PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification © :
`(11; international Publication Number:
`WO 95/22560
`CO07K 14/475, A61K 38/18
`.
`.
`24 August 1995 (24.08.95)
`(43) International Publication Date:
`
`
`
`
`KP, KR, KZ, LK, LR, LT, LU, LV,
`,
`MG,
`,
`MX, NL, NO, NZ, PL, PT, RO, RU, SD, SE,
`
`
`TT, UA, UZ, VN, European patent (AT, BE, CH, DE, DK,
`ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI
`patent (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE,
`
`SN, TD, TG), ARIPO patent (KE, MW, SD, SZ, UG).
`
`
`Published
`With international search report.
`
`
`
`
`
`
`(21) International Application Number:
`
`PCT/US95/02153
`
`(22) International Filing Date:
`
`21 February 1995 (21.02.95)
`
`(30) Priority Data:
`08/200,243
`
`22 February 1994 (22.02.94)
`
`US
`
`(71) Applicant: THE SYNTEX-SYNERGEN NEUROSCIENCE
`_
`JOINT VENTURE[US/US]; 1885 33rd Street, Boulder, CO
`80301-2546 (US).
`
`(72) Inventors: DIX, Daniel, B.; 1183 Twin Peaks Circle, Long-
`mont, CO 80503 (US). KOSKY,Andrew, A.; 2958 Alice,
`Newbury Park, CA 91320 (US). FREUND, Erwin; 801
`South County Road 21, Berthoud, CO 80513 (US).
`
`
`
`
`(74) Agents: SWANSON, Barry, J. et al; Swanson & Bratschun,
`L.L.C., Suite 200, 8400 East Prentice Avenue, Englewood,
`CO 80111 (US).
`
`
`
`(54) Title: PHARMACEUTICAL FORMULATIONS OF CNTF
`
`(57) Abstract
`Pharmaceutical formulationsofciliary neurotrophic factor (CNTF) are described which are useful for therapeutic treatment of damage
`to the peripheral nervous system. Formulations of CNTF ideally suited for intrathecal administration are provided.
`
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`..
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`
`
`
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`
`
`
`
`applications under the PCT.
`
`AT Austria
`Mauritania
`GB United Kingdom
`MR
`GE Georgia
`Malawi
`AU Australia
`MW
`Niger
`NE
`GN Guinea
`Balbados
`BB
`NL Netherlands
`GR Greece
`BE Belgium
`NO Norway
`BF Bwlcina Faso
`HU Hungmy
`NZ New Zealand
`BG Bulgaria
`IE Ireland
`IT Italy
`BJ Benin
`PL Poland
`JP Japan
`PT Portugal
`Brazil
`BR
`KE Kenya
`Romania
`BY Bel8JUS
`RO
`
`RU Russian Federation
`KG Kyrgystan
`
`CA Canada
`
`Democratic People's Republic SD Sudan
`
`CF. Central African Republic
`SE Sweden
`of Korea
`CG Congo
`SI Slovenia
`KR Republic of Korea
`
`CH Switzerland
`KZ Kazakhstan
`Slovakia
`Cl COie d'Ivoire
`SK
`LI Liechtenstein SN Senegal
`CM Cameroon
`LK Sri Lanka
`CN China
`TD Chad
`cs Czechoslovakia LU Luxembourg
`TG Togo
`LV Latvia
`TJ Tajikistan
`CZ Czech Republic
`DE Germany
`TT Trinidad and Tobago
`
`MC Monaco
`UA Ukraine
`MD Republic of Moldova
`Denmark
`DK
`MG Madagascar
`
`United States of America
`ES Spain
`us
`uz Uzbekistan
`FI Finland
`ML Mali
`VN Viet Nam
`MN Mongolia
`FR France
`GA Gabon
`
`KP
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`WO 95/22560
`
`.
