U.S. patent application number 14/090599 was filed with the patent office on 2014-03-27 for compositions and methods for stabilized polysaccharide formulations.
This patent application is currently assigned to DEPUY MITEK, LLC. The applicant listed for this patent is DePuy Mitek, LLC. Invention is credited to Julia Hwang, Brooks J. Story, Dongling Su.
Application Number | 20140088038 14/090599 |
Document ID | / |
Family ID | 46458261 |
Filed Date | 2014-03-27 |
United States Patent
Application |
20140088038 |
Kind Code |
A1 |
Su; Dongling ; et
al. |
March 27, 2014 |
COMPOSITIONS AND METHODS FOR STABILIZED POLYSACCHARIDE
FORMULATIONS
Abstract
Compositions and methods are provided for treating joint
conditions, such as osteoarthritis and/or the pain associated
therewith. The compositions and methods utilize a first component,
namely hyaluronic acid ("HA"), in combination with at least one
stabilizer. The composition can include a stabilizer that increases
the stability and shelf-life of the HA. In another embodiment, the
compositions and methods can also include an additional component,
such as one or more glycosaminoglycans ("GAG") or GAG precursors.
Examples of GAGs or GAG precursors can include chondroitin sulfate
("CS"), dermatan sulfate, heparin, heparan sulfate, keratan
sulfate, and glucosamine ("GlcN").
Inventors: |
Su; Dongling; (Franklin,
MA) ; Hwang; Julia; (Wayland, MA) ; Story;
Brooks J.; (Franklin, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DePuy Mitek, LLC |
Raynham |
MA |
US |
|
|
Assignee: |
DEPUY MITEK, LLC
Raynham
MA
|
Family ID: |
46458261 |
Appl. No.: |
14/090599 |
Filed: |
November 26, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13173658 |
Jun 30, 2011 |
8623839 |
|
|
14090599 |
|
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Current U.S.
Class: |
514/54 |
Current CPC
Class: |
A61L 27/20 20130101;
A61K 9/0019 20130101; A61L 27/505 20130101; A61K 31/737 20130101;
A61K 47/02 20130101; A61P 19/04 20180101; A61K 47/26 20130101; A61K
31/728 20130101; A61P 19/02 20180101; A61K 31/726 20130101; A61P
29/00 20180101; A61K 9/19 20130101; A61K 47/10 20130101; A61K
31/728 20130101; A61K 2300/00 20130101; A61K 31/726 20130101; A61K
2300/00 20130101; A61L 27/20 20130101; C08L 5/08 20130101 |
Class at
Publication: |
514/54 |
International
Class: |
A61K 47/26 20060101
A61K047/26; A61K 47/02 20060101 A61K047/02; A61K 47/10 20060101
A61K047/10; A61K 31/728 20060101 A61K031/728; A61K 31/737 20060101
A61K031/737 |
Claims
1. A composition for treating a joint condition, comprising: a
formulation comprising high molecular weight hyaluronic acid (HA);
and at least one stabilizer, wherein the stabilizer increases
stability of the formulation.
2. The composition of claim 1, wherein the high molecular weight HA
has a molecular weight in the range of about 1 MDa to 6 MDa.
3. The composition of claim 1, wherein the HA is present at a
liquid concentration of at least about 1 mg/ml.
4. The composition of claim 1, wherein the HA is lyophilized.
5. The composition of claim 1, wherein the at least one stabilizer
is selected from the group consisting of tocopherol, tocopherol
derivatives, mannitol, sucrose and trehalose.
6. The composition of claim 1, wherein the at least one stabilizer
is trehalose.
7. The composition of claim 1, wherein the at least one stabilizer
is present in a range of about 0.1% to 50% by weight of the
composition.
8. The composition of claim 1, wherein the formulation is stable at
room temperature.
9. The composition of claim 1, further comprising at least one
additional component.
10. The composition of claim 9, wherein the additional component is
selected from the group consisting of a glycosaminoglycan (GAG) and
a GAG precursor and is present within the composition at a ratio of
HA to additional component in the range of about 1:0.005 to
1:100.
11-22. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to compositions and
methods for treating joints.
BACKGROUND OF THE INVENTION
[0002] Osteoarthritis ("OA"), the most common form of arthritis, is
a type of arthritis that is characterized by degenerative (gradual
deterioration of joint) or abnormal changes in bone, cartilage, and
synovium of the joints. OA is often characterized by a progressive
wearing down of opposing joint surfaces accompanied at times by
inflammation resulting in pain, swelling, and stiffness for the
patient. OA can occur in one or more joints following trauma to the
joint, following an infection of the joint, or simply as a result
of aging. Furthermore, there is emerging evidence that abnormal
anatomy, genetics, and obesity may contribute to early development
of OA.
[0003] Treatment of OA generally involves a combination of
exercise, physical therapy, lifestyle modification, and analgesics.
Acetaminophen is typically the first line treatment for OA. For
mild to moderate symptoms, effectiveness is similar to
non-steroidal anti-inflammatory drugs ("NSAIDs"), such as
ibuprofen. For more severe symptoms NSAIDs may be more effective.
However, while more effective, NSAIDs in severe cases are
associated with greater side effects such as gastrointestinal
bleeding and renal complications. Another class of NSAIDs, COX-2
selective inhibitors (such as Celecoxib), are equally effective to
NSAIDs but no safer in terms of side effects. There are several
NSAIDs available for topical use, including diclofenac. Typically,
they have less systemic side-effects than oral administration and
at least some therapeutic effect. While opioid analgesics, such as
morphine and fentanyl, improve pain this benefit is outweighed by
frequent adverse events and thus they are not routinely used.
Intra-articular steroid injections are also used in the treatment
of OA, and they are very effective at providing pain relief,
especially in patients exhibiting inflammatory elements of OA.
However, the duration of the pain relief is limited to 4-6 weeks
and there are adverse effects that may include collateral cartilage
damage. If pain becomes debilitating, joint replacement surgery may
be used to improve mobility and quality of life. There is no proven
treatment to slow or reverse the disease.
[0004] For patients who do not get adequate pain relief from simple
pain relievers, like acetaminophen or from exercise and physical
therapy, intra-articular injections of hyaluronic acid (HA) provide
another treatment option to address symptomatic pain and may delay
the need for a total joint replacement surgery. It is known that
the concentration and molecular weight of native HA is deficient in
individuals suffering from OA and therefore joint injections of
exogenous HA is believed to replenish these molecules and restore
the viscoelastic properties of synovial fluid. It is this property
that is responsible for lubricating and cushioning the joints.
There is also evidence that HA has biological activity through
binding to cell surface receptors and may have a role in mitigating
inflammation and cartilage degradation. Regardless of the mechanism
of action, pain relief is observed for about six months following a
treatment course. A treatment course for HA products on the US
market can range from a single injection to others that require 3
to 5 weekly injections to attain this durability of pain
relief.
[0005] Currently, hyaluronic acid ("HA") formulations on the market
in the United States are commercialized as ready-to-use liquid HA
solutions in prefilled syringes. They can be stored at room
temperature, and typically have a two year shelf life. While HA of
low to moderate molecular weight can be effective, high molecular
weight HA formulas can provide additional benefits, especially at
higher HA concentrations. However, the HA in solution is known to
degrade over time at room temperature, as measured by reduction in
HA molecular weight which could impact its efficacy as an OA
therapy.
[0006] There remains a need for improved methods and compositions
for treating joints, and in particular to improved methods and
compositions for treating joints using high molecular weight HA,
alone or combined with additional components, to address stability
and shelf-life problems associated with current treatments.
SUMMARY OF THE INVENTION
[0007] Compositions and methods are provided for treating joint
conditions, such as osteoarthritis and/or the pain associated
therewith. The methods and compositions disclosed herein are
generally directed to treatments of joints utilizing a first
component, namely hyaluraonic acid ("HA"), in combination with at
least one stabilizer.
[0008] In one aspect, formulations or compositions are disclosed
for treating a joint condition comprising a formulation. The
formulation or composition can be in liquid form. The formulation
can also be stable at room temperature. Moreover, the formulation
can include a solution of hyaluronic acid (HA). The HA formulation
can be a high molecular weight HA. The molecular weight can be, for
example, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,
1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500,
2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600,
3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700,
4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800,
5900, 6000 kDa or more, or any range derivable therein. In
exemplary embodiments, the HA has a molecular weight in the range
of about 1 MDa to 6 MDa. In another exemplary embodiment, the HA
has a molecular weight greater than 1 MDa.
[0009] Moreover, the HA formulation can be present at particular
concentrations either in liquid, solid or lyophilized form. In one
embodiment, the HA is present at a liquid concentration of at least
about 1 mg/ml. In another exemplary embodiment, the HA has a liquid
concentration of at least about 5 mg/ml, and more preferably at
least about 7 mg/ml, and more preferably at least about 10 mg/ml,
and more preferably at least about 15 mg/ml, and in some
embodiments the concentration can be at least about 25 mg/ml. In
another embodiment, the HA can have a concentration in the range of
about 15 mg/ml to about 25 mg/ml.
[0010] In another aspect, the formulation or composition includes
at least one additional component. The additional component added
to the formulation or composition can be, for example, amino acids,
amino sugars, sugar alcohols, proteins, saccharides,
di-saccharides, oligo-saccharides, poly-saccharides, nucleic acids,
buffers, surfactants, lipids, liposomes, other excipients, and
mixtures thereof. Other useful components can include steroids,
anti-inflammatory agents, non-steroidal anti-inflammatory agents,
analgesics, cells, antibiotics, antimicrobial agents,
anti-inflammatory agents, growth factors, growth factor fragments,
small-molecule wound healing stimulants, hormones, cytokines,
peptides, antibodies, enzymes, isolated cells, platelets,
immunosuppressants, nucleic acids, cell types, viruses, virus
particles, essential nutrients, minerals, metals, or vitamins, and
combinations thereof. Additionally, the formulation or composition
can include a diluent, such as water, saline, or a buffer.
