Note: Descriptions are shown in the official language in which they were submitted.
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A PHAR~ACEUTICAL COMPOSITION FOR ADMINISTRATION OF AN ACTrVE
SUBSTANCE TO OR THROUGH A SKIN OR MUCOSAL SURFACE
~ The present invention relates to a pharmaceutical composition for ~lmini~tratinn of an active
sllh,st~n-e to or through a damaged or nnrl~m~Eed skin or mucosal surface or to the oral cavity
5 including the teeth of an animal such as a human. The composition is particularly suited for
~lmini~ration of snhs~qnr~ which have a very low water solubility and which are to be
supplied in an effective amount in a loc~ ecl region over a period of time.
Bad~lvu~ld of the invention
One important known PY~mrle of a composition for topic 1 ~l~lmini~ration of a ~llhst~nce of very
10 low water solubility is an l~intmf~nt Cont~ining the antiviral nnrler~ lP acyclovir. This ointmPnt
is available under the registered trade mark "ZovirD" or "Zovira2~D". The release rate from this
composition is rather low, and various suggestions for making topical acyclovir compositions
more effective appear from the patent lit~r~tnre, including suggestions for increasing the effect
of acyclovir by means of a potPnti~tor or Pnh~n~er
15 Disclosure of the invention
For many purposes for which acyclovir ointrnPnt~ are used, it would be desirable to have an
nintmPnt which could release the n1l~lP~ ~i(lP at a relatively high release rate for a snffiripntly
long period of time in the region. An important advantage which would be obtained in this
manner would be that the number of daily appli~tion~ of the flintrn~ont could be reduced, such
20 as from the present about five applications to t~,vo or three applic~tinn~
It has now been found that a particular class of systems, notably the so-called liquid crystalline
phases, is capable of ~ . Liv~ly releasing drug substances of a very low solubility. This finding
must be char~ri~ed as surprising because, as it appears from the P~rrlP~n~ti()n which follows,
the active snhst~n-e in question is one having a very low solubility both in water and in the
25 liquid crystalline phase of the composition. Furthermore, the liquid crystalline phase can confer
"hio~-lhP~:iQn" to the composition, which means that the composition will be able to be retained
for a prolonged period of time at its site of applic~tion, e.g. skin or mucosa. Thus, with such
systems, it becomes realistic to considerably reduce the number of applic~ti-)n~ compared to
known compositions.
30 Thus, the invention relates to a pharmaceutical composition for ~lmini~tration of an active
snhst~nre to or through a damaged or nn~lAm~ged skin or mnco,s~l surface of an animal such as
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a human, the comrocition cnmrricinF the active snh~n(e and an effective amount of a fatty
acid ester which, together with a liquid phase, is capable of gPnP~*ng a liquid crystalline phase
in which the con~it-lpnt~c of the composition are çnrlocPrl,
the composition either being one in which the liquid crysWline phase has been gpner~qtpd by the
5 fatty acid ester together with a sufficient amount of a liquid phase originally present in the
composition, or the composition being in a precursor form in which fatl;y acid ester has not
generated the liquid crystalline phase, but is 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 con~tit~ltin~ at least part of the liquid phase
10 the active snh~nre having
i) a solubility in the liquid crystalline phase of at the most 20 mg/g at 20~C, and
ii) a solubility in water of at the most 10 mg/ml at 20~C, the water, where applicable, being
bu~led to a pH snh~t~nti~lly irlPn*r~l to the pH prevailing in the liquid crystalline
phase, determined as described herein, or
15 iii) a minimllm aqueous solubility of at the most 10 mg/ml at 20~C determined at a pH in the
range of 3.6-9, determined as described herein.
TntPrn~tion~l Patent Appli~ tion No. PCT/DK95/00143, pnhli~hed on 12 October, 1995 under
No. WO95/26715 and being in po~P~ion of the same ~ gnee as the present aprlir~ti~)n,
rlosP5 a composition c-~nt~ininF 2% by weight of acyclovir and 98% by weight of a
20 ~ly~ yl~lonooleate and a composition cont~ininE~ 5% by weight of acyclovir and 95% by weight of
a ~ lonooleate product, wherein the glycerylmonooleate product has the composition:
Gl~ llonooleate about 84% w/w
Gl~ ..nnolin-lP~tP about 7% w/w
Saturated monoglycerides about 7% w/w.
25 Therefore, for states in which the present apI lir~tion is co-pending with a national phase of the
above intPrn~tinn~l patent aprlic~ti~n (this is expressed in the claims as "where applicable"), the
following proviso applies to the scope of the present applir~*~.n the cnmp~i*nn is not one
conqi~inF of either 2% by weight of acyclovir and 98% by weight of a ~;ly~ yllllonooleate or 5%
by weight of acyclovir and 95% by weight of a ~ yLllonooleate product, wherein the
30 glycerylmonooleate product has the composition:
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Gl~a~lnlonooleate 80-85% w/w
Gly~ .on~1in~ t~ 5-10% w/w
.~tnr~te~1 monoglycerides 6-10% w/w.
As m~ntioned above, the ph~rm~reutical compositions accoldillg to the invention are intPnrlPd
~ ~ for app1ic~ n n to or through ~ln~l~m~ged or damaged skin or mucosa of an animal such as a
human. The mucosa is preferably selected from oral, nasal, vaginal, rectal, aural, lung, and
1 mucosa. The skin or mucosa may also be infl~mP-1 The comro~ition may also be
A~mini~4red to body cavities such as the oral cavity or by the bucc 1 route.
Furthermore, a rh~rm~r~nti~-~1 composition according to the invention may also be applied to a
10 nail of an animal such as a human.
In the present context the term "active snhst~nce" is intPn(lPd to mean any hio]ngir~11y or
pharm~cologi(~11y active sl1h~nce or antigen-cnmrri.cing m~tPri~l; the term in~ 1(lP.q drug
snhst~ncefi which have utility in the treatment or prevention of diseases or disorders ~ffe~ting
animals or humans, or in the regulation of any animal or human phyqin1Ogir~1 cnn/litinn and it
also includes any hin1Ogic~11y active compound or composition which, when ~llmini~ered in an
effective amount, has an effect on living ceUs or org~nicmg
~.x~mp1Pq of active s11h.st~ncPs of particular importance in the present context are the so-called
antiherpes virus agents which have been or are developed for the trp~tmpnt of herpes virus
infPctinnq [herpes simplex virus types 1 and 2(HSV-1 and HSV-2), varicella zoster virus (VZV),
cyt.nmPg~lnvirus(CMV), Epstein-Barr virus (EBV)]. The antiherpes virus agents include
antiviral drugs and prodrugs thereof, such as nl1rleoqi(lPq, n11r1Po.qi~e analogues, rhoqphQrylated
n11r1enqillPq (nucleotides), nucleotide analogues and salts, cl7mr1P7rPq and prodrugs thereof; e.g.
gn~nnqine analogues, deoxygn~noqin~ analogues, guanine, guanine analogues, thymidine
analogues, uracil analogues and adenine analogues. Especially interesting antiherpes virus agent
for use either alone or in comhin~tion in a composition acco~ g to the present invention are
selected from acyclovir, famciclovir, deciclovir, penciclovir, zidovudin, ganciclovir, (lirl~noqin,
~1rit~hin, valaciclovir, sorivudine, lobucavir, brivudine, cidofovir, n--loco.s~nn1, ISIS-2922, and
prodrugs and analogues thereof. Details concerning active sllhst5ln~pq suitable for use in
cnnnf-~ion with the present invention as well as a description of other interesting active
30 snhst~nr~q are given below.
As m~ntirmed above an important property of a composition according to the present invention
is its ability to generate a liquid crystaUine phase. The term "liquid crystalline phase" as used
herein is used to denote an intermediate state between solid crystals and isotropic liquids,
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rh_r~r~ri~acl by long-range order and short-range properties close to those of a simple liquid or
solution (Keller et al., Handbook of Liquid CrysWs, Verlag Chemie, Wainhçim, Germany, 1980).
~.x~n plP~ of fatty acid esters with an PxrellPnt ability of forming a liquid crystalline phase are
glyceryl mnnop.qter~q of fatty acids. Specific A~mpla.~ include ~ r~ onooleate (monoolein) and
5 ~ ..yl...onolin.~l~~ta 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 a~l; ined in
further detail below, these fatty acid esters also show so-c, lled hiot~ha~ive properties.
Liquid crystalline phases may be a cubic (three cubic phases are known: i) the body-centred
10 lattice, ii) the primitive ~li, mnntl lattice, and iii) the gyroid), hexagonal~ reverse he_agonal or
lamellar phase. By the term "cubic phase" herein is meant a thermodyn~n~ir~lly stable, viscous
and optically isotropic phase made of a fatty acid ester and an aqueous me~ m The term
"aqueous medium" includes media co..~ ;.lg water or another hydrophilic and water-miscible
snh~nce such as, e.g, glycerol. The terms "hexagonal phase" and "reverse hexagonal phase",
15 respectively, are used herein to describe thermodynAmir~lly stable, viscous and optically
anisotropic phases char~l~teri~ed by long-range order in two rlimPn~ionq and made of a fatty acid
ester and an aqueous mpriium The term 'lamellar phase" is char~ . ;serl by a long-range order
in one .liman.qinn The lamellar ~ ,Ult~ iS the origin of liposomes having srharic~l shells of
lipid bilayers. The various liquid crystaUine phases can be detected and irlPn~ifiecl by use of
20 polarized light or by means of X-ray diffraction pattern analysis (see the ~.~r~mplP.c herein). The
cubic phase is normally the preferred phase in the compositions of the invention, but also, e.g,
the reverse hexagonal phase may be an i-~ ing liquid crystalline phase in the compositions
according to the invention.
In accordance with the above-mPntionRd observations, a fatty acid ester for use according to the
25 present invention may be a fatty acid ester which is capable of forming a liquid crystalline phase
on contact with a suitable liquid phase. The liquid of the liquid phase is suitably water or an
aqueous medium. An aqueous medium is a medium c~)nt~ining water at least in part.
Apart from aqueous solutions or dispersions such a marlinm with which the liquid crystalline
30 phase is made may, especially for the p~ ,ur~oramhorlimPnt of the composition, at least in part
be cnn~itntpd by any body fluid or secretion which contains water and with which the
composition comes into contact upon applic~tion~ such as, e.g. in the case of a human body fluid,
saliva, sweat, gastric juice, etc. As inrii(~tPcl above, the body liquid may induce formation of a
liquid crystalline phase when a fatty acid ester is cnnt~l~ted with such a liquid.
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However, in many embo-liment~, the composition according to the invention wiU be one in
which the liquid crystaUine phase is already present, that is, the liquid crystaUine phase has
already been ~hli~h~d by interaction between the liquid phase and the fatty acid ester. In this
~ case, the liquid of the liquid phase may, e.g, typically be water or glycerol or a mixture thereof,
5 water often being a preferred liquid.
As mQntionQd above, the active sl~hst~nce of the composition of the invention is one whose
solubility in the liquid crystaUine phase is low, at the most 20 mg/g at 20~C, such at the most 16
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
10 or 2 mg/g or 1 mg/g at 20~C.
The determin~tion of the solubility of the active snhst~nre in the liquid crysta~line phase of the
composition is, of course, performed on the liquid crystaUine phase as formed. In practice, this
means that when the composition is one in which the liquid crystaUine phase has already been
formed when the comrn~ition is applied, the (l~qrmin~tion of the solubility is pPrfnrm~d on the
15 composition itself. The determin~tion of the solubility is suitably performed by microscopy to
observe any crystals of the active snhst~nce The determin~tion of the conrQntr~tinn 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 ~ets~hlj~::h~(l NormaUy,
a series of tests with varying cc)nrQntrations is pQrformPd to determine the c~nCQntration above
20 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 determin~tion is a liquid
crystaUine phase imit~ting the liquid crystalline phase which will be formed when the
composition absorbs liquid from the site of applic~ti-n This reference liquid crystalline phase is
made up with water (as reprQ~nting the liquid absorbed) in such an amount that the reference
25 liquid crystaUine phase is the same type of liquid crystaUine phase as is generated from the
precursor composition.
While the lower limit of the amount of the fatty acid ester in the composition is determined by
the requirement that the fatty acid ester, in the amount in question, must be able to form and
m:~int~in the liquid crystaUine phase, the composition will in most cases contain at least 20% by
30 weight, calculated on the composition, of the fatty acid ester, normally at least 30% by weight,
and in most cases preferably at least 40% by weight, calculated on the composition, of the fatty
~ acid ester. These numbers apply to the liquid crystalline phase present in the composition; in
precursor compositions, the cnnrentrations will, of course, be higher.
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The pH of the liquid crystalline phase of the comp~ci*on is in the range of 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 llPtormined by a method involving dispersing e.g. 10% of the liquid
5 crystalline phase (cont~ining the active sllh~nnce and any PXripiPntc) in distilled water and
measuring the pH in the water phase, equilibration between the liquid crystalline phase and a
water phase and mP~cnring the pH of the water phase at 20~C. Alternatively, the pH of the
liquid crystalline phase may be measured by means of an suitable pH electrode (see the
~.x~qmrlPC).
10 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 3.6 or higher, and thus, interesting pH ranges for
the liquid crystalline phase are pH 3.6-8, such as 3.7-8, e.g. 3.8-8, such as 3.9-8, e.g. 4.0-8, such
as 4.1-8, eg 4.2-8, e.g 4.3-8, such as 4.5-8, e.g. 4.75-8, such as 5.0-8.
As stated above, the solubility of the active sllh~+nnre in water is very low, at the most 10 mg/g
15 at 20~C and at a pH ,~nhstnn~iqlly i~entir~l to the pH of the liquid cryst~lline phase, ~leterminPd
as (1P.c~rihed 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 sllhstnn- e is meant the water solubility at
the relevant pH, which is a pH snhst~nti~lly iclpntir~l to the pH which will prevail in the
composition, in other words, the pH of the liquid crystalline phase, this pH being determined as
20 described herein. When the pH of the liquid crystnlline phase, determined as described herein, is
di~t~ lt from the pH which will result simply by ~licc~ ltion of the active sllhst~ncç in water,
the water is adjusted to sllhstnntinlly the pH of the liquid crystalline phase by using a suitable
buffer system when determining the s~-lnhility of the active s--hstnncç This buffer system should
of course be so selected that, apart from the pH adj--c~tnPnt~ it has sllhstnntinlly no influence on
25 the solubility of the active snh~nnre in the buffered water.
The composition according to the present invention is very valuable in that it can provide a high
release of active snh~nncPq 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 snhstnnti~lly
idPntir~l to the pH of the liquid crystalline phase, determined as described herein.
30 Of particular interest is also the fact that Px-Pllent. release rates can be obtained of active
~lhst~n~e 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 i~l~ntirs~l to the pH of the liquid crystalline phase, determined as
described herein.
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AltPrn~tively~ the active sllhst~ncR has an minimnm 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
corresponding to 3.6-9. The det~rmin~ti~ln of the minimnm aqueous solubility is pRrfnrmPd 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 obt~ined between the undissolved and
dissolved active snhst~nrP~ i.e. by employment of nltrAeonic treattnPnt and/or stirring for a well-
defined time 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 liql~id crystalline phase apply mutatis mutandis when the aqueous solubility is
the minimum solnhility in a pH range of 3.6-9.
In embo~limante of particular interest a composition according to the invention cont~inc one or
more antiherpes virus agent(s) as an active snhst~nre Relevant antiherpes virus agents are
mRnti~nf?d above and acyclovir is of particular importance. Acyclovir (9-[2-
hy(lr~yt:lhoxy)methyl]-guanine, an acyclic analogue to the natural nllrlP~Iei~le 2'-
deoxygnAn~.sin~, is a widely used agent in the treatment of herpes virus infPctionc Compositions
for oral, topical and intravenous iq(lmini~tration are available. The delivery charArfaristire of
acyclovir following A~lmini~ration 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 1.6 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-rhamir~l
properties, the bioavailability after oral ~mini~ration is rather low (about 16-20%) and highly
variable and the percutaneous panetrAtion is poor.
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 ZovirX or Zovira~D. The object of the present invention has therefore inter
alia been to develop a bioa~lh~ive composition c~-nt~ining e.g. acyclovir or other antiherpes virus
agents with improved release properties so that fewer daily aprlir~qt~on~ are needed to produce
the same therapeutic effect (bioequivalence) or even improve the therapeutic effect.
As appears in more detail in the E~pPrimPntAl section herein, the present inventors have
developed compositions contAining GMO/water 66/35% w/w with acyclovir (crystalline and
micronized, respectively) added in a c. nrPntration of 1-40% w/w. Cubic phases are obtained in
~ these compositions as evidence by polarized light. The results indicate that acyclovir in the
cnnrPntration range inv.~stigAt.~d does not ruin the cubic lattice, and that acyclovir probably is
inert in the cubic system. The distribution of the drug crysta~s in the cubic phase appears as a
35 homl~ganPous distribution (observed by microscopy). The cubic phase without drug is
-
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transparent and has a relatively high viscosity. It is coem~P~ 7lly appealing. When acyclovir is
added, the viscosity is increased with the ~nnrPnt~7tion~ especially for the micronized quality.
When the crystalline quality is added, the composition becomes greyish white. When the cubic
phase is applied to human skin it melts and penetrates the skin.
6 As mPnh~necl above, Zovir~ and Zovira~ cream cont~7ining 6% w/w acyclovir are presently the
drugs of choice for the tre~tmpnt of herpes RimplP~ In order to compare the release rate of
vvir from Zovir~ cream and a cubic phase (GMO/water 65/36 % w/w) cQnt~7ining 5% w/w
acvclovir, the release of acyclovir from these compositions was P~s7minPç7.~ cf. ~7mple 16 herein.
