Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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WO 98/47487 PCTlDK98/00159
A NOVEL BIOADHESIVE DRUG DELIVERY SYSTEM BASED ON LIQUID CRYSTALS
The present invention relates to a drug delivery system containing a liquid
crystalline phase
such as a cubic, a hexagonal, a reverse hexagonal, a lamellar, a micellar and
a reverse micellar
liquid crystalline phase. The compositions are unique in that they as delivery
system contains A)
a substance which is capable of generating a liquid crystalline phase and
providing suitable
biopharmaceutical properties like e.g. suitable release of the active
substance and bioadhesive
properties, and B) at least another substance which without having any
substantially negative
effect on the biopharmaceutical properties provided by the substance mentioned
above under A)
either takes part in the formation of a liquid crystalline phase or dilutes
the proportion of liquid
crystalline phase in the composition while still maintaining suitable
biopharmaceutical properties
and a suitable storage stability.
The present invention also relates to a pharmaceutical composition for
administration of an
active substance to or through a damaged or undamaged skin or mucosal surface
or to the oral
cavity including the teeth of an animal such as a human. The composition is
particularly suited
for administration of substances which have a very low water solubility and
which aye to be
supplied in an effective amount in a localized region over a period of time.
The present invention also relates to a pharmaceutical composition for
administration of an
active substance to a mammal such as a human. The composition is unique in
that it contains i)
a substance which together with a liquid medium such as e.g. water is capable
of forming a
liquid crystalline phase at room temperature and ii) a so-called structurant
which is capable of
participating in the formation of the liquid crystalline phase at room
temperature. Such
compositions are especially useful when it is desired to reduce the
concentration of the substance
mentioned under i) above in the composition without any significant or
negative influence on
the biopharmaceutical properties of the composition.
Furthermore, the invention also relates to a pharmaceutical composition in
which it is possible
to incorporate relatively large amount of certain pharmaceutically acceptable
excipients without
significantly changing the biopharmaceutical properties of the composition.
Background of the invention
From WO 95/26715 it is known that certain fatty acid esters have bioadhesive
properties which
are suitable for use in pharmaceutical compositions. Furthermore, WO 97/13528
(published on
1? April 1997 corresponding to the priority date of the present application)
discloses
pharmaceutical compositions containing active drug substances having a
relatively low solubility
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in water at a pH of between 3.0 and 9.5 such as, e.g., between 3.2 and 9.3,
between 3.4 and 9.1
or between 3.6 and 9 and having a relatively high release rate of the active
drug substance for a
suitable and sufficiently long period of time.
The pharmaceutical compositions disclosed in the above-mentioned international
patent
application are all based on a content of a fatty acid ester which is
sufficient to enable a
formation of a liquid crystalline phase either in the composition itself or in
situ after application
of the composition in the form of a so-called precursor composition.
For various reasons (such as, e.g., i) formulation considerations such as,
e.g., viscosity, dosage
form aspects etc., ii) patient acceptability considerations such as, e.g.,
appearance, taste,
tolerability etc.) it may be desirable to reduce the amount of fatty acid
ester present in the
pharmaceutical compositions. However, the present inventor has found that a
reduction in the
content of a fatty acid ester, i.e. a substance which is capable of generating
a liquid crystalline
phase together with a suitable liquid medium, is not a simple routine for a
person skilled in the
art if the biopharmaceutical properties of the compositions (such as, e.g.,
release properties,
bioadhesive properties and appropriate storage stability properties) still
have to be maintained.
In general, especially stability properties have been observed if such
excipients which normally is
used within the pharmaceutical field have been added to the liquid crystalline
phase.
Notably, in the case where an aqueous medium serves as a liquid medium with
which the fatty
acid ester forms the liquid crystalline phase the inventor has, however,
observed difficulties in
reducing the concentration of fatty acid ester in the composition below a
certain limit (for
glycerylmonooleate. The limit has been found to be about 60-65% by weight in
non-precursor
compositions) without getting a two phase system, e.g. a cubic phase in excess
water.
Disclosure of the invention
In searching for substances which may at least partly function as a suitable
substitute for a
liquid crystalline phase-forming substance like e.g. a fatty acid ester in
such a composition, the
inventor has now surprisingly found that substances which not necessarily are
able to form a
liquid crystalline phase together with water at room temperature but which
together with a
fatty acid ester and a liquid medium is capable of forming a liquid
crystalline phase are capable
of substituting the fatty acid ester and still retaining the required
properties with respect to e.g.
release of active substance, bioadhesion, solubility of active substance in
the fatty acid ester
component etc. Such substances are in the following denoted "structurants" due
to the
observation that such substances most likely together with the liquid
crystalline phase-forming
substance like e.g. the fatty acid ester participate in the formation of the
liquid crystalline phase
structure and, thus, impart a liquid crystalline phase structure to the
composition. A structurant
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is, accordingly, not a substance which merely is enveloped or enclosed in the
composition and
which, accordingly, would result in a dilution of the crystalline liquid
medium which_in turn
may result in a composition having a reduced bioadhesiveness compared with the
parent
composition where no substitution of the liquid crystalline phase-forming
substance content has
taken place. A structurant is capable of ensuring that a substitution of a
certain amount of the
fatty acid ester does not result in a deterioration of the content and
pharmaceutical function of
the~liquid crystalline phase formed by e.g. a fatty acid ester and a liquid
medium.
Thus, the invention relates to a pharmaceutical composition for administration
of an active
substance to or through a nail or a damaged or undamaged skin or mucosal
surface of a
mammal (such as an animal or a human), the composition comprising
i) a first substance which is the active substance,
ii) an effective amount of a second substance which, together with a liquid
medium, is capable of
generating a liquid crystalline phase in which the constituents of the
composition are enclosed,
the liquid crystalline phase being selected from the group consisting of: a
cubic, a hexagonal, a
reverse hexagonal, a lamellar, a micellar and a reverse micellar liquid
crystalline phase,
iii) a structurant which together with said second substance and a liquid
medium is capable of
forming a liquid crystalline phase selected from the group consisting of: a
cubic, a hexagonal, a
reverse hexagonal, a lamellar, a micellar and a reverse micellar liquid
crystalline phase; and
iv) optionally, a liquid medium which is substantially homogeneously
distributed in the
composition,
the composition either being one in which the liquid crystalline phase has
been generated by the
second substance and the structurant together with a sufficient amount of a
liquid medium
originally present in the composition, or the composition being in a precursor
form in which the
second substance and the structurant have not generated the liquid crystalline
phase, but are
capable of forming the liquid crystalline phase in situ with moisture from the
surface on which
the composition is applied, the moisture in this case constituting at least
part of the liquid
medium,
the pH of the liquid crystalline phase being in the range of 3.0-9.5 such as,
e.g., 3.2-9.3, 3.4-9.1
or 3.6-9.0, determined as described herein,
the active substance having
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i) a first solubility in the liquid crystalline phase of at the most 20 mg/g
at 20°C, and
ii) a second solubility in water of at the most 10 mg/ml at 20°C, the
water, where applicable,
being buffered to a pH in a range of 3.0-9.5 such as, e.g., 3.2-9.3, 3.4-9.1
or 3.6-9.
International patent application No. PCT/DK96/00437, published on 17 April
1997, discloses
a composition containing 5% by weight of acyclovir and 95% by weight of a
glycerylmonooleate/water/lecithin (55/35/10% w/w) formulation, wherein the
glycerylmonooleate product is DIMODAN~ GMO-90 and the lecithin is Epikuron
200, and a
composition containing 5% by weight of acyclovir and 95% by weight of a
glycerylmonooleate/water/d-a-tocopherylpolyethyleneglycol 1000 succinate
(65/35% w/w
glycerylmonooleate/water plus 5% w/w d-a-tocopherylpolyethyleneglycol 1000
succinate),
wherein the glycerylmonooleate product is DIMODAN~ GMO-90.
Therefore, for states in which the present application is co-pending with a
national phase of the
above international patent application (this is expressed in the claims as
"where applicable"), the
following proviso applies to the scope of the present application: the
composition is not one
consisting of either a) 5% by weight of acyclovir and 95% by weight of a
glycerylmonooleate/water/lecithin (55/35/10% w/w) formulation, wherein the
glycerylmonooleate product is DIMODAN~ GMO-90 and the lecithin is Epikuron
200, or b) 5%
by weight of acyclovir and 95% by weight of a glycerylmonooleate/water/d-a-
tocopherylpolyethyleneglycol 1000 succinate (65/35% w/w
glycerylmonooleate/water plus 5%
w/w d-a-tocopherylpolyethyleneglycol 1000 succinate), wherein the
glycerylmonooleate product is
DIMODAN~ GMO-90.
However, the present invention is not limited to pharmaceutical compositions
containing drug
substances having a relatively low solubility in water at a pH of between
about 3.0 and 9.5 such
as, e.g. between about 3.6 and 9. For structurants which fulfil a number of
requirements, c~
below, the present inventor has found it possible also to obtain
pharmaceutical compositions
having suitable biopharmaceutical properties as well as a suitable storage
stability, i.e. the
compositions do not separate into at least two distinct phases within a well-
defined period of
time and under well-defined environmental conditions.
Thus, in another aspect, the invention relates to a pharmaceutical composition
for
administration of an active substance to a mammal, the composition comprising
i) a first substance, which is the active substance,
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ii) a second substance which together with a liquid medium is capable of
forming a liquid
crystalline phase at room temperature, the liquid crystalline phase being
selected from the group
consisting of: a cubic, a hexagonal, a reverse hexagonal, a lamellar, a
micellar and a reverse
micellar liquid crystalline phase,
5 iii) a structurant
which - together with said second substance and water - at room temperature is
capable of
forming a liquid crystalline phase selected from the group consisting of: a
cubic, a
hexagonal, a reverse hexagonal, a lamellar, a micellar and a reverse micellar
liquid
crystalline phase,
which in itself together with water can form a liquid crystalline phase
selected from the
group consisting of: a cubic, a hexagonal, a reverse hexagonal and a lamellar,
a micellar
and a reverse liquid crystalline phase,
which - in a two component system wherein the structurant is one of the
components and
water is the other - is not capable of forming a cubic liquid crystalline
phase at room
temperature, and
which has a solubility in said second substance of at least 15°!o by
weight at 60°C; and
iv) optionally, a liquid medium which is substantially homogeneously
distributed in the
composition,
the composition either being one in which the liquid crystalline phase has
been generated by the
second substance in combination with the structurant and together with a
su~cient amount of
the liquid medium originally present in the composition, or the composition
being in a precursor
form in which the second substance and the structurant have not generated the
liquid
crystalline phase, but are capable of forming the liquid crystalline phase in
situ with moisture
from the site at or to which the composition is administered, the moisture in
this case
constituting at least part of the liquid medium, and
the composition being substantially homogeneous and having such a physical
stability that
substantial no irreversible phase separation into two or more distinct phases
can be observed
visually after storage of the composition at 25°C and 60% relative
humidity for one week.
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The present inventor has also found that the content of the substance which is
capable of
generating a liquid crystalline phase (i.e. the substance denoted "second
substance" which e.g. is
a fatty acid ester) can be reduced by incorporating at least 5% by weight of
certain
pharmaceutically acceptable excipients and such a reduction of the content of
e.g. fatty acid ester
does not significantly deteriorate or negatively influence the
biopharmaceutical properties of the
composition. The effect of such pharmaceutically acceptable excipients is most
likely a diluting
effect of the content of the liquid crystalline phase present in the
composition while no
significant diluting effect is observed with respect to the biopharmaceutical
properties of the
composition (e.g. release properties, bioadhesion, storage stability).
Accordingly, in a still further aspect, the invention relates to a
pharmaceutical composition for
administration of an active substance to a mammal, the composition comprising
i) a first substance which is the active substance,
ii) a second substance which together with a liquid medium - at room
temperature is capable of
forming a liquid crystalline phase selected from the group consisting of: a
cubic, a hexagonal, a
reverse hexagonal, a lamellar, a micellar and a reverse micellar liquid
crystalline phase,
iii) a pharmaceutically acceptable excipient in a concentration of at least 5%
by weight based on
the total composition, and
iv) optionally, a liquid medium which is substantially homogeneously
distributed in the
composition,
the composition either being one in which the liquid crystalline phase has
been generated by the
second substance together with a sufficient amount of the liquid medium
originally present in
the composition, or the composition being in a precursor form in which the
second substance
has not generated the liquid crystalline phase, but is capable of forming the
liquid crystalline
phase in situ with moisture from the site at or to which the composition is
administered, the
moisture in this case constituting at least part of the liquid medium,
the composition being substantially homogeneous and having such a physical
stability that
substantial no irreversible phase separation into two or more distinct phases
can be observed
visually after storage of the composition at 25°C and 60% relative
humidity for one week,
the composition containing at the most about 60% by weight of said second
substance.
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As it appears from the aspects of the invention mentioned above, a liquid
medium may
optionally be present. In those case where a liquid medium is not present, the
composition is in
the form of a so-called "precursor composition", i.e. a composition in which a
liquid crystalline
phase has not been generated but which upon application to or on the mammal is
capable of
generating a liquid crystalline phase by means of moisture or body fluid
present on the
application site. The compositions of the invention may also be presented in
precursor form even
if a liquid medium is included in the composition. In such case, either the
concentration of the
liquid medium is too low to enable a formation of the liquid crystalline phase
in the composition
or the liquid medium is of a type with which the second substance does not
form a liquid
crystalline phase. In these cases, the liquid crystalline phase is formed in
situ after application to
the mammal by means of the moisture or body fluid present on the application
site.
In general, the pharmaceutical compositions according to the invention are
intended for
administration to a mammal such as a human. The underlying formulation
principle of
compositions according to the invention is so generally applicable that
composition suitable for
almost any application site or administration route can be prepared by means
of methods well
known in the pharmaceutical practice. Preferably, the pharmaceutical
compositions according to
the invention are intended for application to or through undamaged or damaged
skin or mucosa
of an animal such as a human. The mucosa is preferably selected from oral,
buccal, nasal,
vaginal, rectal, aural, lung, and gastrointestinal mucosa. The skin or mucosa
may also be
inflamed. The composition may also be administered to body cavities such as
the oral cavity or
by the buccal route. Furthermore, the composition may be applied on or at a
tooth or a dental
pocket.
Furthermore, a pharmaceutical composition according to the invention may also
be applied to a
nail of an animal such as a human.
Liquid crystalline phase and suitable substances capable of forming liquid
crystalline
phases
As mentioned above an important property of a composition according to the
present invention
is its ability to generate a liquid crystalline phase. The term "liquid
crystalline phase" as used
herein is used to denote an intermediate state between solid crystals and
isotropic liquids,
characterized by long-range order and short-range properties close to those of
a simple liquid or
solution (Keller et al., Handbook of Liquid Crystals, Verlag Chemie, Weinheim,
Germany, 1980).
The main component in a composition according to the invention - which is
responsible for the
formation of a liquid crystalline phase - is the so-called "second substance".
As appears from the
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above, another component in a composition according to aspects of the
invention may also be
capable of forming a liguid crystalline phase. Such a substance is in the
present context denoted
a "structurant". A structurant takes part in the liquid crystalline phase
generated by the second
substance, but need not be able to generate a liquid crystalline phase in
compositions without
any second substance present. Furthermore, a structurant may generate a quite
different liquid
crystalline phase than the second substance under the same conditions and in
such a case, the
present inventor has observed that the liquid crystalline phase generated by a
composition of the
invention is the one of the second substance. Although further experiments
need to be carried
out in order to confirm that this observation is a general one, it is at
present contemplated that
i) the second substance may be responsible for what kind of liquid crystalline
phase a
composition will generate or ii) the second substance may together with the
structurant generate
a liquid crystalline phase of a different kind than expected taken the nature
of the second
substance into account. Many of the classes of substances and examples of
specific substances
which are suitable for use as second substances may of course also be suitable
for use as
structurants (and vice versa) provided that the specific requirements claimed
are fulfilled.
Examples of suitable substances with an excellent ability of forming a liquid
crystalline phase
are fatty acid esters like, e.g., glyceryl monoesters of fatty acids. Other
substances which have
ability of forming a liquid crystalline phase are found among amphiphilic
substances such as
polar lipids, surfactants and emulsifiers. Specific examples of glyceryl
monoesters of fatty acids
include glycerylmonooleate (monoolein) and glycerylmonolinoleate. Such fatty
acid esters are
capable of forming various crystalline phases upon contact with a hydrophilic
medium such as
water or glycerol. As will be explained in further detail below, these fatty
acid esters also show
so-called bioadhesive properties.
Liquid crystalline phases may be a cubic (three cubic liquid crystalline
phases are well-
characterised: i) the body-centred lattice, ii) the primitive diamond lattice,
and iii) the gyroid),
reverse cubic, hexagonal, reverse hexagonal, lamellar, miceilar or reverse
micellar phase. By the
term "cubic liquid crystalline phase" herein is meant a thermodynamically
stable, viscous and
optically isotropic phase made of a suitable substance such as, e.g., a fatty
acid ester and a liquid
medium such as, e.g., an aqueous medium. The cubic liquid crystalline phase is
contemplated to
be build up of closed reversed micelles. The term "aqueous medium" includes
media containing
water or another hydrophilic and water-miscible substance such as, e.g.,
glycerol. The terms
"hexagonal phase" and "reverse hexagonal phase", respectively, are used herein
to describe
thermodynamically stable, viscous and optically anisotropic phases
characterized by long-range
order in two dimensions and made of a suitable substance such as, e.g., a
fatty acid ester and a
liquid medium such as, e.g., an aqueous medium. The term "lamellar phase" is
characterised by
a Iong-range order in one dimension. The Iamellar structure is the origin of
liposomes having
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spherical shells of lipid bilayers. The various liquid crystalline phases can
be detected and
identified by use of polarized light or by means of X-ray diffraction pattern
analysis (see_the
Examples herein). The cubic liquid crystalline phase is normally the preferred
phase in the
compositions of the invention, but also, e.g., the reverse hexagonal and the
reverse cubic liquid
crystalline phase may be an interesting liquid crystalline phase in the
compositions according to
the invention, notably in compositions which are in precursor form.
In accordance with the above-mentioned observations, the so-called "second
substance" for use in
compositions according to the invention may be a fatty acid ester which is
capable of forming a
liquid crystalline phase on contact with a suitable liquid medium. The liquid
of the liquid
medium is suitably water, glycerol or an aqueous medium. An aqueous medium is
a medium
containing water at least in part.
Apart from aqueous solutions or dispersions, a medium with which the liquid
crystalline phase
is made may, especially for the precursor embodiment of the composition, at
least in part be
constituted by any body fluid or secretion which contains water and with which
upon application
the composition comes into contact, such as, e.g. in the case of a human body
fluid, saliva, sweat,
gastric juice, etc. As indicated above, the body fluid may induce formation of
a liquid crystalline
phase when a second substance such as a fatty acid ester is contacted with
such a liquid.
However, in many embodiments, a composition according to the invention will be
one in which
the liquid crystalline phase is already present, that is, the liquid
crystalline phase has already
been established by interaction between a liquid medium present in the
composition and the
second substance such as a fatty acid ester. In such cases, the liquid of the
liquid medium may
typically be, e.g., water or glycerol or a mixture thereof, water often being
a preferred liquid.
The fatty acid esters capable of generating a liquid crystalline phase as
evidenced by one of the
test methods described herein axe fatty acid esters (i.e. composed of a fatty
acid component and
a hydroxy-containing component) wherein the fatty acid component of the fatty
acid ester is a
saturated or unsaturated fatty acid having a total number of carbon atoms of
from C6 to C26.
Specific examples of saturated fatty acid moieties in the fatty acid esters
according to the
invention are selected from the group consisting of moieties of caproic acid,
caprylic acid, ca.pric
acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid,
and behenic acid.
Specific examples of unsaturated fatty acid moieties in the fatty acid esters
according to the
invention are moieties selected from the group consisting of palmitoleic acid,
oleic acid, linoleic
acid, linolenic acid, and arachidonic acid.
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Particularly suitable fatty acid esters for use in compositions according to
the invention are fatty
acid esters which are selected from the group consisting of fatty acid esters
of polyhydric
alcohols, fatty acid esters of hydroxycarboxylic acids, fatty acid esters of
monosaccharides, fatty
acid esters of glycerylphosphate derivatives, fatty acid esters of
glycerylsulfate derivatives, and
mixtures thereof. In those cases where the hydroxy-containing component of the
fatty acid ester
is polyvalent, the hydroxy-containing component may be partially or totally
esterified with a
fatty acid component or with mixtures of fatty acid components.
The polyhydric alcohol component of the fatty acid ester for use in
compositions according to the
invention is preferably selected from the group consisting of glycerol, 1,2-
propanediol, 1,3-
10 propanediol, diacylgalactosylglycerol, diacyldigalactosylglycerol,
erythritol, xylitol, adonitol,
arabitol, mannitol, and sorbitol. The fatty acid esters formed from such
polyhydric alcohols may
be mono- or polyvalent such as, e.g., divalent, trivalent, etc. In particular
fatty acid monoesters
have proved to have bioadhesive properties and are therefore preferred fatty
acid esters for use
in compositions according to the invention. The position of the polyvalent
alcohol on which the
I5 ester bonds) is(are) established may be any possible position. In those
cases where the fatty acid
ester is a diester, triester, etc. the fatty acid components of the fatty acid
ester may be the same
or different. In a most preferred aspect of the present invention, the
polyhydric alcohol
component is glycerol.
Examples of fatty acid esters for use in compositions according to the
invention and wherein the
hydrogy-containing component is a polyhydric alcohol are glycerylmonooleate,
glycerylmonolinoleate, glycerol monolinoleate, and mixtures thereof. These
fatty acid esters have
especially promising bioadhesive properties, confer the Examples herein.
In those cases where the fatty acid ester for use in compositions according to
the present
invention is formed between a hydroxycarboxylic acid (or a derivative thereof)
and a fatty acid
(or a derivative thereof), the hydroxycarboxylic acid component of the fatty
acid ester is
preferably selected from the group consisting of malic acid, tartaric acid,
citric acid, lactic acid,
and sorbic acid. An interesting example of a fatty acid ester for use in
compositions according to
the invention is a fatty acid monoester of citric acid.
As mentioned above, the hydroxy-containing component of a fatty acid ester for
use in
compositions according to the present invention may also be a saccharide, such
as a
monosaccharide such as, e.g., glucose, mannose, fructose, threose, gulose,
arabinose, ribose,
erythrose, lyxose, galactose, sorbose, altrose, tallose, idose, rhamnose, or
allose. In those cases
where the hydroxy-containing component is a monosaccharide, the fatty acid
ester is preferably
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11
a fatty acid monoester of a monosaccharide selected from the group consisting
of sorbose,
galactose, ribose, and rhamnose.
The hydroxy-containing component of a fatty acid ester for use in compositions
according to the
invention may also be a glycerylphosphate derivative such as, e.g., a
phospholipid selected from
the group consisting of phosphatidic acid, phosphatidylserine,
phosphatidylethanolamine,
phosphatidylcholine, phosphatidylglycerol, phosphatidylinositole, and
diphosphatidylglycerol.
Other interesting phospholipids are DEPE (1,2 dielaidoyl-sn-glycerol-3-
phosphoethanolamine)
and DMPE (PEG 550) (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-
(polyethylene
glycol)550).
Especially interesting compounds having a phospholipid moiety are compounds
wherein the fatty
acid ester is a fatty acid ester of a glycerylphosphate derivative, and the
fatty acid component is
selected from the group consisting of lauric acid, myristic acid, palmitic
acid, stearic acid, oleic
acid, linoleic acid, linolenic acid, and behenic acid. Examples of such useful
fatty acid esters are
dioleyol phosphatidylcholine, dilauryl phosphatidylcholine, dimyristyl
phosphatidylcholine,
dipaimitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dibehenoyl
phosphatidylcholine,
dimyristyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine,
dioleyl
phosphatidylglycerol, dilauryl phosphatidylglycerol, dimyristoyl
phosphatidylglycerol, dipalmitoyl
phosphatidylglycerol, distearoyl phosphatidylglycerol, dipalmitoyl phosphatic
acid and mixtures
thereof.
Most of the fatty acid esters for use in compositions according to the
invention are well-known
chemical compounds which are commercially available or may be prepared by
means of
conventional esterification procedures involving e.g. reaction of a fatty acid
derivative such as,
e.g., the corresponding acid chloride with a hydroxy-containing compound (if
necessary protected
with suitable protection groups) and subsequently isolating the fatty acid
ester, if necessary after
removal of any protecting group. Many of the commercially available fatty acid
esters are
employed in the food industry and in general, no steps are taken in order to
obtain an
approximately 100% pure fatty acid ester. As an example it can be mentioned
that
glycerylmonooleate from Danisco Ingredients A/S, Denmark is a very pure
product containing
about 98% w/w monoesters of which more than about 80% w/w (such as about 92%a
w/w) is
glycerylmonooleate; the remaining monoesters are glycerylmonolinoleate,
glyceryl monopalmitate
and glyceryl monostearate. The fatty acid ester products for use in
compositions according to the
invention may thus be mixtures of fatty acid esters.
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Examples of fatty acid esters with excellent bioadhesive properties as well as
an excellent ability
of forming a liquid crystalline phase are glyceryl monoesters of fatty acids.
Specific examples
include glycerylmonooleate (monoolein) and glycerylmonolinoleate. As mentioned
above, such
fatty acid esters are capable of forming various crystalline phases upon
contact with a
hydrophilic medium such as water or glycerol, a preferred liquid crystalline
phase being the
cubic liquid crystalline phase.
Thus, very interesting compositions according to the invention are
compositions in which the
fatty acid ester is glycerylmonooleate or glycerylmonolinoleate, in particular
glycerylmonooleate.
It has been found that the stability of the composition is considerably
enhanced, such as
resulting in a storage stability of at least 2 years at 25°C, when the
glycerylmonooleate product
(as is well known, fatty acid esters are almost invariably mixed products)
contained in the
product fulfils certain purity standards. Thus, the glycerolmonooleate product
used for the
preparation of the composition should contain at the most 4% of saturated
monoglyceride and
should preferably contain at least 88% of glycerylmonooleate, more preferably
at least 89%, such
as at least 90% or at least 91%, in particular at least 92%, of
glycerylmonooleate.
When the composition is a precursor type composition, the liquid medium is
either not present
at all or is present in small amounts, such as an amount of at least 0.5% by
weight, such as at
least 1% by weight, calculated on the total composition, e.g. at least 2% by
weight, calculated on
the total composition, or up to at least 5% or in certain cases at least 10%,
calculated on the total
composition.
In non-precursor compositions, the liquid medium is normally present in an
amount of at least
20% by weight, calculated on the total composition, such as at least 25% or at
least 30% by
weight, calculated on the total composition, and a preferred amount is often
in the range of 25-
50% such as 25-40% by weight, in particular 25-35%, 27-40%, 27-35% or 30-40%
by weight,
calculated on the total composition.
Normally, the concentration of the second substance in a composition according
to the invention
is at least about IO% by weight such as, e.g., at least 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%,
55%, 60%, 65%, or 70% by weight calculated on the composition.
In other terms, the concentration of the second substance in the composition
is in a range
corresponding to from about 10% to about 90% such as, e.g. about 15%-85%,
about 20%-80%,
about 25%-75%, about 25%-70%, about 25%-65%, about 25%-60%, about 25%-55%,
about 30%-
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50%, about 35%-55%, about 30%-45% or about 30%-40% by weight based on the
total
composition. .
Generally, the maximal concentration of the second substance in the
composition is at the most
about 60% such as, e.g. at the most about 55%, about 50%, about 45%, about
40%, about 35%,
about 30%, about 25% or about 20% by weight based on the total composition.
Structurants
As indicated above the gist of the present invention relates to the use of
certain substances as
substitutes for at least a part of the substance (e.g. a fatty acid ester)
which is capable of forming
a liquid crystalline phase. Surprisingly, the inventor has found that when,
e.g., the fatty acid
ester component is substituted by so-called structurants then the lattice
structure of the liquid
crystalline phase formed in the composition (or in situ if the composition is
a precursor
composition) is not only based on the fatty acid ester itself but the
structurant takes part in this
lattice. Thus, the structurant imparts the lattice structure to the
composition and, thus, the
content of lattice structure in the composition is of the same order of
magnitude as if no
substitution has taken place. When adding active substances or additives to a
composition which
is capable of forming a liquid crystalline phase, the general observation is
that the active
substance or additive is enclosed or enveloped in the composition without
participating in the
formation of the lattice structure. In general, such substances can only be
present in the
composition to a certain extent beyond which the substance will exert a
negative influence on
the formation of a liquid crystalline phase resulting in that either no liquid
crystalline phase is
formed or the biopharmaceutical properties of the composition are negatively
influenced. In
general, substances can be incorporated in a concentration of at the most
about 10% by weight
without any significant change in the ability of the composition to form a
liquid crystalline phase
or to undergo a phase transition.
As mentioned above a structurant participates in the liquid crystalline
structure preferably
formed together with a fatty acid ester. A suitable structurant is typically
an amphiphilic
substance having a molecular weight of at the most 2000 or an emulsifier or a
surfactant.
Tensides (anionic, cationic, non-ionic like e.g. sorbitan esters, sorbitan
macrogol esters
(polysorbates)), polar lipids, glycolipids, lechitins, palmitoyl muramic acid
(PMA), substances
having surface active properties like e.g. certain cellulose derivatives,
sorbitan oleate, sorbitan
laurate, lanolin and derivatives thereof and ethoxylated derivatives of
lanolin (Aqualose W20,
Aqualose L30 and Aqualose L75) are also examples of suitable structurants for
use in
compositions according to the invention.
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Sorbitan esters are a series of mixtures of partial esters of sorbitol and its
mono- and di-
anhydrides with fatty acids. Examples of suitable sorbitan esters for use as
structurants_in a
composition according to the invention are:
Sorbitan di-isostearate
Sorbitan dioleate
Sorbitan monoisostearate
Sorbitan monolaurate
Sorbitan monooleate
Sorbitan monopalmitate
Sorbitan monostearate
Sorbitan sesqui-isostearate
Sorbitan sesquioleate
Sorbitan trioleate
Sorbitan sesquistearate
Sorbitan tri-isostearate
Sorbitan tristearate
Sorbitan tristearate
Polyoxyethylene sorbitan fatty acid esters (polysorbates) are a series of
fatty acid esters or
sorbitol and its anhydrides copolymerized with approximately 20 moles of
ethylene oxide for
each mole of sorbitol and its anhydrides. Examples of suitable polysorbates
for use in the
present context are:
Polysorbate 20
Polysorbate 21
Polysorbate 40
Polysorbate 60
Polysorbate 61
Polysorbate 65
Polysorbate 80
Polysorbate 81
Polysorbate 85
Polysorbate 120
If a liquid medium such as, e.g., water is present in a composition, the
polysorbates may be
dissolved or dispersed therein.
