U.S. patent application number 11/081356 was filed with the patent office on 2005-08-25 for methods for stably incorporating substances within dry, foamed glass matrices and compositions obtained thereby.
This patent application is currently assigned to Elan Drug Delivery Limited. Invention is credited to Gribbon, Enda Martin, Roser, Bruce.
Application Number | 20050186254 11/081356 |
Document ID | / |
Family ID | 34864363 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186254 |
Kind Code |
A1 |
Roser, Bruce ; et
al. |
August 25, 2005 |
Methods for stably incorporating substances within dry, foamed
glass matrices and compositions obtained thereby
Abstract
The invention provides methods for producing foamed glass and
the compositions obtained thereby. The compositions are suitable
for stable storage of a wide variety of substances, particularly
biological and pharmaceutical.
Inventors: |
Roser, Bruce; (Cambridge,
GB) ; Gribbon, Enda Martin; (Cambridge, GB) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Assignee: |
Elan Drug Delivery Limited
Nottingham
GB
|
Family ID: |
34864363 |
Appl. No.: |
11/081356 |
Filed: |
March 15, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11081356 |
Mar 15, 2005 |
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08923783 |
Sep 4, 1997 |
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08923783 |
Sep 4, 1997 |
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08486043 |
Jun 7, 1995 |
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Current U.S.
Class: |
424/440 |
Current CPC
Class: |
A61K 9/2018 20130101;
A61K 9/2095 20130101; Y02A 50/30 20180101; C12N 1/04 20130101; Y02A
50/465 20180101 |
Class at
Publication: |
424/440 |
International
Class: |
A61K 009/68; A61K
031/47 |
Claims
1-77. (canceled)
78. A pharmaceutical composition comprising a foamed glass
matrix.
79. A pharmaceutical composition comprising at least one substance
incorporated into foamed glass matrices (FGMs).
80. A composition obtainable by reconstituting the foamed glass
matrices of claim 79.
81. The composition according to claim 79, wherein the substance is
a bioactive substance.
82. A composition obtainable by reconstituting the foamed glass
matrices (FGMs) of claim 81.
83. A composition comprising a foamed glass matrix and a
therapeutic or diagnostic agent incorporated within the matrix,
wherein the agent is an organic molecule or a virus.
84. A composition comprising a foamed glass matrix and a
therapeutic or diagnostic agent incorporated within the matrix,
wherein the matrix is formed from a carbohydrate.
85. A composition according to claim 84, wherein the agent is a
cell or bacterial microorganism
86. A composition according to claim 83 wherein the agent is a
virus.
87. A composition according to claim 84, wherein the agent is a
virus.
88. A composition according to claim 83, wherein the agent is a
protein, peptide or nucleic acid.
89. A composition according to claim 84, wherein the agent is a
protein, peptide or nucleic acid.
90. A composition according to claim 83 wherein the foamed glass
matrix is formed from a stabilizing polyol.
91. A composition according to claim 86 wherein the foamed glass
matrix is formed from a stabilizing polyol.
92. A composition according to claim 88 wherein the foamed glass
matrix is formed from a stabilizing polyol.
93. A composition according to claim 90, wherein the stabilizing
polyol is a carbohydrate.
94. A composition according to claim 91 wherein the stabilizing
polyol is a carbohydrate.
95. A composition according to claim 92 wherein the stabilizing
polyol is a carbohydrate.
96. A composition according to any of claims 78 to 95, wherein the
matrix is formed from glucose, sucrose or lactose.
97. A composition according to any of claims 78 to 95, wherein the
matrix is formed from trehalose.
98. A composition comprising a foamed glass matrix and,
incorporated within the matrix, a therapeutic or prophylactic
agent, wherein the agent is a virus; a lipid; an organic; a
synthetic protein or peptide; a natural or synthetic protein or
peptide selected from: an enzyme, a growth hormone, a growth
factor, insulin, an antibody, an interferon, an interleukin or a
cytokine; a peptide mimetic; a hormone; a D or L amino acid
polymer; an oligosaccharide; a polysaccharide; a nucleotide; an
oligonucleotide; a nucleic acid; a protein nucleic acid hybrid; or
a small molecule and physiologically active analogues thereof.
99. A composition comprising a foamed glass matrix and,
incorporated within the matrix, a therapeutic or prophylactic
agent, wherein the matrix is formed from a carbohydrate selected
from: a monosaccharide, a disaccharide selected from maltose,
iso-maltose, lactose, maltulose, iso-maltulose, lactulose, sucrose,
cillibiose and mannobiose, a trisaccharide, a sugar alcohol, an
oligosaccharide or its corresponding sugar alcohol, a carbohydfrate
derivative, a chemically modified carbohydrate, hydroxyethyl
starch, a sugar copolymer, a synthetic carbohydrate or a straight
chain polyalcohol.
Description
TECHNICAL FIELD
[0001] 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 OF THE INVENTION
[0002] 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 losses. Pikal (1994) ACS
Symposium 567:120-133. Furthermore, many of the various parameters
within the freeze-drying process remain poorly characterized,
sometimes resulting in the loss of whole batches at the production
level.