`
`PCT/U895/02153
`
`PHARMACEUTICAL FORMULATIONS OF CNTF
`
`TECHNICAL FIELD OF THE INVENTION
`
`This invention relates to pharmaceutical
`formulations of ciliary neurotrophic factor suitable
`for therapeutic treatment of damage to the peripheral
`nervous system.
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`BACKGROUND OF THE INVENTION
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`The peripheral nervous system consists of those
`nerve cells that extend axonal processes outside the
`spinal cord and brain.
`The principle nerve cell types
`in the peripheral nervous system are primary motor
`neurons innervating skeletal muscle and controlling
`movement, autonomic neurons (both sympathetic and
`parasympathetic)
`innervating the cardiovascular system
`and other internal organs and regulating their
`function, and sensory neurons innervating sensory
`receptors throughout the body and conveying sensations
`including pain and proprioception.
`Conditions that compromise the survival and proper
`function of one or more of these types of peripheral
`nerve cells cause peripheral nerve damage.
`Such nerve
`damage may occur from a wide variety of different
`causes. Nerve damage may occur through physical
`injury, which causes the degeneration of the axonal
`processes and/or nerve cell bodies near the site of
`injury. Nerve damage may also occur because of
`
`temporary or permanent cessation of blood flow to parts
`
`of the nervous system, as in stroke. Nerve damage may
`. also occur because of intentional or accidental
`
`exposure to neurotoxins, such as the cancer and AIDS
`chemotherapeutic agents cisplatinum and dideoxycytidine
`(ddc), respectively. Nerve damage may also occur
`because of chronic metabolic diseases, such as diabetes
`or renal dysfunction. Nerve damage may also occur
`because of neurodegenerative diseases such as
`Parkinson’s disease, Alzheimer’s disease, and
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`PCT/US95/02153
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`-2-
`
`Amyotrophic Lateral Sclerosis (ALS), which result from
`the degeneration of specific neuronal populations.
`Neurotrophic factors are naturally occurring
`proteins that promote the survival and functional
`activities of nerve cells. Neurotrophic factors have
`been found in the target cells to which an innervating
`nerve cell connects.
`Such target-derived neurotrophic
`factors regulate the number of contacts formed between
`innervating nerve cells and the target cell population,
`and are necessary for the survival and maintenance of
`
`these nerve cells.
`
`Neurotrophic factors are also found in cells that
`are not innervated. An example of such a neurotrophic
`factor is CNTF. Human CNTF and the gene encoding human
`CNTF are described in detail in U. S. patent numbers
`4,997,929, and 5,141,856, which are specifically
`incorporated herein by this reference.
`Although the biological role of CNTF has not been
`conclusively established, CNTF appears to be released
`upon injury to the nervous system and may limit the
`extent of injury or neuronal damage. Highly-purified
`CNTF has been shown to support the survival in cell
`cultures of chick embryonic parasympathetic,
`sympathetic, sensory, and motor neurons. There is
`significant evidence to support that CNTF is a
`neurotrophic factor for peripheral primary neurons in
`vivo and in vitro.
`U. S. patent application serial
`number 07/735,538 filed July 23, 1991, specifically
`incorporated herein by reference,
`shows the surprising
`effectiveness of systemically administered CNTF to
`accelerate local recovery at the site of peripheral
`nerve damage.
`
`The ability of CNTF to protect motor neurons from
`lesion-induced death may also make it effective in
`preventing nerve cell degeneration associated with such
`neurodegenerative diseases as Parkinson’s disease,
`Alzheimer’s disease, Amyotrophic Lateral Sclerosis
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`/
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`PCT/US95/02153
`
`-3-
`
`(ALS), and Spinal Muscular Atrophy (SMA). U.S. patent
`application serial number 08/015,218 filed February 8,
`1993 and U.S. patent application serial number
`08/116,440 filed September 3, 1993, both of which are
`entitled Methods for Treating Amyotrophic Lateral
`Sclerosis With CNTF, are specifically incorporated
`herein by reference. These patents present evidence
`demonstrating the effectiveness of treating amyotrophic
`lateral sclerosis in humans with CNTF.
