PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THEPATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 6 :
`A61K 9/20
`
`(11) International Publication Number:
`
`WO 96/40077
`
`(43) International Publication Date:
`
`19 December 1996 (19.12.96)
`
`(21) International Application Number:
`
`PCT/GB96/01367
`
`(22) International Filing Date:
`
`7 June 1996 (07.06.96)
`
`(30) Priority Data:
`08/486,043
`
`7 June 1995 (07.06.95)
`
`US
`
`(71) Applicant (for all designated States except US): QUAD-
`RANT HOLDINGS CAMBRIDGE LIMITED [GB/GB];
`Maris Lane, Trumpington, Cambridge CB2 ZSY (GB).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): ROSER, Bruce, Joseph
`[GB/GB]; 4 Archway Court, Barton Road, Cambridge CB3
`9LW (GB). GRIBBON, Enda, Martin [IF/GB]; 18 Southmill
`Road, Bishops Stortford, Hertfordshire CM23 3DP (GB).
`
`(74) Agent: ABLEWHITE, Alan, James; Marks & Clerk, 57-60
`Lincoln’s Inn Fields, London WC2A 3LS (GB).
`
`(81) Designated States: AL, AM, AT, AU, AZ, BB, BG, BR, BY,
`CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL,
`IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV,
`MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU,
`SD, SE, 86, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ,
`VN, ARIPO patent (KE, LS, MW, SD, SZ, UG), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patent (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT,
`LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI,
`CM, GA, GN, ML, MR, NE, SN, TD, TG).
`
`Published
`Without international search report and to be republished
`upon receipt of that report.
`
`for stable storage of a wide variety of substances, particularly biological and pharmaceutical.
`
`(54) Title: METHODS FOR STABLY INCORPORATING SUBSTANCES WITHIN DRY, FOAMED GLASS MATRICES AND
`COMPOSITIONS OBTAINED TI-EEREBY
`
`(57) Abstract
`
`The invention provides methods for producing foamed glass and the compositions obtained thereby. The compositions are suitable
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`Viet Nam
`
`United Kingdom
`Georgia
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Kenya
`Kyrgystan
`Democratic People's Republic
`of Korea
`Republic of Korea
`Kazakhstan
`Liechtenstein
`Sri Lanka
`Liberia
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`Mali
`Mongolia
`Mauritania
`
`AM
`AT
`AU
`BB
`BE
`BF
`86
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`CS
`CZ
`DE
`DK
`EE
`ES
`Fl
`FR
`GA
`
`Armenia
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cbte d’Ivoire
`Cameroon
`China
`Czechoslovakia
`Czech Republic
`Germany
`Denmark
`Estonia
`Spain
`Finland
`France
`Gabon
`
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poiand
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`
`

`

`WO 96/40077
`
`PCT/GB96/01367
`
`NIETHODS FOR STABLY INCORPORATING SUBSTANCES
`
`WITHIN DRY, F OAMED GLASS MATRICES AND
`COMPOSITIONS OBTAINED THEREBY
`
`TECHNICAL FIELD
`
`This invention relates to methods of making foamed glasses and
`
`compositions obtained thereby. More specifically, it relates to methods of stably
`
`incorporating substances, particularly biological substances, into dried foamed
`
`glass matrices (FGMs) and the compositions obtained thereby.
`
`BACKGROUND ART
`
`Traditionally, the most common method of preserving biological
`
`substances which are unstable in solution at ambient temperatures, such as
`
`proteins and DNA, has been freeze-drying. This process involves placing the
`
`substance in solution, freezing the solution, and exposing the frozen solid to a
`
`vacuum under conditions where it remains solid and the water and any other
`
`volatile components are removed by sublimation. The resulting dried formulation
`
`contains the biological substance and any salts or other non-volatile materials
`
`added to the solution before drying. This drying method, conventionally used in
`
`the absence of effective alternatives, often results in significant activity losses.