[0003] 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 567:134-147.
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
substance. This imposes inherent instability upon storage of the
freeze-dried substances.
[0004] 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 al. (1990) Cryobiol. 27:219-231. However, ambient
temperature drying often yields denatured or even inactive
substances unless an appropriate stabilizer 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 are problems in removing residual moisture.
[0005] Trehalose,
.alpha.-D-glucopyranosyl-.alpha.-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
U.S. Pat. Nos. 4,891,319; 5,149,653; 5,026,566; Blakely et al.
(1990) Lancet 336:854; Roser (July 1991) Trends in Food Sci. and
Tech., pp. 166-169; Colaco et al. (1992) Biotechnol. Internat., pp.
345-350; Roser (1991) BioPharm. 4:47; Colaco et al. (1992)
Bio/Tech. 10:1007; Roser and Colaco (1993) New Scientist 138:25-28;
and Crowe (1983) Cryobiol. 20:346-356. Trehalose also stabilizes
lyophilized proteins, such as methanol dehydrogenase (Argall and
Smith (1993) Biochem. Mol. Biol. Int. 30:491), and to confer
thermoprotection to enzymes from yeast. Hottiger et al. (1994) Eur.
J. Biochem. 219:187. Trehalose also inhibits the Maillard reaction
between carbonyl groups of reducing sugars and amino groups of
proteins. Loomis et al. (1979) J. Exp. Zool. 208:355-360; and Roser
and Colaco (1993) New Scientist 138:24-28. Trehalose and a wide
variety of stabilizing polyols have also been found useful in
formulation of solid dosages.
[0006] 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.
[0007] All references cited herein are hereby incorporated by
reference.
SUMMARY OF THE INVENTION
[0008] 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.
[0009] 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 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.
[0010] 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 materials. The methods can be
enhanced by the addition to the solution of various additives such
as volatile salts, decomposing salts, organic solvents and
viscosity modifiers.
[0011] 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.
[0012] 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.
[0013] 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
[0014] FIG. 1 is a photograph depicting FGMs formed in two
differently sized pharmaceutical vials.
[0015] FIG. 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.
[0016] FIG. 3 is a photograph depicting the effect of volatile
salts on FGM formation.
[0017] FIG. 4 is a photograph depicting the effect of varying
viscosity on FGM formation.
[0018] FIG. 5 is a photograph depicting FGMs containing human red
blood cells.
[0019] FIG. 6 is a photograph depicting FGMs of trehalose
octaacetate made from organic solution.
MODES FOR CARRYING OUT THE INVENTION
[0020] 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.
[0021] 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, pheriobarbitals, 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 product that can be
easily determined visually. The methods are straightforward,
standardized, and reproducible.
[0022] 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.
[0023] 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,
flavorings, 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.
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 epinephrin,
erythropoietin, cytokines, growth factors and other
biopharmaceuticals or a single reaction mix such as that required
for ovulation and pregnancy tests and other diagnostic kits.
[0024] 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.
[0025] 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 glass, because of the increased
surface area and the thinness of glass forming the bubble walls of
the foamed glass matrix.
[0026] Preferably, the glass matrix-forming material is a
stabilizing polyol and more preferably it is a carbohydrate and
derivatives thereof, including trehalose, lactitol and palatinit.
Most preferably, the stabilizing polyol is trehalose. Suitable
stabilizing polyols are those in which a desired substance can be
dried and stored without substantial losses in activity by
denaturation, aggregation or other mechanisms.
[0027] 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.
[0028] 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.
[0029] 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 compounds obtained by
reduction of disaccharides such as lactose, maltose, lactulose and
maltulose.
[0030] Particularly preferred carbohydrates are trehalose, maltitol
(4-O-.beta.-D-glucopyranosyl-D-glucitol), lactitol
(4-O-.beta.-D-galactopyranosyl-D-glucitol), palatinit [a mixture of
GPS (.alpha.-D-glucopyranosyl-1.fwdarw.6-sorbitol) and GPM
(.alpha.-D-glucopyranosyl-1.fwdarw.6-mannitol)], and its individual
sugar alcohol components GPS and GPM.
[0031] 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. FIG. 1 is an illustration of FGMs formed in
differently sized vials.
[0032] 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.
[0033] The solvent into which the glass matrix-forming 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 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 material and sufficient organic to dissolve a
hydrophobic substance may be used, allowing the formation of FGMs
incorporating hydrophobic substance(s).
[0034] 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.
[0035] 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 solubilize 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.
[0036] 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 additive contributes to the stability
of the substance incorporated within the FGM. One or more additives
may be present.
[0037] 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
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 3).
[0038] 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.
[0039] Another suitable additive is a foam stabilizing 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 (e.g., phospholipids) or
an agent to increase the viscosity of the foaming syrup, such as a
thickening agent such as guar gum and their derivatives. FIG. 4
illustrates the effect of varying viscosity on FGM formation
(Example 3).
