Note: Descriptions are shown in the official language in which they were submitted.
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SELF-EMULSIFYING FORMULATION
PRODUCING A POSITIVELY CHARGED EMULSION
FIELD OF THE INVENTION
The present invention concerns a self-emulsifying essentially hydrophobic
formuiation, namely, a formulation which upon mixture with water,
spontaneously
disintegrates to form an oil-in-water emulsion. The present invention also
concerns
the pharmaceutical use of such a formulation both (i) as a drug delivery
vehicle of a
lipophilic drug or (ii) as a precursor for the preparation of an oil-in-water
emulsion
useful in turn as a drug delivery vehicle of hydrophobic drugs.
One application of the inventive formulation is in the oral administration of
a drug
intended to be absorbed in the gastro intestinal (GI) tract and then reach the
target
organ via the blood or lymphatic system. Such a form of administration will be
referred to herein as "oral systemic administration".
A particular application of the above preferred embodiment is in the oral
systemic
administration of a drug such as physostigmine, probucol, cyclosporin A,
morphine
base, penclomedine, and others.
The acknowledgement herein of the above prior art should not be interpreted as
an
admission that this art is in any way relevant to the issue of patentability
of the
invention as defined herein.
BACKGROUND OF THE INVENTION
Oral systemic administration of drugs, in general, is the preferred mode of
administration in ambulatory treatment regimens which require repetitive drug
administration over periods of time. While oral systemic administration is
very
effective with respect to water soluble drugs, it proves to be a problematic
administration route for hydrophobic drugs or drugs with limited aqueous
solubility
such as physostigmine base, isradipine, virginiamycine, cyclosporin A,
morphin,
buprenorphine, nalorphine, methorfan, probucol and others.
The poor systemic effect achieved with orally-administered hydrophobic drugs
results from a number of factors. For one, hydrophobic drugs do not dissolve
in
water and form a separate phase in aqueous solutions and are thus not readily
available for absorption through the walls of the GI tract. Furthermore, some
hydrophobic drugs, which are absorbed primarily through the walls of the small
intestine, particularly through the jejunum, may undergo a so-called "first
pass
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effect", i.e passage of the drugs in the liver prior to reaching the blood
system. The
overall effect of these factors is that only low, often non effective, amounts
of orally
administered hydrophobic drugs eventually reach the target organ(s), i.e
orally
administered hydrophobic drugs have generally a low bioavailability. This may
be
overcome by increasing the dose of the drug, but such an increase may,
however,
result in increased incidence of side-effects owing to erratic and variable
inter
subject absorption.
There have been various proposals for increasing bioavailability of
hydrophobic
drugs. For example, previous studies using oleic acid containing a dissolved
lipophilic drug, have demonstrated a beneficial effect on drug bioavailability
(Stella
et al, 1978, J. Pharm. Sci, 67, 1375-1377). Recently, it was shown that the
bioavailability of propranolol following oral administration, can be improved
by
dissolving the drug in a lipid formulation containing mainly oleic acid and
packing it
into a sealed and entero-coated hard gelatine capsule (Barnwell et al, 1992,
Int. J.
Pharmaceutics, $$, 423-432).
Emulsions have been proposed as carriers in oral formulations of hydrophobic
drugs
in general (Pal et al, 1984, J. Int. Pharm, 33. 99-104; Myers et al, 1992,
Int. J.
Pharm. 7$, 217-226) and for drugs such as physostigmine in particular
(Rubinstein
et al, 1991, J. Pharm. Res, $Q, 643-647; Friedman et al, 1989, Drug Design and
Delivery, 4, 135-142; Benita et al, 1989, Drug Delivery Design, 4, 143-153).
Colloid
particles of the emulsion which carry the drug are absorbed in the jejunum and
are
presumably carried away mainly by the lymph through the thoracic duct, thus
bypassing the liver and greatly reducing the first pass effect. Indeed, the
oral
bioavailability of several lipophilic drugs was shown to be somewhat improved
using
emulsions as vehicles for their oral systemic administration.
However, in many cases emulsion formulations offered no improvement in the
bioavailability of hydrophobic drugs versus their administration in aqueous
formulations. This is particularly the case with respect to drugs such as
physostigmine, which is an amphiphilic drug that localizes in the emulsion in
the
oil/water (o/w) interfacial film of the emulsion colloid particles. During
passage of an
emulsion containing physostigmine through the digestive tract, the emulsion,
which
is practically infinitely diluted, quickly releases the drug contained
therein. This
problem is increasingly augmented by the very strong acidity in the stomach
which
has a tendency to reduce the stability of the emulsion's colloid particles.
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Certain lipid solutions, by virtue of their ingredients, have the capacity to
undergo
spontaneous emulsification when introduced into an aqueous phase following
gentle
agitation yielding oil-in-water emulsions. Such lipid solutions are defined in
the
literature as self-emulsifying delivery systems (Charman et al, 1992, Pharm.
