Note: Descriptions are shown in the official language in which they were submitted.
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~.. 218 1390
MODI.P0012
PHOSPHOLIPID FORMULATIONS
Field of the Invention
The present invention relates to improved delivery
of medicinally active compounds into the body. In
particular it relates to production of stable liposomes
for delivery of cosmetics, drugs, enzymes, growth
factors, hormones, interferons, interleukins,
moisturizers, peptides, proteins, and steroids. A
variety of administration techniques, e.g. oral,
injection, topical, may be used, depending on the
medicinally active ingredient.
Background to the Invention
Drugs, to be effective must be present at the site
of action in an effective concentration for a desired
period of time. Oral and parenteral administration of
drugs relies on the blood circulation to carry the drugs
to the target organs. Consequently, the drug is
distributed throughout the body and deposited in all
organs, which often lead to unwanted side effects.
Attempts have been made to favourably influence drug
distribution by combining the drug with a "carrier".
Among the variety of carriers, liposomes, due to their
composition and compatibility with the living organism,
seem to have a good potential for selective drug
delivery.
It is believed that a substance will have a
different destiny when delivered using a liposome rather
than by commercially used carriers (sometimes known as
excipients), which, due to their composition are foreign
to the living organism. Liposomes are made of similar
components of cell membranes, and are compatible with
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the skin superficial layer structure. According to one
source, the horny skin layer can be compared to a wall,
where keratinized cells represent the bricks, and
intracellular lipids the cement which keeps them
together. Lipids, lying in lamellae, show a bilayer
structure similar to that of biological membranes. They
have a lipidic layer enclosed in a water layer. The
function of the lipid layer is double: on the one hand
it represents the most important part of the barrier of
the cutaneous permeability, on the other hand it
maintains the hydration of the skin, indispensable for
the integrity of the skin. It is believed that
liposomes, made of phospholipidic fractions which absorb
water, act both as a water carrier and as a reintegrator
of permeability, since they interact with the lamellar
lipids of the horny skin layer. Moreover, the liposome
vesicle has the function of "carrier", that is, it
releases the encapsulated active substances both through
diffusion from its walls and through spill when it
opens.
Materials like vaccines, hormones, enzymes,
interferons, interleukins, are rapidly inactivated when
injected into body and when they enter systemic
circulation. Accumulation of these active substances in
the body, to generate a superior response or therapeutic
effect, is not satisfactory and systemic side effects
can occur frequently. Incorporating such materials into
liposomes can increase their efficacy several folds and
provides superior therapeutic effect.
Interest in the use of liposomes in dermato-
cosmetology has increased in the recent years, mainly
3 _ 2181390
for two reasons: the particular affinity of liposomes
with the skin, and the fact that they are applied
directly on the part where they are destined. The
advantages of this kind of transportation into the skin
include release of the active ingredients at different
levels in the skin, longer contact with the cutaneous
layers, and reduced systemic absorption. Release of the
active ingredient mainly occurs in the multilamellar
liposomes, which allows a slower release of the active
ingredient in the chosen site, with a so-called depot
effect, which is very useful in order to streamline the
effects on the skin.
The clinical use of liposomes has been delayed
because of difficulties in mass production,
sterilization, stability and safety. The present
invention is aimed at production of stable liposomes for
delivery of medicinally active ingredients.
Molecular weights indicated herein are weight
average molecular weights (Mw)and can be determined by
known light scattering methods or gel filtration
chromatography methods. Light scattering methods are
preferred.
