Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
  • A61K9/1274—Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases, cochleates; Sponge phases

Definitions

• the present invention relates to a drug delivery system containing a liquid crystalline phase such as a cubic, a hexagonal, a reverse hexagonal, a lamellar, a micellar and a reverse micellar liquid crystalline phase.
• a liquid crystalline phase such as a cubic, a hexagonal, a reverse hexagonal, a lamellar, a micellar and a reverse micellar liquid crystalline phase.
• the compositions are unique in that they as delivery system contains A) a substance which is capable of generating a liquid crystalline phase and providing suitable biopharmaceutical properties like e.g.
• the present invention also relates to a pharmaceutical composition for administration of an active substance to or through a damaged or undamaged skin or mucosal surface or to the oral cavity including the teeth of an animal such as a human.
• the composition is particularly suited for administration of substances which have a very low water solubility and which are to be supplied in an effective amount in a localized region over a period of time.
• the present invention also relates to a pharmaceutical composition for administration of an active substance to a mammal such as a human.
• the composition is unique in that it contains i) a substance which together with a liquid medium such as e.g. water is capable of forming a liquid crystalline phase at room temperature and ii) a so-called st ⁇ icturant which is capable of participating in the formation of the liquid crystalline phase at room temperature.
• a liquid medium such as e.g. water
• a so-called st ⁇ icturant which is capable of participating in the formation of the liquid crystalline phase at room temperature.
• the invention also relates to a pharmaceutical composition in which it is possible to incorporate relatively large amount of certain pharmaceutically acceptable excipients without significantly changing the biopharmaceutical properties of the composition.
• WO 95/26715 it is known that certain fatty acid esters have bioadhesive properties which are suitable for use in pharmaceutical compositions.
• WO 97/13528 (published on 17 April 1997 corresponding to the priority date of the present application) discloses pharmaceutical compositions containing active drug substances having a relatively low solubility in water at a pH of between 3.0 and 9.5 such as, e.g., between 3.2 and 9.3, between 3.4 and 9.1 or between 3.6 and 9 and having a relatively high release rate of the active drug substance for a suitable and sufficiently long period of time.
• compositions disclosed in the above-mentioned international patent application are all based on a content of a fatty acid ester which is sufficient to enable a formation of a liquid crystalline phase either in the composition itself or in situ after application of the composition in the form of a so-called precursor composition.
• formulation considerations such as, e.g., viscosity, dosage form aspects etc.
• patient acceptability considerations such as, e.g., appearance, taste, tolerability etc.
• a substance which is capable of generating a liquid crystalline phase together with a suitable liquid medium is not a simple routine for a person skilled in the art if the biopharmaceutical properties of the compositions (such as, e.g., release properties, bioadhesive properties and appropriate storage stability properties) still have to be maintained. In general, especially stability properties have been observed if such excipients which normally is used within the pharmaceutical field have been added to the liquid crystalhne phase.
• an aqueous medium serves as a liquid medium with which the fatty acid ester forms the liquid crystalhne phase
• the inventor has, however, observed difficulties in reducing the concentration of fatty acid ester in the composition below a certain limit (for glycerylmonooleate.
• the limit has been found to be about 60-65% by weight in non-precursor compositions) without getting a two phase system, e.g. a cubic phase in excess water.
• a structurant is, accordingly, not a substance which merely is enveloped or enclosed in the composition and which, accordingly, would result in a dilution of the crystalline liquid medium which jn turn may result in a composition having a reduced bioadhesiveness compared with the parent composition where no substitution of the liquid crystalline phase-forming substance content has taken place.
• a structurant is capable of ensuring that a substitution of a certain amount of the fatty acid ester does not result in a deterioration of the content and pharmaceutical function of the liquid crystalline phase formed by e.g. a fatty acid ester and a liquid medium.
• the invention relates to a pharmaceutical composition for administration of an active substance to or through a nail or a damaged or undamaged skin or mucosal surface of a mammal (such as an animal or a human), the composition comprising
• a second substance which, together with a liquid medium, is capable of generating a hquid crystalline phase in which the constituents of the composition are enclosed, the liquid crystalline phase being selected from the group consisting of: a cubic, a hexagonal, a reverse hexagonal, a lamellar, a micellar and a reverse micellar liquid crystalline phase,
• a structurant which together with said second substance and a hquid medium is capable of forming a liquid crystalhne phase selected from the group consisting of: a cubic, a hexagonal, a reverse hexagonal, a lamellar, a micellar and a reverse micellar hquid crystalline phase; and
• the composition either being one in which the liquid crystalline phase has been generated by the second substance and the structurant together with a sufficient amount of a liquid medium originally present in the composition, or the composition being in a precursor form in which the second substance and the structurant have not generated the liquid crystalline phase, but are capable of forming the hquid crystalhne phase in situ with moisture from the surface on which the composition is applied, the moisture in this case constituting at least part of the liquid medium,
• the pH of the liquid crystalhne phase being in the range of 3.0-9.5 such as, e.g., 3.2-9.3, 3.4-9.1 or 3.6-9.0, determined as described herein,
• the active substance having i) a first solubility in the liquid crystalline phase of at the most 20 mg/g at 20°C, and
• a second solubility in water of at the most 10 mg/ml at 20°C the water, where applicable, being buffered to a pH in a range of 3.0-9.5 such as, e.g., 3.2-9.3, 3.4-9.1 or 3.6-9.
• the composition is not one consisting of either a) 5% by weight of acyclovir and 95% by weight of a glycerylmonooleate/water/lecithin (55/35/10% w/w) formulation, wherein the glycerylmonooleate product is DIMODAN® GMO-90 and the lecithin is Epikuron 200, or b) 5% by weight of acyclovir and 95% by weight of a glycerylmonooleate/water/d- ⁇ - tocopherylpolyethyleneglycol 1000 succinate (65/35% w/w glyceiylmonooleate/water plus 5% w/w d- ⁇ -tocopherylpolyethyleneglycol 1000
• the present invention is not limited to pharmaceutical compositions containing drug substances having a relatively low solubility in water at a pH of between about 3.0 and 9.5 such as, e.g. between about 3.6 and 9.
• a pH of between about 3.0 and 9.5 such as, e.g. between about 3.6 and 9.
• the present inventor has found it possible also to obtain pharmaceutical compositions having suitable biopharmaceutical properties as well as a suitable storage stability, i.e. the compositions do not separate into at least two distinct phases within a well-defined period of time and under well-defined environmental conditions.
• the invention relates to a pharmaceutical composition for administration of an active substance to a mammal, the composition comprising
• a first substance which is the active substance
• a second substance which together with a liquid medium is capable of forming a hquid crystalhne phase at roo n temperature
• the liquid crystalhne phase being selected from the group consisting of: a cubic, a hexagonal, a reverse hexagonal, a lamellar, a micellar and a reverse micellar liquid crystalline phase,
• liquid crystalhne phase selected from the group consisting of: a cubic, a hexagonal, a reverse hexagonal, a lamellar, a micellar and a reverse micellar liquid crystalhne phase,
• liquid crystalline phase selected from the group consisting of: a cubic, a hexagonal, a reverse hexagonal and a lamellar, a micellar and a reverse liquid crystalline phase,
• the composition either being one in which the hquid crystalhne phase has been generated by the second substance in combination with the structurant and together with a sufficient amount of the hquid medium originally present in the composition, or the composition being in a precursor form in which the second substance and the structurant have not generated the liquid crystalhne phase, but are capable of forming the liquid crystalhne phase in situ with moisture from the site at or to which the composition is administered, the moisture in this case constituting at least part of the liquid medium, and
• the composition being substantially homogeneous and having such a physical stability that substantial no irreversible phase separation into two or more distinct phases can be observed visually after storage of the composition at 25°C and 60% relative humidity for one week.
• the content of the substance which is capable of generating a liquid crystalline phase i.e. the substance denoted "second substance" which e.g. is a fatty acid ester
• second substance which e.g. is a fatty acid ester
• the content of the substance which is capable of generating a liquid crystalline phase can be reduced by incorporating at least 5% by weight of certain pharmaceutically acceptable excipients and such a reduction of the content of e.g. fatty acid ester does not significantly deteriorate or negatively influence the biopharmaceutical properties of the composition.
• the effect of such pharmaceutically acceptable excipients is most likely a diluting effect of the content of the liquid crystalline phase present in the composition while no significant diluting effect is observed with respect to the biopharmaceutical properties of the composition (e.g. release properties, bioadhesion, storage stability).
• the invention relates to a pharmaceutical composition for administration of an active substance to a mammal, the composition comprising
• a second substance which together with a liquid medium - at room temperature is capable of forming a liquid crystalhne phase selected from the group consisting of: a cubic, a hexagonal, a reverse hexagonal, a lamellar, a micellar and a reverse micellar hquid crystalhne phase,
• the composition either being one in which the hquid crystalhne phase has been generated by the second substance together with a sufficient amount of the liquid medium originally present in the composition, or the composition being in a precursor form in which the second substance has not generated the hquid crystalline phase, but is capable of forming the liquid crystalline phase in situ with moisture from the site at or to which the composition is administered, the moisture in this case constituting at least part of the liquid medium,
• composition being substantially homogeneous and having such a physical stability that substantial no irreversible phase separation into two or more distinct phases can be observed visually after storage of the composition at 25°C and 60% relative humidity for one week,
• a liquid medium may optionally be present.
• the composition is in the form of a so-called "precursor composition", i.e. a composition in which a liquid crystalline phase has not been generated but which upon application to or on the mammal is capable of generating a liquid crystalhne phase by means of moisture or body fluid present on the apphcation site.
• precursor composition i.e. a composition in which a liquid crystalline phase has not been generated but which upon application to or on the mammal is capable of generating a liquid crystalhne phase by means of moisture or body fluid present on the apphcation site.
• the compositions of the invention may also be presented in precursor form even if a liquid medium is included in the composition.
• the concentration of the liquid medium is too low to enable a formation of the liquid crystalhne phase in the composition or the hquid medium is of a type with which the second substance does not form a liquid crystalhne phase.
• the liquid crystalline phase is formed in situ after application to the mammal by means of the moisture or body fluid present on the apphcation site.
• the pharmaceutical compositions according to the invention are intended for administration to a mammal such as a human.
• the underlying formulation principle of compositions according to the invention is so generally applicable that composition suitable for almost any apphcation site or administration route can be prepared by means of methods well known in the pharmaceutical practice.
• the pharmaceutical compositions according to the invention are intended for application to or through undamaged or damaged skin or mucosa of an animal such as a human.
• the mucosa is preferably selected from oral, buccal, nasal, vaginal, rectal, aural, lung, and gastrointestinal mucosa.
• the skin or mucosa may also be inflamed.
• the composition may also be administered to body cavities such as the oral cavity or by the buccal route.
• the composition may be applied on or at a tooth or a dental pocket.
• composition according to the invention may also be applied to a nail of an animal such as a human.
• liquid crystalhne phase As mentioned above an important property of a composition according to the present invention is its ability to generate a liquid crystalhne phase.
• liquid crystalline phase as used herein is used to denote an intermediate state between solid crystals and isotropic liquids, characterized by long-range order and short-range properties close to those of a simple liquid or solution (Keller et al., Handbook of Liquid Crystals, Verlag Chemie, Weinheim, Germany, 1980).
• the main component in a composition according to the invention - which is responsible for the formation of a liquid crystalhne phase - is the so-called "second substance".
• another component in a composition according to aspects of the invention may also be capable of forming a hquid crystalhne phase.
• Such a substance is in the present context denoted a "structurant”.
• a structurant takes part in the liquid crystalline phase generated by the second substance, but need not be able to generate a liquid crystalhne phase in compositions without any second substance present.
• a structurant may generate a quite different liquid crystalhne phase than the second substance under the same conditions and in such a case, the present inventor has observed that the liquid crystalhne phase generated by a composition of the invention is the one of the second substance.
• the second substance may be responsible for what kind of hquid ciystalline phase a composition will generate or ii) the second substance may together with the structurant generate a liquid crystalhne phase of a different kind than expected taken the nature of the second substance into account.
• the classes of substances and examples of specific substances which are suitable for use as second substances may of course also be suitable for use as structurants (and vice versa) provided that the specific requirements claimed are fulfilled.
• Suitable substances with an excellent ability of forming a liquid crystalhne phase are fatty acid esters like, e.g., glyceryl monoesters of fatty acids.
• Other substances which have ability of forming a hquid crystalhne phase are found among amphiphilic substances such as polar hpids, surfactants and emulsifiers.
• Specific examples of glyceryl monoesters of fatty acids include glycerylmonooleate (monoolein) and glycerylmonolinoleate.
• Such fatty acid esters are capable of forming various crystalhne phases upon contact with a hydrophilic medium such as water or glycerol. As will be explained in further detail below, these fatty acid esters also show so-called bioadhesive properties.
• Liquid crystalhne phases may be a cubic (three cubic liquid crystalhne phases are well- characterised: i) the body-centred lattice, ii) the primitive diamond lattice, and hi) the gyroid), reverse cubic, hexagonal, reverse hexagonal, lamellar, micellar or reverse micellar phase.
• cubic hquid crystalhne phase herein is meant a thermodynamically stable, viscous and optically isotropic phase made of a suitable substance such as, e.g., a fatty acid ester and a liquid medium such as, e.g., an aqueous medium.
• the cubic hquid crystalhne phase is contemplated to be build up of closed reversed micelles.
• aqueous medium includes media containing water or another hydrophilic and water-miscible substance such as, e.g., glycerol.
• hexagonal phase and reverse hexagonal phase are used herein to describe thermodynamically stable, viscous and optically anisotropic phases characterized by long-range order in two dimensions and made of a suitable substance such as, e.g., a fatty acid ester and a liquid medium such as, e.g., an aqueous medium.
• the term “lamellar phase” is characterised by a long-range order in one dimension. The lamellar structure is the origin of liposomes having spherical shells of lipid bilayers.
• the various liquid crystalline phases can be detected and identified by use of polarized light or by means of X-ray diffraction pattern analysis (see, the Examples herein).
• the cubic liquid crystalhne phase is normally the preferred phase in the compositions of the invention, but also, e.g., the reverse hexagonal and the reverse cubic liquid crystalhne phase may be an interesting liquid crystalhne phase in the compositions according to the invention, notably in compositions which are in precursor form.
• the so-called "second substance" for use in compositions according to the invention may be a fatty acid ester which is capable of forming a liquid crystalhne phase on contact with a suitable liquid medium.
• the liquid of the liquid medium is suitably water, glycerol or an aqueous medium.
• An aqueous medium is a medium containing water at least in part.
• a medium with which the liquid crystalline phase is made may, especially for the precursor embodiment of the composition, at least in part be constituted by any body fluid or secretion which contains water and with which upon apphcation the composition comes into contact, such as, e.g. in the case of a human body fluid, saliva, sweat, gastric juice, etc.
• the body fluid may induce formation of a hquid crystalhne phase when a second substance such as a fatty acid ester is contacted with such a hquid.
• a composition according to the invention will be one in which the hquid crystalhne phase is already present, that is, the liquid crystalhne phase has already been established by interaction between a hquid medium present in the composition and the second substance such as a fatty acid ester.
• the hquid of the hquid medium may typically be, e.g., water or glycerol or a mixture thereof, water often being a preferred liquid.
• the fatty acid esters capable of generating a hquid crystalline phase as evidenced by one of the test methods described herein are fatty acid esters (i.e. composed of a fatty acid component and a hydros-containing component) wherein the fatty acid component of the fatty acid ester is a saturated or unsaturated fatty acid having a total number of carbon atoms of from C 6 to C 26 .