`
`Pikal (1994) ACS Symposium 567:120-133. Furthermore, many of the various
`
`parameters within the freeze-drying process remain poorly characterised,
`
`sometimes resulting in the loss of whole batches at the production level.
`
`In spite of the apparent ubiquity of freeze-drying, many freeze-dried
`
`substances are still unstable at ambient temperatures. Pikal (1994); Carpenter et
`
`al. (1994) ACS Symposium 5671134-147. Damage caused by this process may be
`
`circumvented, to a certain degree, by the use of cryoprotectants. Carpenter et al.
`
`(1994). However, cryoprotectants may subsequently react with the dried
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`substance. This imposes inherent instability upon storage of the freeze-dried
`
`substances.
`
`Other methods used to prepare dry, stable preparations of labile biological
`
`and chemical substances such as ambient temperature drying, crystallisation or
`
`co-precipitation also have drawbacks. Ambient temperature drying techniques
`
`eliminate the freezing step and associated freeze-damage to the substance. These
`
`techniques are more rapid and energy—efficient in the removal of water. Crowe et
`
`a1. (1990) Cryobiol. 27:219-231. However, ambient temperature drying often
`
`yields denatured or even inactive substances unless an appropriate stabiliser is
`
`used. Crystallisation or co-precipitation can only be applied to a few substances,
`
`and the products of these methods have poor solubility. Additionally, there may
`
`be problems in removing residual moisture.
`
`Trehalose, oc—D—glucopyranosyl- 0L -D-glucopyranoside, is a naturally
`
`occurring, inert, non-reducing and non-toxic disaccharide which was initially
`
`found to be associated with the prevention of desiccation damage in certain plants
`
`and animals which can dry out without damage and revive when rehydrated.
`
`Trehalose has been shown to be useful in preventing denaturation of a wide
`
`variety of substances such as proteins, viruses and foodstuffs during desiccation
`
`and subsequent storage. Formulations of products air dried in trehalose have been
`
`found to have a remarkably increased storage life. See US. Patent Nos.
`
`4,891,319; 5,149,653; 5,026,566; Blakely et a1. (1990) Lancet 336:854; Roser
`
`(July 1991) Trends in Food Sci. and Tech, pp. 166-169; Colaco et al. (1992)
`
`Biotechnol. Internal, pp. 345-350; Roser (1991) BioPharm. 4:47; Colaco et a1.
`
`(1992) Bio/Tech. 10: 1007; Roser and Colaco (1993) New Scientist 138:25-28; and
`
`Crowe (1983) Cryobiol. 20:346-356. Trehalose also stabilises lyophilised
`
`proteins, such as methanol dehydrogenase (Argall and Smith (1993) Biochem.
`
`M01. Biol. Int. 30:491), and confers thermoprotection to enzymes from yeast.
`
`Hottiger et a1. (1994) Eur. J. Biochem. 219: l 87. Trehalose also inhibits the
`
`Maillard reaction between carbonyl groups of reducing sugars and amino groups
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`of proteins. Loomis et al. (1979) J. Exp. Zool. 208:3 55-3 60; and Roser and
`
`Colaco (1993) New Scientist 138:24-28. Trehalose and a wide variety of
`
`stabilising polyols have also been found useful in formulation of solid dosages.
`
`There is a serious need for a method to inexpensively and stably
`
`incorporate substances into glass matrices with a minimum of residual moisture
`
`remaining in the product. Such a process would provide products exhibiting
`
`increased stability, a longer shelf life, and facile rehydration. Facile rehydration
`
`would be a particular advantage for parenterally administered pharmaceutical
`
`substances.
`
`All references cited herein are hereby incorporated by reference.
`
`DISCLOSURE OF THE INVENTION
`
`The present invention encompasses methods of producing dried foamed
`
`glass matrices (FGMs). The invention also includes methods of stably
`
`incorporating substances, including active substances, within FGMs. Also
`
`included in the present invention are compositions comprising FGMs, as well as
`
`compositions containing substances stably incorporated within FGMs.