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In order to be able to participate in the lattice structure of the liquid
crystalline phase, it is
contemplated that a sta-ucturant should possess a hydrophilic as well as
hydrophobic~or_tion. A
useful structurant normally have the following molecular characteristics:
Chains:
5 alkyl chains (saturated or unsaturated), polyethylene chains and/or
polyoxyethylene chains
and contain at least one of the following functional groups (or a radical
thereof):
sugar, oxyethylene, glycerol, hydroxy, polyhydroxy, amino acid, sulfates,
and/or phosphates.
Suitable structurants are also found among the substances which normally are
denoted
emulsifiers. Preferably, the structurant has a saturated or unsaturated,
branched or unbranched,
10 substituted or unsubstituted C6-C26-alkyl chain, and/or the structurant is
a compound which
contains a polyethylene group.
Furthermore, an important property of a structurant which is suitable for use
in compositions
according to the present invention is its solubility in the second substance
such as e.g. a fatty
acid ester or mixtures of fatty acid esters. It is believed that the two
components (i.e. the second
15 substance and the structurant) should be miscible (or compatible) and "like
each other" in order
to be able to cooperate when the lattice structure is formed. Thus, the
solubility of the
structurants in the second substance like e.g. a fatty acid ester or mixtures
of fatty acid esters
should preferably be at least about I5%, such as at least about 20°!0
or about 253'o by weight at
60°C.
In certain interesting embodiments of the invention the structurant is a
substance which
together with the second substance like a fatty acid ester and a liquid medium
- is capable of
forming a cubic liquid crystalline phase.
It is contemplated that the preferred structurants are substances which - in a
two component
system of the structurant as a first component and water as a second component
- are capable
of forming a non-cubic liquid crystalline phase.
However, a requirement for the structurant is not that it is capable of
forming the same liquid
crystalline phase at room temperature as the second substance. In particular,
most surprising
the inventor has found that a substance like Vitamin E TPGS - which is a very
important
structurant in this context and which in itself together with water is not
capable of forming a
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16
cubic liquid crystalline phase at room temperature - together with fatty acid
esters (as second
substances) and water forms a cubic liquid crystalline phase. _ _
Therefore, interesting examples of structurants are substances which - in a
two component
system of the structurant and water - do not form a cubic liquid crystalline
phase at a
temperature of between 20-40°C.
A composition according to the invention may of course also comprise more than
one structurant
such as, e.g., a combination of two or more structurants.
With respect to the concentration of the structurant(s) in the composition it
is preferred that the
concentration (of the structurants taken either alone or in combination) is at
least 1% by weight
such as, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or
55% by weight
based on the total weight of the composition.
The concentration range is typically in a range of from about 1% to about 60%
such as, e.g.,
from about 5% to about 55%, from about 5% to about 50%, from about 5% to about
45%, from
about 7.5% to about 40%, or from about 10% to about 35% by weight based on the
total weight
of the composition.
Normally, the maximal concentration of a structurant in the composition is at
the most abort
45% by weight such as, e.g., at the most least about 40% or about 35% by
weight based on the
total weight of the composition.
Normally, compositions comprising e.g. glyceryl monooleate (GMO)/water without
any content
of a structurant must have a concentration of the GMO of at least 60-65% by
weight in order to
have a certain suitable and acceptable storage stability and/or to avoid an
excess of water
present in the composition. However, the present inventor has found that when
e.g. GMO is
substituted by a structurant, compositions having a concentration of e.g. GMO
below and well
below 60% by weight have shown to be stable for at least one week at room
temperature and
60% relative humidity. In the present context the stability of the composition
is the physical
stability, i.e. stability with respect to phase separation (i.e. not
necessarily change in liquid
crystalline phase) into two or more distinct phase. The phase separation must
be irreversible, i.e.
no reestablishment of a homogeneous composition can be observed visually by
shaking the
composition at room temperature for 2 days and the distinct phases formed by
either be liquid,
semi-solid or solid phases. In some cases, a physical instability of the
composition may be due to
a change in the liquid crystalline phase present in the composition. Such a
change in the liquid
crystalline phase may either be due to i) a chemical change of either the
second substance or the
structurant, ii) a negative influence on the liquid crystalline phase by any
component included in
,.
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the composition, or iii) the fact that the concentration of the constituents
of the composition is
near a point in the phase diagram where the liquid crystalline phase changes
from one_ phase to
another or is destroyed (i.e. the concentration is near the borderline of the
stable area of the
liquid crystallin phase). In general, a composition according to the invention
is substantially
homogeneous and has such a physical stability that substantial no irreversible
phase separation
into two or more distinct phases can be observed visually after storage of the
composition at
25°C and 60% relative humidity for one week or at least for one week
such as, e.g., at least two
weeks, 1 month, 1 year or preferably at least 2 years at 25°C.
The total concentration of the substances - which together with each other and
an liquid
medium such as, e.g., water are capable of generating a liquid crystalline
phase - in a
composition according the invention is typically at least about 50% by weight
based on the
weight of the total composition. Thus, in general the total concentration of
the second substance
such as, e.g., a fatty acid ester or mixture of fatty acid esters and the
structurant(s) is at least
50% by weight based on the total composition.
Interesting embodiments of the invention are compositions wherein the liquid
crystalline phase
has been generated by a liquid medium present in the composition and wherein
the total
concentration of the second substance like e.g. a fatty acid ester or mixture
of fatty acid esters
and the structurant(s) is at least 50% such as at least 55%, 60%, 65%, 70% or
75% by weight
based on the total composition.
As mentioned above, a composition according to the invention may also be in a
precursor form.
Examples of interesting compositions are those wherein the total concentration
of the fatty acid
ester or mixture of fatty acid esters and the structurant(s) is at least 50%
such as at least 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.5% by weight based on the total
composition.
Specific examples of a suitable structurant for use in a composition according
to the invention
are, e.g., a phospholipid selected from the group consisting of phosphatidic
acid,
phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine,
phosphatidylglycerol,
phosphatidylinositol, and diphosphatidylglycerol.
Other interesting phospholipids are DEPE (1,2 dielaidoyl-sn-glycerol-3-
phosphoethanolamine)
and DMPE (PEG 550) (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-
(polyethylene
glycol)550).
More specifically, the structurant may be a fatty acid ester of a
glycerylphosphate derivative or a
glycerylsulfate derivative, and the fatty acid component is selected from the
group consisting of
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lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic
acid, linolenic acid, and
behenic acid. Specific examples include structurants wherein the fatty acid
ester is selected from
the group consisting of dioleyol phosphatidylcholine, dilauryl
phosphatidylcholine, dimyristyl
phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl
phosphatidylcholine, dibehenoyl
phosphatidylcholine, dimyristyl phosphatidylethanolamine, dipalmitoyl
phosphatidylethanolamine, dioleyI phosphatidylglycerol, dilauryl
phosphatidylglycerol,
dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl
phosphatidylglycerol, dipalmitoyl phosphatic acid and mixtures thereof.
A presently preferred type of structurant is phosphatidylcholine such as,
e.g., a
phosphatidylcholine containing product like Epikuron 200, Epikuron 145, Lipoid
S75 or Lipoid
5100.
In a composition according the invention wherein the structurant is a
phosphatidylcholine or a
derivative or analog thereof, the concentration of the structurant is
preferably in a range of from
about 1 to about 75% such as, e.g., from about 5 to about 55%, from about 5%
to about 35% or
from about 10 to about 20% by weight based on the total weight of the
composition.
Another group of substances which are suitable structurants for use in a
composition according
to the invention is tocopherols. In the present context the term "tocopherols"
is used to broadly
include all Vitamin E or Vitamin E-like substances, derivatives and analogs
thereof. The term
includes all tocol and tocotrienol derivatives such as e.g. methyl tocol. More
specifically, in the
present context, a tocopherol is selected from the group consisting of ø-
tocopherols, sorbitan
esters of tocopherols, d-a-tocopherol, d,l-a-tocopherol, d-a-tocopherol
acetate, d,l-a-tocopherol
acetate, d-a-tocopherol succinate, d,l-a-tocopherol succinate, d-a-tocopherol
nicotinate, d,l-a-
tocopherol nicotinate, tocopherylpolyethylene glycol succinate such as d-a-
tocopherylpolyethylene
glycol succinate or d,l-a-tocopherylpolyethylene glycol succinate, and
derivatives such as fatty
acid ester derivatives and analogues thereof.
The tocopherylpolyethylene glycol succinate is selected from the group
consisting of:
d-a-tocopherylpolyethylene glycol 200 succinate,
d,l-a-tocopherylpolyethylene glycol 200 succinate,
d-a-tocopherylpolyethylene glycol 300 succinate,
d,l-a-tocopherylpolyethylene glycol 300 succinate,
d-a-tocopherylpolyethylene glycol 400 succinate,
d,l-a-tocopherylpolyethylene glycol 400 succinate,
d-a-tocopherylpolyethylene glycol 500 succinate,
d,l-a-tocopherylpolyethylene glycol 500 succinate,
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d-a-tocopherylpolyethylene glycol 600 succinate,
d,l-a-tocopherylpolyethylene glycol 600 succinate, _ _ _
d-a-tocopherylpolyethylene glycol 700 succinate,
d,l-a-tocopherylpolyethylene glycol 700 succinate,
d-a-tocopherylpolyethylene glycol 800 succinate,
d,l-a-tocopherylpolyethylene glycol 800 succinate,
d-a-tocopherylpolyethylene glycol 800 succinate,
d,l-a-tocopherylpolyethylene glycol 800 succinate,
d-a-tocopherylpolyethylene glycol 900 succinate,
d,l-a-tocopherylpolyethylene glycol 900 succinate,
d-a-tocopherylpolyethylene glycol 1000 succinate,
d,l-a-tocopherylpolyethylene glycol 1000 succinate,
d-a-tocopherylpolyethylene glycol 1100 succinate,
d,l-a-tocopherylpolyethylene glycol 1100 succinate,
d-a-tocopherylpolyethylene glycol 1200 succinate,
d,l-a-tocopherylpolyethylene glycol 1200 succinate,
d-a-tocopherylpolyethylene glycol 1300 succinate,
d,l-a-tocopherylpolyethylene glycol 1300 succinate,
d-a-tocopherylpolyethylene glycol 1400 succinate,
d,l-a-tocopherylpolyethylene glycol 1400 succinate,
d-a-tocopheryipolyethylene glycol 1450 succinate,
d,l-a-tocopherylpolyethylene glycol 1450 succinate,
d-a-tocopherylpolyethylene glycol 1500 succinate,
d,l-a-tocopherylpolyethylene glycol 1500 succinate,
d-a-tocopherylpolyethylene glycol 1600 succinate,
d,l-a-tocopherylpolyethylene glycol 1600 succinate,
d-a-tocopherylpolyethylene glycol 1700 succinate and
d,l-a-tocopherylpolyethylene glycol 1700 succinate.
Preferred tocopherols for use in a composition according to the present
invention are d-a-
tocopherylpolyethylene glycol 1000 succinate (in the following denoted vitamin
E TPGS or
simply TPGS) or d,l-a-tocopherylpolyethylene glycol 1000 succinate.
A composition according to the invention containing a tocopherol as a
structurant has typically a
concentration of the tocopherol of at the most about 30% such as at the most
about 25%, 20%,
15%, 10%, 5%, 2.5% or 1% by weight based on the total weight of the
composition.
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Presently preferred compositions according to the invention are those wherein
the structurant is
a combination of vitamin E TPGS and a phosphatidylcholine containing product
such as, e.g.,
Epikuron 200. In such compositions the concentration of vitamin E TPGS is
generally in a range
corresponding to from about 1% to about 30% such as, e.g., from about 5% to
about 25%, from
5 about 5% to about 20% or from about 10% to about 20% by weight and the
concentration of
Epikuron 200 is in a range corresponding to from about 2.5% to about 40% such
as, e.g., about
5% to about 25% or from about 10% to about 20% by weight based on the total
composition.
Pharmaceutically acceptable excipients for use in a composition according to
the
invention
10 As mentioned above, an aspect of the invention relates to compositions
wherein at least a part of
the substance which together with a liquid medium such as, e.g., water is
capable of forming a
liquid crystalline phase at room temperature can be substituted by certain
pharmaceutically
acceptable excipients. As mentioned in the introduction addition of a
pharmaceutically acceptable
excipient to a composition containing a liquid crystalline phase or a
precursor composition will
15 normally lead to a disruption in the liquid crystalline phase. Therefore,
such substance is
generally only added in very small concentrations such as, e.g., about 1-5% by
weight based on
the total composition. The present inventor has surprisingly found that
certain pharmaceutically
acceptable excipients may be added in much larger concentrations without
having any
substantially negative influence on the biopharmaceutical properties of the
composition. Thus,
20 the concentration of such excipients may be at least about 5% by weight
such as, e.g, at least
about 8%, 9%, 10%, 15% or 20% by weight.
Suitable pharmaceutically acceptable excipients may either i) be soluble in
the second substance
or in the liquid crystalline phase, i.e. having a solubility of more than
about than about 15%
such as more than about 25%, 30% or 50% by weight in the second substance (or
liquid
crystalline phase) at 60°C, or ii) have a relative low solubility in
the second substance such as,
e.g., a solubility of less than 15% such as less than about 12.5%, 10%, 7.5%,
5% or 1% by weight
in the second substance at 60°C. More specifically, the
pharmaceutically acceptable excipient may
have a solubility of less than about 15% such as less than about 10%, about
5%, about 2.5%,
about 1%, or about 0.5% by weight in the liquid crystalline phase at room
temperature.
Examples of suitable pharmaceutically acceptable excipients are found e.g.
among inert diluents
or fillers selected from the group consisting of sucrose, sorbitol, sugar,
mannitol, microcrystalline
cellulose, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl
methylcellulose,
ethylcellulose, starches including potato starch, calcium carbonate, sodium
chloride, lactose,
calcium phosphate, calcium sulfate, sodium phosphate, and a polysaccharide
such as, e.g.,
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carmelose, a chitosan, a pectin, xanthan gum, a carrageenan, locust bean gum,
acacia gum, a
gelatin, an alginate, aad dextrans and salts thereof.
Preferably, a in composition according to the above-mentioned aspect of the
invention the
concentration of the second substance in the composition is at the most about
60% such as, e.g.,
at the most about 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% by
weight based on
the total composition.
Examples of suitable pharmaceutically excipients which are soluble in the
second substance or in
the liquid crystalline phase are e.g. sorbitan esters such as, e.g.,
polysorbates; and macrogols. In
the present context, solvents like e.g., water, glycerol, alcohols like e.g.
ethanol and
isopropylalcohol are examples of a liquid medium and are not intended to be
examples of soluble
pharmaceutically acceptable excipients.
Liquid medium
As mentioned above a composition according to the invention may optionally
comprise a liquid
medium. A liquid medium may be present in compositions in which a liquid
crystalline phase
between the second substance and the liquid medium has been generated as well
as in the so-
called precursor compositions in which the liquid crystalline phase has not
been generated in the
composition but is to be formed upon administration of the composition to a
mammal. In the
latter case, any liquid medium present in the precursor composition may or may
not take part in
the formation of a liquid crystalline phase together with any moisture from
the application site
or body fluid present at or on the application site.
In general, a liquid medium is present in a concentration of at least about
0.5% by weight, such
as at least about 1%, at least about 2010, at least about 5%, at least about
10%, at least about
15%, at least about 20%, at least about 25% or at least about 30% by weight,
calculated on the
total composition.
In non-precursor compositions, a liquid medium normally is present in a
concentration of 20%-
50% such as, e.g., about 25%-35% about 25%-30% or about 30%-40% by weight,
calculated on
the total composition.
In preferred non-precursor compositions, a liquid medium is present in a
concentration of 25%-
40% such as, e.g, about 25-35%, about 30%-40% or 27%-37% by weight, calculated
on the total
composition.
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Generally preferred liquid media which participate in the formation of a
liquid crystalline phase
are water, glycerol, alcQhols like e.g. ethanol and mixtures thereof. _ .
Active substances
In the present context the term "active substance" is intended to mean any
biologically or
pharmacologically active substance or antigen-comprising material; the term
includes drug
substances which have utility in the treatment or prevention of diseases or
disorders affecting
animals or humans, or in the regulation of any animal or human physiological
condition and it
also includes any biologically active compound or composition which, when
administered in an
effective amount, has an effect on living cells or organisms.
Examples of active substances of particular importance with respect to all
aspects of the
invention are the so-called antiherpes virus agents which have been or are
developed for the
treatment of herpes virus infections [herpes simplex virus types 1 and 2 (HSV-
1 and HSV-2),
varicella zoster virus (VZV), cytomegalovirus (CI1~I~, Epstein-Barr virus
(EBV)]. The antiherpes
virus agents include antiviral drugs and prodrugs thereof, such as
nucleosides, nucleoside
analogues, phosphorylated nucleosides (nucleotides), nucleotide analogues and
salts, complexes
and prodrugs thereof; e.g. guanosine analogues, deoxyguanosine analogues,
guanine, guanine
analogues, thymidine analogues, uracil analogues and adenine analogues.
Especially interesting
antiherpes virus agent far use either alone or in combination in a composition
according to the
present invention are selected from acyclovir, famciclovir, deciclovir,
penciclovir, zidovudin,
ganciclovir, didanosin, zalcitabin, valaciclovir, sorivudine, lobucavir,
brivudine, cidofovir, n-
docosanol, ISIS-2922, and prodrugs and analogues thereof. Details concerning
active substances
suitable for use in connection with the present invention as well as a
description of other
interesting active substances are given below.
As mentioned above, an important example of an active substance is an
antiviral drug, such as a
nucleoside or a nucleoside analogue, e.g. selected from acyclovir,
famciclovir, deciclovir,
penciclovir, zidovudin, ganciclovir, didanosin, zalcitabin, valaciclovir,
sorivudine, lobucavir,
brivudine, cidofovir, n-docosanol, ISIS-2922 and salts and prodrugs thereof.
However, also a
large number of other drugs which in themselves have a low solubility as
defined herein or the
salts, esters, prodrugs or precursors of which have a low solubility are
important active
substances in the compositions of the invention. Furthermore, there is also a
large number of
drugs which advantageously can be incorporated in a composition according to
the invention,
either as the sole active substance (provided the solubility criteria are
fulfilled) or in combination
with another active substances. In the following is listed a number of active
substances which
either alone or in combination may be incorporated in a composition according
to the present
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invention. In particular a combination of an antiherpes virus agent and a
glucocorticosteroid is of
importance. _
Examples of drugs which are of particular importance in connection with
application to skin or
mucosal surfaces are:
Acyclovir, famciclovir, ribavirin, zidovudin, ganciclovir, didanosin,
zalcitabin, valaciclovir
amantadin, rimantadin
foskarnet
idoxuridin
fluoruracil
interferons and variants thereof, including alpha interferon, beta interferon,
and gamma
interferon,
tromantadin
lentinan
levofloxacin
stavudine
tacrine
vesnarinone
ampligen
atevirdine
delavirdine
hydroxyurea
indinavir sulfate
interleukin-2 fusion toxin, seragen
lamivudine
lidakol
nevirapine
onconase
saquinavir
topotecan
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verteporfin
viraplex _ -
CMV immunoglobulin
efalith
epervudine
podophyllotoxin
proxigermanium
rifabutin
bromovinyldeoxyuridine
ukrain
cidofovir
imiquimod
lamivudine
sorivudine
I5 viraplex
afovirsen
amonafide
hypericin
provir
temoporfin
aphidicolin giycinate
ibobucavir
virend
AL-721
ampligen
arildone
brivudine
CD4
2-deoxy-D-glucose
desciclovir
dichloroflavan
didanosine
ditiocarb Sodium
edoxudine
enviroxime
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fiacitabine
inosine Pranobex _
peptide T
stavudine
5 tribavirin
trifluridine
vidarabine
zalcitabine
miconazol
10 fucidin
erythromycin
macrolides
NSAID's
peptides
15 insulin
polymycin
myperizin
antibiotics
nicotine
20 sucralfate
sucrose octasulfate
salicylic acid
urea
benzoylperoxide
25 minosidil
heparinoid
methotrexate
ciclosporin
A listing of substances of potential interest comprises substances of the
following groups:
_ 30 sodium fluoride
anti-inflammatory drugs such as, e.g., ibuprofen, indomethacin, naproxen,
diclofenac, tolfenamic
acid, piroxicam, and the like;
narcotic antagonists such as, e.g., naloxone, nalorphine, and the like;
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26
antiparkinsonism agents such as, e.g., bromocriptine, biperidin, benzhexol,
benztropine, and the
like; _ _ _ _
antidepressants such as, e.g., imipramine, nortriptyline, pritiptylene, and
the like;
antibiotic agents such as, e.g., clindamycin, erythromycin, fusidic acid,
gentamicin, mupirocien,
amfomycin, neomycin, metronidazole, silver sulphadiazine, sulphamethizole,
bacitracin,
framycetin, polymycin B, acitromycin, and the like;
antifungal agents such as, e.g., miconazol, ketoconazole, clotrimazole,
amphotericin B, nystatin,
mepyramin, econazol, fluconazol, flucytocine, griseofulvin, bifonazole,
amorolfine, mycostatin,
itraconazole, terbenafine, terconazole, tolnaftate, and the like;
antimicrobial agents such as, e.g., metronidazole, tetracyclines,
oxytetracycline, and the like;
antiemetics such as, e.g., metoclopramide, droperidol, haloperidol,
promethazine, and the like;
antihistamines such as, e.g., chlorpheniramine, terfenadine, triprolidine, and
the like;
antimigraine agents such as, e.g., dihydroergotamine, ergotamine, pizotyline,
and the like;
coronary, cerebral or peripheral vasodilators such as, e.g., nifedipine,
diltiazem, and the like;
antianginals such as, e.g., glyceryl nitrate, isosorbide denitrate,
molsidomine, verapamil, and the
like;
calcium channel blockers such as, e.g., verapamil, nifedipine, diltiazem,
nicardipine, and the like;
hormonal agents such as, e.g., estradiol, estron, estriol, polyestradiol,
polyestriol, dienestrol,
diethylstilbestrol, progesterone, dihydroergosterone, cyproterone, danazol,
testosterone, and the
like;
contraceptive agents such as, e.g., ethynyl estradiol, lynestrenol, etynodiol,
norethisterone,
mestranol, norgestrel, levonorgestrel, desogestrel, medroxyprogesterone, and
the like;
antithrombotic agents such as, e.g., heparin, warfarin, and the like;
diuretics such as, e.g., hydrochlorothiazide, flunarizine, minoxidil, and the
like;
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2?
antihypertensive agents such as, e.g., propanolol, metoprolol, clonidine,
pindolol, and the like;
corticosteroids such as, e.g., beclomethasone, betamethasone, betamethasone-I7-
valerate,
betamethasone-dipropionate, clobetasol, clobetasol-17-butyrate, clobetasol-
propionate, desonide,
desoxymethasone, dexamethasone, diflucortolone, flumethasone, flumethasone-
pivalate,
fluocinolone acetonide, fluocinonide, hydrocortisone, hydrocortisone-I7-
butyrate, hydrocortisone-
buteprate, methylprednisolone, triamcinolone acetonide, budesonide,
halcinonide, fluprednide
acetate, alklometasone-dipropionate, fluocortolone, fluticason-propionate,
mometasone-furate,
desoxymethasone, diflurason-diacetate, halquinol, cliochinol, chlorchinaldol,
fluocinolone-
acetonid, and the like;
dermatological agents such as, e.g., nitrofurantoin, dithranol, clioquinol,
hydroxyquinoline,
isotretionin, methoxsalen, methotrexate, tretionin, trioxsalen, salicylic
acid, penicillamine, and
the like;
steroids such as, e.g., estradiol, progesterone, norethindrone,
levonorgestrol, ethynodiol,
levenorgestrel, norgestimate, gestanin, desogestrel, 3-keton-desogestrel,
demegestone,
promethoestrol, testosterone, spironolactone, and esters thereof,
vitro compounds such as, e.g., amyl nitrates, nitroglycerine and isosorbide
nitrates,
opioid compounds such as, e.g., morphine and morphine-like drugs such as
buprenorphine,
oxymorphone, hydromorphone, levorphanol, fentanyl and fentanyl derivatives and
analogues,
prostaglandins such as, e.g., a member of the PGA, PGB, PGE, or PGF series
such as, e.g.,
misoprostol, dinoproston, carboprost or enaprostil,
a benzamide such as, e.g., metoclopramide, scopolamine,
a peptide such as, e.g., growth hormone releasing factors, growth factors
(epidermal growth
factor (EGF), nerve growth factor (NGF), TGF, PDGF, insulin growth factor
(IGF), fibroblast
growth factor (aFGF, bFGF, etc.), and the like), somatostatin, calcitonin,
insulin, vasopressin,
interferons, IL-2, urokinase, serratiopeptidase, superoxide dismutase (SOD),
thyrotropin
releasing hormone (TRH), luteinizing hormone releasing hormone (LH-RH),
corticotrophin
releasing hormone (CRF), growth hormone releasing hormone (GHRH), oxytocin,
erythropoietin
(EPO), colony stimulating factor (CSF), and the like,
a xanthine such as, e.g., cageine, theophylline,
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28
a catecholamine such as, e.g., ephedrine, salbutamol, terbutaline,
a dihydropyridine such as, e.g., nifedipine,
a thiazide such as, e.g., hydrochlorotiazide, flunarizine,
others such as, e.g., propanthelin, silver nitrate, enzymes like
Streptokinases, Streptodases,
vitamins like vitamin A, tretionin, isotretionin, acitretin, vitamin D,
calcipotriol, interferon-a-2b,
selen disulfide, pyrethione.
It will be understood that the compositions of the invention may also comprise
combinations of
active substances, e.g. an active substance together with a potentiator
therefor.
It will of course also be understood that in the aspects of the invention
wherein there is no
specific requirement to the active substance, e.g. with respect to solubility,
any substance which
has a therapeutic or prophylactic activity may be incorporated in the
composition.
Solubility of the active substance in the liquid crystalline phase
As mentioned above, in an aspect of the invention and in some embodiments of
other aspects of
the invention, the active substance of the composition of the invention is a
substance which has
a low solubility in the liquid crystalline phase such as, e.g., at the most
about 20 mg/g at 20°C,
at the most 15 mg/g at 20°C, e.g. at the most 10 mg/g at 20°C or
lower, such as at the most 7
mg/g, 6.5 mg/g, 6 mg/g, 5..5 mg/g, 5 mg/g at 20°C. e.g, at the most 4
mg/g at 20°C or even at
the most 3 mg/g or 2 mg/g or 1 mg/g at 20°C.
The determination of the solubility of the active substance in the liquid
crystalline phase of the
composition is, of course, performed on the liquid crystalline phase as
formed. In practice, this
means that when the composition is one in which the liquid crystalline phase
has already been
formed when the composition is applied, the determination of the solubility is
performed on the
composition itself. The determination of the solubility is suitably performed
by microscopy to
observe any crystals of the active substance. The determination of the
concentration at which
crystals are observed is performed after a period of at least one week after
preparation of the
composition or the liquid crystalline phase, or when equilibrium has been
established. Normally,
a series of tests with varying concentrations is performed to determine the
concentration above
which crystals are found. On the other hand, when the composition is a
precursor composition,
the liquid crystalline phase used as a reference in the solubility
determination is a liquid
crystalline phase imitating the liquid crystalline phase which will be formed
when the
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29
composition absorbs liquid from the site of application. This reference liquid
crystalline phase is
made up with water (~s representing the liquid absorbed) in such an amount
that the -reference
liquid crystalline phase is the same type of liquid crystalline phase as is
generated from the
precursor composition.
While the lower limit of the amount of the second substance such as, e.g., a
fatty acid ester in
the composition is determined by the requirement that the second substance, in
the amount in
question, must be able to form and maintain the liquid crystalline phase, the
composition will in
most cases contain at least 10% by weight, calculated on the composition, of
the fatty acid ester,
such as at least about 15%, 20%, 25%, 30%, or 35% by weight, and in some cases
at least 40%,
45%, 50%, 55%, 60%, 65%, or 70% by weight, calculated on the composition, of
the second
substance. These numbers apply to the liquid crystalline phase present in the
composition; in
precursor compositions, the concentrations will, of course, be higher.
The pH of the liquid crystalline phase of the composition is in the range of
3.0-9.5 such as, e.g.,
3.2-9.3, 3.4-9.1 or 3.6-9. At lower pH values, the composition may be
irritating to the skin or
mucosa on which it is applied; at higher pH values, the composition may be
irritating and may
also directly be etching. The pH of the liquid crystalline phase is determined
by a method
involving dispersing e.g. 10% of the liquid crystalline phase (containing the
active substance and
any excipients) in distilled water and measuring the pH in the water phase,
equilibration
between the liquid crystalline phase and a water phase and measuring the pH of
the water
phase at 20°C (e.g. using a rotomat for 2 hours). Alternatively, the pH
of the liquid crystalline
phase may be measured by means of an suitable pH electrode (see the Examples).
It is generally preferred that the upper limit of the pH of the liquid
crystalline phase is 8. It is
also preferred that the lower limit of the pH is 3.0 or higher, and thus,
interesting pH ranges for
the liquid crystalline phase are pH 3.0-8, such as, e.g., 3.1-8, 3.2-8, 3.3-8,
3.4-8, 3.5-8, 3.6-8, 3.7-
8, 3.8-8, 3.9-8, 4.0-8, 4.1-8, 4.2-8, 4.3-8, 4.5-8, 4.75-8, or 5.0-8.
Furthermore, in an aspect of the invention and in embodiments of other aspects
of the
invention, the solubility of the active substance in water is very low, at the
most 10 mg/g at
20°C and at a pH substantially identical to the pH of the liquid
crystalline phase, determined as
described herein. While a pH range is stated above for the liquid crystalline
phase, it will be
understood that by the water solubility of the active substance is meant the
water solubility at
the relevant pH, which is a pH substantially identical to the pH which will
prevail in the
composition, in other words, the pH of the liquid crystalline phase, this pH
being determined as
described herein. When the pH of the liquid crystalline phase, determined as
described herein, is
different from the pH which will result simply by dissolution of the active
substance in water,
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the water is adjusted to substantially the pH of the liquid crystalline phase
by using a suitable
buffer system when determining the solubility of the active substance. This
buffer system should
of course be so selected that, apart from the pH adjustment, it has
substantially no influence on
the solubility of the active substance in the buffered water.