• saturated fatty acid moieties in the fatty acid esters according to the invention are selected from the group consisting of moieties of caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, and behenic acid.
• unsaturated fatty acid moieties in the fatty acid esters according to the invention are moieties selected from the group consisting of palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid.
• Particularly suitable fatty acid esters for use in compositions according to the invention are fatty acid esters which are selected from the group consisting of fatty acid esters of polyhydric alcohols, fatty acid esters of hydroxycarboxylic acids, fatty acid esters of monosaccharides, fatty acid esters of glycerylphosphate derivatives, fatty acid esters of glycerylsulfate derivatives, and mixtures thereof.
• the hydroxy-containing component of the fatty acid ester is polyvalent, the hydroxy-containing component may be partially or totally esterified with a fatty acid component or with mixtures of fatty acid components.
• the polyhydric alcohol component of the fatty acid ester for use in compositions according to the invention is preferably selected from the group consisting of glycerol, 1,2-propanediol, 1,3- propanediol, diacylgalactosylglycerol, diacyldigalactosylglycerol, erythritol, xylitol, adonitol, arabitol, mannitol, and sorbitol.
• the fatty acid esters formed from such polyhydric alcohols may be mono- or polyvalent such as, e.g., divalent, trivalent, etc.
• fatty acid monoesters have proved to have bioadhesive properties and are therefore preferred fatty acid esters for use in compositions according to the invention.
• the position of the polyvalent alcohol on which the ester bond(s) is(are) established may be any possible position. In those cases where the fatty acid ester is a diester, triester, etc. the fatty acid components of the fatty acid ester may be the same or different. In a most preferred aspect of the present invention, the polyhydric alcohol component is glycerol.
• fatty acid esters for use in compositions according to the invention and wherein the hydroxy-containing component is a polyhydric alcohol are glycerylmonooleate, glycerylmonolinoleate, glycerol monohnoleate, and mixtures thereof. These fatty acid esters have especially promising bioadhesive properties, confer the Examples herein.
• the hydroxycarboxylic acid component of the fatty acid ester is preferably selected from the group consisting of malic acid, tartaric acid, citric acid, lactic acid, and sorbic acid.
• a fatty acid ester for use in compositions according to the invention is a fatty acid monoester of citric acid.
• the hydroxy-containing component of a fatty acid ester for use in compositions according to the present invention may also be a saccharide, such as a monosaccharide such as, e.g., glucose, mannose, fructose, threose, gulose, arabinose, ribose, erythrose, lyxose, galactose, sorbose, altrose, tallose, idose, rhamnose, or allose.
• a saccharide such as a monosaccharide such as, e.g., glucose, mannose, fructose, threose, gulose, arabinose, ribose, erythrose, lyxose, galactose, sorbose, altrose, tallose, idose, rhamnose, or allose.
• the fatty acid ester is preferably a fatty acid monoester of a monosaccharide selected from the group consisting of sorbose, galactose, ribose, and rhamnose.
• the hydroxy-containing component of a fatty acid ester for use in compositions according to the invention may also be a glycerylphosphate derivative such as, e.g., a phospholipid selected from the group consisting of phosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositole, and diphosphatidylglycerol.
• a glycerylphosphate derivative such as, e.g., a phospholipid selected from the group consisting of phosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositole, and diphosphatidylglycerol.
• DEPE dielaidoyl-sn-glycerol-3-phosphoethanolamine
• DMPE PEG 550
• PEG 550 l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol)550
• Especially interesting compounds having a phospholipid moiety are compounds wherein the fatty acid ester is a fatty acid ester of a glycerylphosphate derivative, and the fatty acid component is selected from the group consisting of lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and behenic acid.
• Examples of such useful fatty acid esters are dioleyol phosphatidylcholine, dilauryl phosphatidylcholine, dimyristyl phosphatidylcholine, dipalmitoyl phosphatidylchohne, distearoyl phosphatidylchohne, dibehenoyl phosphatidylcholine, dimyristyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleyl phosphatidylglycerol, dilauryl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dipalmitoyl phosphatic acid and mixtures thereof.
• fatty acid esters for use in compositions according to the invention are well-known chemical compounds which are commercially available or may be prepared by means of conventional esterification procedures involving e.g. reaction of a fatty acid derivative such as, e.g., the corresponding acid chloride with a hydroxy-containing compound (if necessary protected with suitable protection groups) and subsequently isolating the fatty acid ester, if necessary after removal of any protecting group.
• a fatty acid derivative such as, e.g., the corresponding acid chloride
• a hydroxy-containing compound if necessary protected with suitable protection groups
• glycerylmonooleate from Danisco Ingredients A/S, Denmark is a very pure product containing about 98% w/w monoesters of which more than about 80% w/w (such as about 92% w/w) is glycerylmonooleate; the remaining monoesters are glycerylmonolinoleate, glyceryl monopalmitate and glyceryl monostearate.
• the fatty acid ester products for use in compositions according to the invention may thus be mixtures of fatty acid esters. Examples of fatty acid esters with excellent bioadhesive properties as well as an excellent ability of forming a liquid crystalhne phase are glyceryl monoesters of fatty acids.
• glycerylmonooleate (monoolein) and glycerylmonolinoleate.
• fatty acid esters are capable of forming various crystalhne phases upon contact with a hydrophilic medium such as water or glycerol, a preferred liquid crystalhne phase being the cubic hquid crystalhne phase.
• compositions according to the invention are compositions in which the fatty acid ester is glycerylmonooleate or glycerylmonolinoleate, in particular glycerylmonooleate.
• the stability of the composition is considerably enhanced, such as resulting in a storage stability of at least 2 years at 25°C, when the glycerylmonooleate product (as is well known, fatty acid esters are almost invariably mixed products) contained in the product fulfils certain purity standards.
• the glycerolmonooleate product used for the preparation of the composition should contain at the most 4% of saturated monoglyceride and should preferably contain at least 88% of glycerylmonooleate, more preferably at least 89%, such as at least 90% or at least 91%, in particular at least 92%, of glycerylmonooleate.
• the hquid medium is either not present at all or is present in small amounts, such as an amount of at least 0.5% by weight, such as at least 1% by weight, calculated on the total composition, e.g. at least 2% by weight, calculated on the total composition, or up to at least 5% or in certain cases at least 10%, calculated on the total composition.
• the hquid medium is normally present in an amount of at least 20% by weight, calculated on the total composition, such as at least 25% or at least 30% by weight, calculated on the total composition, and a preferred amount is often in the range of 25- 50% such as 25-40% by weight, in particular 25-35%, 27-40%, 27-35% or 30-40% by weight, calculated on the total composition.
• the concentration of the second substance in a composition according to the invention is at least about 10% by weight such as, e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% by weight calculated on the composition.
• the concentration of the second substance in the composition is in a range corresponding to from about 10% to about 90% such as, e.g. about 15%-85%, about 20%-80%, about 25%-75%, about 25%-70%, about 25%-65%, about 25%-60%, about 25%-55%, about 30%- 50%, about 35%-55%, about 30%-45% or about 30%-40% by weight based on the total composition.
• the maximal concentration of the second substance in the composition is at the most about 60% such as, e.g. at the most about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25% or about 20% by weight based on the total composition.
• the gist of the present invention relates to the use of certain substances as substitutes for at least a part of the substance (e.g. a fatty acid ester) which is capable of forming a liquid crystalline phase.
• the fatty acid ester component is substituted by so-called structurants then the lattice structure of the liquid crystalline phase formed in the composition (or in situ if the composition is a precursor composition) is not only based on the fatty acid ester itself but the structurant takes part in this lattice.
• the structurant imparts the lattice structure to the composition and, thus, the content of lattice structure in the composition is of the same order of magnitude as if no substitution has taken place.
• active substances or additives When adding active substances or additives to a composition which is capable of forming a hquid crystalhne phase, the general observation is that the active substance or additive is enclosed or enveloped in the composition without participating in the formation of the lattice structure.
• such substances can only be present in the composition to a certain extent beyond which the substance will exert a negative influence on the formation of a hquid crystalhne phase resulting in that either no hquid crystalhne phase is formed or the biopharmaceutical properties of the composition are negatively influenced.
• substances can be incorporated in a concentration of at the most about 10% by weight without any significant change in the ability of the composition to form a hquid crystalline phase or to undergo a phase transition.
• a structurant participates in the liquid crystalline structure preferably formed together with a fatty acid ester.
• a suitable structurant is typically an amphiphilic substance having a molecular weight of at the most 2000 or an emulsifier or a surfactant.
• Tensides anionic, cationic, non-ionic like e.g. sorbitan esters, sorbitan macrogol esters (polysorbates)), polar lipids, glycolipids, lechitins, palmitoyl muramic acid (PMA), substances having surface active properties like e.g.
• cellulose derivatives sorbitan oleate, sorbitan laurate, lanolin and derivatives thereof and ethoxylated derivatives of lanolin (Aqualose W20, Aqualose L30 and Aqualose L75) are also examples of suitable structurants for use in compositions according to the invention.
• Sorbitan esters are a series of mixtures of partial esters of sorbitol and its mono- and di- anhydrides with fatty acids. Examples of suitable sorbitan esters for use as structurants in a composition according to the invention are:
• Polysorbitan fatty acid esters are a series of fatty acid esters or sorbitol and its anhydrides copolymerized with approximately 20 moles of ethylene oxide for each mole of sorbitol and its anhydrides.
• suitable polysorbates for use in the present context are:
• a liquid medium such as, e.g., water
• the polysorbates may be dissolved or dispersed therein.
• a structurant should possess a hydrophilic as well as hydrophobic portion.
• a useful structurant normally have the following molecular characteristics:
• Chains alkyl chains (saturated or unsaturated), polyethylene chains and/or polyoxyethylene chains
• sugar oxyethylene, glycerol, hydroxy, polyhydroxy, amino acid, sulfates, and/or phosphates.
• Suitable structurants are also found among the substances which normally are denoted emulsifiers.
• the structurant has a saturated or unsaturated, branched or unbranched, substituted or unsubstituted C 6 -C 2 e-alkyl chain, and/or the structurant is a compound which contains a polyethylene group.
• an important property of a structurant which is suitable for use in compositions according to the present invention is its solubility in the second substance such as e.g. a fatty acid ester or mixtures of fatty acid esters. It is believed that the two components (i.e. the second substance and the structurant) should be miscible (or compatible) and "like each other" in order to be able to cooperate when the lattice structure is formed.
• the solubility of the structurants in the second substance like e.g. a fatty acid ester or mixtures of fatty acid esters should preferably be at least about 15%, such as at least about 20% or about 25% by weight at 60°C.
• the structurant is a substance which - together with the second substance hke a fatty acid ester and a liquid medium - is capable of forming a cubic liquid crystalline phase.
• the preferred structurants are substances which - in a two component system of the structurant as a first component and water as a second component - are capable of forming a non-cubic liquid crystalline phase.
• a requirement for the structurant is not that it is capable of forming the same liquid crystalhne phase at room temperature as the second substance.
• a substance hke Vitamin E TPGS - which is a very important structurant in this context and which in itself together with water is not capable of forming a cubic liquid crystalhne phase at room temperature - together with fatty acid esters (as second substances) and water forms a cubic liquid crystalline phase.
• structurants are substances which - in a two component system of the structurant and water - do not form a cubic liquid crystalhne phase at a temperature of between 20-40°C.
• composition according to the invention may of course also comprise more than one structurant such as, e.g., a combination of two or more structurants.
• the concentration (of the structurants) in the composition is at least 1% by weight such as, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% by weight based on the total weight of the composition.
• the concentration range is typically in a range of from about 1% to about 60% such as, e.g., from about 5% to about 55%, from about 5% to about 50%, from about 5% to about 45%, from about 7.5% to about 40%, or from about 10% to about 35% by weight based on the total weight of the composition.
• the maximal concentration of a structurant in the composition is at the most about 45% by weight such as, e.g., at the most least about 40% or about 35% by weight based on the total weight of the composition.
• compositions comprising e.g. glyceryl monooleate (GMO)/water without any content of a structurant must have a concentration of the GMO of at least 60-65% by weight in order to have a certain suitable and acceptable storage stability and/or to avoid an excess of water present in the composition.
• GMO glyceryl monooleate
• the present inventor has found that when e.g. GMO is substituted by a structurant, compositions having a concentration of e.g. GMO below and well below 60% by weight have shown to be stable for at least one week at room temperature and 60% relative humidity.
• the stability of the composition is the physical stability, i.e. stability with respect to phase separation (i.e. not necessarily change in liquid crystalhne phase) into two or more distinct phase.
• phase separation must be irreversible, i.e. no reestablishment of a homogeneous composition can be observed visually by shaking the composition at room temperature for 2 days and the distinct phases formed by either be liquid, semi-solid or solid phases.
• a physical instability of the composition may be due to a change in the liquid crystalline phase present in the composition.
• Such a change in the liquid crystalline phase may either be due to i) a chemical change of either the second substance or the structurant, ii) a negative influence on the liquid crystalhne phase by any component included in the composition, or iii) the fact that the concentration of the constituents of the composition is near a point in the phase diagram where the liquid crystalhne phase changes from one phase to another or is destroyed (i.e.
• a composition according to the invention is substantially homogeneous and has such a physical stability that substantial no irreversible phase separation into two or more distinct phases can be observed visually after storage of the composition at 25°C and 60% relative humidity for one week or at least for one week such as, e.g., at least two weeks, 1 month, 1 year or preferably at least 2 years at 25°C.
• the total concentration of the substances - which together with each other and an liquid medium such as, e.g., water are capable of generating a liquid crystalline phase - in a composition according the invention is typically at least about 50% by weight based on the weight of the total composition.
• the total concentration of the second substance such as, e.g., a fatty acid ester or mixture of fatty acid esters and the structurant(s) is at least 50% by weight based on the total composition.
• compositions wherein the liquid crystalline phase has been generated by a hquid medium present in the composition and wherein the total concentration of the second substance like e.g. a fatty acid ester or mixture of fatty acid esters and the structurant(s) is at least 50% such as at least 55%, 60%, 65%, 70% or 75% by weight based on the total composition.
• the second substance like e.g. a fatty acid ester or mixture of fatty acid esters and the structurant(s) is at least 50% such as at least 55%, 60%, 65%, 70% or 75% by weight based on the total composition.
• compositions according to the invention may also be in a precursor form.
• examples of interesting compositions are those wherein the total concentration of the fatty acid ester or mixture of fatty acid esters and the structurant(s) is at least 50% such as at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.5% by weight based on the total composition.
• a suitable structurant for use in a composition according to the invention are, e.g., a phospholipid selected from the group consisting of phosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, and diphosphatidylglycerol.
• DEPE dielaidoyl-sn-glycerol-3-phosphoethanolamine
• DMPE PEG 550
• PEG 550 l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol)550
• the structurant may be a fatty acid ester of a glycerylphosphate derivative or a glycerylsulfate derivative, and the fatty acid component is selected from the group consisting of lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and behenic acid.
• fatty acid ester is selected from the group consisting of dioleyol phosphatidylcholine, dilauryl phosphatidylcholine, dimyristyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dibehenoyl phosphatidylcholine, dimyristyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleyl phosphatidylglycerol, dilauryl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dipalmitoyl phosphatic acid and mixtures thereof.
• a presently preferred type of structurant is phosphatidylcholine such as, e.g., a phosphatidylcholine containing product hke Epikuron 200, Epikuron 145, Lipoid S75 or Lipoid S100.
• the concentration of the structurant is preferably in a range of from about 1 to about 75% such as, e.g., from about 5 to about 55%, from about 5% to about 35% or from about 10 to about 20% by weight based on the total weight of the composition.