Res, 9,
87-93). Self-emulsifying delivery systems have been formulated using medium-
chain
triglyceride oils and nonionic surfactants which, depending on their exact
nature,
could form the basis of a self-emulsifying drug delivery system (Pouton,
1985a, Int.
J. Pharm. 37, 335-348; Pouton 1985b, Int. J. Pharm. 37, 1 P; Pouton et al,
1987,
Proc. tnt. Symp. Control. Rel. Bioacta. Mater, 14, 113-117; Wakerly et al,
1986,
ACS. Symp. Ser. 311, 242-255; Wakerly et al, 1987, J. Pharm. Sci, _Q7, 1375-
1377).
These formulations may be encapsulated in soft gelatine capsules or sealed
hard
gelatin capsules to yield precise and convenient unit dosage systems.
Early studies in the small intestine clearly established, that the absorptive
cell
interior is negative with respect to mucosal solution (Csaky, IL (Ed.),
Handbook of
Experimental Pharmacology, Vol 70, Springer-Verloy, Berlin, (1984), 324-325).
It has
also been reported that some hydrophobic cationic drugs, completely ionized
over
the pH range of the GI tract, are absorbed rapidly, in spite of their poor
water
solubility (Iseky, K, Hirano, T, Fukushi, Y, Kitamura, Y, Miyazaki, S, Takada,
M;
Sugawara, M, Saiton, H and Miyazaki, K. (1992), J. Pharm. Pharmacol, 44:9, 722-
726; Saiton, H, Kawai, S, lseki, K, Myazaki, K and Arita, T. (1988), J. Pharm.
Pharmacol, 41, 200-202). Moreover, some endogenic compounds bind to
endothelial
surfaces by its NH2-terminal, indicating physiological importance of the
electrostatic
interactions (21).
The formation of oily droplets containing a dissolved drug brings about
distribution of
the drug throughout the Gi tract while providing a large interfacial area for
partitioning of the drug between the oil and the surrounding aqueous phase.
Thus,
for drugs with limited aqueous solubility, which are poorly absorbed in the GI
tract,
the spontaneous disintegration of the lipid phase into very fine dispersed
oily
droplets may offer an improvement in both the rate and extent of absorption.
All the
self-emulsifying drug delivery systems known to date, contain large
concentrations of
surfactants (up to 50%) which were either non-ionic (such as Tween'R' Span"')
or
anionic surfactants (such as phospholipids) resulting in the formation of oily
droplets
having either a neutral or an electronegative charge.
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In a recent report (Elbaz et al, 1993, Int. J. Pharm. 2E, R1-R6) an emulsified
drug
delivery system has been disclosed in which the colloid particles bear a
positive
charge. However, no mention was made in this publication to the possibility of
using
such emulsions as an oral systemic drug delivery vehicle.
It is an object of the present invention toprovide a self-emulsifying
formulation useful
as a drug delivery system of lipophilic drugs.
It is a further object of the invention to provide a novel drug delivery
system for the
systemic oral administration of lipophilic drugs.
It is yet another object of the invention to provide a novel methad for the
administration of lipophiiic drugs.
It is yet still a further object of the invention to provide a method for
production of an
emulsion by the use of a self-emulsifying oiiy preparation.
GENERAL DESCRIPTION OF THE INVENTION
In accordance with the invention, a novel self-emulsifying oily formulation
(SEOF) is
provided. When the formulation of the invention is mixed with an aqueous
solution,
and the mixture is agitated, an oil-in-water emulsion is formed. According to
a
preferred embodiment of the invention the droplets in the so-formed emulsion
have a
diameter below about 0.2 pm. Emulsions having tiny droplets as those
obtainable in
accordance with the invention, were hitherto obtainable only by employing a
complex
homogenization procedure involving the use of intricate equipment (see for
example
Benita et al, 1989, Drug Delivery Design, 4, 143-153).
An additional feature of the SEOF of the invention is that the droplets in the
formed
emulsion are positively charged, unlike the negatively charged emulsions
achieved
with prior art SEOF.
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According to one aspect of the present invention,
there is provided a pharmaceutical composition comprising a
drug and a self-emulsifying oily formulation (SEOF), the
SEOF comprising an oil component and a surfactant, the oil
component comprising an oily carrier, a cationic lipid and a
lipophilic oily fatty alcohol, wherein an oil-in-water
emulsion which forms upon mixture of the SEOF with an
aqueous solution has oily droplets which are positively
charged and are in the submicron range, the drug being
dissolved in the interior of the oily droplets.
The SEOF of the invention comprises an oily
carrier which may be a medium chain triglyceride (MCT) oil,
a long chain triglyceride (LCT) oil, and an oily fatty acid
derivative. In addition, the SEOF comprise also a cationic
lipid. It has been found in accordance with the invention
that the addition of a lipophilic alcohol is required for
obtaining emulsion droplets in the submicron (< 1 pm) range.