5 The composition according to the present invention is very valuable in that
it can provide a high
release of active substances of very low water solubility, such as
a solubility of at the most 7 mg/g, such as at the most 5 mg/g at 20°C
and at a pH substantially
identical to the pH of the liquid crystalline phase, determined as described
herein.
Of particular interest is also the fact that excellent release rates can be
obtained of active
10 substance whose solubility in water is at the most 3 mg/g or even at the
most 2 mg/g at 20°C
and at a pH substantially identical to the pH of the liquid crystalline phase,
determined as
described herein.
Alternatively, the active substance has an minimum aqueous solubility of at
the most 10 mg/ml
such as, e.g., 7 mg/ml, 5 mg/ml, 3 mg/ml and 1 mg/ml at 20°C and at a
pH in a range
15 corresponding to 3.0-9.5 such as, e.g. having a pH between 3.2 and 9.3,
between 3.4 and 9.1 or
between 3.6 and 9Ø The determination of the minimum aqueous solubility is
performed by use
of suitable buffers which are capable of maintaining the pH at the desired
value and measures
are taken to ensure that equilibrium is obtained between the undissolved and
dissolved active
substance, i.e. by employment of ultrasonic treatment and/or stirring for a
well-defined time
20 period. It will be appreciated that the pH-ranges and the aqueous
solubility values given above
when the aqueous solubility is determined at a pH corresponding to the pH
prevailing in the
liquid crystalline phase apply mutatis mutandis when the aqueous solubility is
the minimum
solubility in a pH range of 3.0-9.5.
In embodiments of particular interest a composition according to the invention
contains one or
25 more antiherpes virus agents) as an active substance. Relevant antiherpes
virus agents are
mentioned above and acyclovir is of particular importance. Acyclovir (9-[2-
hydroxyethoxy)methyl]-guanine, an acyclic analogue to the natural nucleoside
2'-
deoxyguanosine, is a widely used agent in the treatment of herpes virus
infections. Compositions
for oral, topical and intravenous administration are available. The delivery
characteristics of
30 acyclovir following administration by these routes are, however, far from
being optimal probably
due to the poor aqueous solubility and/or low lipophilicity of acyclovir. The
solubility of acyclovir
in water is about 1.5 mg/ml at 22°C and the partition coefficient (P)
between octanol and 0.02 M
phosphate buffer pH 7.4 (21°C) is about 0.03. In accordance with the
physico-chemical
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31
properties, the bioavailability after oral administration is rather low (about
15-20%) and highly
variable and the percutaneous penetration is poor.
The active substance of low solubility is normally present in the composition
in an amount in
the range of from 1-20% by weight, usually 1-15% by weight.
A preferred composition according to the invention is a composition in which
the active
substance is acyclovir, the second substance is a the fatty acid ester and a
liquid medium is
present in the composition. In such compositions the fatty acid ester is
preferably a
glycerylmonooleate product having a glycerylmonooleate content of at least 88%
such as, e.g., at
least about 89, 90%, 91% or 92% by weight and a content of saturated
monoglycerides of at the
most 4% by weight such as, e.g., at the most about 2% by weight.
Preferred acyclovir-containing compositions according to the invention
normally has a weight
ratio between the glycerylmonooleate and the liquid medium is in the range
between I:0.3 and
1:2 such as between 1:0.5 and 1:1.5 such as, e.g. 1:1. In those cases where a
structurant is
present, the weight ratio between the combination of the glycerylmonooleate
and the
I5 structurant, and the liquid medium is generally in the range between 60:40
and 75:25 such as
between 63:37 and 73:27.
In such compositions, preferred structurants are Epikuron 200 and Vitamin E
TPGS and
combinations thereof and the weight ratio between Epikuron and Vitamin E TPGS
may be
between about 1:0.5 and 1:2 such as, e.g., between 1:0.75 and 1:1.5 such as,
e.g., about 1:1.
Another preferred acyclovir-containing composition comprises
glycerylmonooleate as second substance, a mixture of Epikuron 200 and Vitamin
E TPGS as
structurants, and water as a liquid medium and the concentration of Epikuron
200 is generally
in a range of from 1%-2b% by weight, the concentration of Vitamin E TPGS is in
a range of
from 1%-25% by weight, and the concentration of water is in a range of from
20%-40% by
weight based on the total composition.
An interesting acyclovir-containing composition may also be presented as a
precursor
composition, wherein the weight ratio between fatty acid ester and any liquid
medium present
in the composition is between 50:50 and 100:0 such as between 60:40 and 99:1,
between 70:30
and 90:10.
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32
In such precursor composition, the weight ratio between the sum of the
glycerylmonooleate and
any structurant(s), and_any liquid medium present in the composition is
between 90:10-and
99:0.5, such as between 90:10 and 99:1.
In precursor compositions a liquid medium like e.g. water or glycerol, or a
mixture of water and
glycerol may be present. If the liquid medium is water containing glycerol,
the glycerol:water
ratio may be up to about 2.5:1 by weight, such as up to corresponding to a
glycerol:water ratio of
1.5:2 such as, e.g., a ratio of about 1:1, 0.5:1, or 0.25:1.
Other interesting compositions are compositions comprising glyceryimonooleate,
phosphatidylcholine (or Vitamin E TPGS) and, optionally, water and the weight
ratio between
the content of phosphatidylcholine (or Vitamin E TPGS) and glycerylmonooleate
is at the most
1, such as e.g. 1:1, 1:2 or 1:4. In such compositions water may be present in
a concentration of at
the most 40% w/w based on the total composition.
Although the active substances of relatively low solubility as discussed above
are of particular
importance for use in compositions according to the invention, the invention
is not limited to
such active substances. Thus, in other aspects of the invention the active
substance can in
principle be any active substance irrespective of its solubility.
Lipophilicity of the active substance
The active substance may have any degree of lipophilicity. In certain
interesting compositions,
the active substance is one which has a lipophilicity of at the most 100, such
as at the most, e.g.,
75, 50, 25, 10, ?.5, 5 or 2.5, expressed as the partition coefficient between
octanol and 0.05M
phosphate buffer, pH 7, at 20°C, in some a partition coefficient of at
the most 10 or even at the
most 1 or at the most 0.75, 0.5, 0.1, 0.075, 0.05 or 0.04.
Alternatively, the lipophilicity may be expressed as the partition coe~cient
between octanol and
an appropriate buffer having a pH corresponding either to the pH of the liquid
crystalline phase
or to the pH at which the active substance has its minimum solubility. In such
cases, the values
mentioned above are also valid.
Factors influencing the absorption or penetration of the active substance
It is known that the active substance must have balanced properties with
respect to aqueous
solubility and partition coefficient.
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With respect to percutaneous absorption of active substances the vehicle in
which the active
substance is located is Qf importance. Thus, the affinity of the active
substance to the vehicle
compared with that of the active substance to the skin or the rate-limiting
barrier of the skin
must be of a less order of magnitude as the active substance otherwise would
predominantly
would be maintained in the vehicle and only slowly be released from the
vehicle and penetrate
the skin and thus enable the active substance to reach the target for the
disease. Initial studies
performed by the inventor show that vehicles on which compositions according
to the invention
are based readily releases the active substances tested (e.g. acyclovir) so
that the active
substances are available for penetration, i.e. a balanced affinity
(vehicle/skin) has been obtained
in these compositions.
Release of active substance from a composition according to the invention
With respect to acyclovir, it is believed that a composition with improved
release properties and
which sticks better to the skin can improve the treatment when compared to
prior art
compositions such as Zovir~ cream or Zovirax~ cream. The object of the present
invention has
therefore inter alia been to develop a bioadhesive composition containing e.g.
acyclovir or other
antiherpes virus agents with improved release properties so that fewer daily
applications are
needed to produce the same therapeutic effect (bioequivalence) or even improve
the therapeutic
effect.
As appears in more detail in the Experimental section herein, the present
inventors have
developed compositions containing GMO/water 65/35% w/w with acyclovir
(crystalline and
micronized, respectively) added in a concentration of 1-40% w/w. Cubic liquid
crystalline phases
are obtained in these compositions as evidence by polarized light. The results
indicate that
acyclovir in the concentration range investigated doss not ruin the cubic
lattice, and that
acyclovir probably is inert in the cubic system. The distribution of the drug
crystals in the cubic
liquid crystalline phase appears as a homogeneous distribution (observed by
microscopy). The
cubic liquid crystalline phase without drug is transparent and has a
relatively high viscosity. It is
cosmetically appealing. When acyclovir is added, the viscosity is increased
with the
concentration, especially for the micronized quality. When the crystalline
quality is added, the
composition becomes greyish white. When the cubic liquid crystalline phase is
applied to human
skin it "melts" (gets softer) and penetrates the skin.
Furthermore, the inventor has developed compositions wherein the GMO has been
substituted
by a structurant and/or by a pharmaceutically acceptable excipient. The
release of acyclovir from
such compositions is described in the Experiments. The results show that the
structurant
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34
and/or certain pharmaceutically acceptable excipients (as defined in the
claims) do not
significantly influence the release rate of acyclovir in a negative manner. -
As mentioned above, Zovir~ and Zovirax~ cream containing 5% w/w acyclovir are
presently the
drugs of choice for the treatment of herpes simples. In order to compare the
release rate of
acyclovir from Zovir~ cream and a cubic liquid crystalline phase (GMO/water
65/35 % w/w)
containing 5% w/w acyclovir, the release of acyclovir from these compositions
was examined, cf.
Example 16 herein. Comparing the rate constants it is seen that the release
rate of acyclovir is
about 5-6 times faster from the cubic liquid crystalline phase than from the
Zovir~ cream. Poor
release properties of the Zovir~ cream are most likely one of the reasons for
its suboptimum
therapeutic effect. The improved release properties from the cubic liquid
crystalline phase must
therefore be seen as a very promising result.
While the present invention is not to be limited to any theory, it is
believed, and supported by
experimental data reported herein, that the capability of the composition to
release the active
substance of very low water solubility and very low solubility in the liquid
crystalline phase at
very satisfactory release rates is due to some kind of efficient dissolution
system for particles,
such as crystals, of the active substance through the liquid medium "channels"
of the liquid
crystalline phase.
The performance of the compositions according to the invention with respect to
releasing the
active substance from the liquid crystalline phase can be adequately expressed
by the slope of
the cumulative release in ~.g as a function of the square root of the release
time in hours in the
release experiment defined in connection with Fig. 13 (in which the
concentration of the
substance is 5%). In preferred compositions according to the invention, the
slope is at least 50,
more preferred at least 100.
An expression of better performance is a slope of at least 200, such as at
least 300, or at least
500 or even at least 700 or at least 900.
Concentration of active substance in a composition according to the invention
Important embodiments of the present invention are compositions in which the
active substance
is present in a concentration which is above the saturation concentration at
20°C so that part of
the active substance, and in many cases the predominant proportion of the
active substance, is
present in the form of particles, such as, e.g., crystals. In such a case,
normally at least 25%,
such as at least 50%'0, by weight of the active substance present in the
composition constitutes a
proportion which is present above the saturation concentration at 20°C.
Very valuable
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compositions according to the invention are compositions, wherein at least
75%, such as at least
90% or even at least 95% or at least 98% by weight of the active substance
present in the -
composition constitutes a proportion which is present above the saturation
concentration at
20°C.
5 In general, the concentration of the active substance in the composition
will depend on the
condition to be treated or prevented and the desired or necessary
administration frequency. The
concentration of the active substance in a pharmaceutical composition depends
on the nature of
the second compound in question, its potency, the severity of the disease to
be prevented or
treated, and the age and condition of the patient. Methods applicable to
selecting relevant
10 concentrations of the active substance in the pharmaceutical composition
are well known to a
person skilled in the art and may be performed according to established
guidelines for good
clinical practice (GCP) or Investigations! New Drug Exemption ("IND")
regulations as described
in e.g. Drug Applications, Nordic Guidelines, NLN Publication No. 12, Nordic
Council on Me-
dicines, Uppsala 1983 and Clinical Trials of Drugs, Nordic Guidelines, NLN
Publication No. 11,
15 Nordic Council on Medicines, Uppsala 1983, or CPMC/E.U. Guidelines far Good
Clinical
Practice 95/135. A person skilled in the art would, by use of the methods
described in standard
textbooks, guidelines and regulations as described above as well as common
general knowledge
within the field, be able to select the exact dosage regimen to be implemented
for any active
substance and dosage form using merely routine experimentation procedures.
20 Bioadhesiveness of the compositions according to the invention
As mentioned above, it is a great advantage of the compositions according to
the invention that
the second substance especially fatty acid esters can confer bioadhesiveness
to the compositions.
During the last decade increased attention has been given to the possibility
of using
bioadhesive/mucoadhesive polymers for drug delivery purposes. It is believed
that several
25 problems associated with conventional controlled release drug delivery
systems may be reduced
or eliminated by using a bioadhesive/mucoadhesive drug delivery system. In
conventional
controlled release drug delivery systems no precautions are made in order to
localize the delivery
system after administration and, furthermore, the contact time in yiyo between
the drug
delivery system and a particular site is often so short that no advantages are
to be expected with
30 respect to, e.g., modifying tissue permeability. Compared with conventional
controlled release
drug delivery systems, bioadhesive drug delivery systems are believed to be
beneficial with
respect to the following features:
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36
i) a bioadhesive drug delivery system localizes a drug substance in a
particular region,
thereby improving and enhancing the bioavailability for drug substances which
may have
poor bioavailability in themselves,
ii) a bioadhesive drug delivery system leads to a relatively strong
interaction between a
bioadhesive substance and a mucosa; such an interaction contributes to an
increasing
contact time between the drug delivery system and the tissue in question and
permits
localization of the drug delivery system to a specific site,
iii) a bioadhesive drug delivery system is contemplated to prolong delivery of
drug substances
in almost any non-parenteral route,
iv) a bioadhesive drug delivery system can be localized on a specific site
with the purpose of
local therapy e.g. treatment of local fungal diseases, permeability
modification, protease
and other enzyme inhibition, and/or modulation of immunologic expression,
v) a bioadhesive drug delivery system may be targeted to specific diseased
tissues, and
vi) a bioadhesive drug delivery system may be employed in those cases where
the
conventional approach to controlled release drug delivery is unsuitable, i.e.
for certain
drug substances or classes of drug substances which are not adequately
absorbed.
Thus, preferred compositions according to the present invention are
compositions in which the
second substance like a fatty acid ester or combination of fatty acid esters
present in the
composition complies with the requirements of bioadhesion defined herein when
tested for
bioadhesion in an in vivo model or any other bioadhesivity model as given in
the experimental
section herein. Especially preferred are compositions which in themselves
comply with the
requirements of bioadhesion defined herein when tested for bioadhesion in an
in vivo model or
other bioadhesivity model as given in the experimental section herein.
Thus, interesting compositions are compositions in which the second substance
like e.g. a fatty
acid ester or combination of fatty acid esters optionally in combination with
the structurant,
when tested in a bioadhesive test system, comprising:
i) placing a segment of longitudinally cut rabbit jejunum on a stainless steel
support in such
a manner that the mucosa layer of the jejunum is placed upside so as to allow
application
of said second substance and any structurant,
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37
ii) placing the resulting support at an angle of -21° t 2° in a
cylindrical cell thermostated at
37°C t 0.5°C and-with the relative humidity kept at about 100%,
iii) flushing the jejunum on the support with 0.02M isotonic phosphate buffer
solution (pH
. 6.5, 37°C) for 5 min at a flow rate of 10 ml/min,
iv) applying an accurately weighed amount of a sample of said second substance
and any
structurant (about 100 mg) on a surface area (about 0.8 x 6 cm) of the mucosa
of the
jejunum on the support,
v) dropping about 0.5 ml of said phosphate buffer solution on the sample
applied,
vi) leaving the resulting sample from step v) for 10 minutes in said cell to
allow the sample to
interact with glycoproteins of the jejunum,
vii) flushing the jejunum with the sample applied with said phosphate buffer
solution (pH 6.5,
37°C) for 30 minutes at a flow rate of 10 ml/min, -
viii) collecting the washings resulting from step vii), and
ix) calculating the residual amount of the sample remaining on the jejunum by
measuring
the amount of the sample in the washings or by measuring the amount remaining
on the
jejunum,
results in a residual amount of at least 60% w/w, in particular a residual
amount of at least
?0% w/w, such as at least 80% w/w, preferably at least 85% w/w and more
preferably at least
90% w/w.
Interesting compositions are also compositions as defined further above which,
when tested in
the jejunum test system defined in claim above, result in a residual amount of
at least 40% w/w
of the second substance such as a fatty acid ester or combination of fatty
acid esters or at least
40% w/w of the active substance.
A measure of the bioadhesivity of a composition itself is that it complies
with the requirements
for bioadhesion defined herein when tested for bioadhesion in the in vivo
model described herein
involving testing the rinsing off ability from skin.
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38
As mentioned above, the biopharmaceutical properties of a composition
according to the
invention are not significantly changed by adding a structurant or a
pharmaceutically-acceptable
excipient. Thus, the score obtained in the test for bioadhesion (washing off
ability from the skin)
is substantially of the same order of magnitude as would have been obtained
for a comparative
composition wherein the structurant(s) and/or pharmaceutically acceptable
excipient has(have)
been replaced with the same amount by weight of said second substance.
As evidenced in the Examples herein, an active substance does not
significantly influence the
bioadhesive properties of a vehicle provided that the concentration of the
active or protective
substance is relatively low such as at the most about 10-15% w/w or at the
most about 8-
10% w/w. The kind of active substance (structure, molecular weight, size,
physico-chemical
properties, loading, pKa, solubility, etc.) will of course be responsible for
the maximal
concentration which can be incorporated in the vehicle without significantly
affecting the
bioadhesive properties of the composition. In the Examples herein, it is also
demonstrated that
the active substance locates in the liquid crystalline phase of the fatty acid
ester and most likely
the solubility of the active substance in this phase has impact on the
bioadhesive properties as
well as on the release properties of the composition.
Administration mutes and pharmaceutical compositions
As mentioned above, the application is intended for skin or mucosa. Other
applications may of
course also be relevant such as, e.g., application on dentures, prostheses and
application to body
cavities such as the oral cavity. The mucosa is preferably selected from oral,
nasal, aural, lung,
rectal, vaginal, and gastrointestinal mucosa.
A bioadhesive composition for administration according to the invention may in
special cases
also be in the form of a multiple unit composition, in the form of, e.g., a
powder. A multiple unit
composition may be administered to skin or mucosa, preferably the mucosa is
selected from oral,
nasal, rectal, aural, vaginal, lung, and gastrointestinal mucosa. Most
preferred is a bioadhesive
composition intended for administration to the gastrointestinal tract.
Bioadhesive compositions according to the invention for application on skin
and especially to
wounds may in certain cases comprise a polysaccharide in a concentration of at
least 15% w/w,
calculated on the total weight of the composition. The polysaccharide is
preferably selected from
the group consisting of carmelose, chitosan, pectins, xanthan gums,
carrageenans, locust bean
gum, acacia gum, gelatins, alginates, and dextrans, and salts thereof. The
compositions are easy
to apply on the wound and are believed to be able to extract water from the
wound and thereby
drying the wound.
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Apart from the active or protective substance and the bioadhesive fatty acid
ester substance, the
bioadhesive compositions for use according to the invention may comprise
pharmaceutically or
cosmetically acceptable excipients or additives normally used in
pharmaceutical compositions.
The bioadhesive compositions may be in form of, e.g., a spray, a solution, a
dispersion, a
suspension, an emulsion, powders, gels including hydrogels, pastes, ointments,
creams, drenches,
delivery devices, suppositories, enemas, implants, aerosols, microcapsules,
microspheres,
nanoparticles, liposomes, dressings, bandages, plasters, tooth paste, dental
care compositions,
and in other suitable form.
The bioadhesive compositions may be formulated according to conventional
pharmaceutical
practice, see, e.g., "R,emington's Pharmaceutical Sciences" and "Encyclopedia
of Pharmaceutical
Technology", edited by Swarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New
York, 1988.
Pharmaceutically acceptable excipients for use in bioadhesive compositions for
use according to
the invention may be, for example,
inert diluents or fillers, such as sucrose, sorbitol, sugar, mannitol,
microcrystalline cellulose,
carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose,
ethylcellulose,
starches including potato starch, calcium carbonate, sodium chloride, lactose,
calcium phosphate,
calcium sulfate or sodium phosphate; and
lubricating agents including glidants and antiadhesives, for example,
magnesium stearate, zinc
stearate, stearic acid, siiicas, hydrogenated vegetable oils or talc.
Other pharmaceutically acceptable excipients can be colorants, flavouring
agents, plasticizers,
humectants, buffering agents, solubilizing agents, release modulating agents,
etc.
For application to the rectal or vaginal mucosa suitable compositions for use
according to the
invention include suppositories (emulsion or suspension type), solutions,
enemas, and rectal
gelatin capsules (solutions or suspensions). Appropriate pharmaceutically
acceptable suppository
bases include cocoa butter, esterified fatty acids, glycerinated gelatin, and
various water-soluble
or dispersible bases like polyethylene glycols and polyoxyethylene sorbitan
fatty acid esters. Vari-
ous additives like, e.g., enhancers or surfactants may be incorporated.
For application to the nasal mucosa, nasal sprays and aerosols for inhalation
are suitable
compositions for use according to the invention. In a typically nasal
formulation, the active
ingredients are dissolved or dispersed in a suitable vehicle. The
pharmaceutically acceptable
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vehicles and excipients and optionally other pharmaceutically acceptable
materials present in the
composition such as diluents, enhancers, flavouring agents, preservatives etc.
are all selected in
accordance with conventional pharmaceutical practice in a manner understood by
the persons
skilled in the art of formulating pharmaceuticals.
5 For application to the oral cavity, teeth, skin or nail, the compositions
for use according to the
invention may contain conventionally non-toxic pharmaceutically acceptable
carriers and
excipients including microspheres and liposomes. The formulations include
creams, ointments,
lotions, liniments, gels, hydrogels, solutions, suspensions, sticks, sprays,
pastes, dressings,
bandages, plasters, tooth paste, dental care compositions, and the like. The
pharmaceutically
10 acceptable carriers or excipients may include emulsifying agents,
stabilizing agents, antioxidants,
buffering agents, preservatives, humectants, penetration enhancers, chelating
agents, gelforming
agents, ointment bases, perfumes and skin protective agents.
Examples of emulsifying agents are naturally occurring gums, e.g. gum acacia
or gum
tragacanth, naturally occurring phosphatides, e.g. soybean lecithin and
sorbitan monooleate
15 derivatives.
Examples of antioxidants are butylated hydroxy anisole (BHA), ascorbic acid
and derivatives
thereof, a-tocopherol and derivatives thereof, vitamin E, salts of sulphur
dioxide, cysteine, citric
acid, ascorbyl palmitate, butylhydroxytoluene, complexing agents, chelating
agents, sodium
pyrosulfite, EDTA and gallic acid esters.
20 Examples of preservatives are parabens, such as methyl, ethyl, propyl p-
hydroxybenzoate,
butylparaben, isobutylparaben, isopropylparaben, potassium sorbate, sorbic
acid, benzoic acid,
methyl benzoate, phenoxyethsnol, bronopol, bronidox, MDM hydantoin,
iodopropynyl
butylcarbamate, EDTA, propyleneglycol (increases the solubility of
preservatives) benzalconium
chloride, benzylalcohol, chlorhe~dine diacetate, chlorhexidine digluconate,
chlorbutol,
25 phenetanol, phenols (phenol, o-cresol, p-cresol, chlorcresol, tricresol),
alkanols (chlorbutanol,
phenetanol), sorbic acid, and mercuri-compounds Iike e.g.
phenylmercurinitrate.
Examples of humectants are glycerin, propylene glycol, sorbitol and urea.
Examples of suitable release modulating agents for use according to the
invention are glycerol,
sesame oil, soybean oil, lecithin and cholesterol.
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Examples of penetration enhancers are oleic acid, propylene glycol, DMSO,
triethanolamine,
N,N-dimethylacetamide,. N,N-dimethylforma.mide, 2-pyrrolidone and derivatives
thereof,- -
tetrahydrofuryl alcohol and Azone.
Examples of chelating agents are sodium EDTA, citric acid and phosphoric acid.
Examples of other excipients for use in compositions for use according to the
invention are
edible oils like almond oil, castor oil, cacao butter, coconut oil, corn oil,
cottonseed oil, linseed oil,
olive oil, palm oil, peanut oil, poppyseed oil, rapeseed oil, sesame oil,
soybean oil, sunflower oil,
and teaseed oil; and of polymers such as carmelose, sodium carmelose,
hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,
chitosane, pectin,
xa.nthan guru, caxrageenan, locust bean gum, acacia gum, gelatin, and
alginates, and solvents
such as, e.g., glycerol, ethanol, propylene glycol, polyethylene glycols such
as PEG 200 and PEG
400, Pluronic, polysorbate, and ethylene glycol.
Examples of ointment bases are beeswax, paraffin, cetyl palmitate, vegetable
oils, sorbitan esters
of fatty acids (Span), Carbopol, polyethylene glycols, and condensation
products between sorbitan
esters of fatty acids and ethylene ode, e.g. polyoxyethylene sorbitan
monooleate (Tween).
A most important composition according to the invention is one in which the
antiviral substance
is acyclovir. Examples of important embodiments hereof and of other
compositions according to
the invention containing nucleosides of low solubility as defined herein are
claimed in the claims
are described in detail in the Examples.
Furthermore, relevant compositions and conditions to be fulfilled for the
individual components
in the compositions are claimed in the claims and described in the Examples.
Other aspects of the invention
The invention also relates to methods for preparing the compositions according
to the invention.
Details concerning the preparation are given in the Examples herein.
Furthermore, the
invention also relates to a method for administering an active substance to
e.g. a human, the
method comprising administering to the human in need thereof a therapeutically
and/or
prophylactically effective amount of the active substance in a pharmaceutical
composition
according to the invention.
As will be understood, details and particulars concerning the composition
aspects of the
invention will be the same as or analogous to the details and particulars
concerning the other
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42
aspects of the invention and the method aspects discussed above, and this
means that wherever
appropriate, the statements above concerning a pharmaceutical composition, a
second-substance,
a structurant, a liquid medium and a pharmaceutically acceptable excipient, as
well as improved
properties and uses apply mutatis mutandis to all aspects of the invention.
MATERIALS
Glycerylmonooleate (monoolein), manufactured by Grindsted Products A/S,
Denmark
DIMODAN~ GMO-90, a distilled monoglyceride
Chemical and physical data
Monoester content min. 95%
Diglycerides max. 3%
Triglycerides max. 0.2%
Free fatty acids max. 0.5%
Free glycerol max. 0.5%
Iodine value approx. 72
Fatty acid composition:
Oleic acid 92%
Linoleic 6%
Saturated (C 16/Cl8) 2%
Melting point 35-37°C
Antio~dants and synergists added:
Ascorbyl palmitate max. 200 ppm
a-Tocopherol max. 200 ppm
Citric acid max. 100 ppm
In the following examples, the term "GMO-90" indicates that the above-
mentioned glycerol
monooleate product is employed, except where otherwise stated.
Another quality of glycerol monooleate has been employed in some of the
following examples,
namely
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RYLO~ MG19 (with a content of about 90% GMO) manufactured by Danisco
Ingredients,
Denmark _
Chemical and physical data
Monoester content i
m
n. 95%
Free fatty acids
max. 0.5%
Free glycerol
max. 1%
Iodine value
approx. ?2
Fatty acid composition:
Oleic acid > 90%
Linoleic and Iinolenic acids < 6%
Antioxidants and synergists added:
Ascorbyl palmitate max. 200 ppm
a-Tocopherol max. 200 ppm
Citric acid max. 100 ppm
Glvcervlmonooleate 84°k "GMO-84" (monoolein), manufactured by Grindsted
Products A/S,
Denmark; the product used has a total content of fatty acid monoesters of at
least about 96%.
The product employed in the examples described herein had the following
composition of fatty
acid monoesters:
Glycerylmonooleate about 84% w/w
Glycerylmonolinoleate about 7% w/w
Glyceryl monopalmitate about 3% w/w
Glyceryl monostearate about 4% w/w
In the following examples, the term "GMO 84" indicates that this glycerol
monooleate product is
employed.
Other commercially available glycerol monooleate products (e.g. Myverol 18-99
and GMOrphic
80 available from Kodak Eastman, U.S.A.) which differ in the composition of
fatty acid
monoesters compared with the products described above may also be applied.
Glycerylmonolinoleate (Dimodan~ LS), manufactured by Grindsted Products A/S;
the product
used has a total content of fatty acid monoesters of at least about 90% such
as about 96% w/w.
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The product employed in the examples described herein had the following
composition of fatty
acid monoesters: - - - -
Glyceryl monopalmitate about 6% w/w
Glyceryl monostearate about 6% w/w
Glycerylmonooleate about 22% w/w
Glycerylmonolinoleate about 63% w/w
Other commercial available glycerylmonolinoleate products (such as, e.g.,
Myverol~ 18-92
available from Kodak Eastman, U.S.A.) which differ in the composition of fatty
acid monoesters
compared with the product described above may also be applied.
Phosohatidvlcholine (Epikuron available from Lucas Meyer, Hamburg, Germany):
Lipoid S100 or S75 (purified Soya phosphatidylcholine available from Lipoid
GmbH, Germany)
Epikuron 200 (purified Soya phosphatidylcholine):
Phosphatidylcholine of soyabean origin
Characteristics: EPIKURON 200 is a purified phosphatidylcholine of soybean
origin.
Composition: The product consists of phosphatidylcholine, a small amount of
lyso-phosphatidylcholine and other phospholipids.
phosphatidylcholine min. 92%
lyso-phosphatidylcholine max. 3%
other phospholipids max. 2%
moisture max. 0.8%
oil content max. 1.0%
a-Tocopherol 0.2%
fatty acids (total content)
including:
palmitic acid/stearic acid 16-22%
. oleic acid 8-12%
-. _._ ..____ . _. . , , . _.
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linoleic acid 62-66%
- linolenic acid 6-8% - -
Epikuron 145 V:
. Deoiled, fractionated soybean lecithin
5 Characteristics: EPIKURON 145 V is a fractionated, wax-like soybean lecithin
with enriched content of phosphatidylcholine for the use in
pharmaceutical industry.
Composition: Mixture polar (phospho- and glyco-) lipids and a small amount of
carbohydrates.