• tocopherols Another group of substances which are suitable structurants for use in a composition according to the invention is tocopherols.
• tocopherols is used to broadly include all Vitamin E or Vitamin E-hke substances, derivatives and analogs thereof.
• the term includes all tocol and tocotrienol derivatives such as e.g. methyl tocol.
• a tocopherol is selected from the group consisting of ⁇ -tocopherols, sorbitan esters of tocopherols, d- ⁇ -tocopherol, d,l- ⁇ -tocopherol, d- ⁇ -tocopherol acetate, d,l- ⁇ -tocopherol acetate, d- ⁇ -tocopherol succinate, d,l- ⁇ -tocopherol succinate, d- ⁇ -tocopherol nicotinate, d,l- ⁇ - tocopherol nicotinate, tocopherylpolyethylene glycol succinate such as d- ⁇ -tocopherylpolyethylene glycol succinate or d,l- ⁇ -tocopherylpolyethylene glycol succinate, and derivatives such as fatty acid ester derivatives and analogues thereof.
• the tocopherylpolyethylene glycol succinate is selected from the group consisting of: d- ⁇ -tocopherylpolyethylene glycol 200 succinate, d,l- ⁇ -tocopherylpolyethylene glycol 200 succinate, d- ⁇ -tocopherylpolyethylene glycol 300 succinate, d,l- ⁇ -tocopherylpolyethylene glycol 300 succinate, d- ⁇ -tocopherylpolyethylene glycol 400 succinate, d,l- ⁇ -tocopherylpolyethylene glycol 400 succinate, d- ⁇ -tocopherylpolyethylene glycol 500 succinate, d,l- ⁇ -tocopherylpolyethylene glycol 500 succinate, d- ⁇ -tocopherylpolyethylene glycol 600 succinate, d,l- ⁇ -tocopherylpolyethylene glycol 600 succinate, d,l- ⁇ -tocopherylpolyethylene glycol 600 succinate, - _ d- ⁇ -tocopherylpolyethylene glycol 700
• Preferred tocopherols for use in a composition according to the present invention are d- ⁇ - tocopherylpolyethylene glycol 1000 succinate (in the following denoted vitamin E TPGS or simply TPGS) or d,l- ⁇ -tocopherylpolyethylene glycol 1000 succinate.
• a composition according to the invention containing a tocopherol as a structurant has typically a concentration of the tocopherol of at the most about 30% such as at the most about 25%, 20%, 15%, 10%, 5%, 2.5% or 1% by weight based on the total weight of the composition.
• Presently preferred compositions according to the invention are those wherein the structurant is a combination of vitamin E TPGS and a phosphatidylcholine containing product such as ⁇ e.g., Epikuron 200.
• the concentration of vitamin E TPGS is generally in a range corresponding to from about 1% to about 30% such as, e.g., from about 5% to about 25%, from about 5% to about 20% or from about 10% to about 20% by weight and the concentration of Epikuron 200 is in a range corresponding to from about 2.5% to about 40% such as, e.g., about 5% to about 25% or from about 10% to about 20% by weight based on the total composition.
• an aspect of the invention relates to compositions wherein at least a part of the substance which together with a liquid medium such as, e.g., water is capable of forming a liquid crystalline phase at room temperature can be substituted by certain pharmaceutically acceptable excipients.
• a pharmaceutically acceptable excipient to a composition containing a liquid crystalhne phase or a precursor composition will normally lead to a disruption in the liquid crystalhne phase. Therefore, such substance is generally only added in very small concentrations such as, e.g., about 1-5% by weight based on the total composition.
• concentration of such excipients may be at least about 5% by weight such as, e.g, at least about 8%, 9%, 10%, 15% or 20% by weight.
• Suitable pharmaceutically acceptable excipients may either i) be soluble in the second substance or in the hquid crystalline phase, i.e. having a solubility of more than about than about 15% such as more than about 25%, 30% or 50% by weight in the second substance (or liquid crystalhne phase) at 60°C, or ii) have a relative low solubility in the second substance such as, e.g., a solubility of less than 15% such as less than about 12.5%, 10%, 7.5%, 5% or 1% by weight in the second substance at 60°C. More specifically, the pharmaceutically acceptable excipient may have a solubility of less than about 15% such as less than about 10%, about 5%, about 2.5%, about 1%, or about 0.5% by weight in the liquid crystalhne phase at room temperature.
• Suitable pharmaceutically acceptable excipients are found e.g. among inert diluents or fillers selected from the group consisting of sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, sodium phosphate, and a polysaccharide such as, e.g., carmelose, a chitosan, a pectin, xanthan gum, a carrageenan, locust bean gum, acacia gum, a gelatin, an alginate, anjd dextrans and salts thereof.
• inert diluents or fillers selected from the group consisting of sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, carboxymethylcellulose sodium, methyl
• a in composition according to the above-mentioned aspect of the invention the concentration of the second substance in the composition is at the most about 60% such as, e.g., at the most about 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% by weight based on the total composition.
• Suitable pharmaceutically excipients which are soluble in the second substance or in the liquid crystalhne phase are e.g. sorbitan esters such as, e.g., polysorbates; and macrogols.
• solvents like e.g., water, glycerol, alcohols like e.g. ethanol and isopropylalcohol are examples of a liquid medium and are not intended to be examples of soluble pharmaceutically acceptable excipients.
• a composition according to the invention may optionally comprise a liquid medium.
• a liquid medium may be present in compositions in which a liquid crystalhne phase between the second substance and the hquid medium has been generated as well as in the so- called precursor compositions in which the liquid crystalhne phase has not been generated in the composition but is to be formed upon administration of the composition to a mammal.
• any hquid medium present in the precursor composition may or may not take part in the formation of a liquid crystalhne phase together with any moisture from the apphcation site or body fluid present at or on the apphcation site.
• a liquid medium is present in a concentration of at least about 0.5% by weight, such as at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25% or at least about 30% by weight, calculated on the total composition.
• a liquid medium normally is present in a concentration of 20%- 50% such as, e.g., about 25%-35% about 25%-30% or about 30%-40% by weight, calculated on the total composition.
• a liquid medium is present in a concentration of 25%- 40% such as, e.g, about 25-35%, about 30%-40% or 27%-37% by weight, calculated on the total composition.
• Generally preferred liquid media which participate in the formation of a liquid crystalhne phase are water, glycerol, alcohols like e.g. ethanol and mixtures thereof.
• active substance is intended to mean any biologically or pharmacologically active substance or antigen-comprising material; the term includes drug substances which have utility in the treatment or prevention of diseases or disorders affecting animals or humans, or in the regulation of any animal or human physiological condition and it also includes any biologically active compound or composition which, when administered in an effective amount, has an effect on living cells or organisms.
• antiherpes virus agents which have been or are developed for the treatment of herpes virus infections [herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV)].
• the antiherpes virus agents include antiviral drugs and prodrugs thereof, such as nucleosides, nucleoside analogues, phosphorylated nucleosides (nucleotides), nucleotide analogues and salts, complexes and prodrugs thereof; e.g.
• guanosine analogues deoxyguanosine analogues, guanine, guanine analogues, thymidine analogues, uracil analogues and adenine analogues.
• Especially interesting antiherpes virus agent for use either alone or in combination in a composition according to the present invention are selected from acyclovir, famciclovir, deciclovir, penciclovir, zidovudin, ganciclovir, didanosin, zalcitabin, valaciclovir, sorivudine, lobucavir, brivudine, cidofovir, n- docosanol, ISIS-2922, and prodrugs and analogues thereof. Details concerning active substances suitable for use in connection with the present invention as well as a description of other interesting active substances are given below.
• an antiviral drug such as a nucleoside or a nucleoside analogue, e.g. selected from acyclovir, famciclovir, deciclovir, penciclovir, zidovudin, ganciclovir, didanosin, zalcitabin, valaciclovir, sorivudine, lobucavir, brivudine, cidofovir, n-docosanol, ISIS-2922 and salts and prodrugs thereof.
• a nucleoside or a nucleoside analogue e.g. selected from acyclovir, famciclovir, deciclovir, penciclovir, zidovudin, ganciclovir, didanosin, zalcitabin, valaciclovir, sorivudine, lobucavir, brivudine, cidofovir, n-docosanol, ISIS-2922 and salts and prodrugs
• interferons and variants thereof including alpha interferon, beta interferon, and gamma interferon,
• a listing of substances of potential interest comprises substances of the following groups:
• anti-inflammatory drugs such as, e.g., ibuprofen, indomethacin, naproxen, diclofenac, tolfenamic acid, piroxicam, and the hke;
• narcotic antagonists such as, e.g., naloxone, nalorphine, and the like; antiparkinsonism agents such as, e.g., bromocriptine, biperidin, benzhexol, benztropine, and the like;
• antidepressants such as, e.g., imipramine, nortriptyline, pritiptylene, and the like;
• antibiotic agents such as, e.g., clindamycin, erythromycin, fusidic acid, gentamicin, mupirocien, amfomycin, neomycin, metronidazole, silver sulphadiazine, sulphamethizole, bacitracin, framycetin, polymycin B, acitromycin, and the like;
• antifungal agents such as, e.g., miconazol, ketoconazole, clotrimazole, amphotericin B, nystatin, mepyramin, econazol, fluconazol, flucytocine, griseofulvin, bifonazole, amorolfine, mycostatin, itraconazole, terbenafine, terconazole, tolnaftate, and the hke;
• antifungal agents such as, e.g., miconazol, ketoconazole, clotrimazole, amphotericin B, nystatin, mepyramin, econazol, fluconazol, flucytocine, griseofulvin, bifonazole, amorolfine, mycostatin, itraconazole, terbenafine, terconazole, tolnaftate, and the hke;
• antimicrobial agents such as, e.g., metronidazole, tetracyclines, oxytetracycline, and the like;
• antiemetics such as, e.g., metoclopramide, droperidol, haloperidol, promethazine, and the like;
• antihistamines such as, e.g., chlorpheniramine, terfenadine, triprolidine, and the like;
• antimigraine agents such as, e.g., dihydroergotamine, ergotamine, pizotyhne, and the like;
• vasodilators such as, e.g., nifedipine, diltiazem, and the like;
• antianginals such as, e.g., glyceryl nitrate, isosorbide denitrate, molsidomine, verapamil, and the hke;
• calcium channel blockers such as, e.g., verapamil, nifedipine, diltiazem, nicardipine, and the like;
• hormonal agents such as, e.g., estradiol, estron, estriol, polyestradiol, polyestriol, dienestrol, diethylstilbestrol, progesterone, dihydroergosterone, cyproterone, danazol, testosterone, and the like;
• contraceptive agents such as, e.g., ethynyl estradiol, lynestrenol, etynodiol, norethisterone, mestranol, norgestrel, levonorgestrel, desogestrel, medroxyprogesterone, and the like;
• antithrombotic agents such as, e.g., heparin, warfarin, and the like;
• diuretics such as, e.g., hydrochlorothiazide, flunarizine, minoxidil, and the like; antihypertensive agents such as, e.g., propanolol, metoprolol, clonidine, pindolol, and the like;
• corticosteroids such as, e.g., beclomethasone, betamethasone, betamethasone-17-valerate, betamethasone-dipropionate, clobetasol, clobetasol-17-butyrate, clobetasol-propionate, desonide, desoxymethasone, dexamethasone, diflucortolone, flumethasone, flumethasone-pivalate, fluocinolone acetonide, fluocinonide, hydrocortisone, hydrocortisone- 17-butyrate, hydrocortisone- buteprate, methylprednisolone, triamcinolone acetonide, budesonide, halcinonide, fluprednide acetate, alklometasone-dipropionate, fluocortolone, fluticason-propionate, mometasone-furate, desoxymethasone, diflurason-
• dermatological agents such as, e.g., nitrofurantoin, dithranol, choquinol, hydroxyquinoline, isotretionin, methoxsalen, methotrexate, tretionin, trioxsalen, salicylic acid, penicillamine, and the like;
• steroids such as, e.g., estradiol, progesterone, norethindrone, levonorgestrol, ethynodiol, levenorgestrel, norgestimate, gestanin, desogestrel, 3-keton-desogestrel, demegestone, promethoestrol, testosterone, spironolactone, and esters thereof,
• nitro compounds such as, e.g., amyl nitrates, nitroglycerine and isosorbide nitrates,
• opioid compounds such as, e.g., morphine and morphine-like drugs such as buprenorphine, oxymorphone, hydromorphone, levorphanol, fentanyl and fentanyl derivatives and analogues,
• prostaglandins such as, e.g., a member of the PGA, PGB, PGE, or PGF series such as, e.g., misoprostol, dinoproston, carboprost or enaprostil,
• a benzamide such as, e.g., metoclopramide, scopolamine,
• a peptide such as, e.g., growth hormone releasing factors, growth factors (epidermal growth factor (EGF), nerve growth factor (NGF), TGF, PDGF, insulin growth factor (IGF), fibroblast growth factor (aFGF, bFGF, etc.), and the like), somatostatin, calcitonin, insulin, vasopressin, interferons, IL-2, urokinase, serratiopeptidase, superoxide dismutase (SOD), thyrotropin releasing hormone (TRH), luteinizing hormone releasing hormone (LH-RH), corticotrophin releasing hormone (CRF), growth hormone releasing hormone (GHRH), oxytocin, erythropoietin (EPO), colony stimulating factor (CSF), and the like,
• growth hormone releasing factors epidermal growth factor (EGF), nerve growth factor (NGF), TGF, PDGF, insulin growth factor (IGF),
• a xanthine such as, e.g., caffeine, theophylline, a catecholamine such as, e.g., ephedrine, salbutamol, terbutaline,
• a dihydropyridine such as, e.g., nifedipine
• a thiazide such as, e.g., hydrochlorotiazide, flunarizine,
• compositions of the invention may also comprise combinations of active substances, e.g. an active substance together with a potentiator therefor.
• any substance which has a therapeutic or prophylactic activity may be incorporated in the composition.
• the active substance of the composition of the invention is a substance which has a low solubihty in the liquid crystalhne phase such as, e.g., at the most about 20 mg/g at 20°C, at the most 15 mg/g at 20°C, e.g. at the most 10 mg/g at 20°C or lower, such as at the most 7 mg/g, 6.5 mg/g, 6 mg/g, 5..5 mg/g, 5 mg/g at 20°C. e.g. at the most 4 mg/g at 20°C or even at the most 3 mg/g or 2 mg/g or 1 mg/g at 20°C.
• a low solubihty in the liquid crystalhne phase such as, e.g., at the most about 20 mg/g at 20°C, at the most 15 mg/g at 20°C, e.g. at the most 10 mg/g at 20°C or lower, such as at the most 7 mg/g, 6.5 mg/g, 6 mg/g, 5..5 mg/
• the determination of the solubihty of the active substance in the hquid crystalhne phase of the composition is, of course, performed on the liquid crystalhne phase as formed. In practice, this means that when the composition is one in which the hquid crystalhne phase has already been formed when the composition is apphed, the determination of the solubihty is performed on the composition itself.
• the determination of the solubihty is suitably performed by microscopy to observe any crystals of the active substance.
• the determination of the concentration at which crystals are observed is performed after a period of at least one week after preparation of the composition or the liquid crystalhne phase, or when equilibrium has been established. Normally, a series of tests with varying concentrations is performed to determine the concentration above which crystals are found.
• the liquid crystalhne phase used as a reference in the solubility determination is a liquid crystalhne phase imitating the liquid crystalhne phase which will be formed when the composition absorbs liquid from the site of apphcation.