[0040] 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 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 noncompetitive 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 noncompetitive inhibitors. These include, but are
not limited to, aminoguanidine and derivatives and amphotericin B.
EP-A-O 433 679 also describes suitable Maillard inhibitors which
include 4-hydroxy-5,8-dioxoquinoline derivatives.
[0041] 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.
[0042] Any substance that can be homogeneously suspended in a
solution of a solvent and glass matrix-forming material can be
processed using these methods. FGMs have a greatly increased
surface area compared to the mixture, a solid dosage form or any
previously described composition. The increased surface area allows
facile dissolution and therefore this invention is applicable to a
large number of substances. Determining whether a substance is
suitable for use herein is within the skill of one in the art, and
the examples provided here are illustrative and non-limiting. By
foaming a homogeneous suspension, areas of unevenly distributed
substance, which could be deleterious for dissolution, are avoided
in FGMs. More preferably, the substance will be solubilized in the
solvent used in the initial mixture.
[0043] Examples of substances that can be incorporated within the
FGMs include any bioactive substances such as pharmaceutically
effective substances, including, but not limited to,
antiinflammatory drugs, analgesics, antiarthritic drugs,
antispasmodics, antidepressants, antipsychotics, tranquilizers,
antianxiety drugs, narcotic antagonists, antiparkinsonism agents,
cholinergic agonists, chemotherapeutic drugs, immunosuppressive
agents, antiviral agents, antimicrobial agents, appetite
suppressants, anticholinergics, antiemetics, antihistaminics,
antimigraine agents, coronary, cerebral or peripheral vasodilators,
hormonal agents, contraceptives, antithrombotic agents, diuretics,
antihypertensive agents, cardiovascular drugs, opioids, and the
like.
[0044] Suitable substances also include therapeutic and
prophylactic agents. These include, but are not limited to, any
therapeutically effective biological modifier. Such substances
include, but are not limited to, subcellular compositions, cells,
bacteria, viruses and molecules including, but not limited to,
lipids, organics, proteins and peptides (synthetic and natural),
peptide mimetics, hormones (peptide, steroid and corticosteroid), D
and L amino acid polymers, oligosaccharides, polysaccharides,
nucleotides, oligonucleotides and nucleic acids, including DNA and
RNA, protein nucleic acid hybrids, small molecules and
physiologically active analogs thereof. Further, the modifiers may
be derived from natural sources or made by recombinant or synthetic
means and include analogs, agonists and homologs. As used herein
"protein" refers also to peptides and polypeptides. Such proteins
include, but are not limited to, enzymes, biopharmaceuticals,
growth hormones, growth factors, insulin, antibodies, both
monoclonal and polyclonal and fragments thereof, interferons,
interleukins and cytokines. Organics include, but are not limited
to, pharmaceutically active moieties with aromatic, carbonyl,
amino, imino and guanidino groups. Suitable steroid hormones
include, but are not limited to, estrogen, progesterone,
testosterone and physiologically active analogs thereof. Numerous
steroid hormone analogs are known in the art and include, but are
not limited to, estradiol, SH-135 and tamoxifen. Many steroid
hormones such as progesterone, testosterone and analogs thereof are
particularly suitable for use in the present invention. Therapeutic
agents prepared by the methods described herein are also
encompassed by the invention. As used herein, "nucleic acids"
includes any therapeutically effective nucleic acids known in the
art including, but not limited to DNA, RNA and physiologically
active analogs thereof. The nucleotides may encode genes or may be
any vector known in the art of recombinant DNA including, but not
limited to, plasmids, retroviruses and adeno-associated
viruses.
[0045] Substances which are prophylactically active and carriers
therefor are further encompassed by the invention. Preferable
compositions include immunogens such as vaccines. Suitable vaccines
include, but are not limited to, live and attenuated viruses,
nucleotide vectors encoding antigens, live and attenuated bacteria,
antigens, antigens plus adjuvants and haptens coupled to carriers.
Particularly preferred are vaccines effective against diphtheria,
tetanus, pertussis, botulinum, cholera, Dengue, Hepatitis A, B, C
and E, hemophilus influenza b, herpes virus, Helicobacterium
pylori, influenza, Japanese encephalitis, meningococci A, B and C,
measles, mumps, papilloma virus, pneumococci, polio, rubella,
rotavirus, respiratory syncytial virus, Shigella, tuberculosis,
yellow fever and combinations thereof. The antigenic component of
vaccines may also be produced by molecular biology techniques to
produce recombinant peptides or fusion proteins containing one or
more portions of a protein derived from a pathogen. For instance,
fusion proteins containing an antigen and the B subunit of cholera
toxin have been shown to induce an immune response to the antigen.
Sanchez et al. (1989) Proc. Natl. Acad. Sci. USA 86:481-485.
Vaccines are particularly suitable for incorporation into the
single-dosage composition. They are stable indefinitely under
ambient conditions and can be redissolved in sterile diluent
immediately before inoculation.