10 phosphatidylcholine min. 45%
phosphatidylethanolamine min. l0%
phosphatidylinositol max. 3%
phosphatidic acid mag. 3%
lyso-phosphatidylcholine max. 4%
15 other phospholipids max. 18%
glycolipids max. 15%
moisture max. 0.6%
oil content max. 2.0%
a-Tocopherol 0.2%
20 fatty acids (total content)
including.
palmitic acid/stearic acid 18-22%
oleic acid 6-10%
linoleic acid 62-66%
25 linolenic acid 6-8%
- Miconazol base available from MedioLast SPA, Milano, Italy
Lidocaine hydrochloride available from Sigma Chemical Co., St. Louis, U.S.A.
. Lidocaine base available from Sigma Chemical Co., St. Louis, U.S.A.
Acvclovir (crystalline) available from Chemo Iberica, Spain, e.g. a quality
where 90-100% of the
30 crystals have a particle size of less than 100 ~m
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Acyclovir (micronized) available from Chemo Iberica, Spain, e.g. a quality
where 100% of the
particles have a particle size under 24 ~m and not less than 90% under l2pm - -
-
Ethanol available from Danisco A/S, Denmark, complies with the DLS standard
(98.8-100% w/w
ethanol)
Sesame oil available from Nomeco, Denmark
Sovbean oil available from Nomeco, Denmark
Glycerol available from Joli Handel ApS, Denmark
Lecithin Epikuron 200 or Epikuron 145 from Lucas Meyer
Benzvl alcohol available from Merck AG, Germany
Water, purified or distilled water
DEAE-dextran (MW = 500,000) available form Sigma Chemical Co., St. Louis,
U.S.A.
Sodium alginate (Sobalg FD 120) available from Grindsted Products A/S, Denmark
Hvdroxvnronylmethylcellulose (Methocel K15MCR Premium USP) available from
Colorcon
Limited, U.S.A.
Carbonol 934 available from The BFGoodrich Company, U.S.A.
Vitamin E TPGS (d-a-tocopherylpolyethyIeneglycol 1000 succinate) available
from Kodak
Eastman (in the following designated TPGS)
Aspirin available from Sigma, Chemical Co., St. Louis, U.S.A.
Propylene alvcol available from BASF Aktiengesellschaft, Germany
a-tocot~herol available from BASF Aktiengeselischaft" Germany
Paraffin oil available from Unichem, Denmark
Polyethylene glycol 200 available from Unichem, Denmark
Lactose available from Inkem
Hydroxvnronylcellulose available from Aldrich Chemical Company, U. S. A.
Lanolin available from Westbrook Lanolin Company
Sorbitan ester available from Masimes ApS, Denmark
Heating and cooling stage, Linkam Peltier Stage and Controller, PE 60 for
microscope
Ultra-Turrax T25 homogenisator
400 Watt Ultrasonic Processors VCX 400
Coulter Multisizer II (Coulter), Malvern 2600 droplet and particle size
analyse (for the
determination of particle size distribution).
Strolein Areameter and Coulter SA3100 for the determination of the surface
area of the
particles.
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4?
METHODS
Test systems for bioadhesion
1. In vitro test system for bioadhesion by means of rabbit jejunum membranes
The test system for bioadhesion described in the following is a modified
system of a method
described by R,anga Rao & Buri (Int. J. Pharm. 1989, 52, 265-270).
Male albino rabbits (3-4 kg, New Zealand white rabbit SSC: CPH) were fasted
for 20 hours
before they were killed by means of a pentobarbital sodium injection. The
intestines of the
rabbits were dissected and placed in an isotonic 0.9% sodium chloride solution
at room
temperature (about 18°C). Within 30 minutes the jejunums were cut and
washed with 0.9%
sodium chloride solution. The lumens were gently rinsed with the saline until
the intestines
were clean. The jejunums were cut into pieces of about 8-9 cm in length and
frozen (-20°C)
immediately. The jejunums were stored up to 3 months before use (when
performing the test
described below it was found that the use of fresh jejunum or, alternatively,
jejunum which had
been frozen for up to 3 months gave reproducible and significantly similar
results). Before
testing, the segment of jejunum was gently thawed out.
The segment of the jejunum was cut longitudinally. It was placed on a
stainless steel support (a
tube of 2 cm in diameter and cut longitudinally at an axis parallel to its
centre) with the mucosa
layer upside, spread and held in position on the support by the adhesive
effect of the jejunum
itself. The support with the jejunum was placed at an angle of from about -
5° to about -25° such
as -7° or -21° (in the F~xamples the angle applied is denoted
"angle" in a cylindrical cell
thermostated at 37°C. A schematic illustration of the cell is shown in
Fig. 1. The relative
humidity in the thermostated cell was kept at about 100%. The jejunum was then
flushed with a
medium of 0.02M isotonic phosphate buffer solution (pH 6.5, 37°C) for 2
or 5 minutes (in the
following denoted "initial rinsing period") at a flow rate of 5 or 1Q ml/min
(in the following
denoted "initial rinsing flow"), respectively, using a peristaltic pump to
equilibrate the jejunum
with the buffer and to rinse off loose mucosa. [Immediately before application
of the sample, the
support was positioned at a horizontal position and after application the
position was changed to
the initial position of -21°.] An accurately weighted amount of the
sample to be tested for
bioadhesive properties (about 50-150 mg) was placed evenly on the mucosa of
the jejunum
(about 0.8 x 6 em). About 1 ml of the buffer solution was carefully dropped
evenly on the sample
applied to ensure forma'on of such a liquid crystalline phase, if possible (in
the case of
monoolein, the liquid crystalline phase may be the cubic, hexagonal, reverse
hexagonal, micellar,
reverse micellar, or lamellar phase). [In those cases where the viscosity of
the test sample are
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48
relatively high or where a precipitation has taken place, the test sample is
gently melted on a
heating plate or in an oven at a temperature of mar. 60°C in the case
of GMO or GML and
cooled to a temperature of at the most about 40°C before application on
the rabbit jejunum.]
Immediately after, the segments were left for 5-20 minutes such as, e.g., 10
minutes in the cell
allowing the sample to interact with the glycoproteins of the jejunum and to
prevent drying of
the mucus. After 10 minutes, the segments were flushed evenly with the
isotonic 0.02M
phosphate buffer solution (pH 6.5, 37°C) for 15-f0 minutes such as,
e.g., 30 minutes at a flow
rate of 5-I5 ml/min such as 10 ml/min (in the Examples denoted "flow rate").
The tip of the
tube carrying the buffer solution was placed 3-4 mm above the jejunum to
ensure an even liquid
flow over the mucosa. The washings were collected into a beaker. The amount of
bioadhesive
component remaining on the jejunum was calculated either by measuring the
amount of sample
in the beaker or by measuring the amount of sample remaining in the jejunum by
means of a
suitable analysis method, e.g. HPLC.
At the end of the experiment, the remaining sample on the jejunum was checked
with a pair of
tweezers to reveal false positive results.
In 1-2 test runs) out of 10, false negative results were observed probably due
to a loose mucosa
layer on the rabbit jejunum.
During testing and validation of the method, the parameters given above were
varied (e.g. the
angle applied, the flow rate, the amount applied, etc.). In order to exclude
false negative and
false positive results it was found that the following conditions were
satisfactory:
Time for prehydration before application of sample:
10 min
Amount applied: about 50-150 mg (tests have shown that a variation in the
amount
applied within a range of from about 25 mg to about 22b mg was
without significant influence on the results obtained)
Angle: -21°
Flow rate: 10 ml/min
Flow period: 30 minutes (it was found that a flow period of at least 10
minutes gives
reproducible results and a prolongation of the period to about 60
minutes does not significantly change the result)
Furthermore, it was found advantageous that the method allows rinsing of the
sample applied
on the jejunum by an aqueous medium, thus allowing a liquid crystalline phase
to be formed.
The method also permits application of fluid samples and pellets.
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49
Determination of the bioadhesiveness of a test sample
In those cases where the test sample is a second substance like a fatty acid
ester optionally in
combination with a structurant, the substances) is/are considered as
bioadhesive if the residual
amount is at least about 60% w/w such as at least about 65% w/w, about 70%
w/w, about
75% w/w, about 80% w/w, about 85% w/w, 90% w/w, or about 95% w/w.
In those cases where the test sample is a composition comprising a combination
of a second
substance like e.g. a fatty acid ester and an active substance and,
optionally, other substances
like structurant and/or excipients, the composition is considered bioadhesive
if the residual
amount (of the second substance like e.g. the fatty acid ester, or the active
substance) is at least
about 40% w/w such as at least about 45% w/w, about 50% w/w, 55% w/w, 60% w/w,
65% w/w, 70% w/w, 75% w/w, or 80% w/w.
In the present context evaluation of the bioadhesive properties of a substance
may also be
performed by use of the test system and test conditions described above but
modified with
respect to type of membrane, amount applied of test sample, test angle, flow
rate, medium, etc.
In this connection, tests have been performed in order to evaluate the
influence of different
membranes on the test results. The following results were obtained using the
above-mentioned
test conditions (angle: -21°, flow rate: 10 ml/min, and flow period: 30
min) and applying GMO
on the membrane:
Membrane Bioadhesion
Residual amount %
rabbit jejunum 90
pig ileum 106*
pig stomach 106*
buccal pig mucosa 88
* the high result is most likely due to an interference from the intestines or
the stomach
2. In vitro test system for bioadhesion by means of tensiometry
The test system for bioadhesion described in the following is a modified
system of a method
described by Tobyn, M., J. Johnson & S. Gibson (in "Use of a TA.XT2 Texture
Analyser in
Mucoadhesive Research", International LABMATE, 1992, XVII (issue VI), 35-38).
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The test system involves measuring the tensile force required to break an
adhesive bond formed
between a model membrane and a test sample (i.e. the sample which is tested
for its bioadhesive
properties).
The test apparatus employed in the following is a TA.XT2 Texture analyser
(Stable Micro
System Ltd., Haslemere, UK) (Fig. 2) equipped with a 5 kg load cell interfaced
with an IBM PC
computer running XT-R.A dimension software, DOS version. The test enables
measuring the
strength of adhesive banding established by contacting a model membrane, i.e.
in this case a pig
intestine segment, and the test sample. An analogous test apparatus may also
be employed.
The TA.XT2 Texture analyser apparatus is equipped with an instrument probe 1
(see Fig. 2)
10 which is movable in a vertical direction at a variable rate. During the so-
called withdrawal phase
of the testing, the instrument probe is moved upwards with a constant rate
until detachment
occurs (see below). Furthermore, the apparatus is equipped with a stationary
plate 2 on which a
first holder 3 is placed. Before and during a test run, a model membrane 4 is
fixed on this
holder, e.g. by means of a cap or double adhesive tape or glue. The area
exposed to the test may
15 be determined by the area of the probe (preferred in this case) or by the
area of the test samples
(e.g. a coated cover glass), or by the area of a holder fixed to the probe.
The accurate size of the
exposed area is used in the calculation of the adhesive strength (see below).
As mentioned above, the test involves employment of a model membrane,
primarily of animal
origin. The membrane could be e.g. rabbit, rat or pig gastric mucosa; a
segment of rabbit, rat or
20 pig intestines, e.g. a segment of rabbit jejunum; a segment of rabbit or
porcine buccal mucosa; or
a segment of rabbit, rat or pig intestines from which the mucosal layer has
been removed prior
to testing, or skin from an animal (after removal of substantially all
subcutaneous fat); or it
could be artificially or commercially available mucin.
In the tests described below, duodenum, jejunum and the upper part of ileum
from freshly
25 slaughtered pigs were used. The gut was stored on ice until it was washed
with 0.9% w/w
sodium chloride solution within 2 hours. The lumens were gently rinsed with
the saline until
the intestines were clean. The gut was cut into pieces of 3-4 cm and
immediately frozen (-20°C).
The intestines were stored up to 2 months before use. Before testing, the
segments were gently
thawed out. The gut segment was opened along the mesenteric border. Serosa and
muscularis
30 layers were removed by stripping with a pair of tweezers, taking care to
maintain the integrity
of the mucus layer. This resulted in a flattening of the originally folded
mucosal surface. Before
use the tissue was equilibrated in the testing medium for about 10 min, which
was sufficient for
the tissue to attain temperature and pH equilibrium as measured by pH paper.
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If the results obtained by use of another membrane than the one mentioned
above are compared
to the bioadhesive propErties of various substances or combinations, the
results of a reference
compound could be included. As discussed below testing of a reference sample
may also be made
as a routine. Polycaxbophil and Carbopol 934 have been found suitable as
reference compounds.
An accurate amount of a test sample (about 25-500 mg) is applied in a uniform
layer either
i) on the luminal side of the model membrane placed on the first holder,
ii) directly on the instrument probe, if necessary by means of a ca.p, a
double adhesive tape
or glue applied on the instrument probe before application of the test sample,
iii) on a cover glass which is placed on the instrument probe with the test
sample pointing
downwards, or
iv) via a probe modified in such a manner that it allows application of a
relatively low viscous
or semi-solid sample, the modified probe also allows the necessary addition of
an aqueous
medium.
In those cases where it is not possible to fig the test sample to the
instrument probe, the
apparatus may be equipped with a second holder 5 on which another model
membrane is fixed.
In such cases, the model membranes employed on the two holders are usually of
the same type.
It is also possible to fix the other model membrane directly to the instrument
probe e.g. by
means of a double adhesive tape, glue, or a cap.
For an adhesion test, a tissue (porcine intestinal mucosa) of about 3 x 3 cm
was fixed on the
tissue holder 3 with the mucosa layer upside. Before application of the
tissue, a piece of gauze
was placed directly on the tissue holder, and thereupon the tissue was placed.
This precaution is
made in order to stabilize the contact force. In order to moist the tissue and
hydrate the sample,
about 0.5 ml isotonic 0.05M phosphate buffer, pH 6.0, was added to the tissue.
Such an addition
also enables a cubic phase to be formed. The instrument probe with sample
(e.g. applied by
smearing 50-80 mg of the sample onto the probe in a thin, smooth layer, see
below) was lowered
with a test speed of 0.1 mm/sec in order to bring the tissue and the sample in
contact under a
constant force. The contact area was either 1.33 cm2 (cover glass) or 1.27 cm2
(probe) depending
on the method of sample preparation. The contact force was set to 0.2N and the
contact time
was 30 min. After 30 min the probe was withdrawn with a rate of 0.1 mm/sec
(post test speed)
for 10 mm. Initial experiments showed that this distance was well beyond the
point where the
sample and mucous separated during withdrawal.
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The peak detachment force and the area under the force/time curve was
calculated
automatically using the XT-R,A dimension software. The work of adhesion (mJ cm-
2), said-to be
the most accurate predictor of mucoa.dhesive performance, was calculated.
Sample preparation
Application method of the polymers used as reference:
Cover glasses having a diameter of 13 mm (area 1.33 cmz) were coated with the
polymers under
investigation by pipetting 100 ~1 of a 1% w/w solution of methanol or water in
the center of the
glass plate. After drying for 2 hours at 60°C in an oven, a thin
polymer film remained. One cover
glass was attached to the probe (diameter of 12.7 mm) with its non-coated side
by means of
double adhesive tape.
Cover glasses and mucosa were only used once (i.e. for one measurement).
Application of compositions:
A. Melting (if possible) of the solid or semi-solid composition and dipping
the probe into it
(this method is only used if the melting procedure does not change the
properties of the
composition). The sample (25-100 mg) was applied to the probe in a smooth
layer by
dipping the probe into melted GMO. The sample was solidified at room
temperature or, if
necessary, by cooling.
B. Smearing 25-100 mg of the sample directly on the probe.
C. Fixing the sample by means of a cap, double adhesive tape, or glue
Test runs are performed after the tissue has equilibrated in an aqueous medium
at room
temperature for 5-20 min. Then the tissue was removed from the aqueous medium
and placed
in the test apparatus and then the test was run.
In some cases, variations of the above-given method may be relevant, e.g.
running the test in an
aqueous medium or running the test at a temperature different from room
temperature such as
37°C.
Furthermore, the test parameters may be varied, e.g, as follows:
Hydration time: 0 - 20 min
Contact time: FO sec - 50 min
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Contact force: 0.05-0.4N
Equilibration medium
Test speed: 0.02-1 mm/sec
Post test speed: 0.02-1 mm/sec
Test run temperature may be changed by employing a suitable temperature
controlled oven such
as a SMTC/04 from Stable Microsystems, Haslemere, UK.
Determination of the bioadhesive properties of a test sample
In order to test whether a test sample is bioadhesive, two test runs are
performed:
1. A test run with the test sample applied (result: work of adhesion WA5),
2. A test run with a known and excellent bioadhesive sample (e.g.
polycarbophil) (result:
work of adhesion WAR).
In both cases the work of adhesion is calculated and the test sample is
considered bioadhesive if
WAS/WAR x 100% is at least 30%, such as 35%, 40%, 45%, 50%, or 55%. In
general, a sample is
graded to be a weak bioadhesive if the result is less than about 30%, a medium
bioadhesive if
the result is about 30%-50%, a strong bioadhesive if the result is at least
50%.
Polycaxbophil (Noveon~ AA-1, BF Goodrich, Hounslow, U.K.) is a high molecular
weight
poly(acrylic acid)copolymer loosely cross-linked with divinyl glycol. On
account of its known
excellent mucoadhesive properties, this polymer serves as a reference. Before
testing in the
above-mentioned tensiometric test, a polycarbophil gel is prepared by mixing
polycarbophil with
water or methanol (resulting concentration about 10-20 mg ml-1) and the
mixture is allowed to
hydrate at room temperature for 24 hours. The polymer solution is periodically
stirred. The
resulting gel is applied on a cover glass and tested as described above and
the result obtained is
used as a reference value for excellent bioadhesive substances.
Similarly, other substances which are known bioadhesive substances are tested
such as, e.g.,
chitosane, tragacanth, hydroxypropylmethylcellulose (HPMC), sodium alginate,
hydroxypropylcellulose (HPC), karaya gam, carboxymethylcellulose (CMC),
gelatin, pectin,
acacia, PEG 6000, povidone, or DEAF-dextran (less bioadhesive than
polycarbophil). By choosing
test substances with various degrees of bioadhesiveness, an evaluation scale
can be made and
the performance of a test sample with respect to bioadhesiveness can be
evaluated. It is
contemplated that the following scale is applicable provided the test
conditions given above are
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applied. It is clear that if the test conditions are changed, another scale
may be more relevant. A
suitable scale is then to be based on the values obtained for the excellent
bioadhesive - -
polycarbophil and the weak bioadhesive such as DEAF-dextran.
Bioadhesive properties Work of adhesion (mJ cm-2)
none less than 0.005
poor about 0.005 - about 0.012
moderate about 0.012 - about 0.020
good about 0.020 - about 0.04
excellent more than 0.04
IO When testing some known bioadhesive substances and GMO, the following
results were
obtained as a mean of six experiments:
Test substance Work of adhesion (mJ
cm-2)
DEAF-dextran 0.010
Sodium alginate 0.015
GMO 84/water 85/15% 0.028
w/w*
I3PMC 0.o3s
Carbopol 934 0.031
GMO 84 0.04?
Polycarbophil 0.060
*: lamellar phase
3. In vivo test system for bioadhesion - washing off ability from the skin
A water soluble dye (Edicol Sunset Yellow, E 110, Amaranth E-123, or Brilliant
Blue E 131)
and/or a lipid soluble dye (Waxoline violet A FW (Maximex), Colur flavus
insolubilis, DAK 63,
or Edilake tartrazin NS) can be added to the test sample and mixed to form a
homogeneous
mixture. In those cases where a water soluble dye is used, the dye is
preferably dissolved in an
aqueous medium before mixing. In most cases, however, a dye is not added as
the result is easily
determined visually. About 0.05-0.5 g (such as 0.2 g) of the resulting mixture
was applied in a
uniform layer on an area of about 4 cm2 of the skin of the hand or of the
wrist. The test
samples could be applied on dry skin as well as on moistened skin. In some
cases, about I0 min
before running the test, a small amount of water could be added to the test
sample applied.
Immediately after application, the test sample on the skin was subjected to
washings with water
from a tip (flow rate corresponding to about 6-8 litres/minute and a
temperature of about 35-
..
_~._~..~.... ~
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40°C). The washings were carried out for about 3 minutes. Then it is
visually assessed in which
degree the test sample is retained on the skin. The visual assessment is done
by use af-a-scale
graded from 1-5, where 5 represents total retainment of the test sample
applied on the skin and
1 represents no retainment of the test sample on the skin.
5 The test sample is evaluated to have bioadhesive properties in the present
context if the result of
the above-described test is at least 4.
The test described above has proved to be suitable when testing compositions
for
bioadhesiveness and the compositions in question have a relatively high
viscosity which makes it
difficult to apply the compositions to the rabbit jejunum model. A
modification of the test
10 described above excluding the addition of a water soluble dye has also
proved suitable for testing
compositions for bioadhesiveness.
Quantitative determinations of glycerylmonooleate and glycerylmonolinoleate by
means of HPLC
The quantitative determination of glycerylmonooleate or glycerylmonolinoleate
was made by
15 high-performance liquid chromatography (HPLC) using a Shimadzu LC-8A HPLC
pump, a
Shimadzu SPD-6A UV detector, a Shimadzu C-5A integrator and a Shimadzu SIL-6B
autosampler.
The column (25 cm x 4 mm i.d.) was packed with Supelcosil LC-18-DM and was
eluted
isocratically at ambient temperature with a mobile phase consisting of
methanol:water:acetate
20 buffer (pH 3.5) (840:120:40 v/v). However, in some cases interference from
other substances
may occur, and then it may be necessary to make minor changes in the
composition of the
eluent.
The size of a sample injected on the column was 20 pI and the flow rate was
1.2 ml/ml. The
column effluent was monitored at 214 nm.
25 Extraction procedure prior to analysis of glycerylmonooleate or
glycerylmonolinoleate in mucosa
The mucosa in question (with a second substance such as a fatty acid ester
like e.g.
glycerylmonooleate) is placed in 50.00 ml of methanol and shaken for 2 hours.
The mixture is
filtered through a 0.45 ~m filter membrane (from Millipore 16555Q) and the
filtrate is subjected
30 to HPLC analysis using the method described above.
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Recovery
In those cases where analysis is performed in order to determine the residual
amount of the
second substance such as the fatty acid ester like e.g. glycerylmonooleate on
the rabbit jejunum
segment in connection with the bioadhesive test No. 1 (above), the calculation
of the residual
amount takes into consideration an appropriate correction in the recovery.
This correction is
found based on determination of the amount of fatty acid ester on the rabbit
jejunum segment
after application of an accurate amount of the second substance (this test is
repeated 5 times
and the recovery is given as the mean value).
The recovery of about 125 mg GMO 84/ethanol 60/40% w/w on rabbit jejunum was
examined.
The recovery was found to be about 95%. The recovery was not determined for
the other
amounts of GMO/ethanol 60/40% w/w nor was it determined for GMO or GML
formulations to
which drug substances or excipients were added.
Solubility of acetylsalicylic acid (aspirin):
Wyatt D.M. and Dorschel O. A cubic phase delivery system composed of glyceryi
monooleate and
water for sustained release of water-soluble drugs, Pharm. Tech. 1992 (Oct.),
p. 116-130, disclose
an experiment in which aspirin is used. Aspirin is not a substance which has a
Iow solubility in
water at a pH prevailing in the composition such as appears from the
following.
Aqueous solubility
The solubility of the weak acid aspirin is 3.3 mg/mI in water (20°C).
It has a pKa value of about
3.5 (25°C) (Analytical Profiles). The solubility of aspirin is strongly
dependent on the pH in the
solution. The degree of ionisation of the acid group in aspirin is favoured
when the pH is around
and above the pKa value of the compound and therefore the solubility is
increased with pH >
3.4. A solubility experiment has shown that the solubility of aspirin is
greater than 10 mg/m1 in
a buffer solution of pH 3.6. The experiment was performed in an 0.5 M acetate
buffer solution
pH 4.0; the buffer was not strong enough to maintain the pH, and the pH in the
final solution
was 3.6. The solubility of aspirin in a buffer solution of pH 4.0 is > 20
mg/m1.
Solubility in GMO/water
The solubility of acetylsalicylic acid in GMO/water 65/35% w/w has been
determined to be > 20
mg/ml. During the experiment, the pH of the aqueous phase at the end of the
experiment was
,.
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4.0 and the aqueous phase used was 0.2 M acetate buffer pH 5.0 (the buffer
used was not strong
enough to maintain the. pH at 5.0) _ .
Determination of the dissolution/release rate of a pharmaceutical formulation
The dissolution rate of acyclovir in various GMO compositions was determined
using Franz
diffusion cells having a diffusion area of 1.77 cm2 and a receptor volume of
6.8 ml. The study
was run at a temperature of 37°C and as diffusion membrane a cellulose
membrane from
Medicell International Ltd. was employed. The membrane employed has a pore
size of about
2.4 nm and it retains particles having a molecular weight larger than about
12,000-14,000.
Before application, the membrane was pretreated and thoroughly rinsed with
distilled water. As
receptor medium was used an isotonic 0.05M phosphate buffer pH 6.5 (Danish
Drug Standards,
DLS) and the medium was magnetically stirred at 100 rpm.
The cellulose membrane was allowed to equilibrate at 37°C for 30 min in
the receptor medium
employed. After placing the membrane in the diffusion cell (the membrane was
supported by a
metallic grating), about 300-400 mg of the composition to be tested was
applied by means of a
syringe or a spatula and care was taken to ensure a homogenous distribution of
the composition
on the total area of the membrane available for diffusion. Alternatively, the
composition to be
tested may be filled into a dish having a well-defined surface area which is
only a little smaller
than that of the cellulose membrane held by a Franz' diffusion cell so that
almost all of the
diffusion area available is used; the dish is turned upside down and placed on
top of the cellulose
membrane. Phosphate buffer was then loaded into the receptor part (time t=0)
and at
appropriate time intervals, samples of 2.0 ml were withdrawn and analyzed for
content of
acyclovir (cf. below). This relatively high volume was withdrawn to ensure
sink condition. The
amount of receptor medium withdrawn was replaced with fresh receptor medium.
Another well-suited dissolution test system for semi-solids is the Hanson p/n-
S7-VC-IS-7 vertical
diffusion cells (12 cells) from Hanson Research Corporation, U.S.A.
Quantitative determination of miconazole and lidocaine hydrochloride,
respectively
_ Samples from Example 88 were analyzed for the content of miconazol and
lidocain
hydrochloride, respectively. The following assays were employed:
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Lidocain HC1
The content of lidocain HCl is determined by a HPLC method.
T: Dissolve the formulation in 30 ml methanol and transfer it quantitatively
to a 50 ml
volumetric flask. Add methanol to 50.00 ml.
R: Weigh out 100.00 mg lidoca.in HCI in a 100 ml volumetric flask. Dilute 1000
~1 to 50.00 ml
with mobile phase.
Analyse T and R on a suitable liquid chromatograph with W-detector and
integrator.
Column: Steel column, length 25 cm x 4.6 mm i.d.
Stationary phase:Nucleosil C-18, 10 ~m
Mobile phase: Methanol R: Acetic acid: Triethylamine:
Water (50:1.5:0.5:48)
Flow: 1.5 ml/min
Temperature: Room temperature
Detection: 254 nm
Injection: 20 ~l loop
Retention time:Lidocain HCI: about 3 min
Calculation:
AT x n(g)
Lidocain HCL recovery, %: x 100%
AR x m(g) x % lidocain HCI
where AT is the area of the test solution T;
AR is the area of the standard solution A;
n is the amount of standard weighed out (g);
m is the amount of formulation applied to the
intestine (g);
% lidocain HCl is the content of lidocain HCl in the formulation determined as
% w/w.
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Miconazol
The content of miconazol is also determined by a HPLC method.
T: Dissolve the formulation in 30 ml methanol and transfer it quantitatively
to a 50 ml
volumetric flask. Add methanol to 50.00 ml.
R: Weigh out 100.00 mg micona2ol in a 100 ml volumetric flask. Dilute 1000 ~1
to 50.00 ml
with mobile phase.
Analyse T and R on a suitable liquid chromatograph with W-detector and
integrator.
Column: Steel column, length 25 cm x
4.6 mm i.d.
Stationary phase: Spherisorb ODS 1, S5
Mobile phase: Methanol R: Buffer (85:15)
Flow: 1.0 ml/min
Temperature: 70C
Detection: 230 nm
Injection: 20 ~1 loop
Retention time:Miconazol: about 8 min
Buffer: 0.05 M NH9HzP04 (5.75 g in 1000
ml H20)
Calculation:
AT x n(g)
Miconazol recovery, %: x 100%
AR x m(g) x % miconazol
where AT is the area of the test solution T;
AR is the area of the standard solution R;
n is the amount of standard weighed out (g);
m is the amount of formulation applied to the
intestine (g);
% miconazol is the content of miconazol in the formulation determined as %
w/w.
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Quantitative determination of acyclovir
Method A
Determination of acyclovir in aqueous media by HPLC
The HPLC method employed was the following.
5 Column: 25 cm x 4.6 mm
i.d.
Stationary phase: Nucleosil C-18
Mobile phase: water:methanol
(85:15)
Temperature: Room temperature
Detection: 254 nm
10 Flow: 1 ml/min
Inj.volume: 20 ~1
Ret. time: ca. 5.4 min
In connection with dissolution/release rate experiments employing Franz
diffusion cells as
described above, the concentration in the test solution (Cn) is calculated as
follows:
15 Reference solution: An accurate amount of about 10.00 mg acyclovir is
diluted to with distilled
water to a concentration of 10.00 ~Sg/ml
Test solution: The sample withdrawn is filtered through a 0.2 pm filter and
injected onto the
column (in some cases it might be necessary to subject the sample to dilution
with water)
- A.1. x amount weieht in (me) of reference x 1000 x 5
20 ~ AR x 100 x 50
in which
AT is the area of the test solution, and
AR is the area of the reference solution.
Calculation of % released:
100 x Cn x Vt +(VS x ~ Cn_1))
n=1 x 00%'0
% of acyclovir in form. x mg of form. apphe~ x 1000
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in which
Cn is the concentratiori of drug in the receptor solution (mg/ml), _ . _
Vt is the receptor volume (unless otherwise stated, Vt = 6.8 ml),
VS is the sample volume withdrawn,
Cn-1 is the concentration in the previous sample (~g/ml).
Method B
Determination of acyclovir in pharmaceutical formulations by HPLC
The HPLC method employed was the following:
Column: Steel column, 25 cm x
4.6 mm i.d.