• This reference liquid crystalhne phase is made up with water (as representing the liquid absorbed) in such an amount that the reference liquid crystalhne phase is the same type of liquid crystalhne phase as is generated from the precursor composition.
• the concentrations will, of course, be higher.
• the pH of the liquid crystalhne phase of the composition is in the range of 3.0-9.5 such as, e.g., 3.2-9.3, 3.4-9.1 or 3.6-9. At lower pH values, the composition may be irritating to the skin or mucosa on which it is apphed; at higher pH values, the composition may be irritating and may also directly be etching.
• the pH of the hquid crystalhne phase is determined by a method involving dispersing e.g. 10% of the liquid crystalhne phase (containing the active substance and any excipients) in distilled water and measuring the pH in the water phase, equilibration between the liquid crystalhne phase and a water phase and measuring the pH of the water phase at 20°C (e.g. using a rotomat for 2 hours).
• the pH of the liquid crystaUine phase may be measured by means of an suitable pH electrode (see the Examples).
• the upper hmit of the pH of the liquid crystalhne phase is 8. It is also preferred that the lower hmit of the pH is 3.0 or higher, and thus, interesting pH ranges for the liquid crystalhne phase are pH 3.0-8, such as, e.g., 3.1-8, 3.2-8, 3.3-8, 3.4-8, 3.5-8, 3.6-8, 3.7- 8, 3.8-8, 3.9-8, 4.0-8, 4.1-8, 4.2-8, 4.3-8, 4.5-8, 4.75-8, or 5.0-8.
• the solubihty of the active substance in water is very low, at the most 10 mg/g at 20°C and at a pH substantially identical to the pH of the liquid crystalhne phase, determined as described herein. While a pH range is stated above for the liquid crystalhne phase, it will be understood that by the water solubihty of the active substance is meant the water solubihty at the relevant pH, which is a pH substantially identical to the pH which will prevail in the composition, in other words, the pH of the liquid crystalhne phase, this pH being determined as described herein.
• the water is adjusted to substantially the pH of the liquid crystalhne phase by using a suitable buffer system when determining the solubihty of the active substance.
• This buffer system should of course be so selected that, apart from the pH adjustment, it has substantially no influence on the solubihty of the active substance in the buffered water.
• composition according to the present invention is very valuable in that it can provide a high release of active substances of very low water solubihty, such as a solubihty of at the most 7 mg/g, such as at the most 5 mg/g at 20°C and at a pH substantially identical to the pH of the hquid crystaUine phase, determined as described herein.
• solubihty such as a solubihty of at the most 7 mg/g, such as at the most 5 mg/g at 20°C and at a pH substantially identical to the pH of the hquid crystaUine phase, determined as described herein.
• the active substance has an minimum aqueous solubihty 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 3.0-9.5 such as, e.g. having a pH between 3.2 and 9.3, between 3.4 and 9.1 or between 3.6 and 9.0.
• the determination of the minimum aqueous solubility is performed by use of suitable buffers which are capable of maintaining the pH at the desired value and measures are taken to ensure that equilibrium is obtained between the undissolved and dissolved active substance, i.e. by employment of ultrasonic treatment and/or stirring for a well-defined time period.
• compositions according to the invention contains one or more antiherpes virus agent(s) as an active substance.
• antiherpes virus agents are mentioned above and acyclovir is of particular importance.
• Acyclovir (9-[2- hydroxyethoxy)methyl]-guanine, an acyclic analogue to the natural nucleoside 2'- deoxyguanosine, is a widely used agent in the treatment of herpes virus infections.
• Compositions for oral, topical and intravenous administration are available. The delivery characteristics of acyclovir following administration by these routes are, however, far from being optimal probably due to the poor aqueous solubihty and/or low lipophilicity of acyclovir.
• the solubility of acyclovir in water is about 1.5 mg/ml at 22°C and the partition coefficient (P) between octanol and 0.02 M phosphate buffer pH 7.4 (21°C) is about 0.03.
• P partition coefficient between octanol and 0.02 M phosphate buffer pH 7.4
• the active substance of low solubihty is normally present in the composition in an amount in the range of from 1-20% by weight, usually 1-15% by weight.
• a preferred composition according to the invention is a composition in which the active substance is acyclovir, the second substance is a the fatty acid ester and a liquid medium is present in the composition.
• the fatty acid ester is preferably a glycerylmonooleate product having a glycerylmonooleate content of at least 88% such as, e.g., at least about 89, 90%, 91% or 92% by weight and a content of saturated monoglycerides of at the most 4% by weight such as, e.g., at the most about 2% by weight.
• Preferred acyclovir-containing compositions according to the invention normally has a weight ratio between the glycerylmonooleate and the hquid medium is in the range between 1:0.3 and 1:2 such as between 1:0.5 and 1:1.5 such as, e.g. 1:1.
• the weight ratio between the combination of the glycerylmonooleate and the structurant, and the hquid medium is generaUy in the range between 60:40 and 75:25 such as between 63:37 and 73:27.
• preferred structurants are Epikuron 200 and Vitamin E TPGS and combinations thereof and the weight ratio between Epikuron and Vitamin E TPGS may be between about 1:0.5 and 1:2 such as, e.g., between 1:0.75 and 1:1.5 such as, e.g., about 1:1.
• Another preferred acyclovir-containing composition comprises glycerylmonooleate as second substance, a mixture of Epikuron 200 and Vitamin E TPGS as structurants, and water as a hquid medium and the concentration of Epikuron 200 is generally in a range of from l%-25% by weight, the concentration of Vitamin E TPGS is in a range of from l%-25% by weight, and the concentration of water is in a range of from 20%-40% by weight based on the total composition.
• An interesting acyclovir-containing composition may also be presented as a precursor composition, wherein the weight ratio between fatty acid ester and any liquid medium present in the composition is between 50:50 and 100:0 such as between 60:40 and 99:1, between 70:30 and 90:10.
• the weight ratio between the sum of the glycerylmonooleate and any structurant(s), and. any liquid medium present in the composition is between 90:10 and 99:0.5, such as between 90:10 and 99:1.
• a liquid medium like e.g. water or glycerol, or a mixture of water and glycerol may be present.
• the liquid medium is water containing glycerol
• the glycerohwater ratio may be up to about 2.5:1 by weight, such as up to corresponding to a glycerohwater ratio of 1.5:2 such as, e.g., a ratio of about 1:1, 0.5:1, or 0.25:1.
• compositions comprising glycerylmonooleate, phosphatidylcholine (or Vitamin E TPGS) and, optionally, water and the weight ratio between the content of phosphatidylchohne (or Vitamin E TPGS) and glycerylmonooleate is at the most 1, such as e.g. 1:1, 1:2 or 1:4.
• water may be present in a concentration of at the most 40% w/w based on the total composition.
• the active substances of relatively low solubihty as discussed above are of particular importance for use in compositions according to the invention, the invention is not hmited to such active substances.
• the active substance can in principle be any active substance irrespective of its solubihty.
• the active substance may have any degree of hpophihcity.
• the active substance is one which has a hpophihcity 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 partition coefficient between octanol and 0.05M phosphate buffer, pH 7, at 20°C, in some a partition coefficient of at the most 10 or even at the most 1 or at the most 0.75, 0.5, 0.1, 0.075, 0.05 or 0.04.
• the hpophihcity may be expressed as the partition coefficient between octanol and an appropriate buffer having a pH corresponding either to the pH of the hquid crystalline phase or to the pH at which the active substance has its minimum solubihty. In such cases, the values mentioned above are also valid.
• the active substance must have balanced properties with respect to aqueous solubihty and partition coefficient.
• the vehicle in which the active substance is located is of importance.
• the affinity of the active substance to the vehicle compared with that of the active substance to the skin or the rate-limiting barrier of the skin must be of a less order of magnitude as the active substance otherwise would predominantly would be maintained in the vehicle and only slowly be released from the vehicle and penetrate the skin and thus enable the active substance to reach the target for the disease.
• Initial studies performed by the inventor show that vehicles on which compositions according to the invention are based readily releases the active substances tested (e.g. acyclovir) so that the active substances are available for penetration, i.e. a balanced affinity (vehicle/skin) has been obtained in these compositions.
• a composition with improved release properties and which sticks better to the skin can improve the treatment when compared to prior art compositions such as Zovir® cream or Zovirax® cream.
• the object of the present invention has therefore inter aha been to develop a bioadhesive composition containing e.g. acyclovir or other antiherpes virus agents with improved release properties so that fewer daily applications are needed to produce the same therapeutic effect (bioequivalence) or even improve the therapeutic effect.
• Cubic hquid crystalhne phases are obtained in these compositions as evidence by polarized hght.
• the results indicate that acyclovir in the concentration range investigated does not ruin the cubic lattice, and that acyclovir probably is inert in the cubic system.
• the distribution of the drug crystals in the cubic hquid crystalhne phase appears as a homogeneous distribution (observed by microscopy).
• the cubic hquid crystalhne phase without drug is transparent and has a relatively high viscosity. It is cosmetically appealing. When acyclovir is added, the viscosity is increased with the concentration, especially for the micronized quality. When the crystalhne quality is added, the composition becomes greyish white. When the cubic liquid crystalhne phase is applied to human skin it “melts” (gets softer) and penetrates the skin.
• compositions wherein the GMO has been substituted by a structurant and/or by a pharmaceutically acceptable excipient The release of acyclovir from such compositions is described in the Experiments. The results show that the structurant and/or certain pharmaceutically acceptable excipients (as defined in the claims) do not significantly influence the release rate of acyclovir in a negative manner. - -
• Zovir® and Zovirax® cream containing 5% w/w acyclovir are presently the drugs of choice for the treatment of herpes simplex.
• acyclovir As mentioned above, Zovir® and Zovirax® cream containing 5% w/w acyclovir are presently the drugs of choice for the treatment of herpes simplex.
• the release rate of acyclovir from Zovir® cream and a cubic liquid crystalhne phase GMO/water 65/35 % w/w
• Example 16 Comparing the rate constants it is seen that the release rate of acyclovir is about 5-6 times faster from the cubic liquid crystaUine phase than from the Zovir® cream. Poor release properties of the Zovir® cream are most likely one of the reasons for its suboptimum therapeutic effect.
• the improved release properties from the cubic liquid crystalhne phase must therefore be seen as a very promising result.
• compositions according to the invention with respect to releasing the active substance from the hquid crystaUine phase can be adequately expressed by the slope of the cumulative release in ⁇ g as a function of the square root of the release time in hours in the release experiment defined in connection with Fig. 13 (in which the concentration of the substance is 5%).
• the slope is at least 50, more preferred at least 100.
• An expression of better performance is a slope of at least 200, such as at least 300, or at least 500 or even at least 700 or at least 900.
• at least 25%, such as at least 50%, by weight of the active substance present in the composition constitutes a proportion which is present above the saturation concentration at 20°C.
• Very valuable compositions according to the invention are compositions, wherein at least 75%, such as at least 90% or even at least 95% or at least 98% by weight of the active substance present in the - composition constitutes a proportion which is present above the saturation concentration at 20°C.
• the concentration of the active substance in the composition will depend on the condition to be treated or prevented and the desired or necessary administration frequency.
• the concentration of the active substance in a pharmaceutical composition depends on the nature of the second compound in question, its potency, the severity of the disease to be prevented or treated, and the age and condition of the patient.
• Methods applicable to selecting relevant concentrations of the active substance in the pharmaceutical composition are well known to a person skilled in the art and may be performed according to established guidelines for good clinical practice (GCP) or Investigational New Drug Exemption ("IND") regulations as described in e.g. Drug Applications, Nordic Guidelines, NLN Publication No. 12, Nordic Council on Medicines, Uppsala 1983 and Clinical Trials of Drugs, Nordic Guidehnes, NLN Publication No. 11, Nordic Counril on Medicines, Uppsala 1983, or CPMC/E.U. Guidehnes for Good Clinical
• the second substance especiaUy fatty acid esters can confer bioadhesiveness to the compositions.
• increased attention has been given to the possibility of using bioadhesive/mucoadhesive polymers for drug dehvery purposes. It is believed that several problems associated with conventional controUed release drug dehvery systems may be reduced or eliminated by using a bioadhesive/mucoadhesive drug dehvery system.
• bioadhesive drug dehvery systems are believed to be beneficial with respect to the following features: i) a bioadhesive drug dehvery system localizes a drug substance in a particular region, thereby improving and enhancing the bioavailability for drug substances which may have poor bioavailability in themselves,
• a bioadhesive drug delivery system leads to a relatively strong interaction between a bioadhesive substance and a mucosa; such an interaction contributes to an increasing contact time between the drug dehvery system and the tissue in question and permits localization of the drug dehvery system to a specific site,
• a bioadhesive drug dehvery system is contemplated to prolong dehvery of drug substances in almost any non-parenteral route
• a bioadhesive drug dehvery system can be locahzed on a specific site with the purpose of local therapy e.g. treatment of local fungal diseases, permeability modification, protease and other enzyme inhibition, and/or modulation of immunologic expression,
• a bioadhesive drug dehvery system may be targeted to specific diseased tissues
• a bioadhesive drug dehvery system may be employed in those cases where the conventional approach to controUed release drug dehvery is unsuitable, i.e. for certain drug substances or classes of drug substances which are not adequately absorbed.
• compositions according to the present invention are compositions in which the second substance like a fatty acid ester or combination of fatty acid esters present in the composition complies with the requirements of bioadhesion defined herein when tested for bioadhesion in an in vivo model or any other bioadhesivity model as given in the experimental section herein.
• compositions in which the second substance hke e.g. a fatty acid ester or combination of fatty acid esters optionally in combination with the structurant, when tested in a bioadhesive test system, comprising:
• step v) leaving the resulting sample from step v) for 10 minutes in said cell to allow the sample to interact with glycoproteins of the jejunum
• 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 85% w/w and more preferably at least 90% w/w.
• compositions as defined further above which, when tested in the jejunum test system defined in claim above, result in a residual amount of at least 40% w/w of the second substance such as a fatty acid ester or combination of fatty acid esters or at least 40% w/w of the active substance.
• a measure of the bioadhesivity of a composition itself is that it complies with the requirements for bioadhesion defined herein when tested for bioadhesion in the in vivo model described herein involving testing the rinsing off ability from skin.
• the biopharmaceutical properties of a composition according to the invention are not significantly changed by adding a structurant or a pharmaceutically-acceptable excipient.
• the score obtained in the test for bioadhesion is substantially of the same order of magnitude as would have been obtained for a comparative composition wherein the structurant(s) and/or pharmaceutically acceptable excipient has(have) been replaced with the same amount by weight of said second substance.
• an active substance does not significantly influence the bioadhesive properties of a vehicle provided that the concentration of the active or protective substance is relatively low such as at the most about 10-15% w/w or at the most about 8- 10% w/w.
• the kind of active substance (structure, molecular weight, size, physico-chemical properties, loading, pKa, solubihty, etc.) wiU of course be responsible for the maximal concentration which can be incorporated in the vehicle without significantly affecting the bioadhesive properties of the composition.
• the active substance locates in the liquid crystalline phase of the fatty acid ester and most likely the solubility of the active substance in this phase has impact on the bioadhesive properties as well as on the release properties of the composition.
• the apphcation is intended for skin or mucosa.
• Other applications may of course also be relevant such as, e.g., apphcation on dentures, prostheses and apphcation to body cavities such as the oral cavity.
• the mucosa is preferably selected from oral, nasal, aural, lung, rectal, vaginal, and gastrointestinal mucosa.
• a bioadhesive composition for administration according to the invention may in special cases also be in the form of a multiple unit composition, in the form of, e.g., a powder.