Comp~7ring the rate con~7nt-~ it is seen that the release rate of acyclovir is about 5-6 times faster
from the cubic phase than from the Zovir~ cream. Poor release properties of the Zovir~lD cream
are most likely one of the reasons for its suboptimum therapeutic effect. The improved release
properties from the cubic phase must therefore be seen as a very promising result.
Important embor7imPnt~ of the present invention are comrositionc in which the active snh.st~n~e
is present in a c~n~ c.Lion which is above the saturation ronr~Qntration at 20~C so that part of
the active substance, and in many cases the prer7.r~min~nt proportion of the active s7lh.s~nrç, 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%, by weight of the active s~lhst~7nrç present in the composition CQnr ~ a
proportion w_ich is present above the saturation con~ ;cn at 20~C. Very va7uablecompositions 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 5nh5t~7nre present in the
composition con~;D~I~s a proportion which is present above the saturation conrPntr~7tion at
20~C.
While the present invention is not to be limited to any theory, it is be7ieved, and supported by
expPrimPnt~l data reported herein, that the c~r~7hility of the composition to release the active
5nhst~7nre of very low water solubility and very low solubility in the liquid crysWline phase at
very s~7ticf~ , release rates is due to some kind of efficient dissolution system for particles,
such as crysta7s, of the active 5llh5t~nce through the 7,iquid phase "rhs7nnPl~" of the liquid
crystalline phase.
The fatty acid esters capable of generating a liquid crystalline phase as evidenced by one of the
test mPt~7or7l~ described herein are fatty acid esters (i.e. composed of a fatty acid componP~nt and
a hydroxy-cont~7ining component) wherein the fatty acid component of the fatty acid ester is a
s~7t7lr~tPd or un~Lu~,ted fatty acid having a toW number of carbon atoms of from C6 to C26.
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Specific Px~mrlPC of saturated fatty acid mniel;~s in the fatty acid esters according to the
invention are selected from the group conRi.cting of mnieti~c of caproic acid, caprylic acid, capric
acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, and behenic acid.
Specific Px~mr~lPc of ~ c~ te~l fatty acid mnietiPc in the fatty acid esters according to the
5 invention are moieties selected from the group concic~ing of pAlmit~ ic acid, oleic acid, linoleic
acid, linnlPnic acid, and ~rs~chi~onic acid.
Particularly suitable fatty acid esters for use according to the invention are fatty acid esters
which are selected from the group Con~ictinF Of fatty acid esters of polyhydric fllcQhol.~, fatty acid
esters of l~ydlo~ ~rboxylic acids, fatty acid esters of monn,c~c-h~rides, fatty acid esters of
10 ~ .h-,sph~te d~ Livt:s, fatty acid esters of glycerylsulfate d~liv~Liv~s, and l~ LUL~S
thereof. In those cases where the hydro_y-cnnt~ining component of the fatty acid ester is
polyvalent, the hydroxy-c--nt~ining component may be partiaUy or totally ~ rified with a fati~y
acid component or with nli~lu~l:s of fatty acid components.
The polyhydric alcohol component of the fatty acid ester for use according to the invention is
15 preferably selected from the group concicting of glycerol, 1,2-prop~3nerlinl, 1,3-prop~n~linl
diacyl~l,qctn~ylglycerol, diacylrli~l~f~t( ~ylglycerol, erythritol, xylitol, s~rlonitc~l, arabitol, m~nnitnl
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 hio~(lhpcive properties and are therefore preferred fatty acid esters for use according to
20 the invention. The position of the polyvalent alcohol on which the ester bond(s) is(are)
P~slhlich~d 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.
~ mplPc of fatty acid esters for use according to the invention and wherein the hydro2~y-
25 ccmt~ining component is a polyhydric alcohol are ~ l,llonooleate, glycerylmonnlinnle~tP,
glycerol monnlinnlP~te, and mi~tures thereof. These fatty acid esters have especially promising
hio~(lhPcive properties, confer the ~ mrlPc herein.
In those cases where the fatty acid ester for use according to the present invention is formed
between a hyd~y~rboxylic acid (or a d~l;v~Liv~ thereof) and a fatty acid (or a d~;ve~liv~
30 thereof), the hyd~ y~rboxylic acid component of the fatty acid ester is preferably selected from
the group concictinF of malic acid, tartaric acid, citric acid, and lactic acid. An interesting
P~nnrle of a fatty acid ester for use according to the invention is a fatty acid monoester of citric
acid.
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As nnf?ntioned above, the hydroxy-cnntAining component of a fatty acid ester for use according to
the present invention may also be a sarrhari(lp~ such as a m--nos~qrrharirlP such as, e.g, glucose,
mann- se, fructose, threose, gulose, arabinose, ribose, erythrose, lyxose, g~alactose~ sorbose,
altrose, tallose, idose, rhamno,sP, or allose. In those cases where the hydroxy-containing
5 componPnt is a m~nt~qacrhari~lp~ the fatty acid ester is preferably a fatty acid m~noPqtpr of a
mon- qarrharifl~ selected from the group conciqtin~ of sorbose, ~alArtocç~ ribose, and rhan~nf~,cç
The hydroxy-containing c--mponPnt. of a fatty acid ester for use according to the invention may
also be a glycerylphosphate derivative such as, e.g, a phospholirill selected from the group
c~n.qiqting of pho~sphra~tirlic acid, phosphatidylserine~ ~hosphati~lylPthanolaminp)
10 phosphatidylrhnlimP, IlhnsphAh(lylglycerol, I~hncphati~ylinocitnlP~ and diphncrhati-lylglycerol.
Especially interesting compounds having a phosphrllipi~l moiety are compounds wherein the fatty
acid ester is a fatty acid ester of a ~ly~ hosphate d~.;v~Livc, and the fatty acid component is
selected from t_e group cnn.ciqting of lauric acid, myristic acid, palmitic acid, stearic acid, oleic
acid, linoleic acid, linc~lenic acid, and behenic acid. ~.xamplP~ of such useful fatty acid esters are
15 dioleyol phosphatidylcholin, dilauryl phosphatirlylcholin, dilyl;sLyl phnsphatit1ylcholin~
llirAlmitoyl pho5phati~ylcholin~ distearoyl phosphatidylcholin, dibehenoyl phncphatif1ylcholin,
dil~lyl;DLyl I~hocphati~lylPtllannlaminP, r1iralmit~lyl phncphati~lylPt~annlsminP, dioleyl
phosrhAtidylglycerol, dilauryl pho5phati~1ylglycerol, dillly~;D~Jyl phnsphati(lylglycerol, llip~a~lmit
phosphatidylglycerol, distearoyl phosrhati(lylglycerol~ ~lipalmitoyl phosphatic acid and mixtures
thereof.
Most of the fatty acid esters for use according to the invention are well-known rhPmicAl
compounds which are commercially available or may be prepared by means of conventional
Pst~rifira~tion procedures involving e.g. reaction of a fatty acid dcl;v~livc~ such as, e.g, the
corresponding add chloride with a hydroxy-contAining compound (if nPcpccAry protected with
suitable protection groups) and subsequently icr)lAting the fatty acid ester, if npcp.ccAry 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
apprnximAtPly 100% pure fatty acid ester. As an P~AmplP it can be mPntionpd thatyllllonooleate from CTrin~l.ctpd Products A/S, Denmark is a very pure product c-~ntaining
about 98% w/w monoesters of which more than about 80% w/w (such as about 92% w/w) is
e.yl~nnor)leAtp; the remAining mc~nop~tprs are ~ly.c.~l...nnolinoleate, glyceryl monopAlmit~ate
and glyceryl mr~nr~te~a~r~t~ The fatty acdd ester products for use according to the invention may
thus be ~i~u~aD of fatty add esters.
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11
F~,rAmrlPc of fatty acid esters with e~rpllpnt hio~ hacive properties as well as an e~rPll~nt ability
of forming a liquid crystalline phase are glycery-l monoesters of fatty acids. Specific P~Amrlac
include ~ hllonooleate (monoolein) and ~,ly~vl~h..nn-linnlPAtP As mantionpd above, such
fatty acid esters are capable of forming various crystalline phases upon contact with a
5 hydrophilic medium such as water or glycerol, a preferred liquid crystalline phase being the
cubic phase.
Thus, very interesting comrociti~nc according to the invention are compositions in which the
fattv acid ester is ~ ce~ nn(u~leAtP or ~ yll.lonf~lin~lP~A~tp~ in particular glycerylmonooleate.
It has been found that the stability of the composition is considerably enhAnce~l such as
10 resulting in a storage stability of at least 2 years at 20~C, when the ~ onooleate product
(as is well known, fatty acid esters are almost invariably mixed products~ cc~ntAined in the
product fulfils certain purity standards. Thus, the glycerolmonooleate product used for the
prepAr~tinn of the composition should contain at the most 4% of stl~uif.Led monoglyceride and
should preferably contain at least 88% of ~ onooleate, more preferably at leact 89%, such
as at least 90% or at least 91%, in particular at least 92%, of ~l~. v.. yl.. lonooleate.
-
When the composition is a precursor type composition, the liquid phase is either not present at
all or is present in small amounts, such as an amount of at least 0.6% by weight, such as at least
1% by weight, rAlc~ tpcl on the total composition, e.g. at least 2% by weight, cAlrlllAtPd 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 phase is normally present in an amount of at least
20% by weight, c~lclllAtpd on the total composition, such as at least 25% or at least 30% by
weight, CAlclllAtPd on the total composition, and a preferred amount is often in the range of 25-
50% such as 30-50% by weight, in particular 27-40%, 27-37% or 30-40% by weight, CAl( lllAtPd on
the total comp~leition
The active snhst~nre may have any degree of lipophilicity. In certain interesting compositions,
the active snhstsnre is one which has a lipophilicity of at the most 100, such as at the most, e.g.,
~ 75, 50, 25, 10, 7.5, 5 or 2.5, expressed as the paffition coQffirient between octanol and 0.05M
phosrh~t-p buffer, pH 7, at 20~C, in some a partition copmripnt of at the most 10 or even at the
~ 30 most 1 or at the most 0.75, 0.5, 0.1, 0.075, 0.05 or 0.04.
Alternatively, the liroI)hilirity may be expressed as the partition copffiripnt between octanol and
an appropriate buffer having a pH corresponding either to the pH of the liquid crystalline phase
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12
or to the pH at which the active snh~ nre has its minimnm solubility. In such cases, the value
manfionf~d above are also valid.
The performance of the compositions according to the invention with respect to releasing the
active sllh~nre from the liquid crystalline phase can be adequately ~l~bsed by the slope of
5 the cumulative release in ~g as a function of the square root of the release time in hours in the
release a~erimPnt defined in connpction v~ith Fig 6 (in which the conrPntrRtion of the
,cnhsfRnre is 5%). In preferred compositions according to the invention, the slope is at least 50,
more preferred at least 100.
An expression of better p~. r -....Rnre 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.
As m~nti~-n~d above, it is a great advantage of the compositions according to the invention that
the fatty acid esters can confer bioadhesivity to the compositions. During the last decade
increased attqntion has been given to the possibility of using hioa~lh~ive/mnroR~lhP~ive
polymers for drug delivery purposes. It is believed that several problems R~ori~tad with
conventional controlled release drug delivery systems may be reduced or PliminRt~d by using a
hioa~lha~ive/mluroR~lhe~ive drug delivery system. In conv~a~Lional controlled release drug delivery
systems no prerRIltinn~ are made in order to localize the delivery system after Rllmini~tration
and, furthermore, the contact time in vivo between the drug delivery system and a particular
site is often so short that no advantages are to be ~ected with respect to, e.g., modifying tissue
perm~Rhility. Compared vwith conventional controlled release drug delivery systems, hi~R(lh~ive
drug delivery systems are believed to be banafiri~l with respect to the following features:
i) a hioa~lha~ive drug delivery system lorRli~ a drug snhstRnce in a particular region,
thereby improving and Pnh~nring the bioavailability for drug snhstRnras which may have
poor bioavailability in themselves,
25 ii) a hioa(lhegive drug delivery system leads to a relatively strong interaction bet~,veen a
bioR~lh~qive snhst~nre and a mucosa; such an interaction contributes to an increasing
contact time between the drug delivery system and the tissue in question and permits
lorRli7Rti~n of the drug delivery system to a specific site,
iu) a hioa(lh~ive drug delivery system is contPmI lRtP~l to prolong delivery of drug snhst~ncac
in almost any non-parenteral route,
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13
iv) a hio~lhpcive drug delivery system can be localized on a specific site with the purpose of
local therapy e.g trp~tm~nt of loc 1 fungal ~lice~cPc~ perme~hility n~orlifi~tir~n~ protease
and other enzyme inhihition~ and/or modulation of immnnnlogic expression,
v) a hif~llhPcive drug delivery system may be targeted to specific fli~Q~ced tissues, and
.
vi) a hioar1hPcive drug delivery system may be employed in those cases where theconventional approac_ to controlled release drug delivery is unsuitable, i.e. for certain
drug snhst~nçPc or classes of drug sllhst~ncec which are not adequately absorbed.
Thus, preferred compocitionc according to t_e present invention are comp-7,sitic nc in which the
fatty acid ester or comhin~tion of fatty acid esters present in the composition cnmrliPc with the
requirements of hina~h~ion defined herein when tested for hio~llhPcinn in an in vivo model or
any other bi~ hPCivity model as given in the experiment~l section herein. Especially preferred
are compositions which in themselves comply with the requirements of hio~llhPcion defined
herein when tested for bio~-lh~cion in an in vivo model or other hio~h~civity model as given in
the exp~riment~l section herein.
Thus, interesting compositions are compositions in which the fatty acid ester or comhinsltion of
fatty acid esters, when tested in a hioaflh~cive test system, compricing
i) placing a segm~nt of lollgit~ in~lly cut rabbit jejunum on a st~inl~cc steel support in such
a manner that the mucosa layer of the jejunum is placed upside so as to allow application
of said fatty acid ester,
20 u) placing the resulting support at an angle of -21~ + 2~ in a cylinrlri~l cell thermnst~t~d at
37~C + 0.5~C and with the relative humidity kept at about 100%,
iii) flushing the jejunum on the support with 0.02M isotonic I~hosrh~tp buffer solution (pH
6.5, 37~C) for 5 min at a flow rate of 10 ml/min,
iv) applying an ac~ ately weighed amount of a sample of said fatty acid ester (about 100 mg)
~25 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 phncrh~tP buffer solution on the sample applied,
vi) leaving the resulting sample from step v) for 10 minntPc in said cell to aUow the sample to
interact with glycoproteins of the jejunum,
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14
vii) flnqhing the jejunum with the sample applied with said phocph~t~ buffer solution (pH 6.6,
37~C) for 30 minutes at a flow rate of 10 ml/min,
viii) collecfing the washings resulting from step vii), and
i_) r~ nl~hn~ the residual amount of the sample r~m~ining on the jejunum by measuring
the amount of the sample in the washings or by measuring the amount rem~ining on the
jejunum,
results in a residual amount of at least 60% w/w, in particular a residual amount of at least
70% w/w, such as at least 80% w/w, preferably at least 8~% w/w and more preferably at least
90% w/w.
10 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 fatty acid ester or c-lmhin~tion of fatty acid esters or at least 40% w/w of the active
snh~n~e
A measure of the hio~rlh~qivity of a composition itself is that it comrli~q with the requirements
1~ for hio~hecion defined herein when tested for hioa-lheqion in the in vivo model described herein
involving testing the rinsing off ability from skin.
The active sllh~nce 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.
As m~nti-necl above, an important f-x~mrlf? of an active sllhst~nce is an antiviral drug, such as a
20 nucleoside or a nnrll?o~qi(le analogue, e.g selected from acyclovir, famciclovir, deciclovir,
penciclovir, zidovudin, ganciclovir"~ noqin~ it~hin~ valaciclovir, sorivudine, lobucavir,
brivudine, cidofovir, n-~locQs~nol 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 snlllhility are important active
25 sllhst~ncPc 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 sllhst~nce (provided the solubility criteria are fulfilled) or in comhin:~ti~n
with another active sllh~ncPq In the following is listed a number of active snhst~nce which
either alone or in cQmhin~ti~m may be incorporated in a composition according to the present
30 invention. In particular a comhin~tion of an antiherpes virus agent and a glucocorticosteroid is of
importance.