`
`Accordingly, one aspect of the invention is methods for producing FGMs,
`
`comprising preparing a mixture comprising at least one glass matrix-forming
`
`material in at least one suitable solvent, evaporating bulk solvent from the
`
`mixture to obtain a syrup, exposing the syrup to a pressure and temperature
`
`sufficient to cause boiling of the syrup, and optionally removing residual
`
`moisture.
`
`In another aspect of the invention, methods are provided for stably
`
`incorporating at least one substance within the FGMs. These methods include
`
`preparing a mixture comprising at least one solvent, at least one glass matrix-
`
`forming material and at least one substance to be incorporated, evaporating bulk
`
`solvent from the mixture to obtain a syrup, exposing the syrup to a pressure and
`
`temperature sufficient to cause boiling of the syrup, and optionally removing
`
`residual moisture. The substances that can be incorporated encompass active
`
`

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`WO 96/40077
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`PCT/GB96/01367
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`4
`
`materials. The methods can be enhanced by the addition to the solution of
`
`various additives such as volatile salts, decomposing salts, organic solvents,
`
`surface active agents and viscosity modifiers.
`
`Another aspect of the invention encompasses methods for producing
`
`stable, dried, readily soluble single dosages of a substance which is unstable in
`
`solution. These methods include preparing a mixture comprising at least one
`
`glass matrix-forming material and a substance in at least one solvent, evaporating
`
`bulk solvent from the mixture to obtain a syrup, exposing the syrup to a pressure
`
`and temperature sufficient to cause boiling of the syrup, and optionally removing
`
`residual moisture.
`
`The invention encompasses compositions obtained by the methods
`
`described herein. The invention further encompasses compositions comprising
`
`FGMs and compositions comprising FGMs and any substance(s) stably
`
`incorporated therein.
`
`In another aspect, the invention includes methods for reconstituting
`
`substances that are incorporated into the FGMs. The methods include adding a
`
`suitable solvent to the FGMs in an amount sufficient to attain the desired
`
`concentration of the substances incorporated therein.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Figure 1 is a photograph depicting FGMs formed in two differently sized
`
`pharmaceutical vials.
`
`Figure 2 is a photograph depicting the effect of varying pressures on FGM
`
`formation (Fig. 2A) and comparison to freeze-drying (Fig. 2B). The samples in
`
`Fig. 2B were of identical composition to those of Fig. 2A, except that the samples
`
`in Fig. 2A were formed into FGMs by the methods described herein, while the
`
`samples in Fig. 2B were freeze-dried.
`
`Figure 3 is a photograph depicting the effect of volatile salts on FGM
`
`formation.
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`WO 96140077
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`Figure 4 is a photograph depicting the effect of varying viscosity on FGM
`
`formation.
`
`Figure 5 is a photograph depicting FGMs containing human red blood
`
`cells.
`
`Figure 6 is a photograph depicting FGMs of trehalose octaacetate made
`
`from organic solution.
`
`Figure 7 is a photograph depicting the formation of FGMs in two pre-
`
`filled syringes.
`
`BEST MODE F OR CARRYING OUT THE INVENTION
`
`It has now been found that glass matrix-forming materials can be
`
`processed into foamed glass matrices (FGMs) that are particularly useful for
`
`stably incorporating substances, such as active substances, particularly including
`
`bioactive substances. As used herein, a "substance" is any substance having an
`
`intended use that can be stored in a dry, non-liquid state.
`
`The methods of this invention result in products with markedly reduced
`
`residual moisture content compared to thick, unfoamed glasses, resulting in a
`
`drier product with increased stability and higher glass transition temperatures.