`In addition,
`Sendtner et al.
`(1990) Nature 345:440-441,
`showed that
`local application of CNTF prevents lesion-induced death
`of motor neurons in the rat facial brain stem nucleus.
`Oppenheim et al.
`(1991) Science 251:1616-1618,
`showed
`that CNTF promoted the in vivo survival of chick spinal
`motor neurons.
`
`A major problem with the delivery of a
`therapeutically effective amount of CNTF to a patient
`for treatment or prevention of peripheral nerve damage
`is the instability of CNTF in solution.
`CNTF in
`solution rapidly precipitates when agitated or
`subjected to thermal incubation.
`To the extent that
`
`the effective amount
`any of the protein is denatured,
`of biologically active CNTF is diminished. Protein
`integrity must therefore be maintained during
`manufacture and storage as well as during
`administration. Proteins are particularly prone to
`degradation at elevated temperatures. Excipients known
`to physically stabilize proteins in solutions appear to
`negatively affect the thermal stability of CNTF.
`In addition, CNTF is subject to loss from solution
`by nonspecific adsorption to the surface areas of
`storage containers and dispensing devices.
`Such
`nonspecific binding may occur to a variety of materials
`including glass and plastics, for example, polyethylene
`or polypropylene. These materials may be in the form
`of vials,
`tubing, syringes,
`inplantable infusion
`devices, or any other surface which may come in contact
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`WO 95/22560
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`PCT/US95/02153
`
`-4-
`
`with CNTF during its manufacture, storage or
`administration.
`
`For certain applications for the treatment or
`prevention of peripheral nerve damage in humans it is
`desirable to administer the CNTF,
`in formulation,
`intrathecally.
`Intrathecal administration necessitates
`that CNTF must be maintained in an aqueous solution for
`relatively long periods of time at elevated
`
`In certain such administrations the CNTF
`temperatures.
`formulations will be maintained at room temperature,
`while other administrations will actually require that
`the CNTF formulation will be held at body temperature.
`These are obviously relatively harsh conditions for a
`sensitive protein such as CNTF.
`
`In addition to the problems associated with the
`administration of CNTF,
`there are also problems
`associated with its long term storage from the time of
`Manufacture to administration.
`Lyophilization is one method of enabling the long
`term storage of biological proteins,
`impeding
`degradation, aggregation, and/or nonspecific
`adsorption. However,
`the lyophilization process itself
`often presents difficulties. As the volume of liquid
`decreases during the freezing process,
`the effective
`salt concentration increases dramatically, which tends
`to denature the protein, reducing effective therapeutic
`activity upon reconstitution.
`In addition,
`formation
`of ice crystals during the freezing process may cause
`denaturation and also decrease the effective amount of
`bioactive CNTF available.
`The most desired formulation
`for CNTF must be able to prevent loss of bioactivity of
`the CNTF during the lyophilization process.
`The present invention includes formulations of
`CNTF which physically and thermally stabilize CNTF
`against degradation and precipitation under a wide
`variety of storage and administration regimes.
`This invention further includes formulations of
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`WO 95/22560
`
`.
`
`PCT/US95/02153
`
`-5-
`
`CNTF in which bioactivity is maintained after
`lyophilization and reconstitution, and methods of
`storing biologically active CNTF in solution.
`
`
`BRIEF SUMMARY OF THE INVENTION
`
`The present invention includes therapeutically
`useful formulations of CNTF which greatly reduce the
`prior art problems of loss of CNTF from solutions due
`to chemical degradation, precipitation, and adsorption.
`The present invention includes three types of
`stabilized CNTF formulations, characterized as
`stabilized aqueous formulations, aqueous formulations
`suitable for intrathecal administration and
`
`lyophilization formulations suitable for lyophilization
`and subsequent reformulations. Also included within
`the scope of this invention are CNTF containing
`mixtures that are the result of lyophilization of the
`lyophilization formulations of the present invention,
`as well as the same after reconstitution.