[0011] In an exemplary embodiment, the formulation or composition
includes at least one stabilizer or stabilizing excipient, such as
tocopherol, tocopherol derivatives, mannitol, glucose, sucrose and
trehalose. The stabilizers can be present in a range of about
0.1-70% by weight; about 0.1-50% by weight, about 0.1-20% by
weight; or about 0.5-20% by weight. Alternatively, the excipient
can be present at a concentration in a range of about 1 mg/ml to
about 700 mg/ml, and more preferably in a range of about 1 mg/ml to
about 500 mg/ml, and more preferably in a range of about 1 mg/ml to
about 200 mg/ml, and more preferably in a range of about 5 mg/ml to
about 200 mg/ml.
[0012] In another aspect, the formulation or composition includes
at least one additional component, such as a glycosaminoglycan
(GAG), including GAGs like chondroitin sulfate ("CS"), dermatan
sulfate, heparin, heparan sulfate, and keratan sulfate, or a GAG
precursor such as glucosamine ("GlcN"). In one embodiment, a GAG or
GAG precursor can be present in the composition at a concentration
of at least about 0.1 mg/ml, and more preferably at least about 2
mg/ml, and more preferably at least about 5 mg/ml, and more
preferably at least about 7 mg/ml, and more preferably at least
about 20 mg/ml. In another embodiment, the GAGs can have various
molecular weights, but in certain exemplary embodiments the
molecular weight is in the range about 5 to 1,000 kDa, more
preferably in the range of about 6 to 500 kDa, more preferably in
the range of about 7 to 300 kDa, more preferably in the range of
about 8 to 200 kDa, more preferably in the range of about 9 to 100
kDa, and most preferably in the range of about 10 to 80 kDa. In
other embodiments, the molecular weight of a GAG fragment can be
below about 5 kDa and even more preferably below about 3 kDa. In
another embodiment, the GAG precursor can have a molecular weight
of about 180 Da. In other embodiments, the GAG precursor can have a
molecular weight of at least about 100 Da. In an exemplary
embodiment, the GAG precursor can have a molecular weight in the
range of about 100 Da to about 250 Da.
[0013] Moreover, the formulation or composition can have a weight
ratio of HA to stabilizer in the range of about 1:0.001 to about
1:100, and more preferably at a ratio in the range of about
1:0.0125 to about 1:10 about 1:0.00125 to about 1:100, and more
preferably at a ratio in the range of about 1:0.01 to about 1:10.
Alternatively, the formulation or composition can include about 1%
to about 75% or more by weight of each of the individual
components, e.g. HA and stabilizer.
[0014] One more aspect can include a kit and methods of using the
kit. The kit can include a solution of high molecular weight
hyaluronic acid (HA) and at least one stabilizer. The kit can be
stored at room temperature. Additionally, the kit can include a
syringe containing the HA solution and the stabilizer in a single
chamber. Another embodiment can include a single chamber with the
HA solution and stabilizer and a separate chamber with an
additional component, such as a GAG or GAG precursor. For example,
a glycosaminoglycan (GAG) can be present in the kit in a separate
chamber from the HA/stabilizer solution. The additional component
can be present in liquid, solid or lyophilized form. The kit can
further include a diluent, such as water, saline, and a buffer, to
solubilize one of the components, the additional component.
[0015] In an additional aspect, a method for treating joints is
disclosed that includes administering a liquid formulation
comprising high molecular weight hyaluronic acid to a subject in
need. The method can include the high molecular weight hyaluronic
acid in the presence of a stabilizer. In addition, the high
molecular weight hyaluronic acid can be stored in a liquid
formulation at room temperature prior to administration. The method
can also include administering the solution of HA present in the
kit or present in the formulation or composition of high molecular
weight HA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The appended drawings have been included herein so that the
above-recited features, advantages and objects will become clear
and can be understood in detail. These drawings form a part of the
specification. It is to be noted, however, that the appended
drawings illustrate exemplary embodiments and should not be
considered to limit the scope.
[0017] FIG. 1 is a schematic view of one embodiment of a mixing and
delivery system for use with the compositions and methods of the
present invention;
[0018] FIG. 2 is a perspective view of another embodiment of a
mixing and delivery system for use with the compositions and
methods of the present invention;
[0019] FIG. 3 shows the degradation of HA as determined by size
exclusion chromatography-high performance liquid chromatography
(SEC-HPLC) when in a HA and CS saline solution stored at 5.degree.
C. and 40.degree. C./75% RH for 5 months;
[0020] FIG. 4 shows the stabilizing effect trehalose has on HA when
in solution with CS stored at 5.degree. C. and 40.degree. C./75% RH
for 5 months as determined by SEC-HPLC;
[0021] FIG. 5 is a graph comparing cartilage lesion widths in a rat
medial meniscal tear (MMT) model that received intra-articular
injections of trehalose to those that received no treatment;
[0022] FIG. 6 is a graph comparing cartilage lesion depth ratio in
the rat MMT model that received the intra-articular injections of
trehalose to those that received no treatment;
[0023] FIG. 7 is a graph comparing collagen degradation in the rat
MMT model that received the intra-articular injections of trehalose
to those that received no treatment;
[0024] FIG. 8 shows the effect trehalose had on cartilage
degeneration of the medial tibial plateau; and
[0025] FIG. 9 shows the effect trehalose had on cartilage
degradation in a rabbit anterior cruciate ligament transection
(ACLT) model of osteoarthritis.
DETAILED DESCRIPTION OF THE INVENTION
[0026] In general, the present invention provides compositions and
methods for treating joint conditions, such as osteoarthritis
and/or the pain associated therewith. The compositions and methods
utilize a first component, namely high molecular weight hyaluronic
acid ("HA"), alone or in combination with an additional component,
combined with a stabilizer to prolong shelf-life. In an exemplary
embodiment, the composition includes a stabilizer or stabilizing
excipient to stabilize HA and, if present, a GAG or GAG
precursor.
[0027] Hyaluronic Acid
[0028] Hyaluronic acid (HA) can have various formulations and can
be provided at various concentrations and molecular weights. The
terms "hyaluronic acid," "hyaluronan," "hyaluronate," and "HA" are
used interchangeably herein to refer to hyaluronic acids or salts
of hyaluronic acid, such as the sodium, potassium, magnesium, and
calcium salts, among others. These terms are also intended to
include not only pure hyaluronic acid solutions, but hyaluronic
acid with other trace elements or in various compositions with
other elements. The terms "hyaluronic acid," "hyaluronan," and "HA"
encompass chemical or polymeric or cross-linked derivatives of HA.
Examples of chemical modifications which may be made to HA include
any reaction of an agent with the four reactive groups of HA,
namely the acetamido, carboxyl, hydroxyl, and the reducing end. The
HA used in the present application is intended to include natural
formulations (isolated from animal tissue) or synthetic
formulations (derived from bacterial fermentation) or combinations
thereof. The HA can be provided in liquid form or solid
formulations that is reconstituted with a diluents to achieve an
appropriate concentration
[0029] HA is a glycosaminoglycan (GAG), and in particular HA is an
unbranched polysaccharide made up of alternating glucuronic acid
and N-acetyl glucosamine units. In liquid form, the HA has
viscoelastic properties. HA is also found in the extracellular
matrix of cartilage as an important structural component of
aggrecan, which make up the proteoglycan complex. The major
function of the proteoglycan complex is to retain water in the
cartilage matrix, imparting its characteristic turgidity and
mechanical resiliency.
[0030] HA not only helps to maintain healthy mechanical properties
of cartilage that cushions joints, but it is also a major component
of synovial fluid.
[0031] HA abnormalities are a common thread in connective tissue
disorders. HA can thus be used to prevent, treat, or aid in the
surgical repair of connective tissue disorders.
[0032] HA can be used in the compositions and methods of the
present invention at various molecular weights. Since HA is a
polymeric molecule, the HA component can exhibit a range of
molecular weights, and almost any average of modal molecular weight
formulation of HA can be used in the compositions and methods of
the present invention, including Low Molecular Weight ("LWM")
Hyaluronan (about 500 to 700 kilodaltons (kDa), Medium Molecular
Weight ("MMW") Hyaluronan (700-1000 kDa), and High Molecular Weight
("HMW") Hyaluronan (1.0-6.0 million daltons (MDa)). In certain
exemplary embodiments, the HA has a molecular weight of at least
about 700 kDa, and in certain embodiments, the HA is a High
Molecular Weight ("HWM") HA having a molecular weight of at least
about 1 MDa. The molecular weight can be, for example, 500, 600,
700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800,
2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900,
4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000,
5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000 kDa or
more, or any range derivable therein. It is expected that
chemically modified HA's could have very different molecular
weights than described above. A crosslinked HA can likewise have
much higher molecular weight than noted above. Regardless, these
materials are also applicable in this invention.
[0033] HA can be present in solid, lyophilized or liquid form. When
in liquid form, solvents can be used to solubilize HA. Solvent can
include, but are not limited to, water, saline or other salt
solutions, buffer solutions such as phosphate buffered saline,
histidine, lactate, succinate, glycine, and glutamate, dextrose,
glycerol, as well as combinations thereof.
[0034] The concentration of HA present in the formulation can also
vary, but in an exemplary embodiment HA is provided at a
pharmaceutically effective amount. In one embodiment, the HA has a
concentration of at least about 1 mg/ml. In an exemplary
embodiment, the HA has a concentration of at least about 5 mg/ml,
and more preferably at least about 7 mg/ml, and more preferably at
least about 10 mg/ml, and more preferably at least about 15 mg/ml,
and in some embodiments the concentration can be at least about 25
mg/ml. In another embodiment, the HA can have a concentration in
the range of about 15 mg/ml to about 25 mg/ml. Suitable
concentrations of HA include about 1 mg/ml, 2 mg/ml, 3 mg/ml, 4
mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11
mg/mg, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml,
18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24
mg/ml, 25 mg/ml, 26 mg/ml, 27 mg/ml, 28 mg/ml, 29 mg/ml, 30 mg/ml,
31 mg/ml, 32 mg/ml, 33 mg/ml, 34 mg/ml, 35 mg/ml, 36 mg/ml, 37
mg/ml, 38 mg/ml, 39 mg/ml, 40 mg/ml, 41 mg/ml, 42 mg/ml, 43 mg/ml,
44 mg/ml, 45 mg/ml, 46 mg/ml, 47 mg/ml, 48 mg/ml, 49 mg/ml, 50
mg/ml, 51 mg/ml, 52 mg/ml, 53 mg/ml, 54 mg/ml, 55 mg/ml, 56 mg/ml,
57 mg/ml, 58 mg/ml, 59 mg/l, 60 mg/ml or more or any range
derivable therein.
