U.S. patent application number 17/151790 was filed with the patent office on 2021-07-22 for liposome formulation of vilanterol trifenatate.
This patent application is currently assigned to Cai Gu Huang. The applicant listed for this patent is Cai Gu Huang, Xiao Ting Huang. Invention is credited to Cai Gu Huang, Xiao Ting Huang.
Application Number | 20210220275 17/151790 |
Document ID | / |
Family ID | 1000005401713 |
Filed Date | 2021-07-22 |
United States Patent
Application |
20210220275 |
Kind Code |
A1 |
Huang; Cai Gu ; et
al. |
July 22, 2021 |
LIPOSOME FORMULATION OF VILANTEROL TRIFENATATE
Abstract
The present invention is directed to a liposomal formulation
comprising a lipid ingredient encapsulating vilanterol trifenatate,
and a method for administering the liposomal formulation by
nebulizing the liposomal formulation in an inhaler. The liposomal
formulation comprises vilanterol or a salt thereof, and lipid
ingredients comprising a pharmaceutical lipid and/or a sterol. The
drug to lipid mass ratio is in the range of about 1:20 to about
1:100. Additionally, the present invention is directed to the use
of the liposomal formulation for the prevention or treatment of
respiratory diseases such as Chronic Obstructive Pulmonary Disease
and/or asthma.
Inventors: |
Huang; Cai Gu; (Shrewsbury,
MA) ; Huang; Xiao Ting; (Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Huang; Cai Gu
Huang; Xiao Ting |
Shrewsbury
Shanghai |
MA |
US
CN |
|
|
Assignee: |
Huang; Cai Gu
Shrewsbury
MA
|
Family ID: |
1000005401713 |
Appl. No.: |
17/151790 |
Filed: |
January 19, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62963531 |
Jan 20, 2020 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/138 20130101;
A61K 9/1277 20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/138 20060101 A61K031/138 |
Claims
1. A formulation comprising a plurality of liposomes, wherein the
liposomes comprise a lipid ingredient encapsulating vilanterol
trifenatate, the lipid ingredient comprises a lipid and a sterol,
and wherein the vilanterol trifenatate and lipid ingredient are
present in a mass ratio of between about 1:10 and about 1:150
(vilanterol trifenatate:lipid ingredient).
2. The formulation according to claim 1, wherein the liposomes have
an average size of about 50 nm to about 500 nm.
3. The formulation according to claim 1, wherein the lipid is
selected from the group consisting of phosphatidylcholine (PC),
phosphatidic acid (PA), phosphatidylethanolamine (PE),
phosphatidylglycerol (PG), phosphatidylserine (PS),
phosphatidylinositol (PI), dimyristoyl phosphatidyl choline (DMPC),
distearoylphosphatidyl choline (DSPC), dipalmitoyl phosphatidyl
choline (DPPC), dimyristoyl phosphatidyl glycerol (DMPG),
distearoylphosphatidyl glycerol (DSPG), dioleoyl phosphatidyl
glycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG),
dimyristoyl phosphatidyl serine (DMPS), distearoyl phosphatidyl
serine (DSPS), dioleoyl phosphatidyl serine (DOPS), dipalmitoyl
phosphatidyl serine (DPPS), dioleoyl phosphatidyl ethanolamine
(DOPE), palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE),
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyp-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidylethanolamine (DSPE), di
stearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine
(DOPC), dipalmitoylphosphatidylcholine (DPPC), and combinations
thereof.
4. The formulation according to claim 1, wherein the sterol is
selected from the group consisting of cholesterol, ergosterol,
lanosterol, and combinations thereof.
5. The formulation according to claim 1, wherein the liposomes have
a D50 value of less than about 10 .mu.m.
6. The formulation according to claim 1, further comprising an
antioxidant selected from the group consisting of a water-soluble
antioxidant and an oil-soluble antioxidant.
7. The formulation according to claim 1, wherein the lipid
ingredient comprises DPPC, DSPG, and cholesterol, in a molar ratio
of about 9:1:5 (DPPC:DSPG:cholesterol).
8. The formulation according to claim 1, wherein the lipid
ingredient comprises DPPC and cholesterol, in a molar ratio of
about 1:5 (DPPC:cholesterol).
9. A method for preparing a liposomal formulation, comprising: (1)
preparing a vilanterol trifenatate solution, (2) preparing a lipid
solution, (3) mixing the vilanterol trifenatate solution with the
lipid solution to provide a mixture, and (4) extruding the
mixture.
10. A method for preparing a liposomal formulation, comprising: (1)
preparing a drug solution, (2) injecting a lipid solution into the
drug solution to provide a mixture, and (3) adjusting the pH of the
mixture.
11. The method according to claim 9, wherein the liposomal
formulation has a pH of about 4.0 to about 7.5.
12. The method according to claim 10, wherein the liposomal
formulation has a pH of about 1.0 to about 4.0.
13. The method according to claim 9, wherein the lipid solution
comprises a lipid ingredient, and wherein the vilanterol
trifenatate and the lipid ingredient are present in a mass ratio
that ranges from about 1:10 to about 1:150.
14. The method according to claim 10, wherein the lipid solution
comprises a lipid ingredient, and wherein the vilanterol
trifenatate and the lipid ingredient are present in a mass ratio
that ranges from about 1:10 to about 1:50.
15. The method according to claim 9, wherein the extruding is
carried out at a temperature between about 60.degree. C. and about
70.degree. C.
16. The method according to claim 10, wherein the injecting is
carried out at a temperature between about 40.degree. C. and about
80.degree. C.
Description
PRIORITY STATEMENT
[0001] This application claims the benefit of the filing date of
U.S. Provisional patent Application No. 62/963,531, filed on Jan.
20, 2020, the contents of which are incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a liposome formulation and
a preparation method for the liposome formulation.
BACKGROUND OF THE INVENTION
[0003] Vilanterol trifenatate, chemically known as
4-{(1R)-2-[(6-(2-((2,6-dichlorobenzyl) oxy) ethoxy) hexyl)
amino]-1-hydroxyethyl}-2-(hydroxymethyl) phenol mono
(2,2,2-triphenylacetate) has been described in WO03/024439.