Disclosure of the Invention
Accordingly the present invention provides a
formulation comprising i) at least one medicinally
active ingredient, ii) at least three compounds selected
from the group consisting of egg phosphatidylcholine
(PC) dilauryl phosphatidylcholine (DLPC), dimyristoyl
phosphatidylcholine (DMPC), dipalmitoyl
phosphatidylcholine(DPPC), dioleoyl phosphatidylcholine
(DOPC), dimyristoyl phosphatidylglycerol (DMPG),
s S
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dipalmitoyl phosphatidylglycerol(DPPG), dimyristoyl
phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid
(DPPA), dipalmitoyl phosphatidylethanolamine (DPPE),
distearoyl phosphatidylcholine (DSPC), brain
phosphatidylserine (PS), brain sphingomyelin (SM),
cholesterol (C), cardiolipin (CL), trioctanoin (TC),
triolein (TO), soy phosphatidylcholine, poly(adenylic
acid), phosphatidylethanolamine (PE), phosphatidyl
glycerol (PG), phosphatidyl inositol (PI), sphingosine,
cerebroside (glycolipid), and iii) at least one
biodegradable polymer selected from the group consisting
of copolymers of sucrose and epichlorohydrin having
molecular weights of from 70 000 to 400 000, glucose
polymers having molecular weights of from 10 000 to
300 000, polyethylene glycols having molecular weights
of from 1000 to 100 000, polyvinyl alcohols having
molecular weights of from 70 000 to 110 000,
hydroxymethyl cellulose, hydroxyethyl cellulose,
hydroxypropyl cellulose, hydroxypropyl-methyl cellulose,
hydroxypropyl-ethyl cellulose, sodium carboxymethyl
cellulose, gelatin, starch, crosslinked starch,
polyethyleneimine, methoxypolyethylene glycol,
ethoxypolyethylene glycol, polyethylene oxide,
polyoxyethylene, polyoxypropylene, cellulose acetate,
sodium alginate, N,N-diethylaminoacetate, block
copolymers of polyoxyethylene and polyoxypropylene,
polyvinyl pyrrolidone, polyoxyethylene X-lauryl ether
wherein X is from 9 to 20, and polyoxyethylene sorbitan
esters.
The letter combinations in parentheses are
abbreviations used elsewhere in this specification.
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It is to be understood that the term "egg
phosphatidylcholine" comprises combinations of
substantially saturated phosphatidylcholines, e.g.
combinations of 99% saturated Phospholipon H (trade
mark) and 90% saturated Phospholipon G (trade mark) in
ratios of 70:30 to 95:5 Phospholipon H:Phospholipon G.
In another embodiment the formulation has at least
two biodegradable polymers.
In yet another embodiment the combinations of
compounds are selected from the group consisting of:
i) brain phosphatidylserine, cholesterol, triolein and
phosphatidylethanolamine, ii) egg phosphatidylcholine,
distearoyl phosphatidylcholine, cholesterol and
triolein, iii) cholesterol, cardiolipin, poly(adenylic
acid) and triolein, iv) egg phosphatidylcholine,
distearoyl phosphatidylcholine, cholesterol and
trioctanoin, v) egg phosphatidylcholine, cholesterol,
trioctanoin and phosphatidyl glycerol, vi) egg
phosphatidylcholine, cholesterol, cardiolipin and
triolein, vii) dioleoyl phosphatidylcholine,
cholesterol, cardiolipin and triolein, viii) egg
phosphatidylcholine, cholesterol, brain
phosphatidylserine and triolein, ix) dioleoyl
phosphatidylcholine, brain phosphatidylserine,
cholesterol and triolein, x) egg phosphatidylcholine,
dipalmitoyl phosphatidylglycerol, cholesterol and
trioctanoin, xi) dimyristoyl phosphatidylcholine,
dipalmitoyl phosphatidylcholine, distearoyl
phosphatidylcholine and triolein, xii) egg
phosphatidylcholine, dipalmitoyl phosphatidylcholine,
distearoyl phosphatidylcholine and trioctanoin, xiii)
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phosphatidylcholine, cholesterol, brain
phosphatidylserine and triolein, xiv)
phosphatidylethanolamine, cholesterol cardiolipin and
triolein, xv) phosphatidylcholine, cholesterol,
trioctanoin and dipalmitoyl phosphatidylethanolamine,
xvi) dimyristoyl phosphatidylcholine, dipalmitoyl
phosphatidylcholine, distearoyl phosphatidylcholine and
triolein, xvii) phosphatidylcholine,
phosphatidylethanolamine, cholesterol, brain
phosphatidylserine and triolein, xviii)
phosphatidylcholine, phosphatidylthanolamine,
cholesterol, cardiolipin and triolein, xix)
phosphatidylcholine, dioleoyl phosphatidylcholine,
cholesterol, cardiolipin and triolein, xx)
phosphatidylcholine, dioleoyl phosphatidylcholine,
cholesterol, brain phosphatidylserine and triolein, xxi)
phosphatidylcholine, dimyristoyl phosphatidylcholine,
dipalmitoyl phosphatidylcholine, distearoyl
phosphatidylcholine and triolein and xxii)
phosphatidylcholine, cholesterol, phosphatidylcholine
ethanolamine and triolein.