[0035] In one embodiment, the first component comprises an HA
having a high molecular weight (1 to 6 MDa) having a concentration
in the range of about 5-40 mg/ml. One such product is
Orthovisc.RTM. manufactured by Anika Therapeutics, Inc. of Bedford,
Mass. Orthovisc.RTM. is a sterile, non-pyrogenic, clear,
viscoelastic solution of hyaluronan. Orthovisc.RTM. consists of
high molecular weight (1.0-2.9 MDa), ultra-pure natural hyaluronan
dissolved in physiological saline and having a nominal
concentrations between 12.5-17.5 mg/ml. Orthovisc.RTM. is isolated
through bacterial fermentation. One skilled in the art will
recognize that there are companies such as Shiseido and Lifecore
who can produce high molecular weight HA through a bacterial
fermentation process. Another example of an HA product available in
the United States with these characteristics is Euflexxa.RTM..
[0036] Stabilizers
[0037] The HA can be combined with at least one stabilizer.
Stabilizers can be used in the methods and compositions of the
present invention. Stabilizers can be sugars or derivatives, such
as saccharides, disaccharides, modified saccharides, sugar
alcohols, or polysaccharides. In an exemplary embodiment, the
stabilizer can be tocopherol, tocopherol derivatives, glucose,
mannitol, sucrose and/or trehalose.
[0038] Common stabilizers or stabilizing excipients used in the
pharmaceutical industry are saccharides, disaccharides, modified
saccharides, sugar alcohols and polysaccharides. Some of these
molecules are commonly used as excipients because they can also
serve as a sweetening agent when the drug is in a tablet form for
oral delivery. According to U.S. Pat. No. 7,351,798, hyaluronic
acid and glycosaminoglycans can also be excipients. It has long
been believed that these polysaccharides are stable molecules. In
fact, all HA materials that are marketed in the US are stored at
room temperature. Some of these HA products are in buffered
solutions or in saline solutions but none have any other excipient
included. These HA products are approved in the US as
viscosupplementation devices and are injected into the joint to
relieve the pain that results from osteoarthritis. In addition, a
product on the market called Viscoat (sold by Alcon) is a
formulation that incorporates hyaluronic acid and chondroitin
sulfate in a buffered solution and contains no other excipients.
This product is used in ophthalmic surgery during the implantation
of intraocular lenses. It is known that storage of HA at room
temperature results in gradual loss of stability and degradation to
lower molecular weights, especially in the presence of other
components, such as chondroitin sulfate, hence the refrigerated
storage requirement for Viscoat
[0039] Tocopherol belongs to a class of chemical compounds that
encompass mono, di and trimethyltocols. Many of the tocopherols
demonstrate vitamin E activity. Beta, gamma and delta are
stereoisomers of alpha-tocopherol. Esters of tocopherol are often
used in cosmetic and personal care products. These esters include,
tocopheryl acetate, the acetic acid ester of tocopherol; tocopheryl
linoleate, the linoleic acid ester of tocopherol; tocopheryl
linoleate/oleate, a mixture of linoleic and oleic acid esters of
tocopherol; tocopheryl nicotinate, the nicotinic acid ester of
tocopherol; and tocopheryl succinate, the succinic acid ester of
tocopherol. potassium ascorbyl tocopheryl phosphate, a salt of both
vitamin E (tocopherol) and vitamin C (ascorbic acid) may also be
used in cosmetic products.
[0040] Other tocopherol-derived ingredients that may be found in
cosmetic products include dioleyl tocopheryl methylsilanol, which
is the dioleyl ether of tocopheryl acetate monoether with
methylsilanetriol, and tocophersolan, which is also called
tocopheryl polyethylene glycol 1000 succinate. The addition of
succinic acid and an average of 22 ethylene oxide groups to
tocopheryl makes tocophersolan a water-soluble form of
tocopherol.
[0041] Mannitol is also an exemplary stabilizer due to its low
hygroscopicity, excellent chemical and physical drug compatibility,
better sweetness and relatively slower dissolution kinetics. It
also has relatively low aqueous solubility and good dispersibility
and often used to enhance formulation stability where other
excipients have failed. Mannitol can be used in a wide array of
dosage forms, including but not limited to, tablets, capsules,
sachets, pastilles, liquids, emulsions, suspensions, ointments,
paste, lotions and intravenous solutions.
[0042] Mannitol also serves as a matrix forming additive for
lyophilization. When used at concentrations up to 10% w/v, mannitol
forms an amorphous (non-crystalline) matrix which supports proteins
and other biomolecules for freeze drying. It is generally inert and
once freeze dried, rehydrates rapidly. Its amorphous structure
while frozen prevents it from disrupting proteins while providing
channels for water sublimation during processing.
[0043] Similar to mannitol, sucrose is also widely used in tablet
form for oral delivery due to its sweetness and palatability.
Sucrose, is a non-reducing disaccharide (glucose linked by its
anomeric carbon to fructose) that is widely used as a
lyoprotectant.
[0044] Trehalose (.alpha.-D-glucopyranosyl
.alpha.-D-glucopyranoside), a disaccharide known for its
antioxidant properties, has been known as a non-reducing saccharide
consisting of glucoses. As is described in Advances in Carbohydrate
Chemistry, Vol. 18, pp. 201-225 (1963), published by Academic
Press, USA, and Applied and Environmental Microbiology, Vol. 56,
pp. 3,213-3,215 (1990), trehalose widely exists in microorganisms,
mushrooms, insects, etc., though the content is relatively low.
Trehalose is a non-reducing saccharide, so that it neither reacts
with substances containing amino groups such as amino acids and
proteins, induces the amino-carbonyl reaction, nor deteriorates
amino acid-containing substances. Thus, trehalose can be used
without a fear of causing an unsatisfactory browning and
deterioration.
[0045] Trehalose can also inhibit the inflammatory cascade, thereby
suppressing cytokine production. (Minutoli, et al, SHOCK, Vol. 27,
No. 1, pp. 91-96, 2007; and Chen Q, Haddad G G: Role of trehalose
phosphate synthase and trehalose during hypoxia: from flies to
mammals. J Exp Biol 207:3125-3129, 2004.) Trehalose is a unique
sugar capable of protecting biomolecules against environmental
stress and may inhibit the inflammatory cascade that in turn causes
oxidative damage and cytokines production. Trehalose has also been
shown to preserve cell viability, during exposure to a range of
environmental stress, such as heat shock, dehydration and
hypoxia.
[0046] Trehalose is also a common food additive because it is a
strong antioxidant and sweetener, and it is often used as a
stabilizing agent in pharmaceutical preparations. Trehalose, like
sucrose, is a non-reducing disaccharide (two glucose molecules
linked by the anomeric carbon) that can act as an effective
lyoprotectant for the freeze drying of proteins and other
biomolecules. During the freeze drying process, proteins can
denature as water is removed unless a substitute molecule is
available to support the structure of the protein. Trehalose fills
the void left by exiting water and prevents this denaturation. When
used at concentrations as low as 2% it can effectively protect
proteins and other biomolecules.
[0047] Useful forms of trehalose can include trehalose dihydrate
(TD) which is crystalline, amorphous trehalose (AT) which is a
vitreous form, and the anhydrous forms of trehalose, anhydrous
amorphous trehalose (AAT) and anhydrous crystalline trehalose
(ACT). Powdered anhydrous trehalose may contain AAT and/or ACT. The
term "trehalose," as used herein, refers to any physical form of
trehalose including anhydrous, partially hydrated, fully hydrated
and mixtures and solutions thereof. The manufacture and use of
anhydrous trehalose from TD can be found in International
Publication No.: PCT/GB97/00367, the disclosure of which is
incorporated into this specification by reference.
[0048] The addition of trehalose to the formulation or composition
can help stabilize high molecular weight HA as well as inhibit
damaging inflammatory cascades. Trehalose can be present in liquid,
solid, lyophilized or crystalline forms. When present in liquid
form, trehalose can be in a buffered solution. Solvents that can be
used to solubilize trehalose can include, but are not limited to,
water, saline or other salt solutions, buffer solutions such as
phosphate buffered saline, histidine, lactate, succinate, glycine,
and glutamate, dextrose, glycerol, as well as combinations thereof.
Particularly, trehalose can be present in the HA formulation as a
solution.
[0049] At least one stabilizer, can be present in the HA
formulation. Solvents that can be used to solubilize the stabilizer
can include, but are not limited to, water, saline or other salt
solutions, buffer solutions such as phosphate buffered saline,
histidine, lactate, succinate, glycine, and glutamate, dextrose,
glycerol, as well as combinations thereof. Particularly, the
stabilizer can be present in the HA formulation as a solution.
[0050] The concentration of the at leaste one stabilizer present in
the HA formulation can vary, but in an exemplary embodiment at
least one excipient is provided at a pharmaceutically effective
amount. In an exemplary embodiment, the at least one stabilizer has
a concentration of at least about 1 mg/ml, and more preferably at
least about 5 mg/ml, and more preferably at least about 50 mg/ml,
and more preferably at least about 100 mg/ml, and in some
embodiments the concentration can be at least about 200 mg/ml.