[0046] Preferably, the immunogenic composition contains an amount
of an adjuvant sufficient to enhance the immune response to the
immunogen. Suitable adjuvants include, but are not limited to,
aluminum salts, squalene mixtures (SAF-1), muramyl peptide, saponin
derivatives, mycobacterium cell wall preparations, monophosphoryl
lipid A, mycolic acid derivatives, nonionic block copolymer
surfactants, Quil A, cholera toxin B subunit, polyphosphazene and
derivatives, and immunostimulating complexes (ISCOMs) such as those
described by Takahashi et al. (1990) Nature 344:873-875. For
veterinary use and for production of antibodies in animals,
mitogenic components of Freund's adjuvant can be used.
[0047] As with all immunogenic compositions, the immunologically
effective amounts of the immunogens must be determined empirically.
Factors to be considered include the immunogenicity, whether or not
the immunogen will be complexed with or covalently attached to an
adjuvant or carrier protein or other carrier, route of
administration and the number of immunizing dosages to be
administered. Such factors are known in the vaccine art and it is
well within the skill of immunologists to make such determinations
without undue experimentation.
[0048] The substance can be present in varying concentrations in
the FGMs. Typically, the minimum concentration of the substance is
an amount necessary to achieve its intended use, while the maximum
concentration is the maximum amount that will remain in solution or
homogeneously suspended within the initial mixture. For instance,
the minimum amount of a therapeutic agent is preferably one which
will provide a single therapeutically effective dosage.
Super-saturated solutions can also be used if the FGM is formed
prior to crystallization. For bioactive substances, the minimum
concentration is an amount necessary for bioactivity upon
reconstitution and the maximum concentration is the point at which
a homogeneous suspension cannot be maintained. In the case of
single-dosage units, the amount is that of a single therapeutic
application. For instance, Neupogen.RTM. is delivered at a dosage
of 300 .mu.g (1.+-.0.6.times.10.sup.8 U/mg; 5 .mu.g/kg/day). Thus,
300 .mu.g would be processed per vial to provide a single dosage
format. The preferred amount of the substance varies from substance
to substance but is easily determinable by one of skill in the
art.
[0049] In the primary drying step, the solvent is evaporated to
obtain a syrup. Typically, a "syrup" is defined as a solution with
a viscosity in the region of 10.sup.6-10.sup.7 Pascal seconds. The
syrup is not defined as a fixed concentration, but is a result of
the bulk of the solvent evaporating from the mixture. Typically, a
syrup is a viscous mixture containing the glass matrix-forming
material and/or additives and/or substances, in a significantly
higher concentration than that of the initial mixture. Typically,
the evaporation step is conducted under conditions sufficient to
remove about 20% to 90% of the solvent to obtain a syrup. The
viscosity of the syrup is preferably such that when the syrup
boils, evaporation from the increased surface area, provided by
extensive bubble formation, results in its vitrification.
[0050] The preferred consistency of the syrup is dependent on the
FGM desired for a particular application. The bubble size is
controlled by the viscosity, rate of boiling and volatile
component(s) or foam stabilizer if used.
[0051] The length of the initial drying step depends on the volume
of solvent and the concentrations of the glass matrix-forming
material(s) and any additives and/or substance(s) in the initial
mixture, as well as the external temperature and pressure. For a
given pressure, the rate of solvent evaporation increases with
external temperature. Because the evaporative process has a cooling
effect on the sample itself, the external temperature can be raised
to increase the evaporation rate without affecting sample
temperature. However, the rate of evaporation within the sample is
inversely proportional to viscosity. As solvent is removed from the
sample, the rate of evaporation thus decreases. This in turn allows
an increase in sample temperature to the boiling point at reduced
pressure.
[0052] The initial drying step can be performed under pressure less
than ambient. Preferably, the pressure is 0.1 to 30 Torr/mm Hg.
Even more preferably, the pressure is 5 to 20 Torr/mm Hg. Most
preferably, the pressure is 7.5 to 12.5 Torr/mm Hg and the external
temperature is 40.degree. C. Aqueous or organic solutions, or
mixtures thereof can be processed under these conditions. Dilute
solutions with concentrations of 10-50% (w/v) are also suitable for
processing under these conditions.
[0053] Reduction of the external pressure has at least two
desirable effects. Firstly, it reduces the vapor pressure of the
solvent in the gas phase, thus accelerating evaporation and drying.
The increased rate of evaporation causes evaporative cooling of the
samples unless external heat is applied to replace the latent heat
of evaporation. Under vacuum, the rate of drying is limited by this
energy input. Thus, the effect of increasing the external
temperature is, surprisingly, to accelerate the rate of drying and
not to increase the sample temperature. The second effect of
reduced external pressure is to drastically lower the boiling point
of the sample. Boiling can therefore be conducted by a very modest
rise in sample temperature which does not have a deleterious effect
on the product.