Vilanterol trifenatate has the following chemical structure:
##STR00001##
[0004] Vilanterol trifenatate has been described as a long-acting
muscarinic antagonist that activates beta-2 adrenoreceptors on
airway smooth muscle, causing broncho-dilation. Beta-2 receptors
are the adrenergic receptors in bronchial smooth muscle. Vilanterol
trifenatate can provide a therapeutic benefit in the treatment of
asthma or chronic obstructive pulmonary disease (COPD), including
chronic bronchitis and emphysema.
[0005] Vilanterol trifenatate has been administered by means of dry
powder inhalation. However, administration by dry powder inhalation
is difficult or unpleasant for some patients, in particular for
children and the elderly. As an aspect of the present invention, to
increase the delivery of vilanterol trifenatate to the desired
targets and reduce the incidence of side effects associated with
it, vilanterol trifenatate is encapsulated in a lipid bilayer to
make it available only at the site of action while minimizing the
effect on other tissues.
[0006] Liposomes are microscopic closed vesicles which have an
internal phase enclosed by one or more lipid bilayers. Liposomes
can entrap an active agent. Vilanterol trifenatate can be entrapped
in liposomes with high efficiency, wherein the vilanterol
trifenatate is retained in liposome constituents so that the
vilanterol trifenatate can be delivered to a target tissue.
Liposomes can improve protection of an encapsulated drug, increase
drug stability, change the in vivo distribution behavior of the
drug, and carry the drug to a diseased region by passive or active
targeting, as well as improve drug efficiency and reduce drug
toxicity.
[0007] Accordingly, the present invention relates to liposomal
formulations, which are particularly suitable for administering
vilanterol trifenatate by nebulization inhalation, and which are
especially suitable for the treatment of asthma and chronic
obstructive pulmonary disease.
[0008] Moreover, the present invention relates to liposomal
formulations having particle size uniformity, high drug-loaded
capacity as well as high encapsulation efficiency. The liposomal
formulations disclosed in the present invention are suitable for
administration by nebulization inhalation.
[0009] Furthermore, administration of a liposomal formulation by
inhalation is advantageous compared to administration by
conventional dry powder inhalation. For example, administration by
dry powder inhalation is more difficult, particularly for children
and elderly patients. Also, dry powder inhalation may cause side
effects on the lung. The liposomal formulations of the present
invention are particularly suited for administering vilanterol
trifenatate by nebulization inhalation, especially for treating
asthma and chronic obstructive pulmonary disease.
[0010] It is now surprisingly found that a novel liposomal
formulation of vilanterol trifenatate can be prepared and that this
liposomal formulation is very stable and suitable for nebulization
inhalation.
SUMMARY OF THE INVENTION
[0011] Aspects and advantages of the current invention will be set
forth in part in the following description, or may be obvious from
the description, or may be learned through practice of the
invention.
[0012] The present invention relates to formulations comprising
vilanterol trifenatate encapsulated in liposomes and methods for
their preparation. One aspect of the present invention provides
liposomes having a high uniformity, which results in minimizing
side effects, high drug-loading capacity, high encapsulation
efficiency, and good stability, which are suitable for
administration by nebulization inhalation.
[0013] The liposomal formulation is characterized by liposomes
having desirable compositions and physical characteristics. The
liposomal formulations of the present invention comprise one or
more lipid ingredient encapsulating vilanterol trifenatate.
[0014] The liposome formulations of the present invention comprise
liposomes comprising one or more lipid ingredients and vilanterol
trifenatate, wherein the mass ratio of the vilanterol trifenatate
to that of the lipid ingredient(s), called the drug to lipid ratio,
is in the range of about 1:10 to about 1:150 by weight, such as
about 1:10 to about 1:50 by weight. This drug to lipid ratio
enhances stability and effectiveness of liposome and also has an
impact on drug release and liposome integrity. These and other
structural characteristics impart unexpected benefits to the
instant formulation.
[0015] The liposomes of the present invention have a size in the
range from about 50 to about 500 nm, more preferably in the size
range of about 100 to about 400 nm, depending on the type of
vilanterol trifenatate and/or the direct carrier used. In one
embodiment, the liposomes are in the size range of about 150 to
about 170 nm.
[0016] Another aspect of the present invention is to provide two
preparation methods, preparation method 1 and preparation method 2,
for preparing liposome formulations. Liposomes formulated by these
process have desirable characteristics. Preparation method 1 for
preparing the liposomes includes: (1) preparation of vilanterol
trifenatate solution, (2) mixing the as-prepared vilanterol
trifenatate solution with a lipid and a sterol to provide a
mixture, and (3) extruding the mixture. Preparation method 2 for
the preparing the liposomes includes: (1) preparing a drug
solution, (2) injecting a solution containing lipid ingredients
into the drug solution to provide a mixture, and (3) adjusting the
pH of the resulting mixture. Each of these methods produces a
solution containing liposome vesicles comprising a drug, such as
vilanterol trifenatate.
[0017] If desired, a further step of ultrafiltration and
concentration of the resulting liposome vesicle-containing solution
may be included in the preparation processes.
[0018] The preparation methods of the current invention are
suitable for commercial production by scaling up preparation of a
liposomal formulation of vilanterol trifenatate.
[0019] In one embodiment, a formulation is prepared by (1)
preparing a drug solution; (2) injecting into the drug solution a
lipid solution made from lipid ingredients comprising DPPC, DSPG,
and cholesterol in a molar ratio of about 1:5:1 to about 1:10:6,
more particularly about 1:9:5, and wherein the mass ratio of
vilanterol trifenatate to lipid is in the range of about 1:10 to
1:150; (3) adding water to the desired volume; and (4) adjusting
the pH of the resulting solution.
[0020] Yet another aspect of the present invention is a liposomal
formulation made in accordance with the methods described above.
The formulation comprises a plurality of liposomes, composed of an
amount of one or more lipid ingredients encapsulating vilanterol
trifenatate. In one embodiment, the lipid ingredients comprise one
or more of DPPC, DSPG, and cholesterol, and the mass ratio of
vilanterol trifenatate to lipid ingredient(s) is in the range of
about 1:10 to about 1:150. In an embodiment, the formulation is
prepared by: (1) preparing a vilanterol trifenatate solution, (2)
preparing a lipid and cholesterol solution, (3) mixing the
vilanterol trifenatate solution with the lipid and cholesterol
solution to provide a mixture, and (4) extruding the mixture.