`
`Further, the high surface area afforded by FGMs results in significantly increased
`
`dissolution rates on reconstitution. This is especially useful for low solubility
`
`substances such as organic substances, including, but not limited to, Cyclosporin
`
`A, lipids, esterified sugars, beta blockers, H2 agonists and antagonists, steroids,
`
`sex hormones, phenobarbitals, analgesics, antimicrobials, antivirals, insecticides,
`
`pesticides and the like.
`
`These methods produce products which provide all of the benefits and
`
`none of the drawbacks of freeze—drying. These drawbacks include, but are not
`
`limited to, long and energy-intensive drying processes using extremely low
`
`temperatures and increased product dissolution times. The products encompassed
`
`by the present invention are rapidly dissolved, with complete solubilization of the
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`

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`WO 96/40077
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`PCT/GB96/01367
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`6.
`
`product that can be easily determined visually. The methods are straightforward,
`
`standardised, and reproducible.
`
`Any material that can be formed into a glass matrix is suitable in this
`
`invention. Suitable materials include, but are not limited to, all polyols, including
`carbohydrate and non-carbohydrate polyols. Particularly suitable materials
`
`include sugars, sugar alcohols and carbohydrate derivatives.
`
`FGMs are useful for storing any substance. FGMs are particularly useful
`
`for poorly soluble substances such as organic substances. Additionally, FGMs are
`
`particularly suitable for dyes, flavourings, biomolecules, molecular assemblies,
`
`cells and other unstable substances.
`
`In accordance with this invention, it is now
`
`possible to produce single-dosage units of bioactive substances which are storage
`
`stable at ambient and even elevated temperatures. For instance, the single dosage
`
`form can be produced in a syringe to form prefilled syringes. This eliminates
`
`steps that can cause contamination of substances prior to injection and eliminates
`
`dosage errors. Upon reconstitution, a single dosage of the bioactive substance is
`
`obtained. Single dosages can be, for instance, a single therapeutic dosage of a
`
`biological substance such as epinephrine, erythropoietin, cytokines, growth
`
`factors and other biopharrnaceuticals or a single reaction mix such as that required
`for ovulation and pregnancy tests and other diagnostic kits. Due to the increased
`
`stability of the biological substances, storage and shipping are greatly improved.
`
`‘
`
`The present invention encompasses methods of producing FGMs. The
`
`methods comprise the steps of preparing a mixture of at least one glass matrix-
`
`forming material in a solvent therefor, evaporating bulk solvent from the mixture
`
`to obtain a syrup, exposing the syrup to a pressure and temperature sufficient to
`
`cause boiling of the syrup and optionally removing residual moisture.
`
`As used herein, "foamed glass matrix" ("FGM") is a high surface area
`
`foamed glass matrix. FGMs can be of varying thickness, including thin or ultra-
`thin. Typically, the FGM is much less dense than the solid dosage amorphous
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`glass, because of the increased surface area and the thinness of glass forming the
`
`bubble walls of the foamed glass matrix.
`
`Preferably, the glass matrix-forming material is a stabilising polyol and
`
`more preferably it is a carbohydrate and derivatives thereof, including trehalose,
`
`lactitol and palatinit. Most preferably, the stabilising polyol is trehalose. Suitable
`
`stabilising polyols are those in which a desired substance can be dried and stored
`
`without substantial losses in activity by denaturation, aggregation Or other
`
`mechanisms.
`
`As used herein, the term "carbohydrates" includes, but is not limited to,
`
`monosaccharides, disaccharides, trisaccharides, oligosaccharides and their
`
`corresponding sugar alcohols, polyhydroxy compounds such as carbohydrate
`
`derivatives and chemically modified carbohydrates, hydroxyethyl starch and
`
`sugar copolymers. Both natural and synthetic carbohydrates are suitable for use
`
`herein. Synthetic carbohydrates include, but are not limited to, those which have
`
`the glycosidic bond replaced by a thiol or carbon bond. Both D and L forms of
`
`the carbohydrates may be used. The carbohydrate may be non-reducing or
`
`reducing.