[0054] The syrup obtained from the primary drying step is exposed
to a reduced pressure to effect boiling of the syrup. As used
herein, "boiling" is defined as the point at which the vapor
pressure of the mixture is equal to or exceeds the external
pressure to which the sample is exposed. Boiling is evidenced
visually by bubbling as the solvent and/or other volatile
components rapidly vaporize. Typically, the most important factor
determining sample boiling temperature is the external pressure. If
a lower boiling point is desired to preserve the integrity of the
substance, the external pressure is chosen is less than atmospheric
(i.e., a vacuum), thus lowering the temperature required for
boiling. Because the boiling step is thus achieved at lower
temperatures, the integrity of the substance is not
jeopardized.
[0055] If reduced pressure is used, rapid drying continues until
the viscosity of the sample begins to increase. At this point, the
reduced mobility of water molecules through the viscous syrup
reduces the rate of evaporative cooling and the sample temperature
rises until it reaches the boiling point at the reduced pressure.
On boiling, a large increase in the area of the liquid/gas
interface occurs due to the bubbling of the syrup. This increased
evaporative surface causes a sharp increase in the drying rate and
the liquid foam dries into solid glass foam. Typically, this occurs
soon after boiling.
[0056] Temperatures for the boiling step can be above or below
ambient temperature. Preferably, the external temperature for the
boiling step is 5 to 80.degree. C. Most preferably, the external
temperature is 15 to 60.degree. C.
[0057] Preferably, the external pressure during the boiling step is
20 to 0.01 Torr/mm Hg. More preferably, the external pressure is
0.1 to 0.05 Torr/mm Hg. FIG. 2 shows the effect of varying vacuum
pressure on FGM formation. For creation of a vacuum, any vacuum
drier with control, preferably programmable control, of the vacuum
pressure and external temperature can be used. The pump must be
capable of providing a vacuum of 0.01 Torr/mm Hg and evacuating the
product chamber down to 0.2-0.01 Torr/mm Hg in 15-20 mins. The
machines used in the present work were the FTS Systems Inc. (Stone
Ridge, N.Y.) Model TDS 00078-A with a VP-62P vacuum pump and a
FD-00057-A condenser module or the Labconco, Inc. (Kansas City)
Model No 77560 with a Lyph-Lock 12 condenser unit and an Edwards
E2M8 two-stage vacuum pump.
[0058] The boiling step results in formation of bubbles which
greatly increases the evaporative surface area of the syrup. This
allows increased evaporation of residual solvent and the FGM
vitrifies as a solid foam of the bubbles which result from the
boiling step. The endpoint of the boiling step can be determined by
an increase in sample temperature, which is preferably maintained
for a period to ensure complete drying. This varies from sample to
sample but is easily determinable by one of skill in the art.
[0059] Residual moisture may be optionally removed to assure
complete drying. This step typically occurs at elevated temperature
and/or reduced pressure. Preferably, the final product should have
a residual moisture content of approximately 0.1-5% (w/w).
Preferably, the residual moisture is removed within 1-15 hours. The
residual moisture is removed in shorter times at elevated
temperatures.
[0060] Because the formation of the FGMs occurs via bubble
formation, the random bubble arrangement and size may give rise to
regions of variable residual moisture content. Thus, during the
secondary drying step, some regions will dry much more readily than
others. As has been discussed above, the presence of a volatile or
decomposing salt and/or volatile organic solvent results in an FGM
with small, uniform bubble size, which leads to lower residual
moisture content and a more homogeneous distribution thereof.
[0061] Materials incorporated in trehalose glasses can be stored at
ambient temperatures for at least 3 years. Active substances
incorporated in glasses formed from other polyols can also show
extended storage stabilities.
[0062] The FGMs can be reconstituted immediately upon addition of
suitable solvent. Thus, the invention includes methods of
reconstituting substances that have been incorporated into the
FGMs. The nature and amount of the solvent will depend upon the
type and amount of substance to be reconstituted, as well as the
intended use of the reconstituted substance. Typically, a minimum
amount of solvent, in an amount necessary to effect solubilization
of the glass matrix and the substance will be added. If the
substance is a pharmaceutical or bioactive, reconstitution is
preferably with a biologically acceptable buffer. Reconstitution
can be performed at any temperature, provided it does not
substantially harm the activity of the substance. Preferably,
reconstitution is at ambient temperatures.
[0063] The invention also encompasses single-dosage units of active
substances which are storage stable at ambient and even elevated
temperatures (in some instances up to 100.degree. C.) and which
upon reconstitution with a premeasured aliquot of a suitable,
preferably sterile solvent, forms a therapeutically effective
dosage of the substance. This is especially effective for use with
therapeutics, including purified and recombinant proteins and
active substances, such as bioactives, which are normally stable in
solution only at or below 4-8.degree. C. Compositions of
single-dosage (or multiple dosage) formats for more stable
products, units and kits containing one or more single-dosage units
and aliquots (preferably premeasured) of suitable solvent are also
encompassed by this invention. Active substances which would be
particularly suitable for storage and reconstitution using the
method of this invention include, but are not limited to, Factor
VIII, Neupogen.RTM., Epogen.RTM., TPA, cytokines, growth hormones,
growth factors, vaccines, lipids, enzymes and other
biopharmaceuticals, as well as other parenterally administered
active substances.