[0021] The above described methods can produce a liposomal
formulation, which has useful characteristics, by, for example,
varying the ratio of the lipid ingredient(s), the drug to lipid
ratio, and the pH value, so as to be suitable for administration by
nebulization inhalation or soft mist inhalation.
[0022] These and other features, aspects and advantages of the
present invention will become better understood with reference to
the following description and appended claims. The accompanying
drawings, which are incorporated in and constitute a part of this
specification, illustrate embodiments of the invention and,
together with the description, serve to explain the principles of
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a graph of the size distribution of the vilanterol
trifenatate liposomes of sample 1 prepared as described in example
2.
[0024] FIG. 2 is a graph of the size distribution of the vilanterol
trifenatate liposomes of sample 2 prepared as described in example
2.
[0025] FIG. 3 is a graph of the size distribution of the vilanterol
trifenatate liposomes of sample 3 prepared as described in example
2.
[0026] FIG. 4 is a graph of the size distribution of the vilanterol
trifenatate liposomes of sample 4 prepared as described in example
2.
[0027] FIG. 5 is a graph of the size distribution of the vilanterol
trifenatate liposomes of sample 5 prepared as described in example
6.
[0028] FIG. 6 is a graph of the size distribution of the vilanterol
trifenatate liposomes of sample 6 prepared as described in example
6.
[0029] FIG. 7 is a graph of the size distribution of the vilanterol
trifenatate liposomes of sample 7 prepared as described in example
6.
[0030] FIG. 8 is a graph of the particle size distribution of
droplets of sample 1 formed with a compressed air nebulizer;
[0031] FIG. 9 is a graph of the particle size distribution of
droplets of sample 2 formed with a compressed air nebulizer;
[0032] FIG. 10 is a graph of the particle size distribution of
droplets of sample 3 formed with a compressed air nebulizer;
[0033] FIG. 11 is a graph of the particle size distribution of
droplets of sample 1 formed with an ultrasonic vibrating mesh
nebulizer;
[0034] FIG. 12 is a graph of the particle size distribution of
droplets of sample 2 formed with an ultrasonic vibrating mesh
nebulizer;
[0035] FIG. 13 is a graph of the particle size distribution of
droplets of sample 3 formed with an ultrasonic vibrating mesh
nebulizer;
[0036] FIG. 14 is a graph of the particle size distribution of
droplets of sample 5 formed with a compressed air nebulizer;
[0037] FIG. 15 is a graph of the particle size distribution of
droplets of sample 6 formed with a compressed air nebulizer;
[0038] FIG. 16 is a graph of the particle size distribution of
droplets of sample 7 formed with a compressed air nebulizer;
[0039] FIG. 17 is a graph of the particle size distribution of
droplets of sample 5 formed with an ultrasonic vibrating mesh
nebulizer; and
[0040] FIG. 18 is a graph of the particle size distribution of
droplets of sample 7 formed with an ultrasonic vibrating mesh
nebulizer.
DETAILED DESCRIPTION OF THE INVENTION
[0041] For purposes of the describing the invention, reference now
will be made in detail to embodiments and/or methods of the
invention, one or more examples of which are illustrated in or with
the drawings. Each example is provided by way of explanation of the
invention, not limitation of the invention. In fact, it will be
apparent to those skilled in the art that various modifications and
variations can be made in the present invention without departing
from the scope or spirit of the invention. For instance, features
or steps illustrated or described as part of one embodiment can be
used with another embodiment or steps to yield still further
embodiments or methods. Thus, it is intended that the present
invention covers such modifications and variations as come within
the scope of the appended claims and their equivalents.
[0042] The present invention relates to a liposomal formulation and
two methods for preparing the liposomal formulation. The
formulation comprises a plurality of liposomes encapsulating
vilanterol trifenatate. The physical characteristics of each
liposome facilitate stability and effectiveness of the liposomal
formulation. The formulation is characterized by liposomes which
are substantially uniform in size and shape distribution.
[0043] Additionally, the invention provides an efficient method for
preparing the liposomal formulation, which can meet the needs of
large-scale preparation.
[0044] As used herein, the term "liposome" refers to microscopic
closed vesicles having an internal phase enclosed by a lipid
bilayer. In the present invention, liposome includes, but is not
limited to, small single-membrane liposome, large single-membrane
liposome, still larger single-membrane liposome, multilayer
liposome having multiple concentric membranes, liposome having
multiple membranes that are not concentric, but irregular.
[0045] The term "liposome internal phase" refers to an aqueous
region enclosed in the lipid bilayer of the liposome, and has the
same meaning as "internal water phase" and "liposome internal water
phase."
[0046] The present invention relates to a liposomal formulation.
Different liposome ingredients may be used to form the liposomes of
the invention. In an embodiment, the lipid ingredient comprises one
or more non-toxic biocompatible lipids, for example, lipids
prepared from phosphatidyl-choline, phosphoglycerol, and/or
cholesterol. In one embodiment, the lipid ingredient comprises
dipalmitoylphosphatidylcholine (DPPC),
diastearoylphosphatidylcholine (DSPC),
diastearoylphosphatidylglycerol (DSPG) and cholesterol, or
combinations thereof, In another embodiment, the lipid ingredient
comprises DPPC, DSPG, and cholesterol, which may be present in a
molar ratio of about 9:1:5 (DPPC:DSPG:cholesterol).
[0047] As used herein, the term "lipid ingredients" refers to one
or more sterols and/or one or more lipids. Exemplary lipid
ingredients include, for example, but are not limited to,
cholesterol and diastearoylphosphatidylglycerol (DSPG), cholesterol
and dipalmitoylphosphatidylcholine (DPPC).
[0048] The liposome is not particularly limited in terms of form as
long as it is a liposome capable of encapsulating a drug.
[0049] The term "encapsulating" means taking a form in which a drug
is contained in an inner water phase enclosed by the liposome
membrane. For example, the liposome may be a form where drug is
encapsulated within a closed space formed of a membrane, a form
where a drug is included in the membrane itself, or a combination
thereof.