`
`Prevention of losses of activity can be enhanced by the addition of various
`
`additives such as inhibitors of the Maillard reaction as described below. Addition
`
`of such inhibitors is particularly preferred in conjunction with reducing
`
`carbohydrates.
`
`Reducing carbohydrates suitable for use in the present invention are those
`
`known in the art and include, but are not limited to, glucose, maltose, lactose,
`
`fructose, galactose, mannose, maltulose, and lactulose. Non-reducing
`
`carbohydrates include, but are not limited to, non-reducing glycosides of
`
`polyhydroxy compounds selected from sugar alcohols and other straight chain
`
`polyalcohols. Other useful carbohydrates include raffinose, stachyose,
`
`melezitose, dextran, sucrose, cellibiose, mannobiose and sugar alcohols. The
`
`sugar alcohol glycosides are preferably monoglycosides, in particular the
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`WO 96/40077
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`8
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`compounds obtained by reduction of disaccharides such as lactose, maltose,
`
`lactulose and maltulose.
`
`Particularly preferred carbohydrates are trehalose, maltitol (4-O—B-D-
`
`g]ucopyranosyl-D-glucitol), lactitol (4-0- B-D-galactopyranosyl-D-glucitol),
`
`palatinit [a mixture of GPS (a-D-glucopyranosyl-l—>6-sorbitol) and GPM (or-D-
`
`glucopyranosyl-l—>6-mannitol)], and its individual sugar alcohol components
`
`GPS and GPM.
`
`Different mixtures and various container shapes and sizes can be
`
`processed simultaneously. Ideally, the container size used is sufficient to contain
`
`the initial mixture and accommodate the‘volume of the FGM formed thereof.
`
`Typically, this is determined by the mass of the glass matrix-forming material, the
`surface area of the container and the conditions of FGM formation. The mass of
`
`glass matrix—forming material must be sufficient to give a viscous syrup to be
`
`foamed which translates practically as a minimal mass per unit area of container
`
`surface. This ratio varies from mixture to mixture and container used but is easily
`
`determined empirically by one skilled in the art by following the procedures set
`
`forth herein. Any such vials can be used, including Wheaton moulded and tube-
`
`cut vials. Figure 1 is an illustration of FGMs formed in differently sized vials.
`
`Although singular forms may be used herein, more than one glass matrix-
`
`forming material, more than one additive, and more than one substance may be
`
`present. Effective amounts of these components are easily determined by one of
`
`skill in the art.
`
`The solvent into which the glass matrix-fuming material is mixed can be
`
`aqueous, organic, or a mixture of both. The use of combinations of organic and
`
`aqueous solvents can provide an additional benefit, as the use of a volatile organic
`solvent enhances the foamed glass formation. Enhanced foamed glass formation
`
`can also be achieved by using a volatile or decomposing salt as discussed below.
`
`Additionally, sufficient aqueous solvent to dissolve the glass matrix-forming
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`9
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`material and sufficient organic solvent to dissolve a hydrophobic substance may
`be used, allowing the formation of FGMs incorporating hydrophobic substance(s).
`
`The choice of solvent will depend upon the nature of the material chosen
`
`for glass matrix formation, as well as the nature of any additive and/or substance
`
`to be incorporated. The solvent should be of a nature and of sufficient volume to
`
`effect adequate solubilization of the glass matrix—forming material as well as any
`
`additive and/or substance. If the substance is a hydrophilic material, the liquid
`
`will preferably be aqueous to avoid any potential loss of activity due to
`
`deleterious solvent interactions. Preferably, the aqueous solvent includes any
`
`suitable aqueous solvent known in the art, including, but not limited to, water and
`
`biological buffer solutions. Preferably, the aqueous solvent is present in an
`
`amount of 5 to 95% by volume.