Suitable concentrations of at least one stabilizer can include
about 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6
mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml,
4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11
mg/mg, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml,
18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24
mg/ml, 25 mg/ml, 26 mg/ml, 27 mg/ml, 28 mg/ml, 29 mg/ml, 30 mg/ml,
31 mg/ml, 32 mg/ml, 33 mg/ml, 34 mg/ml, 35 mg/ml, 36 mg/ml, 37
mg/ml, 38 mg/ml, 39 mg/ml, 40 mg/ml, 41 mg/ml, 42 mg/ml, 43 mg/ml,
44 mg/ml, 45 mg/ml, 46 mg/ml, 47 mg/ml, 48 mg/ml, 49 mg/ml, 50
mg/ml, 51 mg/ml, 52 mg/ml, 53 mg/ml, 54 mg/ml, 55 mg/ml, 56 mg/ml,
57 mg/ml, 58 mg/ml, 59 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90
mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150
mg/ml, 160 mg/ml, 160 mg/ml, 170 mg/ml, 180 mg/ml, 190 mg/ml, 200
mg/ml, 300 mg/ml, 400 mg/ml, 500 mg/ml, 600 mg/ml or more or any
range derivable therein. Stabilizers can also be in a concentration
in a range of about 0.1-60% by weight; about 0.1-50% by weight,
about 0.1-45% by weight; or about 0.1-20% by weight. Other suitable
concentrations of at least one excipient can include about 0.1%,
0.5%, 1%, 2.5%, 5%, 6%, 7%, 8%, 9% 10%, 11%, 12%, 13%, 14%, 15%,
16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or
about 60% by weight.
[0051] Additional Components
[0052] In an exemplary embodiment, at least one additional
component can also be combined with the HA formulation or
composition. The additional component can be a lyophilized
glycosaminoglycan (GAG) or GAG precursor. The term
"glycosaminoglycan," or "GAG," refers interchangeably to the family
of sulfated mucopolysaccharides that typically include heparin,
heparin sulfate, chondroitin, chondroitin sulfate, keratan sulfate,
dermatan sulfate, and their respective derivatives.
[0053] Glycosaminoglycans (GAGs) are long unbranched
polysaccharides containing a repeating disaccharide unit. The
disaccharide units contain either of two modified sugars,
N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc), and
a uronic acid such as glucuronate or iduronate. GAGs are highly
negatively charged molecules, with extended conformation that
imparts high viscosity to the mixture. GAGs are located primarily
on the surface of cells or in the extracellular matrix (ECM). Along
with the high viscosity of GAGs comes low compressibility, which
makes these molecules ideal for a lubricating fluid in the joints.
GAGs can thus help slow down the inflammatory process. At the same
time, their rigidity provides structural integrity to cells and
provides passageways between cells, allowing for cell migration. In
addition, a GAG such as CS has been found in the synovial fluid and
may play a role in health of the joint. Therefore it can be
advantageous to have a therapy that can deliver CS at the same time
as HA. It is believed that CS can enhance the efficacy of HA.
However, it has been observed that HA in the presence of CS can
degrade over time. Therefore, in order to prevent this degradation,
HA must be refrigerated until use. However, most clinics and
hospitals do not have the capacity to store these types of products
in a refrigerator, so it would be beneficial to provide products
that combine HA and GAGs that are stable and that are capable of
being stored at room temperature.
[0054] The GAGs can have various molecular weights, but in certain
exemplary embodiments the molecular weight is in the range about 5
to 1,000 kDa, more preferably in the range of about 6 to 500 kDa,
more preferably in the range of about 7 to 300 kDa, more preferably
in the range of about 8 to 200 kDa, more preferably in the range of
about 9 to 100 kDa, and most preferably in the range of about 10 to
80 kDa. In other embodiments, the molecular weight of the GAG
fragment is below about 5 kDa and even more preferably below about
3 kDa.
[0055] The concentration of GAGs present in mixture can also vary,
but in an exemplary embodiment the GAG is provided at a
pharmaceutically effective amount. In an exemplary embodiment, the
GAG has a concentration of at least about 0.1 mg/ml, and more
preferably at least about 2 mg/ml, and more preferably at least
about 5 mg/ml, and more preferably at least about 7 mg/ml, and more
preferably at least about 20 mg/ml. In another embodiment, the GAG
or GAG precursor can be present in the composition at a
concentration in the range of about 0.1 mg/ml to about 20 mg/ml.
Suitable concentrations of GAGs include about 0.1 mg/ml, 0.2 mg/ml,
0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml,
0.9 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7
mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/mg, 12 mg/ml, 13 mg/ml, 14
mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml,
21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml, 26 mg/ml, 27
mg/ml, 28 mg/ml, 29 mg/ml, 30 mg/ml, 31 mg/ml, 32 mg/ml, 33 mg/ml,
34 mg/ml, 35 mg/ml, 36 mg/ml, 37 mg/ml, 38 mg/ml, 39 mg/ml, 40
mg/ml, 41 mg/ml, 42 mg/ml, 43 mg/ml, 44 mg/ml, 45 mg/ml, 46 mg/ml,
47 mg/ml, 48 mg/ml, 49 mg/ml, 50 mg/ml, 51 mg/ml, 52 mg/ml, 53
mg/ml, 54 mg/ml, 55 mg/ml, 56 mg/ml, 57 mg/ml, 58 mg/ml, 59 mg/ml,
60 mg/ml or more or any range derivable therein.
[0056] Chondroitin sulfate (CS), which is an essential component of
cartilage, is composed of an alternating sequence of sulfated
and/or unsulfated D-glucuronic acid (GlcA) and
N-acetyl-D-galactosamine (GalNAc) residues linked through
alternating .beta.(1,3) and .beta.(1,4) bonds. These compounds each
have a polymeric structure consisting mainly of about 40 to 100
times repetition of the disaccharide units. CS can be used at
various molecular weights and concentrations, as discussed above
with respect to the GAG component, but in an exemplary embodiment,
the CS has a molecular weight of in the range of about 10,000 to
80,000 kDa and a concentration in the range of about 0.1 to 100
mg/ml. CS can be isolated from bovine or marine sources. A
chondroitin chain can have over 100 individual sugars, each of
which can be sulfated in variable positions and quantities.
Chondroitin-4 sulfate, also carbon 4 of the N-acetylgalactosamine
(GalNAc) sugar, is found in nasal and tracheal cartilages of
bovines and porcines. It is also found in the bones, flesh, blood,
skin, umbilical cord, and urine of these animals. Chondroitin-6
sulfate, also carbon 6 of the GalNAc sugar, has been isolated from
the skin, umbilical cord, and cardiac valves of these animals.
Chondroitin-6 sulfate has the same composition, but slightly
different physical properties from chondroitin-4 sulfate.
Chondroitin sulfate is involved in the binding of collagen and is
also directly involved in the retention of moisture. These are both
properties that aid the healing process. A person skilled in the
art will appreciate that the terms "chondroitin sulfate," "CS,"
"chondroitin," "chondroitin sulfuric acid," and "chonsurid" are
used interchangeably herein and also encompass chemical or isomeric
or cross-linked derivatives throughout this application.
[0057] Dermatan sulfate (DS), also called chondroitin sulfate B, is
mainly made up disulfated and/or trisulfated disaccharide units of
L-iduronic acid and N-acetyl-D-galactosamine joined by .beta.1,4 or
1,3 linkages, but there is a case where some of the repeating units
contain sulfated L-iduronic acid or D-glucuronic acid as uronic
acid, or contain non-sulfated N-acetylgalactosamine or
4,6-disulfated N-acetylgalactosamine instead of
N-acetylgalactosamine-4-sulfate. DS is defined as a chondroitin
sulfate by the presence of GalNAc. The presence of iduronic acid
(IdoA) in DS distinguishes it from chondroitin sulfates-A
(4-O-sulfated) and -C (6-0-sulfated) and likens it to heparin and
HS, which also contain this residue. It is considered that dermatan
sulfate is absorbed by the body when orally taken. The molecular
weight and concentration of DS can vary, as discussed above with
respect to the GAG component, but in an exemplary embodiment the
molecular weight is in the range of about 10 to 80 kDa and a
concentration in the range of about 0.1-100 mg/ml. A person skilled
in the art will appreciate that, unlike HA which is bacterially
fermented and therefore has a molecular weight that can be
controlled, dermatan sulfate is isolated from animal tissue and may
contain fragments. The molecular weight of the dermatan sulfate,
and any fragments contained therein, can therefore significantly
vary. A person skilled in the art will also appreciate that the
terms "dermatan sulfate," "DS," and "dermatan" are used
interchangeably herein and also include sulfated derivatives of
dermatan sulfate, the dermatan sulfate benzethonium salt, the
persulfated derivatives of dermatan sulfate benzethonium salts, and
also the dermatan sulfate sodium salt.
[0058] Heparin and heparan sulfate (HS) are composed of a
glucuronic acid (GlcA) linked to N-acetylglucosamine. They are
composed of .alpha.1-4 linked disaccharide repeating units
containing a uronic acid and an amino sugar. Heparan sulfate
proteoglycans are an integral part of the basement membrane. HS
proteoglycan is a large biomolecule with a molecular mass as great
as 400 kDa, composed of a core protein covalently bound to heparan
sulfate chains. The number of the polysaccharide chains and the
size of the core protein may vary according to the source. Heparan
sulfate proteoglycan is a multifunctional molecule binding to
fibroblast growth factors, vascular endothelial growth factor
(VEGF), and VEGF receptors through the sugar moiety, acting as a
docking molecule for matrilysin (MMP-7) and other matrix
metalloproteinases and playing important roles in cell
proliferation and differentiation. Heparan sulfate proteoglycans
also promote attachment of cells by binding to a variety of
molecules found in the extracellular matrix including laminin,
fibronectin, collagen type IV, and FGF-basic. The molecular weight
and concentration of HS can vary, as discussed above with respect
to the GAG component, but in an exemplary embodiment the molecular
weight is in the range of about 3-30 kDa (when isolated from
tissues) and a concentration in the range of about 0.1-100
mg/ml.
[0059] Keratan sulfate, also keratosulfate (KS), is
highly-negatively charged and found principally in aggrecan, the
most abundant proteoglycan in the extracellular matrix of hyaline,
fibrous and elastic cartilage. KS is composed of disaccharide
repeating unit, -4GlcNAc.beta.1-3Gal.beta.1-. Sulfation occurs at
carbon position 6 (C6) of either or both the galactose (Gal) or
GlcNAc. Specific KS types are composed of three regions: alinkage
region, at one end of which the KS chain is linked to the core
protein, the repeating disaccharide unit, and chain capping region,
occurring at the opposite end of the KS chain to the protein
linkage region. The molecular weight and concentration of KS can
vary, as discussed above with respect to the GAG component, but in
an exemplary embodiment the molecular weight is in the range of
about 5-10 kDa (when isolated from tissues) and a concentration in
the range of about 1-100 mg/ml.