`,
`This invention provides physically and chemically
`stable aqueous formulations of CNTF. Preferred
`formulation embodiments comprise aqueous solutions of:
`(a)
`ciliary neurotrophic factor;
`{b)
`stabilizing agents;
`(c)
`a sufficient amount of biologically
`acceptable salt to maintain isotonicity;
`(d)
`a buffer to maintain the pH of the
`formulation from about 4.5 to about 6.0; and
`(e)
`optionally, a biologically acceptable water
`soluble carrier.
`
`The pharmaceutical CNTF formulation embodiments of
`
`this invention suitable for intrathecal administration
`
`comprise aqueous solutions of:
`
`ciliary neurotrophic factor;
`(a)
`two or more stabilizing agents;
`(b)
`a sufficient amount of a biologically
`(c)
`acceptable salt to maintain isotonicity; and
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`PCT/US95/02153
`
`-6-
`
`(d)
`
`a buffer in an amount sufficient to maintain
`
`the pH of the formulation at about 6.0.
`This invention further includes pharmaceutical
`CNTF formulation embodiments suitable for
`lyophilization that comprise aqueous solutions of:
`(a)
`ciliary neurotrophic factor;
`(b)
`a biologically acceptable bulking agent;
`(c)
`one or more stabilizing agent (s);
`(d)
`a buffer in an amount sufficient to maintain
`
`the pH of the formulation at about 5.0 to about 7.6;
`and
`
`a biologically acceptable salt.
`(e)
`Further embodiments of this invention are the
`
`lyophilized formulations from which the water has been
`
`substantially removed. Upon reconstitution with a
`reconstituting vehicle, optionally including a
`biologically acceptable carrier,
`the lyophilized
`formulations of the present invention are suitable for
`administration to patients in need thereof.
`Such
`
`reconstituted solutions of CNTF are also included
`
`within the scope of this invention.
`Another embodiment of the present invention is a
`method for the prevention and treatment of peripheral
`nerve damage by administering a therapeutically
`effective amount of a human protein ciliary
`neurotrophic factor formulation as described herein to
`
`the
`In particular,
`a patient in need thereof.
`invention provides formulations for administering
`therapeutically effective amounts of CNTF in order to
`prevent and treat peripheral nerve damage,
`including
`neurodegenerative diseases such as Parkinson's disease,
`Alzheimer’s disease, ALS, and SMA.
`The present
`invention also includes methods for preventing and
`treating peripheral nerve damage by administering to a
`patient in need thereof a therapeutically effective
`amount of CNTF formulated as described herein.
`It is to be understood that both the foregoing
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`WO 95/22560
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`.
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`PCT/US95/02153
`
`-7-
`
`general description and the following detailed
`description are exemplary and explanatory only and are
`not restrictive of the invention as claimed.
`
`
`DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
`
`Reference will now be made in detail to the
`
`presently preferred embodiments of the invention,
`which,
`together with the following examples, serve to
`explain the principles of the invention.
`The present invention includes therapeutically
`useful formulations of CNIF for the prevention and
`treatment of peripheral nerve damage,
`including
`neurodegenerative diseases such as Parkinson’s and
`
`Alzheimer’s diseases, ALS, and SMA.
`
`The present invention includes the use of CNTF as
`a therapeutic agent by administering a therapeutic
`composition whose active ingredient consists of CNTF.
`Such therapeutic compositions include compositions
`suitable for use in a wide variety of administration
`regimes which are stable under a variety of storage and
`administration conditions. When included in most
`
`standard prior art formulations, CNTF is chemically
`unstable even when stored at 4°C.
`The therapeutic use
`of CNTF in the treatment of peripheral nerve damage
`requires formulations which are acceptable for rapid
`and easy administration to patients in need thereof,
`readily manufacturable, and stable for a prolonged
`period of time over a variety of storage conditions.