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16
l~c of drugs which are of particular importance in connection with aprlic~t~on to skin or
mucosal surfaces are:
~ Acyclovir, f~mrirlovir, ribavirin, zidowdin, ganciclovir, ~ nocin~ ~Alçit~hin~ valaciclovir
~nnr~nt~lin, rim~nt~(lin
6 fio~k~rn~t
rirlin
fluoruracil
interferons and variants thereof, in~ (ling alpha interferon, beta interferon, and gamma
interferon,
10 trom~nt~lin
l~ntins~n
lev ~flr~in
stavudine
tacrine
16 vesnarinone
,s3mrligf-n
atevirdine
delavi~ e
hyd~v~yuiea
20 indinavir suLfate
interleukin-2 fusion toxin, seragen
lamivudine
lidakol
nevirapine
26 ~nç~n~qe
saquinavir
~ topotecan
verteporfin
viraplex
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16
CMV immnnoglobulin
efalith
epervudine
podophyllotoxin
pro~igPrmslninm
rifabutin
bromovinyldeoyuridine
ukrain
cidofovir
imiquimod
lamivudine
sorivudine
vlraplex
~fuvil~e
15 ~m~nAfi~
hyperlcin
provir
temoporfin
aphi-licolin glycinate
20 ibobucavir
virend
AL-721
ampligen
s~rilrlonP
26 brivudine
CD4
2-deoxy-D-glucose
desciclovir
dichl oroflavan
30 (lirl~no~ine
ditiocarb Sodium
ednsnl-1in~
~llvil~x~e
fi~i~hin~
35 inosine Pranobe
peptide T
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17
stavudine
tribavirin
trifluridine
vidarabine
7,~1rit~hinP
micon,~r~l
fucidin
erythromycin
macrolides
NSAID's
peptides
insulin
polymydn
myperizin
antibiotics
nicotine
sllcrfllfs~tP
sucrose octasulfate
salicylic acid
urea
benzoylperoxide
minflxi~il
heparinoid
methotrexate
rirlo~sporin
A listing of snhstqnrP.s of potential interest comrri~P~ sllhRtqnc~.c of the following groups:
anti-infl7lmm~qt?ry drugs such as, e.g., ibuprofen, in~lomPths~rin, naproxen"lirlofPn~c, tolf~n~mic
acid, piroxicam, and the like;
~ narcotic antagonists such as, e.g., n~ nnP, nalorphine, and the like;
~ 30 ~ntir~rkin~l~ni.~m agents such as, e.g., bromocriptine, biperidin, bçn~hP~nl, benztropine, and the
like;
,~nti~lepressants such as, e.g, imipramine, nolLl;~Lylille, pritiptylene, and the like;
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18
antibiotic agents such as, e.g., clindamycin, e~ythromycin, fusidic acid, gent~mi~in, mupirocien,
a~fomyw~ neomycin, metronidazole, silver sulphRlliR~ina~ snlrhRmetllisole, bRritrarin,
framycetin, polymycin B, aw~ .y~ll, and the like;
antifungal agents such as, e.g., mirnnR~ol k~toconR~nla, clotrimA7Ole~ Rmrhot~ririn B, nystatin,
mepyramin, econA7ol, fl~lconR7f~l~ ilucytocine, griseofulvin, kifonR7~-le; amorolfine,
itr~con~nla, terbenafine, terconR7nle, toln~ft~tA, and the like;
Rntimirrobial agents such as, e.g, metroniclR701a, t~L.~ es, 0xytt:Lla~ e~ and the like;
slntiam~;r.~ such as, e.g., metoclopramide, droperidol, hRloperidol, prom~hR7ine> and the like;
~ntihi~tslminRc such as, e.g., chlorI)hanir~min~ terf~nR~line~ triprolidine, and the like;
10 ~ntimigrR~ina agents such as, e.g., dihydroergotamine, ergotamine, ~ iyli~e, and the like;
coronary, cerebral or peripheral vasodilators such as, e.g, nif~o~lirinf~, rlilti~m~ and the like;
~ntignginRlc: such as, e.g, glyceryl nitrate, isosorbide denitrate, molci~lomin~ varRrRmil, and the
like;
cRlcium channel blockers such as, e.g., vf~rRrRmil, nifP(lipinf~, ~lilti~am, nicardipine, and the like;
15 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, ethinyl estradiol, lynestrenol, etynodiol, norethisterone,
mestranol, norgestrel, levonorgestrel, deso~llel, med.~y~Lc~ Lel..ne, and the like;
20 antithrombotic agents such as, e.g., hepRrin, warfarin, and the like;
diuretics such as, e.g., hydrochlorothiR~ a flunarizine, min~ and the like;
antihypertensive agents such as, e.g., propanolol, metoprolol, rlnnillin~, pin(lolol~ and the like;
corticosteroids such as, e.g., berlomethRcoma~ ket~methRc( na, bet~m~hRcone-17-valerate,
bet~methRconP-dipropinnRta, clobetasol, rlobetRcrll-l7-butyrate~ clobetasol-propionate, ~laqr~ni
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19
desoxym~~h~cl-nP, ~lP~r~m~h~cnnP, difiucortolone, finmpthAcomp~ fiumpth:~cona-pivalate~
fl~lorinC~lone ~cetoni~, fillocinnni(7H~ hyd~uco.Liso~-e, hydrocortisone-17-buly-~Le, hydlùc~lLisone-
buteprate, methylpre(lni~olnne, trjqmrin~ np ~retoni~ bn(l~cQni-lP, hsllrin~ ni~l~ fluprednide
acetate, ~lklomptp~cf~nR-diprc!pion~tQ~ fluocortolone, flnti~con-propion~tQ~ mompt~one-furate~
5 desoxymethasone, diflurason-~ ret~te, halquinol, cliorhin~ clorrhin~lclnl, fl~ rinolonP
~retoni~l~ and the like;
ll~PrmAt~logiç~l agents such as, e.g, nitrofilr~ntnin, rlitllr,qnol, clioquinol, LyLu~y4..h.tlinP
isotretionin, met~n~q~lPn, methotrexate, tretionin, tril ~c~l~n, salicylic acid, pPnirill~minH, and
the like;
10 steroids such as, e.g., estradiol, plu~ elu..e, norethindrone, levonorgestrol, ethynodiol,
levenorgestrel, no~H,~ t~, gestanin, deso~H~L~el, 3-keton-deso~H~Llel, demegestone,
prnm~thoestrol, tpsto~terone~ spir nnl~rt~mP, and esters thereof,
nitro compounds such as, e.g., amyl nitrates, niLl~ le and isosorbide nitrqtP~,
opioid compounds such as, e.g, morphine and morphine-like drugs such as buprenorphine,
15 oxymorphone, hydromorphone, levorphanol, fentanyl and fentanyl de.;v~Livr~s and anAlogues,
prn~gl~n(linc such as, e.g., a member of the PGA, PGB, PGE, or PGF series such as, e.g.,
misoprostol, dinoproston, carboprost or enaprostil,
a ben7~mi(1~P such as, e.g., metoclopramide, scop-llsmin~P,
a peptide such as, e.g, grov~th hl rmon~o releasing factors, growth factors (epidermal growth
20 factor (EGF), nerve growth factor (NGF), TGF, PDGF, insulin growth factor (IGF), fibroblast
grov~th factor (aFGF, bFGF, etc.), and the like), som~t-)st~t;n, r~lrit-~nin, insulin, vasopressin,
interferons, IL-2, urokinase, serratiopeptidase, superoxide tlicmllt~ce (SOD), LhylvL~ in
releasing h~rmonP (TRE), lnt~ini7ing hormone releasing hormone (LH-R~I), corticotrophin
releasing hc~rm~me (CRF), gro~-vth hormone releasing hormone (GHRH), oxytocin, erythropoietin
25 (EPO), colony stimulating factor (CSF), and the like,
a ~r~nthine such as, e.g, caffeine, theophylline,
a r~terholsminp such as, e.g., ephe-lrinp~ salbutamol, terbutaline,
a dihydlu~y~;dine such as, e.g, nife~1irinP,
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a thiazide such as, e.g, hydrochlorotiazide, flunarizine,
others such as, e.g, propAnthPlin, silver nitrate, enzymes like Streptnkin~cPc~ Streptodases,
vitamins like vitamin A, tretionin, isotretionin, acitretin, vitamin D, calcipotriol, interferon-a-2b,
selen disulfide, pyrethione.
5 It will be l1ntlprstQod that the compositions of the invention may also cnmpriRe comhin~tions of
active snh~nr~s, e.g. an active snh~nre together with a pot~nti~tor therefor.
As evidenced in the ~.x~mplPc herein, an active or protective snhs~nce does not cignifir~ntly
influence the bio~tlhpcive properties of a vehicle provided that the con....i dlion of the active or
protective snhst~nce is relatively lo~v such as at the most about 10-15% w/w or at the most
10 about 8-10% w/w. The kind of active snhst~nre (structure, molPclll~r weight, size, physico-
rhemir~l properties, loading, pKa, etc.) will of course be responsible for the m~xim~l
concPntration which can be incorporated in the vehicle without ,cignifirantly AffPcting the
bio~tlhPeive properties of the composition. In the ~.x~mpl~ herein, it is also demonstrated that
the active snhst~nre locates in the liquid crystalline phase of the fatty acid ester and most likely
15 the solubility of the active snhst~nrP in t_is phase has impact on the hir~rlhPcive properties as
well as on the release properties of the composition.
As manti()nRd above, the applir~tinn is intPndPd for skin or mucosa. Other applirationc may of
course also be relevant such as, e.g., aprlir.~t;on on dentures, prostheses and applic~tion to body
cavities such as the oral cavity. The mucosa is preferably selected from oral, nasal, aural, lung,
20 rectal, vaginal, and ga jt~ h~l mucosa.
A hio~lhp~sive composition for ~ mini~ration 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 mnltirle unit
composition may be ~rlmini~t4red to skin or mucosa, preferably the mucosa is selected from oral,
nasal, rectal, aural, vaginal, lung, and gastrointpstin~l mucosa. Most preferred is a hio2~rlhpcive
25 composition intandpd for ~rlmini~tration to the ga~ P~ l tract.
Rio~(lhPRive compositions according to the invention for aprlicatinn on skin and especially to
wounds may in certain cases comrri~e a polys~rrhAri-lP in a cnnCpnt~tion of at least 15% w/w,
c~lrlll~t~l on the total weight of the composition. The poly~rrh~ri~lP is preferably selected from
the group cnnci~ing of carmelose, rhito,c~n, pectins, x~nth~n gums, carr~eçn~nc, locust bean
30 gum, acacia gum, gelatins, ~lgin~tpc~ 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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21
Apart from the active or protective ~lhst~nre and the hjoArlh~cive fatty acid ester snhst~nrP the
hA~h~ge compositions for use according to the invention may comrrice pharmAcelltirs.lly or
cl 5meti~11y acceptable exripientq
The hi~A~lhQcive comrogitiong may be in form of, e.g., a spray, a s~lnt;on, a dispersion, a
5 suspension, an emulsion, powders, gels inr~ in~ hydrogels, pastes, ~intmPntc, creams, drenches,
delivery devices, suppocitoriAg~ enemas, implAntg, aerosols, microcapsules, microspheres,
n~nr~p~qrticles, lipos--mPe, dressings, bandages, plasters, tooth paste, dental care compositions,
and in other suitable form.
The hit~A~lhPcive compositions may be formulated according to co~-v~lllional pharmaceutical
10 prArtirA, see, e.g, "Remington's PharmAce~ltir~l ~ripnresll and "Encyclopedia of PhArmAreutical
Terhnt-logy", edited by Swarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., Ne~,v York, 1988.
Ph~rmArelltir~lly acceptable PxripiAnt~c for use in hioArlhAcive compncitiong for use according to
the invention may be, for ~x5lmrlA
inert diluents or fillers, such as sucrose, sorbitol, sugar, m~nnitol, mic~ ,L~, i,l~lline cPlhllose,
15 carboxymethylcellulose sodium, methylcPlllllQsP, hyd~ y~ropyl methylcellulose, ethylcellulose,
starches including potato starch, calcium carbonate, sodium rhlori~e, lactose, c~cium phosphate,
calcium sulfate or sodium phosrhAt~p; and
lubricating agents inrlnlling glidants and Anti ~lhAgives~ for P~r~mplP, mA~nPcium stearate, zinc
stearate, stearic acid, silicas, hydrogenated vegetable oils or talc.
20 Other phArmAreutically acceptable P~ripiAntC can be cnlorAnt~c, navou.;~g agents, plActi
hnmPct~ntc, buffering agents, solnhili7ing agents, release modulating agents, etc.
For applirP~tion to the rectal or vaginal mucosa suitable compositions for use according to the
invention include suppocit~riPs (emlllgion or suspension type), solutions, enemas, and rect 1
gelatin capsules (s(lllltion~ or suspensions). Appropriate ph~rmAreutically acceptable suppository
25 bases include cocoa butter, esterified fatty acids, gly-rprinAted gelatin, and various water-soluble
or dispersible bases like poly~hylene glycols and polyoxyethylene sorbitan fatty acid esters. Vari-
ous additives like, e.g., enh~nrers or sllrfArt~nt~c may be incorporated.
For applir~h-~n 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 phA~ I;rAlly acceptable
-
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ve_icles and PXriri~nte and optionally other ph~ r-reuLically acceptable m~teri~le present in the
comroeition such as (lilllantc, çnh~nççr,q, lldvuul;llg agents, preservatives etc. are all selected in
accordance with conventional rh~rm~reutical practice in a manner understood by t_e persons
skilled in the art of form~ ting pharmaceuticals.
5 For applirAtion to the or 1 cavity, teeth, skin or nail, the compositions for use according to the
invention may contain conven~ion~lly non-toxic ph~rm~reutically ~rcephhlç carriers and
Rx(iriantA including microspheres and liposomes. The formulations include creams, f)intrnent.e,
lotions, linimPnt.e, gels, hy-l~v~ ls, solutions, snepen~ione, sticks, sprays, pastes, dressings,
bandages, r~ tere, toot_ paste, dental care compositions, and the like. The pharm~reliltic~lly
10 acceptable carriers or Pxriri~nt.q may include emulsifying agents, ~nti~ nt.e, buffering agents,
Selv~Liv~S~ hllmP~rtf~nt~e~ penPtr~ m Pnhflnrers~ chP~ ngagents~ gPlformingagents~ ointmpnt
bases, perfumes and skin protective agents.
Fx~mrle,q of emulsifying agents are naturally occurring gums, e.g. gum acacia or gum
tra~-~nth, naturally occurring phosph~ti~l~.c, e.g soybean lecithin and sorbitan mnnor)lP~tç
15 del;v~Lives.
F.x~mples of slnti~xiflsint.c are butylated hydroxy anisole (BHA), ascorbic acid and del;v~lives
thereof, tocopherol and del;v~Liv~s thereof, vitamin E, salts of sulphur dioxide, butylated
hydroxy anisole and cysteine.
F.x~mrlPq of p~eselvdLive:i are parabens, such as methyl, ethyl, propyl p-hylllvAybenzoate,
20 butylr~r~hPn, isobutylparaben, isopropylparaben, pot~q~ m sorbate, sorbic acid, benzoic acid,
methyl benzoate, phenoxyethanol, bronopol, bronido~, MDM hydantoin, iodopropynylbutylcarbamate, EDTA, propyleneglycol (increases the solubility of pres~l v~Lives) bPn~lçnnium
chlori-lç, and benzyl~lroh-ll
FxslmrlP,q of hnmPctSIntq are glycerin, propylene glycol, sorbitol and urea.
25 F.x~mrlPq of suitable release modulating agents for use according to the invention are glycerol,
sesame oil, soybean oil, lecithin and rholestprol.
F.x~mplP5 of penetration Pnh~nçers are propylene glycol, DMSO, triethanol~minP, N,N-
dimethyl~çets,mi~, N~N-dimeLhylru~ mi(lp~ 2-pyrrolidone and de-;vdLivt:s thereof,
tetrahydlvrulyl alcohol and Azone.
30 F.x~mrk.q Of rhPls~*ng agents are sodium EDTA, citric acid and phosphori~ acid.
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~,x~ml~las of other Px~ipient.c for use in compositions for use according to the invention are
edible oils like almond oil, castor oil, cacao butter, coconut oil, corn oil, cottc neee-l 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,
5 hydlo yl~ropylmethylcellulose, hyd~u~yt:Lhylcellulose~ l-yd-~y~ropylcellulose~ ~hitoe~ne~ pectin,
~rAnth~n gum, carrageenan, locust bean gum, acacia gum, gelatin, and ~lgin~tPe, and solvents
such as, e.g., glycerol, ethanol, propylene glycol, polyethylene glycols such as PEG 200 and PEG
400, Pluronic, polysorbate, and ethylene glycol.
~.xslmplae of ~intmPnt. bases are bees~,vax, p:l~ffin, cetyl p~lmit~te, vegetable oils, sorbitan esters
10 of fatty acids (Span), Carbopol, polyethylene glycols, and con(lPn.c~qtion products between sorbitan
esters of fatty acids and ethylene o_ide, e.g. polyu~yt7Lhylene sorbitan monooleate (Tween).
A most important composition according to the invention is one in which the antiviral snhst~n-e
is acyclovir. li'.x~mplP.e of important embo~limPnt-e hereof and of other compositions according to
the invention cont~ininf~ nucleosides of low solubility as defined herein are claimed in claims 75-
15 91 and are described in detail in the ~x~mplae
Description of the drawing
Fig. 1 shows a s~ hPmsltic diagram of the apparatus used in the test method denoted test method1 described in detail in the experimental section herein. The reference numbers illustrate th
e
following
20 1. Thermf ~t~tic water flow (4û~C)
2. Reservoir cont~ining the washing solution (37~C)
3. A peristaltic pump
4. A stainless steel support
5. A model membrane
25 6. Receiver for c~llacting the washings
Fig. 2A shows a srhPm~ic diagram of the apparatus used in the test method denoted test
method 2 rlpe~rihed in detail in the experimental section herein. The reference numbers
illustrate the following:
.
1. Instrument probe 6. Sliding stand
30 2. ~t~tion~y plate 7. Diepl~- PmPnt transducer
3. A first holder 8. Control unit
SUBSTITUTE SHEET
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4. A model mPmhrsln~ 9. Person~l computer
5. A second holder
Fig 2B shows a s-h~mAtic diagram of a variation of the apparatus used in the test method
denoted test method 2 described in detail in the e~rerim~nt~l section herein. The reference
5 numbers illustrate the following:
1. Instrument probe 8. Sliding stand
2. ,~t~tir~nAry plate 9. Di~plAr~m~nt transducer
3. A first holder 10. Control unit
4. A model m~mhr~n~ 11. Personal computer
10 5. A second holder
6. A thermost~ticAlly controlled heater/stirrer
7. A vessel
Fig 3 illu~ les the pH-solubility profile for acyclovir.