`
`The volume of the solvent can vary and will depend upon the glass
`
`matrix-forming material and the substance to be incorporated as well as any
`
`additives. The minimum volume required is an amount necessary to solubilise the
`
`various components. However, homogeneously dispersed suspensions of the
`
`substance(s) can also be used. Suitable amounts of the components in specific
`
`embodiments are easily determinable by those skilled in the art in light of the
`
`examples provided herein.
`
`Various additives can be put into the glass matrix-forming material.
`
`Typically, the additives enhance foam formation and/or the drying process or
`
`contribute to the solubilization of the substance. Alternatively, the additives
`
`contribute to the stability of the substance incorporated within the FGM. One or
`
`more additives may be present.
`
`As an example, addition of volatile salts allows larger initial volumes and
`
`results in a higher surface area within the FGMs, thus effecting superior foam
`
`formation and more rapid drying. As used herein, volatile salts are salts which
`
`volatilize under the conditions used to produce FGMs. Examples of suitable
`
`volatile salts include, but are not limited to, ammonium acetate, ammonium
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`bicarbonate and ammonium carbonate. Salts that decOmpose to give gaseous
`
`products also effect enhanced foam formation and more rapid drying. Examples
`
`of such salts are sodium bicarbonate and sodium metabisulphite. Preferably, the
`
`volatile salts are present in an amount of from abOut 0.01 to 5 M. Concentrations
`
`of up to 5 M are suitable for use herein. The resultant FGMs have uniform foam
`
`conformation and are significantly drier compared to FGMs in which volatile salts
`
`are not used. The effect of volatile salts on FGM formation is shown in Fig. 3
`
`(see Example 4a).
`
`Volatile organic solvents can also be used in the initial mixture in order to
`
`improve the formation of FGMs. Examples of suitable volatile organic solvents
`
`include, but are not limited to, alcohols, ethers, oils, liquid hydrocarbons and their
`
`derivatives. While the volatile organic solvent may be used as the sole solvent for
`
`the glass matrix-forming material and/or substance, they are more commonly used
`
`in aqueous/organic mixtures. Preferably, the aqueous component of the mixture
`
`comprises between 5-80% by weight of the mixture, and more preferably
`
`comprises 10-50% by weight.
`
`Another suitable additive is a foam stabilising agent, which can be used in
`
`combination with either the volatile or decomposing salt and/or organic solvent.
`
`This may either be a surface active component such as an amphipathic molecule
`
`(i.e., such as phospholipids and surfactants) or an agent to increase the viscosity
`
`of the foaming syrup, such as a thickening agent such as guar gum and their
`
`derivatives. Figure 4 illustrates the effect of varying viscosity on FGM formation
`
`(Example 40).
`
`Another additive is an inhibitor of the Maillard reaction. Preferably, if the
`
`substance and/or glass matrix-forming material contains carbonyl and amino,
`
`imino or guanidino groups, the compositions further contain at least one
`
`physiologically acceptable inhibitor of the Maillard reaction in an amount
`
`effective to substantially prevent condensation of amino groups and reactive
`
`carbonyl groups in the composition. The inhibitor of the Maillard reaction can be
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`WO 96140077
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`any known in the art. The inhibitor is present in an amount sufficient to prevent,
`
`or substantially prevent, condensation of amino groups and reactive carbonyl
`
`groups. Typically, the amino groups are present on the substance and the
`
`carbonyl groups are present on the glass matrix forming material, or the converse.
`
`However, the amino and carbonyl groups may be intramolecular within either the
`
`substance or the carbohydrate.
`
`Various classes of compounds are known to exhibit an inhibiting effect on
`
`the Maillard reaction and hence to be of use in the compositions described herein.
`
`These compounds are generally either competitive or non-competitive inhibitors
`
`of the Maillard reaction. Competitive inhibitors include, but are not limited. to,
`
`amino acid residues (both D and L), combinations of amino acid residues and
`
`peptides. Particularly preferred are lysine, arginine, histidine and tryptophan.