[0060] The concentration of GAGs or GAG precursors present in
mixture can also vary, but in an exemplary embodiment the GAG or
GAG precursor is provided at a pharmaceutically effective amount.
In an exemplary embodiment, the GAG or GAG precursor has a
concentration of at least about 0.1 mg/ml, and more preferably at
least about 2 mg/ml, and more preferably at least about 5 mg/ml,
and more preferably at least about 7 mg/ml, and more preferably at
least about 20 mg/ml. In another embodiment, the GAG or GAG
precursor can be present in the composition at a concentration in
the range of about 0.1 mg/ml to about 20 mg/ml. Suitable
concentrations of GAGs or GAG precursors include about 5 mg/ml, 6
mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/mg, 12 mg/ml, 13
mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml,
20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml, 26
mg/ml, 27 mg/ml, 28 mg/ml, 29 mg/ml, 30 mg/ml, 31 mg/ml, 32 mg/ml,
33 mg/ml, 34 mg/ml, 35 mg/ml, 36 mg/ml, 37 mg/ml, 38 mg/ml, 39
mg/ml, 40 mg/ml, 41 mg/ml, 42 mg/ml, 43 mg/ml, 44 mg/ml, 45 mg/ml,
46 mg/ml, 47 mg/ml, 48 mg/ml, 49 mg/ml, 50 mg/ml, 51 mg/ml, 52
mg/ml, 53 mg/ml, 54 mg/ml, 55 mg/ml, 56 mg/ml, 57 mg/ml, 58 mg/ml,
59 mg/ml, 60 mg/ml or more or any range derivable therein.
[0061] A person skilled in the art will appreciate that, while
lyophilized GAGs or GAG precursors are particularly useful in
exemplary embodiments, liquid GAGs or GAG precursors can also be
used in the composition. For example, CS can be obtained in powder
form and mixed with a solvent, such as water, to form a solution.
The solution can be combined with liquid HA to form a mixture
having a reduced viscosity, as compared to the HA alone. While
effective to reduce the viscosity of the HA, the resulting mixture
will have a reduced concentration due to the presence of water.
Thus, while liquid, non-lyophilized GAGs can be used with the
present invention, in an exemplary embodiment the GAG is
lyophilized to allow for the use of HA at a high concentration.
[0062] Glucosamine (C.sub.6H.sub.13NO.sub.5) ("GlcN") or its
derivatives or other GAG precursors can also be included in the HA
formulation to enhance synthesis of key components of cartilage and
synovial fluid by feeding both reactions necessary for the
production of hyaluronan as well as for proteoglycans. GlcN is an
amino sugar carrying four hydroxyl groups and an amine group, and
it is a prominent precursor in the biochemical synthesis of
glycosylated proteins and lipids. GlcN is a naturally occurring
molecule that has nutritive and effector functions. For example,
GlcN is compatible with and promotes stem cell growth and
differentiation of mesenchymal stem cells to form chondrocytes.
GlcN can have a role in tissue development and repair, such as
cartilage growth and development, in general. It is used as a
nutritional supplement to combat the symptoms of OA, and has been
shown slow cartilage destruction in clinical studies. In one
embodiment, the GlcN can by lyophilized together with the CS. Salt
forms of glucosamine can have limited stability in liquid phase. In
addition, HCl salt of GlcN can lower the pH sufficiently to degrade
the HA once combined. For this reason, in order to retain stability
of the components, it is advantageous to have GlcN in the
lyophilized form to be solubilized with HA gel prior to injection.
As used herein, "glucosamine" includes glucosamine salts, such as
glucosamine hydrochloride and glucosamine sulfate, as well as
non-salt forms such as N-acetylglucosamine.
[0063] The concentration of the GlcN can vary. A suitable local
concentration can be at least about 10 mg/ml, about 9 mg/ml, about
8 mg/ml, about 7 mg/ml, about 6 mg/ml, 5 mg/ml, 4.5 mg/ml, 4 mg/ml,
3.5 mg/ml, 3 mg/ml, about 2.9 mg/ml, about 2.8 mg/ml, about 2.7
mg/ml, about 2.6 mg/ml, about 2.5 mg/ml, about 2.4 mg/ml, about 2.3
mg/ml, about 2.2 mg/ml, about 2.1 mg/ml, about 2.0 mg/ml, about 1.9
mg/ml, about 1.8 mg/ml, about 1.7 mg/ml, about 1.6 mg/ml, about 1.5
mg/ml, about 1.4 mg/ml, about 1.3 mg/ml, about 1.2 mg/ml, about 1.1
mg/ml, about 1.0 mg/ml, about 0.9 mg/ml, about 0.8 mg/ml, about 0.7
mg/ml, about 0.6 mg/ml, about 0.5 mg/ml or so on. A person skilled
in the art can determine a suitable local concentration of GlcN
practicing methods known in the pharmaceutics art, and that
determination will govern the nature and composition of the GlcN
composition of interest to obtain the desired concentration of
GlcN.
[0064] Buffering agents can also be added to the HA formulation to
control pH. Examples of buffering agents can be any one or more of
the following agents, and is not limited to, acetic acid, ammonium
carbonate, ammonium phosphate, boric acid, citric acid, glycine,
lactic acid, phosphoric acid, potassium citrate, potassium
metaphosphate, potassium phosphate monobasic, sodium acetate,
sodium citrate, sodium lactate solution, dibasic sodium phosphate,
monobasic sodium phosphate, TRIS and sodium carbonate.
[0065] Additionally, isotonic agents can be added to control ionic
concentration and/or osmotic pressure of the HA formulation.
Examples of isotonic agents can be any one or more of the following
agents, and is not limited to, dextrose, sucrose, trehalose,
glycerin, mannitol, potassium chloride, sodium chloride.
[0066] A person skilled in the art will appreciate that the
compositions and methods of the present invention can include
various other joint treatment components, including, for example,
amino acids, proteins, saccharides, di-saccharides,
poly-saccharides, lipids, nucleic acids, buffers, surfactants, and
mixtures thereof. Other useful components can include steroids,
anti-inflammatory agents, non-steroidal anti-inflammatory agents,
analgesics, cells, antibiotics, antimicrobial agents,
anti-inflammatory agents, growth factors, growth factor fragments,
small-molecule wound healing stimulants, hormones, cytokines,
peptides, antibodies, enzymes, isolated cells, platelets,
immunosuppressants, nucleic acids, cell types, viruses, virus
particles, essential nutrients or vitamins, and combinations
thereof.
[0067] Lyophilization
[0068] Any one or more of the components present in the
compositions and methods of the present invention can be
lyophilized using various techniques known in the art.
Lyophilization is a dehydration process that is typically used to
preserve a perishable material, and it works by freezing the
material and then reducing the surrounding pressure and adding
enough heat to allow the frozen water in the material to sublime
directly from the solid phase to the gas phase. Standard
lyophilization techniques known in the art can be used to
lyophilize any one or more of the components. In an exemplary
embodiment, the HA is lyophilized.
[0069] Prior to lyophilization, various solvents can be used to
form an aqueous mixture containing the component(s) to be
lyophilized. In an exemplary embodiment, the aqueous mixture is
prepared by combining water with one or more of the components. In
an exemplary embodiment, the composition is filter sterilized, such
as with a 0.2 .mu.m filter, prior to lyophilization.
[0070] In one embodiment, the component(s) can be lyophilized using
the following cycle:
[0071] Freezing: from ambient temperature to 5.degree. C. in 15
minutes [0072] Hold at 5.degree. C. for 100 minutes [0073] Down to
-45.degree. C. in 50 minutes [0074] Hold at -45.degree. C. for 180
minutes
[0075] Primary Drying: set pressure at 50 mTorr [0076] Shelf Up to
-15.degree. C. in 175 minutes [0077] Hold at -15.degree. C. for
2300 minutes
[0078] Secondary Drying: set pressure at 75 mTorr [0079] Shelf Up
to 25.degree. C. in 200 minutes [0080] Hold for 900 minutes
[0081] Cycle end: backfill with nitrogen to .about.730 Ton [0082]
Capping and crimping
[0083] Variations to the temperatures, times and settings can be
made in accordance to practices used by a person of skilled in the
art. Variations may include, but are not limited to, cycling
temperatures for the freezing cycle, drying temperatures and end
cycles. Variations may also include differences in holding times
for the freezing, drying and capping/crimping cycles. Variations
may also include differences in set pressures for the drying cycles
and capping/crimping cycles. In addition, the number of drying
cycles may be increased or decreased depending the machine used or
component(s) to be lyophilized.
[0084] The addition of a buffering agent can provide for improved
solubility and stability of the protein in lyophilized HA
formulations. Biocompatible buffering agents known in the art can
be used, such as glycine; sodium, potassium, or calcium salts of
acetate; sodium, potassium, or calcium salts of citrate; sodium,
potassium, or calcium salts of lactate; sodium or potassium salts
of phosphate, including mono-basic phosphate, di-basic phosphate,
tri-basic phosphate and mixtures thereof. The buffering agents can
additionally have sugar added to the composition to function as a
bulking agent. The pH preferably can be controlled to be between
pH5 and pH10, and more preferably between pH 6 to 8.
[0085] Formulations
[0086] In one embodiment, the HA and at least one additional
component, such as an excipient, can be configured to be combined
and administered intra-articularly as part of a surgical procedure
involving an articulating joint, either immediately before, during
or immediately after the surgical procedure. Alternatively, the HA
and at least one additional component could be previously combined
and present as a combination formulation at the time of the
surgical procedure. The other components, when combined, can form a
resulting composition or mixture having each component present in
the composition at various amounts. The amount of each component in
the composition can vary, but in an exemplary embodiment, the
mixing ratio between HA and at least one additional component, such
as an excipient, can have a weight ratio of HA to additional
component in the range of about 1:0.001 to about 1:100, and more
preferably at a ratio in the range of about 1:0.01 to about 1:10.