[0050] As used herein, the term "average particle size" refers to
an average value of diameters of liposomes as measured by a light
scattering method.
[0051] The liposome is preferably in the form of spherical shape or
a morphology close thereto.
[0052] The term "step" as used herein includes not only an
independent step, but also a step which may not be clearly
separated from another step, insofar as an expected outcome of the
step can be attained.
[0053] The liposomal formulation is characterized by liposomes
having a desirable composition and physical characteristics. The
liposomes of the present invention comprise lipid ingredients
encapsulating vilanterol trifenatate. In an embodiment, the lipid
is selected from the group consisting of phosphatidylcholine (PC),
phosphatidic acid (PA), phosphatidylethanolamine (PE),
phosphatidylglycerol (PG), phosphatidylserine (PS),
phosphatidylinositol (PI), dimyristoyl phosphatidyl choline (DMPC),
distearoylphosphatidyl choline (DSPC), dipalmitoyl phosphatidyl
choline (DPPC), dimyristoyl phosphatidyl glycerol (DMPG),
distearoylphosphatidyl glycerol (DSPG), dioleoyl phosphatidyl
glycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG),
dimyristoyl phosphatidyl serine (DMPS), distearoyl phosphatidyl
serine (DSPS), dioleoyl phosphatidyl serine (DOPS), dipalmitoyl
phosphatidyl serine (DPPS), dioleoyl phosphatidyl ethanolamine
(DOPE), palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE),
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyp-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidylethanolamine (DSPE), di
stearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine
(DOPC), dipalmitoylphosphatidylcholine (DPPC),
palmitoyloleoylphosphatidylcholine (POPC), and
palmitoyloleoyl-phosphatidylethanolamine (POPE).
[0054] In an embodiment, the sterol is selected from the group
consisting of cholesterol, ergosterol, and lanosterol, and
combinations thereof.
[0055] The phrase "drug to lipid ratio" as used herein refers to
the relative amounts of the drug to the lipid ingredient(s) by
mass. In one embodiment, the liposome has a drug to lipid ratio in
the range of about 1:10 to about 1:150 by weight. In another
embodiment, the liposome has a drug to lipid ratio in the range of
about 1:20 to 1:100 by weight.
[0056] The pH affects the properties of the liposomal formulation.
The pH affects the stability, drug leakage rate from the liposome,
and drug encapsulation capability of the liposomal formulation.
[0057] In an embodiment, the liposomal formulation comprises a
plurality of liposomes which have the characteristics described
above and are substantially uniform in size and shape. The
liposomes may be in the size range of about 50 to about 500 nm. In
an embodiment, the size range is about 100 to about 400 nm.
[0058] In another embodiment, the size range is about 100 to about
300 nm. In a particular embodiment, the size of the liposome is
about 110 to about 140 nm.
[0059] In another particular embodiment, the size of the liposome
is about 150 to about 170 nm.
[0060] In one embodiment, the liposomal formulation is formulated
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries known in the art.
[0061] The liposomal formulation may be administered by any route
which effectively transports the liposomes to the appropriate site
of action. One effective route of administration is inhalation.
Other suitable routes of administration include, but are not
limited to injection, such as, for example, intramuscular,
subcutaneous, or intra-peritoneal injection.
[0062] In an embodiment, the liposome formulation comprises an
antioxidant selected from the group consisting of water-soluble
antioxidants and oil-soluble antioxidants. Examples of oil-soluble
antioxidants include, but are not limited to, alpha-tocopherol,
alpha-tocopherol succinate, alpha-tocopherol acetate, and mixtures
thereof. Examples of water-soluble antioxidants include, but are
not limited to, ascorbic acid, sodium bisulfite, sodium sulfite,
sodium pyrosulfite, L-cysteine, and mixtures thereof. The amount of
the antioxidant added ranges from about 0 to about 1.0% (w/v) of
the formulation. In one embodiment, the antioxidant is omitted
entirely.
[0063] The processes for making the liposomes and liposomal
formulations permit manipulation of the physical characteristics
described above, as well as allow control of certain process
parameters, for example, solvent composition, solvent ratios, and
vesicle preparation temperature. Preparation method 1 for the
liposomal formulation comprises: (1) preparing a vilanterol
trifenatate solution, (2) preparing a lipid solution, (3) mixing
the vilanterol trifenatate solution with the lipid solution to
provide a mixture, and (4) extruding the mixture. Preparation
method 2 for the liposomal formulation comprises: (1) preparing a
drug solution, (2) injecting a lipid solution into the drug
solution to provide a mixture, (3) adjusting the pH of the
mixture.
[0064] If desired, a further step of ultrafiltering and
concentrating the resulting liposome vesicle-containing solution
may be used.
[0065] The preparation methods have the advantage of allowing
control and monitoring of the physiological and chemical features
of the liposomes. For example, the drug to lipid ratio may be
managed by selection of the lipid ingredients used to form the
liposome or the amount of lipids added to the dissolved active
agent. Increasing the amount of lipid ingredient decreases the drug
to lipid ratio, and vice versa.
[0066] The first two steps of method 1 comprise preparing a
vilanterol trifenatate solution and a lipid solution separately. In
an embodiment, the ratio of vilanterol trifenatate to solvent is
between about 1:5 to about 1:10 (w/v). In an embodiment, DPPC and
DSPG, are dissolved in chloroform and methanol in a ratio of about
5:1 to about 10:1 (v/v).
[0067] The third step of method 1 comprises mixing the vilanterol
trifenatate solution with the lipid solution.
[0068] The fourth step of method 1 involves extruding the mixture.
In an embodiment, the extruder is a heated stainless steel extruder
at a temperature in the range of about 60.degree. C. to about
70.degree. C. In one embodiment, the extrusion process requires
about 5 to about 15 passes to achieve the desired liposome particle
size, for example, a vesicle size in the range from about 80 nm to
about 500 nm.
[0069] The process of extrusion reduces the size of the
multi-lamellar vesicles. The method uses a single extrusion step,
and low pressure to produce liposomes that are highly-uniform in
size and shape.