`This application describes CNTF formulations
`
`The aqueous CNTF
`suitable for certain situations.
`formulations described herein are suitable for
`
`maintaining the stability and bioactivity of CNTF in
`aqueous solution.
`The aqueous formulations suitable
`
`for intrathecal administration are suitable for
`maintaining the stability and bioactivity of CNTF in
`situations where the formulation is maintained at
`elevated temperatures for long periods of time and is
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`PCT/US95/02153
`
`-8-
`
`Such
`
`The
`contained in high surface area containers.
`lyophilization formulations of CNTF are suitable for
`maintaining the stability and bioactivity of CNTF when
`subjected to lyophilization and subsequent
`reconstitution.
`The most preferred embodiments of the
`present invention are aqueous formulations that are
`stable prior to lyophilization and that have retained
`their bioactivity following lyophilization,
`long term
`storage, and reconstitution.
`According to the present invention, CNTF may be
`formulated for use as a therapeutic agent when included
`in solution with agents that are effective in
`stabilizing the CNTF against precipitation from
`solution,
`thermal degradation and adsorption.
`agents include non-ionic surfactants such a
`polysorbate-80, as well as propylene glycol,
`polyethylene glycol,
`lysine, arginine, cysteine,
`glutathione, ethanol and other alcohols.
`The preferred
`formulations of the present invention also may include
`other ingredients that function to improve the
`therapeutic capabilities of the CNTF.
`Such other
`ingredients include sodium chloride, glycerol, human
`serum albumin,
`sodium phosphate, and tris
`(hydroxymethyl) aminomethane.
`As used in the specification, "pharmaceutically
`effective amount" means an amount of CNTF which is
`therapeutically effective in various administration
`regimes in the prevention and treatment of peripheral
`nerve damage.
`"Biologically acceptable" applies to
`‘materials characterized by the absence of significant
`adverse biological effects in vivo.
`"Room temperature"
`is between about 22°C to about 250°C.
`"Body
`temperature" is between about 36°C to about 400C.
`"Lyophilizable formulation" refers to an aqueous
`formulation of CNTF which may be freeze dried to a
`moisture content of less than about 2% and which
`retains at least about 70% of the initial CNTF
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`-9-
`
`"Isotonic" refers to
`bioactivity upon reconstitution.
`a solution having approximately the same osmotic
`A
`pressure as blood serum, about 300 mM per liter.
`"carrier" is any biologically acceptable emulsifier,
`dispersing agent, surfactant, or protein which
`decreases adsorption of CNTF to a surface.
`The preferred form of CNTF is human CNTF (hCNTF).
`The most preferred form of hCNTF is recombinant hCNTF
`(YhCNTF). Methods of obtaining CNTF suitable for use
`in the formulations of this invention are known to
`those skilled in the art.
`For example, suitable rhCNTF
`may be produced by the recombinant DNA procedures
`described in U.S. patent number 5,141,856, specifically
`incorporated herein by this reference.
`The CNTF should
`be at least 65% pure, and most preferably at least 98%
`pure.
`The purity of isolated CNTF for use in the
`formulations may be determined by silver-stained SDs-
`PAGE or other means known to those skilled in the art.
`
`A. Aqueous CNTF formulations.
`In the aqueous CNIF formulations provided, CNTF is
`present in therapeutically effective amounts.
`Preferably CNTF comprises from about 0.01 to about 8.0
`mg/ml.
`The aqueous CNTF formulations optionally include
`carriers.
`The presence of the carrier in the
`formulation reduces or prevents CNTF adsorption to
`various surfaces.
`The need for a carrier depends upon
`the concentration of CNTF in the aqueous formulation.
`Suitable carriers include, but are not Limited to,
`polysorbates such as Tween® 80 and proteins such as
`serum albumin. Further, amino acids such as lysine and
`arginine have been shown to prevent adsorptive losses
`of CNTF.