Stationary Nucleosil C-18, 5 ~m
phase:
Mobile phase: water:methanol (20:80)
Temperature: Room temperature
Detection: 254 nm
Flow: 0.8 ml/min
Iqj.volume: 20 ~1
Ret. time: ca. 3.5 min
Reference solution: Weigh out an accurate amount of about 20.00 mg acyclovir
and dilute it with
mobile phase to a concentration of about 0.008 mg/ml
Test solution: Weight out 100.00 mg of the GMO/acyclovir formulation in a 50
ml volumetric
flask. Dilute with mobile phase to 50.00 ml. Dilute 5.00 ml to 50.00 ml with
mobile phase.
From the areas of the test solution and reference solution, respectively, the
percentage of
acyclovir present in the formulation is calculated.
Method C
Recovery of acyclovir on intestine
The HPLC method employed is the same as described under Method B. The test
solution is
prepared as follows:
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The intestine is shaken for 2 hours with 50.00 ml of the mobile phase. The
test solution is
filtered through a 0.2 ~tm filter. Dilute 1000 ~1 to 10.00 ml with mobile
phase.
From the areas of the test solution and reference solution, respectively, the
percentage of
acyclovir present in the formulation is calculated.
Determination of pH in the liquid crystalline phase
pH in the crystalline liquid phase is determined in a 10% w/w dispersion of
the liquid crystalline
phase (containing the active substance and any excipients) in distilled water.
Prior to
determination the dispersion is subjected to ultrasonic treatment for 30
minutes in order to
ensure that an equilibrium between the liquid crystalline phase and the
distilled water has
taken place. The pH is measured by employment of a HAMILTON FLUSHTRODE which
is a
suitable pH-electrode for measurement of pH in the dispersions. The procedure
followed was in
accordance with the instructions given by the manufacturer of the electrode.
The method described above ca.n be employed for various compositions, i.e. for
composition
wherein the concentration of the active ingredient in the liquid crystalline
phase may be varied
(e.g. from 1-20% w/w or in any range relevant for compositions according to
the invention.
Modifications of the method described above may also be employed e.g. i) the
dispersion
mentioned above may obtained by diluting the liquid crystalline phase in a
range corresponding
to from about 1:20 to about 1:5 with distilled water, ii) ultrasonic treatment
may he omitted or
substituted by stirring or treatment in a rotomat provided that measures are
taken to ensure
that equilibrium takes place or, alternatively, that measurement of pH takes
place after a well-
defined time period, and iii) other suitable electrodes may be employed.
Most important is it to ensure that for comparative purposes the test
conditions (stirring,
ultrasonic treatment, time, electrodes) should be essentially the same when
determining pH in
the liquid crystalline phase of compositions.
In order to determine when an equilibrium between the liquid crystalline phase
and the distilled
water has taken place a number of experiments were performed varying the time
period for
ultrasonic treatment (0-5 hours) and measuring the pH immediate after the end
of the ultra
sonic treatment and 24 hours later. The experiments are performed on a
GMO/water 65/35
containing 5% w/w of acyclovir. Based on the results of these experiments a
time period of 30
minutes proved suitable, i.e. there is only an insignificant difference in the
pH measured
immediately after the end of ultra sonic treatment and 24 hours later.
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Determination of drug solubility
The determination of the solubility of the active substance in the liquid
crystalline phase of the
composition is, of course, performed on the liquid crystalline phase as
formed. In practice, this
means that when the composition is one in which the liquid crystalline phase
has already been
formed when the composition is applied, the determination of the solubility is
performed on the
composition itself. The determination of the solubility is suitably performed
by microscopy to
observe any crystals of the active substance. Suitable test conditions involve
a magnification of
about 250 x and e.g. room temperature (20°C or 37°C may also be
employed). The determination
of the concentration at which crystals are observed is performed after a
period of at least one
week after preparation of the composition or the liquid crystalline phase to
ensure that
equilibrium has taken place. Normally, a series of tests with varying
concentrations is performed
to determine the concentration above which crystals are found. On the other
hand, when the
composition is a precursor composition, the liquid crystalline phase used as a
reference in the
solubility determination is a liquid crystalline phase imitating the liquid
crystalline phase which
will be formed when the composition absorbs liquid from the site of
application. This reference
liquid crystalline phase is made up with water (as representing the liquid
absorbed) in such an
amount that the reference liquid crystalline phase is the same type of liquid
crystalline phase as
is generated from the precursor composition.
In order to determine the aqueous solubility of the active substance at the pH
prevailing in the
liquid crystalline phase, the pH is determined in the liquid crystalline phase
as described above
to determine the pH conditions when determining the solubility. [Many
experiments with GMO
have revealed that the pH of the liquid crystalline phase predominantly is
about 4.5, however,
the pH depends on the quality of GMO employed.] The solubility of the active
substance is then
determined by stirring an excess amount of the active substance in water,
where applicable,
being buffered to a pH substantially identical to the pH prevailing in the
liquid crystalline phase
for a time period of at least 24 hours (to ensure that equilibrium has taken
place) and at a
constant temperature (e.g. 20°C, room temperature or 37°C). In
some case the samples initially
were subjected to ultrasonic treatment for half an hour in order to accelerate
the time for
equilibrium. The concentration of the active substance in the supernatant
(i.e. the aqueous
solubility at the given pH) is then determined by an appropriate assay (e.g,
by HPLC or LJV
spectroscopy).
As mentioned above, when the pH of the liquid crystalline phase, determined as
described
herein, is different from the pH which will result simply by dissolution of
the active substance in
water, the water is adjusted to substantially the pH of the liquid crystalline
phase by using a
suitable buffer system when determining the solubility of the active
substance. This buffer
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system should of course be so selected that, apart from the pH adjustment, it
has substantially
no influence on the solubility of the active substance in the buffered water.
pH-solubility profile
Alternatively, the aqueous solubility is determined as a function of pH, i.e.
by determining the
aqueous solubility in buffer systems having a pH in a range of about 3 to
about 9.5 such as
about 3.6 to about 9. Suitable buffer systems include acetate, citrate,
phosphate, borate etc. and
the concentration of the buffer is sufficient to ensure a constant pH during
the experiments. A
concentration of at least 0.01 M is normally suitable. This method is
applicable when
determining the minimum aqueous solubility of a specific active substance at a
given
temperature and at a given pH range. The test conditions described (pH,
temperature, ultrasonic
treatment, stirring, time for ensuring that equilibrium has taken place) above
are also valid
when determining the minimum solubility.
Determination of liquid crystalline structure
Phase transitions of GMO 84 and/or GMO 90 containing compositions
In the following tests are described which make it possible to determine the
crystalline structure
of suitable compositions for use according to the invention. The tests allow
determination of the
presence of, e.g., the GMO 84 or GMO 90 in a lamellar, hexagonal or cubic
phase, and it is
possible to test the compositions before and after application to an
appropriate application site.
With respect to the various liquid crystalline phases formed by GMO or other
glycerol fatty acid
esters, an excellent review is given by Ericsson et al. in ACS Symp. Ser.
(1991), pp 251-265,
American Chemical Society and by Larsson in Chapter 8 (part 8.2.1 entitled
"Lamellar and
hexagonal liquid-crystalline phases") in The Lipids Handbook edited by
Gunstone et al. In short,
the lamellar phase is the dominating one at a relatively low water content
(below 20% w/w) and
at a temperature of about 37°C, whereas the cubic phase dominates as
the water content
increases (more than about 20°lo w/w).
A. Phase transition of GMO 84 and/or GMO 90 compositions determined by
differential
scanning calorimetry (DSC)
The DSC measurements were performed using a Perkin Elmer Unix DSC model 7
Differential
Scanning Calorimeter. The heating rate was 5°C/min and the scanning
temperature was from
5°C to 70°C. Samples were contained in sealed aluminium pans
(Perkin Elmer No. B014-3017)
and as a reference empty aluminium pans were employed. The phase transitions
caused only a
- , ,
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relatively small enthalpy change and, therefore, the amount of sample tested
was optimized to
about 30-40 mg. The prepared pans were sealed and stored for two days at
5°C prior~o analysis.
B. Phase transition of GMO 84 and/or GMO 90 compositions determined by
polarimetry
The liquid crystalline phase can also be determined using polarized light and
e.g. employing a
5 stereomicroscope (Leitz, Diaplane) equipped with polarization filters. The
appearance of reversed
micelles (L2) are seen as a liquid oil, the lamellar phase (La) is mucous-like
and in polarized
light it is birefringent. The appearance of the cubic phase is as a very
viscous and glass-clear
sample. In polarized light the cubic phase (Q) is optically isotropic and
gives a black background
with no details indicating that it does not reflect the light. The Iamellar
and hexagonal phases
IO are optically anisotropic. The lamellar phase gives a structure like a pipe
cleaner on a black
background or, expressed in another manner, could be identified from the oily
streak texture
and the spherical, positive maltase cross-units visible between crossed
polarisers. The reversed
hexagonal phase gives different patterns but in most cases it resembles a
mosaic-like structure
or gives angular or fan-like textures.
15 The method can be employed in testing the phase behaviour of various
bioadhesive
compositions.
C. Phase transition of GMO 84 and/or GMO 90 compositions determined by X-ray
diffraction
A modified diffraction thermal pattern (DTP) camera was employed. The source
was an X-ray
tube equipped with a Cu-anode emitting Ka-rays at a wavelength of 1.54181. The
X-ray
20 generator was a Philips PW 1729.
The liquid crystalline state can be identified by low angle X-ray diffraction
and its appearance in
polarized light. The characteristic X-ray diffraction pattern for the three
liquid crystalline phases
(lamellar, hexagonal, cubic) will give rise to diffraction lines in the
following orders:
1:1/2:1/1:4...Qamellar)
25 1:I/J3:1/4:1/J7...(hexagonat)
1:1/J2:1/J3:1/J4:1/J5:1/J6:1/JB...(cubic)
In the case of the cubic form, the 3 different lattices will give rise to
three different diffraction
lines.
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EXAMPLES
The following examples 1-80 relate to the preparation and structure of
compositions according to
the invention.
Unless otherwise stated, all percentages are by weight.
In all examples, the gIycerylmanooleate (abbreviated as GMO in the following)
(and whenever
relevant glycerylmonolinoleate (Dimodan~ LS)) is gently melted on a heating
plate or in an oven
and the liquid obtained (max. temperature of the melted liquid is about
60°C) is cooled to about
40°C before mixing with other ingredients. The monoglyceride mixtures
and the ingredients
were mixed by stirring or shaking.
More specifically, compositions of GMO/Vitamin E TPGS, GMO/lecithin, and
GMO/lecithin/Vitamin E TPGS, respectively, are prepared as follows:
GMO/Vitamin E TPGS:
GMO and Vitamin E TPGS are melted together at a temperature of max.
60°C. Alternatively,
GMO and Vitamin E TPGS, respectively, are individually melted before mixing of
the two
components. Then the liquid phase is added.
GMO/lecithin:
Lecithin is dissolved in GMO at a temperature of about 60°C. Then the
liquid phase is added. If
the content of lecithin is > 50% by weight of the GMO content (lecithin/GMO >
0.5) then
lecithin and the GMO may be dissolved in ether or ethanol followed by
evaporation of the
solvent by vacuum distillation. Then the liquid phase is added.
GMO/lecithin/Vitamin E TPGS:
Lecithin is dissolved in a GMO-Vitamin E TPGS phase at 60°C. The method
described above
involving ether or ethanol as solvent ca.n also be employed.
GMO/lecithin/Vitamin E TPGS/acyclovir:
GMO and Vitamin E TPGS are melted at max. 60°C. Acyclovir is suspended
into the melt under
stirring. Epicuron 200 is dispersed in an aqueous medium by means of a
homogenizer and is
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then added to the GMO/Vitamin E TPGS/acyclovir mixture in a mortar under
vigorous stirring
until a homogeneous mixture is obtained. The mixture is subjected to
ultrasonic treat-meat for 1
hour and left in an oven (37°C) for 2 days to ensure that equilibrium
has taken place.
To the compositions described above any active drug substance and or other
excipients are
added or, alternatively, these substances are dissolved in the liquid phase
before these two
phases are mixed together.
In those cases where the composition contains an active substance in a
GMO/ethanol or
GML/ethanol vehicle or a lipid phase/ethanol vehicle, one of the following
methods can be
applied:
1, the active substance was dissolved or dispersed in ethanol and then mixed
with melted
GMO under stirring,
2. the active substance was dissolved or dispersed in melted GMO and then
ethanol was
added under stirring,
3. the active substance was dissolved or dispersed in a GMO/ethanol mixture.
When storing at room temperature (22°C) some formulations become
inhomogeneous. In
relevant cases the formulations were melted and stirred to obtain a
homogeneous mixture before
use.
An acyclovir ointment composition was prepared as follows:
In general, the acyclovir was suspended in the melted lipid phase and the
other ingredients were
added. The monoglyceride mixtures and the ingredients were mixed by stirring
or shaking. The
compositions were subjected to ultrasound treatment for about 1 h and were
stored for at least
two days at 3?°C before use to ensure that equilibrium had been
obtained (e.g. that the stable
liquid crystalline phase has been formed in the total formulation and that
equilibrium between
the solid and dissolved substance has taken place). As an alternative to
adding the acyclovir to
the melted GMO, the acyclovir can be suspended in the liquid phase before
combining the liquid
phase with the melted GMO.
In those cases where a bioadhesive test is performed, the values given are
mean values of the
results of 2-4 tests. It should be noted that the values given in the Examples
are not corrected
for recovery, i.e. the values are minimum values. If a correction for recovery
is made the values
will become larger.
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EXAMPLES 1-80
>rx l:ompysition Visual appearanceIdenh capon o
at qm
GMO-90/Epilturon room t~peraturecrystalline phase
100IHZ0 behaviour*
1 BGH 93: GMO-90IEpikurontransparent, black background,
yellow, cubic cubic
200/H20 consistency
60I1OJ30 % w/w
2 BGH 94: GMO-901F.pikurontransparent, black background,
yellow, cubic cubic
200/Hz0 consistency
50120/30 % wlw
-
3 BGH 110: aM0- Transparent, lack background,
yellow cubic
90/Epikuron2001Hz0
45125130 % w/w
4 BGH 95: GMO-90IEpikuroncloudy, ye low,b ack bac ground,
cubic cubic
200/H20 consistency
40/30/30 % w/w
BGH 96: GMO-90/Epikuroncloudy, yellow,non-cubic
softer than
200/H20 cubic consistency
30140130 % w/w
6 BGH 97: GMO-90/Epikuroncloudy, yellow,non-cubic
very soft
200/H20
20/50/30 % w/w
1~ BGH 9 : GMO- Epikurontransparent, black background,
amber yellow, cubic
145/Hy0 cubic consistency
60/10130 % wlw
8 BGH 99: GIvIO-9~lEpikurontransparent, cubic
amber ye low,
1451H20 cubic consistency
50/20/30 % w/w
9 BGH 100: GMO-90lfipikuroncloudy, amber non-cubic
yellow,
45/H20 cubic consistency
40130/30 % wlw
BGH 101: GMO-90/Epikuroncloudy, amber non-cubic
yellow,
451H20 cubic consistency
30/40130 % w/w
11 BGH 102: GMO~OTEpikuroacloudy, amber non-cu is
ye ow,
451H20 softer than
cubic
20/50/30 % wlw consistency
I BG I 1 : GMO- PGS/H20transparent, blac hac ground,
cubic cu ~c
60/10/30 % wlw consistency
13 BGH 119: GMO-90ITPGS/Hz0transparent, black background,
cubic cubic
50/20/30 % wlw consistency
14 BGH X20: GMO- lTPCiS1H20ciou y, shg non-cubic
tly yellow,
40130/30 % w/w much soRer than
cubic
consistency
(lamellar
consistency)
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69
ompostdon tsu appearance t ~d radon o qut
at
GMO-901Epikuron room temperaturecrystalline phase
200/Hi0 behaviours'
15 BGH 121: GMt?.90fTPGS/H20cloudy, slightlynon-cubic
yellow,
30/40/30 % wlw much softer
than cubic
consistency
(lamollar
consistency)
16 BGH 122: GMO-9 cloudy, slightlynon-cubic
l1'PGS/HZO yellow,
20/50130 % w/w much softer
than cubic
consistency
(Iamellar
consistency)
17 BaH 123: GMO-90/Epiturontransparent/cloudy,black background,
cubic
200/H20 + 5% TPOS yellow, cubic
consistency
50!20/30 % wlw
1H BGH 124: GMO-90/Epikurontransperentlcloudy,black background,
cubic
200/H20 + 5 % TPGSyellow, cubic
consistency
50/20/30 % w/w
GH 1 : GMO- transparent, blac background,
yellow, cubic cubic
90/Epikuron200/H20consistency,
excess water
43,?124,3/32 %
wlw
20 BGH 13 : GMO- transparent, black ackground,
yellow, cubic cubic
90/Epikuron200/H20consistency,
excess water
41,8123,2135 %
w/w
21 BGIi 128: OMO-90/Epikurontransparent, black background,
yellow, cubic cubic
200IH20 consistency,
excess water
39,9/22,1/38 %
w/w
22 BGH 12 -1: GMO-9 cloudy, yellow,black a~ekg~round,
/Epikttron cubic cubic, with
200/H20 consistency close crystal structure
45/25/30 % w/w
+ 5 % acyclovir
(cryet)
23 BaH 126-1: GMO-90 cloudy, yellow,black background,
Epikuron cubic cubic, with
~M20 consistency, even and close
excess water cryetsl structure
43,7124,3/32 %
w/w + 5%
acyclovir (tryst)
24 BGH i27-I : GMO- cloudy, yellow,black background,
/Epikuron cubic cubic, with
2~~20 consistency, close crystal structure,
disintegrates little
41,8123,2/35 % slightly, excesslumpy
w/w + 5% water
ecyclovir (tryst)
25 BGH 128-I : GMO-90/Epikuroncloudy, yellow,black background,
cubic cubic, with
200/H20 consistency, close crystal structure,
excess water little
39,9122,1/38 % lumpy
w/w + 5%
acyclovir (tryst)
26 BGH 130: OMO- /Epikurontransparent, black background,
yellow, cubic cubic with
2001H20 consistency, grid structure
ether smell
38,9129,1/32 %
w/w
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Fat- Gomposadoa is appearance en don o qmd
at
GMO-90/Epikuron room t~peraturecrysta>Une phase
2001H20 bebaviour*
27 BGH 131: GMO-90901fipikurontransparent, black background,
yellow, cubic cubic
200/H20 consistency,
ether smell
37,1/27,9/35 % w/w
28 BGH 132: (HMO-90/F.pikurontransparent, black background,
yellow, cubic cubic
200/H20 consistency,
slightly
35,4126,6138 b w/w disintegrates,
ether smelt
29 BGH 129-1GMO-90/F.pikuroncloudy, yellow,black background,
cubic cubic with
200 /HZO consistency, even and close
ether smell crystal structure
40/30/30 Y6 w/w
+ 5% acyclovir
(tryst)
30 BGH 130-1: GMO-90/F.pikuroncloudy, yellow,black background,
cubic cubic with
200 /H20 consistency, even and close
ether smell crystal structure
38,9129,1/32 % w/w
+ S%
acyclovir (tryst)
31 BAH 131-1: GMO-901F.pikuroncloudy, yellow,black background,
cubic cubic, with
200 /H20 consistency, close crystal structure,
ether smell little
37,1127,9135 % wlw lutttpy
+ 5%
acyclovir (tryst)
32 BGIi 132-1: GMO-90/F.pikuroncloudy, yellow,black background,
cubic cubic with
200 /H20 consistency, even and close
ether smell crystal stntcture
35,4/26,6/38 96
wlw + 59E
acyclovir (tryst)
33 BGH 133: GMO-90/Epikurontransparent, black background,
200 cubic cubic
IH20lTPG5 consistency
38,3121,3125,5/15
% wlw
(tI20 is added after
1 day with heat
at b0 C + stirring
34 BGH 134: GMO-90/Epikurontransparent, black bnckground,
200 cubic cubic
/HZOffPGS consistency
36/20/24120 % w/w
(H20 is added aRer
I day with heat
at 60 C + stirring
35 BGH 135: GMO- F.pikurontransparent, tack ackground,
2 0 cu tc cubic
/HZOrTPGS consistency
35,5/19,7/29,8/15
% w/w
(HZO is added after
1 day with heat
at 60 C + stirring
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71
omposthoa is appea' en tcahon o qmt
GMO-901Ep;koron room t~nperaturecrystalline phase
ZOO/HZO behavionr*
36 BGH 136: GMO- /Epikurontransparent, black background,
200 cubic cubic
/HZOITPGS consistency
33,4118,6128120
% w/w
(H20 is added after
I day with heat
at 60 C + slitting
37 BGH 13'T: GMO- Epikuroncloudy/transparent,black background,
: cu ~c
/HZOrTPGS yellow, cubic
consistency
34125,5/25,5/15 (cloudiness
% wlw due to air)
(Hy0 is added after
3 days with
heat at 60 C + stirring
38 BGH 138: GMO-90/Epikuroncloudyltransparent,black background,
200 cubic, plus
/H20lTPGS yellow, softersomething else
than cubic
32!24/24!20 % wlw consistency
(cloudiness
(HZO is added afterdue to air)
3 days with
heat at 60 C + stirring
39 B(3H 139: GMO-90/Epikuroncloudyltransparent,black background,
200 cubic
1H20/TPGS yellow, cubic
consistency
31,s/23,7129,811s (cloudiness
% Ww due to air)
(HZO is added after
l day with heat
at 60 C + stirring
40 BGH 1 : GMO- ikuroncloudy, yellow,ac background,
0 cubic cubic, wi
IHZOITPGS consistency HIt and La-patterns
(cloudiness
29,7/22,3128120 due to air)
% w/w
(H20 is added after
3 days with
heat at 60 C + stirring
41 BGH 133-1:GM - Epikuroncloudy, yellow,b eck backgroun
cu is , cubic, with
lHyO/TppS consistency even and close
trysts! structure
38,3121,3125,5/15
% w/w + 5%
acyclovir (tryst)
(H20 is added
after I day with
heat at 60 C +
stirring
42 BGH 134-1:GM0-90lEpikuron200cloudy, yellow,black background,
cubic cubic, with
/HZOrI'pG5 consistency even and close
crystal structure
36120/24/20 % wlw
+ s %
acyclovir (tryst)
(H20 is added
after l day with
heat at 60 C +
stirring
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72
omposs4on Visual appearanceen ahon o qwt
at
GMO-90/Epikuran room temperaturecrystaliine phase
200/Hz0 bebaviour*
43 BGH 135-1:GMO-90/Epikuron200cloudy, yellow,black background,
cubic cubic, with
/HZO/TP(iS consistency even and close
crystal structure
35,5/19,7/29,8/15
% w/w + 5%
acyclovir (tryst)
(H20 is added
after i day with
heat at 60 C +
8
BGH 13 -1:GM0- /Epikuron2cloudy, yellow,black background,
cubic cubic, with
IHZOITPGS consistency, even and close
lumpy crystal structure
33,4/18,6/28/20
% w/w + S%
acyclovir (tryst)
(1120 is added
after 1 day with
heat at 60 C +
stirring
45 BGH 137-1GM0-90lEpikuroncloudy, yellow,black background,
200 cubic cubic, with
IHZO/Tl'GS consistency even and close
crystal structure
34/25,5/25,5115 and occasional
% wlw + 5% lumps
acyclovir (tryst)
(H20 is added
after 1 day with
heat at 60 C +
stirring
46 BGH 138-1:GM0.9~i1-uroa200cloudy, yellow,ack ac ground,
cu rc cubic, wah
/H20/fPGS consistency, even and close
lumpy crystal structure
32124/24120 % wlw
+ 5 %
acyclovir (tryst)
(HZO is added
after 3 days with
heat at 60 C +
stirring
47 GH 9-1:GM0- Eprkuron2clou y, yellow,blac background,
cubic cu tc, wrth
IHZOffPGS consistency even and close
crystal structure
31,5123,7f29,8/15%wlw
+ S%
acyclovir (cryet)
(H20 is added
after 1 day with
heat at 60 C +
stirring
48 BGH 144-1:GM0-90/Epikuron200cloudy, yellow,black background,
slightly cubic, with
/HZOITPGS softer than even and close
cubic crystal structure
29,7/22,3/28120%wlwconsistency
+ 5%
acyclovir (tryst)
(H20 is added
after 3 days with
heat at 60 C +
stirring
49 BGH 141: GMO- ikurontransparent, black background,
ye low, cubic
200/H20 slightly softer
than cubic
47,9119, I /33 % consistency
w/w (lump)
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73
W:omposidon W st appeseanceand cadon o qw
GMO-90/Epilcuron at crystalline phase _
200IH20 room temperaturebehaviour*
51 BGH 142: GMO-90/F.pikurontransparent, black background,
200/H20 yellow, cubic cubic
46,4/18,613596 w/w consistency
52 8OH 143: GMO-90/F.pikuron(lump) black background,
2001H20 cubic
44,3/17,7/38 96 transparent,
w/w yellow, cubic
BGH 1 1-1: GMO- consistency black backgroun
Ept turon (lump) , cubic, mth
2001H20
47,9119,1 /33 % cloudy, yellow,even and close
wlw + S % cu is crystal structure
acyclovir (tryst.) consistency
53 BGH 142-1: GMO- cloudy, yellow,black ackground,
/F.ptkuron cubic cubic, m
200/H20 consistency, even and close
excess water crystal structure
46,4!18,6/35% wlw
+ 5%
acyclovir (tryst.)
BG 1 -1: GMO- ~kuroncloudy, yel black background,
ow, cubic cubic, with
2001H2O consistency, even and close
excess liquid crystal structure
44,3/17,7/38 % wlw (lump)
+ 5 %
acyclovir (tryst.)
55 BGH 1 : G Ep,kuron transparent, b eck background,
yeiiow, cubic cu ~c
H20lfp(iS consistency
45/18/27110 % w/w
56 BG . MO- t ron transparent, b ack background,
yellow, cubic c b c
H20rfPGS consistency
41,8/16,7/31,5/10
% wlw
57 BGH 146: GMO-90/Epikurontransparent, black background,
200/ yellow, cubic cubic
H20/Z'PGS consistency
42,5117125,5115
% w/w
58 BOH 147: GMO-90/Epikurontransparent, black background,
2001 yellow, cubic cubic
HZOrI'P(3S consistency
39,4/I5,8I29,8/i5
% wlw
GH 1 . O- ,In,ron transparent, lac ackground,
ye ow, cubic cu is
H20/1'PGS consistency
40116124120 % w/w
60 BGH 14 : GMO- Ep~kurontransparent, lac c ac ground,
yellow, cubro cubic
HZOrTPGS consistency
37,1114,9128120
% w/w
61 BGH 1 0: GMO-90/Ep'ikurontransparent, black background,
200/ yellow, cubic cubic
HZOfTPGS consistency
37,1120,7/27,2115
% wlw
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Ex omposWom su appeara~e cadoa o qm
at
GMO-90IEpiJntroa room femperaturecrystalline phase
2001H20 behaviour*
62 BGH 151: GMO-901fipikurontransparent, black background,
200/ yellow, cubic cubic
HZOITPGS consistency
33,9/18,8/32,3/15
% w/w
63 BGH 144-1: GMO-90/Epikuroncloudy, yellow,black background,
cubic cubic, with
200/ H20f1"PGS consistency even and close
crystal structure
45/18/27110 % w/w
+ 596
acyclovir (cryst.)
64 8GH 145-1: GMO-90JEpikuroncloudy, yellow,black background,
cubic cubic, with
200/ H20/T'PGS consistency even and ciose
crystal structure
41,8116,7/31,5110
% wlw + 5%
acyclovir (cryst.)
65 BGH 146-1: GMO-90/Epikuroncloudy, yellow,black background,
cubic cubic, with
200/ HZO/TPGS consistency even and close
crystal structure
42,5117/25,5/15
96 w/w + 5%
acyclovir (cryst.)
6 BGH -1: GMO- Eptcuroncloudy, yellow,black background,
cubic cubic, with
200/ HZO/TPGS consistency even and close
crystal structure
39,4/15,8/29,8115
% wlw + 5%
ecyclovir (cryst.)
67 BGH 148-1GM0- Eptkuronc oudy, yel ack bac ground,
ow, cubic cubic, with
200/ H20rfPGS consistency even and close
crystal structure
40/16/24/20 % w/w
+ 5%
acyclovir (cryst.)
68 BGH 149-1: GMO- cloudy, yellow,b ack bac ground,
Eptkuron cubic cubic, with
200/ H20fTPGS consistency even and close
crystal structure
37,1/14,9/28/20
% w/w + 5%
acyclovir (cryst.)
69 BGH 150-1: aM0- c oudy, ye black bac ;ground,
Eptkuron low, cu ~c cubic, wit
200/ H20/'fPGS consistency even and close
crystal structure
37,1120,7!27,2/15
% wlw + 596
acyclovir (cryst.)
70 BGH 151-1GMO- /Eptkuronc oudy, yellow,black backgroun
cubic , cubic, with
200/ H20JTPGS consistency even and close
crystal structure
33,9118,8/32,3/15
% w/w + 5%
acyclovir (cryst.)
BGH 1 : GMO- Eptkuronc oudy/transparent,black background,
2 cubic
HZO/TPGS yellow, cubic
consistency
35/19,4/25,6/20
% w/w
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omposthoa rsual appearanceen aUoa o 4qm
at
GMO-90/Epikuron room temperaturecrystalline phase _
20Q/Hi0 bebaviour*
72 BGH 153: GMO-90/Epikuroncloudy/tranaparent,black background,
200/ cubic,
HZOITPGS yellow, cubic
consistency
31,9/17,7/30,4/20
% wlw
73 BGH I 4: GMO-90lTPGS/H20cloudy/transparent,black background,
grey, cubic
55/15/30 % w/w cubic consistency
74 BGH 155: oM0- P clou y/transparent,b ac c background,
S ZO grey, cubic
50115/35 % w/w cubic consistency
75 BGH 156: GMO-90/TPGS/H20cloudy/transparent,black background,
grey, cubic,
45/20135 % w/w cubic consistency
76 BGH 15 -1: GMO- c oudy, yellow,b ack background,
v rron cubic cu rc, wrth
2001 HZOITPGS consistency even and close
(gritty) crystal structure
35119,4/25,6!20
% wlw + 5%
acyclovir (cryat.)
77 BGH 15 -1: GMO- cloudy, yellow,beginning La-lines
prkuron cubic with even
200/ H20fTPGS consistency and close crystal
(gritty) stnrcture
31,91/7,7/30,4!20 cubic with traces
% w/w + 5% of !smaller
acyclovir (cryet.) phase
78 BGH IS -1: GMO- clou y, whrtrsh,black backgroun
PGS H20 cubic , cubic, with
55/15!30 %w/w + consistency even and close
5% acyclovir crystal structure
(cryat.)