`
`Lysine and arginine are the most effective. There are many known non-
`
`competitive inhibitors. These include, but are not limited to, aminoguanidine and
`
`derivatives and amphotericin B. EP-A-0 433 679 also describes suitable Maillard
`
`inhibitors which include 4-hydroxy-5,8-dioxoquinoline derivatives.
`
`Substances to be incorporated into the FGMs are added to the mixture
`
`before the foaming step. A wide variety of substances can be incorporated. For
`
`example, bioactive substances such as pharmaceutical agents and biological
`
`modifiers, as well as whole cells such as red blood cells and platelets, can be
`
`processed according to the methods described herein.
`
`Any substance that can be homogeneously suspended in a solution of a
`solvent and glass matrix-forming material can be processed using these methods.
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`FGMs have a greatly increased surface area compared to the mixture, a solid
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`dosage form or any previously described composition. The increased surface area
`allows facile dissolution and therefore this invention is applicable to a large
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`number of substances. Determining whether a substance is suitable for use herein
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`is within the skill of one in the art, and the examples provided herein are
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`illustrative and non-limiting. By foaming a homogeneous suspension, areas of
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`unevenly distributed substance, which could be deleterious for dissolution, are
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`avoided in FGMs. More preferably, the substance will be solubilized in the
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`solvent used in the initial mixture.
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`Examples of substances that can be incorporated within the FGMs include
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`any bioactive substances such as pharmaceutically effective substances, including,
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`but not limited to, antiinflammatory drugs, analgesics, antiarthritic drugs,
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`antispasmodics, antidepressants, antipsychotics, tranquillisers, antianxiety drugs,
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`narcotic antagonists, antiparkinsonism agents, cholinergic agonists,
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`chemotherapeutic drugs, immunosuppressive agents, antiviral agents,
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`antimicrobial agents, appetite suppressants, anticholinergics, antiemetics,
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`antihistaminics, antimigraine agents, coronary, cerebral or peripheral vasodilators,
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`hormonal agents, contraceptives, antithrombotic agents, diuretics,
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`antihypertensive agents, cardiovascular drugs, opioids, and the like.
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`Suitable substances also include therapeutic and prophylactic agents.
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`These include, but are not limited to, any therapeutically effective biological
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`modifier. Such substances include, but are not limited to, subcellular
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`Compositions, cells, bacteria, viruses and molecules including, but not limited to,
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`lipids, organics, proteins and peptides (synthetic and natural), peptide mimetics,
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`hormones (peptide, steroid and corticosteroid), D and L amino acid polymers,
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`oligosaccharides, polysaccharides, nucleotides, oligonucleotides and nucleic
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`acids, including DNA and RNA, protein nucleic acid hybrids, small molecules
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`and physiologically active analogues thereof. Further, the modifiers may be
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`derived from natural sources or made by recombinant or synthetic means and
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`include analogues, agonists and homologs.
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`As used herein "protein" refers also to peptides and polypeptides. Such
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`proteins include, but are not limited to, enzymes, biopharmaceuticals, growth
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`hormones, growth factors, insulin, antibodies, both monoclonal and polyclonal
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`and fragments thereof, interferons, interleukins and cytokines.
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`Organics include, but are not limited to, pharrnaceutically active moieties
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`with aromatic, carbonyl, amino, imino and guanidino groups.
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`Suitable steroid hormones include, but are not limited to, oestrogen,
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`progesterone, testosterone and physiologically active analogues thereof.
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`Numerous steroid hormone analogues are known in the art and include, but are
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`not limited to, estradiol, SH-135 and tamoxifen. Many steroid hormones such as
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`progesterone, testosterone and analogues thereof are particularly suitable for use
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`in the present invention.