[0064] The invention further encompasses compositions comprising
the glass matrix obtained by the methods described herein. The
compositions include, but are not limited to FGM(s); FGM(s) with
various substances incorporated therein; and reconstituted
substances obtained from FGM(s).
[0065] The following examples are provided to illustrate by not
limit the present invention.
EXAMPLE 1
Effect of Vacuum Pressure and External Temperature on Primary
Drying Times
[0066] 2 ml aliquots of 10% (w/v) trehalose in deionized distilled
water were placed in 10 ml Wheaton pharmaceutical vials and dried
in an FTS drier at various vacuum pressures and shelf temperature
settings. The sample temperatures and the time taken to remove
approximately 90% of the water (i.e. primary drying to give a
syrup) were determined. The results are shown in Table 1.
EXAMPLE 2
Formation of FGMs
2a. Formation from an Aqueous Solution of Glass Matrix-Forming
Material
[0067] Aliquots of 250, 410 .mu.l and 500 .mu.l of a 50% (w/v)
solution of trehalose in 3 ml, 5 ml and 10 ml pharmaceutical vials
respectively, were dried in an FTS drier for 16 hrs. The shelf
temperature was maintained at 25.degree. C. throughout the run and
the vacuum pressure dropped to 0.03 Torr/mm Hg within the first 15
mins of the run and maintained at 0.03 Torr/mm Hg throughout the
run. The FGMs formed are shown in FIG. 1A. The foam-like appearance
is due to the instantaneous drying of the bubbles that form during
the boiling step.
2b. Formation From an Aqueous Solution of Glass Matrix-Forming
Materials Incorporating Active in Solution
[0068] Recombinant Hepatitis B Surface Antigen in 20% (w/v)
trehalose.+-.0.5% (w/v) Byco A in PBS was dried in 300 .mu.l
volumes in 3 ml pharmaceutical vials. The FTS drying protocol
involved a pressure of 0.03 Torr/mm Hg with shelf temperature
maintained at 40.degree. C. throughout a drying cycle of 18 hours.
The mean residual moisture contents of FGMs was in the region of 4%
w/w.
2c. Formation From an Organic Solution of Glass Matrix-Forming
Material
[0069] 500 .mu.l aliquots of 50% (w/v) trehalose octaacetate in
dichloromethane were dried in 10 ml pharmaceutical vials. Shelf
temperature and pressure were maintained at 30.degree. C. and 0.03
Torr/mm Hg respectively, throughout the 16 hour drying cycle. The
FGMs formed are shown in FIG. 6. Rapid dissolution of the FGMs was
observed on reconstitution.
2d. Formation From an Aqueous/Organic Mixture Containing Glass
Matrix-Forming Material and Active Substance
[0070] 750 .mu.l aliquots of a 2:1 mixture of 50% (w/v) trehalose
in deionized distilled water and 100 mg/ml of an organic active
substance, the anaesthetic atracurium, in ethanol was dried in 10
ml pharmaceutical vials in the FTS drier. Shelf temperature and
pressure were maintained at constant values of 40.degree. C. and
0.03 Torr/mm Hg respectively, throughout the 18 hour drying cycle.
Reconstitution of the FGMs in 20% v/v ethanol in deionized
distilled water, resulted in rapid dissolution to give a
homogeneous solution of the anaesthetic.
2e. Formation From an Aqueous Solution of Glass Matrix-Forming
Material, Plus Additive, Incorporating Active Substance in
Homogeneous Suspension
[0071] The inorganic active substance, the adjuvant aluminium
hydroxide, was dried at suspension concentrations of either 2.5 or
6 mg/ml, in either PBS or 0.9% (w/v) saline as the solvent for the
glass matrix-forming material, using the following formulations
which contained a volatile salt additive to improve FGM formation
(see Example 4);
[0072] i) 20% (w/v) trehalose.+-.ammonium bicarbonate
[0073] ii) 50% (w/v) trehalose.+-.ammonium bicarbonate
[0074] iii) 38.5% (w/v) maltose.+-.ammonium bicarbonate
[0075] iv) 25% (w/v) trehalose.+-.ammonium bicarbonate
[0076] 250 and 300 .mu.l samples, containing a range of ammonium
bicarbonate concentrations from 0.05-0.75 M, were dried in 3 ml
pharmaceutical vials using one of the two following FTS
protocols:
[0077] 1) pressure was reduced to 0.03 Torr/mm Hg and shelf
temperature raised at 2 hour intervals from 35.degree. C. to
50.degree. C. and finally 60.degree. C. Total cycle time was
approximately 18 hours. Resultant FGMs had residual moisture
contents in the range of 1.5-2.9% w/w. Reconstitution of the FGMs
was instantaneous.
