
sodium acetate or phosphate. To prepare the "dust", the composition
prepared as described above, is lyophylized, and recovered as a dry
powder.
If to be used in aerosol administration, the composition is
supplied in finely divided form along with a propellant. Useful
propellents are typically gases at ambient conditions, and are
condensed under pressure. Lower alkanes and fluorinated alkanes,
such as Freon.sup.R, may be used. The aerosol is packaged in a
container equipped with a suitable valve so that the ingredients
may be maintained under pressure until released.
Administration of liquid suspension, dry powder "dust", or aerosol
is through inhalation of the composition into the lung via the
trachea.
The compositions of the present invention may also be administered
during bronchoscopy procedures.
The compositions of the present invention are administered in an
amount suitable for the condition and the subject being treated and
the subject. The amount of pharmaceutically active substance can
vary widely depending on the particular pharmaceutically active
substance and its use. Generally, the effective amount for any
given pharmaceutically active substance is known. Hormones are
usually administered in the nanogram range, while it is not
uncommon for chemotherapeutics to be administered in the range of
10 milligrams. Amounts of pharmaceutically active substance between
about 1 ng and 10 mg are administered in one dose. The number of
doses necessary is dependent on the condition being treated.
D. Uses
The compositions of the present invention can be used to administer
a wide range of pharmaceutically active substances efficiently
across pulmonary surfaces. The compositions of the invention can be
useful for systemic delivery of pharmaceutically active substances
as well as local delivery of pharmaceutically active
substances.
A variety of lung-specific diseases such as infant respiratory
distress syndrome, adult respiratory distress syndrome, viral
pneumonia, bacterial pneumonia, Group B streptococcal infection,
oxygen toxicity, alpha-1-anti-protease deficiency, emphysema,
asthma, tuberculosis, lung cancer, bronchitis, etc. could be
treated successfully with an administration system which could
deliver pharmaceutically active substances directly across the
pulmonary surfaces. The pharmaceutically active substances that can
be administered for these diseases include, but are not limited to,
antivirals such as acyclovir, zidovudine, and ribavarin;
antibacterials such as sulfamethoxazole and nalidixic acid;
fungicides such as fungizone and mycostatin; antibiotics such as
cephalosporins, penicillins, tetracyclines and aminoglycosides:
protease inhibitors such as alpha-1-antiprotease; anti-oxidants
such as vitamin E, vitamin C, superoxide dismutase and catalase:
anti-inflammatory agents such as prostaglanins, salicylates,
pyrazolons, propionic acid derivatives and para-aminophenol
derivatives; anti-allergics such as antihistamines, including
terfenadine, diphenhydramine, chloropheniramine and promethazine:
methyl xanthines such as theopbylline and .beta.-adrenergic
agonists: sympathomimetic amines such as epinephrine,
phenylephrine, pseudoephedrine, isoproterenol and albuterol;
mucolytics such as acetyl cysteine; corticosteroids such as
dexamethasone and triamcinolone and; chemotherapeutic agents such
as alkylating agents (nitrogen mustards, alkyl sulfonates,
nitrosoureas and triazenes) and antimetabolites (folic acid
derivatives, pyrimidine derivatives, and purine derivatives).
Many of the indications that may find utility in the present
invention are a direct result and/or effect of infant respiratory
distress syndrome or adult respiratory distress syndrome. In these
circumstances the transport of pharmaceutically active substances
across a pulmonary surface could be used as an adjunct to
conventional lung surfactant replacement therapy. Viral pneumonia
can be treated with antiviral agents. Bacterial pneumonia can be
treated with antibacterial agents and antibiotics. Group B
streptococcal infection can be treated with antibiotics. Oxygen
toxicity can be treated with vitamin E, vitamin C, superoxide
dismutase and catalase. Superoxide dismutase and catalase are the
enzymes involved with the metabolism of superoxide anion and
hydrogen peroxide, respectively.
Pneumonia not associated with infant respiratory distress syndrome
may also benefit from delivery of anti-virals and/or
antibiotics.
Therapy for emphysematous disease as well as congenital
alpha-1-antiprotease deficiency could be treated with
anti-proteases.
Another lung-specific use would be bacterial infections which are
difficult to treat with systemic administration of antibiotics or
with antibiotics that have substantial side effects. An example of
this would be pentamidine isethionate treatment of Pneumocystis
carinii. Pentamidine isethionate is quite toxic if given
systemically. Also lung-specific administration yields higher local
doses.
Chemotherapeutic agents can be administered in a lung-specific
fashion which would yield higher local doses. The administration
may be given during bronchoscopy.
Methyl xanthines, including theophylline and .beta.-adrenergic
agonists, can be delivered for asthma.
Since this administration system can also transport
pharmaceutically active substances directly into the general
circulation, a variety of pharmaceutically active substances which
would act in a systemic fashion can also utilize this system. These
pharmaceutically active substances include insulin, growth hormone,
other peptide hormones, thrombolytics, fibroblast growth factor,
calcitonin, vasopresin, renin, prolactin, thyroid stimulating
hormone, corticotropin, follicle stimulating hormone, luteinizing
hormone, chorionic gonadotropin, atrial peptides, interferon,
tissue plasminogen activator, gammaglobulin, and Factor VIII, to
name but a few.