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`Therapeutic nucleic acid-based agents prepared by the methods described
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`herein are also encompassed by the invention. As used herein, "nucleic acids"
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`includes any therapeutically effective nucleic acids known in the art including,
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`'but not limited to DNA, RNA and physiologically active analogues thereof. The
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`nucleotides may encode genes or may be any vector known in the art of
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`recombinant DNA including, but not limited to, plasmids, retroviruses and adeno-
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`associated viruses.
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`Substances which are prophylactically active and carriers therefor are
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`further encompassed by the invention. Preferable compositions include
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`immunogens such as vaccines. Suitable vaccines include, but are not limited to,
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`live and attenuated viruses, nucleotide vectors encoding antigens, live and
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`attenuated bacteria, antigens, antigens plus adjuvants and haptens coupled to
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`carriers. Particularly preferred are vaccines effective against diphtheria, tetanus,
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`pertussis, botulinum, cholera, Dengue, Hepatitis A, B, C and E, Haemophilus
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`influenza b, herpes virus, Helicobacterium pylori, influenza, Japanese
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`encephalitis, meningococci A, B and C, measles, mumps, papilloma virus,
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`pneumococci, polio, rubella, rotavirus, respiratory syncytial virus, Shigella,
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`tuberculosis, yellow fever and combinations thereof. The antigenic component of
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`vaccines may also be produced by molecular biology techniques to produce
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`recombinant peptides or fusion proteins containing one or more portions of a
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`protein derived from a pathogen. For instance, fusion proteins containing an
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`antigen and the B subunit of cholera toxin have been shown to induce an immune
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`response to the antigen. Sanchez et al. (1989) Proc. Natl. Acad Sci. USA 86:48]-
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`.485. Vaccines are particularly suitable for incorporation into the single-dosage
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`composition. They are stable indefinitely under ambient conditions and can be
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`redissolved in sterile diluent immediately before inoculation.
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`Preferably, the immunogenic composition contains an amount of an
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`adjuvant sufficient to enhance the immune response to the immunogen. Suitable
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`adjuvants include, but are not limited to, aluminium salts, squalene mixtures
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`(SAF-l), muramyl peptide, saponin derivatives, mycobacterium cell wall
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`preparations, monophosphoryl lipid A, mycolic acid derivatives, non-ionic block
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`copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and
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`derivatives, and immunostimulating complexes (ISCOMs) such as those described
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`by Takahashi et al. (1990) Nature 344:873-875. For veterinary use and for
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`production of antibodies in animals, mitogenic components of Freund's adjuvant
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`can be used.
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`As with all immunogenic compositions, the immunologically effective
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`amounts of the immunogens must be determined empirically. Factors to be
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`considered include the immunogenicity, whether or not the immunogen will be
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`complexed with or covalently attached to an adjuvant or carrier protein or other
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`carrier, route of administration and the number of immunising dosages to be
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`administered. Such factors are known in the vaccine art and it is well within the
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`skill of immunologists to make such determinations without undue
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`experimentation.
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`The substance can be present in varying concentrations in the FGMs.
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`Typically, the minimum concentration of the substance is an amount necessary to
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`achieve its intended use, while the maximum concentration is the maximum
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`amount that will remain in solution or homogeneously suspended within the
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`initial mixture. For instance, the minimum amount of a therapeutic agent is
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`preferably one which will provide a single therapeutically effective dosage.
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`Super-saturated solutions can also be used if the FGM is formed prior to
`crystallisation For bioactive substances, the minimum-concentration is an
`
`amount necessary for bioactivity upon reconstitution and the maximum
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`concentration is the point at which a homogeneous suspension cannot be
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`maintained.
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`In the case of single-dosage units, the amount is that of a single
`
`therapeutic application. For instance, Neupogen® is delivered at a dosage of
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`300ug (li0.6 x 108 U/mg; Sug/kg/day). Thus, 300ug would be processed per
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`via] to provide a single dosage format, The preferred amount of the substance
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`varies from substance to substance but is easily determina