Fig. 4 shows a thermogram in~ic~qting the phase transition L~-to-Q (lAmPllAr to cubic) for a
15 GMO/water composition (85/15% w/w)
Fig. 5 shows the cumulative release of acyclovir (test con~lition~ as described under Fig 6)
Fig. 6 shows the release of acyclovir (1-5% micronized) delivered from a cubic phase
(GMO/water 65/35% w/w) and Zovi}~D cream, respectively, into isotonic 0.05 M ~ho~phAtf-
buffer solution, pH 6.5 (37~C) [% acyclovir released as a function of time]
20 Fig 7 shows a Higuchi plot of the release of acyclovir (test contlitiQn~ as described under Fig. 6)
Fig. 8 shows the release of a~ vir (1%) delivered from GMO/water 65/35% w/w into isotonic
0.05M phosrhAte buffer solution, pH 6.5 (37~C). A cnmpAri~on of the release from 1% of
micronized acyclovir and 1% of crystalline acyclovir shows that there is no ~igni~cAnt difference
in the release of the two different qualities of acyclovir using 1% acyclovir
25 Fig. 9 shows the release of acyclovir (1%) delivered from GMO/water/lecithin 55/35/10% w/w
into isotonic 0.05M phosphAte buffer solution, pH 6.5 (37~C). It will be seen that in this case, the
crystalline acyclovir is released slightly faster than the micronized acyclovir
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Fig. 10 shows the release of acyclovir (5%) delivered from GMO/water 65/35% w/w into isotonic
0.05M phnsphsltp buffer solutions, pH 6.5 (37~C). It will be seen that in this case the micronized
acyclovir is released slightly faster than the crysWline acyclovir
Fig 11 illustrates the release of acyclovir that is micronized from various GMO form~ tion~
çr)ntAining 1% acyclovir into isotonic 0.05M phn~ph~te buffer sollltion~, pH 6.5 (37~).
Fig 12 illustrate the cumulative amount of acyclovir permeated through pig skin; the
GMO/water is 65/35% w/w cnntAining 5% acyclovir (for details see ~mpl~ 20).
MATERLALS
Glycerylmonooleate (monool-;n)~ manufactured by Grin-l~ted Products A/S, Denmark
10 DIMODAN'ID GMO-90, a dis~lled monoglyceride
Chemical and physical data
MonoP~qr content min. 95%
Diglycerides ma~. 3%
Triglycerides ma_. 0.2%
Free fatty acids maX. 0.5%
Free glycerol ma2~. 0.5%
Iodine value - approx. 72
Fatty acid composition:
Oleic acid 92%
Linoleic 6%
~t~lr~tPd (Cl6/Cl8) 2%
Melting point 35-37~C
Anti~xi~l~ntc and synergists added:
Ascorbyl p~lmitAte max. 200 ppm
~-Tocopherol max. 200 ppm
Citric acid ma_. 100 ppm
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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 int~s~inps of the
rabbits were ~ .ce~te~l 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 int-o~tin~q
were clean. The jejunums were cut into pieces of about 8-9 cm in length and frozen (-20~C)
immP~ tPly. The jejunums were stored up to 3 months before use (when pPrforming 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 ~ignific~ntly similar results). Before
testing, the sPgmPnt. of jejunum was gently thawed out.
The sçgm~nt. of the jejunum was cut lon~itll~lin~lly It was placed on a st~inlPq~ steel support (a
tube of 2 cm in (li~mPtPr and cut longit~ in~lly 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 l~ mrhP5 the angle applied is denoted "angle" in a cylin~lrir~l cell
thermost~ted at 37~C. A srh~m~t~ trfltion of the cell is shown in Fig. 1. The relative
humidity in the thermn~tPd cell was kept at about 100%. The jejunum was then flushed with a
medium of 0.02M isotonic phosph~tP 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 10 mVmin (in the following
denoted "initial rinsing flow"), respectively, using a peri.ct~ltic pump to eqllilihr~qte the jejunum
vwith the buffer and to rinse off loose mucosa. [TmmP(li~tPly before applir~tion of the sample, the
support was pnsitir~necl at a hori7nnt~1 position and after applir~ti~n the position was changed to
the initial position of -21~.] An a~ d~ely weighted amount of the sample to be tested for
hio~rlhP~ive properties (about 60-150 mg) was placed evenly on the mucosa of the jejunum
(about 0.8 x 6 cm). About 1 ml of the buffer solution was carefully dropped evenly on the sample
applied to ensure fnrm~tion 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,
or l~m~ r phase). [In those cases where the visco~ y of the test sample are relatively high or
where a precipit~tion has taken place, the test sample is gently melted on a heating plate or in
an oven at a temperature of max. 60~C in the case of GMO or GML and cooled to a temperature
of at the most about 40~C before aprlir~tion on the rabbit jejunum.] TmmP~i~tPly after, the
segmpnt~ were left for ~-20 minnt~Ps such as, e.g, 10 minutes in the cell allowing the sample to
interact with the glycoproteins of the jejunum and to prevent d~ying of the mucus. After 10
minutes, the segment-c were flushed evenly with the isotonic 0.02M phosphAtP buffer solution
(pH 6.5, 37~C) for 15-60 minutes such as, e.g., 30 minutes at a flow rate of 5-1~ ml/min such as
10 ml/min (in the T~ mrlP~ denoted "flow rate"). The tip of the tube carlying the buffer solution
was placed 3-4 mm above the jejunum to ensure an even liquid flow over the mucosa. The
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26
In the following a~Qmrlac, the term "GMO-90" inrlir-Qt~c that the above-mPntionçtl glycerol
monooleate product is employed, except where otherwise stated.
Gly~ nnonlçQte 84% "GMO-84" (monoolein), manufactured by ~rin~ d Products A/S,
DçnmQrk; the product used has a total content of fatty acid monna~+~r~ of at least about 96%.
5 The product employed in the ~ Qmpla,c ~la.crrihed herein had the following composition of fatty
acid monoesterc:
Gly~ lonooleate about 84% w/w
GlycerylmnnnlinnlçQta about 7% w/w
Glyceryl monopQlmit~te about 3% w/w
10 Glyceryl monosteQr~t~ about 4% w/w
In the following P~zQmplac~ the term "GMO 84" inflicQtç,c that this glycerol monooleate product is
employed.
Other commercially available glycerol monooleate products (e.g Myverol 18-99 and GMOrphic
80 available from Kodak T~Q~+ nQn, U.S.A.) which differ in the composition of fatty acid
15 monoçcters compared with the products described above may also be applied.
Glycervlm~mnlinr)l~Qt.o (Dimodan~ LS), manufactured by ~rin(l~tçd 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.
The product employed in the P~Qmpl~Ps described herein had the following composition of fatty
acid mnnop~+~r!
20 Glyceryl monl~pQlmitQte about 6% w/w
Glyceryl monn~teQrate about 6% w/w
Glycerylmonooleate about 22% w/w
Gl.~ onnlinnlaQt~ about 63% w/w
Other commercial available ~ ,l--.onnlinolaQte products (such as, e.g., Myverol~ 18-92
25 available from Kodak T;~.A~+ nQn, U.S.A.) which differ in the composition of fatty acid monoesters
compared with the product described above may also be applied.
MjconQ7ol base available from MPrlioT.Q~t SPA, Milano, Italy
T.i(lorQine hydrochloride available from Sigma Chemical Co., St. Louis, U.S.A.
I,i~lot Qinç base available from Sigma Ch~mirQl Co., St. Louis, U.S.A.
-
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Acyclovir (crystalline) available from Chemo Iberica, Spain, e.g a quality where 90-100% of the
crystals have a parlicle size of less than 100 ~m
Acvclovir (micronized) available from Chemo Iberica, Spain, e.g a quality where 100% of the
~ particles have a particle size under 24 I~m and not less than 909~o under 12~m
Ethanol available from Danisco A/S, D~nm~rk, complies with the DLS standard (98.8-100% w/w
ethanol)
Sesame oil available from Nomeco, Denmark
SoYbean oil available from Nomeco, Denmark
Glycerol available from Joli Handel ApS, Denmark
T.~itl-in Epicuron 200 from Lucas Meyer
Benzyl alcohol available from Merck AG, Germany
Water, purified or distilled water
DEAE-dextran (MVV = 600,000) available form Sigma Chemical Co., St. Louis, U.S.A.
Sodium al~inate (Sobalg FD 120) available from Grin~l~te(l Products A/S, DenmarkIIvdl~y~ropvlmethvlc~ lose (Metl oc~ol K15MCR Premium USP) available from Colorcon
T.imi~d~ U.S.A.
Carbo~ol 934 available from The BFGoodrich Company, U.S.A.
Vitamin E TPGS (d-a-tocopherylpolyethyleneglycol 1000 el~rcin~t~o) available from Kodak
~ ~m~n (in the following ~eci~n~ted TPGS)
AsPirin available from Sigma, Chemical Co., St. Louis, U.S.A.
Propvlene ~lycol available from BASF Aktieng~llsch~f~j Germany
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 determin~tion of the surface area of the
particles.
METHODS
Test systems for b ~ ~lh .,s r~n
1. In vitro test system for l.i A-lhç~ion by means of rabbit jejunum membranes
- The test system for hio~lh~ion described in the follo~,ving is a modified system of a method
described by Ranga Rao & Buri (Int. J. Pharm. 1989, ~, 265-270).
.
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washings were c~ cted into a beaker. The amount of hio~-lh~cive component rem~ining on the
jejunum was r~lr~ t~d either by measuring the amount of sample in the beaker or by
me~7lring the amount of sample r~m~ining in the jejunum by means of a suitable analysis
~ method, e.g HPLC.
- 5 At the end of the expariment~ the r~m~ining sample on the jejunum was checked with a pair of
~w~ to reveal false positive results.
In 1-2 test run(s) out of 10, false negative results were observed probably due to a loose mucosa
layer on the rabbit jejunum.
During testing and v~litl~tic n of the method, the p~r~m~fqrs given above were varied (e.g the
10 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 cc Tl-litinn~ were ~p~ti~f~c)ry
Time for prehydration before applir~ti~n of sample:
10 min
Amount applied: about 60-150 mg (tests have shown that a variation in the amount16 applied within a range of from about 25 mg to about 225 mg was
without signific~nt 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
reprodnrihl~ results and a proll n~ion of the period to about 60
minutes does not ~ignifir~ntly 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 applir~hon of fluid sa-m-ples and pellets.
25 Determin~on of the hio~(lhp~iveness of a test samPIe
In those cases where the test sample is a fatty acid ester, the fatty acid ester is considered as
hio~-lh~ive 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.
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In those cases where the test sample is a composition comrri~in~ a comhinPtion of a fatty acid
ester and an active or protective sllhst~nre, the compQsition is considered hio~llh~eive if the
residual amount (of fatty acid ester or active/protective snh~nre) 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,
6 76% w/w, or 80% w/w.
In the present context ev~ln~hnn of the bio~flh~cive properties of a ~nh~nce may also be
performed by use of the test system and test con-litionc described above but mo(lifi~d with
respect to type of membrane, amount applied of test sample, test angle, flow rate, medium, etc.
In this cc nnec~inn~ tests have been perFormed in order to evaluate the infll7~nre of different
10 membranes on the test results. The following results were obtained using the above-m~ntinnecl
test con-litirnc (angle: -21~, flow rate: 10 ml/min, and ilow period: 30 min) and applying GMO
on the m~mhr~nf.-
Membrane Rinslrlh~cion
16 % w/w T~ irll-cl amount %
rabbit jejunum 90
pig ileum 106*
pig ct~)m~rh 106*
buccal pig mucosa 88
* the high result is most likely due to an interference from the intf~chnf~c or the stom~rh
2. In vitro test sy6tem for hios~lhe~ion by means of t~ ~iomet~y
The test system for hin~q-lh~ci- n described in the follovwing is a modified system of a method
25 described by Tobyn, M., J. Johnson & S. Gibson (in "Use of a TA.XT2 Texture Analyser in
Mnco~lh~ocive Research~, Tntern~hon~ql LABMATE, 1992, XVII (issue VI), 35-38).
The test system involves me~cllring 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 hio~lh~cive
properties).
30 The test apparatus employed in the follovving is a TA.XT2 Texture analyser (Stable Micro
System Ltd., ~elPm~re, UK) (Fig 2) equipped with a 5 kg load cell int~rf~ced vwith an IBM PC
computer running XT-RA (limencinn software, DOS version. The test enables measuring the
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31
strength of adhesive bonding Pet~hli~hPd by cont~rting a model membrane, i.e. in this case a pig
intPstinp segment, and the test sample. An analogous test apparatus may also be employed.
The TA.XT2 Texture analyser apparat,us is equipped with an instrument probe 1 (see Fig. 2)
which is movable in a vertical direction at a variable rate. During the so-called vvithdrawal phase
- 5 of the testing, the instrument probe is moved upwards with a con~t~nt rate until ~Pt~rhmPnt
occurs (see below). Furthermore, the apparatus is equipped with a st~tion~ry 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
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 fi~ed to the probe. The ~-cnr~tP size of the
exposed area is used in the c~ tio~ of the adhesive strength (see below).
As mPntionPd 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 spgmpnt of rabbit, rat or
pig intp~tinps~ e.g. a segmpnt of rabbit jejunum; a ~PgrnPnt of rabbit or porcine buccal mucosa; or
15 a segment of rabbit, rat or pig intp~tinp~ from which the mnl~l s~l layer has been removed prior
to testing, or skin from an animal (after removal of sllh~t~nti~lly all s~lhcnt~neous fat); or it
could be artificially or commercially available mucin.
In the tests rlP~rrihed below, duodenum, jejunum and the upper part of ileum from freshly
nghtered pigs were used. The gut was stored on ice until it was washed with 0.9% w/w
20 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 immPrli~tPly frozen (-20~C).
The intp~inp~ were stored up to 2 months before use. Before testing, the segmPnt-c were gently
thawed out. The gut segmPnt was opened along the melspntpri~ border. Serosa and mll~c~ ri~
layers were removed by stripping with a pair of tweezers, taking care to m~int~in the integrity
25 of the mucus layer. This resulted in a fl~ttpning 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 eqnilihrillm as measured by pH paper.
If the results obtained by use of another membrane than the one mPntinmPd above are compared
to the hio~lhpcive properties of various snhst~nrPs or comhin~tions, the results of a reference
30 compound could be included. As ~ c~ Pd below testing of a reference sample may also be made
- as a routine. Polycarbophil and Carbopol 934 have been found suitable as reference compounds.
An ~ccllr~tP amount of a test sample (about 25-500 mg) is applied irl a uniform layer either
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32
i) on the luminal side of the model membrane placed on the first holder,
ii) directly on the instrument probe, if n~c~qc~ry by means of a cap, a double adhesive tape
or glue applied on the instrument probe before appli~tion 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 mo~ifi~d in such a manner that it allows appli~tinn of a relatively low viscous
or semi-solid sample, the mo~ified probe also allows the nRc~.cc~ry arl~liti()n of an aqueous
medium.
In those cases where it is not possible to ~ the test sample to the instrument probe, the
10 apparatus may be equipped with a second holder 5 on which another model memhr~n~ is fixed.
In such cases, the model membranes employed Oll 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 .q~lhPcion test, a tissue (porcine intpctin~l mucosa) of about 3 x 3 cm was fixed on the
15 tissue holder 3 with the mucosa layer upside. Before appli~hon of the tissue, a piece of gauzewas placed directly on the tissue holder, and thereupon the tissue was placed. This preca~ltion
is
made in order to St~hili7P the contact force. In order to moist the tissue and hydrate the sample,
about 0.5 ml isotonic 0.05M phosph~te 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
20 cme~ring 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
c~ n~nt 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)
25 for 10 mm. Initial exppriment~c showed that this distance was well beyond the point where the
sample and mucous separated during withdrawal.
The peak rlet~rhm~nt force and the area under the force/time curve was r~lrlllAted
~ntom~ti~ lly using the XT-RA 11im~ncion software. The work of ~lh~cif~n (mJ cm~2), said to be
the most accurate predictor of m--- o~lhRcive pRrform~nre~ was ~ lcnl~te-l
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Sample preparation
ApplicAti-~n method of the polymers used as reference:
Cover glasses having a rliAmetqr of 13 mm (area 1.33cm2) were coated with the polymers under
invos'i~Ation by pipetting 100 ~l of a 1% w/w solution of methanol or water in the center of the
5 glass plate. After drying for 2 hours at 60~C in an oven, a thin polymer film rPmAinP~l One cover
glass was Att~rhed 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).
ApplicAtif~n of fatty acid ester compositions:
10 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 e~ Pd at room temperature or, if
neCPee~ry~ by cooling.
B. ~mPAring 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 perform~Pd 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.
20 In some cases, vAriAtic!ne 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: 60 sec - 50 min
Contact force: 0.05-0.
Equilibration mPrlillm
Test speed: 0.02-1 mm/sec
Post test speed: 0.02-1 mm/sec
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Test run temperature may be changed by employing a suitable temperature controlled oven such
as a SMTC/04 from Stable Microsystems, ~AqlPmPre, UK
Determin~ti~n of the hin~-lhPcive properties of a test sample
In order to test whether a test sample is hioA-lhPcive, two test runs are performed: ~
1. A test run with the test sample applied (result: work of AtlhPcion WAS),
2. A test run with a known and PxrP~lPnt hioAIlhPcive sample (e.g. polycarbophil) (result:
work of A~h~ci~n WAR).
In both cases the work of Aflhl?qinn is c AlrnlAted and the test sample is considered hioArlh~cive if
WAS/WAR x 100% is at least 30%, such as 35%,40%,45%, 50%, or 55%. In general, a sample is
10 graded to be a weak hioA(lhecive if the result is less than about 30%, a medium hioA-lhPcive if
the result is about 30%-50%, a strong hioAIlhecive if the result is at least 50%.