Preferred weight ratios of these combinations are
shown in parentheses: PE/C/PS/TO (4.5/4.5/1/1);
PC/C/DSPC/TC (4.1/1.9/1/1); PE/C/CL/TO (4.5/4.5/1/1);
PC/C/TC/DPPE (4.1/1.9/6.6/1); PC/C/PS/TO (4.5/4.5/2/1);
PC/C/CL/TO (4.5/4.5/1/1); DOPC/C/CL/TO (4.5/4.5/1/1);
PC/C/PG/TC (5/4/1/1); DOPC/C/PS/TO (5/5/1/1);
PC/C/DPPG/TC (5/4/1/1); DMPC/DPPC/DSPC/TO (5/6/10/2);
PC/DSPC/C/DPPC/TC (5/5/1/5/1); PC/PE/C/PS/TO
(4.5/4.5/4.5/1/1); PC/PE/C/CL/TO (4.5/4.5/4.5/4.5/1/1);
PC/DOPC/C/CL/TO (4.5/4.5/4.5/1/1); PC/DOPC/C/PS/TO
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(5/5/5/1/1); PC/DMPC/DPPC/DSPC/TO (5/5/6/10/2); and
PC/C/PE/TO (4/2/1/1).
The above ratios refer to specific preferred
ratios. It is to be understood that variations in the
ratios may be made to effect the desired long release
characteristics. For example, each of the compounds in
the combinations are preferably present in
concentrations of between about 5 and about 555 of the
combination. For example phosphatidylcholine,
phosphatidylcholine ethanolamine, dioleoyl
phosphatidylcholine, trioctanoin and cholesterol
typically may be present in concentrations of 5 to 55~
of the compound combination; dimyristoyl
phosphatidylcholine, dipalmitoyl phosphatidylcholine and
distearoyl phosphatidylcholine typically may be present
in concentrations of 15 to 50~ of the compound
combination; and brain phosphatidylserine, triolein,
cardiolipin, dipalmitoyl phosphatidylethanolamine,
dipalmitoyl phosphatidylglycerol, and phosphatidyl
glycerol typically may be present in concentrations of 5
to 15~ of the compound combination.
Those skilled in the art will be aware of the
general effect of individual phospholipids and this is
useful in determining the selection of compounds in any
particular composition. For example long chain fatty
acids such as phosphatidyl glycerol or triolein are
useful for enhancing the penetration of the composition
into the skin and for solubilization of drugs.
Substantially saturated phosphatidylcholine is useful in
stabilizing liposomes as they are less prone to
oxidation. The presence of phosphatidylcholine also
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makes manufacture easier because it is not necessary to
make the formulation in a nitrogen atmosphere.
Additionally, for example, cardiolipin, brain
phosphatidylserine and brain sphingomyelin are charged
lipids and tend to concentrate the formulation on the
desired site of attack.
The preferred liposome formulation is prepared
using stabilizers, absorption enhancers, and
antioxidants in addition to the compounds, biodegradable
polymer, and medicinally active ingredients.
The formulations of the present invention may be in
lamellar, vesicle or other form, depending on the
particular composition of the formulation. The
lamellae, vesicles or other forms may be coated with at
least one biodegradable polymer, preferably two polymers
selected from the group consisting of copolymers of
sucrose and epichlorohydrin having molecular weights of
from 70 000 to 400 000, glucose polymers having
molecular weights of from 10 000 to 300 000,
polyethylene glycols having molecular weights of from
1000 to 100 000, polyvinyl alcohols having molecular
weights of from 70 000 to 110 000, hydroxymethyl
cellulose, hydroxyethyl cellulose, hydroxypropyl
cellulose, hydroxypropyl-methyl cellulose,
hydroxypropyl-ethyl cellulose, sodium carboxymethyl
cellulose, , starch, crosslinked starch,
polyethyleneimine, methoxypolyethylene glycol,
ethoxypolyethylene glycol, polyethylene oxide,
polyoxyethylene, polyoxypropylene, cellulose acetate,
sodium alginate, N,N-diethylaminoacetate, block
copolymers of polyoxyethylene and polyoxypropylene,
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polyvinyl pyrrolidone, polyoxyethylene X-lauryl ether
wherein X is from 9 to 20, and polyoxyethylene sorbitan
esters.