The inclusion of a lipophilic alcohol in the SEOF of the
invention is thus preferred.
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The present invention thus provides a self-emulsifying oily formulation (SEOF)
comprising an oil component and a surfactant, the SEOF being characterized in
that
the oil component comprises an oily carrier and a cationic lipid and
optionally, a
lipophilic alcohol, the oil-in-water emulsion which forms upon mixture of the
SEOF,
having oily droplets which are positively charged.
The SEOF of the invention can be used as a delivery vehicle for hydrophobic
drugs.
When such a delivery vehicle, with the drugs dissolved therein, comes into
contact
with a body fluid, it spontaneously emulsifies forming tiny oiiy droplets with
the drug
contained therein.
One specific example of such a drug delivery vehicle is one which is intended
for the
oral systemic administration of hydrophobic drugs. For such administration,
the
SEOF with the dissolved drugs are preferably encapsulated.
The present -invention thus provides a pharmaceutical preparation comprising
an
effective amount of a hydrophobic drug dissolved in a liquid carrier, the
preparation
being characterized in that said carrier is the above SEOF. Specific
embodiments of
said preparation is oral systemic preparations.
The term "effective arnount" should be understood as meaning a dose of the
drug
effective in exerting a therapeutic effect. For an oral preparation of the
invention, the
term "effective amount" means a dose of the drug which after its absorption
into the
body through the walls of a GI tract, yield a drug concentration in the blood
which is
effective in exerting a therapeutic effect on a target organ.
The invention also provides a method for administration of a hydrophobic drug,
to
locations in the body, e.g GI tract, into the blood, etc..., where the
preparation comes
into contact with body fluids, comprising administering the drug in an oily
vehicle
being the above SEOF.
Another use of the SEOF of the invention is in a process for the production of
emulsions, particularly such having droplets in the submicron range (submicron
emulsions). A specific embodiment in the preparation of emulsions comprising a
hydrophobic drug and intended to be used as pharmaceutical preparations. Thus,
the present invention provides a process for the production of an emulsion,
comprising mixing said SEOF with an aqueous solution and agitating the
mixture.
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Where the emulsion is to be used as a carrier of hydrophobic drugs, the SEOF
will
have said drug dissolved therein.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the invention, a novel self-emulsifying oily formulation
has been
prepared. This SEOF may be used in pharmaceutical preparations as a carrier of
hydrophobic drugs or may be used as a precursor for the preparation of
emulsions,
particularly submicron emulsions.
The SEOF of the invention comprises a surfactant and an oil component
comprising
a cationic lipid and at least one of MCT, LCT and an oily fatty acid
derivative. The oil
component comprises an oily fatty alcohol as an optional ingredient. In
addition, the
oil component may also comprise other ingredients as will be specified below.
Obviously, all ingredients used should be physiologically compatible.
MCT oil is a triglyceride oil in which the carbohydrate chain has an average
of about
8-12 carbon atoms. Examples of MCT oils are TCMTM (Societe des Oleagineux,
France) which is a mixture of triglycerides wherein about 95% of the fatty
acid chains
have either 8 or 10 carbon atoms; or MYGLYOL 812Tm (Dynamit Nobel, Sweden)
which is a mixture of triesters of glycerine and of caprylic and capric acid.
LCT oil is a triglyceride oil in which the carbohydrate chain has an average
chain
length above 12 carbon atoms. Examples of LCT oils which can be used in
accordance with the present invention are arachis oil, safflower oil, sesame
oil,
soybean oil, cotton seed oil, olive oil.
Oily fatty acid derivatives may be various lipophilic substituted fatty acids,
e.g esters
with alkyl alcohols, examples being methyl or ethyl esters of fatty acids,
such as
ethyl oleate.
Cationic lipids are lipids which have a positively charged polar group.
Examples of
cationic lipids are C-10-C24 fatty alkylamines and C12-C24 fatty
alkanoylamines,
C12-C18 fatty alkylamines and C12-C18 fatty alkanoylamines being preferred.
Specific examples of cationic lipids are stearylamine and oleylamine. In
addition to
cationic lipids, the oil component may also comprise cationic surfactants such
as
cationic cholesterol ester and cationic cholesterol derivatives, e.g
cholesterol
betainate.
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Oily fatty alcohols include various lipophilic alcohols such as fatty acid
alcohols, e.g,
oleyl alcohol, or aryl alcohols, e.g, benzyl alcohol, ethyl alcohol or any
other
lipophilic non-toxic alcohol.
In addition to the above ingredients, said oil component may also comprise
various
other oily ingredients including other types of vegetable oil, mineral oil and
isopropyl
myristate. In addition, the oil component may also comprise neutral or anionic
lipids,
the amount of the anionic lipids being such so as not to fully eliminate the
positive
charge of the emulsion droplets.