Polycarbophil (Noveon~ AA-1, BF Goodrich, Hounslow, U.K) is a high mnlpclllAr weight
poly(acrylic acid)copolymer loosely cross-linked with divinyl glycol. On account of its knawn
PxrP~lf?nt. mllcoA-lhPcive properties, this polymer serves as a reference. Before testing in the
15 above-mPntioned tpncinmetric test, a polycarbophil gel is prepared by mixing polycarbophil with
water or methAnol (resulting conr~ntration about 10-20 mg ml~l) and the mixture is allowed to
hydrate at room temperature for 24 hours. The polymer solution is periorlirAlly 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 PxrPllent hioAIlhf~Rive snhst~ncPR
Similarly, other snhst~nceR which are known hioA~lh~cive snhst~nr~Pc are tested such as, e.g.,
rhit~RAne trA~ArAntll~ l,y~ yl,ropylmethylc~lllllnRe (HPMC), sodium Al~inAt~P,
Lydlu~y~ropylcellulose (HPC), karaya gum, carboxymethylcPllnlnRe (CMC), gelatin, pectin,
acacia, PEG 6000, povidone, or DEAE-dextran (less hioAflh~cive t_an polycarbophil). By choosing
test sllh.ctAn~Pc with various degrees of hio~A~IlhPRiveness~ an evaluation scale can be made and
25 the performance of a test sample with respect to bioA~lhPRiveness can be evaluated. It is
contemrlAted that the following scale is applicable provided the test conllitinn.C given above are
applied. It is clear that if the test con(litionR are changed, another scale may be more relevant. A
suitable scale is then to be ba ed on the values obtained for the PxrPllPnt hioArlhPRive
polycarbophil and the weak hioA~lhPcive such as DEAE-dextran.
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Rio~r~hPcive properties Work of ~(lhe~il)n (mJ cm 2)
none less than 0.005
poor about 0.005 - about 0.012
morl~ts about 0.012 - about 0.020
- 5 good about 0.020 - about 0.04
P~PllPnt more than 0.04
When testing some known bio~lhp~ive snhst~nces and GMO, the following results were
obtained as a mean of si~ experiments:
Test snhs~p,nc~p Work of ~dhPsion (mJ cm~2)
DEAE-de~tran 0.010
Sodium ~lginate 0.015
GMO 84/water 85/15% w/w* 0.028
HPMC 0.036
Carbopol 934 0.031
GMO 84 0.047
Polycarbophil 0.060
*: lamellar phase
3. In vivo test 6y6tem for bioadhe6ion - washing off ability from the 6kin
A water soluble dye (Edicol Sunset Yellow, E 110, Amaranth E-123, or Brilliant Blue E 131)
20 and/or a lipid soluble dye (Waxoline violet A FW (MA~r;mP~), 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
25 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 moi~PnPd skin. In some cases, about 10 min
before running the test, a small amount of water could be added to the test sample applied.
TmmPrli~tPly after app~ ti~n, the test sample on the skin was subjected to washings with water
from a tip (flow rate corrP.sponding to about 6-8 litres/minute and a temperature of about 35-
30 40~C). The washings were carried out for about 3 minutes. Then it is visually ~e~ed in whichdegree the test ~Pmple is retained on the skin. The visual ~Pq~mpnt is done by use of a scal
e
graded from 1-5, where 5 represents total ret~inmant of the test sample applied on the skin and
1 represents no ret~inmPnt of the test sample on the skin.
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The test sample is evaluated to have hio<qtlhPgive properties in the present conte2~t if the result of
the above-(lPcrrihe(l test is at least 4.
The test described above has proved to be suitable when testing compositions forhio~llh~civene5s and the comrocitione in question have a relatively high visco~iLy which makes it
5 difficult to apply the compositions to the rabbit jejunum model. A motlifir~tion of the test
described above excluding the flrllli*on of a water soluble dye has also proved suitable for testing
compositions for hio~(lh~civeness.
Q-l~nt;tstive deter ninAtio--~; of ~ l...onooles~t~ and ~ onolinol~te bymeans of HPLC
10 The q~ l;vv determin~tion of ~ ,v~ onooleate or ~ly~,v.yh--cm-lin~ tt? was made by
high-performance liquid chromatography (HPLC) using a ~him~ u LC-6A HPLC pump, a~him~ u SPD-6A W clete~or~ a ~him~rl7u C-5A i-lle~ ur and a ~him~ u SIL-6B
fl7lt~c~mrlpr~
The column (25 cm x 4 mm i.d.) was packed with Snrpl(~ogil LC-18-DM and was eluted
15 isocratically at Pmhient temperature with a mobile phase congi~ing of mPtl-~nr)l water:acetate
buffer (pH 3.5) (840:120:40 v/v). However, in some cases interference from other sllhgt~ncPc
may occur, and then it may be necpc~p~ly to make minor changes in the composition of the
eluent.
The size of a sample injected on the column was 20 ~l and the flow rate was 1.2 ml/ml. The
20 column effluent was monitored at 214 nm.
F~Y~ L:~n 1J1~C~ U~ prior to analysis of ~ e.,~l~onooleate or
Kl~ l...nnolin~es~e in ...-
~
The mucosa in question (with a fatty acid ester, e.g. ~ ~~ ,lonooleate) is placed in 50.00 1 of
m~th~nol and shaken for 2 hours. The mixture is filtered through a 0.45 ~m filter mf~mhr~n~
25 (from Millipore 16555Q) and the filtrate is subjected to HPLC analysis using the method
rles~rihed above.
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Recovery
In those cases where analysis is pPrform~Pd in order to dPt~prminp the residual amount of fatty
acid ester (e.g ~ ..nno~lla~ta) on the rabbit jejunum sPgtn~nt in c-mnP~ti~.n with the
hin~lhpqive test No. 1 (above), the ~ tinn of the residual amount takes into cnn~ Pr~ti~n an
5 appropriate correction in the l~,~,VVt71,~. This correction is found based on determin~tinn of the
amount of fatty acid ester on the rabbit jejunum segmant after appli-~tion of an accurate
amount of fatty acid ester (this test is repeated 5 times and the rc~,vv~l~y is given as the mean
value).
The l~vv~l y of about 125 mg GMO 84/ethanol 60/40% w/w on rabbit jejunum was e~Amined
10 The l~vv~ was found to be about 95%. The rt7cvvt~l~ was not deter_ined for the other
amounts of GMO/ethanol 60/40% w/w nor was it lletPrminPcl for GMO or GML formnl~tinnq to
which drug sllhqt~nrPq or P~r-iriPntq were added.
,~lnh;lity of ~c_~,yl~alicylic acid (aspirin):
Wyatt D.M. and Dorschel O. A cubic phase delivery system composed of glyceryl, monooleate
and water for sn~t~in~Pd release of water-soluble drugs, Pharm. Tech. 1992 (Oct.), p. 116-130,
disclose an experiment in which aspirin is used. Aspirin is not a sllhst~nce which has a low
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/ml in water (20~C). It has a pKa value of about
20 3.5 (25~C) (Analytical Profiles). The solubility of aspirin is strongly dependent on the pH in the
solution. The degree of inni~tion 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/ml in
a buffer solution of pH 3.6. The experiment was performed in an 0.5 M acetate buffer solution
25 pH 4.0; the buffer was not strong enough to m~int~in 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/ml
llhility in GMO/water
The solubility of acetylsalicylic acid in GMO/water 65/35% w/w has been (iPtprmined to be >20
mg/ml. During the exppriment~ 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)
Dete- mins-t;Qn of the ~ olnt;on/release rate of a pharmaceutical fo~rnl~ f;on
The dissolution rate of acyclovir in various GMO compositions was determined using Franz
diffusion cells having a ~iffil~ion 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 r1iffn~ion mRmhrAne a cPll~llose membrane from
MP~1;CP11 Tnterns~tion~l Ltd. was employed. The membrane employed has a pore size of about
2.4 nm and it retains particles having a m~llPculAr weight larger than about 12,000-14,000.
Before apr)li(Ation~ the mRnnhrAnR was pretreated and thoroughly rinsed with distilled water. As
10 receptor medium was used an isotonic 0.05M phosphate buffer pH 6.5 (Danish Drug Standards,
DLS) and the medium was mAgneti( Ally 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, about 300-350 mg of the composition
to be tested was applied by means of a syringe or a spatula and care was taken to ensure a
15 homogenous distribution of the composition on the total area of the membrane available for
lli~lcion 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 cPllnlnsR 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 memhrAne Phnsl)hAtR buffer was then loaded
20 into the receptor part (time t- 0) and at appropriate time intervals, samples of 2-3 ml were
withdrawn and analyzed for content of acyclovir (cf. bel~v). This relatively high volume was
withdrawn to ensure sink con~liti~m The amount of receptor medium withdrawn was replaced
with fresh receptor medium.
Q~ nt;tstjve deternlin~t;o of Inicons~7ol~ and lirlocs~ine hyd~ochloride, re6pectively
~Amplps from T~'.x~mpl~ 12 were analyzed for the content of mi~r~nA~l and lidocain
hydro-~hlori~le, respectively. The following assays were employed:
T.iclocAin HCl
The content of lidocain HCl is determined by a HPLC method.
T: Dissolve the formulation in 30 ml mPthAn~ll and transfer it qnAntitAtively to a 50 ml
30 volnm~ flask. Add mPthAn-l to 50.00 ml.
-
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R: Weigh out 100.00 mg lidocain HCl in a 100 ml Vt~ mptri~ flask. Dilute 1000 ~l to 60.00 ml
with mobile phase.
Analyse T and R on a suitable liquid chrnm~togr~rh with W-detector and integrator.
Column: Steel column, length 25 cm x 4.6 mm i.d.
5 St~tion~ry phase: Nucleosil C-18, 10 llm
Mobile phase: M~ nol R: Acetic acid: Triethylamine: Water (50:1.6:0.5:48)
E~ow: 1.5 ml/min
Temperature: Room temperature
Detecfi~n- 254 nm
10 Injection: 20 Ill loop
~etPntion time: Lidocain HCl: about 3 min
Calculation:
T.i~loc~in HCL lecov~l ~, %: AT x n(g) x 100%
AR x m(g) x % lidor~in HCl
-
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 intes+ine (g);
% lidocain HCl is the content of lidocain HCl in the form~ ti-~n determined as
% w/w.
MirQn~7~1
The content of micnn~7~1 is also determined by a HPLC method.
T: Dissolve the formulation in 30 ml mpth~nol and transfer it qn~ntit~tively to a 50 ml
volllmPtri~ flask. Add meth~n-l to 50.00 ml.
R: Weigh out 100.00 mg mil~on~ol in a 100 m~ v-~lumetric flask. Dilute 1000 ~Ll to 50.00 ml
with mobile phase.
Analyse T and R on a suitable liquid chrl m~togr~rh with W-detector and integrator.
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Column: Steel column, length 25 cm x 4.6 mm i.d.
~t~tif~n~ry phase: ~pherisorb ODS 1, S5
Mobile phase: M~llAnol R: Buffer (85:16)
E~ow: 1.0 ml/min
5 Temperature: 70~C
Dete~on 230 nm
Injection: 20 ~l loop
l?et~ntinn time: Mi(~on~7r~1 about 8 min
Buffer: 0.05 M NH4H2PO4 (5.75 g in 1000 ml H20)
10 C',~lrnl~tic-n
Mi~on~7-~1 recovery, %: AT x n(g) x 100%
AR x m(g) x % micon~7cl1
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 inte~ine (g);
% mi~on~7:r~1 is the content of mir on~7~1 in the formnlRti~,n determined as % w/w.
Q~ nt;f-~tive deter nin~t.inn of acyclovir
20 Method A
DeterminP.tion of acyclovir in aqueous media by EIPLC
The HPLC method employed was the following
Column: 25 cm x 4.6 mm i.d.
,C~tsltion~7y phase: Nucleosil C-18
Mobile phase: W~ .. eth~nol (85:15)
Temperature: Room temperature
Dete~n 254 nm
Flow: 1 ml/min
Inj.volume: 20 ~l
Ret. time: ca. 5.4 min
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In c~-nnP~inn with dissolution/release rate experiment-q employing Franz diffusion cells as
described above, the conrRntrqtion in the test solution (Cn) is ~ .nl~t.od as follows:
- Referellce solution: An accurate amount of about 10.00 mg acyclovir is diluted to with distilled
water to a concPntr~tion of 10.00 ~g/ml
5 Test sr~lntic)n The sample withdrawn is filtered through a 0.2 ~m filter and injected onto the
column (in some cases it might be n~ceeq~ry to subject the sample to dilution with water)
T x amount wei~ht in (m~) of reference x 1000 x 5~g/m1
AR X10OX60
in which
10 AT is the area of the test solution, and
AR is the area of the reference snlution
Calculation of % released:
n
100 x Cn x Vt + (V5 X ~ Cn-l))
15 n=l x 100%
% of acyclovir in form. x mg of form. applied x 1000
in which
Cn is the con~ntration of drug in the receptor solution (mg/ml),
Vt is the receptor volume (unless otherwise stated, Vt = 6.8 ml),
20 Vs is the sample volume withdrawn,
Cn l is the concentration in the previous sample (~g/ml).
Method B
Determin~tion of acyclovir in pharnnA-ellti-~l formulations by HPLC
The ~LC method employed was the following
25 Column: Steel column, 25 cm x 4.6 mm i.d.
StD~ti~n~ly phase: Nucleosil C-18, 5,Um
Mobile phase: water-meth~nol (20:80)
Temperature: Room temperature
Det~;On- 254 nm
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Flow: 0.8 ml/min
Inj.volume: 20 ~l
Ret. time: ca. 3.5 min
Reference solution: Weigh out an Arc11r~tP amount of about 20.00 mg acyclovir and dilute it with
5 mobile phase to a conc~sntration 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 snh1tiQn, respectively, the percentage of
acyclovir present in the formulation is c~1c~ te(1
10 Method C
Recovery of acyclovir on inte~ine
The HPLC method employed is the same as described under Method B. The test solution is
prepared as follows:
The intestine is shaken for 2 hours with 50.00 ml of the mobile phase. The test solution is
15 filtered through a 0.2 ~m filter. Dilute 1000 I.l to l0.00 ~ with mobile phase.
From the areas of the test solution and reference solution, respectively, the percentage of
acyclovir present in the formulation is r~q1r~ tel1
Determin:.f;on of pH in the liquid cry6t~11ine pha~e
pH in the crystalline liquid phase is determined in a 10% w/w dispersion of the liquid crystalline
20 phase (cnnt~ining the active ~1hs~nce and any ~Yr.ipi~nte) in distilled water. Prior to
determin~ti~)n the dispersion is subjected to n1tr~gr~ni~ tre?/tment for 30 minutes in order to
ensure that an equilibrium between the liquid cryst~lline 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
25 accordance with the instructions given by the m~nnf~l~tnrer of the electrode.
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The method described above can be employed for various compositions, i.e. for composition
wherein the cnncçntration 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.
Morlific~tirnc of the method described above may also be employed e.g i~ the dicpPrcion
5 mPntionP~l 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, u) ultrasonic treatment may be omitted or
s~ led by stirring 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.
10 Most important is it to ensure that for cnmr~r~qtive purposes the test con-lit-onc (stirring,
nltr~.coni~ treQt nPnt~ time, electrodes) should be ~ccPn*~lly 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 dis~lled
water has taken place a number of expprimpnte were perfc)rmod varying the time period for
15 uItrasonic treQtrnPnt (0-5 hours) and meQ~llrin~ the pH imme~ t~P after the end of the ultra
sonic tre~tmont and 24 hours later. The Q~rpqrimçnte are performed on a GMO/water 65/35
c~mtQining 5% w/w of acyclovir. Based on the results of these expPrimont.e a time period of 30
minutes proved suitable, i.e. there is only an ineignifil~nt difference in the pH measured
immefli~toly after the-end of ultra sonic treQttnçnt and 24 hours later.
20 Deterrninat;on of drug ~olllhility
The detorminQtion of the solubility of the active snhst~n~e in the liquid crystalline phase of the
composition is, of course, porformod 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 determinQtinn of the solubility is performed on the
25 composition itself. The determinQtion of the solubility is suitably porformetl by microscopy to
observe any crystals of the active sllhet~n( e Suitable test conrlition~ involve a m~gnific~tion of
about 250 x and e.g. room temror:~t-lre (20~C or 37~C may also be employed). The determinQti- n
of the conrentrQtion at which crystQls are observed is pPrformpcl after a period of at least one
week after preparation of the cQmroeition or the liquid crystalline phase to ensure that
30 eq~lilihrinm has taken place. Normally, a series of tests with varying conrontrations is performed
to detorminR the con(~ontration above which crystals are found. On the other hand, when the
comrositi(--is a precursor composition, the liquid crystalline phase used as a reference in the
solubility determin~tion is a liquid crystalline phase imitQting the liquid crystalline phase which
CA 0223l273 l998-03-04
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44
will be formed when the composition absorbs liquid from the site of applir~hon This reference
liquid crystalline phase is made up v~lith water (as repr~o~pnting the liquid absorbed) in such an
amount that the reference liquid crystalline phase is the same type of liquid crystalline phase as
is genQr~tecl from the p~ or romrogitiQn
In order to detprminp the aqueous solubility of the active ~lhst~nce at the pH prevailing in the
liquid crystalline phase, the pH is determined in the liquid cryst~lline phase as described above
to determine the pH con~lihonC when flPtermining the c( l-lhility. [Many P~perimPnt~c with GMO
have revealed that the pH of the liquid crystalline phase pre~omin~nt1y is about 4.5.] The
solubility of the active snhst~nre is then determined by stirring an excess amount of the active
10 s--hst~nre in water, where applicable, being buffered to a pH s--hs~ntislly idPnh~l 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 con~t~nt temperature (e.g 20~C, room temperature or
37~C). In some case the samples initially were subjected to nltr~conic tre~t~nPnt. for half an hour
in order to accelerate the time for equilibrium. The con~Rntration of the active sllhst~nre in the
15 supernatant (i.e. the aqueous solubility at the given pH) is then determined by an appropriate
assay (e.g by HPLC or UV spe_LLvscvpy).