EXAMPLES
Many of the techniques which are used to make liposomes and assay
performance are widely practiced in the art, and most practitioners
are familiar with the standard resource materials which describe
specific conditions and procedures. The examples are written in
observation o; such knowledge and incorporate by reference
procedures considered conventional in the art. The following
examples are intended to illustrate the invention, without acting
as a limitation upon its scope.
A. Liposome Preparation
The liposomes of examples 1-5 were prepared using the following
general procedure. All lipids were obtained from Avanti Polar
Lipids (Birmingham, AL). Unilamellar liposomes were prepared in
minimal essential medium containing Kreb's improved salts (Dawson,
R. M. C., et al., Data for Biochemical Research (1969), p. 507
Clarendon Press, Oxford), 2 mM sodium pyruvate, 13 mM glucose, and
25 mM HEPES, pH 7.4 (MEM-Krebs) as described by the French pressure
cell method of Hamilton, et al., J. Lipid Res. (1980) 21:981-992.
Briefly, liposomes were made by admixing in a chloroform solution
dipalmitoyl phosphatidyl choline, phosphatidyl choline,
phosphatidyl glycerol and cholesterol . in the weight ratios of
5:10:2:1.5, respectively. The chloroform is evaporated from the
mixture by rotary evaporation. The dried lipids were rehydrated in
MEM-Krebs and sheared to form unilamellar liposome vesicles by
French pressure cell. Alternatively sonication can be employed to
form the unilamellar vesicles.
B. In Vitro Assay
Type II Cell Isolation and Incubation
The in vitro assay employed in examples 1 and 4 was performed by
the following general procedure. Freshly isolated type II cells
were prepared by the method of Dobbs, et al., Am. Rev. Respir. Sid.
(1986) 134:141-145. Briefly, lungs of specific-pathogen-free male
Sprague-Dawley rats weighing 160-180 g (Bantin-Kingman, Fremont,
CA) were digested with elastase (Cooper Biomedical, Malverne, PA).
The minced digest was filtered through a series of nylon meshes and
cells were collected by centrifugation. The cells were then plated
on IgG-coated plates and the non-type II cells, which were
primarily macrophages and lymophocytes, adhered to the plate over a
period of one hour at 37.degree. C. in a 10% CO.sub.2 /air
incubator. The non-adherent cells were removed by gentle panning
and were centrifuged at 130.times.g for 8 minutes. This cell pellet
contained 85.+-.1% type II cells. The viability of the cells as
determined by erythrosin B exclusion averaged 97.+-.1%.
2.5.times.10.sup.6 freshly isolated type II cells were suspended in
1 ml of MEM-Krebs. The concentration of calcium was 2 .mu.M. The
cells were incubated at 37 or 4.degree. C. in 15 ml centrifuge
tubes tilted at an angle of approximately 25.degree. from
horizontal in order to enhance mixing. Liposomes which contain
radiolabelled protein were added. After mixing, the cells were
incubated at 37.degree. or 4.degree. C. for various lengths of
time. At the end of the incubation period, 4 ml of MEM-Krebs at
4.degree. C. was added to cells, and the cells and media were
separated immediately by centrifugation at 140.times.g for 10
minutes in a Damon International CRU-5000 Centrifuge using a number
269 rotor (International Equipment Co., Needham Heights, MA). The
medium was removed and the cells were gently resuspended. The cells
were transferred to a fresh tube and washed twice more by
centrifugation. Zero time values were determined by centrifugation
and washing of the cells immediately after addition of liposomes.
The actual elapsed time before the beginning of the first
centrifugation was 2-3 minutes. The final cell pellet was
resuspended in 0.5 ml of MEM-Krebs; 0.3 ml was analyzed for
radioactivity in a Beckman LS-75000 scintillation counter. A cell
count was obtained using a Neubauer counting chamber.
C. In Vivo Assay
Anesthetized Rat Model
The in vivo assay employed in examples 2 and 3 was performed by the
following general procedure. The compositions were administered to
the lungs of anesthetized adult Sprague-Dawley rats by endotracheal
cannula. At various times after administration of the compositions,
the animals were sacrificed by overdosing with anesthetics. The
lungs and serum were obtained from each rat for analysis. The lung
tissue was minced and extracted with 2:1 chloroform:methanol. The
serum was extracted with 2:1 chloroform:methanol. The serum was
sampled to get an indication of the amount of pharmaceutically
active substance being transported into general circulation.
EXAMPLE 1
LIPOSOMES CONTAINING VITAMIN E IN VITRO
Vitamin E is a water-insoluble pharmaceutically active substance
with antioxidant properties. Premature infants require supplements
of Vitamin E. Furthermore, administration of vitamin E may be a
beneficial adjunct to conventional lung surfactant therapy for
premature infants because it may protect against oxidant lung
injury.
Liposomes were prepared as described in section A with the addition
of .sup.3 H-Vitamin E in the lipid mixture before evaporation. The
following weight ratios of lipid and Vitamin E were used to prepare
the liposomes:
DPPC:PC:PG:Cholesterol:Vitamin E ##STR1## The chloroform was
evaporated from the lipid/Vitamin E mixture by rotary evaporation.