[0078] 2) pressure was held at 15 Torr/mm Hg for 30 minutes prior
to decreasing to 10 Torr/mm Hg for 30 minutes. Shelf temperature
was raised from 10.degree. C. to 25.degree. C. Pressure was reduced
to 0.03 Torr/mm Hg/mm Hg and held at this for approximately 18
hours. During this stage shelf temperature was raised from
25.degree. C. to 45.degree. C. and 2 hours later to 60.degree. C.
The resultant FGMs resembled freeze-dried plugs and rehydration was
again instantaneous. These results also illustrate the effect of
shelf temperature and vacuum pressure (see Example 3) and volatile
salt additive (see Example 4) on FGM formation, appearance and
residual moisture contents.
EXAMPLE 3
Effect of Vacuum Pressure/Shelf Temperature on FGM Formation
3a. Formation From Solution of Glass Matrix-Forming Material Plus
Additive
[0079] Aliquots of 1 ml or 500 .mu.l of 25% (w/v) trehalose
containing either 0.25 or 0.5 M ammonium bicarbonate, were dried in
10 ml pharmaceutical vials in the FTS drier. The 1 ml samples were
dried at a constant vacuum pressure of 0.03 Torr/mm Hg for 14 hrs,
with shelf temperature initially 25.degree. C., raised to
45.degree. C. after the first 2 hours (i.e., syrup formed). The 500
.mu.l samples were dried at a constant shelf temperature of
25.degree. C. and a constant vacuum-pressure of 0.01 Torr/mm Hg for
14 hr. The FGMs formed (FIG. 2A) occupied larger volumes than
identical samples processed by freeze-drying (FIG. 2B).
3b. Formation From Solution of Glass Matrix-Forming Material
Incorporating an Active
[0080] 300 .mu.l aliquots of a solution of 43.4 mg/ml trehalose
containing 66 mg/ml of an antimicrobial peptide was dried in 10 ml
polypropylene tubes (10 mm diameter) in the FTS drier. Samples, at
25.degree. C., were loaded onto a shelf that had been preheated to
35.degree. C. The vacuum pressure in the chamber was progressively
reduced to 20 Torr/mm Hg over 10 minutes. This pressure was held
for a further 30 minutes before the pressure was further reduced to
0.03 Torr/mm Hg. After 981 minutes the shelf temperature was
increased to 50.degree. C. This shelf temperature was maintained
for 190 minutes after which the cycle was stopped. The FGMs
produced have an open plug-like structure similar to freeze-dried
materials. Moisture content was 1.1 to 1.3% (w/w). Dissolution was
instantaneous on reconstitution. Similar FGMs were produced by the
use of sucrose or GPS instead of trehalose. Elevated temperature
storage of the FGMs containing trehalose as the glass
matrix-forming material at 60.degree. C. and at ambient humidity
showed no shrinking over a period of more than 30 days and the FGM
structure remained intact. Dissolution of samples remained
instantaneous even after storage.
EXAMPLE 4
Effect of Additives on FGM Formation
4a. Effect of Volatile Salt Additive on FGM Formation
[0081] 500 .mu.l aliquots of 3-60% (w/v) trehalose in deionized
distilled water containing a range of concentrations of 0-4 M
Ammonium acetate or bicarbonate were dried in the FTS drier. Shelf
temperature was maintained constant at 20.degree. C. and vacuum
pressure at 0.03 Torr/mm Hg for the 18 hours drying cycle. Residual
moisture contents of the FGMs formed were in the range of 2-5.5%
(w/w) and rehydration was instantaneous on reconstitution. An
example of the FGMs formed is shown in FIG. 3.
4b. Effect of Decomposing Salt Additive on FGM Formation
[0082] 500 .mu.l aliquots of 50% (w/v) trehalose.+-.1M Sodium
metabisulphite were dried in an FTS drier for either 12 or 18
hours. Shelf temperature and pressure were maintained at a constant
40.degree. C. and 0.03 Torr/mm Hg respectively, throughout the
drying cycle. At the shorter drying time of 12 hours, the FGMs
formed from solutions containing the decomposing salt showed
significantly lower residual moisture contents. Rapid dissolution
of all the FGMs formed was observed on reconstitution.
4c. Effect of Viscosity Modifying Additive on FGM Formation
[0083] 500 .mu.l aliquots of 50-90% (w/v) trehalose solutions in
deionized distilled water or PBS, containing 0.5-2% (w/v) guar gum
(Jaguar HP60), were dried in 5 or 10 ml vials in the FTS drier for
16 hours. Initial shelf temperature and vacuum pressure of
30.degree. C. and 30 Torr/mm Hg, respectively, were raised to
60.degree. C. and 0.03 Torr/mm Hg after 2 hours and maintained at
these values for the next 14 hours of the drying cycle.
Representative examples of the FGMs formed are shown in FIG. 4. All
FGMs again showed rapid dissolution on reconstitution in either
water or PBS.