As mentirmed above, when the pH of the liquid crystalline phase, detqrminPd as dP,crrihed
herein, is di~.e..l from the pH which will result simply by dissolution of the active ,c~lhst~nce in
water, the water is adjusted to snhst~nti~lly the pH of the liquid crystalline phase by using a
20 suitable buffer system when determining the solubility of the active snh~nce This buffer
system should of course be so selected that, apart from the pH adjn~tnnent, it has sllhct~nfi~lly
no influence on the solubility of the active snhst~nre in the buffered water.
pH-solubility profile
Alternatively, the aqueous solubility is detarmin~d as a function of pH, i.e. by tlPtPrmining the
25 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, ph~l.srh~tP, borate etc. and
the cc~nrpntration of the buffer is sufficient to ensure a cQn.~nt pH during the expçrimRnt~c A
con(Pntration of at least 0.01 M is normally suitable. This method is applicable when
determining the minimum aqueous solubility of a specific active snhs+~nre at a given
30 temperature and at a given pH range. The test cnn-litionc described (pH, temperature, llltr~c(mic
trPiqtlnRnt, stirring, time for ensuring that equilibrium has taken place) above are also valid
when detprmining the minimum solubility.
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Deterrnins~t;on of liquid clysts1lin~ structure
Phase tr~ncitionc of GMO 84 and/or GM0 90 co~t~ining compositions
.
In the following tests are described which make it possible to lleterminQ the crystalline structure
~ of suitable compo~eitionc for use according to the invention. The tests allow determinQhnn of the
5 presence of, e.g., the GM0 84 or GMO 90 in a lamellar, hexagonal or cubic phase, and it is
possible to test the compositions before and after apr1ir~ti~ n to an appropriate applir~tion site.
With respect to the various liquid crystalline phases formed by GM0 or other glycerol fatty acid
esters, an P~r~llPnt review is given by ~.rirccnn et al. in ACS Symp. Ser. (1991), pp 251-265,
AmPrirsln ~h~mirAl Society and by Larsson in Chapter 8 (part 8.2.1 entitled "T.~m~ r and
10 hexagonal liquid-crystalline phases") in The Lipids T-T~nrlhonk edited by Gunstone et al. In short,
the l~mall~r phase is the dnmin~ing one at a relatively low water content (below 20% w/w) and
at a temperature of about 37~C, whereas the cubic phase domin~tR.~ as the water content
increases (more than about 20% w/w).
A. Phase transition of GMO 84 and/or GMO 90 compositions determined by differential
sr~nning r~lnrimPt-y (DSC)
The DSC measurements were performed using a Perkin Elmer Uni~ DSC model 7 Di~e.~uLal
Sc~nning ~lnrimetqr The heating rate was 5~Clmin and the sr~nning temperature was from
5~C to 70~C. ~C~amI lPs were cr)nt~in~sd in sealed ~lnmini~lm pans (Perkin Elmer No. B014-3017)
and as a reference empty aluminium pans were employed. The phase transitions caused only a
20 relatively small enthalpy change and, therefore, the amount of sample tested was optimized to
about 25 mg. The prepared pans were sealed and stored for two days at ~~C prior to analysis.
B. Phase transition of GMO 84 and/or GMO 90 compositions ~letarmine~ by pnlqrimPfry
The liquid crystalline phase can also be determined using polarized light and e.g employing a
stereomicroscope (Leitz, Diaplane) equipped wit,h po~ri7~tion filters. The appearance of reversed
2~ micelles (L2) are seen as a liquid oil, the lamellar phase (L~) 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 bac~ u~.d
with no details indic~ting that it does not reflect the light. The lamellar and hexagonal phases
are optically anisotropic. The lamellar phase gives a structure like a pipe cleaner on a black
30 background or, expressed in another manner, could be idPntifiPd from the oily streak texture
and the ,spherir~l, positive maltese cross-units visible between crossed pnlsri~Prs~ The reversed
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he2~agonal phase gives different patterns but in most cases it r~mhl~c a mosaic-like structure
or gives angular or fan-like t~l u-~s.
The method can be employed in testing the phase behaviour of various hio~rlh~cive
c~mp-~citi~nc
5 C. Phase transition of GMO 84 and/or GMO 90 composi~*ions 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 ~mit~ing Ka-rays at a wavelength of 1.5418 A. The X-ray
generator was a Philips PW 1729.
The liquid crystalline state can be i(len*fiRd by low angle X-ray ~ *on and its appearance in
10 polarized light. The char~ teri.c*~- X-ray diffraction pattern for the three liquid crystalline phases
(l~rn~ r, hexagonal, cubic) will give rise to diffraction lines in the following orders:
1:1/2:1/1:4...aamellar)
1:1/~/3:1/4:1/~/7...(hexagonal)
1:1/~/2:1/~/3:1/~4:1/~!5:1/~/6:1/~/8...(cubic)
15 In the case of the cubic form, the 3 different lattices will give rise to three di~lallt diffraction
lines.
EXAMPLES
The follovving ex~mrl~c 1-11 relate to the prepara~*on and structure of h;o~lhR,cive compositions
or hif ~h~qive vehicles for use therein.
20 Unless otherwise stated, all percentages are by weight.
In all ~x~ml~lR.c, the ~ al.~l-..onool~qta (abbreviated as GMO in the following) (and whenever
relevant ~;ly~ l...onolin- lR~tP (Dimodan~ LS)) is gently melted on a heating plate or in an oven
and the liquid obtained (ma-x-. 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
25 were mi_ed by stirring or .ch~king In those cases where the composi~*on cont~inc an active
snhst~nce in a GMO/ethanol or GML/ethanol vehicle, one of the following methods can be
applied:
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1. the active snhst~nce was dissolved or ~ persed 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,
5 3. the active ~lh~t~nre was dissolved or ~licpprsed in a GMO/ethanol mixture.
When storing at room temperature (22~) some formulations become inhomogpnpous. In
relevant cases the formulations were melted and stirred to obtain a hom.)g~nPous i2~ture before
use.
The acyclovir ~lint nRn1: composition was prepared as follows:
10 In general, the acyclovir was suspended in the melted GMO and the other ingredients were
added. The monoglyceride mixtures and the ingredients were mixed by stirri~g or sh~king In
the case of comro~it;~m~ cont~ining TPGS, the acyclovir was added to the TPGS solution before
mixing with GMO. The compositions were subjected to ultrasound trP~t~nPnt for about 1 h and
were stored for at least two days at 37~C before use to ensure that eqnilihrillm had been
15 obtained (e.g. that the stable liquid crystalline phase has been formed in the total formulation
and that equilibrium between the solid and dissolved sllhst~ncQ has taken place). As an
alternative to adding the acyclovir to the melted GMO, the acyclovir can be suspended in the
liquid phase before comhinin~ the liquid phase with the melted GMO.
In those cases where a bio~-lhP~ive test is pPrforme/l the values given are mean values of the
20 results of 2-4 tests. It should be noted that the values given in the ~.x~mples are not corrected
for recovery, i.e. the values are minimnm values. If a correction for rec.~v~l y is made the values
will become larger.
The test conllition~ for performing Test No. 1 for hir~lhP~iveness are:
angle: -21~
initial rinsing period: 5 min~ltl~c
initial rinsing flow: 10 ml/min
flow rate: 10 ml/min
flow period: 30 minutes
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EXAMPLE 1
Prep-q-r.q-t;on of a semi-601id (colourless gel) cQ~nro~it;~n without any active sllh~ ~qnce
The composition was prepared from the following ingredients:
GM0 65 g
5 Water 35 g
The GM0 and water were mixed by ~h~king The liquid crystal structure of the gel obtained is
cubic as evidenced by polarized light.
The composition was tested for hinsl/lhl~iveness in test system No. 3 (washing off ability). A
score of 4-5 was found in(lic~ting that the composition is hioA~lh~ive.
10 EXAMPLE 2
Prepqr~t;on of a semi-solid (grey white~ acyclovir o;nt~nent comrncit;on
GM0 61.8g
Water 33.3 g
Acyclovir (crystalline) 5.0 g
The liquid crystal structure of the gel obtained is cubic as evidenced by both polarized light and
X-ray diffract,ion (see below).
The composition was tested for hin~lh~iveness in test system No. 3 (washing off ability). A
score of 4-5 was found inrlir<qting that the composition is hio~-lh~ive. A similar result was
obtained by employment of test system No. 2 (ten~:iom~ry).
20 EXAMPLE 3
Preparation of a semi-solid (milk white) acyclovir oint~nent compocition
GM0 61.8 g
Water 33.3 g
Acyclovir (micronized) 5.0 g
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The liquid crystal structure of the gel obtained is cubic as evidenced by both polarized light and
X-ray diffraction (see below).
The composition was tested for hio~rlhç~iveness in test system No. 3 (washing off ability). A
score of 4-6 was found in~ n~ that the composition is hio~dh~ive.
6 Prelimin~ty t~nSiomPtri~ measurements (test system No. 2) confirmPd that compositions
cont~ining 5% w/w c~ystalline acylovir were hi~rlhPsive. The comp- ~it;ons tested were GMO 90
with 5% w/w acyclovir and GMO 90/water 65/35% w/w with 5% w/w acyclovir.
EXAMPLE 4
Preparation of a semi-fiolid comroeitinn without an active s~h~tst~e
10 The composition was prepared from the following ingredients:
GMO 85 g
Water 15 g
-
The GMO and water were mixed by shaking and a lameUar phase of GMO was obtained as
evidenced by polarized light.
15 The composition was tested for hio~q~he~iveness in test system No. 1. A residual amount of
about 84% w/w GMO was found after testing
A composition of GMO/water 90/10% w/w was prepared in the same manner and gave aresidual amount of about 87% after testing in test system No. 1.
The compositions were also tested for hio~h~iveness in test ~ystem No. 3 (washing off ability).
20 A score of 4 was found in(lir.qting that the compositions are hic ~-lhP~ive.
EXAMPLE 5
/
Preps-r~t;on of a semi-solid c.~ o~ on (colourless gel~ without an active sllhs~n~e
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The composition was prepared from the following ingredients:
GMO 65 g
Glycerol 35 g
The GMO and glycerol were mixed by .qh~king
5 The liquid crystal structure of the gel obtained is cubic as evidenced by polarized light.
The composition was tested for hio~llheqiveness in test system No. 3 (was_ing off ability). A
score of 4-5 was found inrlir~ting that the composition is hio~rlh~qive.
E;XAMPLE 6
Preps~r~t;o of a liquid c.~..q-~ :I.;on without an active s~h tsnce
10 The composition was prepared from the following ingredients:
GMO 50 g
Ethanol 30 g
Glycerol 20 g
The GMO was mixed with ethanol and glycerol was added to the resulting mixture while
16 stirring.
The composition was tested for bio~h~qiveness in test system No. 1. A residual amount of
about 81% w/w GMO was found after testing.
EXAMPLE 7
Prep~r~t; ~n of a liquid compo6ition without an active E;nh~t~nce
20 The composition was prepared from the following ingredients:
GMO 60 g
Ethanol 30 g
Benzyl alcohol 10 g
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The GMO was mixed with ethanol, and benzyl alcohol was added to the resulting mi~ture while
stirring
The composition was tested for bio~h~siveness in test system No. 1. A residual amount of
about 87% w/w GMO was found after testing.
5 EXAMPLE 8
Prep~r~t;~n of a fiemi-~olid comroR;t~n without an active s~h~s -e
The composition was prepared from the following ingredients:
Dimodan~ LS 65 g
Water 35 g
10 Water was added to the Dimodan~D LS under vigorous stirring.
The composition was tested for hi~ lhPgiveness in test system No. 3 (washing off ability). A
score of 4-5 was found in~lic~bng that the composition is hio~rlh~.cive.
EXAMPLE 9
.
Prep~ on of a ~prayable comrolcit;on without an active ~nh~sn~e
15 The composition was prepared from the following ingredients:
Dimodan~lD LS 60 g
Ethanol 40 g
Ethanol was added to DimodanX LS and mixed.
The composition was tested for hio~-lheciveness in test system No. 1. A residual amount of
20 about 95% w/w GMO was found after testing.
~ EXAMPLE 10
Acyclovir cont~ining C~.~ ff;i ;o
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In the following table is listed a number of acyclovir cnnt~ining co~nrncitinng according to the
invention. The compositions were prepared as (l~g-~rihed above. 5% w/w a~clovir was added to
all the compositions listed in the table below.
Comrogition Yow/w
5 GMO 90/water 98/2
GMO 90/water 95/5
GMO 90/water 90/10
GMO 90/water 85/15
GMO 90/water 80/20
GMO 90/water 75/25
GMO 90/water 72/28
GMO 90/water 71/29
GMO 90/water 70/30
GMO 90/water 69/31
GMO 90/water 68/32
GMO 90/water 67/33*
GMO 90/water 66/34
GMO 90/water 65/35
GMO 90/water 64/36
GMO 90/water 63/37
GMO 90/water 62/38
GMO 90/water 61/39
GMO 90/water 60/40
GMO 90/water 55/45
* To this composition of GMO/water 10%, 20% and 30% w/w acyclovir, respectively, was added
The compositions having from about 55 to about 80% w/w GMO (based on the content of GMO
and water) are cubic at room temperature.
The compositions having from about 95 to about 98% w/w GMO (based on the content of GMO
and water) are probably the reversed micellar phase (L2) (precursor of the cubic phase).
30 The compositions having from about 80 to about 90% w/w GMO (based on the content of GMO
and water) are the lamellar phase (La) (pl~llSO~- of the cubic phase).
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The presently most promising comr~citions are those having a content of from about 55 to about
80% such as, e.g, from about 60 to about 75%, from about 65 to about 70% w/w GMO (based
on the content of GMO and water).
Compositions cont~inin~ glycerol and/or lecithin were also prepared as described above. 5% w/w
~ 5 acyclovir was added to all the c-~mpo,citionc listed in the table below. All c-)mroA~iffonc are cubic.
Composition ~ow/w
GMO 90/water/glycerol 60/20/20
GMO 90/water/glycerol 65/20/15
GMO 90/water/glycerol 65/25/10
10 GMO 90/water/glycerol 67/20/13
GMO 90/water/glycerol 70/10/20
GMO 90/water/glycerol 70/15/15
GMO 90/water/glycerol 70/20/10
GMO 90/water/glycerol 75/15/10
15 GMO 90/water/lecithin 55/35/10
-
All the listed cubic compositions (both the compositions based on GMO 90/water, GMO
90/water/glycerol and GMO 90/water/lecithin, respectively) were hio~-lh~cive (evidenced by
employment of test system No. 3?. A score of 4-5 was generally obtained. Furthermore, the cubic
phase of all the compositions are stable at 25~C (60% relative humidity) and at 40~C (75%
20 relative humidity). Under the test con(1itionc acyclovir has been found to be stable in the cubic
phase and, furthermore, GMO has also proved to be stable. The stability m.ontioned above is
valid for at least 1 year (the term "stability" in the present context is generally nn~r~tood by a
person skilled within the pharmaceutical field as ll~noting that a decrease in content of a specific
sllhst~nce of at the most 10% based on the initial value has been observed).
25 EXAMPLE ll
CO~;I SOnS C~n~qinin~ antiviral sl~hAtsnces
In the following table is listed a number of interesting compositions. The compositions are
~ prepared as described above. 6% w/w of an antiviral sllh~n~e is added to all the compositions
listed in the table below.
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Vehicle Composition % w/w
GML/water 65/35+5% acyclovir
GMO 90/water 67/33+5% penciclovir
GMO 90/water 67/33+5% famciclovir
5 GMO 90/water 67/33+5% ganciclovir
GMO 90/water 67/33+5% valaciclovir
GMO 90/water 65/35+10% acyclovir
GMO 90/water 65/35+10% cidofovir
GMO 90/water 65t36+10% lobucavir
10 GMO 90/water 65/35+10% sorivudine
GMO 90/water 65/35+20% ~ noCin~
Other compositions are also relevant, i.e. comrneitinne having other active sllhct~nc~c or having
a drug conc~ntration of about 1-10% w/w and compositions having a composition of the vehicle
as given in ~ mI)le 10 above.
FY~mrl~ll
-
pH-sr~ hility profile for acyclovir
Experiment~l
To a 100 ml li~rlenm~yer 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 monoh~cic sodium phosphate and
dibasic sodium pho,cph~te (pH adjnetment with phosphorit acid). The buffers in the pH range
6.0 to 9.6 were prepared using monobasic pu~ l... phosph~te (pH adjustment with dodium
hydroxide). The molarity of the phosph~te salts was 0.05M; the pH of the medium was
mea~eured with a pH-meter.
Each mixture was stirred with a m~gn~tic 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 so~nhility 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 minimnm solubility of acyclovir is at a pH in a range of from about 4
to about 6.
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Acyclovir/Solubility at (li~ ,t pH
pH (buffer) Acyclovir, m~/ml
3.6 1.9
4.2 1.8
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
E~nnPLE 13
15 Inverti~tien of the inflll~nce of different active ~l~h~fsnce~ on the liquid clystsllinP,
phase
Mirfm~7n1~ is an PYqmplP of an active sllh~t~nrp which is insoluble in water but has a solubility
of more than 2% w/w in the liquid crystalline phase. However, the release of mironA7olP is very
slowly from the cubic phase. The table given below shows the solubility of and the crystalline
20 phase obtained for mic-~n~7olP in a GMO/water 70/30% w/w vehicle.
Mi~nn~7~ (% w/w) Solubility Liquid crystalline phase
soluble cubic
2 soluble cubic
25 3 soluble cubic
4 soluble cubic
soluble cubic
6 crysWs lamellar
.