`In one embodiment of the invention, human
`serum albumin (HSA)
`is particularly preferred.
`In
`another embodiment of the invention, Tween® is
`preferred.
`In yet another embodiment of the invention,
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`
`-10-
`
`a combination of lysine and arginine is preferred.
`The CNTF formulations may also contain a
`sufficient amount of a chemically stabilizing agent
`such as glycerol. Glycerol has been shown to
`physically stabilize CNTF formulations from degradation
`resulting from repeated freezing and thawing cycles,
`and from degradation when exposed to high temperatures
`(37°C or above).
`In the absence of a physically
`Stabilizing agent such as glycerol, CNTF in solution
`must be stored at
`
`-200C to maintain biological activity.
`The aqueous CNTF formulations further contain a
`biologically acceptable buffer to maintain solution pH.
`The preferred stable CNTF formulation is buffered with
`a biologically acceptable buffer to a pH between 4.5 to
`about 8.0, most preferably between about 5.0 and 7.6.
`Depending on the specific formulation, suitable buffers
`include citric acid, tris (hydroxymethyl) aminomethane,
`citrate, acetate, phosphate,
`tromethamine, and
`histidine.
`The preferred amount of buffer will vary
`depending on the type of buffer used and its buffering
`capacity.
`The buffer should be present in the
`formulation in sufficient quantity to maintain the
`preferred pH range.
`The preferred concentration of
`buffer for stable CNTF formulations is 1 to 50 mM.
`In one embodiment of the aqueous CNTF formulation
`of the present invention, a non-ionic surfactant is
`added to stabilize CNTF in solution upon agitation.
`one embodiment of formulated CNTF,
`the non-ionic
`surfactant is polysorbate-80 (Tween® 80).
`The
`preferred concentration of Tween® 80 was found to
`increase with increasing CNTF concentration.
`In one
`embodiment of the present invention, CNTF is present in
`a concentration range of between 0.01 - 8.0 mg/ml and
`the non-ionic surfactant is polysorbate-80 (Tween® 80)
`in the concentration range of between 0.1 - 1.0%
`weight/volume (w/v).
`In a more preferred embodiment of
`
`In
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`PCT/US95/02153
`
`-11-
`
`the present invention,
`
`the formulation further
`
`comprises glycerol in the concentration range between 4
`- 20%.
`In a further preferred embodiment of the
`
`the formulation comprises 0.01 - 8.0
`present invention,
`mg/ml CNTF, 0.2 - 0.8% (w/v) Tween® 80,
`5
`- 15% (w/v)
`
`glycerol, human serum albumin (HSA)
`
`in the
`
`concentration range of 0.01 -
`
`5% (w/v), 1-50 mM sodium
`
`1 - 50 mM tris (hydroxymethyl)
`phosphate, pH 6-8,
`aminomethane, and 100 - 400 mM sodium chloride.
`In a second embodiment of the aquecus CNTF
`formulation of the present invention, propylene glycol
`is added to prevent precipitation of CNTF in solution.
`
`In one embodiment of the present invention, CNTF is
`
`present in the formulation in the concentration range
`of between 0.01 - 8.0 mg/ml and propylene glycol is
`present in the concentration range of between 1 - 30%
`(w/v).
`In a more preferred embodiment of the present
`invention,
`the formulation is further comprised of
`glycerol in the concentration range between 4 - 20%
`(w/v).
`In a further preferred embodiment of the
`present invention,
`the formulation is comprised of 0.01
`~ 8.0 mg/ml CNTF, 20 -25% (w/v) propylene glycol, 10 -
`20% (w/v) glycerol, HSA in the concentration range of
`0.01 -
`5% (w/v),
`1 - 50 mM sodium phosphate, pH 6-8,
`
`1
`
`- 50 mM tris (hydroxymethyl) aminomethane, and 100 -
`400 mM sodium chloride.
`
`In a third embodiment of the aqueous CNTF
`
`formulation of the present invention, an alcohol is
`
`added to stabilize CNTF in solution upon agitation.