The dried lipid/Vitamin E mixture was rehydrated in MEM-Krebs to a
concentration of 87 .mu.g Vitamin E/ml. Alveolar surfactant protein
SP-A was added to the liposome mixture to a concentration of 100
.mu.g/ml. 100 .mu.l of the liposome mixture in the presence and
absence of alveolar surfactant protein SP-A was added to 1 ml of
freshly isolated type II cells and assayed as described above in
section A. The washed cell pellets were analyzed for radiolabelled
Vitamin E incorporation at 0 and 60 minutes by counting in a
Beckman LS-7500 scintillation counter with the following
results:
______________________________________ TIME 0 min* 60 min*
______________________________________ Liposomes + alveolar
surfactant SP-A 17.9 85.9 Control Liposomes 6.8 28.4 (no alveolar
surfactant SP-A) ______________________________________ *data
expressed as ng Vitamin E/10.sup.6 cells
This data indicates that the alveolar surfactant protein SP-A
stimulates the uptake of liposomes containing Vitamin E.
EXAMPLE 2
LIPOSOMES CONTAINING VITAMIN E IN VITO 60 MINUTE TIMECOURSE
The liposomes containing Vitamin E prepared as in Example I above
were tested in vivo by administration to rats according to the
procedure outlined above in section C. The animals were sacrificed
at 0 and 60 minutes and the lungs were tested for incorporation of
radiolabelled Vitamin E as described above, with the following
results:
______________________________________ TIME 0 min* 60 min*
______________________________________ Liposomes + alveolar
surfactant SP-A 505 10,409 n = 2 534 3,719 Control Liposomes 299
914 (no alveolar surfactant SP-A) 235 926 n = 2
______________________________________ *data expressed as dpm
The data indicates that alveolar surfactant protein SP-A stimulates
the transport of the liposomes containing Vitamin E to lung tissue
in a rapid fashion.
EXAMPLE 3
LIPOSOMES CONTAINING VITAMIN E IN VIVO 24 HOUR TIMECOURSE
The liposomes containing Vitamin E prepared as in Example I above
were tested in vivo by administration to rats according to the
procedure outlined above in section C. The animals were sacrificed
at 1 and 24 hours. The lungs and serum were tested for
incorporation of radiolabelled Vitamin E with the following
results:
______________________________________ TIME 1 hour* 24 hours*
______________________________________ LUNG Liposomes + alveolar
surfactant SP-A 2.8 .times. 10.sup.6 1.4 .times. 10.sup.6 Control
Liposomes 2.4 .times. 10.sup.5 1.2 .times. 10.sup.6 (no alveoloar
surfactant SP-A) Serum Liposomes + alveolar surfactant SP-A 2.1
.times. 10.sup.4 5.8 .times. 10.sup.3 Control Liposomes 8.6 .times.
10.sup.3 4.5 .times. 10.sup.3 (no alveolar surfactant SP-A)
______________________________________ *data expressed as cpm
The data indicates not only that the alveolar surfactant protein
SP-A enhances transport to the lung, but it also enhances serum
levels of radiolabelled vitamin E. The transport of Vitamin E into
the serum probably reflects transport through pulmonary surfaces
into the general circulation. At 24 hours, the amount of
radiolabelled Vitamin E in the lung is comparable in experimental
and control animals, reflecting continued non-specific uptake after
the initial increase due to the addition of alveolar surfactant
protein SP-A. The same non-specific effect is also seen in the
serum.
EXAMPLE 4
LIPOSOMES CONTAINING WATER SOLUBLE SUBSTANCE IN VITRO
The water soluble substance 6-carboxy-fluorscein was encapsulated
in liposomes and tested for transport into type II cells in vitro.
The liposomes were prepared as described above in section A. The
dried lipids were rehydrated in MEM-Krebs to which 1% (w/v)
6-carboxy-fluorscein had been added. The 6-carboxy-fluorscein not
contained within the liposome was removed by gel filtration
chromatography. Alveolar surfactant protein SP-A was added to the
liposome mixture to a concentration of 100 .mu.g/ml. 100 .mu.l of
the liposome mixture in the presence and absence of alveolar
surfactant protein SP-A is administered to 1 ml of freshly isolated
type II cells and assayed as described above in section B. The
cells were analyzed for ability to fluoresce after 60 minutes as
follows:
______________________________________ Liposomes + alveolar
surfactant SP-A* 2.5 n = 2 3.3 Control Liposomes* 0.9 (no alveolar
surfactant SP-A) 0.4 n = 2 ______________________________________
*data expressed as AFU (arbitrary fluorescence units)
6-carboxy-fluorscein was used as a model system for the assessment
of delivery of water soluble pharmaceutically active substances. In
this model system alveolar surfactant protein SP-A also stimulates
the uptake of liposomes containing the water soluble substance
6-carboxy-fluorscein.
Modifications of the above described modes for carrying out the
invention that are obvious to those of skill in the art are
intended to be within the scope of the following claims.
* * * * *