It will be recognized by those skilled in the art
that for many pharmaceutical compositions it is usual to
add at least one antioxidant to prevent degradation and
oxidation of the medicinally active ingredients.
Preferred antioxidants may be selected from the group
consisting of ascorbic acid, tocopherol and deteroxime
mesylate.
In another embodiment, an antifungal, antimicrobial
agent selected from the group consisting of ethyl
paraben, methyl paraben, propyl paraben and combinations
thereof may also be added to the composition.
In another embodiment the formulation contains at
least one absorption enhancer, especially absorption
enhancers selected from the group consisting of Na-
salicylate-chenodeoxy cholate, Na deoxycholate,
polyoxyethylene 9-lauryl ether, chenodeoxy cholate-
deoxycholate and polyoxyethylene 9-lauryl ether,
monoolein, Na tauro-24,25-dihydrofusidate, Na
taurodeoxycholate, Na glycochenodeoxycholate, oleic
acid, linoleic acid, linolenic acid.
Polymeric absorption enhancers may also be added to
the formulation, e.g. polyoxyethylene ethers,
polyoxyethylene sorbitan esters, polyoxyethylene 10-
lauryl ether, polyoxyethylene 16-lauryl ether, azone (1-
dodecylazacycloheptane-2-one), and sodium chloride,
sodium bicarbonate in combination with the above
mentioned materials.
Examples of the medicinally active ingredients
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include insulin, heparin, hirugen, hirulos, hirudin;
vaccines such as influenza virus vaccine, pneumonia,
hepatitis A vaccine, hepatitis B vaccine, and hepatitis
C vaccine, cholera toxin B-subunit, influenza vaccine
virus, typhoid vaccine, plasmodium falciparum vaccine,
diphtheria vaccine, tetanus vaccine, herpes simplex
virus vaccine, tuberculosis vaccine, HIV vaccine,
bordetella pertussis vaccine, measles vaccine, mumps
vaccine and rubella vaccine (MMR), bacterial toxoids,
vaccinia virus, adenovirus, canary pox, polio vaccine
virus, bacillus calmette geurin (BCG), klebsiella
pneumonia, HIV envelope glycoproteins,; hormones such as
bovine somatropin, oestrogen, androgens, prostaglandins,
somatotropins, enzymes such as thyroids, pituitary,
digestive, a-, ~- and y-interferons, tuftsin,
interleukins, insulin and insulin like growth factors,
roes, steroids, terpenoids, triterpenes, retinoids;
anti-ulcer H2 receptor antagonists, anti-ulcer drugs,
hypoglycaemic agents, moisturizers, cosmetics and drugs.
Examples of bacterial toxoids are tetanus,
diphtheria, pseudomonas A, mycobacterium tuberculosis.
Examples of HIV envelope glycoproteins are gp 120 and gp
160 for AIDS vaccines. Examples of anti-ulcer H2
receptor antagonists are ranitidine, cimetidine and
famotidine, and other anti-ulcer drugs are omparazide,
cesupride and misoprostol. An example of a
hypoglycaemic agent is glizipide. Insulin is used for
the control of diabetes.
As will be understood by those skilled a.n the art,
two or more pharmaceutical agents may be combined for
specific effects. The necessary amounts of active
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ingredient can be determined by simple experimentation.
Specific pharmaceutical agents which may be used in this
invention are insulin, heparin, low molecular weight
heparin, hirugen, hirulos and hirudin.
The method of making the formulation is
straightforward. Typically the composition of compounds
is dissolved in an organic solvent such as ethanol or
chloroform/methanol. The drug is dissolved in water or
a buffer solution and vigorously mixed. The liposome
forms instantaneously on vigorous stirring, sonication
or agitation. The medicinally inactive ingredients,
e.g. any antioxidants, inorganic salts, protective
polymers, protease inhibitors, absorption enhancers and
other ingredients, e.g. colouring matter, flavourings,
are then added and mixed until the solution is
homogeneous. Typically the compound to medicinally
active ingredient ratio may vary from 50:1 to 1:1
In the selection of a suitable absorption enhancing
compound combination, it has been found that the amount
of total absorption enhancing compound should be less
than about 10 wt./wt.~ of the formulation and preferably
from 1 to 5 wt./wt.~. Frequent use or prolonged use of
higher concentrations of absorption enhancing compounds
are likely to be harmful to linings and tissues in the
gastrointestinal tract, and may cause diarrhoea. It is
believed that the optimum range for most combinations is
from 1.5 to 3.5 wt./wt.~
The invention may also be better understood by
reference to the following examples:
Example I:
Compositions containing Ponceau-S dye were prepared
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using following procedure:
Compositions shown in Table I were dissolved in 15
ml of chloroform/methanol (2:1 vol/vol). The resulting
clear solution was introduced into a 250 ml round bottom
flask with a ground-glass neck. The flask was attached
to a rotary evaporator, rotated at about 100 rpm to
remove solvent at a reduced pressure. The rotation of
flask was continued until all the liquid evaporated and
dry uniform film was obtained on the walls of the flask.