[0070] Extrusion of liposomes using a polycarbonate membrane is an
effective method for reducing the size of the liposomes to a
relatively well-defined size distribution. In one embodiment, the
extruder temperature is above the lipid transient-phase
temperature, so as to enable size reduction. For example, in the
case of DPPC, DSPG, and cholesterol, a temperature in the range of
about 60.degree. C. to about 70.degree. C. may be suitable. Other
lipids may be used and their known transient-phase temperature
determines the desired temperature for the extrusion step. Multiple
passes may be required to achieve the desired vesicle size and
homogeneity.
[0071] In one embodiment, the extrusion process requires about 9 to
about 11 passes to achieve liposome vesicles of in the size range
of about 120 nm to about 270 nm.
[0072] The first step of method 2 involves preparing a drug
solution. In an embodiment, vilanterol trifenatate is dissolved in
NaCl solution.
[0073] In an embodiment, a lipid solution is prepared by dissolving
lipid ingredient(s) at a temperature in the range of about
40.degree. C. to about 80.degree. C. The second step involves
injecting the lipid solution into the drug solution, and the third
step, is to make the final volume and pH adjustment.
[0074] If desired, a step of ultrafiltration and concentration may
be conducted. Different types of filtration membranes may be used
during the ultrafiltration process. In one embodiment, the
ultrafiltration step uses a hollow fiber membrane, where the
formulation is pushed through the open hollow cores of the fiber
while the relatively larger liposomes remain within the fiber.
[0075] In one embodiment, when a hollow fiber cartridge is used for
ultrafiltration, the cartridge is filled with 100% alcohol for
about 40 minutes to about 80 minutes. In an embodiment, the
cartridge is soaked for over one hour. In an embodiment, the
alcohol is pumped through the cartridge at about 3 to about 8 psi.
In an embodiment, the alcohol is removed, and the cartridge is
rinsed with clean water.
[0076] In an embodiment, a reused hollow fiber cartridge is used.
In such an embodiment, before ultrafiltration, the fiber cartridge
may be washed with pure water, and then the sample may be pumped
through the cartridge for ultrafiltration, until the liposome
sample is concentrated to a desired concentration. After finishing
ultrafiltration, the cartridge may be washed by pure water, and
then the cartridge may be soaked with about 3% to about 10% NaOH
solution.
[0077] In an embodiment, the ultrafiltration and concentration may
be accomplished using a peristaltic pump connected with a hollow
fiber cartridge. In such embodiment, before ultrafiltration, the
fiber cartridge may be washed by pure water, and then the sample of
liposome formulation may be pumped through the cartridge for
ultrafiltration, until the sample is concentrated.
[0078] In an embodiment, the liposome formulation may be filtered
by a hollow fiber cartridge for concentrating to a volume of, for
example, about 10 mL and removing ethanol and free vilanterol
trifenatate.
[0079] In an embodiment, after ultrafiltration, the hollow fiber
cartridge may be washed by pure water, and then soaked with 5%
NaOH.
[0080] After ultrafiltration, the process may further include a
dialyzing step wherein the formulation is dialyzed against a volume
of buffered solution. In one embodiment, the buffer solution is
normal saline. Other buffer additives are known in the art,
including, but not limited to, sucrose, glycine, sodium and/or
succinate. In an embodiment, the buffer solution reflects the
environment of the final formulation that is external to the
liposomes. In an embodiment, the buffer solution is isotonic and
non-toxic to cells. The buffer solution may be filtered to further
reduce contaminants and may be prepared in advance of the
preparation process.
[0081] The lipid ingredients may be in the form of a solution
containing the desired starting amount of the lipid ingredient(s)
in a volume of one or more lipid solvents. Any suitable lipid
ingredient(s) and lipid solvent may be used. For example, the lipid
ingredients may comprise DPPC and cholesterol in a molar ratio
ranging from about 1:3 to about 1:8 prior to liposome formation.
The resultant liposome formed according to this combination of
lipids may also have about a 1:5 molar ratio of DPPC and
cholesterol.
[0082] Examples of lipid solvents include, but are not limited to,
ethanol, t-butanol, water, and mixtures thereof. In an embodiment,
the lipid ingredients are dissolved in the lipid solvent. The lipid
solvent may be heated to a temperature that facilitates
solubilization of the lipid ingredients, for example, ranging from
about 40.degree. C. to about 80.degree. C. to generate the lipid
solution.
[0083] In an embodiment, the initial concentration of lipid
ingredients dissolved in the ethanol is in the range of about 0.20
g/L to about 1.3 g/L. The lipid solution may be prepared apart from
the manufacturing process discussed herein.
[0084] The liposomal formulation of the present invention may
comprise a pH adjusting agent, such as a pharmacologically
acceptable base or a pharmacologically acceptable acid.
Pharmacologically acceptable bases include, but are not limited to,
alkali metal hydroxides and alkali metal carbonates. In an
embodiment, the alkali metal ion is sodium. If bases of this kind
are used, it should be ensured that the resulting salts, which are
then present in the finished pharmaceutical formulation, are
pharmacologically compatible with the above mentioned acid. In one
embodiment, the pH adjusting agent is sodium hydroxide.
[0085] In an embodiment, the pH of the liposomal formulation
prepared according to preparation method 1 is between about 4.0 and
about 7.5, such as between about 5.0 and about 7.5.
[0086] In one embodiment, the pH of the formulation is adjusted
with a pH adjusting agent to a pH between about 6.5 and about
7.0.
[0087] In another embodiment, the pH adjusting agent is a
pharmaceutically acceptable acid, such as for example, hydrochloric
acid, citric acid, ascorbic acid, or a combination thereof.
[0088] In one embodiment, the pH adjusting agent is hydrochloric
acid.
[0089] In an embodiment, the pH of the liposomal formulation
prepared according to preparation method 2 is between about 1.0 and
about 4.0, such as between about 1.0 and about 3.0, or more
specifically about 2.0.
[0090] In an embodiment, the drug to lipid ratio in the liposomal
formulation may be controlled by varying the amounts of lipid
ingredients and vilanterol trifenatate. Optionally, mildly heating
the lipid solution or the mixture resulting from combining the drug
solution with the lipid solution may aid in mixing together the
lipid ingredients and vilanterol trifenatate. This mixing process
can result in efficient encapsulation of vilanterol trifenatate
into multi-lamellar vesicles.