The surfactant used in the SEOF of the invention may be any of those known per
se.
The surfactant is preferably a non-ionic surfactant and may, for example, be
Tween,
e.g Tween 80 and Tween 85; Span, e.g Span 80; or a glycosylated
polyoxyethylenized glyceride, e.g LABRAFIL M 1944 CSTM (Gattefosse Corp, USA).
Other examples of non-ionic surfactants are esters of sorbitol and fatty acids
such as
sorbitan monoleate, and oily sucrose esters such as sucrose mono-, di-, or tri-
paimitate. In addition to non-ionic surfactants also ionic surfactants such as
phospholipids may be used, the amount of such ionic surfactants should be such
so
as not to eliminate the positive charge of the emulsion droplets. At times it
is useful
to use a combination of different surfactants such as a combination of Tween
80 and
Span 80 or Tween 85 and Span 80.
Typical pharmaceutical application of the SEOF of the invention is in oral
systemic
administration of hydrophobic drugs. For this administration the SEOF with the
drug
dissolved therein will preferably be encapsulated in sealed soft or hard
gelatin
capsule. The capsule is typically of a kind which is dissolved in a particular
region of
the GI tract releasing its content there. An example of such a capsule is an
entero-
coated soft or hard gelatin capsule. Enteric coating, as known per se, is a
coating
with a substance or a combination of substances that resists dissolution in
gastric
fluid but disintegrates in the intestine. Examples of enteric coatings are
hydroxypropylmethylcellulosephthalate B.P. As a result of the enteric coating,
the
capsule is resistant to dissolution in upper parts of the digestive tract and
thus
dissolves only in the intestine, e.g as a result of contact with bile acids
and salts in
the jejunum. Emulsion droplets particularly such comprising LCT, e.g soybean
oil,
are likely absorbed in the jejunum via the lymphatic system in chylomicrons
which
are carried away from the small intestine through the thoracic duct, thus
bypassing
the liver. Such an absorption route thus significantly reduces the first pass
effect of
drug degradation in the liver.
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Commercially available capsules range in size up to such having an internal
volume
of about 0.7 ml. From experiments carried out in accordance with the present
invention it became evident that this volume is sufficient for the delivery of
an
effective amount of drug, such as physostigmine, so as to achieve a systemic
effect.
For pharmaceutical use of the SEOF of the invention preservatives such a
methyl
paraben, propyl paraben, butyl paraben or a combination of these, as well as
anti-
oxidants such as propyl gallate, BHT or dimercaprol can be used. Furthermore,
prior
to use, the preparation is preferably sterilized by filtration.
The invention will now be further illustrated in the following examples and
the
annexed drawing.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings :
Figure 1 : shows the mean droplet size as a function of the level of dilution
of the
SEOF in an aqueous phase. The SEOF consisted of 25% Tween 80, 12.5% Benzyl
alcohol, 2.5% Oleylamine and Ethyl oleate to -100%. The particulars of these
experiments are described in example IV.
Figure 2 : shows the results of a similar experiment to that shown in Figure
1, also
reported in example IV, with the difference being in that the Tween 80
concentration
was 35%.
Figure 3: shows the results of an experiment, described in example IV, in
which the
mean droplet size was tested over time in three different dilutions of the
SEOF in an
aqueous phase. The SEOF consisted of 25% Tween 80, 12% Benzyl alcohol, 2.5%
Oleylamine and Ethyl oleate -100%. The pH level of the aqueous phase was 5Ø
Figure 4: shows the results of a similar experiment to that of Figure 3, also
described in example IV, with the difference being in that the pH level of the
aqueous phase was 2.5. (adjusted with phtala buffer)
Figure 5 : shows the results of an experiment in which the inhibition of
cholinesterase versus time following a PO administration of three different
formulations of physostigmine.
Figure 6 : shows Progesterone serum concentrations, following administration
of
positive and negative SEOF, containing the drug, as well as Exp.Progesterone
suspension in water and blank formulation.
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EXPERIMENTAL METHODS
(i) Particle Size Evaluation :
Particle droplet size of disintegrating SEOF was determined using a super
nanosizer
apparatus (Coulter'"" N4) in which the particle sizes are evaluated by a
photon
correlation spectroscopy. Prior to evaluation, the preparations were diluted
using
filtered and sonicated 2.25% glycerin solution.
(ii) Emulsion Charge :
The charge on the emulsion was determined by electrophoretic mobility. A
standard
negatively charged fat emulsion, Intralipid(R) (Kabi-Vitrum, Sweden) immersed
in
2.25% glycerin solution moves in the direction of the positive electrode clue
to the
negative charge of the dispersed oil droplets. The various emulsified oil
formulations
(in gastric or intestinal fluid, USP) migrated in the opposite direction
towards the
negative electrode validating the positive charge on the oil droplets.