Alternatively, the formulation can include about 1% to about 75% or
more by weight of each of the individual components, such as HA and
at least one additional component (e.g. an excipient), in the total
composition, alternatively about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70% or more by weight of HA and at least one additional
component, such as an excipient, in the total formulation. In an
exemplary embodiment, the amount of HA present in the disclosed
formulation can be about 1-20% by weight of the total formulation,
and the amount of at least one additional component, such as an
excipient, can be present in the disclosed formulation at about
0.01-20% by weight of the total formulation.
[0087] Solvents that can be used to solubilize one or more of the
components include, for example, water, acidic solvents,
hydrochloric acid, acetic acid, benzoic acid, phosphate buffered
saline, dextrose, glycerol, ethanol and the like, as well as
combinations thereof. Solvents that can be used to solubilize HA
and/or the additional component can include, but are not limited
to, water, saline or other salt solutions, buffer solutions such as
phosphate buffered saline, histidine, lactate, succinate, glycine
and glutamate, dextrose, glycerol, hydrochloric acid, acetic acid,
benzoic acid, acidic solvent and other suitable solvents, as well
as combinations thereof. The formulation can also include other
components, such as dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. Isotonic agents include,
for example, sugars, polyalcohols such as mannitol, sorbitol, or
sodium chloride in the composition. The formulation can also
include minor amounts of auxiliary substances such as wetting or
emulsifying agents, preservatives or buffers, which enhance the
shelf life or effectiveness of the formulation.
[0088] The formulations can be incorporated into pharmaceutical
compositions suitable for administration to a subject. Typically,
the pharmaceutical composition comprises HA and another component,
such as at least one excipient, and a pharmaceutically acceptable
carrier. As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Examples of pharmaceutically acceptable carriers include one or
more of water, hydrochloric acid, acetic acid, benzoic acid, acidic
solvent, saline, phosphate buffered saline, dextrose, glycerol,
ethanol and the like, as well as combinations thereof. In many
cases, it can be useful to include isotonic agents, for example,
sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride
in the composition. Pharmaceutically acceptable carriers may
further comprise minor amounts of auxiliary substances such as
wetting or emulsifying agents, preservatives or buffers, which
enhance the shelf life or effectiveness of the composition.
[0089] The compositions can be in a variety of forms. These
include, for example, liquid, semi-solid and solid dosage forms,
such as liquid solutions (e.g., injectable and infusible
solutions), dispersions or suspensions, tablets, pills, and
powders. An exemplary form can depend on the intended mode of
administration and therapeutic application. Typical compositions
can be in the form of injectable or infusible solutions, such as
compositions similar to those used for in vivo injection. In one
embodiment, an exemplary mode of administration is parenteral
(e.g., intra-articular, subcutaneous, intraperitoneal,
intramuscular). In another embodiment, the composition can be
administered by infusion or injection directly into the target
area, such as a joint. In yet another embodiment, the composition
can be administered by intramuscular or subcutaneous injection.
[0090] Sterile injectable solutions can be prepared by
incorporating the active compound in a therapeutically effective or
beneficial amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle which
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
embodiments can include methods of preparation of vacuum drying and
freeze-drying which yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0091] Delivery Systems
[0092] The methods and compositions encompass kits for treating
articular disorders, such as joints. The kits can comprise HA, and
at least one stabilizer. Both and/or all the components can be
housed in a single chamber. The kit can also include HA with at
least one stabilizer and an additional component. The additional
component can be housed in a single or separate chamber of a
syringe for injecting with the HA mixture of HA and at least one
stabilizer. The HA and at least one stabilizer can be lyophilized.
In one exemplary embodiment, the HA is combined with at least one
stabilizer in a single chamber. In another embodiment, a kit is
provided having HA and the at least one stabilizer housed in the
same chamber and an additional component in a second chamber. In
another exemplary embodiment, the HA is combined with at least one
stabilizer in the same chamber and a GAG or GAG precursor is housed
in a second chamber. The HA component can comprise up to about 10
ml of Orthovisc.RTM., which contains about 150 mg of hyaluronan, 90
mg of sodium chloride, and up to about 10.0 mL of water for
injection and can be scaled appropriately for the specific
indication. Examples can be for the knee, shoulder and hip, the
volume of injection for a single injection product can be in the
range of about 3 ml to 10 ml and the hand can be in the range of
about 500 .mu.l and 1.5 ml. The HA can have a molecular weight in
the range of about 1.0 to 6 MDa. The at least one stabilizer
present in the HA formulation or composition can have a
concentration in the range from about 0.1 mg/ml to about 200 mg/ml.
The at least one stabilizer can be lyophilized or, alternatively
can be in a solution, as noted above, in combination with the HA
component.
[0093] Compounds can be stored separately to increase shelf-life.
The individual compounds can be lyophilized or in solid form in one
syringe/cartridge with diluent or a second compound in a second
syringe/cartridge. In one embodiment, one of the compounds is in
lyophilized form or in solid form and the second compound is a
solution capable of combining with the lyophilized/solid compound.
An example can be at least one lyophilized or solid component can
be stored in a first chamber and a solubilized HA can be stored in
a second chamber. In another embodiment, both compounds can be
lyophilized or in solid form and housed in a single or separate
chambers of a syringe/cartridge. In another embodiment, compounds
can be lyophilized directly in the syringe or cartridge.
[0094] Pre-filled dual-chamber syringes and/or cartridges can also
be utilized with the components and compositions. Pre-filled
dual-chamber syringes enable the sequential administration of two
separate compositions with a single syringe push, thereby replacing
two syringes with one. The benefits of a single delivery capability
include increasing the speed and ease of drug administration;
reducing risk of infection by reducing the number of connections;
lowering the risk of drug administration or sequence errors, and
quicker delivery of compositions requiring combination prior to
administration. The dual-chamber syringe can accommodate
lyophilized, powder or liquid formulations in the front chamber
combined with diluents, saline or buffer in the rear chamber.
[0095] Prefilled syringes can contain the exact deliverable dose of
desired compounds and diluents. The prefilled syringes can contain
volumes from about 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml,
0.7 ml, 0.8 ml, 0.9 ml, 1.0 ml, 1.5 ml, 2 ml, 2.5 ml, 3 ml, 3.5 ml,
4 ml, 4.5 ml, 5 ml, 5.5 ml, 6 ml, 6.5 ml, 7 ml, 7.5 ml, 8 ml, 8.5
ml, 9 ml, 9.5 ml, 10 ml or more or any derivative therein.
[0096] The dual syringe and/or cartridge can be side-by-side
chambers with separate syringe plungers that mix into a single
chamber or linear chambers with one plunger. The dual chamber
syringe and/or cartridges can also have a stopper or connector in
the middle to serve as a barrier between the two chambers. The
stopper or connector can be removed to allow mixing or combining of
the compounds in the two chambers.
[0097] FIG. 1 illustrates one embodiment of a mixing and delivery
system that is in the form of a dual chamber syringe 10. As shown,
the dual chamber syringe 10 generally includes a housing having
proximal and distal chambers 14, 12 separated by a valve 16. A
plunger 18 is slidably disposed within the proximal chamber 14 and
is configured to inject fluid present within the proximal chamber
14 into the distal chamber 12 to thereby mix the components. In one
embodiment, the first component, e.g., liquid HA with at least one
stabilizer, can be present in the proximal chamber 14 and an
additional component, e.g., one or more additional components, can
be present in the distal chamber 12. Alternatively, the first
component, e.g., liquid HA, can be present in the proximal chamber
14 with at least one stabilizer, such as trehalose, and at least
one additional component, e.g., chondroitin sulfate, can be present
in the distal chamber 12. The plunger 18 can be advanced through
the proximal chamber 14 to inject the first component, e.g., liquid
HA with at least one stabilizer, into the distal chamber 12
containing the second component, e.g., one or more additional
components. In another embodiment, the proximal chamber 14 can
contain a solvent, such as water or saline, and the distal chamber
12 can contain all of the components in solid form. For example,
the distal chamber 12 can contain lyophilized or solid HA with at
least one stabilizer. The plunger 18 can be advanced through the
proximal chamber 14 to inject the solvent into the distal chamber
12, thereby solubilizing the components in the distal chamber 12.
Once all components are combined in the distal chamber 12, the
mixture can be delivered to tissue, for example by attaching a
needle to the distal end of the dual chamber syringe.
[0098] FIG. 2 illustrates another embodiment of a mixing and
delivery system 20, which is sold commercially under the trade name
MixJect.RTM.. In this embodiment, the system includes a fluid
control assembly 22 that is coupled between a syringe 24 and a vial
26. The syringe 24 defines a first chamber 24a (not labeled in
figure) which can contain a liquid, such as liquid HA or a solvent,
and the vial defines a second chamber 26a (not labeled on figure)
which can contain a solid, such as one or more additional
components. Deployment of the plunger 28 through the syringe 24
will inject the liquid through the control system and into the vial
26, where the solid will be solubilized by the liquid. Once the
components are fully solubilized, the vial 26 can be inverted and
the plunger 28 can be retracted to draw the mixture back into the
chamber 24a in the syringe 24. The vial 26 can then be removed from
the system, and the mixture can be injected from the syringe
through a needle 29 and into tissue.
[0099] A person skilled in the art will appreciate that any dual
chamber systems known in the art can be used, and that the chambers
can be side-by-side chambers with separate syringe plungers that
mix into a single chamber or linear chambers with a single
plunger.
[0100] Treatments
[0101] The method and compositions can be administered, for in vivo
applications, parenterally by injection or by gradual perfusion
over time. Administration may be intraarticular, intravenous,
intraperitoneal, intramuscular, subcutaneous, intracavity, or
transdermal. For in vitro studies the agents may be added or
dissolved in an appropriate biologically acceptable buffer and
added to a cell or tissue.
[0102] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's intravenous vehicles include fluid and nutrient
replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like. Preservatives and other additives
may also be present such as, for example, anti-microbials,
anti-oxidants, chelating agents, growth factors and inert gases and
the like.