79 BGH 15 -1: GM0.90lTPCiS/H20cloudy, whitish,black background,
cubic cubic, with
50115/35 %w/w + consistency, even and close
5% acyclovir lumps crystal structure
BGH 156-1: GMO- clou y, whitish,b ack background,
'PGS H20 cubic cubic, with
45/20/35 %wlw + consistency, even and close
5% acyclovir lumps crystal stricture
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EXAMPLE 81
Stability studies of compositions according to the invention
The purpose of the study is to examine the stability of compositions in which
a cubic liquid crystalline
phase has been generated and wherein structurants like Epikuron 200 and
Vitamin E TPGS are included.
The compositions included in the study are left in transparent glass
containers in a climate cabinet and the
temperature is maintained at 15°C, 25°C or 40°C.
Samples are withdrawn at time t = 1 day after start of the experiment and at
time t = 14 days and the
samples are examined visually and by microscopy to investigate any change in
the appearance of the
compositions with respect to homogenicity, lipid precipitations, agglomerates
of drug substance, etc.
i0 Presence of excess water and/or separation into two or more liquid phases
are visual signs of instability
and are therefore especially noted.
Each composition investigated is prepared using GMO-90 (with a content of GMO
of about 90% w/w)
or, alternatively, by using RYLO MG19 (with a content of about 90°~b
GMO). The compositions which
are prepared using both qualities of GMO have been marked with a * in the
following.
The following studies are performed:
1. GMO/Vitamin E TPGS/water
Investigation of the influence of the concentration of vitamin E TPGS on the
stability of the cubic liquid
crystalline phase
Composition No. GMO/Vitamin E TPGS/water composition
1 55/15/30
2 SO/i5/35
3 50/20/30
4* 46.4/18.6/35
55/i5/30 + 5l wlw acyclovir
6 SO/ 15/35 + 5 % w/w acyclovir
7 50!20130 + 5 ~ w/w acyclovir
8* 46.4/18.6/35 + 5l w/w acyclovir
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2. GMO/Epikuron 200/water
Investigation of the influence of the concentration of phophatidylcholine
(Epikuron 200 is employed in
this study) on the stability of the cubic liquid crystalline phase
Composition No. GMO/Epikuron 200/water composition
9 50/20/30
10* 46.4/18.6/35
11 45/25/30
12 41.8/23.2/35
13(ether method) 40/30/30
14 (ether method)37.1/27.9/35
50/20/30 + 5 % w/w acyclovir
16* 46.4/18.6/35 + 5% w/w acyclovir
I7 45/25/30 + 5 % w/w acyclovir
18 41.8/23.2/35 + 5~ w/w acyclovir
15 19 (ether method)40/30/30 + 5 6 w/w acyclovir -
(ether method) 37.1 /27.9/35 + 5 b w/w acyclovir
3. GMO/Epikuron 145 (about 45°~o phosphatidylcholine)Iwater
Investigation of the influence of the concentration of phophatidylcholine
(Epikuron 145 is employed in
this study) on the stability of the cubic liquid crystalline phase
20 Composition No. GMO/Epikuron 145/water composition
21 46.4/18.6/35
22 46.4/18.6!35 + 5°r& w/w acyclovir
4. GMO/Epikuron 200/water/Vitamin E TPGS
Investigation of the influence of the concentration of phophatidylcholine
(Epikuron Z00 is employed in
this study) and vitamin E TPGS on the stability of the cubic liquid
crystalline phase
Composition No. GMO/Epikuron 200/waterlVitamin E TPGS composition
23 45/18/27/10 = 50/20/30 + 109b w/w TPGS
24 41.8/16.7/31.5/10 = 46.4/18.6/35 + 10°6 wlw TPGS
42.5/17/25.5/ 15 = 50/20/30 + 15 °6 w/w TPGS
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26 41.3/16.5/27.2//5 = 48.6/19.4/32 + l5~fo w/w
TPGS
27* _ 39.4/15.8/29.8/15 = 46.4/18.6/35 + 159b w/w
TPGS
28 37.7/15/32.3/15 = 44.3/17.7/38 + 15% w/w TPGS
29 40/ 16/24/20 = 50120/30 + 20 lo w/w TPGS
30* 37.1/14.9/28/20 = 46.4/18.6/35 + 20lo wlw
TPGS
31 34.3/13.7/32/20 = 42.9//7.1/40 + 20% w/w TPGS
32 38.3/21.3/25.5/15 =45/25/30 + 15fo w/w TPGS
33 35.5/19.7/29.8/15 =41.8/23.2/35 + 15 % wlw
TPGS
34 36/20/24120 = 45/25/30 + 206 w/w TPGS
35 33.4/i8.6/28/20 = 41.8/23.2/35 + 20/ w/w TPGS
36 45/ 18/27/ 10 = (50/20/30 + 10 ~ w/w TPGS)
+ 5 % w/w
acyclovir
37 41.8/16.7/31.5/10 = (46.4/18.6/35 + 10b w/w
TPGS) +
5 r6 w/w acyclovir
38 42.5117/25.5/ 15 = (50/20/30 + 15 % w/w TPGS)
+ 5 ~
w/w acyclovir
39 41.3!16.5/27.2115 = (48.6/19.4/32 + IS% w/w
TPGS) +
5 96 wlw acyclovir
40* 39.4/15.8/29.8/15 = (46.4/18.6/35 + 1596 w/w
TPGS) +
5 ~ acyciovir
41 37.7/15/32.3/15 = (44.3/17.7/38 + 156 TPGS)
+ 5% w/w
acyclovir
42 40/16/24/20 = (50/20/30 + 20 % w/w TPGS) +
5 b w/w
acyclovir
43* 37.1/14.9/28/20 = (46.4/18.6/35 + 20~ wlw
TPGS) + 5
w/w acyciovir
44 34.3/13.7/32/20 = (42.9/17.1/40 + 20% w/w
TPGS) + 5%
w/w acyciovir
45 38.3/21.3/25.5/15 = (45/25/30 + 159'o w/w
TPGS) + Sl
w/w acyciovir
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46 35.5/19.7/29.8/15 = (41.8/23.2/35 + 15~ wlw TPGS) +
°r& wlw acyclovir _ _ _
47 36/20/24/20 = (45/25/30 + 20% w/w TPGS) + 5% w/w
acyclovir
5 48 33.4/ 18.6/28/20 = (41.8/23.2/35 + 20 ~o w/w TPGS) + 5
w/w acyclovir
Results: The stability studies showed that all the compositions tested were
stable for at least 1 month at
15°C, 25°C and 40°C at a relative humidity according to
the accepted guidelines. In polarized Iight it
was observed that a cubic liquid crystalline phase was still present in all
compositions tested.
5. GMOlEpikuron 145/water/Vitamin E TPGS (°do wlw)
Investigation of the influence of the concentration of lecithin (Epilcuron 145
is employed in this study)
and vitamin E TPGS on the stability of the cubic liquid crystalline phase
Composition No. GMO/Epikuron 145lwater/Vitamin E TPGS composition
49 39.4/15.8/29.8/15 = 46.4/18.6!35 + 15°6 w/w TPGS
50 39.4/15.8/29.8!15 = (46.4/18.6/35 + 15 % w/w TPGS) +
5% w/w acyclovir
51 37.1114.9/28/20 = 46.4/18.6/35 + 20~Y w/w TPGS
52 37.1/14.9/28/20 = (46.4/18.6/35 + 20~n w/w TPGS) + 5
w/w acyclovir
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6. Stability at 25°C and b0°Xo relative humidity
In the following stability tests, the liquid crystalline phase was
investigated by means of polarised light at
22°C. The compositions investigated was prepared by dissolving Epikuron
200 in melted GMO/Vitamin
E TPGS (max. 60°C).
Batch Formulation Months Appearance Phase
(22C)
no. description*
BGH i78 0 clear ye cubic
GM090/epicuron200ITPGSIweter low, cubic
consistency
41,8/16,7110131,5 ,5
I .5 clear -yellow,cubic
cubic
consistency
3 clear - cubic
yellow,
cubic
consistency
8.5 clear yellow,cubic
cubic
consistency
BGH GMO clear ye cubic
epicuro ow, cu
PGSlwater is
consistency
42,5117115125,5 0.5
1.5 clear yellow,cu ~c
cubic
consistency
3 clear yellow,cubic
cubic
consistency
8.5 clear yellow,cubic
cubic
consistency
BGH 180 0 clear yellow,cubic
GM090/epicuron2001TPGSlwater cubic
consistency
41,3/16,5/15!27,2 .
1.5 clear yellow,cubic
cubic
consistency
3 clear yellow,cubic
cubic
consistency
8.5 clear yellow,cubic
cubic
consistency
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B F 1 1 GMO ep~curo PGS water clear ye cubic
ow, cubic
consistency
39
4/15
8/
, 0.5
,
1
/29.8
1.5 c ear yellow,cubic
cubic
consistency
3 clear yellow,cubic
cubic
consistency
a.~ clear yellow,cubic
cu tc
consistency
BGH 182 GM090/epicuron2 /TPGS/water clear yellow,cubic
cubic
consistency
37
7II5115132
3
,
,
1.5 clear yellow,cu is
cu is
consistency
3 clear yellow,cubic
cubic
consistency
a,~ clear yellow,cubic
cubic
consistency
BGH 186 GM0901epicuron20 1TPGS/water0 clear yellow,cubic
cubic
consistency
38
3I21
3115J25
5
,
.
,
I .5 clear yellow,cubic
cubic
consistency
3 clear yellow,cu is
cubic
consistency
8.5 yellow, a cubic
little
unclear,
cubic
consistency
BGH I$ GMO Oleptcuro C~9Slwater clearye~w, cubic
cubic
consistency
35
5/19
7/151
. 0.5
,
29,8
1.5 c ear yellow,cubic
cubic
consistency
3 clear yellow,cubic
cubic
consistency
clear yellow,cu is
cubic
consistency
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BGH 188 GM090fep~curon200lTP~S/water0 clear ye cubic
ow, cubic
consistency
36!20!20!24 0.5
1.5 clear yellow,cubic
cubic
consistency
3 clear yellow,cubic
cubic
consistency
8,5 clear yellow,cubic
cubic
consistency
BGH 189 GIvI0901epicuron2001TPGSlwater0 clear yellow,cubic
cubic
consistency
33,4/18,6120!28 ,
1.5 clear yellow,cubic
cubic
consistency
3 clear yellow,cubic
cubic
consistency
8,5 clear yellow,cubic
cubic
consistency
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Stability of various GMO/PC/TPGS/water formulations added 5~ w/w acyclovir
(AC) at 25°C/60~RH.
Batch no. Formulation Monthsappearance Phase
description
BGH 191 GMO epicuron2 0 unclear yellow,cubic
cubic
GS/water consistency
41,8/16,7/10/31,5 .5 unclearyeliow,cubic
cubic
consistency
+ 5 % AC 1, whit~yellow,cu is
cu ~c
consistency
3 unclear yellow,cubic
cubic
consistency
yellow, so
r an
cubic consistency
BGH 192 GM090/epicuron200ITP0 unclear yellow,cubic
cubic
GS/weter consistency
42,5/17/25,5/15 . uncearyellow,cub~ccu ~c
consistency
+ 5 % AC 1. white-yellow,cubic
cubic
consistency
unclear yellow,cubic
cubic
consistency
~t,S yellow, cubiccubvc
consistency
BG 3 GMO epicuro P unclear yel cubic
ow, cubic
GS/water consistency
41,3/16,5115127,2 0.5 uaclearyellow,cubic
cubic
consistency
+ 5% AC i. whrto-y ow, cubic
cubic
' consistency
3 unclear yellow,cubic
cubic
consistency
yellow, cubiccubic
consistency
BGH 196 GMO O/opicuro 0 unclear yellow,cubic
.00lTP cubic
GSlwater consistency
40116120/24 , uncearye cu ~c
ow, cubic
consistency
+ 5% AC 1. white-yellow,cubic
cubic
consistency
3 unclear yellow,cubic
cubic
consistency
8.5 yellow, cubiccubic
consistency
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BGH 197 GM09~Tepicuron2001T'P unclear yellow,cubic
cubic
GS/water consistency
37, LI14,9/20128 0.5 unclear yellow,cubic
cubic
consistency
+ 5 ~ AC , whrta-yellow,cu is
cubic
consistency
3 unclear yellow,cubic
cubic
consistency
S,5 yellow, cubiccu t~
consistency
BGH 198 GM090/epicuron200lTP0 unclear yellow,cubic
cubic
GS/water consistency
34,3113,7120/32 , unclearye cubic
ow, cubic
consistency
+ S ~ AC 1. whit~yeliow,cubic
cubic
consistency
3 unclear yellow,cubic
cubic
consistency
8,5 yellow, cubiccubic
consistency
BGH 199 OMO ep~curo ~0 unclear ye cubic
P low, cubic
GS/water consistency
38,3/21,3/i5/25,5 0.5 unclear yellow,cubic
cubic
consistency
+ 596 AC 1, white-ye cubic
low, cu
is
consistency
3 unclear yellow,cubic
cubic
consistency
8,5 yellow, cubiccubic
consistency
BGH 200 aM09 epicuron2001TP0 unclear yellow,cubic
cubic
GS/water consistency
35,5/19,7/15/29,8 . uncearyellow,cucu tc
tc
consistency
+ 5% AC 1. white-yellow,cubic
cubic
consistency
3 unclear yellow,cubic
cubic
consistency
8,5 yellow, cubiccubic
consistency
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BGH 201 GMO /epicuron2 unclear yellow,cu is
P cubic
GS/water consistency
36/20120/24 0.5 unclear yellow,cubic
cubic
consistency
+ 596 AC , w ~to-ye ow, cubic
cubic
consistency
3 unclear yellow,cubic
cubic
consistency
e,~ yellow, cu cubic
tc
consistency
BGH 202 GM090/epicuron200lTP0 unclear yellow,cubic
cubic
GS/water consistency
33,4!18,6/20128 , unc ear yellow,cu ~c
cu is
consistency
+ 5~ AC 1.5 unciearyellow,cubic
cubic
consistency
3 unclear yellow,cubic
cu is
consistency
8.5 yellow, cubiccubic
consistency _
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Batch Forma uon Months Appearance Phase
no. , (22C) de~cription*
BGH 157 0 clear to week grey,cubic
GM090/TPGS/water cubic
consistency
55/15/30 , clearto wee grey, cubic
cubic
consistency
3 clear to weak grey,cubic
cubic
consistency
9 clear brownish, cubic
cubic
consistency
BGH 158 clear to week grey,cubic
- C3M09 cubic
TPGS
water
consistency
50/15135 0.75 clearto week grey,cubic
cubic
consistency
I.
3 clear to week grey,cubic
cubic
consistency
9 clear brownish cubic
, cubic
consistency
BOH 159 clear to wee grey,cubic
(3M0 cu tc
S water
consistency
50120130 0.75 Clearto week grey,cubic
cubic
consistency
1.
3 clearto week grey,cubic
cubic
consisteacy
9 clear marblod discoloucubic
, cubic
consistency
BGH 160 0 clear to week grey,cubic
GM090/TPGSIwater cubic
consistency
46.4118,6/35 . clearto wee grey, cubic
cubic
consistency
1.
3 ciearto week grey,cubic
cubic
consistency
9 clear marbled discocubic
oared , cubic
consistency
BGH 161 0 white-unclear, cubic
GM090/TPCiS/water cubic consistency
55!15130 , whue-unclear, cubiccubic
+ 596 consistency
AC
3 white-uncloer, cubic
cubic consistency
9 white, cubic consistencycubic
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BC31I GMO PO water ~ w its-unc ear, cu is
cu ~c cona~stancy
50/15/35-+ 5% AC 0.75 white-unclear, cubic-
cubic consistency
1.5
w u~unc ear, cubiccu ~c
BC3H 163 aM0 S water consistency cu tc
w tie-unclear,
cu is constatoncy
50/20/30 + 5% AC 0.75 white-unclear, cubic
cubic consistency
1.5
white-unclear, cu ~c
cubic consistency
9 white, cubic consistencycu tc
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7. Stability at 40°C and 75°!o relative humidity
In the following stability tests, the liquid crystalline phase was
investigated by means of polarised light at
22°C. The compositions investigated was prepared by dissolving Epikuron
200 in melted GMO/Vitamin
E TPGS {max. 60°C).
Batch Formulation Months Appearance pytase
no. (22C) description*
BGH GM090Iepicuron200lTPGS clear yellow,cubic
water cubic
178 consistency
41,8/16,7110/31,5 0.5
1-5 clear yellow,cubic
cubic
consiste~y
3
8.S fluid unstable
BGH GM090/epicuron200/TPGSlwater0 clear yellow,cubic
cubic
179 consistency
42,511711S/25,5 ,
1.5 clear yellow,cubic
cubic
consistency
. uid unstable
BGH GM090/epicunon200/TPGSlwater0 clear yellow,cubic
cubic
180 consistency
41,3/16,SI1SI27,2 .
1.5 clear yellow,cu ~c
cubic
consistency
3
uuta unstable
BGH GMO 0 eptcuro . TPGS clear ye ow, cu tc
water cubic
181 consistency
39,4/lS,8/15129.8 0.5
clear yellow,cubic
cubic
consistency
3
8.S fluid unstable
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8GH GMO epicuron2 S water c ear ye ow, cubic
cubic
182 _ consistency
37
?!15!15
, 0.5
132,3
clear yel cubic
ow, cubic
consistency
3
8 5 fluid unstable
BGH- GMO epicuro PGS water clear yellow,cu ~c
cu tc -
186 consistency
38
3121
3/
, 0.
.
15125,5
I .5 clear yellow,cubic
cubic
consistency
8, fluid unstable
BGH GM0901epicuro 1TPGSlwater0 clear yellow,cubic
cubic
187 consistency
35
5!19
7/15!29
8
. .
,
,
1.5 clear yellow,cubic
cubic
consistency
8,5 utd unstable
BGH GM090 ePicuron200fCPGSlwater0 clear yellow,cubic
cub'r'~
188 consistency
36120/20!24
I .5 clear ye low,cu ~c
cu ~c
consistency
e.o unclear yellow,rev.
excess
water, softerhexagonal
than
cubic consistency
BGH GM090/epicurott200lTPGSlwater0 clear yellow,cubic
189 cubic
consistency
33
4!18
6/20128
,
,
I .5 clear yellow,cubic
cubic
consistency
8. unc ear yellow,rev.
excess
water, softerhexagonal
than
cubic consistency
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GMO/PC/TPGS/Water formulations containing 5% w/w % acyclovir
Batch no. Formulation Months Appearance Phase
(22C) description'
BGH 191 GM090/epicuron200lTPGS/water0 unclear yellow,cubic
cubic
consistency
41,8/16,7/10/31,5 0. unclear yellow,cu tc
cubic
consistency
+ 5 % AC 1.5 whit~yellow,cubic
cubic
consistency
BGH 192 GM090/epicuron200ITPGSIwater unclear ye cubic
ow, cubic
consistency
42,5117115/25,5 0. unclear yellow,cubic
cubic
consistency
+ 5% AC 1. w its-yel cubic
ow, cubic
consistency
3
9 pale ye low,rev. hexagonal
softer
than cubic
consistency
BGH 193 GM090/epicuron2001TPGSlwater0 unclear yellow,cubic
cubic
coasistency
41,3116,5/15127,2 . unc ear ye cubic
ow, cubic
consistency
+ 5% AC 1. white-yellow,cubic
cubic
consistency
9 pale yellow,rev. hexagons
softer
than cubic
consistency
BGH 194 GM090Iepicuron200lfPGSlwater0 unclear yellow,traces
softer of
than cubic something
consistency else
than the
cubic
phase
39,4/15.8!29,8/15 0.5 unclear yellow,phase
softer change
then cubic
consistency
+ 596 AC 1. two phase unstable
system,
fluid
3
9 fluid unstable
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GH GMO epicuron PGS water unclear ye cubic
ow, cu ~c
_ consistency _
r
40/16/20/24 0.5 unclear yellow,cubic
cubic
consistency
+ 5% AC , white-ye cubic
low, cubic
consistency
3
pale yellow,rev. hexagonal
eo er
than cubic
consistency
BGH 197 GM090/epicuron2 n'PGSlwater0 unclear yellow,cubic
cubic
consistency
37,1114,9/20/28 . unclear yellow,cubic
cu is
consistency
+ 5% AC 1.5 white-yellow,cubic
cubic
consistency
pale yellow,rev. hexagonal
softer
than cubic
consistency
BGH 198 GM0901epicuron2 0!1'PGSlwater0 unclear yellow,cubic
cubic
consistency
34,3/13,7!20/32 , unc ear yellow,cu ~c
cubic
consistency
+ 5% AC
i. white-yellow,cubic
cubic
consistency
9 pale yellow,rev. hexagonal
softer
than cubic
consistency
BGH 1 G 09 epicuro PGS water unclear yellow,cu tc
cu ~c
consistency
38
3121
3115/25
, 0. unclear yellow,cubic
, cubic
,
consistency
+ 596 AC 1. white-y low,cu tc
cubic
consistency
3
9 uid unstable
BGH 2 GMO ep~curo T S water unc ear yellow,cu ~c
cu ~c
consistency
35,5/19,7/15/29,8 0.5 unclear yellow,cubic
cubic
consistency
+ 5% AC
, w ate-yellow,cubic
cubic
consistency
3
uid unstable
CA 02286052 1999-10-15
WO 98147487 PCT/DK98/00159
92
BOH~OI aM0901epicuron200/TPGSJwater unc ear ye cubic
low, cu ~c
consistency _ _.
36/20120/24 p.5 unclear yellow,cubic
cubic
consistency
+ 5G AC 1. white-yellow,cu tc
cubic
consistency
3
9 flm unsta
a
B(3H 202 (iM090/epicuron200lTP(3Slwater0 unclear yellow,cubic
cubic
consistency
33,4/18,6120128 0.5 unclear yellow,cubic
cubic
consistency
+ 5 ~6 AC . unclear ye cubic
ow, cu tc
consistency
3
9 pale yellow rev. hexagonal
with few
dark yellow
spots,
separates,
soRer then
cubic consistency
CA 02286052 1999-10-15
WO 98147487 PCTIDK98/00159
93
Batch Formulation Mon Appearance Phase
s
no. (12C) discription"_
._
BGI~ I G O GS water c ear to wee grey,cubic
cuWc
consistency
55115/30 , clear to w~
grey, cubic cu ~c
consistency
3
a~ unclear brownie rev.
, cubic
consistency hexagonal
BGH 158 G O IT S water clear to wee grey,cu is
cubic
consistency
50/15/35 . Clear to week cubic
grey, cubic
consistency
3
a.~ unclear brownish rev.
, cu is
consistency hexagonal
GH 1 9 OMO PCiS water clear to week cubic
grey, cu ~c
consistency
50120130 . clear to week cubic
grey, cubic
consistency
3
a.~ clear marbled- cu ~c
iscoloured ,
cubic consistency
BGH 1 GMO ITPGS water c ear to wee grey,cubic -
cubic
consistancy _
46.4/18,6135 . clear to wee grey,cubic
cu ~c
consistency
3
a.~ clear marbled cubic
discolou ,
cubic consistency
BGH 16 G11 O S water ~ w ttteunc ear, cubic
cu tc
consistency
55/15/30 + 5% AC . w rto-unclear, cubic
cubic
consiste~xy
3
w te, cu is cotwetencycu tc
_ 0 w ito-uaclear, cubic
BGH 162 GM09 !I'PGS/watar cubic
consistency
50115/35 + 5% AC , whit~unclear, cubic
cubic
coaeistency
1.5
8,5 white, cubic consistencycubic
BGH 1 GMO ITPGS water w te-unc ear, cubic
cu ~c
consistency
50/20/30 + 5% AC . white-unclear, cubic
cubic
consistency
1.
3 white-unclear, cubic
cubic
consistency
8. white, cubic consistencycubic
1~
I G O S water w ue.unc ear, cu tc
cu ~c
consistency
46,4/18,6/35 + 5% , white-unclear
cubic
, cu is
AC
consistency
1.
3
w ite, cu is consistencycu ~c
CA 02286052 1999-10-15
WO 98147487 94 PCTIDK98100159
8. Stability at 15°C (cooling cabinet)
Method: polarized light at 22°C
t orm Uoa o ppesrance ptron
110. (22'C)
-
water c ear to wee c cu tc
grey, cu ~c
consistency
55115!30 . - ear to wee grey, c nc
c is
co~istency
c ear to w grey, c
is
consistency
9 clear to week 'c
grey, cubic
consistency
water ciwr to week grey,~c
cubic
coasi9tenry
50I1SI35 . leer to wee grey,cu uc
cubic
consistency
ear to wee grey, a
cubic
co~stmcy
c r to w grey, ~c
c ~c
consistency
UGH 159 ear to wee grey, cu ~c
GM0901TPGSlwater cu is
consistency
50120130 , ear to wee c grey,cu uc
cu uc
consistency
- c ear to week
grey, cubic
consistency
8.5 clear to week is
grey, cubic
- cat~sisteacy
water ~ clear to week cubic
grey, cubic
consistency
46.4/18,6/35 . clear to week cubic
grey, cubic
co~istency
to w grey, c is cu a
con.Ristency
S.s clear to week cubic
grey, cubic
consistency
S/ w te-un ear, cu cu is
water is
consiRtency
55!15/30 , w te-unc ear, cu ~c
+ SRfo c~ tc
AC consistency
3 white-nuclear, cu 'c
c is
consistency
8.5 yellow-brown, cubic
cubic
coaRiatency
1 2 9 w uteun ear, cu cubic
water uc
consistency
50115135 . w 'te-unc r, cu cubic
+ 5~ ac
AC consistency
w te- ear, cu cu is
is
consistency
white, softer a c a
than cubic
consistency
SUBSTITUTE SHEET (RULE 26)
CA 02286052 1999-10-15
WO 98147487 9b PCTIDK98100159
~~~3 w , cu c
water consateacy
50/20130 + 596 AC
. w te-unc ear, cu c
cu nc _ _ _
confisteacy
1.5
3 whito-unclear, a
cubic
consistency
a,5 white, softer phase change
thaan 'c
co~istency
SUBSTITUTE SHEET (RULE 26)
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WO 98/47487 96 PCTIDK98/00159
tc onnu uon on s ppearance ase escnpaon
no. (22C)
riCiH a dear yeuow, tunic tunic
CiMtriU/epicurociluuW consistency
rci~r
178
water
41,8116,7110131,5
1. clear yellow, cubiccu is
consistency
c ear ye ow, cu cu is
is consistency
clear yellow, cubiccu is
consistency
~J epicuro ~ c ear ye ow, cu cu to
179 is consistency
water
42,5117/i5/25,5
clear yel ow, cubiccubic
consistency
c ear ye ow, cu cu is
is consistency
clear yellow, cubiccubic
consistency
BGH c ear ye ow, cu cu is
(iMUHU/ep~cutonlUU/ is consistency
1-Y~iS/
180
water
41,3/16,5!15/27,2
1.5 clear yellow, cubiccu is
consistency
c ear ye ow, cu cu is
is consistency
9 clear yellow, cubiccu is
consistency
8GH c ear ye ow, cu cu is
CiMU9Uleptcuronluul is consistency
lr~iSr
181
water
39,4!15,8/15/29.8 ,
1. clear yellow, cubiccubic
consistency
c ear ye ow, cu cu is
is consistency
9 clear yellow, cubiccubic
consistency
epicumn200ITPG5/ c ear ye low, cu cu is
182 is consistency
water
37,7!15115!32,3
1.5 clear yellow, cubiccubic
consistency
c ear ye ow, cu cu is
is consistency
9 clear ye low, cubiccubic
consistency
BGH a clear yeciow, cuoiccu is
GM0901epicuron2UUl'l consistency
YCiJI
184
water
37,1114,9120/28
clear yellow, cubiccubic
consistency
c ear ye ow, cu cu is
is consistency
clear yellow, cubiccubic
consistency
1~J epicuro c ear ye ow, cu cubic
186 is consistency
water
38,3/2I.3/15/25,5
1.5 clear yellow, cubiccubic
consistency
c ear yellow, cu cu is
is consistency
9 clear ye low, cubiccubic
consistency
BGH a treat yeuow, cuoic cuoic
GMUHOIeptcuronluUl consistency
rrG~r
187
water
35.5!19,7/15/29,8
1.5 clear yellow, cubiccubic
consistency
c ear ye ow, cubic cu is
consistency
9 clear yellow, cubiccubic
consistency
8GH a clear yeuow, cubic conic
GM09u/eptcuronllxJflYG~ consistency
188
/water
36!20120/24
.5 clear yellow, cubiccubic
consistency
c ear ye ow, cu cu is
is consistency
clear yellow, cubiccubic
consistency
SUBSTITUTE SHEET (RULE 26)
CA 02286052 1999-10-15
WO 98/47487 9? PCT/DK98100159
epm~ c ear ye ow, cu cu ~c
189 /water ~c consistency
33,4118,6/20128 ,
clear ye ow, cubiccu is ' --
consistency
c ear ye ow, cu cu ~c
~c cons~srency
unclear yellow, p ase change
softer than cubic
consistency
SU8ST1TUTE SHEET (RULE 26)
CA 02286052 1999-10-15
WO 98147487 98 PCTlDK98/00159
GMO/PC/TFGS/V~later formulations containing 5% w/w % acyclovir
15°C cooling cabinet
atc ~ ocmu anon on s Appearance escnption
n0.
epicuro lTPGS U unclear yellow, cu is
191 /water cubic consistency
41,8116,7/10/31,5 . unc ear ye ow, cu cu is
is conssstency
+5%AC 1.
unc ear ye ow, cu cu is
lc consistency
9 unclear yellow, cubic
cubsc consistency
if0lepicuronZOUITPGSU unclear yellow, cu is
192 /water cubsc conssstency
42,5/17!15/25,5 . unc ear ye ow, cu cu is
is consistency
+ 5 9 AC 1.5
unc ear ye ow, cu cu is
tc consistency
unclear yellow, cubic
cubic consistency
epicuron2001TPG5 0 unclear yellow, cuoic
193 /water cubic consistency
41,3/16,5/15/27,2 . unc ear ye oiv, cu is
cu sc consistency
+ 5 'i' AC 1.
unc ear ye ow, cu cu is
is consistency
9 unclear yellow, cubic
cubic consistency
GM090/epicuron2UUITPGSU unclear yellow, cuoic
196 /water cubsc consistency
40116/20124 . unc ear ye ow, cu cu is
sc consistency
+ 5% AC 1.5
unc ear yei ow, cu is
cu is consistency
unc ear yellow, cubic
- cubic consistency
GM090Iepicuron2WITPGSU unclear yellow, cutsic
197 /water cubic consistency
37,1114,9/20/28 . unc ear ye ow, cu cu is
is consistency
+596 AC
unc ear ye ow, cu cu is
is consistency
9 unclear yellow, cubx
cubic consistency
epscuro unclear, cubic consistencycubic
198 !water
34,31I3,7/20132 . unc ear yel ow, cubic
cu is consistency
--
+SqoAC 1.
unc ear ye ow, cu cu is
is consistency
9 unclear yellow, cubic
cubic consistency
epscuro 0 unclear yellow, cubic
199 /water cubic consistency
38,3/21,3/15/25,5 , unclear ye ow, cu cu is
is consistency
+ 5 'lo AC 1.5
unc ear ye ow, cu cu is
is consistency
9 unclear yellow, cubic
cubic consistency
GM0901epicuron2UUl'1'PGS unc ear ye ow, cu cu tc
200 !water is consistency
35,5119,7115/29,8 . unc ear ye ow, cu cubic
is consistency
+ 5% AC 1.5
unc ear ye ow, cu cu is
is consistency
unclear yellow, cubic
- cu is consistency
GM090/epicuron2UUl'I'YGS unc ear ye ow, cu cu sc
BGH sc consistency
201 !water
36120!20124 . - unc ear ye ow, cu cubic
is consistency
+S~OAC 1.5
unc ear ye ow, cubiccu is
consistency
unc ear yellow, cubic
cubic consistency
SUBSTITUTE SHEET (RULE 26)
CA 02286052 1999-10-15
WO 98/47487 99 PCT/DK98/00159
BGH GM090lepicuron7, unc ear ye ow, cu cmc
is consy
202 /water
33
4/18
6120128
, , une ear ye ow, cu cu ~c
, tc consistency
+5%AC
unc ear ye ow, cu cu ~c
tc consistency
9 unc ear ye ow, cu cu ~c
is consistency
*uniform crystal distribution is observed in the cubic phase
SUBSTITUTE SHEET (RULE 26)
CA 02286052 1999-10-15
WO 98!47487 PCTlDK98l00159
100
EXAMPLE 82
Investigation of the solubility of acyclovir in compositions according to the
invention
The purpose with the study is to investigate whether the presence of
structurants like
phosphatidylcholine and Vitamin E TPGS has any influence on the solubility of
acyclovir in the
liquid crystalline phase.