[0091] The present invention will be described in further detail
with reference to the following examples. The following examples
are intended to illustrate and exemplify the various aspects of
carrying out the present invention and are not intended to limit
the scope of the present invention in any way.
EXAMPLES
[0092] Materials and reagents:
Vilanterol trifenatate is commercially available and may be
purchased from Shengde Pharmaceutical Co., Ltd.
Dipalmitoylphosphatidylcholine (DPPC) is commercially available and
may be purchased from AVT Pharmaceutical Technology Co., Ltd.
Distearoyl phosphatidylglycerol (DSPG) is commercially available
and may be purchased from AVT Pharmaceutical Technology Co., Ltd.
Similarly, cholesterol is commercially available and may be
purchased from AVT Pharmaceutical Technology Co., Ltd.
Example 1
[0093] Preparation Method 1:
Preparation of vilanterol trifenatate solution: NaCl (0.9 g) was
added to a beaker containing 100 ml purified water. To this NaCl
solution, 117 .mu.Lof hydrochloric acid was added. Vilanterol
trifenatate (7.03 mg) was added to 70 ml of the solution, then
sonicated for 10 min to dissolve. Then, the solution containing
vilanterol trifenatate was filtered using a 0.22 .mu.m-filter
membrane.
[0094] Mixing vilanterol trifenatate solution with lipid solution:
DPPC (44.57 mg), DSPG (5.49 mg), and cholesterol (13.20 mg) were
combined in a 50-ml round-bottom flask followed by the addition of
18 ml of chloroform and 2 ml of methanol to dissolve the DPPC and
DSPG with gentle shaking. The solvent was removed using a rotary
evaporator at 65.degree. C., until a thin film of dried lipid was
deposited on the walls of the flask. Finally, 20 ml of the prepared
vilanterol trifenatate solution was added to the round-bottom flask
containing the thin layer of dried lipid. The temperature was
maintained at 65.degree. C. for approximately 30 minutes.
[0095] Extrusion: The formulation was subjected to extrusion using
a heated stainless steel extruder assembled with one polycarbonate
membrane at 65.degree. C., to reduce the size of the liposomes and
improve homogeneity. The extrusion process was repeated for 9-11
passes to achieve 80-200 nm liposomes. After extrusion, NaOH was
added to the liposomal solution to adjust the pH to 6.5-7.0.
Finally, the liposomal solution was filtered using a 1-.mu.m GF
membrane to obtain liposomes encapsulating vilanterol
trifenatate.
Example 2
[0096] In accordance with the preparation method described above,
four different samples were prepared with high encapsulation
efficiency and different drug to lipid ratios. The encapsulation
efficiency of each of the four samples was over 97%, and the
encapsulation efficiency of each of sample 2 and sample 4 was 100%.
The average particle sizes of the liposomes were in the range of
about 120 nm to about 320 nm.
[0097] Sample 1: NaCl (0.9 g) was added to a beaker containing 100
ml purified water. To this NaCl solution, 117 .mu.Lof hydrochloric
acid was added. Vilanterol trifenatate (7.03 mg) was added to 70 ml
of the solution, then sonicated for 10 min to dissolve. Then, the
solution containing vilanterol trifenatate was filtered using a
0.22-.mu.m filter membrane. Then DPPC (178.95 mg), DSPG (21.78 mg),
and cholesterol (52.90 mg) were combined in a 50-ml round-bottom
flask, followed by addition of 18 ml of chloroform and 2 ml of
methanol to dissolve the DPPC and DSPG with gentle shaking. The
solvent was removed using a rotary evaporator at 65.degree. C.,
until a thin film of dried lipid was deposited on the walls of the
flask. 20 ml of the prepared vilanterol trifenatate solution was
added to the round-bottom flask containing the thin layer of dried
lipid. The temperature was maintained at 65.degree. C. for
approximately half hour. The formulation was then subjected to
extrusion using a heated stainless steel extruder assembled with
one polycarbonate membrane at 65.degree. C., to reduce the size of
the liposomes and improve homogeneity. The extrusion process was
repeated for 9-11 passes to achieve 80-200 nm liposomes. After
extrusion, NaOH was added to the liposomal solution to adjust the
pH to 6.5-7.0. Finally, the liposomal solution was filtered using a
1-.mu.m GF membrane to obtain liposomes encapsulating vilanterol
trifenatate.
[0098] Sample 2: NaCl (0.9 g) was added to a beaker containing 100
ml purified water. To this NaCl solution, 117 .mu.Lof hydrochloric
acid was added. Vilanterol trifenatate (7.03 mg) was added to 70 ml
of the solution, then sonicated for 10 min to dissolve. Then, the
solution containing vilanterol trifenatate was filtered using a
0.22-.mu.m filter membrane. Then DPPC (44.57 mg), DSPG (5.49 mg),
and cholesterol (13.20 mg) were combined in a 50 ml round-bottom
flask followed by the addition of 18 ml of chloroform and 2 ml of
methanol to dissolve DPPC and DSPG with gentle shaking. The solvent
was removed using a rotary evaporator at 65.degree. C., until a
thin film of dried lipid was deposited on the walls of the flask.
20 ml of the prepared vilanterol trifenatate solution was added to
the round-bottom flask containing the thin layer of dried lipid.
The temperature was maintained at 65.degree. C. for approximately
30 minutes. The formulation was then subjected to extrusion using a
heated stainless steel extruder assembled with one polycarbonate
membrane at 65.degree. C., to reduce the size of the liposomes and
improve homogeneity. The extrusion process was repeated for 9-11
passes to achieve 80-200 nm liposomes. After extrusion, NaOH was
added to the liposomal solution to adjust the pH to 6.5-7.0.
Finally, the liposomal solution was filtered using a 1-.mu.m GF
membrane to obtain liposomes encapsulating vilanterol
trifenatate.