(iii) Zeta-Potential :
The zeta-potential was measured using the moving boundary electrophoresis
technique. The zeta-potential value of the spontaneously formed oil droplets
was
determined using a ZetasizerTM (DELSA 440 Coulter) using a 2.250/00 glycerin
solution as diluent.
(iv) Visual Observations :
The degree of stabiiity of the SEOF formulation and the degree of the phase
separation in the emulsion derived from such formulations was assessed
visually at
given time intervals. Any visible change'was recorded.
EXAMPLE I
SEOF Preparation
The SOEFs were prepared by the following steps :
1. The fatty alcohol and most of the oily carrier were mixed in a flask
2. A surfactant was then added and solubilized by stirring
3. The cationic lipid was then added and dissolved in the above mixture. In
the case
of stearylamine, a light heating to about 37 C was required in order to
solubilize
this ingredient.
4. The final weight of the composition was completed by the addition of the
remaining oily carrier.
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5. = The drug may be dissolved either in fatty alcohol prior its addition to
the mixture
or added after incorporation of other ingredients of the formulation before
completing to the final weight with the oily carrier.
6. Only in examples VI - X drug was included. Examples II - V contained no
drug.
For emulsion formation the prepared SEOF was mixed with double distilled water
or
simulated USP gastric and intestinal fluids.
EXAMPLE II
SEOFs which disintegrate into oil droplets having a diameter less than 250 pm
A range of SEOFs was prepared from the following ingredients : Span, Tween 85,
Stearylamine and arachis oil. The following Table lists these four ingredients
showing the tested concentration range and the preferred concentration range
and a
typical concentration for each ingredient which yields a minimal particle
size.
Table I
Preferred Conc. Conc. Range Typical Conc.
I n redient (%, w/w (%, w/w (%, w/w
Span 80 25 - 30 15 - 40 25
Tween 85 4-5 1- 10 3
Stearylamine 2.5 - 3 1-3 3
Arachis oil to 100
Each of the formulation was transparent in liquid at 37 C and became
opalescent
and viscous at room temperature. The emulsions which were formed from these
formulations, remained stable at room temperature for a few hours. The
particle size
of the oil droplets in the emulsions following gentle agitation ranged from 50
to
250 pm.
In addition to arachis oil, other LCT oils including safflower oil, sesame
oil, soybean
oil, cotton seed oil and olive oil were tested. Emulsions within the above
range (50-
250 pm) were achieved with all these LCT oils. Optimal emulsions, having
droplets
at the lower end of the above range were obtained with arachis and safflower
oils.
In addition, also MCT oil was tested in place of arachis oil and showed to
yield also
droplets of a size within the same range.
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EXAMPLE III
SEOFs which disintegrate into-submicron oil droplets
SEOFs were prepared from the following ingredients : Tween 80, Span 80, oleyl
alcohol, oleyl amine and ethyl oleate. The ingredients of a typical
formulation, the
tested concentration range for each ingredient, the preferred concentration
range
and a typical concentration yielding droplets of minimal size are shown in the
following Table II.
Table It
Preferred Conc. Conc. Range Typical Conc.
I n red i ent (%, w/w (%, w/w (%, w/w
Tween 80 25 - 30 15 - 40 25
Span 80 1.5-2 0.5-3 1.5
Oleyl alcohol 5-8 0- 10 7.5
Oleyl amine 2- 2.5 1-3 2.5
Ethyl oleate to 100 771
The droplet size is of the emulsions which were formed from these SEOFs ranged
in
diameter between 160 to 200 nm with a standard deviation of 60-70 nm. The oily
droplets were positively charged.
The emulsions were all stable at room temperature for several weeks.
The concentration of oleyl alcohol in the lipid solution was found to affect
the final
droplet size. The results are shown in the following Table Ill.
Table Ili
Oleyl alcohol conc. (%, w/w) Droplet diameter (nm)
160 60
200 + 63
12.5 650 + 900
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When Tween 85 was used instead of Tween 80, an increase in concentration of
oleylamine up to 7-8% was needed to achieve a positive emulsion. The final
particle
size of the resulting dispersed droplets in this case ranged from 1 to 2 pm.
Ethyl oleate was the only solvent of those tested which was able to produce
emulsions iri the submicron range.
EXEMPLE IV
SEOFs which disintegrate into oil droplets ranging from 20 to 200 nm
SEOFs were prepared from the following ingredients : Tween 80, benzyl alcohol,
oleyl amine and ethyl oleate. The ingredients in the tested concentration
range of
each are shown in the following Table IV.
Table IV
Ingredients Conc. Ran e%, w/w)
Tween 80 25 - 40
Benz I alcohol 0.5 - 50
Oleyl amine 2.5
Ethyl oleate to 100
A formulation of which contained less than 0.5% benzyl alcohol was not stable.