The compositions are treated for 1 hours in an ultrasonic bath and stored at
37°C for at least 2
days before each composition is subjected to polarized light to see whether
acyclovir crystals are
present in the cubic phase. If crystals are observed then the solubility is
judged to be less than 2
mg/g or 5 mg/g.
The solubility of acyclovir at room temperature (22°C) is 0.5-I
mg/g.
The following compositions are tested:
1. 46.4/18.6/35 (GMO/Vitaxnin E TPGS/water) % w/w + 0.5% w/w acyclovir
2. 46.4/18.6/35 (GMO/Epikuron 200/water) % w/w + 0.5% w/w acyclovir
3. 34.3/13.7/32/20 (GMO/Epikuron 200/water/TPGS) % w/w + 0.5% w/w acyclovir
4. 46.4/18.6/35 (GMO/Vitamin E TPGS/water) % w/w + 0.2% w/w acyclovir
5. 46.4/18.6/35 (GMO/Epikuron 200/water) % w/w + 0.2% w/w acyclovir
6. 34.3/13.7/32/20 (GMO/Epikuron 200/water/TPGS) % w/w + 0.2% w/w acyclovir
The solubility of all the compositions is found to be less than 0.2% as
acyclovir crystals are
observed in the microscope. The results indicate that the lecithin and the
TPGS do not increase
the solubility of acyclovir compared with a GMO/water cubic phase.
EXAMPLE 83
A. Investigation of whether phosphatidylcholin together with Vitamin E TPGS
form a cubic liquid crystalline phase
The following compositions are studied:
1. Epikuron 200/Vitamin E TPGS/water (% w/w)
1. 10/60/30
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WO 98/47487 PCT/DIC98100159
101
2. 20/50/30
3. 30/40/80
4. 40/30/30
5. 50/20/30
2. Epikuron 145/Vitamin E TPGS/water (% w/w)
6. 10/80/30
7. 20/50/30
8. 30/40/30
9. 40/30/30
10. 50/20/30
The compositions are prepared as described above (Epikuron 145 is
dispersed/dissolved in
Vitamin E TPGS at 60°C for up to 3 days). The compositions are
inspected in polarized light as
described under the heading "Methods".
The results show that no cubic phase has been formed in any of the
compositions investigated.
B. Investigation of the bioadhesiveness of phosphatidylcholine/Vitamin E TPGS
based compositions
The compositions described above under heading "A" are subjected to a test for
bioadhesion
employing test system No. 3 - washing og ability from the skin described under
the heading
"Methods". The results are that at least some of the compositions are
bioadhesive.
EXAMPLE 84
Phase transitions of GMO 84 or GMO 90 containing compositions
A. Compositions without any drug substance
A composition of GMO 84/water 85/15% w/w is tested employing the DSC method
described
under the heading "Methods" above. The results are given in Fig. 4. DSC
experiments give
information about at which temperature a phase conversion takes place. DSC
measurements
alone give no information of the particular phases involved (e.g. lamellar,
cubic hexagonal etc.).
However, if the DSC results as in the present case are compared with e.g.
results from
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102
observation of the compositions in polarized light (see above under the
heading "Methods")
information on the crystalline phases as well as the transition temperature is
obtained. .
For the composition investigated, the results from the DSC and polarized light
measurement
show that the lamellar phase is present at room temperature and the lamellar
phase is changed
to the cubic phase when the temperature increases (Fig. 4). The transition
temperature is about
37°C.
Compositions containing GMO 90 and Vitamin E TPGS and/or Epikuron 200 as
structurants
are subjected to X-ray diffraction measurements (as described under the
heading "Methods") in a
temperature scan at 20-80°C. The aim of the study is to investigate the
influence structurants
like Vitamin E TPGS and/or phosphatidylcholin have on the phase behaviour of
GMO/water
based composition. Furthermore, the aim of the study is also to obtain an
indication of whether
the structurants participate in the cubic structure formed due to the
GMO/water content in the
compositions or whether the function of the structurants more are like
diluents, i.e. they do not
in themselves participate in the formation of the cubic structure but may
merely just be
incorporated in the composition and the cubic structure is more or less alone
based on the
GMO/water content.
The compositions are:
1. GMO/Vitamin E TPGS/water (50/20/30 %'o w/w)
2. GMO/Vitamin E TPGS/water (50/15/35 °lo w/w)
3. GMO/Epikuron 200/water/Vitamin E TPGS (39.4/15.8/29.8/15 % w/w)
In the following results from the compositions 1-3 mentioned above at
37°C are given for
illustrative purposes:
d-Spacings:
comp.l ratio comp.3 ratio
and 2
Lipid phase 65.6A 1 75.2A 1
58.1A o.ss so.SA o.80
36.3A 0.55 36.7A 0.48
The results show that the compositions 1-3 are cubic at 37°C and give
an indication of that
Vitamin E TPGS and/or Epikuron 200 participate in the cubic structure, i.e.
the cubic liquid
CA 02286052 1999-10-15
WO 98/47487 PCT/DK98/00159
103
crystalline phase formed is based on the GMO/Vitamin E TPGS/water or
GMO/Epikuron
200/water/Vitamin E TPGS compositions and not on the GMO/water content alone.
These
indications are supported by the observation that rather high concentrations
(above 10% by
weight) of Vitamin E TPGS and/or lecithin (both are soluble in GMO) can be
incorporated in
the GMO without any or any substantial deterioration or the cubic lattice
structure. This is in
contrast to what is generally observed, namely that if the lattice structure
should not be
substantially disturbed by the presence of an auxiliary substance, the
auxiliary substances in
general only can be incorporated into the GMO cubic lattice structure in a
relatively low
concentration. If Vitamin E TPGS and/or lecithin do not participate in the
cubic lattice structure
then an excess of water would have been expected, i.e. a phase separation of
the composition
into at least two distinct liquid phases would have been observed and no such
observation is
made.
The compositions Nos. 1-3 above are also subjected to DSC experiments. No
peaks is observed
(see Figs. 5-7) indicating that the cubic phase do not transform to another
liquid crystalline
phase in the temperature range investigated (20-?0°C). These results
are in consistency with the
results obtained from the X-ray measurements given above.
B. Compositions containing acyclovir
DSC experiments as described above were also performed on compositions
containing
GMO/water 65/35% w/w with 5% w/w acyclovir (crystalline and micronized,
respectively). The
samples were stored at 5°C for two days to ensure equilibration of the
sample. The lipids in the
sample solidified at this temperature. The DSC was run at 5-70°C. The
thermograms obtained
showed only a clear melting peak at about 16-17 °C for both the
reference sample (GMO/water
65/35% w/w) and the samples containing 5% w/w acyclovir. The solidified sample
transfers to
the cubic phase (reversible process). No phase transition of the cubic phase
seemed to have
taken place. The results are in well agreement with the results obtained by
use of X-ray
diffraction measurement described in the following.
Compositions containing GMO/water 65/35% w/w and GMO/water 65/35% w/w with
acyclovir
(crystalline and micronized, respectively) added in concentrations 2.5, 5.0
and 10% w/w were
subjected to X-ray diffraction measurements (as described under the heading
"Methods") in a
temperature scan at 20-70°C. The aim of the study was to examine if the
cubic phase of
GMO/water 65/35% w/w is changed when acyclovir is added. In the following
results from the
compositions mentioned above at 37°C are given for illustrative
purposes:
CA 02286052 1999-10-15
WO 98!47487 PCT/DK98100159
104
d-Spacings:
_ crystalline micronized ratio _ --
Lipid phase 61.7~r 61.7A 1
50.5~r 50.5.4 0.81
36.3A 36.3 0.58
29.7 29.71 0.48
Acyclovir 12.9~r - -
8.44~r - _
3.74A 3.74A -
3.42A 3.42 -
The results show that the compositions are cubic at 37°C.
The results obtained for all the tested compositions in the temperature range
20-70°C show that
all the tested compositions are cubic in the temperature interval 20-
70°C. The diffraction lines
from acyclovir do not interfere with the lines from the cubic phase. In
conclusion, the results
indicate that acyclovir both in its crystalline and micronized form is inert
in the cubic phase
(probably because acyclovir has a low solubility in the cubic phase). Thus, no
influence of
acyclovir on the phase behaviour has been observed in the concentration range
investigated and
the cubic phase containing acyclovir is rather stable against temperature
fluctuations.
Moreover, compositions containing GMO 90 and Vitamin E TPGS and/or Epikuron
200 as
structurants and 5% w/w acyclovir (crystalline) are also subjected to X-ray
diffraction
measurements (as described under the heading "Methods") in a temperature scan
at 20-80°C.
The aim of the study is to investigate the influence of acyclovir on the phase
behaviour of
compositions wherein phosphatidylcholine and/or Vitamin E TPGS are added as
structurants.
Acyclovir is added to the compositions denoted No. 2 and 3 above under the
heading "A.
Compositions without any drug substance", i.e. 5% w/w acyclovir is added to
GMO/Vitamin E
TPGS/water (50/15/35 % w/w), composition No. 2A, and to GMO/Epikuron
200/water/Vitamin
E TPGS (46.4/18.6/35 % w/w), composition No. 3A.
In the following results from the compositions 2A and 3A mentioned above at
37°C are given:
d-Spacings:
comp.2A ratio comp.3A ratio
Lipid phase 67.0 1 85.6A I
r
CA 02286052 1999-10-15
WO 98/47487 PCTlDK98/00159
105
0
58.1A 0.86 64.2A 0.75
a
- 35.OA 0.53 37.6A - 0:43
The results show that the compositions 2A and 3A are cubic at 37°C.
Thus, acyclovir in a
concentration of 5°!o w/w does not seem to result in a change in the
phase behaviour and the
cubic phase seems to be rather stable within the temperature ranges
investigated. These results
are supported by results from DSC experiments where no peaks is observed (see
Figs. 8-9)
indicating that the cubic phase do not transform to another liquid crystalline
phase in the
temperature range investigated (20-70°C).
Furthermore, compositions containing GMO/water 65/35% w/w with acyclovir
(crystalline and
micronized, respectively) added in a concentration of 1-40% were tested in
polarized light at 22°C
and 37°C, respectively, as described above under the heading "Methods'.
The results show the
presence of cubic phases in all compositions indicating that acyclovir
probably is inert in the
cubic phase.
EXAMPLE 85
Investigation of the bioadhesiveness of compositions according to the
invention
The following compositions (with or without 5010 crystalline acyciovir) are
tested in the washing
off ability test system for bioadhesiveness described under the heading
"Methods".
1. GMO/Vitamin E TPGS/water (60/20/80 °lo w/w)
2. GMO/Vitamin E TPGS/water (50/15/35 9'o w/w)
3. GMO/Epikuron 200/water/Vitamin E TPGS (39.4/15.8/29.8/15 % w/w)
4. GMO/Epikurnn 200/water (55/15/30 °!o w/w)
5. GMO/Lipoid S75/water/Vitamin E TPGS (39.4/15.8/29.8/15 °!o w/w)*
6. GMO/Lipoid S75/water/Vitamin E TPGS (33.4/18.6/28/20 % w/w)*
7. GMO/Lipoid S75/water/Vitamin E TPGS (40/10/30/15 9'o w/w)*
* was added 0.1% w/w a-tocopherol as an antioxidant
AlI 14 compositions are bioadhesive and scores of about 4-5 are obtained.
CA 02286052 1999-10-15
WO 98147487 PCT/DIC98/00159
106
EXAMPLE 86
Investigation of the dissolution/release of acyclovir from various
compositions
according to the invention
The dissolution rate of acyclovir in various GMO compositions is determined
using Franz
diffusion cells as described under the heading "Methods".
A series of GMO compositions containing Epikuron 200 (phosphatidylcholin)
and/or Vitamin E
TPGS and acyclovir are prepared as described above, and they are subjected to
the above
dissolution/release rate determination. All compositions were suspensions of
acyclovir, that is,
they contain acyclovir which has not dissolved. The solubility of acyclovir in
the compositions
investigated is less than 0.2% w/w according to the solubility experiments
performed.
The following compositions are investigated:
Composition No. composition
1 67/33 (GMO/water) % w/w + 5% w/w acyclovir
2 46.4/18.6/35 (GMO/Vitamin E TPGS/water} + 5% w/w
acyclovir
3 46.4/18.6/35 (GMO/Epikuron 200/water) % w/w + 5% w/w
acyclovir
4 34.3/13.7/32/20 (GMO/Epikuron 200/water/TPGS) % w/w +
5% w/w acyclovir
5 70/10/10/10 (GMO/Epikuron 200/TPGS/water) % w/w + 5%
w/w acyclovir
6 GMO/oleic acid/water/acyclovir (33/5/27/5) % w/w
The release profiles for composition Nos. 1-4 are shown in Figs. 10 and 11 and
the following
slopes (Higuchi plots) are found:
Composition No. slope (a)
1 1107
2 1616
3 1407
4 1254
...,. r ,. ,
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The results show that the release of acyclovir from composition No. 4
containing Epikuron 200
as well as Vitamin E TPGS and only about 34% by weight of GMO is almost of the
same-order
of magnitude as the release from composition No. 1 without any content of
structurant and with
a concentration of GMO of about 65% by weight.
With respect to compositions Nos. 5-6, the liquid crystalline phase present in
composition No
is the lamellar liquid crystalline phase. The composition is in the form of a
precursor
composition and a phase conversion to the cubic liquid crystalline phase takes
place during the
testing. The release rate of acyclovir (cf. Fig. 11A) is of the same order of
magnitude as the
reference cubic liquid crystalline phase-containing composition (GMO/water)
and thus, confirms
that the cubic liquid crystalline phase is formed during the test.
Furthermore, at the end of the
experiment a tested sample was investigated by polarised light and a cubic
liquid crystalline
phase was observed.
Composition No. 6 contains oleic acid as an enhancer. The liquid crystalline
phase present in the
composition is the reverse hexagonal crystalline phase. The release rate of
acyclovir (see Fig.
11A) is of the same order of magnitude as the reference cubic liquid
crystalline phase-containing
composition (GMO/water) and thus, indicates that the cubic liquid crystalline
phase is formed
early during the test. It is most likely that oleic acid is rapidly released
and then the conditions
present in the composition favours a formation of the cubic liquid crystalline
phase.
Furthermore, at the end of the experiment a tested sample was investigated by
polarised light
and a cubic liquid crystalline phase was observed. Also, after 1 hour of
testing a sample was
withdrawn and the cubic liquid crystalline phase was observed.
In conclusion, it has been shown that i) the rate limiting step in the
dissolution process is
diffusion of acyclovir molecules, ii) a precursor composition is capable of
generating a cubic
liquid crystalline phase, and iii) a phase conversion may take place after
application of a
composition.
EXAMPLE 87
Compositions containing antiviral substances
In the following table is listed a number of interesting compositions. The
compositions are
prepared as described above. 5% w/w of an antiviral substance is added to all
the compositions
listed in the table below.
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Vehicle Composition % w/w
GMO/TPGS/water 45/50/35 + 5% acyclovir
GMO/TPGS/water 45/50/35 + 5% peniciclovir
GMO/TPGS/water 45/50/35 + 5% famiciclovir
GMO/TPGS/water 45/50/35 + 5% ganciclovir
GMO/TPGS/water 45/50/35 + 5% valaciclovir
GMO/TPGS/water 45/50/35 + 5% cidofovir
GMO/TPGS/water 45/50/35 + 5% lobucavir
GMO/TPGS/water 45/50/35 + 5% sorivudine
GMO/TPGS/water 45/50/36 + 5% didanosine
GMO/Epikuron 200/water 50/20/30 + 59b acyclovir
GMO/Epikuron 200/water 50/20/30 + 5% peniciclovir
GMO/Epikuron 200/water 50/20/30 + 5% famiciclovir
GMO/Epikuron 200/water 50/20/30 + 5% ganciclovir
GMO/Epikuron 200/water 50/20/30 + 5% valaciclovir
GMO/Epikuron 200/water 50/20/30 + 5% cidofovir
GMO/Epikuron 200/water 60/20/30 + 5% lobucavir
GMO/Epikuron 200/water 50/20/30 + 5% sorivudine
GMO/Epikuron 200/water 50/20/30 + 5% didanosine
GMO/Epikuron 200/water/TPGS 39.4/15.8/29.8/16 + 5% acyclovir
GMO/Epikuron 200/water/TPGS 39.4//5.8/29.8/15 + 5% peniciclovir
GMO/Epikuron 200/water/TPGS 39.4/15.8/29.8/16 + 5% famiciclovir
GMO/Epikuron 200/water/TPGS 39.4/15.8/29.8/15 + 5% ganciclovir
GMO/Epikuron 200/water/TPGS 39.4/15.8/29.8/15 + 5% valaciclovir
GMO/Epikuron 200/water/TPGS 39.4/15.8/29.8/15 + 5% cidofovir
GMO/Epikuron 200/water/TPGS 39.4/15.8/29.8/15 + 5% lobucavir
GMO/Epikuron 200/water/TPGS 39.4/15.8/29.8/15 + 5% sorivudine
GMO/Epikuron 200/water/TPGS 39.4/15.8/29.8/15 + 5% didanosine
In the following is listed a number of suitable precursor compositions
containing 5% by weight of
acyclovir.
GMO/Epikuron 200/TPGS/paraffin oil + 5% w/w acyclovir:
70/10/10/10
85/15/15/5
73/10/15/2
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GMO/Epikuron 200/TPGS/paraffin oil/water + 5% w/w acyclovir:
69/10/10/10/1
64/15/15/5/1
72/10/15/2/1
GMO/Epikuron 200/TPGS/sorbitan ester + 5% w/w acyclovir:
70/10/10/10
65/15/15/5
?3/10/15/2
GMO/Epikuron 200/TPGS/sorbitan ester/water + 5% w/w acyclovir:
69/10/10/10/1
64/15/15/5/1
72/10/15/2/1
GMO/Epikuron 200/TPGS/lanolin/water + 5°!o w/w acyclovir:
69/10/10/10/1
64/15/15/5/1
72/10/15/2/1
GMO/Epikuron 200/TPGS/water + 5% w/w acyclovir:
70/10/10/10
70/12/16/2
71/12/16/1
GMO/Epikuron 200/TPGS/sesame oil/water + 5% w/w acyclovir:
76/10/10/2/2
?8/10/10/1/1
68/15/15/1/1
GMO/Epikuron 200/TPGS/sunflower oil/water + 5% w/w acyclovir:
76/10/10/2/2
78/10/10/1/1
68/15/15/1/1
Comments to the precursor compositions: Oil (e.g. olive oil, ricinus oil etc.)
or another substance
which decreases the melting point of the lipid formulations may be added to
obtain a cream or
an ointment at room temperature. After application on the skin or mucosa, the
compositions are
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able to absorb e.g. sweat or exudate from a wound and a liquid crystalline
phase like e.g. the
cubic liquid crystalline phase is formed. -
Alternatively, a liquid crystal phase inhibitor may be added and exert its
function in the solid
state (e.g. sugar alcohols like trehalose, or PVP).
Addition of water in small amounts (corresponding to a concentration of 0.1-5%
by weight) may
reduce the viscosity and hinder crystallization of the lipids.
Other compositions are also relevant, i.e. compositions having other active
substances or having
a drug concentration of about 1-20% w/w and compositions having a composition
of the vehicle
as given in Example 1-80 above.
EXAMPLE 88
pH-solubility profile for acyclovir
Experimental
To a 100 ml Erlenmeyer flask were added 50 ml buffer solution and 250 mg
acyclovir.
The buffers with pH 3.6, 4.2 and 5.3 were prepared using monobasic sodium
phosphate and
dibasic sodium phosphate (pH adjustment with phosphoric acid). The buffers in
the pH range
6.0 to 9.6 were prepared using monobasic potassium phosphate (pH adjustment
with sodium
hydro~de). The molarity of the phosphate salts was 0.05M; the pH of the medium
was
measured with a pH-meter.
Each mixture was stirred with a magnetic stirrer for 24 hours, and after
equilibrium to room
temperature, the sample was passed through a membrane filter. The solution was
diluted to
appropriate volume and the amount of acyclovir dissolved was determined by
HPLC.
The solubility of acyclovir as a function of pH is given in the table below
and in Fig. 3. From the
results, it is seen that the minimum solubility of acyclovir is at a pH in a
range of from about 4
to about 6.
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Acyclovir/Solubility at different pH
LFi (buffer) Acyclovir me/ml
3.6 1.9
4.2 l,g
5.3 1.8
6.0 1.8
6.6 1.9
7.6 1.9
8.5 2.2
8.8 2.5
9.0 2.5
9.2 2.9
9.6 3.5
EXAMPLE 88
Investigation of the influence of different active substances on the liquid
crystalline
phase
Miconazole is an example of an active substance which is insoluble in water
but has a solubility
of more than 2°k w/w in the liquid crystalline phase. However, the
release of miconazole is very
slowly from the cubic phase. The table given below shows the solubility of and
the crystalline
phase obtained for miconazole in a GMO/water 70/30°k w/w vehicle.
Miconazole (% w/w) Solubility Liquid crystalline phase
soluble cubic
2 soluble cubic
3 soluble cubic
soluble cubic
5 soluble cubic
6 crystals lamellar
For miconazole (as well as for some other substances which are soluble in the
cubic liquid
crystalline phase in certain concentrations) experiments have shown that the
bioadhesiveness of
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compositions containing the substances varies with the concentration of the
substance. In the
table below results are given from testing various miconazole compositions in
a GMO/ethanol
60/40%a w/w vehicle or in a GML/ethanol 60/40% w/w vehicle, respectively, for
bioadhesiveness
employing Test system No. 1.
Concentration of Bioadhesion*
miconazol (% w/w) (residual amount %)
GMO-based GML-based
0 85 95
2
3
4 72 86
I55 41
6 72
8 33
10
2025
*: In the tests runs the following test conditions were employed: initial
rinsing period: 5 min,
initial rinsing flow: i0 ml/min, angle: -21°, flow rate: 10 ml/min,
flow period: 30 min
From the results given above for the GMO-based composition it is seen that
there is a dramatic
fall in bioadhesiveness when the concentration of miconazole exceeds 6% w/w,
i.e. when the
liquid crystalline phase changes from the cubic phase to the lamellar phase
and when
miconazole in the liquid crystalline phase is present as crystals, i.e. when
the concentration
exceeds the solubility of miconazole.
The results support the results of other experiments performed by the
inventors, namely that
there is a close correlation between the presence of a cubic phase and
occurrence of a high
degree of bioadhesiveness. The other experiments performed by the inventors
involved
application of GMO, GMO/ethanol mixtures, GML on Test system No. 1 for
bioadhesiveness. It
was found that the samples applied in contact with the mucosa and washing
medium all had
converted into the cubic phase and that the samples were bioadhesive. The same
applies for
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compositions containing indomethacin (5% w/w) in a GMO/ethanol 60/40% w/w
vehicle and
other bioadhesive compositions containing an active substance. _ _
From the results given above in the table it is seen that when the
concentration of miconazol
exceeds a certain level, the bioadhesion is severely impaired. This indicates
that when the
concentration of the active substance in the cubic phase exceeds a certain
level, the cubic phase
structure is disturbed, or another liquid crystalline phase may perhaps have
been formed (the
active substance and/or any excipients may alter the phase diagram).
In the case of acyclovir, however, this reduction in bioadhesiveness with
increased content of
acyclovir, beyond the saturation point, does not seem to influence the cubic
phase and does not
seem to impair the bioadhesiveness (tested by means of Test system No. 3).
Experiments
showing this were performed with acyclovir ointment compositions, prepared
with GMO 90,
with concentrations of crystalline acyclovir of 2%, 5%, 10%, 20% and 30% by
weight, respectively.
These compositions were found to be highly bioadhesive, indicating that with
substances having
a very low solubility in the liquid crystalline phase, the liquid crystalline
phase remains less
disturbed by the presence of particles of the active substance and retains its
bioadhesive
properties.
EXAMPLE 90
Investigation of the influence of different excipients or solvents on the
bioadhesiveness of GMO or GML based compositions
The influence of various excipients and solvents was investigated. The various
compositions were
prepared as described above and the bioadhesiveness was tested using the test
system No. 1.
The following results were obtained:
Composition % w/w Bioadhesion
Residual amount %
GMOa
GMLa 65*
GMO/GMLa 40/60*** 56*
Mixtures with solvents:
GMO/water 85/16b 94
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GML/ethanol 60/40 95*
GMO/ethanol/propylene glycol/water: _ __
45/30/10/15 93
Mixtures with solubilizing
agents or preservatives:
GMO/ethanol/benzyl alcohol:
60/30/10 8?**
GMO/ethanol/benzyl alcohol/water:
60/20/5/15 80
50/20/10/20 89
Mixtures with release modulating
agents:
GMO/ethanol/glycerol:
50/30/20 97
GMO/ethanol/sesame oil:
59/40/1 96
58/40/2 93
50/40/10 14
50/30/20 0**
GMO/ethanol/soybean oil:
59/40/1 98
58/40/2 93
50/40/10 22
40/20/40 0**
GMO/ethanol/lecithin:
55/40/5 99
45/40/15 97
melted gently before application
6 lamellar phase
* lower results than expected; probably due to the reference values used in
the analysis of
the mixture
** test conditions: angle: -21°; initial rinsing period: 5 min; initial
rinsing flow: 10 ml/min;
flow rate: 10 ml/min; flow period: 30 min.
*** the GMO/GML mixture corresponds to about equal amounts of glycerol
monooleate and
glycerol monolinoleate
,.
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The results given above show that addition of relevant excipients or solvents
such as, e.g., agents
which are known solubi~izers for active substances or agents which are known
as release
modulating agents (i.e. agents which when added make it possible to adjust or
control the
release of the active substance from a composition) do not significantly
influence the
bioadhesiveness of the composition when the agents (excipients or solvents)
are added in
relatively low concentrations (less than about 10°lo w/w). Thus, the
release of an active
substance from a composition which has proved to possess bioadhesive
properties can be
. controlled at least to a limited extent by adjusting the amount of a release
modulating agent
such as, e.g., glycerol, sesame oil, soybean oil, sunflower oil, lecithin,
cholesterol, ete. A
modulating agent may influence the pore size of the water channels in the
cubic phase and/or
alter the partition coeffient of the active substance between the lipid
domains and the aqueous
phase at least to a limited extent. Furthermore, if necessary, solubilisation
of an active substance
or a fatty acid ester for use in a bioadhesive composition ca.n be effected by
use of e.g. benzyl
alcohol without significantly influencing the bioadhesive properties of the
composition. In
conclusion, the bioadhesive principles described herein have a high potential
with respect to
developing bioadhesive drug compositions having such a drug localization, such
a drug release
profile, and such a drug duration which are desirable or necessary under the
given -
circumstances. Thus, the present inventors have found an advantageous
bioadhesive drug
delivery system.
EXAMPLE 91
Investigation of the presence of an active substance in a liquid crystalline
phase of
glycerol monooleate
The methodology described herein is a methodology which is generally useful
for investigating
whether mixing or dissolving of an active substance in a vehicle capable of
forming a liquid
crystalline phase also leads to incorporation of the active substance in the
liquid crystalline
phase. While miconazol and lidocain hydrochlorides have been used as model
substances in the
description of the experiments, the same measures as described herein can be
used for
substances which have a very low solubility in both water and ethanol such as,
e.g., acyclovir.
Furthermore, the study was performed in order to examine the recovery of the
samples applied.
. 30 A lipophilic (miconazol) and a hydrophilic active substance (lidocain
hydrochloride), respectively,
were applied on the rabbit jejunum test model for bioadhesiveness (test system
No. 1). A vehicle
of GMO 84/ethanol 60/40% w/w incorporating 2% w/w of either miconazol or
lidocain
hydrochloride was employed. The GMO 84/ethanol vehicle is bioadhesive in
itself. After a flow
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period of 10 sec (corresponding to t=0), and a flow period of 30 minutes
(corresponding to the
end of the experiment) the samples applied were removed from the mucosa and
the cabie phase
was quantitatively examined by HPLC for the content of active substance. As
seen from the
table below almost all miconazole was found after 10 sec and 30 minutes. These
results indicate
that the lipophilic miconazole is incorporated in the cubic phase formed and
the result at 30
minutes indicates that the drug is very slowly released from the cubic phase.