[0099] Sample 3: NaCl (0.9 g) was added to a beaker containing 100
ml purified water. To this NaCl solution, 117 .mu.Lof hydrochloric
acid was added. Vilanterol trifenatate (7.03 mg) was added to 70 ml
of the solution, then sonicated for 10 min to dissolve. Then, the
solution containing vilanterol trifenatate was filtered using a
0.22-.mu.m filter membrane. Then DPPC (27.05 mg), DSPG (3.25 mg),
and cholesterol (7.84 mg) were combined in a 50-ml round-bottom
flask followed by the addition of 18 ml of chloroform and 2 ml of
methanol to dissolve DPPC and DSPG with gentle shaking. The solvent
was removed using a rotary evaporator at 65.degree. C., until a
thin film of dried lipid was deposited on the walls of the flask.
20 ml of the prepared vilanterol trifenatate solution was added to
the round-bottom flask containing the thin layer of dried lipid.
The temperature was maintained at 65.degree. C. for approximately
30 minutes. The formulation was then subjected to extrusion using a
heated stainless steel extruder assembled with one polycarbonate
membrane at 65.degree. C., to reduce the size of the liposomes and
improve homogeneity. The extrusion process was repeated for 9-11
passes to achieve 80-200 nm liposomes. After extrusion, NaOH was
added to the liposomal solution to adjust the pH to 6.5-7.0.
Finally, the liposomal solution was filtered using a 1-.mu.m GF
membrane to obtain liposomes encapsulating vilanterol
trifenatate.
[0100] Sample 4: NaCl (0.9 g) was added to a beaker containing 100
ml purified water. To this NaCl solution, 117 .mu.Lof hydrochloric
acid was added. Vilanterol trifenatate (10.4 mg) was added to 70 ml
of the solution, then sonicated for 10 min to dissolve. Then, the
solution containing vilanterol trifenatate was filtered using a
0.22-.mu.m filter membrane. Then DPPC (44.68 mg), DSPG (5.44 mg),
and cholesterol (13.11 mg) were combined in a 50-ml round-bottom
flask followed by the addition of 18 ml of chloroform and 2 ml of
methanol to dissolve DPPC and DSPG with gentle shaking. The solvent
was removed using a rotary evaporator at 65.degree. C., until a
thin film of dried lipid was deposited on the walls of the flask.
20 ml of the prepared vilanterol trifenatate solution was added to
the round-bottom flask containing the thin layer of dried lipid.
The temperature was maintained at 65.degree. C. for approximately
30 minutes. The formulation was then subjected to extrusion using
heated stainless steel extruder assembled with one polycarbonate
membrane at 65.degree. C., to reduce the size of the liposomes and
improve homogeneity. The extrusion process was repeated for 9-11
passes to achieve 80-200 nm liposomes. After extrusion, NaOH was
added to the liposomal solution to adjust the pH to 6.5-7.0.
Finally, the liposomal solution was filtered using a 1-.mu.m GF
membrane to obtain liposomes encapsulating vilanterol
trifenatate.
[0101] The particle size of each of the above four samples of
liposomal formulation was analyzed using a Malvern Nano ZS90
particle size analyzer.
Example 3
[0102] All four samples prepared in above example 2 were evaluated
for drug-loading using HPLC, and the particle size was measured
using a Malvern Nano ZS90 particle size analyzer. As shown in table
1, the encapsulation efficiency of each of the four samples was
above 95%. Furthermore, the average particle size recorded was in
the range of about 120 nm to about 320 nm.
TABLE-US-00001 TABLE 1 Parameter list of the samples Parameter
Sample 1 Sample 2 Sample 3 Sample 4 Drug to lipid ratio 1:100 1:50
1:30 1:50 Drug-loading (.mu.g/ml) 100.18 86.58 105.72 110.28
Average particle size 261.6 286.07 311.23 125.27 (nm) Encapsulation
efficiency 97.67 100 98.21 100 (%)
Example 4
[0103] The drug release profile of sample 2, as described in
example 2, was studied by taking 10 ml of the liposomal formulation
and loading it into a dialysis bag. The dialysis bag containing the
liposomal formulation was kept in 400 ml PBS solution and stirred.
The dialysate (PBS solution) was taken every half hour to measure
the vilanterol trifenatate content. The results are tabulated in
Table 2.
TABLE-US-00002 TABLE 2 Drug release profile of drug-loaded liposome
Time (h) 0.5 1 2 3 4 6 24 Cumulative 19.54 35.16 50.59 62.74 74.62
82.98 97.90 Release (%)
Example 5
[0104] Preparation Method 2:
Preparation of drug solution: NaCl (0.9 g) and 100 ml of purified
water were combined in a beaker. The pH was adjusted using 1174, of
hydrochloric acid solution. Vilanterol trifenatate (10.04 mg) was
added to 80 ml of the solution, then sonicated for 10 min to
dissolve. The solution containing vilanterol trifenatate was
filtered using a 0.22-.mu.m MCE membrane.
[0105] Injecting lipid solution into the drug solution: 29.91 mg of
DPPC and 15.61 mg of cholesterol were combined in a beaker
containing 15 ml ethanol, and then the beaker was placed in a water
bath at 50.degree. C. to fully dissolve. 30 mL of the vilanterol
trifenatate solution prepared above was added to a beaker and
stirred continuously. The prepared lipid solution was injected into
the vilanterol trifenatate solution in the beaker. After the
addition of the lipid solution, the resulting solution was stirred
for at least 20 minutes.
[0106] pH adjustment: Following stirring, the resulting solution
was transferred to a 50-ml volumetric flask, and the pH was
adjusted to 2.0. Finally, the volume was adjusted to 50 ml using
purified water.
Example 6
[0107] In accordance with the preparation method described in
example 5, three different samples were prepared with high
encapsulation efficiency and different drug to lipid ratios. The
encapsulation efficiency of each of the three samples was over 95%,
and the encapsulation efficiency of each of sample 5 and sample 7
was 100%. The average particle sizes of the liposomes were in range
of about 120 nm to about 260 nm.