• a multiple unit composition may be administered to skin or mucosa, preferably the mucosa is selected from oral, nasal, rectal, aural, vaginal, lung, and gastrointestinal mucosa. Most preferred is a bioadhesive composition intended for administration to the gastrointestinal tract.
• Bioadhesive compositions according to the invention for application on skin and especially to wounds may in certain cases comprise a polysaccharide in a concentration of at least 15% w/w, calculated on the total weight of the composition.
• the polysaccharide is preferably selected from the group consisting of carmelose, chitosan, pectins, xanthan gums, carrageenans, locust bean gum, acacia gum, gelatins, alginates, and dextrans, and salts thereof.
• the compositions are easy to apply on the wound and are believed to be able to extract water from the wound and thereby drying the wound.
• the bioadhesive compositions for use according to the invention may comprise pharmaceutically or cosmeticaUy acceptable excipients or additives normally used in pharmaceutical compositions.
• the bioadhesive compositions may be in form of, e.g., a spray, a solution, a dispersion, a suspension, an emulsion, powders, gels including hydrogels, pastes, ointments, creams, drenches, dehvery devices, suppositories, enemas, implants, aerosols, microcapsules, microspheres, nanoparticles, liposomes, dressings, bandages, plasters, tooth paste, dental care compositions, and in other suitable form.
• a spray e.g., a spray, a solution, a dispersion, a suspension, an emulsion, powders, gels including hydrogels, pastes, ointments, creams, drenches, dehvery devices, suppositories, enemas, implants, aerosols, microcapsules, microspheres, nanoparticles, liposomes, dressings, bandages, plasters, tooth paste, dental
• bioadhesive compositions may be formulated according to conventional pharmaceutical practice, see, e.g., "Remington's Pharmaceutical Sciences” and “Encyclopedia of Pharmaceutical Technology", edited by Swarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988.
• compositions for use according to the invention may be, for example,
• inert dfluents or fiUers such as sucrose, sorbitol, sugar, mannitol, microcrystalline ceUulose, carboxymethylceUulose sodium, methylceUulose, hydroxypropyl methylceUulose, ethylceUulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate; and
• lubricating agents including ghdants and antiadhesives, for example, magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable ofls or talc.
• pharmaceuticaUy acceptable excipients can be colorants, flavouring agents, plasticizers, humectants, buffering agents, solubilizing agents, release modulating agents, etc.
• suitable compositions for use according to the invention include suppositories (emulsion or suspension type), solutions, enemas, and rectal gelatin capsules (solutions or suspensions).
• Appropriate pharmaceutically acceptable suppository bases include cocoa butter, esterified fatty acids, glycerinated gelatin, and various water-soluble or dispersible bases hke polyethylene gl cols and polyoxyethylene sorbitan fatty acid esters.
• Various additives hke, e.g., enhancers or surfactants may be incorporated.
• nasal sprays and aerosols for inhalation are suitable compositions for use according to the invention.
• the active ingredients are dissolved or dispersed in a suitable vehicle.
• suitable vehicles and excipients and optionally other pharmaceutically acceptable materials present in the composition such as diluents, enhancers, flavouring agents, preservatives etc. are all selected in accordance with conventional pharmaceutical practice in a manner understood by the persons skilled in the art of formulating pharmaceuticals.
• compositions for use according to the invention may contain conventionally non-toxic pharmaceutically acceptable carriers and excipients including microspheres and liposomes.
• the formulations include creams, ointments, lotions, liniments, gels, hydrogels, solutions, suspensions, sticks, sprays, pastes, dressings, bandages, plasters, tooth paste, dental care compositions, and the like.
• the pharmaceutically acceptable carriers or excipients may include emulsifying agents, stabilizing agents, antioxidants, buffering agents, preservatives, humectants, penetration enhancers, chelating agents, gelforming agents, ointment bases, perfumes and skin protective agents.
• emulsifying agents are naturally occurring gums, e.g. gum acacia or gum tragacanth, naturally occurring phosphatides, e.g. soybean lecithin and sorbitan monooleate derivatives.
• antioxidants examples include butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof, ⁇ -tocopherol and derivatives thereof, vitamin E, salts of sulphur dioxide, cysteine, citric acid, ascorbyl palmitate, butylhydroxytoluene, complexing agents, chelating agents, sodium pyrosulfite, EDTA and gallic acid esters.
• BHA butylated hydroxy anisole
• ⁇ -tocopherol and derivatives thereof examples include ascorbic acid and derivatives thereof, ⁇ -tocopherol and derivatives thereof, vitamin E, salts of sulphur dioxide, cysteine, citric acid, ascorbyl palmitate, butylhydroxytoluene, complexing agents, chelating agents, sodium pyrosulfite, EDTA and gallic acid esters.
• preservatives are parabens, such as methyl, ethyl, propyl p-hydroxybenzoate, butylparaben, isobutylparaben, isopropylparaben, potassium sorbate, sorbic acid, benzoic acid, methyl benzoate, phenoxyethanol, bronopol, bronidox, MDM hydantoin, iodopropynyl butylcarbamate, EDTA propyleneglycol (increases the solubility of preservatives) benzalconium chloride, benzylalcohol, chlorhexidine diacetate, chlorhexidine digluconate, chlorbutol, phenetanol, phenols (phenol, o-cresol, p-cresol, chlorcresol, tricresol), alkanols (chlorbutanol, phenetanol), sorbic acid, and mercuri-compounds like e.
• humectants examples include glycerin, propylene glycol, sorbitol and urea.
• Suitable release modulating agents for use according to the invention are glycerol, sesame oil, soybean oil, lecithin and cholesterol.
• penetration enhancers are oleic acid, propylene glycol, DMSO, triethanolamine, N,N-dimethylacetamide r N,N-dimethylformamide, 2-pyrrolidone and derivatives thereof, - tetrahydrofuryl alcohol and Azone.
• chelating agents examples include sodium EDTA, citric acid and phosphoric acid.
• excipients for use in compositions for use according to the invention are edible oils like almond oil, castor oil, cacao butter, coconut oil, corn oil, cottonseed oil, linseed oil, olive oil, palm oil, peanut oil, poppyseed oil, rapeseed oil, sesame oil, soybean oil, sunflower oil, and teaseed oil; and of polymers such as carmelose, sodium carmelose, hydroxypropylmethylcellulose, hydroxyethylceUulose, hydroxypropylcellulose, chitosane, pectin, xanthan gum, carrageenan, locust bean gum, acacia gum, gelatin, and alginates, and solvents such as, e.g., glycerol, ethanol, propylene glycol, polyethylene glycols such as PEG 200 and PEG 400, Pluronic, polysorbate, and ethylene glycol.
• edible oils like almond oil, castor oil, cacao butter, coconut oil, corn oil, cottonseed oil, l
• ointment bases are beeswax, paraffin, cetyl palmitate, vegetable oils, sorbitan esters of fatty acids (Span), Carbopol, polyethylene glycols, and condensation products between sorbitan esters of fatty acids and ethylene oxide, e.g. polyoxyethylene sorbitan monooleate (Tween).
• composition according to the invention is one in which the antiviral substance is acyclovir.
• examples of important embodiments hereof and of other compositions according to the invention containing nucleosides of low solubUity as defined herein are claimed in the claims are described in detaU in the Examples.
• compositions and conditions to be fulfiUed for the individual components in the compositions are claimed in the claims and described in the Examples.
• the invention also relates to methods for preparing the compositions according to the invention. Details concerning the preparation are given in the Examples herein. Furthermore, the invention also relates to a method for administering an active substance to e.g. a human, the method comprising administering to the human in need thereof a therapeutically and/or prophylactically effective amount of the active substance in a pharmaceutical composition according to the invention.
• composition aspects of the invention will be the same as or analogous to the details and particulars concerning the other aspects of the invention and the method aspects discussed above, and this means that wherever appropriate, the statements above concerning a pharmaceutical composition, a second-substance, a structurant, a liquid medium and a pharmaceutically acceptable excipient, as well as improved properties and uses apply mutatis mutandis to all aspects of the invention.
• Glycerylmonooleate (monoolein), manufactured by Grindsted Products A/S, Denmark
• Fatty acid composition Oleic acid 92% Linoleic 6% Saturated (C 16 /C 18 ) 2%
• Antioxidants and synergists added Ascorbyl palmitate max. 200 ppm ⁇ -Tocopherol max. 200 ppm Citric acid max. 100 ppm
• GMO-90 indicates that the above-mentioned glycerol monooleate product is employed, except where otherwise stated.
• glycerol monooleate Another quality of glycerol monooleate has been employed in some of the following examples, namely RYLO® MG19 (with a content of about 90% GMO) manufactured by Danisco Ingredients, Denmark
• the product employed in the examples described herein had the following composition of fatty acid monoesters:
• Glycerylmonooleate about 84% w/w Glycerylmonolinoleate about 7% w/w
• Glyceryl monostearate about 4% w/w
• GMO 84 indicates that this glycerol monooleate product is employed.
• Glycerylmonolinoleate (Dimodan® LS), manufactured by Grindsted Products A/S; the product used has a total content of fatty acid monoesters of at least about 90% such as about 96% w/w.
• the product employed in the examples described herein had the following composition of fatty acid monoesters:
• Glyceryl monopalmitate about 6% w/w
• Glyceryl monostearate about 6% w/w
• Glycerylmonooleate about 22% w/w
• Glycerylmonolinoleate about 63% w/w
• Phosphatidylchohne (Epikuron available from Lucas Meyer, Hamburg, Germany):
• Lipoid SlOO or S75 purified soya phosphatidylcholine available from Lipoid GmbH, Germany
• EPIKURON 200 is a purified phosphatidylcholine of soybean origin.
• composition The product consists of phosphatidylchohne, a small amount of lyso-phosphatidylcholine and other phospholipids.
• phosphatidylchohne min. 92% lyso-phosphatidylcholine max. 3% other phosphohpids max. 2%
• fatty acids including: palmitic acid/stearic acid 16-22% oleic acid 8-12% linoleic acid 62-66% linolenic acid 6-8%
• EPIKURON 145 V is a fractionated, wax-like soybean lecithin with enriched content of phosphatidylcholine for the use in pharmaceutical industry.
• Composition Mixture polar (phospho- and glyco-) lipids and a small amount of carbohydrates.
• phosphatidylcholine min. 45% phosphatidylethanolamine min. 10% phosphatidylinositol max. 3% phosphatidic acid max. 3% lyso-phosphatidylcholine max. 4% other phosphohpids max. 18% glycolipids max. 15%
• fatty acids including: palmitic acid/stearic acid 18-22% oleic acid 6-10% linoleic acid 62-66% linolenic acid 6-8%
• Acyclovir (crystalhne) available from Chemo Iberica, Spain, e.g. a quality where 90-100% of the crystals have a particle size of less than 100 ⁇ m
• Acyclovir (micronized) available from Chemo Iberica, Spain, e.g. a quahty where 100% of the particles have a particle size under 24 ⁇ m and not less than 90% under 12 ⁇ m - -
• Ethanol available from Danisco A/S, Denmark complies with the DLS standard (98.8-100% w/w ethanol) Sesame oil available from Nomeco, Denmark
• Vitamin E TPGS (d- ⁇ -tocopherylpolyethyleneglycol 1000 succinate) available from Kodak
• Coulter Multisizer II (Coulter), Malvern 2600 droplet and particle size analyse (for the determination of particle size distribution).
• test system for bioadhesion is a modified system of a method described by Ranga Rao & Buri (Int. J. Pharm. 1989, 52, 265-270).
• jejunums were stored up to 3 months before use (when performing the test described below it was found that the use of fresh jejunum or, alternatively, jejunum which had been frozen for up to 3 months gave reproducible and significantly similar results). Before testing, the segment of jejunum was gently thawed out.
• the segment of the jejunum was cut longitudinally. It was placed on a stainless steel support (a tube of 2 cm in diameter and cut longitudinaUy at an axis paraUel 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 Examples the angle apphed is denoted "angle" in a cylindrical cell thermostated at 37°C.
• a schematic Ulustration of the ceU is shown in Fig. 1. The relative humidity in the thermostated cell was kept at about 100%.
• the jejunum was then flushed with a medium of 0.02M isotonic phosphate buffer solution (pH 6.5, 37°C) for 2 or 5 minutes (in the foUowing denoted “initial rinsing period”) at a flow rate of 5 or 10 ml/min (in the following denoted "initial rinsing flow”), respectively, using a peristaltic pump to equUibrate the jejunum with the buffer and to rinse off loose mucosa.
• the support was positioned at a horizontal position and after apphcation the position was changed to the initial position of -21°.
• An accurately weighted amount of the sample to be tested for bioadhesive properties (about 50-150 mg) was placed evenly on the mucosa of the jejunum (about 0.8 x 6 cm).
• About 1 ml of the buffer solution was carefully dropped evenly on the sample apphed to ensure formation of such a hquid crystalhne phase, if possible (in the case of monoolein, the liquid crystalhne phase may be the cubic, hexagonal, reverse hexagonal, micellar, reverse micellar, or lamellar phase).
• 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 apphcation on the rabbit jejunum.
• the segments were left for 5-20 minutes such as, e.g., 10 minutes in the cell allowing the sample to interact with the glycoproteins of the jejunum and to prevent drying of the mucus.
• the segments were flushed evenly with the isotonic 0.02M phosphate buffer solution (pH 6.5, 37°C) for 15-60 minutes such as, e.g., 30 minutes at a flow rate of 5-15 ml/min such as 10 ml/min (in the Examples denoted "flow rate").
• the tip of the tube carrying the buffer solution was placed 3-4 mm above the jejunum to ensure an even liquid flow over the mucosa.
• the washings were collected into a beaker.
• the amount of bioadhesive component remaining on the jejunum was calculated either by measuring the amount of sample in the beaker or by measuring the amount of sample remaining in the jejunum by means of a suitable analysis method, e.g. HPLC.
• Amount apphed about 50-150 mg (tests have shown that a variation in the amount applied within a range of from about 25 mg to about 225 mg was without significant influence on the results obtained)
• the method allows rinsing of the sample apphed on the jejunum by an aqueous medium, thus allowing a liquid crystalline phase to be formed.
• the method also permits apphcation of fluid samples and pellets. Determination of the bioadhesiveness of a test sample
• the substance(s) is/are considered as bioadhesive if the residual amount is at least about 60% w/w such as at least about 65% w/w, about 70% w/w, about 75% w/w, about 80% w/w, about 85% w/w, 90% w/w, or about 95% w/w.
• the test sample is a composition comprising a combination of a second substance like e.g. a fatty acid ester and an active substance and, optionally, other substances like structurant and/or excipients
• the composition is considered bioadhesive if the residual amount (of the second substance like e.g. the fatty acid ester, or the active substance) is at least about 40% w/w such as at least about 45% w/w, about 50% w/w, 55% w/w, 60% w/w, 65% w/w, 70% w/w, 75% w/w, or 80% w/w.
• evaluation of the bioadhesive properties of a substance may also be performed by use of the test system and test conditions described above but modified with respect to type of membrane, amount apphed of test sample, test angle, flow rate, medium, etc.
• tests have been performed in order to evaluate the influence of different membranes on the test results.
• the foUowing results were obtained using the above-mentioned test conditions (angle: -21°, flow rate: 10 ml/min, and flow period: 30 min) and applying GMO on the membrane:
• test system for bioadhesion is a modified system of a method described by Tobyn, M., J. Johnson & S. Gibson (in "Use of a TA.XT2 Texture Analyser in Mucoadhesive Research", International LABMATE, 1992, XVTI (issue VI), 35-38).