When more than 50% benzyl alcohol was included, no stable submicron emulsion
was achieved.
The following Table V shows the particle size as a function of Tween 80 and
Benzyl
alcohol concentration (under constant dilution with the aqueous phase of 1:1).
Table V
Tween 80 (%, w/w) 25 25 25 25 35 40
Benz. Alc. (%, w/w) 5- 12 25 37.5 50 45 40
Particle size nm 191 60 175 63 110 38 86 30 37 14 < 12
As can be seen, the smallest droplet diameter was achieved with a 40% Tween 80
and a 40% Benzyl alcohol formulation.
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Figure 1 shows results obtained with a SEOF which consisted of 25% Tween 80,
12.5% benzyl alcohol, 2.5% oleylamine and ethyl oleate added to a total of
100%.
As can be seen in Figure 1, the particle size decreased with increased aqueous
phase dilution until dilution of about 1:500. Further dilution to 1:1000
brought to a
slight increase in the droplet size.
As can be seen in Figure 2, the same type of behavior was also seen with a
different
SEOF comprising 35% Tween 80 instead of 25% as in the SEOF of Figure 1.
Figure 1, Figure 2 and table VI demonstrate, that effective self-
emulsification does
not show high pH dependency, but some tendency of droplet size enlarging as a
function of pH increase was observed.
Influence of pH on mean droplet size as a function of pH and time.
Table VI
pH dilution 0 day 1 day 7 days 34 days
2.5 1/500 105 36 113 25 120 33 137 45
1/1000 125+45 127 37 134 42 157 52
5.0 1/500 112 34 120 36 125 36 136 43
1/1000 130+46 139+46 150+44 157+52
7.4 1/500 127+44 137+44 145+44 198+b ro a d
1/1000 137 48 146 52 151 56 188 broad
Reference is made to Figures 3 and 4 showing results from an experiment in
which
the stability of emulsions, as reflected by their mean droplet size, was
tested over
time. The SEOF in both cases consisted of 25% Tween 80, 12.5% benzyl alcohol
(BA), 2.5% oleylamine (OA) and ethyl oleate (EO) added to 100%. In the
experiment
shown in Figure 3, the pH was 5.0 whereas the pH in the experiment of Figure 4
was
2.5. As can be seen, the emulsions which were formed were stable for over 30
days
under the tested conditions. It should be noted that under a dilution of less
than
1:100, the emulsions were stable for only about one week. Furthermore, under a
pH
of the aqueous phase of above 7.4, the emulsions were also stable for a period
of
only one week.
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EXEMPLE V
Zeta-potential of the c9roplets, produced from SEOF
The quantitative Zeta-potential measurements have been composed with
emulsions,
prepared from SEOF, that were described in example 3. The emulsions comprised
different amounts of Oleylamine (0.5%). Table VII shows zeta-potentials of
these
emulsions, the emulsions resulting from the dilution of the SEOF (1:10)
comprised
different amounts of oleylamine (0-0,5%), measured in 2.25% glycerin solution.
As
can be seen, SEOF, that included Oleylamine were capable of producing
droplets,
bearing a positive charge.
Table VII
Zeta potential measurements of
emulsions produced by dilution of
SEDDS with 2.25% glycerin solution
Formulation Zeta-potential value at 25 C
(%, w/w m V
Tween 80 25
BA 12.5 -8.4
EO to 100
Tween 80 25
BA 12.5 + 15.8
OA 2.5
EO to 100
Tween 80 25
BA 12.5 + 33.3
OA
EO to 100
EXAMPLE 1/1
SEOFs with the Drugs
Physostigmine, cyclosporin A, probucol and insulin (insulin was finely
dispersed in
the oil formulation by sonication for 5 min using a bath sonicator) were
incorporated
into SEOFs and the formulations are shown in the following Table VIII
CA 02215800 1997-09-18
Table VIII
Ingredients Conc. %, w/w)
1 Physostigmine Salicylate 0.6% 25 %
Span 80 25 %
Tween 85 5 %
Stearylamine 3 %
Arachis oil to 100 %
2 Cyclosporine A 5 %
Tween 80 25 %
Span 80 1.5 %
Oleyl alcohol 7.5 %
Oleyl amine 2.5 %
Ethyl oleate to 100 %
3 Probucol 4%
Tween 80 25 %
Benzyl alcohol 50 %
Oleyl amine 2.5 %
Ethyl oieate to 100 %
4 Insulin 0.5 %
Tween 80 25 %
Benzyl alcohol 12.5 %
Oleyl amine 2 5 %
Ethyl oleate to 100 %
5 Progesterone 10%
Tween 80 25 %
Benzyl alcohol 30 %
Oleyl amine 2.5 %
Eth I oleate to 100 %
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EXAMPLE Wii
Inhibition of Cholinesterase by Orally Administered Physostigmine in rats
The pharmacological effect of orally administered physostigmine (PS) on rats
was
tested by a modification of a method previously reported by Steffens (Steffens
A.B
1969, Physiology and Behavior, 4, 833-836). Male Sabra rats were cannulated
chronically through the jugular vein one day prior to experimentation allowing
frequent blood sampling from a non-anaesthetized rat. Each group of awake rats
(3-
4 animals in each group) were administered per os (PO) with one of three PS
formulations. All formulations were administered at a dose of PS of 0.5 mg/kg.