`
`Such alcohols that may be used include ethanol,
`propanol,
`t-butanol, and other simply alkyl alcohols.
`
`In one embodiment of the formulation of the present
`
`invention,
`the alcohol is ethanol.
`In a preferred
`embodiment of the present invention, CNTF is present in
`the concentration range of between 0.01 - 8.0 mg/ml and
`ethanol is present in the concentration range of 0.1 -
`20% (v/v).
`In a more preferred embodiment of the
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`
`the formulation further comprises
`present invention,
`glycerol in the concentration range between 4 - 20%
`(w/v).
`In a further preferred embodiment of the
`present invention,
`the formulation is comprised of 0.01
`- 8.0 mg/ml CNTF,
`8 - 13% (v/v) ethanol, and 5
`- 20%
`(w/v) glycerol, HSA in the concentration range of 0.01
`-
`5% (w/v),
`1 - 50 mM sodium phosphate, pH 6-8,
`1 - 50
`mM tris (hydroxymethyl) aminomethane, and 100 - 400 mM
`sodium chloride.
`
`B.
`
`Aqueous CNTF formulations suitable for Intrathecal
`
`Administration.
`
`/
`
`Several embodiments of the CNTF formulation cf the
`present invention are useful for intrathecal delivery.
`The intrathecal CNTF formulations of the present
`invention represent a major advance in solving the
`problems of CNIF instability and surface adsorption
`upon incubation and agitation. An intrathecal
`formulation requires chemical, physical, and thermal
`stability under all conditions associated with
`intrathecal delivery via an external or inplantable
`pump. Further, biological activity must not be
`decreased through surface adsorption upon incubation.
`CNTF formulations appropriate for intrathecal
`delivery must also contain a sufficient amount of
`biologically acceptable salt to maintain fluid
`tonicity. Preferably,
`the CNTF formulation contains
`sufficient salt to be isotonic, within physiologically
`acceptable limits, with human blood or cerebral spinal
`fluid.
`The preferred salt is sodium chloride (NaCl),
`but other biologically acceptable salts may be used,
`such as potassium chloride (KCl), calcium chloride
`(CaCl,), and magnesium chloride (MgCl,).
`The salt may
`be one salt or a combination of salts.
`A preferred
`formulation comprises 100 to 400 m™ salt of the aqueous
`formulation.
`
`One intrathecal formulation of the present
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`-13-
`
`invention is comprised of 0.01 - 8.0 mg/ml CNTF, 10-13%
`(v/v) ethanol, and 5% lysine.
`In a preferred
`embodiment,
`the intrathecal formulation is further
`comprised of 10 mM phosphate (pH 7.0), 150 mM NaCl, and
`0.1 - 20% glycerol.
`An additional intrathecal formulation is comprised
`of 0.01 - 8.0 mg/ml CNTF, 20-25% propylene glycol, and
`10% glycerol.
`In a more preferred embodiment,
`the
`intrathecal formulation is further comprised of 3-5%
`lysine.
`In a preferred embodiment,
`the formulation is
`further comprised of 10 mM citrate acid (pH 6.0) and
`150 mM Nacl.
`
`An additional intrathecal formulation is comprised
`of 0.01 - 8.0 mg/ml CNTF,
`4% polyethylene glycol 8000
`(PEG 8000), and 10% glycerol.
`Ina preferred
`embodiment,
`the formulation is further comprised of 5%
`lysine.
`In a more preferred embodiment,
`the
`formulation is further comprised of 10 mM citrate acid
`(pH 6.0) and 150 mM NaCl.
`A final intrathecal formulation is comprised of
`0.1 - 8.0 mg/ml CNTF,
`2% PEG 8000,
`10% propylene
`glycol, and 10% glycerol.
`The preferred embodiment is
`further comprised of 5% lysine, 10 mM citrate acid (pH
`6.0), and 150 mM NaCl.