30 For micon~7r~ (as well as for some other snh~sln-~Ps which are soluble in the cubic liquid
crysWline phase in certain roncpntrations) expPrim~nt~ have shown that the bio~rlhP.~iveness of
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compositions cont~ining the sllhfit~nc~q varies with the conc.,.~l ~dLion of the cnh~n(~e In the
table below results are given from testing various micon~701a compositions in a GMO/ethanol
60/40% w/w vehicle or in a GML/ethanol 60/40% w/w vehicle, respectively, for hio~(lhaciveness
employing Test system No. 1.
Concentration of Rin~llh~qion*
mif on~7nl (% w/w) (residual amount 9'o)
GMO-based GML-based
O 85 95
4 72 86
16 5 41
6 72
33
*: In the tests runs the following test con~litionq were employed: initial rinsing period: 5 _ in,
initial rinsing flow: 10 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 rlr~ms~ti
fall in hio~llheqiveness when the concpntration of miron~7nlP exceeds 6% w/w, i.e. when the
liquid crystalline phase changes from the cubic phase to the l~m~ r phase and when
micnn~7nle in the liquid crystalline phase is present as crystals, i.e. when the cnncPntration
exceeds the solubility of mi(~on~7~1a
The results support the results of other expPrimPntq performed by the inventors, namely that
30 there is a close correlation between the presence of a cubic phase and occurrence of a high
degree of h~io~ hpqiveness~ The other eXppriment-q parformad by the inventors involved
apr~ tion of GMO, GMO/ethanol mixtures, GML on Test system No. 1 for hio~llh~qiveness. It
was found that the samples applied in contact with the mucosa and washing m~-linm all had
converted into the cubic phase and that the samples were hio~lh~qive. The same applies for
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compositions cont~ining indnmet1lArin (5% w/w) in a GMO/ethanol 60/40% w/w vehicle and
other hioA~lhP~ive compositions contAining an active snhstqnre
From the results given above in the table it is seen that when the ron~Pnt~tion of mirnnA7nl
e~ceeds a certain level, the hioAflhR~ion is severely impaired. This inllir~AtP~ that when the
- ~; con~entration of the active sllhst~nce 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 sllhst~nce and/or any P~rripiant~ may alter the phase lliAgrAm).
In the case of acyclovir, however, this reduction in hioA~lhpciveness with increased content of
acyclovir, beyond the sAt~lr~tion point, does not seem to inflllenre the cubic phase and does not
10 seem to impair the hioA~lhpciveness (tested by means of Test system No. 3). E~perinmPnt.c
showing this were performed with acyclovir ointrn~nt compositions, prepared with GMO 90,
with cnn~antrations of crystalline acyclovir of 2%, 6%, 10%, 20% and 30% by weight, respectively.
These compositions were found to be highly bioadhesive, in~ Ating that with ~nhst~n~P~ having
a very low solubility in the liquid crystalline phase, the liquid crystalline phase remains less
15 disturbed by the presellce of particles of the active s~lh~nre and retains its hioA~hP~ive
properties.
EXA~IPLE: 14
Inve~i~t;on of the inflnenoe of different ~Y~irient~ or solvents on the
l~io~llheRiveness of GMO or GML based cOIl~O~;~ ;on~
20 The infl~lPnce of various P~rripiPnt~ and solvents was inves~igAtP~l The various compositions were
prepared as described above and the hioflllhP~iveness was tested using the test system No. 1.
The following results were obtained:
Composition % w/w RioA/1hP!::inn
26 Residual amount %
GMOQ 90
GMLa 65*
GMO/GMLa 40/60*** 56*
Mi~tures with solvents:
GMO/water 85/15b 94
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G~LL/ethanol 60/40 95**
GMO/ethanol/propylene glycol/water:
45/30/10/15 93
Mi2~tures with soll1hili~ing
agents or pl~S~' vdLiVt~
GMO/ethanol/benzyl alcohol:
60/30/10 87~*
GMO/ethanol/ben2yl alcohol/water:
60/20/5/15 80
10 50/20/10/20 89
Mi~tures with release modulating
agents:
GMO/ethanol/glycerol:
50/30/20 97
15 GMO/ethanol/sesame oil:
59/40/1 96
58/40/2 93
50/40/10 14
50/30/20 0**
20 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
a melted gently before application
b lamellar phase
* lower results than expected; probably due to the reference values used in the analysis of
the mixture
** test con~itionc as in ~x~mple.~1-12
*** the GMO/GML mixture corresponds to about equal amounts of glycerol monooleate and
glycerol mcmnlin~ t~
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The results given above show that ~r.7~.7iti.)n of relevant P~ripir ntq or solvents such as, e.g., agents
which are known .cn1nhi1i~erfi for active sl1h~t~nreq 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 açtive ~nh~ncp from a composition) do not .cignific~nt1y inflnPnce the
hio~r1hP$iveness of the composition when the agents (P~ripiPntq or solvents) are added in
relatively low conçpntr~s7t~ q (less than about 10% w/w). Thus, the release of an active
s11hst~nre from a composition which has proved to possess ~;o~r7hP~ive properties çan 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, rhnle~st~orol~ etc. A
10 motl711s7ting agent may inflnPnrP the pore size of the water rh~nnPlq in the cubic phase and/or
alter the partition coefflent of the active snhqtS7nce between the cubic phase and the aqueous
phase at least to a limited e2~tent. Furthermore, if nPcpqq~ry~ sn1nhi1i~s~tion of an active snh,st~nre
or a fatty acid ester for use in a hio~f1h~cive composition can be effected hy use of e.g benzyl
alcohol wit_out ~i~nifi~ntly influencing the hio~-lhPRive properties of the composition. In
15 cnn(111gion, the hio~(lheqive prinl~ip1Pq ~lPsrrihed herein have a _igh potential wit_ respect to
developing kio?~hPqive drug compositions having such a drug lcr~li7~tion, such a drug release
profile, and such a drug durat,ion which are desirable or npcpcq~qry under the given
Thus, the present inventors have found an advantageous hin~rlhP~ive drug
delivery system.
20 EXAMPLE 15
Inverti~ n of the ~_c~c~ of an active ~llh~t~nçe in a liquid ~ry~sllin~ phase ofglycerol m~rccleate
The metho~ Ogy (lpqrrihe~l herein is a mPthc)dnlo~y which is generally useful for investi~ting
whether mixing or dissolving of an active snhfit~nre in a vehicle capable of forming a liquid
25 crystalline phase also leads to incorporation of the active snhst~nra in the liquid crystalline
phase. While miron~7O1 and lidocain hydrorhlori-l~q have been used as model snhst~n~c in the
description of the expPrimPntq, the same measures as (lPqrrihed herein can be used for
snhst~lnrPq which have a very low solubility in both water and ethanol such as, e.g., acyclovir.
Furthermore, the study was pPrformPd in order to P~mine the recv~ of the samples applied.
- 30 A lipophilic (mil~nn~ 1) and a hydrophilic active s11hst~nre (li~sr~in hydrorhlnri-le), respectively,
were applied on the rabbit jejunum test model for bioa(lh~qiveness (test system No. 1). A vehicle
of GMO 84/ethanol 60/40% w/w incorporating 2% w/w of either miron~o1 or lidocainhydroch1nri~1~ was employed. The GMO 84/ethanol vehicle is bio~h~qive 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 exp~rim~nt) the samples applied were removed from the mucosa and the cubic phase
was q11~r~ lively .o~minf~cl by HPLC for the content of active ~nhst~nrç As seen from the
table below almost all miron~ was found after 10 sec and 30 minutes. These results indicate
5 that the lipophilic micf)nA~--1e is incorporated in the cubic phase formed and the result at 30
minntf~.q inllic,~t~ that the drug is very slowly released from the cubic phase. This is Concietc,nt
with release expr?rimpnt-c of mir-~n~7O1~ delivered from a cubic phase into a 0.05 M phosphslt~
buffer solut,ion, pH 6.5 (37~C). Micon~7~lle 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 snh~anre %
mean of two det~ormin~ti-mR
GMO 84/ethanol/mir-ns~
15 58.8/39.2/2 10 sec 85
30 min 93
GMO 84/ethanol/lidocain HCl:
58.8/39.2/2 10 sec 37
30 min 7
20 GMO 90/acyclovir
95/5 10 sec 87
30 min 65
In the experiment with li(lor~in~ hydrorh1r)ri~1~, barely half the content of the drug was
25 recovered after a flow period of 10 sec and only a neg1i~ih1.9 amount after 30 minnt.o.c Because of
its high water solubility (about 0.7 g/ml at 25~C), the greater part of the 1i~r~ine hydrorhlori-l~
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
had been released at the end of the experiment. Other studies have shown that 1i~1Or~ine
30 hydrochloride is released rather quickly from the cubic phase probably through the water
rh5-nnf~1c cont~ined 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 llemnn~rate that ac~vclovir is en~losed in the cubic liquid crystalline phase formed
and some of it may have been released during the exp~rim~ont
In con~ cinn, the çypprimentc reported above indicate that formlllAhon~s in which GMO and an
~ 5 active substance are dissolved in ethanol or the active lDhstpnce sllgI)Pn~le~l in GMO 90, serve as
a precursor for the formAtinn of a cubic phase formed in situ, and that the active snhcPnce is
incorporated in the cubic phase formed.
EXAMPLE 16
Phase tr~n~iti~n~ of GMO 84 c~nt~in;n~ con~rocit;nnc
A. Compositions without any drug sllh.st~n~e
The composition of ~Y~mrlR 4, i.e. a composition of GMO 84/water 85/15% w/w, is tested
employing the DSC method described under the heading "MPt~loll~" above. The results are given
in Fig 4. DSC expPrimPnt-c give information about at which tempe-dl u~ a phase conversion
takes place. DSC measurements alone give no information of the particular phases involved (e.g.
1~ lamellar, cubic hexagonal etc.). However, if the DSC results as in the present case are compared
v.~ith e.g. results from observation of the comro~i*nn~ in pnlf~ri7ed light (see above under the
heading "MPt~ncl~") information on the c~stalline phases as well as the trAn~ition temperature
is obtained.
For the composition from P~rAmple 4, the results from the DSC and polarized light measurement
show that the lamellar phase is present at room tpmpprAtllre and the lamellar phase is changed
to the cubic phase when the temperature increases (Fig. 4). The transition temperature is about
37~C.
B. Compositions cnnt~ining acyclovir
DSC PxperimPntc as described above were also pRrforme(l on compositions cont~ining
~MO/water 65/35% w/w with 5% w/w acyclovir (crystalline (~.YAmple 2) and micronized
(~Y~mplP 3), 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 tllPrmogrAm~ 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 cnntAining ~% w/w acyclovir.
The snlillifiRd sample trAn~fRrs to the cubic phase (reversible process). No phase transition of the
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62
cubic phase seemed to have taken place. The results are in well agreement with the results
obtained by use of X-ray ~liffr~rtion measurement ~l~srrihed in the following
c~mrncitiQnc c- nt~ining GMO/water 66/35% w/w and GMO/water 65/35% w/w with acyclovir
(cryst~lline and micronized, respectively) added in conr~ontrations 2.5, 6.0 and 10% w/w were
5 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 f~ mine if the cubic phase of
GMO/water 65/35% w/w is changed when acyclovir is added. In the following results from the
compositions of ~r~mple 2 and 3, respective, at 37~C are given for illu~ t.livt: purposes:
d-Spacings:
E~. 2 Ex. 3 Ratio
Lipid phase 61.7A 61.7A
50.5A 50.5A 0.81
36.3A 36.3A 0.58
29.7A 29.7A 0.48
15 Acyclovir 12.9A
8.44~ -
3.74A 3.74A
3.42A 3.42A
The results show that the compositions are cubic at 37~C.
20 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 i~ r~le with the lines from the cubic phase. In conrlll~ion, the results
indicate that acyclovir both in its crystalline and micronized form is inert in the cubic phase.
Thus, no inflll~nce of acyclovir on the phase behaviour has been observed in the conr~ntration
25 range invr~ig~ted and the cubic phase cont~inin~ acyclovir is rather stable against temperature
fl~lctns~tion~
Furthermore, compositions cont~ining GMO/water 65/35% w/w with acyclovir (crystaUine and
micronized, respectively) added in a concentrp/tion of 1-40% were tested in polarized light at 22~C
and 37~C, respectively, as described above under the heading "M~o~e". The results show the
30 presence of cubic phases in all compositions in-lic~lting that acyclovir probably is inert in the
cubic phase.
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EXAMPLE 17
Di~Ql~lt;nr~/release ~ate of a bios~rlhP~ive co~l,oLiLion contq;nin~ acyclovir
The rliqgollltir~n rate of acyclovir in various GMO compositions was determined using Frarlz
diffusion cells as described under the heading "Methods".
6 A series of GMO comrogition~e cont~inin~ acyclovir were prepared as described above, and they
were subjected to the above dissolution rate determin~tion All compositions were suspensions of
acyclovir, that is, they contain acyclovir which was not dissolved. The solubility of acyclovir in
the connroqition.s inve~ig~tP(l was less than 0.1% w/w (0.05% w/w<the solubilit,y of
acyclovircO.1% w/w).
10 The results appear from Figures 5-11. The results indicate that the release of acyclovir from a
GMO based vehicle is ~epen-lPnt on the cnncPntration of acyclovir in the composition, provided
that the release takes place from a cubic phase system. Furthermore, the results indicate the
c~r7lhi1ity of a GMO-based vehicle to function as a very effective drug delivery system.
Figs. 5-7 show the release of acyclovir (1-5% micronized) from a cubic phase (GMO/water
65/35% w/w) and Zovir~ crearn, respectively, into isotonic 0.05 M phoqrh~te buffer solution, pH
6.5 (37~C). As appears from the graph of Fig. 6 showing the cllmnl~tive release of acyclovir, the
release of acyclovir increases with increasing conccntration of acyclovir over the range
investi~t~fl There is not proportionality between the rate of release and the conrentration; this
appears from the fact that the graphs of % released (Fig. 6) do not coincide and the slope of the
20 Higuchi plots (Fig 7); the release is dependent on the conl-Arll~dlion.
It is justified to refer to rate c~7n~n~ herein as the release of acyclovir from the liquid
crystalline formnl,~ti~n.e according to the invention which can be described by means of the so-
called Higuchi equation (Higuchi, T., Rate of release of m~ m~?ntq from ointrn~nt. base
cont~ining drug in suspension. J. Pharm. Sci., 50 (1961) 874-875): on linear regression, the
26 cumulative amount of acyclovir released plotted versus the square root of time results in a
straight line with the slope k (rate con~nt ~g/hh). This appears from Fig 7 which shows the
plots for a number of compositions Cont~ining acyclovir in concentrations from 0.99% by weight
to 4.76% by weight in cl~mrslrigon with Zovir~ cream cnnt~ining 5% by weight of acyclovir. The
- slopes of the graphs in Fig. 7 are as follows:
30 Zovir~ cream, 5%: 155
Acyclovir 0.99% w/w: 410
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Acyclovir 1.96% w/w: 687
Acyclovir 2.91% w/w: 717
Acyclovir 3.8~% w/w: 773
Acyclovir 4.76% w/w: 1016
5 The higher the acyclovir con~ LLion is, the smaller the percentage of acyclovir released. This
would indicate that acyclovir must first be dissolved before it is released from the cubic phase,
probably through the water rhAnn~lR In other words, the rate of dissolution seems to be a rate
limiting factor, to the rate of release in the diffusion process. 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
10 composition according to the invention is drAm~*rAlly increased compared to the Zovir~ cream.
Thus, a cc~mrAri~on of the rate con~nt for acyclovir (5%) released from Zovir~ cream and
GMO/water 65/35% W/W shows that the rate cnn~t~nt is about 6 times larger for the latter
(Fig. 7).
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 ~AminPIl Figures
8, 9 and 10 show an i(l~nti~ Al release pattern for crystalline and micronized acyclovir,
e. Liv~ly, from a formulation con~i~ing 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 65/35/10% w/w + 1% acyclovir) compared
20 to the same composition contAining micronized acyclovir (Figs. 8-9). By compAring the rele_se
profiles for compositions con~ ng of GMO/water 65/35% W/W contAining5% crystAlline and
5% micronized acyclovir, respectively (Fig 10), it seems that the release rate has increased
somewhat with the micronized quality. On the other hand other studies have in~lirAt~d. that the
release rate of acyclovir from a composition con~i~ing of GMO/water/glycerol % w/w + 5%
25 acyclovir is id.ontirAl for the crystallinic and the micronized quality. Whether the release is
improved by applir~tion of a micronized quality as opposed to a cryst lline quality depends on
the composition of the cubic phase. However, more experim~nt-~ have to be carried out to
exclude that the differences observed arise from e~perim~nt~l variation.
The micronized quality increases the viscosity of the cubic phase more that the crystalline phase.
30 This con(lition 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, r~b~e~ Liv~ly, contAining release modulating or solllhili~ing compounds was P~Amined
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and compared with the release from a cubic phase ~on~ n~ of 65 parts of GMO and 35 parts of
water (figs. 9-10). All the comrogition~ except the compositions cont~ining sesame oil and the
composition cont~ining 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. 11 for the compositions
5 cnnt~inin~ 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 cnntslinin~ sesame oil. In the
latter case the release speed is drastically reduced, which could mean that the compositions
consist of the re,v~lsed hexagonal phase, but this has not been confirm~l1 It should be noted that
the composition cnn~ n~ of 65 parts of GMO and 35 parts of glycerol, have the same release
10 profile as the reference composition, although both the visual and the pnl~ri~ed 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 PxperimPnt, through its contact
with the dissolution medium (37~C). A~rlitinn of the release modulating sllhst~ncPs glycerol and
lecithin to the cubic phase has not ~ignific~ntly changed the release of acyclovir in the
15 concpntr~ti~mc e~mine~l Neither does the TPGS seem to have increased the dissolution of
acyclovir in the cubic phase nor changed the partitions' co~mriPnt between the cubic phase and
the release medium, as the release profile is irlPnffr~l with the profile of the reference
composition. Fig 11 shows the release profiles of composition cont~ining 5% acyclovir. The
release proles for the compositions cont~ining glycerol and lecithin are i~Pntir~l while the
20 release profile of the lc,rel- ~ce composition is somewhat smaller. This infli~tP~ that the release
of acyclovir is slightly increased from the compositions added release mo~nl~ti~n agents,
however, the improvement is modest. The tests indicate that it is diflicult to change the release
of acyclovir si~nifir~qntly There are limited pos~ihilitiPc for chiqnEing the release if the cubic
structure is to be preserved.