0.1 mi blood samples were taken at specific time intervals up to five hours
following
the drug administration and analyzed for cholinesterase activity according to
the
radiometric assay of Johnson & Russel (Johnson C.D and Russel R.L, 1975, Anal.
Biochem, 64, 229-238).
The tested formulations were the following :
Formulation 1 :
9 mg P.S, 8.5 gm soybean oil, 8.5 gm oleic acid, 1.0 gm labrafil M1944Cs, 0.02
gm
isopropyl gallate, 0,02 gm PHT, 0.02 gm dimercaptol and 0.02 gm butyl paraben.
Formulation 2 :
mg P.S, 1 ml Ethanol (95%), 1.0 gm Lipoid-E80, 0.02 gm a-Tocopherol, 0.21 gm
stearylamine and 8.0 gm soybean oil.
Formulation 3 :
6 mg P.S, 3 mg Span 80, 0.6 gm Tween 85, 0.36 gm Stearylamine and Arachis oil
to
12 gm.
The results of cholinesterase inhibition with each of the formulations are
shown in
figure 5. As can be seen, P.S dissolved in soybean oil without the addition of
a
cationic lipid (Formulation 1) was able to inhibit about 20% of the
cholinesterase
activity but the inhibition was not maintained for long periods of time. P.S
dissolved
in soybean oil formulation containing the cationic lipid continued to maintain
above
20% inhibition of cholinesterase activity even three hours after
administration of the
drug. The most pronounced inhibition, of above 20% for over more than five
hours
was achieved with the use of Arachis oil as the oily carrier in combination
with the
cationic lipid.
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17
EXAMPLE VIII
Effect of orally administered insulin on glucose level in diabetic rats
Experimental methods :
Sabra male rats weighing 200 g were injected i.p with streptozocin at a dosage
of
100 mg/kg, prepared by dissolving streptozocin in saline. The pH of the saline
was
adjusted to 4,4 with 0.1 N citric acid solution.
Streptozocin affects the glucose metabolism, and at this dose depletes
endogenous
insulin rendering the rats diabetic. The diabetogenic effect was assessed 24
hours
after injection, by monitoring glucose blood levels using Reagent Strips test
: a drop
of rat blood from the tail was applied on the GlucostixTM (Miles Ltd, Ames
Division,
TM
Slough, England) test pads. The glucose level was determined using Glucometer
fl
(Miles Lab, Ames Division, Elkhart, In USA) according to manufacturer
instructions.
It should be emphasized that the maximum limit of the glucometer is 400 mg/dl.
Rats were anesthetized with ether and the abdominal area was shaven. Using a
scalpel, a small opening was made in the abdomenal muscles and peritoneum, and
the jejunal area of the intestines was exposed and wetted with saline.
The various lipid formulations were first diluted with double distilled water
(1:5),
gently mixed and immediately injected into the jejunum (1 ml of the resulting
final
emulsion containing 1 mg of insulin). The preparations were diluted prior to
administration in view of the lack of fluids in the rat. The peritoneum
muscles and
skin were then sutured. Time '0' was taken before drug administration. The
remainder of the experiment was carried out while animals were awake and blood
was withdrawn from the rat tail at given time intervals.
The tested formulations were the following :
Positive formulation : the SEOF insulin formulation shown in Table VI (n 4),
which
yielded positively charged emulsion droplets.
Negative formulation : the same formulation as the positive formulation
without the
oleylamine, which yielded negatively charged emulsion droplets.
Blank formulation : a formulation similar to the positive formulation without
insulin.
Insulin aqueous suspension : a suspension of insulin in saline
Marketed insulin preparation : insulin preparation marketed by Novo
CA 02215800 1997-09-18
18
The results are shown in the following Table IX
Table IX
The effect of the various lipid formulations on blood
glucose levels following intra-jejunal administration
Giucose blood levels, mg/dl (% reduction*1
Marketed regular
Novo -
Time (min) Positive Blank Insulin aqueous Insulin 8 Units
formulation emulsion suspension i.p injection
N=6 N=3 N=4 N=1
0 > 400 > 400 > 400 > 400
15 253(37 %
45 325 18 % 165 59 %
60 260 29 % 390 2.5 % 164 59 %
90 246(31 % 392 2% 312 13 %
120 237 30 % 374 6.5 % 339 10 % 105 74 %
150 375 6 %
180 255 25 % 365 9% 332 12 % 109 73 %
240 350 10 % 396 1% 387 5% 100 75 %
(") the percentage of blood glucose levels were calculated on the basis of 400
mg/dl
at time 0, even is actual levels were higher.