EXAMPLE 5
Illustrative Examples of FGM Formation
5a. Formation of FGM From Glass Matrix-Forming Materials, Plus
Additive, Incorporating Molecular Active Substance in Homogeneous
Solution
[0084] Formulations containing an active, alkaline phosphatase (1
mg/ml) in solution with a mixture of glass matrix-forming
materials, trehalose [in a range of concentrations from 20-50%
(w/v)] and HSA (2% w/v) plus volatile salt additive, ammonium
bicarbonate (50 mM) were prepared in PBS or HEPES buffer. 250 .mu.l
volumes were aliquoted into 3 ml pharmaceutical vials and dried in
the FTS drier. The shelf temperature was initially set at
30.degree. C. and the vacuum pressure altered at 2 minute intervals
from 30 Torr/mm Hg down to 25, 20, 15, 10 and finally 0.03 Torr/mm
Hg before increasing the shelf temperature to 40.degree. C. and
finally 60.degree. C. Total cycle time was approximately 20 hours.
Residual moisture contents of resultant FGM's were approximately 1%
w/w.
5b. Formation of FGM of Glass Matrix-Forming Material Incorporating
Mixtures of Molecular Active Substances in Homogeneous
Suspension
[0085] The commercial vaccine formulation of Hepatitis B surface
antigen adsorbed onto the inorganic adjuvant aluminium hydroxide
was dried in 300 .mu.l volumes of 20% (w/v) trehalose in PBS, in 3
ml pharmaceutical vials. The FTS drying protocol involved a
pressure of 0.03 Torr/mm Hg with shelf temperature maintained at
40.degree. C. throughout a drying cycle of approximately 18 hours.
Mean residual moisture contents of FGMs were approximately 4-4.5%
w/w.
5c. Formation of FGM of Glass Matrix-Forming Materials
Incorporating Macromolecular Active Substances
[0086] Formulations dried to obtain FGMs contained Measles or Oral
Polio Virus at the required dosages and comprised of:
[0087] i) 50% (w/v) trehalose.+-.2% (w/v) HSA.+-.50 mM ammonium
bicarbonate
[0088] ii) 50% (w/v) lactitol.+-.2% (w/v) HSA.+-.50 mM ammonium
bicarbonate
[0089] iii) 40% (w/v) trehalose.+-.10% (w/v) sorbitol.+-.2% (w/v)
HSA.+-.50 mM ammonium bicarbonate
[0090] Samples were prepared using either PBS or HEPES buffer and
250 .mu.l aliquots were dispensed into 3 ml pharmaceutical vials
and dried in the FTS drier using two protocols.
[0091] a) For samples containing the volatile salt additive,
ammonium bicarbonate vacuum pressure was altered at 2 minute
intervals from 30, 25, 20, 15, 10 and finally 0.03 Torr/mm Hg. The
shelf temperature was set at 30.degree. C. initially before
increasing to 40.degree. C. Total cycle time was approximately 20
hours. Residual moisture contents were approximately 2% (w/v) and
all showed rapid dissolution on reconstitution.
[0092] b) For samples that contained no volatile salt additive,
vacuum pressure was set immediately for 0.03 Torr/mm Hg and
maintained throughout the 20 hour drying cycle. The shelf
temperature was set at 30.degree. C. initially before increasing to
40.degree. C. The residual moisture contents of the FGMs formed
were approximately 4% (w/w) and all showed rapid dissolution on
reconstitution.
5d. Formation of FGM of Glass Matrix-Forming Materials
Incorporating Cellular Substances
[0093] Drying Human Red Blood Cells
[0094] 5% (v/v) hematocrit concentration of erythrocytes were
formulated in either:
[0095] i) 50% (w/v) trehalose.+-.50 mM ammonium bicarbonate
[0096] ii) 25% (w/v) trehalose.+-.10% (w/v) hydroxyethyl starch
(HES) in PBS
[0097] 200 .mu.l aliquots were dried in 3 ml pharmaceutical vials
in the FTS drier. The drying protocol used a constant shelf
temperature of 37.degree. C. and vacuum pressure was immediately
reduced to 0.03 Torr/mm Hg. Cycle time was 18 hours. The resulting
FGM's had residual moisture contents of 2.5-3% (w/w) and rehydrated
rapidly on reconstitution.
[0098] Drying Human Blood Platelets
[0099] Platelets at an initial concentration of
500.times.10.sup.9/L were dried in a formulation of 5% (w/v)
trehalose in HEPES buffered saline containing 5 mM potassium
chloride, 1 mM magnesium sulphate, 0.05 U/ml hirudin, 0.0125 U/ml
apyrase, 10 .mu.M indomethacin and 250 mM ammonium bicarbonate. 200
.mu.l aliquots were dried in 3 ml pharmaceutical vials in the FTS
drier. The drying protocol used a constant shelf temperature of
37.degree. C. and vacuum pressure was reduced immediately to 0.03
Torr/mm Hg. Cycle time was 18 hours. The resulting FGM's had mean
residual moisture contents of 1% (w/w) and rehydrated rapidly on
reconstitution.
[0100] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be apparent to those skilled in the art
that certain changes and modifications may be practiced. Therefore,
the description and examples should not be construed as limiting
the scope of the invention, which is delineated by the appended
claims.
* * * * *