The flask was removed from the rotary evaporator
equipment and was attached to a vacuum line for the
complete removal of the solvent. After releasing the
vacuum, the flask was flushed with nitrogen, and 0.1~
Ponceau-S dye solution prepared in 5 ml of distilled
water containing 0.3~ polyethylene glycol having a
molecular weight of 10 000, 0.3~ ethylhydroxy cellulose
(1:1), 0.1~ monoolein, 0.1~ linoleic acid, and 0.2~
tocopherol. The solution was poured in the flask and 0.5
g glass beads were added. The flask was rotated slowly
for 30 minutes until all the compound was removed from
the walls of the flask. A homogenous milky white
suspension was formed. The suspension was allowed to
stand at room temperature for 2 hrs in order to allow
swelling to take place. The resulting suspension was
spun at 12 000 G for 10 minutes in a bench centrifuge. A
pellet was formed, which was then removed and stored at
room temperature for analysis.
The composition containing Ponceau-S dye was
analyzed for ~ encapsulation using Protamine aggregation
method:
0.1 ml of liposomal suspension (20 mg/ml lipid) in
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ml 0.9% NaCl solution in a glass centrifuged tube. To
this suspension was added 0.2 ml protamine solution and
allowed to stand for 5 min and was spun at 2000 G for 20
minutes at room temperature. Supernatant was analyzed
5 for free, untrapped materials using UV spectroscopy (dye
concentration was measured at 510 and 560 nm). The
resulting pellet was collected and 0.6 ml 10% Triton-X
100 added to dissolve liposomal material. The
solubilized material was further diluted with 5 ml
10 saline solution and the resulting liquid was assayed for
entrapped material (dye) by W spectroscopy at 510 and
560 nm. Table I outlines % Ponceau-S dye in various
liposomes.
The percent dye entrapped is calculated as (the dye
concentration in the liposome composition minus the dye
concentration in supernatant) divided by the dye
concentration in the liposome composition x 100.
TABLE I
Liposome Composition % dye entrapped
PC/PE/C/PS/TO (4.5/4.5/4.5/1/1) 87%
PE/C/PS/TO (4.5/4.5/1/1) 87%
PC/C/DSPC/TC (4.1/1.9/1/1) 89%
PC/PE/C/CL/TO (4.5/4.5/4.5/1/1) 87%
PE/C/CL/TO (4.5/4.5/1/1) 8~~
PC/C/TC/DPPE (4.1/1.9/6.6/1) 84%
PC/C/PS/TO (4.5/4.5/2/1) 84%
PC/C/CL/TO (4.5/4.5/1/1) 85%
PC/DOPC/C/CL/TO (4.5/4.5/4.5/1/1) 82%
DOPC/C/CL/TO (4.5/4.5/1/1) 82%
PC/C/PG/TC (5/4/1/1) 78%
PC/DOPC/C/PS/TO (5/5/5/1/1) 80%
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DOPC/C/PS/TO (5/5/1/1) 80%
PC/C/DPPG/TC (5/4/1/1) 75%
PC/DMPC/DPPC/DSPC/TO (5/5/6/10/2) 89%
DMPC/DPPC/DSPC/TO (5/6/10/2) 89%
PC/DSPC/C/DPPC/TC (5/5/1/5/1) 77%
The release characteristics of one of the compound
composition was investigated by solubilizing the
compound composition in a dilute (1-2%) Triton-X 100
(trade mark) solution. The composition containing dye
(100 ~L) was diluted with 3 ml saline and placed in a
glass tube. To this solution 100 ~,L of 1-2% Triton-X 100
solution was added. The resulting solution was mixed to
obtain a homogenous solution, and centrifuged. The
supernatant was placed in a glass W cuvette and
absorbance readings were taken at 510 and 560 nm at
regular time intervals. The results are shown in Table
II.