• the test system involves measuring the tensile force required to break an adhesive bond formed between a model membrane and a test sample (i.e. the sample which is tested for its bioadhesive properties).
• the test apparatus employed in the foUowing is a TA.XT2 Texture analyser (Stable Micro System Ltd., Haslemere, UK) (Fig. 2) equipped with a 5 kg load cell interfaced with an IBM PC computer running XT-RA dimension software, DOS version.
• the test enables measuring the strength of adhesive bonding established by contacting a model membrane, i.e. in this case a pig intestine segment, and the test sample.
• An analogous test apparatus may also be employed.
• the TA.XT2 Texture analyser apparatus is equipped with an instrument probe 1 (see Fig. 2) which is movable in a vertical direction at a variable rate. During the so-called withdrawal phase of the testing, the instrument probe is moved upwards with a constant rate until detachment occurs (see below). Furthermore, the apparatus is equipped with a stationary plate 2 on which a first holder 3 is placed. Before and during a test run, a model membrane 4 is fixed on this holder, e.g. by means of a cap or double adhesive tape or glue. The area exposed to the test may be determined by the area of the probe (preferred in this case) or by the area of the test samples (e.g. a coated cover glass), or by the area of a holder fixed to the probe. The accurate size of the exposed area is used in the calculation of the adhesive strength (see below).
• the membrane could be e.g. rabbit, rat or pig gastric mucosa; a segment of rabbit, rat or pig intestines, e.g. a segment of rabbit jejunum; a segment of rabbit or porcine buccal mucosa; or a segment of rabbit, rat or pig intestines from which the mucosal layer has been removed prior to testing; or skin from an animal (after removal of substantially all subcutaneous fat); or it could be artificiaUy or commercially avaUable mucin.
• the membrane could be e.g. rabbit, rat or pig gastric mucosa; a segment of rabbit, rat or pig intestines, e.g. a segment of rabbit jejunum; a segment of rabbit or porcine buccal mucosa; or a segment of rabbit, rat or pig intestines from which the mucosal layer has been removed prior to testing; or skin from an animal (after removal of substantially all subcutaneous fat); or it could
• duodenum, jejunum and the upper part of Ueum from freshly slaughtered pigs were used.
• the gut was stored on ice until it was washed with 0.9% w/w sodium chloride solution within 2 hours.
• the lumens were gently rinsed with the saline until the intestines were clean.
• the gut was cut into pieces of 3-4 cm and immediately frozen (-20°C).
• the intestines were stored up to 2 months before use. Before testing, the segments were gently thawed out.
• the gut segment was opened along the mesenteric border. Serosa and muscularis layers were removed by stripping with a pair of tweezers, taking care to maintain the integrity of the mucus layer.
• test sample about 25-500 mg
• the modified probe also aUows the necessary addition of an aqueous medium.
• the apparatus may be equipped with a second holder 5 on which another model membrane is fixed.
• the model membranes employed on the two holders are usuaUy 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.
• a tissue (porcine intestinal mucosa) of about 3 x 3 cm was fixed on the tissue holder 3 with the mucosa layer upside. Before apphcation of the tissue, a piece of gauze was placed directly on the tissue holder, and thereupon the tissue was placed. This precaution is made in order to stabilize the contact force. In order to moist the tissue and hydrate the sample, about 0.5 ml isotonic 0.05M phosphate buffer, pH 6.0, was added to the tissue. Such an addition also enables a cubic phase to be formed.
• the instrument probe with sample e.g.
• Cover glasses having a diameter of 13 mm were coated with the polymers under investigation by pipetting 100 ⁇ l of a 1% w/w solution of methanol or water in the center of the glass plate. After drying for 2 hours at 60°C in an oven, a thin polymer film remained.
• One cover glass was attached to the probe (diameter of 12.7 mm) with its non-coated side by means of double adhesive tape.
• Test runs are performed after the tissue has equUibrated 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.
• variations of the above-given method may be relevant, e.g. running the test in an aqueous medium or running the test at a temperature different from room temperature such as 37°C.
• test parameters may be varied, e.g. as follows:
• Test run temperature may be changed by employing a suitable temperature controlled oven such as a SMTC/04 from Stable Microsystems, Haslemere, UK.
• a suitable temperature controlled oven such as a SMTC/04 from Stable Microsystems, Haslemere, UK.
• test runs In order to test whether a test sample is bioadhesive, two test runs are performed:
• test run with the test sample apphed (result: work of adhesion WA S ),
• the test sample is considered bioadhesive if WA S /WA R x 100% is at least 30%, such as 35%, 40%, 45%, 50%, or 55%.
• a sample is graded to be a weak bioadhesive if the result is less than about 30%, a medium bioadhesive if the result is about 30%-50%, a strong bioadhesive if the result is at least 50%.
• Polycarbophil (NoveonTM AA-1, BF Goodrich, Hounslow, U.K.) is a high molecular weight poly(acrylic acid)copolymer loosely cross-linked with divinyl glycol. On account of its known exceUent mucoadhesive properties, this polymer serves as a reference.
• a polycarbophil gel is prepared by mixing polycarbophil with water or methanol (resulting concentration about 10-20 mg ml "1 ) and the mixture is allowed to hydrate at room temperature for 24 hours. The polymer solution is periodically stirred. The resulting gel is applied on a cover glass and tested as described above and the result obtained is used as a reference value for excellent bioadhesive substances.
• bioadhesive substances such as, e.g., chitosane, tragacanth, hydroxypropylmethylcellulose (HPMC), sodium alginate, hydroxypropylcellulose (HPC), karaya gum, carboxymethylcellulose (CMC), gelatin, pectin, acacia, PEG 6000, povidone, or DEAE-dextran (less bioadhesive than polycarbophil).
• a water soluble dye (Edicol Sunset YeUow, E 110, Amaranth E-123, or Brilliant Blue E 131) and/or a lipid soluble dye (Waxohne violet A FW (Maximex), Colur flavus insolubUis, DAK 63, or EdUake tartrazin NS) can be added to the test sample and mixed to form a homogeneous mixture.
• 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.
• test samples were applied in a uniform layer on an area of about 4 cm 2 of the skin of the hand or of the wrist.
• the test samples could be applied on dry skin as well as on moistened skin. In some cases, about 10 min before running the test, a small amount of water could be added to the test sample apphed.
• test sample on the skin was subjected to washings with water from a tip (flow rate corresponding to about 6-8 litres/minute and a temperature of about 35- 40°C). The washings were carried out for about 3 minutes. Then it is visually assessed in which degree the test sample is retained on the skin. The visual assessment is done by use of a scale graded from 1-5, where 5 represents total retainment of the test sample apphed on the skin and 1 represents no retainment of the test sample on the skin.
• test sample is evaluated to have bioadhesive properties in the present context if the result of the above-described test is at least 4.
• test described above has proved to be suitable when testing compositions for bioadhesiveness and the compositions in question have a relatively high viscosity which makes it difficult to apply the compositions to the rabbit jejunum model.
• a modification of the test described above excluding the addition of a water soluble dye has also proved suitable for testing compositions for bioadhesiveness.
• glycerylmonooleate or glycerylmonolinoleate was made by high-performance hquid chromatography (HPLC) using a Shimadzu LC-6A HPLC pump, a Shimadzu SPD-6A UV detector, a Shimadzu C-5A integrator and a Shimadzu SIL-6B autosampler.
• the column (25 cm x 4 mm i.d.) was packed with SupelcosU LC-18-DM and was eluted isocraticaUy at ambient temperature with a mobUe phase consisting of methanol:water:acetate buffer (pH 3.5) (840:120:40 v/v).
• a mobUe phase consisting of methanol:water:acetate buffer (pH 3.5) (840:120:40 v/v).
• pH 3.5 methanol:water:acetate buffer
• interference from other substances may occur, and then it may be necessary to make minor changes in the composition of the eluent.
• the size of a sample injected on the column was 20 ⁇ l and the flow rate was 1.2 ml/ml.
• the column effluent was monitored at 214 nm.
• the mucosa in question (with a second substance such as a fatty acid ester like e.g. glycerylmonooleate) is placed in 50.00 ml of methanol and shaken for 2 hours.
• the mixture is filtered through a 0.45 ⁇ m filter membrane (from Millipore 16555Q) and the filtrate is subjected to HPLC analysis using the method described above.
• the calculation of the residual amount takes into consideration an appropriate correction in the recovery. This correction is found based on determination of the amount of fatty acid ester on the rabbit jejunum segment after apphcation of an accurate amount of the second substance (this test is repeated 5 times and the recovery is given as the mean value).
• the solubihty of the weak acid aspirin is 3.3 mg/ml in water (20°C). It has a pKa value of about 3.5 (25°C) (Analytical Profiles).
• the solubihty of aspirin is strongly dependent on the pH in the solution. The degree of ionisation of the acid group in aspirin is favoured when the pH is around and above the pKa value of the compound and therefore the solubUity is increased with pH > 3.4.
• a solubility experiment has shown that the solubihty 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 pH 4.0; the buffer was not strong enough to maintain the pH, and the pH in the final solution was 3.6.
• the solubihty of aspirin in a buffer solution of pH 4.0 is > 20 mg/ml.
• the solubihty of acetylsalicylic acid in GMO/water 65/35% w/w has been determined to be >20 mg/ml.
• the pH of the aqueous phase at the end of the experiment was 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 tha pH at 5.0) - -
• the dissolution rate of acyclovir in various GMO compositions was determined using Franz diffusion cells having a diffusion area of 1.77 cm 2 and a receptor volume of 6.8 ml. The study was run at a temperature of 37°C and as diffusion membrane a cellulose membrane from Medicell International Ltd. was employed. The membrane employed has a pore size of about 2.4 nm and it retains particles having a molecular weight larger than about 12,000-14,000. Before application, the membrane was pretreated and thoroughly rinsed with distiUed water. As receptor medium was used an isotonic 0.05M phosphate buffer pH 6.5 (Danish Drug Standards, DLS) and the medium was magnetically stirred at 100 rpm.
• DLS isotonic 0.05M phosphate buffer pH 6.5
• the cellulose membrane was allowed to equilibrate at 37°C for 30 min in the receptor medium employed.
• the membrane was supported by a metallic grating
• about 300-400 mg of the composition to be tested was applied by means of a syringe or a spatula and care was taken to ensure a homogenous distribution of the composition on the total area of the membrane available for diffusion.
• the composition to be tested may be filled into a dish having a weU-defined surface area which is only a little smaller than that of the cellulose membrane held by a Franz' diffusion cell so that almost all of the diffusion area avaUable is used; the dish is turned upside down and placed on top of the cellulose membrane.
• the content of lidocain HCI is determined by a HPLC method.
• T Dissolve the formulation in 30 ml methanol and transfer it quantitatively to a 50 ml volumetric flask. Add methanol to 50.00 ml.
• a ⁇ is the area of the test solution T
• a E is the area of the standard solution R; n is the amount of standard weighed out (g); m is the amount of formulation applied to the intestine (g); % lidocain HCI is the content of lidocain HCI in the formulation determined as
• the content of miconazol is also determined by a HPLC method.
• T Dissolve the formulation in 30 ml methanol and transfer it quantitatively to a 50 ml volumetric flask. Add methanol to 50.00 ml.
• a ⁇ is the area of the test solution T
• a E is the area of the standard solution R; n is the amount of standard weighed out (g); m is the amount of formulation apphed to the intestine (g);
• % miconazol is the content of miconazol in the formulation determined as % w/w. Quantitative determination of acyclovir
• HPLC method employed was the following:
• Reference solution An accurate amount of about 10.00 mg acyclovir is diluted to with distiUed water to a concentration of 10.00 ⁇ g/ml
• Test solution The sample withdrawn is filtered through a 0.2 ⁇ m filter and injected onto the column (in some cases it might be necessary to subject the sample to dUution with water)
• a R is the area of the reference solution.
• C n is the concentration, of drug in the receptor solution (mg/ml), - -
• HPLC method employed was the following:
• Reference solution Weigh out an accurate amount of about 20.00 mg acyclovir and dilute it with mobile phase to a concentration of about 0.008 mg/ml
• Test solution Weight out 100.00 mg of the GMO/acyclovir formulation in a 50 ml volumetric flask. DUute with mobUe phase to 50.00 ml. DUute 5.00 ml to 50.00 ml with mobile phase.
• 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 filtered through a 0.2 ⁇ m filter. Dilute 1000 ⁇ l to 10.00 ml with mobile phase. - -
• pH in the crystalline hquid phase is determined in a 10% w/w dispersion of the liquid crystalline phase (containing the active substance and any excipients) in distiUed water. Prior to determination the dispersion is subjected to ultrasonic treatment for 30 minutes in order to ensure that an equihbrium between the liquid crystaUine phase and the distilled water has taken place.
• the pH is measured by employment of a HAMILTON FLUSHTRODE which is a suitable pH-electrode for measurement of pH in the dispersions. The procedure followed was in accordance with the instructions given by the manufacturer of the electrode.
• compositions i.e. for composition wherein the concentration of the active ingredient in the liquid crystalhne phase may be varied (e.g. from 1-20% w/w or in any range relevant for compositions according to the invention.
• Modifications of the method described above may also be employed e.g. i) the dispersion mentioned above may obtained by diluting the liquid crystaUine phase in a range corresponding to from about 1:20 to about 1:5 with distiUed water, ii) ultrasonic treatment may be omitted or substituted by stirring or treatment in a rotomat provided that measures are taken to ensure that equihbrium takes place or, alternatively, that measurement of pH takes place after a well- defined time period, and hi) other suitable electrodes may be employed.
• test conditions (stirring, ultrasonic treatment, time, electrodes) should be essentially the same when determining pH in the liquid crystaUine phase of compositions.
• the determination of the solubihty of the active substance in the liquid crystalline phase of the composition is, of course, performed on the liquid crystalhne phase as formed. In practice, this means that when the composition is one in which the liquid crystalhne phase has already been formed when the composition is apphed, the determination of the solubihty is performed on the composition itself.
• the determination of the solubihty is suitably performed by microscopy to observe any crystals of the active substance. Suitable test conditions involve a magnification of about 250 x and e.g. room temperature (20°C or 37°C may also be employed).
• the determination of the concentration at which crystals are observed is performed after a period of at least one week after preparation of the composition or the liquid crystaUine phase to ensure that equilibrium has taken place.
• the liquid crystalline phase used as a reference in the solubility determination is a liquid crystaUine phase imitating the liquid crystalline phase which will be formed when the composition absorbs hquid from the site of apphcation.
• This reference liquid crystalhne phase is made up with water (as representing the hquid absorbed) in such an amount that the reference liquid crystaUine phase is the same type of hquid crystalhne phase as is generated from the precursor composition.
• the pH is determined in the liquid crystaUine phase as described above to determine the pH conditions when determining the solubUity.
• the solubUity of the active substance is then determined by stirring an excess amount of the active substance in water, where applicable, being buffered to a pH substantially identical to the pH prevaUing in the liquid crystalhne phase for a time period of at least 24 hours (to ensure that equUibrium has taken place) and at a constant temperature (e.g.
• the samples initially were subjected to ultrasonic treatment for half an hour in order to accelerate the time for equUibrium.
• concentration of the active substance in the supernatant i.e. the aqueous solubility at the given pH
• an appropriate assay e.g. by HPLC or UV spectroscopy.
• the water is adjusted to substantially the pH of the liquid crystalline phase by using a suitable buffer system when determining the solubihty of the active substance.
• This buffer system should of course be so selected that, apart from the pH adjustment, it has substantially no influence on the solubihty of the active substance in the buffered water.