[0103] Frequently used "carriers" or "auxiliaries" include
magnesium carbonate, titanium dioxide, lactose, mannitol and other
sugars, talc, milk protein, gelatin, starch, vitamins, cellulose
and its derivatives, animal and vegetable oils, polyethylene
glycols and solvents, such as sterile water, alcohols, glycerol and
polyhydric alcohols, and dimethyl sulfoxide. Intravenous vehicles
include fluid and nutrient replenishers. Preservatives include
antimicrobial agents, antioxidants, chelating agents and inert
gases. Other pharmaceutically acceptable carriers include aqueous
solutions, non-toxic excipients, including salts, preservatives,
buffers and the like, as described, for instance, in Remington's
Pharmaceutical Sciences, 15th ed. Easton: Mack Publishing Co.:
1405-1412, 1461-1487, 1975 and The National Formulary XIV., 14th
ed. Washington: American Pharmaceutical Association, 1975 the
contents of which are hereby incorporated by reference. The pH and
exact concentration of the various components of the pharmaceutical
composition are adjusted according to routine skills in the art.
See Goodman and Gilman's The Pharmacological Basis for Therapeutics
(7th ed.).
[0104] Examples of symptoms or diseases, for which the composition
and methods disclosed herein can be useful, encompass treating
articular disorders, such as arthritis caused by infections,
injuries, allergies, metabolic disorders, etc., rheumatoids such as
chronic rheumatoid arthritis, and systemic lupus erythematosus;
articular disorders accompanied by gout, arthropathy such as
osteoarthritis, internal derangement, hydrarthrosis, stiff neck,
lumbago, etc. Varying the effects depending on the use of the
composition or the types of diseases to be treated, the agent can
exert desired prophylactic and alleviative effects, or even
therapeutic effects on swelling, pain, inflammation, and destroying
of articulations without seriously affecting living bodies. The
composition for treating articular disorder can be used to prevent
the onset of articulation disorders, as well as to improve,
alleviate, and cure the symptoms after their onsets.
[0105] The methods of treatment can include directly injecting the
compositions into the target area, such as a joint. Injections can
be performed as often as daily, weekly, several times a week, bi
monthly, several times a month, monthly, or as often as needed as
to provide relief of symptoms. For intra-articular use, from about
1 to about 30 mg/ml of HA per joint, depending on the size of the
joint and severity of the condition, can be injected. The frequency
of subsequent injections into a given joint are spaced to the time
of recurrence of symptoms in the joint. Illustratively, dosage
levels in humans of the composition can be: knee, about 1 to about
30 mg/ml per joint injection; shoulder, about 1 to about 30 mg/ml
of HA per joint injection; metacorpal or proximal intraphalangeal,
about 1 mg/ml to about 30 mg/ml of HA per joint injection; and
elbow, about 1 to about 30 mg/ml per joint injection. The total
amount of injection can range from about 1 mg/ml to 200 mg/ml of
HA.
[0106] It will be understood, however, that the specific dosage
level for any particular patient will depend upon a variety of
factors including the activity of the specific compound employed,
the age, body weight, general health, sex, diet, time of
administration, route of administration, rate of excretion, drug
combination and the severity of the particular disease undergoing
therapy. The pharmaceutical compositions can be prepared and
administered in dose units. Under certain circumstances, however,
higher or lower dose units may be appropriate. The administration
of the dose unit can be carried out both by single administration
of the composition or administration can be performed in several
smaller dose units and also by multiple administrations of
subdivided doses at specific intervals.
[0107] In one embodiment, the medical condition is osteoarthritis
(OA) and the composition is administered in a joint space, such as,
for example, a knee, shoulder, temporo-mandibular and
carpo-metacarpal joints, elbow, hip, wrist, ankle, and lumbar
zygapophysial (facet) joints in the spine. The viscosupplementation
may be accomplished via a single injection or multiple
intraarticular injections administered over a period of weeks into
the knee or other afflicted joints. For example, a human subject
with knee OA may receive one, two, three, four, or five injections
of about 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 ml or more per
knee. For other joints, the administered volume can be adjusted
based on the size on the joint.
[0108] It will be understood, however, that the specific dosage
level for any particular patient will depend upon a variety of
factors including the activity of the specific compound employed,
the age, body weight, general health, sex, diet, time of
administration, route of administration, rate of excretion, drug
combination and the severity of the particular disease undergoing
therapy.
[0109] Experimental Data
Example 1
Stability of Liquid HA and CS Formulations with Various
Stabilizers
[0110] HA at 2.5 mg/mL and Chondroitin sulfate (CS) at 2.5 mg/mL
were formulated with different stabilizers as outlined in Table 1.
The formulations were tested for HA stability at 5.degree. C. and
40.degree. C./75% RH storage conditions (FIG. 3). After five
months, the test samples were analyzed by a size exclusion
chromatography (SEC)-high performance liquid chromatography method.
Briefly, the test sample was diluted with a mobile phase (100 mM
sodium phosphate buffer, pH 7) to 0.1 mg/mL HA. The diluted sample
was then injected onto a HPLC column (phenomenex, Poly Sep-GFC-P
linear column, catalog number 00H-3147-KO) with a flow rate of 0.6
mL/min. The eluted HA peaks were monitored with 207 nm wavelength
and the retention time was compared with HA molecular weight
standards to determine the molecular weight of the test sample.
[0111] As shown in Table 1, the saline formulation was the least
stable formulation at 40.degree. C. as compared to the other
40.degree. C. samples. FIG. 3 show the chromatograms of the saline
formulation. The HA peak of the saline sample shifted a lot to the
right, indicating HA degraded to smaller molecules. In contrast,
the 40.degree. C. trehalose formulation had a much smaller shift as
shown in FIG. 4. Similar results were observed with the sucrose and
mannitol formulations. These formulations should have better
shelf-life at ambient temperature as compared to the saline
formulation.
TABLE-US-00001 TABLE 1 Stability Study of Liquid HA and CS
Formulation at five month time point Molecular weight, Excipients
and concentration Sample ID Dalton Fresh prepared in mobile
Standard 2014438 phase 0.9% 9 (w/v) NaCl Saline formulation,
5.degree. C. 2000484 in water Saline formulation, 40.degree. C.
487429 0.9% (w/v)NaCl with 3 mM PBS formulation, 5.degree. C.
1984656 phosphate buffer, pH 7 PBS formulation, 40.degree. C.
783509 5% (w/v)glucose with 3 mM Glucose formulation, 5.degree. C.
2034540 phosphate buffer, pH 7 Glucose formulation, 40.degree. C.
511731 5% (w/v)mannitol with 3 mM Mannitol formulation, 5.degree.
C. 2030504 phosphate buffer, pH 7 Mannitol formulation, 40.degree.
C. 1765220 10% (w/v)sucrose with 3 mM Sucrose formulation,
5.degree. C. 2024464 phosphate buffer, pH 7 Sucrose formulation,
40.degree. C. 1688080 10% (w/v)trehalose with 3 Trehalose
formulation, 5.degree. C. 2089824 mM phosphate buffer, pH 7
Trehalose formulation, 40.degree. C. 1706619 5% (w/v) mannitol in
water Mannitol, no phosphate 2112776 formulation, 5.degree. C.
Mannitol, no phosphate 734536 formulation, 40.degree. C.
[0112] HA at 8 mg/mL and Chondroitin sulfate at 8 mg/mL were also
formulated with various stabilizers as outlined in Table 2. The
formulated solutions were filled into 3-mL glass vials at 0.5
mL/vial and lyophilized. The dried samples were placed at
40.degree. C./75% RH for stability study. After 4 months, the test
samples were reconstituted with WFI and analyzed by the SEC-HPLC
method as mentioned in Example 1. As shown in Table 2, the
formulations containing saline had less stability as compared to
the formulations containing glucose, mannitol, sucrose and
trehalose. Actually no significant changes in molecular weight were
detected in these lyophilized formulations when stored at
40.degree. C. for four months.
TABLE-US-00002 TABLE 2 Stability of Lyophilized HA and CS
Formulation at four month time point Excipients and Molecular
concentrations Pre- weight lyophilization Sample ID kDa 0.9% (w/v)
saline with 3 mM PBS formulation A, 40.degree. C. 873 phosphate
buffer, pH 7 869 5% (w/v) glucose with 3 mM Glucose formulation B,
2070 phosphate buffer, pH 7 40.degree. C. 2191 5% (w/v)mannitol
with 3 mM Mannitol formulation C, 2020 phosphate buffer, pH 7
40.degree. C. 2028 2.5% (w/v) mannitol/5% Mannitol/sucrose formula-
2034 (w/v) sucrose with 3 mM tion D, 40.degree. C. 2046 phosphate
buffer, pH 7 10% (w/v) trehalose with 3 Trehalose formulation E,
1939 mM phosphate buffer, pH 7 40.degree. C. 1968 10% (w/v) sucrose
with 3 mM Sucrose formulation F, 40.degree. C. 2036 phosphate
buffer, pH 7 2024 10% trehalose with 10 mM Trehalose formulation G,
1962 histidine buffer, pH 7 40.degree. C. 1960 0.9% saline in water
Saline formulation H, 40.degree. C. 964 978
[0113] High concentrations of HA were also tested. HA at 20 mg/mL
and CS at 20 mg/mL formulated with various stabilizers. The
formulations were tested for stability at 5.degree. C. and
40.degree. C./75% RH storage conditions as outlined in Table 3.
After three months, the test samples were analyzed using the
SEC-HPLC method. As shown in Table 3, the formulation containing
saline only had less stability as compared to the formulations
containing trehalose, sucrose and mannitol.
TABLE-US-00003 TABLE 3 Stability of liquid high concentration HA
and CS formulations at three month time point HA MW, Excipients and
concentrations Sample ID kDa Fresh in mobile phase std 2077 10%
(w/v) trehalose with 3 Trehalose formulation A, 2041 mM phosphate
buffer, pH 7 5.degree. C. formulation A, 40.degree. C. 1565 10%
(w/v) sucrose with 3 mM Sucrose formulation B, 5.degree. C. 2039
phosphate buffer, pH 7 formulation B, 40.degree. C. 1590 5% (w/v)
mannitol with 3 mM Mannitol formulation C, 5.degree. C. 1965
phosphate buffer, pH 7 formulation C, 40.degree. C. 1611 0.9% (w/v)
saline in water Saline formulation D, 5.degree. C. 1967 formulation
D, 40.degree. C. 785 5% (w/v) trehalose/0.23% Trehalose/PBS
formulation, 2023 (w/v)saline with 3 mM 5 C. phosphate buffer, pH 7
Trehalose/PBS formulation, 1852 40 C.