TABLE II
Time (Minutes) % Dye Released
10 7.5%
14.3%
60 18.9%
120 25%
180 33.3%
25 240 40%
300 47.5%
Example II
In order to demonstrate the efficacy of the
formulation of the present invention in the field of
30 dermato-cosmetology, a composition containing PE/C/PS/TO
in ratio of (5/5/2/1) was dissolved in 15 ml of
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chloroform/methanol (2:1 vol/vol). The resulting clear
solution was introduced into a 250 ml round bottom flask
with a ground-glass neck. The flask was attached to a
rotary evaporator, rotated at about 100 rpm to remove
solvent at a reduced pressure. The rotation of flask was
continued until all the liquid evaporated and dry
uniform film was obtained on the walls of the flask. The
flask was removed from the rotary evaporator equipment
and was attached to vacuum line for the complete removal
of the solvent. After releasing the vacuum, the flask
was flushed with nitrogen and 500 mg lidocaine solution
prepared in 5 ml of distilled water containing 0.3%
polyethylene glycol having a molecular weight of 10 000,
0.3% ethylhydroxy cellulose (1:1), 0.1% monoolein, 0.1%
linoleic acid, and 0.2% tocopherol. The solution was
poured in the flask and 0.5 g glass beads were added.
The flask was rotated slowly for 30 min until all the
PE/C/PS/TO was removed from the walls of the flask to
give a homogenous milky white suspension. The suspension
was allowed to stand at room temperature for 2 hrs in
order to allow swelling to take place. The resulting
suspension was spun at 12 000 G for 10 minutes in a
bench centrifuge. A pellet was formed, which was then
removed and stored at room temperature for analysis.
The lidocaine concentration was calculated by
estimating amount of lidocaine in the PE/C/PS/TO
solution and untrapped lidocaine in supernatant. The
liposomal lidocaine suspension was then stored in a
bottle at room temperature. Ten healthy male guinea pigs
weighing 500-700 g were anaesthetized by intraperitonial
injection of 0.2 ml of sodium pentobarbitol. Lidocaine
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cream (5 guinea pigs) and the present PE/C/PS/TO
lidocaine (5 guinea pigs) were applied to tail, leg and
back areas. After about 4.5 hours tissue samples
(epidermis, dermis, and subcutaneous layers) were
collected and processed for lidocaine concentration
estimation. Tissue samples from each layer were weighed
and approximately equal amounts were placed in a
phosphate buffer (pH 7.4), homogenized, and centrifuged
at 20 000 G for 30 minutes. Supernatant was collected
and was analyzed for lidocaine concentration using
standard HPLC technique. The results for tissue
concentration of lidocaine are shown in Table III,
overleaf, which compares 2~ Lidocaine Cream (control
sample, not of the present invention) and 2~ liposomal
Lidocaine Cream (an embodiment of the present invention)
of the above composition.
TABLE III
Tissue Sample Regular Lidocaine Liposomal Lidocaine
(after 4.5 Hrs) (~,g/g tissue) (~.g/g tissue)
Epidermis 75.87 198.8
Dermis 22.61 48.1
Subcutaneous 5.32 3.9
Plasma 0.077 0.023
Example III
In order to further demonstrate a method of making
a formulation of the present invention, which is
suitable for scale-up to industrial quantities, 8 g of a
composition with compounds containing PC/C/
Phospholipon G/PE/TO in ratio of (2/2/2/1/1) was
dissolved in 6 g of 100 ethanol at a temperature of
-1~- 218130
60°C to 65°C, in a beaker. 0.2 g of tretinoin-A (trade
mark: Ratinyl-A) was added to the beaker and dissolved
at high speed using a magnetic stirrer rotating at about
1000 rpm. 86.8 g distilled water was added at 60°C
slowly to the alcohol solubilized mixture, while
stirring at 1000 rpm at a temperature of 60°C to 65°C.
The resulting reaction mixture was cooled to room
temperature after completion of the addition of water,
while continuing to stir at 1000 rpm. A multi-lamellar
liposome suspension formed spontaneously. The size of
the liposome suspension was measured by flow cytometer,
using laser light scattering techniques, and the sizes
measured were from 50 nm to 1 ~.m. The percent
encapsulation of tretinoin was determined using HPLC
equipped with a W detector and found to be 91.5°s
,.~