This is consistent
with release experiments of miconazole delivered from a cubic phase into a
0.05 M phosphate
buffer solution, pH 6.5 (3?°C). Miconazole seems to prefer the
lipophilic part of the cubic phase.
The results are given in the following table; results for an acyclovir
composition are also given.
Composition Flow period Recovery of active
substance % mean of
two determinations
GMO 84/ethanol/miconazol:
58.8/39.2/2 10 sec 85
30 min 93
GMO 84/ethanol/lidocain HC1:
58.8/39.2/2 10 sec 37
30 min 7
GMO 90/acyclovir
95/5 10 sec 87
min 65
25 In the experiment with lidocaine hydrochloride, barely half the content of
the drug was
recovered after a flow period of 10 sec and only a negligible amount after 30
minutes. Because of
its high water solubility (about 0.7 g/ml at 25°C), the greater part of
the lidocaine hydrochloride
is probably dissolved and washed away in the buffer solution during the
prehydration time (10
min) and only some is incorporated in the cubic phase formed. Most of the
incorporated drug
30 had been released at the end of the experiment. Other studies have shown
that lidoca.ine
hydrochloride is released rather quickly from the cubic phase probably through
the water
channels contained in the cubic phase.
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Results for acyclovir, which is poorly soluble in both water and the cubic
phase, given in the
table clearly demonstrate that acyclovir is enclosed in the cubic liquid
crystalline phase formed
and some of it may have been released during the experiment.
In conclusion, the experiments reported above indicate that formulations in
which GMO and an
active substance are dissolved in ethanol or the active substance suspended in
GMO 90, serve as
a precursor for the formation of a cubic phase formed in situ, and that the
active substance is
incorporated in the cubic phase formed.
EXAMPLE 92
Dissolution/release rate of a bioadhesive composition containing acyclovir
The dissolution rate of acyclovir in various GMO compositions was determined
using Franz
diffusion cells as described under the heading "Methods".
A series of GMO compositions containing acyclovir were prepared as described
above, and they
were subjected to the above dissolution rate determination. All compositions
were suspensions of
acyclovir, that is, they contain acyclovir which was not dissolved. The
solubility of acyclovir in
the compositions investigated was less than 0.1% w/w (0.05% w/w<the solubility
of
acyclovir<0.1% w/w).
The results appear from Figures 12-18. The results indicate that the release
of acyclovir from a
GMO based vehicle is dependent on the concentration of acyclovir in the
composition, provided
that the release takes place from a cubic phase system. Furthermore, the
results indicate the
capability of a GMO-based vehicle to function as a very effective drug
delivery system.
Figs. 12-14 show the release of acyclovir (1-5% micronized) from a cubic phase
(GMO/water
65/35% w/w) and Zoviro cream, respectively, into isotonic 0.05 M phosphate
buffer solution, pH
6.5 (37°C). As appears from the graph of Fig. 12 showing the cumulative
release of acyclovir, the
release of acyclovir increases with increasing concentration of acyclovir over
the range
investigated. There is not proportionality between the rate of release and the
concentration; this
appears from the fact that the graphs of % released (Fig. 13) do not coincide
and the slope of the
Higuchi plots (Fig. 14); the release is dependent on the concentration.
However, at a
concentration of 3-5% w/w acyclovir, no significant difference in release
rates was found.
It is justified to refer to rate constant herein as the release of acyclovir
from the liquid
crystalline formulations according to the invention which can be described by
means of the so-
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called Higuchi equation (Higuchi, T., Rate of release of medicaments from
ointment base
containing drug in suspension. J. Pharm. Sci., 50 (1961) 874-875): on linear
regression; the
cumulative amount of acyclovir released plotted versus the square root of time
results in a
straight line with the slope k (rate constant ~g/h''~). This appears from Fig.
14 which shows the
plots for a number of compositions containing acyclovir in concentrations from
0.99% by weight
to 4.76% by weight in comparison with Zovir~ cream containing 5% by weight of
acyclovir. The
slopes of the graphs in Fig. 14 are as follows:
Zovir~ cream, 5%: 155
Acyclovir 0.99% w/w: 410
Acyclovir 1.96% w/w: 587
Acyclovir 2.91% w/w: ?17
Acyclovir 3.85% w/w: 7?3
Acyclovir 4.76% w/w: 1016
The higher the acyclovir concentration is, the smaller the percentage of
acyclovir released.
Acyclovir must first be dissolved before it is released from the cubic phase,
probably through the
water channels. Comparing the release profiles for 1% micronised and
crystalline acyclovir,
respectively, gives identical release profiles and indicates that the rate
limiting step is diffusion
of dissolved acyclovir rather than dissolution of the suspended acyclovir. In
spite of this, and in
spite of the low solubility of acyclovir both in water and in the cubic phase,
the release of
acyclovir from the composition according to the invention is dramatically
increased compared to
the Zovir~ cream. Thus, a comparison of the rate constant for acyclovir (5%)
released from
Zovira cream a.nd GMO/water 65/35% w/w shows that the rate constant is about 6
times larger
for the latter (Fig. 14).
With a view to testing if the rate of release can be improved by means of
micronized acyclovir,
as opposed to crystallinic acyclovir, the release from various compositions
was examined. Figures
15, 16 and 17 show an identical release pattern for crystalline and micronized
acyclovir,
respectively, from a formulation consisting of GMO/water 65/35% w/w + 1%
acyclovir. On the
other hand it appears that the release rate of crystalline acyclovir is
slightly improved from a
composition containing lecithin (GMO/water/lecithin 55/35/10% w/w + 1%
acyclovir) compared
to the same composition containing micronized acyclovir (Figs. 15-16).
However, no significant
difference is found. By comparing the release profiles for compositions
consisting of GMO/water
65/35% w/w containing 5% crystalline and 5% micronized acyclovir, respectively
(Fig. 17), it
seems that the release rate has increased somewhat with the micronized
quality. On the other
hand other studies have indicated that the release rate of acyclovir from a
composition consisting
of GMO/water/glycerol % w/w + 5% acyclovir is identical for the crystallinic
and the micronized
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quality. Whether the release is improved by application of a micronized
quality as opposed to a
crystalline quality depends on the composition of the cubic phase. However,
more exper-invents
have to be carried out to exclude that the differences observed arise from
experimental variation.
The results from the release experiments with micronized and crystalline
acyclovir, respectively,
indicate that the rate limiting step is diffusion of dissolved acyclovir and
not dissolution of
suspended acyclovir.
The micronized quality increases the viscosity of the cubic phase more that
the crystalline phase.
This condition alone favours the use of the crystalline quality in a potential
product so that
product of suitable and not too high viscosity can be obtained. Furthermore,
the use of the
crystalline form is favourable from a stability point of view.
The release of acyclovir from various GMO compositions containing 1% w/w and
5% micronized
acyclovir, respectively, containing release modulating or solubilising
compounds was examined
and compared with the release from a cubic phase consisting of 65 parts of GMO
and 35 parts of
water (Figs. 16-17). All the compositions except the compositions containing
sesame oil and the
composition containing GMO/glycerol 65/35 % w/w were the cubic phase, as
evidenced in
polarised light. As can be seen from the release profiles in Fig. 18 for the
compositions
containing 1% acyclovir, the profile of GMO/water 65/35 % w/w (reference) has
a shape similar
to the others with the exception of the profiles for the compositions
containing sesame oil. In the
latter case the release rate is drastically reduced, which could mean that the
compositions
consist of the reversed hexagonal phase, but this has not bean confirmed. It
should be noted that
the composition consisting of 65 parts of GMO and 35 parts of glycerol, have
the same release
profile as the reference composition, although both the visual and the
polarized light do not
indicate that they consist of the cubic phase. It is possible however, that
the cubic phase is
created on the surface of the formulation during the release experiment,
through its contact
with the dissolution medium (37°C). Addition of the release modulating
substances glycerol and
lecithin (lecithin is as described above also a structurant) to the cubic
phase has not significantly
changed the release of acyclovir in the concentrations examined. Neither does
the TPGS seem to
have increased the dissolution of acyclovir in the cubic phase nor changed the
partition
coe~cient between the cubic phase and the release medium, as the release
profile is identical
with the profile of the reference composition. Fig. I1 shows the release
profiles of composition
containing 5% acyclovir. The release profiles for the compositions containing
glycerol and lecithin
are identical while the release profile of the reference composition is
somewhat smaller. This
indicates that the release of acyclovir is slightly increased from the
compositions added release
modulation agents, however, the improvement is modest and the difference
observed is not
significant. The tests indicate that it is difficult to change the release of
acyciovir significantly.
There are limited possibilities for changing the release if the cubic
structure is to be preserved.
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EXAMPLE 93
Investigation of the influence of various pharmaceutically acceptable
excipients i) on
the formation of a cubic liquid crystalline phase and ii) on the
bioadhesiveness of
GMO-containing compositions
In the table below is given the various compositions tested and the results of
the tests employed.
Composition Phase condition Bioadhesion
(polarized light, (washing-off
22°C) ability)
GMO/lactose/water
50/20/30 % w/w cubic, undiss.+ (score 4)
GMO/HPC/water (hydroxypropyl-
cellulose is dissolved in
water)
50/20/30 % w/w +
GMO/TiPC/water (hydrogypropyl-
cellulose is dissolved
in GMO)
50/20/30 % w/w
GMO/sorbitan ester/water
50/20/30 % w/w cubic +
GMO/polyethylene glycol 200/water
50/20/30 % w/w non-cubic + (score 4)
GMO/propylene glycol/water
50/20/80 % w/w non-cubic not bioadhesive
GMO/paraffin oil/water
50/20/30 % w/w non-cubic not bioadhesive
GMO/lanolin/water
50/20/30 % w/w not bioadhesive
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EXAMPLE 94
Phase diagram for GMO/Vitamin E TPGS/water systems
Initial investigation of the phase diagram for E TPGS/water systems
GMO/Vitamin at mom
temperature shows the following:
Composition of GMO/Vitamin E TPGS/water Phase
(~o w/w)
60//0/30 cubic
45/20/35 cubic
46.4/18.6/35 cubic
50/15/35 cubic
55/15/30 cubic
50/20/30 cubic
41.8/23.2/35
45/25/30
40/30/30 non-cubic
30/40/30 non-cubic
20/50/30 non-cubic
The results are shown in Fig. 19.
EXAMPLE 95
Case stories on treatment of cold sores - preclinical study in humans
A composition of GMO/water 65/35% w/w with 5% w/w acyclovir has been used for
the
treatment of cold sores in humans.
Treatment was started with a maximum of 24 hours delay from start of symptoms.
In one case,
treatment with Zovirag~ cream was tried for 4.5 days before switch to GMO
acyclovir cream.
GMO acyclovir cream was applied 3 times daily (range 2-4) for 2.5 days (range
1.5-4).
The results of the study are given in the Table below.
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WO 98/47487 122 PC'T~DK98~00159
o m
+' x
d
'V
3
~,a~ _ _
o '
'~ o
a
b
~1 ~ ' '~w t~-
a ~
~
z
~.
a a a ~, ~, a a
~ ~
~, ~
w z z z ~ H~ z z
H ~
.~
U
~x
~n
3
v~ a, '
o
H
V ~ d ~
~ +~
O W dl
O y 0
.,
O O O ~ ~3 O by t3,
;~
0 0. c. sa.u, u. ;~ .~
~ a
\i
r
n
H
b
v
b
Q
11J
'C
d
G~l
~ G~ d
H N c7 N 'd'd' M .-iGsl
m
a
o _
~ ' V~
c ~ N
?,
~
o
a M ~ ~ ~ ~ ~ ~ z
0
A C~ ~ ~ ~ M M C'~~C~rJ
cd
d
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~ c~ c~ er ~W n ca r rl
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,,
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In 7 of 8 treatments symptoms ceased or improved very much. In one case,
ulceration occurred
and treatment was stopped. Healing was only reported in one case, probably
because the -
treatment prevented the typical ulceration of a cold sore.
Side effects were noted by 2 to 7 persons. One of tliese persons received two
treatments and in
both cases, treatment was stopped due to side effects. The side effects
reported were ulceration,
transient erythema and dry skin. No severe or serious side effects were
reported.
The reported case stories do not represent scientific evidence of the efficacy
of GMO acyclovir
cream. They do, however, indicate that the characteristics of GMO/water 65/35%
w/w with 5%
acyclovir on certain points differ from those of Zovirax~ cream from Glaxo
Wellcome.
GMO/water 65/35% w/w with 5% acyclovir adheres firmly to skin. Therefore fewer
daily
applications of GMO/water 65/35% w/w with 5% acyclovir were administered than
what is
recommended for Zovirax~ cream. In 6 out of 8 cases, treatment could
successfully be stopped
after 2-3 days. This is shorter than the normally recommended treatment period
for Zovirax~
cream of 5 to 10 days.
Application frequency and treatment duration for GMO acyclovir cream in these
case reports are
less than recommended for Zovirage cream. By the persons treated, the efficacy
was judged to
be equivalent or better than that of Zoviraxa cream.
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EXAMPLE 96
A. Skin irritation of GMO-containing compositions
Purpose
Evaluation of skin irritation level for four GMO-based test formulations by a
cumulative
evaluation performed on intact human skin compared with four commercially
available products.
Natusan ointment (negative control)
HIorhexidin cream 1% (positive contrnl)
Zovira cream (acyclovir cream, control)
GMO in para~n 20% (w/w) (test) Batch no. BGH 271
GMO/Epicuron 200/water 50/20/30 (w/w) (test) Batch no. BGH 269
GMO/Epicuron 00/water/TPGS 35.5/19.7/29.8/15 (w/w) (test) Batch no. BGH 270
The test was performed on nine healthy volunteers.
The test was performed over five working days with four applications and four
recordings after
24 hours. The test material was applied onto the volunteers on volar next to
their non-dominant
underarms.
Approx. 30-35 mg of each test material was placed under the skin under a 8 mm
~ aluminum
chamber (Finn Chamber, Epitest Ltd O~ positioned on a sticking plaster of non-
occlusive type
(Scanpor, Norges Plaster). The volunteer removed the plaster the morning after
application. The
result was recorded approximately two hours later in connection with a fresh
application of the
test material. On day 5 only one records! was made. In connection with the
recordal, a photo
was also taken.
The skin reaction was evaluated both in respect of erythema and oedema on a
scale ranging
from 0 to 3. The scoring was made by the same person for all the volunteers.
Each product was applied four times in the same place and was left for 21-23
hours. If a product
caused a third degree irritation, the product in question was taken out of the
testing of the
volunteer in question.
Evaluated on total score, Zovire cream, GMO/Epicurean/TPGS and GMO/Epicuron
were at the
same low level. GMO 20% and Natusan caused no irntation.
r
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In this test, GMO formulations containing Epikuron 200 have caused reactions
at the same level
as the commercially avajlable Zovir~ cream. Consequently, they are considered
suitable far
pharmaceutical formulations.
B. Skin irritation of compositions with 5% acyclovir
The following compositions are tested:
1. Paraffin oil (negative control)
2. 0.5 Sodium lauryl sulfate (positive control)
3. HIorhesidin~ cream 1% (positive control and reference)
4. Zovirax~ cream 5%
5. Vectavir cream 1%
6. GMO/Epikuron 200/water 50/20/30 + 5% acyclovir (% w/w)
7. GMO/Vitamin E TPGS/water 50/20/30 + 5% acyclovir (% w/w)
8. GMO/Epikuron 200/TPGS/water *33.4/18.6/20/28 + 5% acyclovir (% w/w)
9. GMO/Epikuron 200/TPGS/water *41.3/16.5/15/27.2 + 5% acyclovir (% w/w)
10. GMO-90/water 65/35 % w/w
11. GMO/Epikuron 200/TPGS 45/10/15/30 + 5% acyclovir (% w/w)
*: Stability testing at 25°C and 60% relative humidity has shown that
the composition is in
the form of a cubic liquid crystalline phase which is stable for at least 9
months.
The Chamber Scarification Test is used in order to evaluate the skin
irritation profile of the
above-mentioned compositions.
The Chamber Scarification Test is developed to investigate and compare
cosmetics, cosmetic
ingredients and consumer products intended for repeated use on normal or
diseased skin. The
assay amplifies irritant reactions to the test products by scarification of
the test area prior to the
first application. The test is carried out as described by K E. Andersen in
Contact Dermatitis
1996 (34), pp. 181-184 by P. J. Frosch & A. M. Kligman in Contact Dermatitis
1976 (2), pp. 314-
324.
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EXAMPLE 97
In vitro permeability of compositions according to the invention across human
skin
Test compositions
1. GMO/Vitamin E TPGS/water 45/20/35% w/w + 5% w/w acyclovir
2. GMO/Epikuron 200/water 50/20/30% w/w + 5% w/w acyclovir
3. GMO/Epikuron 200/water/TPGS 39.4/15.8/29.8/15% w/w + 5% w/w acyclovir
4. Zovir~ 5%, Wellcome (containing 5% w/w acyclovir) BFJ15-6
5. GMO/water 65/35% w/w + 5% w/w acyclovir
Preparation of skin membranes
Excised abdominal skin from humans was obtained from The Clinic of Plastic
Surgery. The
hairs were removed from the epidermal side by clipping. Subcutaneous fat on
the dermal side
was removed. The skin was washed with distilled water and stored at -
18°C until use.
Apparatus
Franz diffusion cells having an available diffusion area of L77 cm2 were used.
The epidermal
side of the skin was exposed to ambient laboratory conditions while the dermal
side was bathed
with the receptor medium consisting of 6.8 ml of 0.05 M phosphate buffer, pH
6.5. Each cell was
placed on a magnetic stirrer. The temperature of the water flowing in the
closed circulatory
system was kept at 37°C.
Permeation procedure
The skin membranes were thawed and mounted in Franz diffusion cells. The
receptor chambers
were filled with receptor medium and the epidermal side of the skin was wetted
with a few
drops of receptor medium. The skin was then allowed to equilibrate for about
24 hours. Blood
and soluble enzymes were at the same time washed out of the skin, and thereby
could not
disturb analysis of the receptor medium for acyclovir. The integrity of the
individual skin
samples was ensured by measuring the capacitance of the skin. Skin samples
with a capacitance
of less than about 0.055 wF were considered intact, whereas skin samples with
a higher
capacitance were considered damaged. The water permeability (3H) may also be
determined as a
measure of the integrity of the skin. Before application of the test
substances, the receptor
medium was replaced by fresh media. 300-350 mg of the test substance was
spread across the
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entire epidermal surface in an even layer. At appropriate intervals (t=0, 6
hours, 1, 2, 3, 4 and 5
days) 2 ml samples werE withdrawn and replaced by fresh receptor medium
keeping an--infinite
sink. Due to variation when using biological membranes, at least six
permeation studies were
performed on each test substance.
The results from the permeation study (see Fig. 21) shows that the permeation
profiles for
acyclovir from the GMO/water composition (cubic liquid crystalline phase) and
Zovir~,
respectively, are not significantly different. The lag time is about 1 day.
The release profile
shows tht acyclovir delivered from a cubic liquid crystalline phase of GMO
permeates the skin
and, accordingly, a cubic liquid crystalline phase is an excellent drug
delivery system for
acyclovir and most likely also for other active substances especially
antiviral substances. During
the experiment which lasts for 4-5 days, the compositions remain on the skin.
However, release tests in vitro have shown that acyclovir incorporated into a
cubic phase of
GMO (GMO/water 65/35% w/w + 5% acyclovir) is released approx. 5-6 times faster
than
acyclovir from Zovir~ cream.
I5 Permeation experiments across epidermis (pig) isolated by heat separation
gave the same
results.
EXAMPLE 98
In vitro permeability of compositions according to the invention across human
skin
A. Wholly skin
In order to evaluate the influence of the compositions on the ability of
acyclovir or other
antiviral compounds to penetrate the stratum corneum and to accumulate in the
epidermis and
the dermis, the following experiments can be performed using wholly intact
human skin excised
from cosmetic surgery. The skin is obtained from clinics for plastic surgery.
The skin is treated
as mentioned in the Example above and stored at -18°C. Skin from other
mammals than
humans may also be employed such as, e.g. guinea pigs, mice and pigs. The skin
may be
separated into epidermis and dermis by exposing the skin to hot water
(60°C) for e.g. 45 seconds
(heat separation) or by slicing with a dermatome (mechanical separation) for
permeation or
penetration studies. The stratum corneum can be isolated by tape stripping. If
the drug
substance is insoluble in water, the epidermis can be separated by dry heat
separation e.g. at
60°C for 2 min by placing the sample (contained in a closed package) in
a water bath or in a
heat cabinet. The test conditions are generally as described in the Example
above, but other test
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times (e.g. from 1 hour to 7 days), amounts of sample applied (e.g. 50-350 mg)
etc. may be
appropriate. To avoid intra-individual variations the same donor is used to
testing different
compositions and the skin specimens were taken from the same skin area. In
order to simulate
injured skin, the skin can be injured by applying a skin enhance or by
stripping the skin with
tape.
The amount of drug substance within the skin can be calculated by measuring
the concentration
of the drug substance in i) the receptor medium, ii) the skin, and/or iii) the
remaining
composition. By measuring i) and iii}, the amount of drug substance in the
skin ca.n be
calculated.
B. Different layers of the skin
The herpes virus replicate in the living epidermis. The basal layer of the
epidermis appears to be
the primarily site of antiviral activity in cutaneous HSV-1 infections, i.e.
the epidermis appears
to be the target site for antiviral drug substances.
Permeation (i.e. penetration into and through the skin) of acyclovir or other
antiviral substances
can be investigated across isolated epidermis by diffusion (as described
above). In this manner, a
measure is obtained of the amount of acyclovir having permeated the epidermis.
Alternatively, a
picture is obtained of the penetration (i.e. the entry into the skin but not
through the skin) of
acyclovir (or other antiviral substances) in the skin by means of diffusion
test using wholly skin
which at the end of the experiment is divided into stratum corneum (e.g.
enzymatic degradation
or tape stripping; tape stripping. 10-20 x, e.g. using Scotch Brand Magic Tape
No. 810 from 3M,
Minneapolis, U.S.A.), epidermis and dermis by means of a dermatome. The
individual layers are
analyzed for acyclovir (or other antiviral substances), e.g. by liquid
scintillation.
Before the tape stripping the compositions are removed by use of a spatula and
the skin is dried
using HIeenex dipped in a ethanol-water (3:1) solution.
In those cases where radioactive acyclovir (or other radioactive antiviral
drugs) are used, the
amount of acyclovir penetrating the tissue was measured by a liquid
scintillation technique (3H-
acyclovir is commercially available in form of a ethanol/water 30/70 solution
(Sigma); e.g. 21 ~1
3H-acyclovir corresponding to about 0.8 ~Ci/ml is added to 1 g composition).
Due to the fact that the main part of the acyclovir is present in the form of
undissolved particles
in a concentration of about 1-10% by weight, it is initially necessary to
dissolve cold acyclovir
and add the hot radioactive form on dissolved form in order to obtain a
homogeneous mixture of
cold and hot molecules. Cold acyclovir is dissolved in 0.1 N NaOH and the hot
acyclovir is added
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under stirring. Hydrochloric acid (to adjust pH to about 7) is rapidly added
under vigorous
stirring to obtain a uniform precipitation of hot and cold acyclovir crystals.
The precipitation is
enhanced by maintaining the mixture at 5°C for e.g. 24 hours. Then the
acyclovir crystals are
filtered off and washed 3 times with a small amount of water. The crystals are
dried in an
excicator. By using this procedure it is possible to control the ratio between
hot and cold
acyclovir in dissolved and undissolved form, respectively.
In order to examine the content of acyclovir in different skin
sections/layers, the skin sections
were placed in scintillation vials with e.g. Soluene 350 over night to
dissolve the skin
components. Scintillation cocktail (e.g. Hionic-Flour) was subsequently added
and the samples
were assayed for content of acyclovir (or the appropriate antiviral drug) by
liquid scintillation
spectrometry. The drug metabolizing enzyme activity in the epidermis is
greatly dependent on
tissue viability. Therefore, it should be stressed that the determination of
skin absorption
described above does not distinguish between the intact antiviral drug and its
metabolites. It
cannot be excluded that excised skin (usually stored) will loose some of its
original enzyme
activity. However, acyclovir exhibits no known metabolism in the skin.
By extracting acyclovir from the skin components, acyclovir can also be
quantified by HPLC.
EXAMPLE 99
Permeation of compositions containing acyclovir or other drugs by means of an
in
vitro cell culture model
The permeation of acyclovir or other antiviral drugs delivered from various
compositions
according to the invention can be examined using in vitro cell cultures as a
model of e.g. human
oral epithelium. A model involving e.g. TR 146 cell (from the Royal Danish
School of Pharmacy,
Copenhagen, Denmark) is suitable for sensitivity and permeability studies of
antiviral drugs.
Other cell culture models are also available, e.g. for the testing of the
efficacy of drugs.
EXAMPLE 100
Permeation of compositions containing acyclovir or other antiviral drugs by
means
of an in vivo animal model
The herpes virus replicate in the living epidermis. The basal layer of the
epidermis appears to be
the primary site of antiviral activity in cutaneous HSV-1 infections, i.e. the
target for antiviral
drugs. Methods - using hairless mouse or guinea pig as an animal model - are
available. The
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methods allow calculation of the target site concentration of the antiviral
(e.g. acyclovir) drug
applied and allow an estimation of the efficacy of the antiviral compositions
tested (see.--e:g. Lee,
P.H. et al., Pharm. Res. 9_, 8, pp 979-988, 1992 and Su, M.-H. et al., Drug
Develop. Ind. Pharm.
20 (4), 685-718, 1994). In the following is described model systems suitable
for testing the
antiviral effect of the compositions according to the invention.
Animal models often used are the hairless mouse model (5-7 weeks old) and the
guinea pig
model. The guinea pigs are shaved on their back before the start of the
experiment in order to
make a hairless test area.
The animals are anaesthetized before inducing skin lesions, e.g. on the
lateral side of the body or
in the lumbosacral area. 0.005-0.2 ml of a virus suspension [herpes simplex
virus type 1 (HSVl),
e.g. strain E-377 or E-115 (titer usually in a range of 106 - 108 plaque
forming units (PFU)/ml),
stored at -70°C until use] was injected or rubbed on the skin with a
cotton swab saturated with
the virus (a drop of the virus suspension is applied on the test area and then
6 small holes are
made by means of a scalpel. The test area on the skin of the test animal can
be divided into
several test areas, e.g. six areas, thereby allowing e.g. two different
compositions (2x2) and their
controls (1x2), placebo) to be tested at the same time on the same animal.
Usually 10-30 animals
are used for each composition (the number of animals depends on the number of
applications).
The infection induced by the virus generated skin lesions which appeared at
the area of
inoculation. Shortly after virus inoculation (e.g. 24-48 hours) compositions
with antiviral drugs
were applied on the test areas at the skin e.g. with a 1 ml syringe and
samples are blindly
randomized. The lesions are treated with the compositions for 2-10 days
(applied 2-5 times daily)
and then the effect of the treatment was investigated. The lesions were scored
for each animal
and two distinct antiviral assessments can be made: i) topical e~cacy is
determined by
measuring the antiviral activity of the antiviral drug substance (e.g.
acyclovir) delivered from the
compositions tested, and ii) systemic efficacy is determined by measuring the
antiviral activity of
the antiviral drug substance (e.g. acyclovir) in the circulatory system which
delivers the antiviral
substance to the target site (presumably the epidermal basal layer).
In order to quantify the effect of the different compositions, a score system
is used. Different
score systems may be employed based on the appearance of the skin lesions at
various times
after inoculation. The score system could be that of Alenius and Oberg,
Archives of Virology
1978, 58, 27?-288, where the course of infection is divided into a phase of
progression denoted by
scores with Arabic numerals and into a phase of regression denoted by scores
with Roman
numerals. E.g. the inoculated areas can be scored for symptoms daily, starting
24 hours after
inoculation and ending after 4-20 days, giving scores during the development
of vesicles and
their subsequent drying and crusting. The length and size of skin lesions can
also be measured.
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A low cumulative score of a composition indicates a good efficacy compared to
a placebo
composition (control) vyhich generally gives a high score. _ _
During the test HSV-1 virus may be isolated from the lesions and the number is
counted. The
results give an indication of i) inactivation of virus, ii) effect of the
antiviral composition applied
etc.
- EXAMPLE 101
Clinical development programme of GMO acyclovir cream for herpes labialis
The following parameters are suggested for all clinical studies:
Setting
Outpatients from GPs, dermatologists or hospital clinics. Primary recruitment
possible in
connection with a Herpes simplex eruption that are not included in the study.
Patients receive
study medication and are instructed to start treatment immediately upon
recurrence of
prodromes and to return to investigator after start of treatment.
Inclusion criteria
Clinically confirmed history of recurrent Herpes Iabialis, 2-3 annual
recurrences. Present
prodromal symptoms of Herpes Labialis eruption.
Exclusion criteria
Herpes labialis with ulceration or crusts
Immunodeficiency
Allergy to acyclovir/GMO
Efficacy parameters
Duration days/hours from start of treatment to cessation of symptoms caused by
virus
replication, including pain, weal, numbness and erythema.
Duration days/hours form start of treatment to crust formation.
Duration days from start of treatment to complete skin healing.
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Safety parameters
Local reactions to cream administration, including a 28-30 day follow-up. _ _
_
Dose finding
The experience from individual case reports indicates that fewer daily
applications of GMO
acyclovir compared to Zovirax~ are required to obtain efficacy. The optimal
administration
frequency will have to be determined.
Study groups:
Placebo
Once daily
Twice daily
Three times daily
If more than three daily applications is required, GMO acyclovir is not
considered to have any
advantage compared to Zovirax~ cream.
At present no data are available on the statistical variation of efficacy
parameters, therefore a
proper dimensioning of the study has not been possible. It is assumed that
between 100 and 200
patients ger study group is required.
Pivotal studies
It is assumed that two identical or at least very similar studies must be
performed.
Study groups
Placebo
GMO acyclovir x times daily
Zoviraxa 5 times daily
The argument for including a placebo group in the pivotal study is to document
that the
expected clinical equivalence between Zovirax~ and GMO acyclovir is not a
consequence of both
products inefficiency.
At present no data are available on the statistical variation of efficacy
parameters, therefore a
proper dimensioning of the studies has not been possible. It is assumed that
between 100 and
200 patients per study group is required.
,,