25 EXAMPLE 18
C~ase stories on tr~ t~nçnt of cold sores - pre~ lini~ tudy in hnn~nR
A composition of GMO/water 65/35% W/W with 5% w/w acyclovir has been used for the
trea~rnPnt of cold sores in hllms~n~:
Tre~hn~nt was started with a maximum of 24 hours delay from start of symptoms. In one case,
tre?(trnpnt with Zovira2~D 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.~ days (range 1.~-4).
The results of the study are given in the Table below.
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o ~
r8 ~ ~ C~ ~
C
a a a 1:~ ~ a R
.. 3 ~
C ~D
o Q' ~ c~
o
C
-
~.
C~
o C
~ C
~ o c~ o O
D~
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In 7 of 8 treiqtm~nt-c symptoms ceased or improved very much. In one case, ulceration occurred
and treat_ent was stopped. Healing was only reported in one case, probably because the
treatment prevented the 1 ypical ulceration of a cold sore.
Side effects were noted by 2 to 7 persons. One of these persons received two tr~trnPntC and in
both cases, tre~t~n~snt 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 srient;~c evidence of the efficacy of GMO acyclovir
cream. They do, however, indicate that the char~tqri~irq of GMO/water 65/35% w/w with 5%
acyclovir on certain points differ from those of Zovira~ cream from Gla2~o Wellcome.
GMO/water 65/35% w/w with 5% acyclovir adheres firmly to skin. Therefore fewer daily
appliratinnc of GMO/water 65/35% w/w with 5% acyclovir were ~-lministered than what is
recommen~ed for Zovirax6 cream. In 6 out of 8 cases, tre~trn~nt could successfully be stopped
after 2-3 days. This is shorter than the nor_ally rerommend~d tr~t~nent period for Zovirax~
cream of 5 to 10 days.
Appli~tion frequency and tre~tm~nt duration for GMO acyclovir cream in these case reports are
less than recommPn-led for Zovirax!~D cream. By the persons treated, the efficacy was judged to
be equivalent or better than that of Zovira~ cream.
EXAMPLE 19
Skin irritation of GMO/water 65/35% w/w with 5% acyclovir
The Chamber ~r~rifir~tion Test has been used in order to evaluate the skin irrit~tion profile of
GMO/water 65/35% w/w with 5% acyclovir.
The Chamber ~r~rific,4ti~n Test is developed to investigate and compare c~m~ti~ c, co~metir
ingredients and consumer products jnt~qnll~d for repeated use on normal or diseased skin. The
assay ~mplifi~s irritant reactions to the test products by sc~rific~tion of the test area prior to the
first applic~*on
The implic~tion~ of results of a Chamber ~r~rifirlltinn Test using 20 volunteers (ProDERM
study # 94,011-05) with GMO 70% in water showing a mean sum of score of about 8 are
evaluated with respect to the potential for causing unacceptable skin irrit~tir~n
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An average score around 8 is in the high range and cnmp~r~hla to the irritation caused by
known products such as Chlorha~ n~ cint~nant 1% and the old formulation of ~alosqn
nintment These products may be used for a short period of time v~ithout causing subjective or
visible skin reactions.
5 A high score in a Chamber .~c~rifir~ti~n Test is problemqtir for products intQn(le~ for daily use
over a long period, on sensitive skin areas and in individuals with hyperirritable skin.
Rc~ herpes simplex infe~;nnc are a nuisance for the patients berause of itching, oozing,
parq~a~tlla~iq and skin eruptions lasting from several days to a fe~,v weeks. If an improved
acyclovir cream clears the eruption in a few days, it may be regarded as a very good therapeutic
10 effect compared to the spontaneous course of the disease, irrespective of a certain degree of skin
irritation that may be caused by the topical drug. This possible irrit~tion may not be detected at
all due to the herpes symptoms.
The new cream is meant to be applied on the herpes simplex infected skin area without
occlusion t~,vice daily for a fe~,v days. In the vast majority of p~qtiantC~ it will probably be tolerated
15 without any problems. After the herpes attack, no further applir~tionc of the drug will be
performed until the next herpes attack months later.
The product has several favourable rh~r~Pri~i~c i.a. increased hio~-lh-Pciveness and increased
bioavailability for a~.l~ vir. Furthermore, pilot experimPnt.c in volunteers with recurrent herpes
simple2~ have shown that the product is well tolerated and leads to rapid clearing of the herpes
20 attack.
.
EXAMPLE 20
In vitro perme~hility of co~o~jil ;on~ acc~ li~g to the invention across porcine skin
Test sllhs~nrP~:
1. GMO/Water 65/35% w/w added 5% w/w micronized
acyclovir BFJ30-1
2. GMO/Water 65/35% w/w added 5% w/w crystalline
acyclovir BFJ30-3
3. Zovir8 5%, Wellcome (Cont~ining 5%
acyclovir) BFJ15-6
,
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Preparation of skin membranes
Excised abdominal skin from pigs was obtained from University of Cop~nhA~Pn, The Panum
Tn! t;~ltP, Department of E~cperlmçntA1 Surgery. The hairs were removed from the epidermal
side by clipping. Subcutaneous fat on the dermal side was removed. The skin was washed with
~ 5 distilled water and stored at -18~C until use.
Apparatus
Franz diffusion cells having an available (liffil.cion area of 1.77 cm2 were used. The eri-l~rmAl
side of the skin was exposed to Amhient laboratory con~iti~nc while the dermal side was bathed
with the receptor mQ~ m con.qicting of 6.81 of 0.05 M ph~sph~t~ buffer, pH 6.5. Each cell was
10 placed on a mAgnetir stirrer. The t~mper~tnre of the water flowing in the closed circulatory
system was kept at 37~C.
Perm~bon procedure
The skin membranes were thawed and mounted in Franz diffusion cells. The receptor chambers
were filled with r~ Lu- medium and the epi~lermAl side of the skin was wetted with a few
15 drops of rec~,pluL- me~i~lm 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 mellillm for f~,luvir. The in~ ;Ly of the individual skin
samples was ensured by measuring the rApA~ritAnce of the skin. Skin samples with a rAparitAnre
of less than about 0.066 ~F were considered intact, whereas skin samples with a higher
20 rApAritAnce were considered damaged. The water permeAhility may also be determined as a
measure of the integrity of the skin. Before applirAtinn of the test s~lb~nr.os, the receptor
medium was replaced by fresh media. 300-360 mg of the test snhst~nre was spread across the
entire er)i~lermAl surface in an even layer. At appropriate intervals (t=0, 6, 24, 30, 48 hours) 2
ml samples were withdrawn and replaced by fresh receptor medium keeping an infinite sink.
Due to variation when using bi~logirAl membranes, at least eight permeation studies were
performed on each test 5nhs~1~nr~o
~ Pernn~ation tests over pig skin have shown that acyclovir from Zovir~ cream and the cubic
phase permeates more or less at the same rate during the first 48-hours periods (see Fig. 12).
However, release tests in vitro have shown that acyclovir incorporated into a cubic phase of
30 GMO (GMO/water 66/36% w/w + 6% acyclovir) is released approx. 6-6 times faster than
a~ ,luvir from Zovir~ cream.
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E~ PLE 21
In vitro perm~hility of c~ o~ on6 according to the invention across human skin
A. Wholly skin
5 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 ArCl1mlllAt~P in the epidermis and
the dermis, the following Pxperimpntq can be performed using wholly intact human skin excised
from co~qm~Ptir surgery. The skin is obtained from clinics for plastic surgery. The skin is treated
as mentioned in the F~xAmrle above and stored at -18~C. Skin from other m~mmAlg than
10 humans may also be employed such as, e.g. guinea pigs, mice and pigs. The skin may be
separated into epi~Prmig and dermis by exposing the skin to hot water (60~C) for e.g. 30 seconds
(heat separation) or by slicing with a microtome (merhAni( Al sPpArAtion) The st,ratum corneum
can be isolated by tape stripping The test con~litiong are generally as described in the Fxs-mrlP
above, but other test times (e.g from 1 hour to 7 days), amounts of sample applied (e.g. 50-350
lb mg) etc. may be appropriate. To avoid intra-individual vAriA*nng the same donor is used to
testing different compositions and the skin srerimçng 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 snhg-t~nce within the skin can be c~l~nlAte(l by mPAgllring the con~ Pntration
20 of the drug sllhgt~nce in i) the receptor medium, ii) the skin, and/or iii) the rPmAining
composition. By measuring i) and iii), the amount of drug substance in the skin can be
('Al('lllAtPrl
B. Different layers of the skin
The herpes virus replicate in the living epirlprmig The basal layer of the epidermis appears to be
25 the primArily site of antiviral activity in cutaneous HSV-1 infectinng, i.e. the epi~lPrmig appears
to be the target site for antiviral drug sllhgt~nrPg
PermPAtion (i.e. penpt~tinn into and through the skin) of acyclovir or other antiviral cnhst~ncPg
can be invPstigAtPd across isolated epidermis by lliffnginn (as described above). In this manner, a
measure is obtained of the amount of acyclovir having permeated the epidermis. ~ltprnAtively~ a
30 picture is obt,ained of the penptrfllinn (i.e. the entry into the skin but not through the skin) of
acyclovir (or other antiviral snhst~ncPg) in the skin by means of diffusion test using wholly skin
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which at the end of the e2~perimènt is divided into stratum corneum, epidermis and dermis by
means of a microtome. The individual layers are analysed for acyclovir (or other antiviral
5llhst~nrF-s)~ e.g. by liquid s~intillAti~n
In those cases where rAIlioArtive acyclovir (or other rA~ioA~tive antiviral drugs) are used, the
amount of acyclovir pPn~trAting the tissue was measured by a liquid scintillAtion technique (3H-
acyclovir is commercially available in form of a ethanol/water 30/70 solllti--n). In order to
P2Aminf~ the content of acyclovir in ~ L skin sections/layers, the skin sections were placed
in ~rintillAtion vials with e.g Soluene 350 over night to dissolve the skin components.
.~rintillAtion cocktail was subsequently added and the samples were assayed for content of
10 acyclovir (or the appropriate antiviral drug) by liquid srintillA~ n spectrometry. The drug
metabolizing enzyme activity in the epidermis is greatly derPn-lQnt on tissue viability. Therefore,
it should be stressed that the determinAtinn of skin absorption /lpsrrihed above does not
disting~ush between the intact antiviral drug and its mPt~olitQ~ It cannot be Px~l~(lPd that
excised skin (usually stored) will loose some of its original enzyme activity. However, acyclovir
1~ exhibits no known metabolism in the skin.
By R~trAl~ting acyclovir from the skin components, acyclovir can also be qnAntifie~l by HPLC.
-
EXAMPLE 22
Pern e~t;on of cQ nrn~ ;nn~ contsining acyclovir or other drugs by means of an invitro cell culture model
20 The permeation of acyclovir or other antivir_l drugs delivered from various compo~i~ion~:
according to the invention can be Q2Amined 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, D~nmArk) is suitable for sensitivity and perme~Ahility studies of antiviral drugs.
Other cell culture models are also available, e.g. for the testing of the efficacy of drugs.
2~ EXAMPLE 23
~ Per ne~;on of connro~;t;on~ cnnt~;n;ng acyclovir or other antivi~al drugs by means
of an in vivo animal model
The herpes virus replicate in the living epidermis. The basal layer of the epider_is appears to be
the primaIy site of antiviral activity in cutaneous HSV-1 infect~n.c, i.e. the target for antiviral
30 drugs. M~t)~o~ - using hairless mouse as an animal model - are available. The met~ul~ allow
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calculation of the target site con-entration of the antiviral (e.g acyclovir) drug applied and aUow
an astim~tinn of the efficacy of the antiviral compositions tested (see. e.g. Lee, P.H. et al.,
Pharm. Res. ~, 8, pp 979-988, 1992 and Su, M.-H. et al., Drug Develop. Ind. Pharm. 20 (4), 685-
718, 1994). In the foUowing is described model systems suitable for testing the antiviral effect of
5 the compositions according to the invention.
Animal models often used are the hairless mouse model (5-7 weeks old) and the g unea pig
model. The g unea pigs are ~haved on their back before the start of the experiment in order to
make a hairless test area.
The animals are ~n~-~ethefi7çd before in~ ing skin lesions, e.g on the lateral side of the body or
10 in the lumbosacral area. 0.005-0.2 ml of a virus suspension [herpes simplex virus type 1 (HSV1),
e.g. strain E-377 or E-115 (titer usuaUy in a range of 106 108 plaque for_ing 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 sllqpenqion is applied on the test area and then 6 smaU holes are
made by means of a sc lpel. The test area on the skin of the test animal can be divided into
15 several test areas, e.g. si~ areas, thereby allowing e.g two di~t~ L compositions (2x2) and their
controls (1x2), placebo) to be tested at the same time on the same animal. UsuaUy 10-30 animals
are used for each comroqi*~n (the number of animals depends on the number of applir~tion~). 1
day prior to (and also after) inocnl~tir n the area can advantageously be treated with an
çnhslncPr such as, e.g., Azone, ethanol, sodium laurylsulfate or propylene glycol. The inf~ctil~n
20 induced by the virus generated skin lesions which appeared at the area of inoc~ tion Shortly
after virus inocnl~tion (e.g 24 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 r~nllnmi~.od (if desirable,
pretreated with an PnhslncPr like Azone). The lesions are treated with the compositions for 2-10
days (appied 2-5 times daily) and then the effect of the tre~t~ant was invaqtig~t~-l The lesions
25 were scored for each animal and two distinct antiviral ~qqagqmantq can be made: i) topical
efflcacy is determined by mç~qllrinE~ the antiviral activity of the antiviral drug snhqtSInce (e.g.
acyclovir) delivered from the compositions tested, and ii) systemic efficacy is rletarminad by
measuring the antiviral activity of the antiviral drug snhst~ncç (e.g. acyclovir) in the circulatory
system which delivers the antiviral snhst~nce to the target site (presumably the epidermal basal
30 layer).
In order to quantify the effect of the diLl~ compositions, a score system is used. Different
score systems may be employed based on the appearance of the skin lesions at various times
after innclll~tinn The score system could be that of Alenius and Oberg, Archives of Virology
1978, ~, 277-288, where the course of infection is divided into a phase of plv~le~ion denoted by
35 scores with Arabic numerals and into a phase of regression denoted by scores with Roman
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nllmerAl~ E.g the innc~ t~d areas can be scored for symptoms daily, stsrting 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.
~ A low cumulative score of a composition indir~tPs a good efficacy compared to a placebo
5 composition (control) which 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 in~ tion of i) inactivation of virus, ii) effect of the antiviral composition applied
etc.
13XAMPLE 24
Clinical dev~lopment pl~mme of GMO acyclovir cream for herpes Is~h;~
The following parameters are suggested for all clinical studies:
Setting
OntI~qhpnts from GPs, derms~t ~logi~s or hospitPl clinics. Primary r.3..~ nent possible in
15 cnnmPrti~-n with a Herpes simplex eruption that are not inrll~ Pd in the study. Patients receive
study mprlir2~tion and are instructed to start treatment immPr7i~tPly upon recurrence of
prodromes and to return to invP~i~tnr after start of trPatmpnt
Inclusion criteria
Clinic lly confirmPd history of recurrent Herpes T~hi~lic, 2-3 annu~l recurrences. Present
20 prodromal symptoms of Herpes Labialis eruption.
E2~clusion criteria
Herpes labialis with l7lcpr~qti~n or crusts
Tmmnnl~p.firi~pncy
Allergy to acyc7lovir/GMO
25 Efficacy p~rAm~Pt~rs
Duration days/hours from start of treatment to cP~tion of symptoms caused by virus
replic~ti~n, including pain, wea7., nl7mhnP~s and erythema.
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Duration days/hours form start of trefltrnent to crust forms-ti.~n
Duration days from start of treQtmpnt~ to complata skin healing
Safet,y p~rslmet~r!:
Local reactions to cream ~llmini~trQtion, including a 28-30 day follow-up.
6 Dose finding
The a~erianre from individual case reports inflil~tqc that fewer daily appli~Qtir~n~ of GMO
acyclovir compared to Zovira2~ are required to obtain efficacy. The optimal ~lminictration
frequency will have to be (letarminP~l
Study groups:
10 Placebo
Once daily
Twice daily
Three times daily
If more than three daily applications is required, GMO acyclovir is not considered to have any
15 advantage compared to Z;ovira2~ cream.
At present no data are available on the st,Q*ct~ l variation of efficacy parameters, therefore a
proper dimensioning of the study has not been p~cihle It is assumed that between 100 and 200
patients per study group is required.
Pivotal studies
20 It is Q~llmed that two iclan*~Ql or at least very similar studies must be pPrform~
Study groups
Placebo
GMO acyclovir x times daily
Zovira2~ 6 times daily
25 The argument for inrlll~ling a placebo group in the pivotal study is to docnm~nt that the
expected clinical equivalence between Zovira~D and GMO acyclovir is not a consequence of both
products inaffi~iqn~y.
-
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At present no data are available on the s~ff~ic~l variation of efficacy p~r~meters~ therefore a
proper rlim~n.~inning of the studies has not been possible. It is assumed that bet~,veen 100 and
200 patients per study group is required.