It can be noted from the results shown in Table IX, that the aqueous
dispersion of
insulin was not significantly active although some absorption did occur as a
result of
the huge administered dose. It should be noted that the marketed insulin
solution,
when injected i.p to the diabetic rats (8 units/rat) markedly reduced the
blood
glucose level showing the validity of the use of these rats as a comparative
model.
The blank lipid formulation which comprises all the excipients except insulin
did not
induce any hypoglycemic effect, as expected. The positive formulation which
had
positively charged emulsion droplets elicted a much more effective
hypoglycemic
effect that those of the aqueous insulin suspension.
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19
EXAMPLE IX
Serum progesterone concentrations, following administration of various
formulations.
Progesterone is the endogenic lipophilic compound that is not administered
orally
because of significant first pass effect. SEOF ability of improving its oral
bioavailability was tested in the present study.
Four groups of sabra female rats at the age of 24-28 days (6 rats in a group)
received 3 formulations, containing Progesterone and the blank formulation.
Every
animal was feeded with 0.5 ml of the appropriate formulation, which contained
either
no drug or 4 mg of Progesterone per kg weight.
The tested formulations were following :
Blank formulation : SEOF, described in example VI (n 5), diluted 1:50 with
double
distilled water (DDW), without the drug.
Suspensioin : Progesterone, suspended in DDW
Negative formulation : SEOF, described in example VI (n 5), diluted 1:50 with
DDW, without Oleyl amine
Positive formulation : SEOF, described in example V! (n 5), diluted 1:50 with
DDW.
Progesterone concentrations were determined by RIA, using
ImmuChemTM Coated tube kit, ICN Biomedicals, Inc, Costa Miesa, California, USA
The results are shown in Figure 6. The pharmacokinetic parameters of
Progesterone, obtained following its administration in various formulations,
as well
as relative bioavailability of the SEOF's in comparison to suspension are
presented
in Table X
CA 02215800 1997-09-18
Table X
Pharmacokinetic data of ro esterone formulations
Suspension Ne ative emulsion Positive emulsion
tmax hr 4 2 1
Cmax n/ml 29 + 12 47 + 23 62 + 21 *
AUC** n.hr/ml 69 + 34 99 + 58 131 + 47*
F*** relative
bioavailability 1.43 1.88
(*) Statistically significant, p<0.05, compared to suspension formulation,
according to
Mann-Whitney test.
(**) AUC was calculated using the level of 13 ng/ml as "0" level
(**~) AUC emulsion (test)
F' = -------------------------------------
AUC suspension (reference)
EXEMPLE :iC
Acute toxicity of SEOF in BALB/c mice
Acute toxicity of the formulations described in examples 2 - 4, was examined
in
BALB/c mice during 30 days. 0.5 ml of each SEOF, diluted 1:10 (more than 300-
fold
overdosage) and 1:100 (more than 30-fold overdosage) were injected i.p to the
group of 4-6 mice. Animal's condition was inspected during several hours after
the
procedure and then every day up to month.
Mice that received both dilutions of SEOF, described in example II, and
dilution
1:100 of formulations, described in example IV, (containing 12.5% and 30% of
BA)
showed no sign of intoxication. Dilution 1:10 of SEOF comprising 12.5% BA also
did
not cause any toxic effects. On the other hand, dilution 1:10 of the
formulation,
containing 30% of Benzyl alcohol was found to induce immediate neurologic
shock
with further partial improvement and death of the animals after several days.
A half
dose of the same formulation still caused neurotoxic symptoms, but mice stayed
alive.
As can be seen from table Xi, the same toxic effects were observed also after
injection of 1.5% and 3% BA water solutions, confirming its role in
formulation's 300 -
fold overdose toxicity.
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Table Xi
Acute toxicity of emulsified SEDDS
in BALB/c mice followin i. injection
SEDDS dilution Number of % survival after
Formulation rate in water animals per 30 days Side effects
(%, w/w) for group
injection (vol*)
Tw.80 25 1:10 (0.5) 6 84 none
BA 12.5
OA 3 1:100 (0.5) 6 100 none
EO to 100
Tw.80 25 1:10 (0.5) 6 0 neurologic shock
BA 30
OA 3 1:100 (0.5) 6 100 none
EO to 100 1:10 (0.25) 4 100 neurologic shock
BA 3 1:10 (0.5) 4 15 neurologic shock
in water for
injection
BA 1.5 1:10 (0.5) 6 100 neurologic shock
in water for
injection
(*) Vol : volume of the resulting emulsion injected i.p
(**) Dilution 1:10 : a dose 300 times larger than the expected administered
dose