[0108] Sample 5: NaCl (0.9 g) and 100 ml of purified water were
combined in a beaker. The pH was adjusted using 117 .mu.L of
hydrochloric acid solution. Vilanterol trifenatate (10.04 mg) was
added to 80 ml of the solution, then sonicated for 10 min to
dissolve. The solution containing vilanterol trifenatate was
filtered using a 0.22-.mu.m MCE membrane. Then, 29.91 mg DPPC and
15.61 mg cholesterol were dissolved in 15 ml ethanol, which was
heated to temperature of 50.degree. C. in a beaker, and mixed until
completely dissolved to provide a lipid solution. 30 mL of the
vilanterol trifenatate solution prepared above was added to a
separate beaker and stirred continuously. The prepared lipid
solution was added drop-wise to the beaker containing the
vilanterol trifenatate solution. After the addition of the lipid
solution, the resulting solution was stirred for at least 20
minutes. Following stirring, the resulting solution was transferred
to a 50-ml volumetric flask, and the pH was adjusted to 2.0. The
volume was adjusted to 50 ml with normal saline.
[0109] Sample 6: NaCl (0.9 g) and 100 ml of purified water were
combined in a beaker. The pH was adjusted using 117 .mu.L of
hydrochloric acid solution. Vilanterol trifenatate (7.03 mg) was
added to 80 ml of the solution, then sonicated for 10 min to
dissolve. The solution containing vilanterol trifenatate was
filtered using a 0.22-.mu.m MCE membrane. Then, 32.6 mg DPPC and
17.62 mg cholesterol were dissolved in 15 ml ethanol, which was
heated to temperature of 50.degree. C. in a beaker, and mixed until
completely dissolved to provide a lipid solution. 30 mL of the
vilanterol trifenatate solution prepared above was added to a
separate beaker and stirred continuously. The prepared lipid
solution was added drop-wise to the beaker containing the
vilanterol trifenatate solution. After the addition of the lipid
solution, the resulting solution was stirred for at least 20
minutes. Following stirring, the resulting solution was transferred
to a 50-ml volumetric flask, and the pH was adjusted to 2.0. The
volume was adjusted to 50 ml with normal saline.
[0110] Sample 7: NaCl (0.9 g) and 100 ml of purified water were
combined in a beaker. The pH was adjusted using 117 .mu.L of
hydrochloric acid solution. Vilanterol trifenatate (10.40) was
added to 80 ml of the solution, then sonicated for 10 min to
dissolve. The solution containing vilanterol trifenatate was
filtered using a 0.22-.mu.m MCE membrane. Then, 74.94 mg DPPC and
39.67 mg cholesterol were dissolved in 15 ml ethanol, which was
heated to temperature of 50.degree. C. in a beaker, and mixed until
completely dissolved to provide a lipid solution. 30 mL of the
vilanterol trifenatate solution prepared above was added to a
separate beaker and stirred continuously. The prepared lipid
solution was added drop-wise to the beaker containing the
vilanterol trifenatate solution. After the addition of the lipid
solution, the resulting solution was stirred for at least 20
minutes. Following stirring, the resulting solution was transferred
to a 50-ml volumetric flask, and the pH was adjusted to 2.0. The
volume was adjusted to 50 ml with normal saline.
[0111] The drug-loading was evaluated using HPLC, and the particle
size was measured using a Malvern Nano ZS90 particle size analyzer.
As shown in Table 2, the encapsulation efficiency of each of the
three samples was above 95%. Furthermore, the average particle size
recorded is in the range of 120 nm-250 nm.
TABLE-US-00003 TABLE 2 Parameter list of the samples Parameter
Sample 5 Sample 6 Sample 7 Drug to lipid ratio 1:20 1:30 1:50
Drug-loading (.mu.g/ml) 24.84 29.32 20.57 Average particle size
132.3 247.1 160.43 (nm) Encapsulation efficiency 100 96 100 (%)
Example 7
[0112] Sample 1, sample 2, and sample 3 were sprayed using each of
an ultrasonic vibrating mesh nebulizer and a compressed air
nebulizer. A Malvern Spraytec (STP5311) particle size analyzer was
used to measure the particle size distribution of the resulting
droplets. The particle size distribution of the droplets is
expressed in terms of D10, D50, and D90. The results are shown in
table 3 and table 4. The D50 values of the droplets formed using
each of the compressed air nebulizer and the ultrasonic vibrating
mesh nebulizer were less than 10 .mu.m, and similarly D90 values of
droplets formed using each of the compressed air nebulizer and the
ultrasonic vibrating mesh nebulizer are less than 15 .mu.m.
TABLE-US-00004 TABLE 3 Particle size distribution using compressed
air nebulizer Sample Number Sample 1 Sample 2 Sample 3 Drug to
lipid ratio 1:100 1:50 1:30 D.sub.10 1.93 1.45 1.78 D.sub.50 4.85
4.34 4.71 D.sub.90 10.8 10.3 10.4
TABLE-US-00005 TABLE 4 Particle size distribution using ultrasonic
vibrating mesh nebulizer Sample Number Sample 1 Sample 2 Sample 3
Drug to lipid ratio 1:100 1:50 1:30 D.sub.10 2.49 2.60 2.59
D.sub.50 4.68 4.86 4.91 D.sub.90 8.20 9.06 9.74
Example 8
[0113] Sample 5, sample 6, and sample 7 were also sprayed using
each of an ultrasonic vibrating mesh nebulizer and a compressed air
nebulizer. A Malvern Spraytec (STP5311) particle size analyzer was
used to measure the particle size distribution of the resulting
droplets. The particle size distribution of the droplets is
expressed in terms of D10, D50, and D90. As shown in table 5 and
table 6, the D50 values of the droplets formed using each of the
compressed air nebulizer and the ultrasonic vibrating mesh
nebulizer are less than 10 and D90 values of the droplets formed
using each of the compressed air nebulizer and ultrasonic vibrating
mesh nebulizer were less than 15
TABLE-US-00006 TABLE 5 Particle size distribution using compressed
air nebulizer Sample Number Sample 5 Sample 6 Sample 7 Drug to
lipid ratio 1:20 1:30 1:50 D.sub.10 1.55 1.77 1.26 D.sub.50 4.33
4.58 4.26 D.sub.90 10.2 10.6 10.5
TABLE-US-00007 TABLE 6 Particle size distribution using ultrasonic
vibrating mesh nebulizer Sample Number Sample 5 Sample 6 Sample 7
Drug to lipid ratio 1:20 1:30 1:50 D.sub.10 2.43 2.45 2.54 D.sub.50
4.64 4.77 497 D.sub.90 8.51 9.52 9.97
* * * * *