Example 2
Stability of HA Formulation with Various Concentrations of
Trehalose
[0114] HA at different concentrations was formulated with various
concentrations of trehalose as outlined in Table 4. Some of the
formulated solutions were divided into two groups. One was placed
at stability chambers for liquid stability study and the other was
lyophilized for solid stability assessment. After three months of
storage at 5.degree. C. and 40.degree. C./75% RH, the test samples
were analyzed using the SEC-HPLC method. As shown in Table 4, no
significant differences in molecular weight were observed among the
samples with different concentrations of trehalose. However,
without any excipients HA formulated with water only was much less
stable in liquid form and lyophilized form.
TABLE-US-00004 TABLE 4 Stability of HA formulation with various
concentrations of trehalose Molecular Weight Formulation
composition Formulation ID kDa 10 mg/mL HA with 5% Liquid 177A,
5.degree. C. 2103 (w/v)trehalose in water Liquid 177A, 40.degree.
C. 1902 2 mg/mL HA with 2% (w/v) Liquid 146A, 5.degree. C. 2050
trehalose in water Liquid 146A, 40.degree. C. 1713 Lyo 146A,
5.degree. C. 2090 Lyo 146A, 40.degree. C. 2036 2 mg/mL HA with
0.67% (w/v) Liquid 146B, 5.degree. C. 2052 trehalose in wter Liquid
146B, 40.degree. C. 1690 Lyo 146B, 5.degree. C. 2112 Lyo 146B,
40.degree. C. 2013 No any excipients Liquid 171E, 5.degree. C. 2057
Liquid 171D, 40.degree. C. 666 Lyo 171E, 5.degree. C. 2038 Lyo
171E, 40.degree. C. 731
Example 3
Rat MMT Model of Osteoarthritis
[0115] In the rat medial mensical tear (MMT) model of
osteoarthritis, transection of the medial meniscus results in joint
deterioration and reduced weight bearing that mimics human
osteoarthritis. A unilateral medial meniscal tear in 300-400 gram
rats results in rapidly progressive cartilage degenerative changes
characterized by chondrocyte and proteoglycan loss, fibrillation,
osteophyte formation and chondrocyte cloning. Such changes are
typically observed to be substantial by day 21 following the
meniscal surgery. The extent of joint deterioration, as determined
primarily by the extent of cartilage lesions formed, can be
measured using a semi-quantitative histological scoring system.
[0116] To test the utility of trehalose as a vehicle for
intra-articularly injected compounds, a solution of trehalose was
formulated as a 5% solution in 3 mM glycine-HCl buffer, having a pH
of approximately 3. The solution was tested in the rat MMT model.
Transection of the medial collateral ligament just below attachment
to the meniscus was performed on 15 male Lewis rats. The meniscus
was cut at its narrowest point (away from the ossicles) taking care
not to damage the tibial surface.
[0117] Beginning three days after MMT surgery, intra-articular
injections of the trehalose solution were given weekly for five
weeks, followed by euthanasia at week six.
[0118] Histology results showed that the trehalose solution gave an
unexpected improvement in cartilage preservation. As shown in FIG.
5, trehalose gave a significant reduction in the width of
significant cartilage lesions, as defined by the width of tibial
cartilage lesions in which chondrocyte and proteoglycan loss extend
through 50% or more of the original cartilage thickness. Such
measurements are taken using an ocular micrometer from stained
histological cross sections of the medial compartment of the
joint.
[0119] Another measure of cartilage preservation is shown in FIG.
6. Trehalose demonstrated significant improvement in lesion Depth
Ratio. which is calculated by comparing the depth of the lesion
with the original cartilage thickness, which is estimated by
measuring the distance from the extrapolated original tibial
plateau to the tidemark. These measurements are taken using an
ocular micrometer from stained histological cross sections of the
medial compartment of the joint.
[0120] Cartilage preservation also correlated to the extent of
collagen degeneration. FIG. 7 shows that trehalose significantly
reduced the width of cartilage lesions which exhibit collagen
depletion characterized as either marked or severe, as measured
using an ocular micrometer to assess the cross sectional thickness
of cartilage exhibiting reduced collagen staining. The extent of
collagen degeneration is defined as follows: [0121] Severe damage
(total or near total loss of collagen to tidemark, >90%
thickness) [0122] Marked damage (extends through 61-90% of the
cartilage thickness) [0123] Moderate damage (extends thru 31-60% of
the cartilage thickness) [0124] Mild damage (extends through 11-30%
of the cartilage thickness) [0125] Minimal damage (very
superficial, affecting upper 10% only)
[0126] Trehalose also improved cartilage degeneration scores, which
is a measure of the cellular viability of the remaining cartilage.
Cartilage degeneration scores were determined histologically by
estimating the area of non-viable (significant chondrocyte and
proteoglycan loss but collagen retention) and completely absent
cartilage matrix, then estimating the percentage of that area
compared to the area of the extrapolated original cartilage cross
sectional area. A score of 1-5 is assigned, with a score of 1
indicating cartilage degeneration of 10% or less, and a score of 5
indicating complete cartilage degeneration. In FIG. 8, Zone 1
refers to the one third of the medial tibial plateau closest to the
transected meniscus. Zone 1 is the area having the most severe
cartilage damage in the MMT model, and was considered extremely
challenging as a target for OA therapies. Zone 3 is the one third
of the tibial plateau furthest from the surgical site, and
sustained the least amount of cartilage damage. Zone 2 was
considered to be the area of intermediate cartilage damage and
represented a reasonable target for OA therapies. Trehalose
statistically improved cartilage degeneration score in all three
zones.
Example 4
Rabbit ACLT Model of Osteoarthritis
[0127] The trehalose formulation (a 5% trehalose solution in 3 mM
glycine-HCl buffer having a pH of approximately 3) described above
was also tested in the rabbit anterior cruciate ligament
transection (ACLT) model of OA. The ACLT surgery destabilized the
rabbit knee to a greater extent than meniscal transection,
resulting in not only substantial cartilage deterioration but also
osteophyte formation. Studies investigating the development and
regulation of osteophyte formation in the rabbit model of OA have
detected plate-like formations as early as 4 weeks post-ACLT, and
osteophyte growth in all compartments of the knee by 12 weeks
post-ACLT.
[0128] Rabbits were anesthetized. The patellar fat pad was
retracted, and the ACL was isolated. The anterior cruciate ligament
was severed with a surgical blade. The fibrous capsule and
subcutaneous tissue were closed prior to the skin being sutured.
Beginning one week after surgery, intra-articular injections were
given weekly for seven weeks, followed by euthanasia at 8 weeks.
Cartilage degradation was determined histologically using a
modified Mankin scale. Higher scores indicated more extensive
cartilage degradation, with a maximum score of 18.
[0129] As shown in FIG. 9, the trehalose formulation gave a
significant improvement in modified Mankin score compared to
untreated animals. (n=15), indicating decreased cartilage
degeneration as a result of the intra-articular trehalose
injections.
[0130] Terminology
[0131] A "therapeutically effective amount" or "effective amount"
is that amount of an agent to achieve a pharmacological effect. The
term "therapeutically effective amount" includes, for example, a
prophylactically effective amount. An "effective amount" is an
amount effective to achieve a desired pharmacologic effect or
therapeutic improvement without undue adverse side effects. For
example, an effective amount refers to an amount that increases
operativity, quality of life, increases the ability of the patient
to increase mobility, or increases weight bearing load, or
decreases pain, or increases growth in the bone and cartilage of
one or more joints, or reduces joint distortion, pain, swelling, or
stiffness. The effective amount of an agent will be selected by
those skilled in the art depending on the particular patient and
the disease level. It is understood that "an effect amount" or "a
therapeutically effective amount" can vary from subject to subject,
due to variation in metabolism of therapeutic agents and/or
prokinetic agents, age, weight, general condition of the subject,
the condition being treated, the severity of the condition being
treated, and the judgment of the prescribing physician.
[0132] "Treat" or "treatment" refers to any treatment of a disorder
or disease associated with bone, cartilage, of soft tissue (such as
the synovium) disorder, such as preventing the disorder or disease
from occurring in a subject which may be predisposed to the
disorder or disease, but has not yet been diagnosed as having the
disorder or disease; inhibiting the disorder or disease, e.g.,
arresting the development of the disorder or disease, relieving the
disorder or disease, causing regression of the disorder or disease,
relieving a condition caused by the disease or disorder, or
stopping the symptoms of the disease or disorder. Thus, as used
herein, the term "treat" is used synonymously with the term
"prevent."
[0133] By "co-administered" is meant simultaneous administration in
the same formulation or in two different formulations that are
combined into one formulation for administration. In one
embodiment, the HA and the other component are co-administered via
delivery in the same formulation.
[0134] The term "subject" as used herein refers to an animal, in
one embodiment, a mammal and in another embodiment, a human, who
can benefit from the compositions and methods of the present
invention. There is no limitation on the type of animal that could
benefit from the present methods. A subject regardless of whether a
human or non-human animal may be referred to as an individual,
subject, animal, host or recipient. The methods of the present
invention have applications in human medicine, veterinary medicine
as well as in general, domestic or wild animal husbandry. In one
embodiment, the candidate subject is a mammal such as a human,
laboratory test animal, such as a mouse, rat, rabbit, guinea pig,
hamster or avian species, such as a poultry bird and veterinary
medical animal, such as dog, cat, horse, cow, sheep, etc.
[0135] One of ordinary skill in the art will appreciate further
features and advantages of the invention based on the
above-described embodiments. Accordingly, the invention is not to
be limited by what has been particularly shown and described,
except as indicated by the appended claims. All publications and
references cited herein are expressly incorporated herein by
reference in their entirety.
* * * * *