• the aqueous solubihty is determined as a function of pH, i.e. by determining the aqueous solubihty in buffer systems having a pH in a range of about 3 to about 9.5 such as about 3.6 to about 9.
• Suitable buffer systems include acetate, citrate, phosphate, borate etc. and the concentration of the buffer is sufficient to ensure a constant pH during the experiments. A concentration of at least 0.01 M is normally suitable.
• This method is applicable when determining the minimum aqueous solubihty of a specific active substance at a given temperature and at a given pH range.
• the test conditions described pH, temperature, ultrasonic treatment, stirring, time for ensuring that equilibrium has taken place) above are also valid when determining the minimum solubihty.
• the lameUar phase is the dominating one at a relatively low water content (below 20% w/w) and at a temperature of about 37°C, whereas the cubic phase dominates as the water content increases (more than about 20% w/w).
• the DSC measurements were performed using a Perkin Elmer Unix DSC model 7 Differential Scanning Calorimeter. The heating rate was 5°C/min and the scanning temperature was from 5°C to 70°C. Samples were contained in sealed aluminium pans (Perkin Elmer No. BO14-3017) and as a reference empty aluminium pans were employed. The phase transitions caused only a relatively small enthalpy change and, therefore, the amount of sample tested was optimized to about 30-40 mg. The prepared pans were sealed and stored for two days at 5°C prior -to analysis.
• the liquid crystalhne phase can also be determined using polarized light and e.g. employing a stereomicroscope (Leitz, Diaplane) equipped with polarization filters.
• the appearance of reversed 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.
• the cubic phase (Q) is optically isotropic and gives a black background with no details indicating 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 background or, expressed in another manner, could be identified from the oily streak texture and the spherical, positive maltese cross-units visible between crossed polarisers.
• the reversed hexagonal phase gives different patterns but in most cases it resembles a mosaic-like structure or gives angular or fan-like textures.
• the method can be employed in testing the phase behaviour of various bioadhesive compositions.
• a modified diffraction thermal pattern (DTP) camera was employed.
• the source was an X-ray tube equipped with a Cu-anode emitting K ⁇ -rays at a wavelength of 1.5418 A.
• the X-ray generator was a Philips PW 1729.
• the liquid crystalhne state can be identified by low angle X-ray diffraction and its appearance in polarized hght.
• the characteristic X-ray diffraction pattern for the three liquid crystalline phases (lameUar, hexagonal, cubic) will give rise to diffraction lines in the following orders:
• the glycerylmonooleate (abbreviated as GMO in the following) (and whenever relevant glycerylmonolinoleate (Dimodan® LS)) is gently melted on a heating plate or in an oven and the liquid obtained (max. temperature of the melted liquid is about 60°C) is cooled to about 40°C before mixing with other ingredients.
• GMO glycerylmonooleate
• Dimodan® LS glycerylmonolinoleate
• compositions of GMO/Vitamin E TPGS, GMO/lecithin, and GMO/lecithin/Vitamin E TPGS, respectively, are prepared as follows:
• GMO and Vitamin E TPGS are melted together at a temperature of max. 60°C.
• GMO and Vitamin E TPGS, respectively are individuaUy melted before mixing of the two components. Then the hquid phase is added.
• Lecithin is dissolved in GMO at a temperature of about 60°C. Then the hquid phase is added. If the content of lecithin is >50% by weight of the GMO content (lecithin/GMO >0.5) then lecithin and the GMO may be dissolved in ether or ethanol followed by evaporation of the solvent by vacuum distillation. Then the hquid phase is added.
• Lecithin is dissolved in a GMO-Vitamin E TPGS phase at 60°C.
• the method described above involving ether or ethanol as solvent can also be employed.
• GMO and Vitamin E TPGS are melted at max. 60°C.
• Acyclovir is suspended into the melt under stirring.
• Epicuron 200 is dispersed in an aqueous medium by means of a homogenizer and is then added to the GMO/Vitamin E TPGS/acyclovir mixture in a mortar under vigorous stirring until a homogeneous mixture is obtained.
• the mixture is subjected to ultrasonic treatment for 1 hour and left in an oven (37°C) for 2 days to ensure that equihbrium has taken place.
• compositions described above any active drug substance and or other excipients are added or, alternatively, these substances are dissolved in the liquid phase before these two phases are mixed together.
• composition contains an active substance in a GMO/ethanol or GML/ethanol vehicle or a lipid phase/ethanol vehicle
• one of the following methods can be apphed:
• the active substance was dissolved or dispersed in ethanol and then mixed with melted GMO under stirring,
• the active substance was dissolved or dispersed in a GMO/ethanol mixture.
• An acyclovir ointment composition was prepared as foUows:
• the acyclovir was suspended in the melted lipid phase and the other ingredients were added.
• the monoglyceride mixtures and the ingredients were mixed by stirring or shaking.
• the compositions were subjected to ultrasound treatment for about 1 h and were stored for at least two days at 37°C before use to ensure that equUibrium had been obtained (e.g. that the stable hquid crystaUine phase has been formed in the total formulation and that equihbrium between the sohd and dissolved substance has taken place).
• the acyclovir can be suspended in the hquid phase before combining the liquid phase with the melted GMO.
• compositions in which a cubic liquid crystalline phase has been generated and wherein structurants like Epikuron 200 and Vitamin E TPGS are included.
• compositions included in the study are left in transparent glass containers in a climate cabinet and the temperature is maintained at 15°C, 25°C or 40°C.
• composition investigated is prepared using GMO-90 (with a content of GMO of about 90% w/w) or, alternatively, by using RYLO MG19 (with a content of about 90% GMO).
• the compositions which are prepared using both qualities of GMO have been marked with a * in the following.
• the liquid crystalline phase was investigated by means of polarised light at 22°C.
• the compositions investigated was prepared by dissolving Epikuron 200 in melted GMO/Vitamin E TPGS (max. 60°C).
• the liquid crystalline phase was investigated by means of polarised light at 22 °C.
• the compositions investigated was prepared by dissolving Epikuron 200 in melted GMO/Vitamin E TPGS (max. 60°C).
• the purpose with the study is to investigate whether the presence of structurants hke phosphatidylcholine and Vitamin E TPGS has any influence on the solubihty of acyclovir in the liquid crystalline phase.
• compositions are treated for 1 hours in an ultrasonic bath and stored at 37°C for at least 2 days before each composition is subjected to polarized light to see whether acyclovir crystals are present in the cubic phase. If crystals are observed then the solubility is judged to be less than 2 mg/g or 5 mg/g.
• the solubihty of acyclovir at room temperature (22°C) is 0.5-1 mg/g.
• the solubihty of all the compositions is found to be less than 0.2% as acyclovir crystals are observed in the microscope.
• the results indicate that the lecithin and the TPGS do not increase the solubility of acyclovir compared with a GMO/water cubic phase.
• compositions are prepared as described above (Epikuron 145 is dispersed/dissolved in Vitamin E TPGS at 60°C for up to 3 days).
• the compositions are inspected in polarized light as described under the heading "Methods”.
• compositions described above under heading “A” are subjected to a test for bioadhesion employing test system No. 3 - washing off ability from the skin described under the heading “Methods”. The results are that at least some of the compositions are bioadhesive.
• compositions without any drug substance are provided without any drug substance
• the results from the DSC and polarized hght measurement show that the lamellar phase is present at room temperature and the lameUar phase is changed to the cubic phase when the temperature increases (Fig. 4).
• the transition temperature is about 37°C.
• compositions containing GMO 90 and Vitamin E TPGS and/or Epikuron 200 as structurants are subjected to X-ray diffraction measurements (as described under the heading "Methods") in a temperature scan at 20-80°C.
• the aim of the study is to investigate the influence structurants like Vitamin E TPGS and/or phosphatidylcholin have on the phase behaviour of GMO/water based composition.
• the aim of the study is also to obtain an indication of whether the structurants participate in the cubic structure formed due to the GMO/water content in the compositions or whether the function of the structurants more are like diluents, i.e. they do not in themselves participate in the formation of the cubic structure but may merely just be incorporated in the composition and the cubic structure is more or less alone based on the GMO/water content.
• compositions are:
• GMO/Vitamin E TPGS/water 50/15/35 % w/w
• GMO/Epikuron 200/water/Vitamin E TPGS 39.4/15.8/29.8/15 % w/w
• compositions 1-3 are cubic at 37°C and give an indication of that Vitamin E TPGS and/or Epikuron 200 participate in the cubic structure, i.e. the cubic liquid crystalhne phase formed is based on the GMO/Vitamin E TPGS/water or GMO/Epikuron 200/water/Vitamin E TPGS compositions and not on the GMO/water content alone.
• the cubic liquid crystalhne phase formed is based on the GMO/Vitamin E TPGS/water or GMO/Epikuron 200/water/Vitamin E TPGS compositions and not on the GMO/water content alone.
• auxiliary substances in general only can be incorporated into the GMO cubic lattice structure in a relatively low concentration. If Vitamin E TPGS and/or lecithin do not participate in the cubic lattice structure then an excess of water would have been expected, i.e. a phase separation of the composition into at least two distinct liquid phases would have been observed and no such observation is made.
• compositions Nos. 1-3 above are also subjected to DSC experiments. No peaks is observed (see Figs. 5-7) indicating that the cubic phase do not transform to another liquid crystalline phase in the temperature range investigated (20-70°C). These results are in consistency with the results obtained from the X-ray measurements given above.
• compositions containing GMO/water 65/35% w/w and GMO/water 65/35% w/w with acyclovir (crystalline and micronized, respectively) added in concentrations 2.5, 5.0 and 10% w/w were subjected to X-ray diffraction measurements (as described under the heading "Methods") in a temperature scan at 20-70°C.
• Methods X-ray diffraction measurements
• compositions containing GMO 90 and Vitamin E TPGS and/or Epikuron 200 as structurants and 5% w/w acyclovir are also subjected to X-ray diffraction measurements (as described under the heading "Methods") in a temperature scan at 20-80°C.
• the aim of the study is to investigate the influence of acyclovir on the phase behaviour of compositions wherein phosphatidylchohne and/or Vitamin E TPGS are added as structurants.
• Compositions without any drug substance i.e. 5% w/w acyclovir is added to GMO/Vitamin E
• compositions 2A and 3A are cubic at 37°C.
• acyclovir in a concentration of 5% w/w does not seem to result in a change in the phase behaviour and the cubic phase seems to be rather stable within the temperature ranges investigated.
• compositions containing GMO/water 65/35% w/w with acyclovir (crystalline and micronized, respectively) added in a concentration of 1-40% were tested in polarized hght at 22°C and 37°C, respectively, as described above under the heading "Methods”.
• Methods The results show the presence of cubic phases in all compositions indicating that acyclovir probably is inert in the cubic phase.
• compositions (with or without 5% crystalhne acyclovir) are tested in the washing off ability test system for bioadhesiveness described under the heading "Methods”.
• GMO/Vitamin E TPGS/water 50/15/35 % w/w
• GMO/Epikuron 200/water/Vitamin E TPGS 39.4/15.8/29.8/15 % w/w
• the dissolution rate of acyclovir in various GMO compositions is determined using Franz diffusion cells as described under the heading "Methods”.
• a series of GMO compositions containing Epikuron 200 (phosphatidylcholin) and/or Vitamin E TPGS and acyclovir are prepared as described above, and they are subjected to the above dissolution/release rate determination. All compositions were suspensions of acyclovir, that is, they contain acyclovir which has not dissolved. The solubihty of acyclovir in the compositions investigated is less than 0.2% w/w according to the solubility experiments performed.
• composition Nos. 1-4 The release profiles for composition Nos. 1-4 are shown in Figs. 10 and 11 and the following slopes (Higuchi plots) are found:
• the hquid crystalhne phase present in composition No. 5 is the lamellar liquid crystalhne phase.
• the composition is in the form of a precursor composition and a phase conversion to the cubic liquid crystalline phase takes place during the testing.
• the release rate of acyclovir (cf. Fig. 11 A) is of the same order of magnitude as the reference cubic liquid crystaUine phase-containing composition (GMO/water) and thus, confirms that the cubic liquid crystaUine phase is formed during the test.
• GMO/water reference cubic liquid crystaUine phase-containing composition
• Composition No. 6 contains oleic acid as an enhancer.
• the liquid crystalhne phase present in the composition is the reverse hexagonal crystaUine phase.
• the release rate of acyclovir (see Fig. 11A) is of the same order of magnitude as the reference cubic liquid crystalline phase-containing composition (GMO/water) and thus, indicates that the cubic hquid crystaUine phase is formed early during the test. It is most likely that oleic acid is rapidly released and then the conditions present in the composition favours a formation of the cubic hquid crystaUine phase.
• GMO/water reference cubic liquid crystalline phase-containing composition
• the rate limiting step in the dissolution process is diffusion of acyclovir molecules
• a precursor composition is capable of generating a cubic liquid crystaUine phase
• a phase conversion may take place after apphcation of a composition.
• compositions containing antiviral substances are provided.
• compositions are prepared as described above. 5% w/w of an antiviral substance is added to all the compositions listed in the table below.
• Oil e.g. olive oil, ricinus oil etc.
• another substance which decreases the melting point of the lipid formulations may be added to obtain a cream or an ointment at room temperature.
• the compositions are able to absorb e.g. sweat or exudate from a wound and a liquid crystalline phase like e.g. the cubic liquid crystalhne phase is formed.
• a hquid crystal phase inhibitor may be added and exert its function in the solid state (e.g. sugar alcohols hke trehalose, or PVP).
• Addition of water in small amounts may reduce the viscosity and hinder crystallization of the lipids.
• compositions are also relevant, i.e. compositions having other active substances or having a drug concentration of about 1-20% w/w and compositions having a composition of the vehicle as given in Example 1-80 above.
• the buffers with pH 3.6, 4.2 and 5.3 were prepared using monobasic sodium phosphate and dibasic sodium phosphate (pH adjustment with phosphoric acid).
• the buffers in the pH range 6.0 to 9.6 were prepared using monobasic potassium phosphate (pH adjustment with sodium hydroxide).
• the molarity of the phosphate salts was 0.05M; the pH of the medium was measured with a pH-meter.
• Miconazole is an example of an active substance which is insoluble in water but has a solubility of more than 2% w/w in the hquid crystaUine phase. However, the release of miconazole is very slowly from the cubic phase.
• the table given below shows the solubihty of and the crystaUine phase obtained for miconazole in a GMO/water 70/30% w/w vehicle.
• compositions were found to be highly bioadhesive, indicating that with substances having a very low solubihty in the hquid crystalhne phase, the liquid crystalhne phase remains less disturbed by the presence of particles of the active substance and retains its bioadhesive properties.
• the GMO/GML mixture corresponds to about equal amounts of glycerol monooleate and glycerol monolinoleate
• relevant excipients or solvents such as, e.g., agents which are known solubijizers for active substances or agents which are known as release - modulating agents (i.e.
• agents which when added make it possible to adjust or control the release of the active substance from a composition do not significantly influence the bioadhesiveness of the composition when the agents (excipients or solvents) are added in relatively low concentrations (less than about 10% w/w).
• the release of an active substance from a composition which has proved to possess bioadhesive properties can be controlled at least to a hmited extent by adjusting the amount of a release modulating agent such as, e.g., glycerol, sesame oU, soybean oil, sunflower oil, lecithin, cholesterol, etc.
• a modulating agent may influence the pore size of the water channels in the cubic phase and/or alter the partition coeffient of the active substance between the lipid domains and the aqueous phase at least to a hmited extent.
• solubUisation of an active substance or a fatty acid ester for use in a bioadhesive composition can be effected by use of e.g. benzyl alcohol without significantly influencing the bioadhesive properties of the composition.
• the bioadhesive principles described herein have a high potential with respect to developing bioadhesive drug compositions having such a drug localization, such a drug release profile, and such a drug duration which are desirable or necessary under the given circumstances.
• the present inventors have found an advantageous bioadhesive drug dehvery system.
• the methodology described herein is a methodology which is generally useful for investigating whether mixing or dissolving of an active substance in a vehicle capable of forming a hquid crystaUine phase also leads to incorporation of the active substance in the liquid crystalhne phase. While miconazol and lidocain hydrochlorides have been used as model substances in the description of the experiments, the same measures as described herein can be used for substances which have a very low solubUity in both water and ethanol such as, e.g., acyclovir.
• lidocaine hydrochloride In the experiment with lidocaine hydrochloride, barely half the content of the drug was recovered after a flow period of 10 sec and only a neghgible amount after 30 minutes. Because of its high water solubihty (about 0.7 g/ml at 25°C), the greater part of the lidocaine hydrochloride is probably dissolved and washed away in the buffer solution during the prehydration time (10 min) and only some is incorporated in the cubic phase formed. Most of the incorporated drug had been released at the end of the experiment. Other studies have shown that lidocaine hydrochloride is released rather quickly from the cubic phase probably through the water channels contained in the cubic phase. Results for acyclovir, which is poorly soluble in both water and the cubic phase, given in the table clearly demonstrate that acyclovir is enclosed in the cubic liquid crystalline phase formed and some of it may have been released during the experiment.
• a series of GMO compositions containing acyclovir were prepared as described above, and they were subjected to the above dissolution rate determination.
• AU compositions were suspensions of acyclovir, that is, they contain acyclovir which was not dissolved.
• the solubUity of acyclovir in the compositions investigated was less than 0.1% w/w (0.05% w/w ⁇ the solubihty of acyclovir ⁇ 0.1% w/w).
• the results appear from Figures 12-18.
• the results indicate that the release of acyclovir from a GMO based vehicle is dependent on the concentration of acyclovir in the composition, provided that the release takes place from a cubic phase system. Furthermore, the results indicate the capability of a GMO-based vehicle to function as a very effective drug delivery system.
• Figs. 12-14 show the release of acyclovir (1-5% micronized) from a cubic phase (GMO/water 65/35% w/w) and Zovir® cream, respectively, into isotonic 0.05 M phosphate buffer solution, pH 6.5 (37°C).
• acyclovir (1-5% micronized) from a cubic phase (GMO/water 65/35% w/w) and Zovir® cream, respectively, into isotonic 0.05 M phosphate buffer solution, pH 6.5 (37°C).
• the release of acyclovir increases with increasing concentration of acyclovir over the range investigated. There is not proportionality between the rate of release and the concentration; this appears from the fact that the graphs of % released (Fig. 13) do not coincide and the slope of the Higuchi plots (Fig. 14); the release is dependent on the concentration. However, at a concentration of 3-5% w/w acyclovir, no significant difference in release rates was found.
• rate constant herein as the release of acyclovir from the liquid crystaUine formulations according to the invention which can be described by means of the so-called Higuchi equation (Higuchi, T., Rate of release of medicaments from ointment base containing drug in suspension. J. Pharm. Sci., 50 (1961) 874-875): on linear regression; the cumulative amount of acyclovir released plotted versus the square root of time results in a straight line with the slope k (rate constant ⁇ g/h' ⁇ ). This appears from Fig.
• FIGs 15, 16 and 17 show an identical release pattern for crystalhne and micronized acyclovir, respectively, from a formulation consisting of GMO/water 65/35% w/w + 1% acyclovir.
• the release rate of crystalline acyclovir is slightly improved from a composition containing lecithin (GMO/water/lecithin 55/35/10% w/w + 1% acyclovir) compared to the same composition containing micronized acyclovir (Figs. 15-16).
• micronized quality increases the viscosity of the cubic phase more that the crystalline phase. This condition alone favours the use of the crystalhne quahty 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 profile of GMO/water 65/35 % w/w has a shape simUar to the others with the exception of the profiles for the compositions containing sesame oU. In the latter case the release rate is drastically reduced, which could mean that the compositions consist of the reversed hexagonal phase, but this has not been confirmed. It should be noted that the composition consisting of 65 parts of GMO and 35 parts of glycerol, have the same release profile as the reference composition, although both the visual and the polarized hght do not indicate that they consist of the cubic phase.
• the cubic phase is created on the surface of the formulation during the release experiment, through its contact with the dissolution medium (37°C).
• Addition of the release modulating substances glycerol and lecithin flecithin is as described above also a structurant) to the cubic phase has not significantly changed the release of acyclovir in the concentrations examined.
• TPGS seem to have increased the dissolution of acyclovir in the cubic phase nor changed the partition coefficient between the cubic phase and the release medium, as the release profile is identical with the profile of the reference composition.
• Fig. 11 shows the release profiles of composition containing 5% acyclovir.
• a composition of GMO/water 65/35% w/w with 5% w/w acyclovir has been used for the treatment of cold sores in humans.
• Treatment was started with a maximum of 24 hours delay from start of symptoms. In one case, treatment with Zovirax® cream was tried for 4.5 days before switch to GMO acyclovir cream. GMO acyclovir cream was apphed 3 times daily (range 2-4) for 2.5 days (range 1.5-4).
• Apphcation frequency and treatment duration for GMO acyclovir cream in these case reports are less than recommended for Zovirax® cream.
• the efficacy was judged to be equivalent or better than that of Zovirax® cream.
• test was performed over five working days with four applications and four recordings after 24 hours.
• the test material was apphed onto the volunteers on volar next to their non-dominant underarms.
• the skin reaction was evaluated both in respect of erythema and oedema on a scale ranging from 0 to 3. The scoring was made by the same person for aU the volunteers.
• the Chamber Scarification Test is used in order to evaluate the skin irritation profile of the above-mentioned compositions.
• the Chamber Scarification Test is developed to investigate and compare cosmetics, cosmetic ingredients and consumer products intended for repeated use on normal or diseased skin.
• the assay amplifies irritant reactions to the test products by scarification of the test area prior to the first application.
• the test is carried out as described by K. E. Andersen in Contact Dermatitis 1996 (34), pp. 181-184 by P. J. Frosch & A. M. Khgman in Contact Dermatitis 1976 (2), pp. 314- 324.
• compositions according to the invention in vitro permeability of compositions according to the invention across human skin
• Excised abdominal skin from humans was obtained from The Chnic of Plastic Surgery. The hairs were removed from the epidermal side by clipping. Subcutaneous fat on the dermal side was removed. The skin was washed with distilled water and stored at -18°C until use.
• the skin membranes were thawed and mounted in Franz diffusion cells.
• the receptor chambers were filled with receptor medium and the epidermal side of the skin was wetted with a few drops of receptor medium.
• the skin was then allowed to equilibrate for about 24 hours. Blood and soluble enzymes were at the same time washed out of the skin, and thereby could not disturb analysis of the receptor medium for acyclovir.
• the integrity of the individual skin samples was ensured by measuring the capacitance of the skin. Skin samples with a capacitance of less than about 0.055 ⁇ F were considered intact, whereas skin samples with a higher capacitance were considered damaged.
• the water permeability ( 3 H) may also be determined as a measure of the integrity of the skin.
• the results from the permeation study shows that the permeation profiles for acyclovir from the GMO/water composition (cubic liquid crystalhne phase) and Zovir®, respectively, are not significantly different.
• the lag time is about 1 day.
• the release profile shows tht acyclovir delivered from a cubic liquid crystalline phase of GMO permeates the skin and, accordingly, a cubic liquid crystalhne phase is an excellent drug delivery system for acyclovir and most likely also for other active substances especially antiviral substances.
• the compositions remain on the skin.
• compositions according to the invention in vitro permeability of compositions according to the invention across human skin
• the foUowing experiments can be performed using wholly intact human skin excised from cosmetic surgery.
• the skin is obtained from clinics for plastic surgery.
• the skin is treated as mentioned in the Example above and stored at -18°C.
• Skin from other mammals than humans may also be employed such as, e.g. guinea pigs, mice and pigs.
• the skin may be separated into epidermis and dermis by exposing the skin to hot water (60°C) for e.g.
• the stratum corneum can be isolated by tape stripping. If the drug substance is insoluble in water, the epidermis can be separated by dry heat separation e.g. at 60°C for 2 min by placing the sample (contained in a closed package) in a water bath or in a heat cabinet.
• the test conditions are generally as described in the Example above, but other test times (e.g. from 1 hour to 7 days), amounts of sample apphed (e.g. 50-350 mg) etc. may be appropriate.
• the same donor is used to testing different compositions and the skin specimens were taken from the same skin area. In order to simulate injured skin, the skin can be injured by applying a skin enhance or by stripping the skin with tape.
• the amount of drug substance within the skin can be calculated by measuring the concentration of the drug substance in i) the receptor medium, ii) the skin, and/or iii) the remaining composition. By measuring i) and iii), the amount of drug substance in the skin can be calculated.
• the herpes virus replicate in the living epidermis.
• the basal layer of the epidermis appears to be the primarily site of antiviral activity in cutaneous HSV-1 infections, i.e. the epidermis appears to be the target site for antiviral drug substances.
• Permeation (i.e. penetration into and through the skin) of acyclovir or other antiviral substances can be investigated across isolated epidermis by diffusion (as described above). In this manner, a measure is obtained of the amount of acyclovir having permeated the epidermis.
• a picture is obtained of the penetration (i.e. the entry into the skin but not through the skin) of acyclovir (or other antiviral substances) in the skin by means of diffusion test using wholly skin which at the end of the experiment is divided into stratum corneum (e.g. enzymatic degradation or tape stripping; tape stripping: 10-20 x, e.g. using Scotch Brand Magic Tape No.
• compositions are removed by use of a spatula and the skin is dried using Kleenex dipped in a ethanol-water (3:1) solution.
• acyclovir or other radioactive antiviral drugs
• the amount of acyclovir penetrating the tissue was measured by a liquid scintillation technique ( 3 H- acyclovir is commercially avaUable in form of a ethanol/water 30/70 solution (Sigma); e.g. 21 ⁇ l H-acyclovir corresponding to about 0.8 ⁇ Ci/ml is added to 1 g composition).
• acyclovir Due to the fact that the main part of the acyclovir is present in the form of undissolved particles in a concentration of about 1-10% by weight, it is initially necessary to dissolve cold acyclovir and add the hot radioactive form on dissolved form in order to obtain a homogeneous mixture of cold and hot molecules.
• Cold acyclovir is dissolved in 0.1 N NaOH and the hot acyclovir is added under stirring.
• Hydrochloric acid (to adjust pH to about 7) is rapidly added under vigorous stirring to obtain a uniform precipitation of hot and cold acyclovir crystals. The precipitation is enhanced by maintaining the mixture at 5°C for e.g. 24 hours.
• the acyclovir crystals are filtered off and washed 3 times with a small amount of water. The crystals are dried in an excicator. By using this procedure it is possible to control the ratio between hot and cold acyclovir in dissolved and undissolved form, respectively.
• acyclovir In order to examine the content of acyclovir in different skin sections/layers, the skin sections were placed in scintillation vials with e.g. Soluene 350 over night to dissolve the skin components. Scintillation cocktail (e.g. Hionic-Flour) was subsequently added and the samples were assayed for content of acyclovir (or the appropriate antiviral drug) by liquid scintillation spectrometry.
• the drug metabolizing enzyme activity in the epidermis is greatly dependent on tissue viability. Therefore, it should be stressed that the determination of skin absorption described above does not distinguish between the intact antiviral drug and its metabolites. It cannot be excluded that excised skin (usually stored) will loose some of its original enzyme activity. However, acyclovir exhibits no known metabolism in the skin.
• acyclovir By extracting acyclovir from the skin components, acyclovir can also be quantified by HPLC.
• compositions containing acyclovir or other drugs by means of an in vitro cell culture model
• acyclovir or other antiviral drugs delivered from various compositions according to the invention can be examined using in vitro ceU cultures as a model of e.g. human oral epithehum.
• a model involving e.g. TR 146 cell is suitable for sensitivity and permeability studies of antiviral drugs.
• Other cell culture models are also avaUable, e.g. for the testing of the efficacy of drugs.
• compositions containing acyclovir or other antiviral drugs by means of an in vivo animal model
• the basal layer of the epidermis appears to be the primary site of antiviral activity in cutaneous HSV-1 infections, i.e. the target for antiviral drugs.
• Methods - using hairless mouse or guinea pig as an animal model - are avaUable.
• the methods allow calculation of the target site concentration of the antiviral (e.g. acyclovir) drug applied and allow an estimation of the efficacy of the antiviral compositions tested (see. e.g. Lee, P.H. et al., Pharm. Res. 9, 8, pp 979-988, 1992 and Su, M.-H. et al., Drug Develop. Ind. Pharm. 20 (4), 685-718, 1994).
• model systems suitable for testing the antiviral effect of the compositions according to the invention.
• Animal models often used are the hairless mouse model (5-7 weeks old) and the guinea pig model. The guinea pigs are shaved on their back before the start of the experiment in order to make a hairless test area.
• the animals are anaesthetized before inducing skin lesions, e.g. on the lateral side of the body or in the lumbosacral area.
• 0.005-0.2 ml of a virus suspension [herpes simplex virus type 1 (HSVl), e.g. strain E-377 or E-115 (titer usually in a range of 10 6 - 10 8 plaque forming units (PFU)/ml), stored at -70°C until use] was injected or rubbed on the skin with a cotton swab saturated with the virus (a drop of the virus suspension is apphed on the test area and then 6 small holes are made by means of a scalpel.
• HSVl herpes simplex virus type 1
• E-377 or E-115 titer usually in a range of 10 6 - 10 8 plaque forming units (PFU)/ml
• the test area on the skin of the test animal can be divided into several test areas, e.g. six areas, thereby aUowing e.g. two different 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 composition (the number of animals depends on the number of applications).
• Shortly after virus inoculation (e.g. 24-48 hours) compositions with antiviral drugs were apphed on the test areas at the skin e.g. with a 1 ml syringe and samples are bhndly randomized.
• the lesions are treated with the compositions for 2-10 days (applied 2-5 times daUy) and then the effect of the treatment was investigated.
• the lesions were scored for each animal and two distinct antiviral assessments can be made: i) topical efficacy is determined by measuring the antiviral activity of the antiviral drug substance (e.g. acyclovir) delivered from the compositions tested, and ⁇ ) systemic efficacy is determined by measuring the antiviral activity of the antiviral drug substance (e.g. acyclovir) in the circulatory system which delivers the antiviral substance to the target site (presumably the epidermal basal layer).
• topical efficacy is determined by measuring the antiviral activity of the antiviral drug substance (e.g. acyclovir) delivered from the compositions tested
• systemic efficacy is determined by measuring the antiviral activity of the antiviral drug substance (e.g. acyclovir) in the circulatory system which delivers the antiviral substance to the target site (presumably the epidermal
• a score system is used. Different score systems may be employed based on the appearance of the skin lesions at various times after inoculation.
• the score system could be that of Alenius and Oberg, Archives of Virology
• HSV-1 virus may be isolated from the lesions and the number is counted. The results give an indication of i) inactivation of virus, ii) effect of the antiviral composition applied etc.
• ChnicaUy confirmed history of recurrent Herpes Labialis, 2-3 annual recurrences. Present prodromal symptoms of Herpes Labialis eruption.
• GMO acyclovir is not considered to have any advantage compared to Zovirax® cream.

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