`the therapeutic compositions
`As described above,
`of this invention may be administered intrathecally by
`continuous infusion from an implanted or an external
`pump. Other effective administration forms, such as
`parenterally slow-release formulations, parentally by
`injection,
`inhalant mists, orally active formulations,
`or suppositories, are also envisioned.
`
`Cc.
`
`Lyophilizable CNTF formulation.
`The most preferred embodiments of the CNTF
`formulation of the present invention are specifically
`formulated to remain stable and bioactive during and
`after lyophilization, and upon reconstitution of the
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`
`The aqueous lyophilized
`lyophilized material.
`formulations of this invention are particularly useful
`for providing long term storage of CNTF.
`The
`lyophilizable formulations of this invention comprise
`CNTF, a biologically acceptable bulking agent, a buffer
`to maintain the pH of the formulation from about 5.0 to
`about 7.5, biologically acceptable salt, and optionally
`a biologically acceptable water soluble carrier.
`CNIF is present in the lyophilizable formulations
`over the same concentration range as in the aqueous
`formulations described above.
`The bulking agent
`generally provides mechanical support by allowing the
`Matrix to maintain its conformation during and after
`the freeze drying process. One or more sugars may be
`used as the bulking agent.
`Sugars, as used herein,
`include but are not limited to, monosaccharides,
`oligosaccharides and polysaccharides. Examples of
`suitable sugars include fructose, glucose, mannose,
`ribose, xylose, maltose,
`lactose, sucrose, and dextran.
`Sugar also includes sugar alcohols, such as Mannitol,
`sorbitol,
`inositol, dulcitol, xylitol, and arabitol.
`Mixtures of sugars may also be used in accordance with
`this invention.
`In one embodiment of the
`lyophilization formulation of the invention,
`polyethylene glycol of average molecular weight 3500
`(PEG 3500)
`is preferred.
`The most preferred bulking
`agent of the present invention is sucrose.
`
`the choice of buffer takes into account
`Ideally,
`potential pH shifts during lyophilization caused by
`sequential crystallization of buffer components.
`For
`example, with phosphate buffers,
`the basic component
`has a higher eutectic point than the acidic component,
`hence it crystallizes out first and the pH drops. This
`behavior may be acceptable and perhaps even beneficial
`in the formulations of the present invention.
`In one
`embodiment of the invention, citric acid buffer is
`preferred because it is thought that both buffer
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`
`components have about the same eutectic point,
`resulting in very little pH fluctuation as the
`
`temperature drops. Other suitable buffers are the
`amino acids histidine and cysteine.
`The lyophilizable formulations may also comprise a
`biologically acceptable salt.
`The salt, which may be
`selected from the same salts useful in the aqueous
`formulations described above,
`is present in the
`lyophilizable formulations at the same or reduced
`concentrations as that in the aqueous formulations.
`Because the salt concentration may increase during
`lyophilization, it may be desirable to reduce the
`concentration of salt present in the lyophilizable
`formulations to prevent protein denaturation.
`Reductions in salt concentration in the lyophilizable
`formulations may be compensated for during
`reconstitution so as to provide a final formulation
`
`sufficiently isotonic to be suitable for administration
`
`into an individual.
`
`the lyophilizable formulations
`Optionally,
`comprise a biologically acceptable water soluble
`carrier.
`The carriers and the concentration of
`
`carriers which may be used in the lyophilizable
`formulations of this invention are the same as those
`
`that are suitable for use in the aqueous formulations.
`
`The lyophilizable formulations of this invention
`
`are generally lyophilized to a residual moisture
`
`content of less than about 2%; however,
`
`formulations
`
`which retain CNTF biological activity at higher or
`lower amounts of moisture content are also
`
`contemplated.
`
`An example of a formulation of CNTF which retains
`
`bioactivity after lyophilization and reconstitution is
`comprised of 0.5 mg/ml CNTF, 85 mg/ml sucrose, 2.0
`mg/ml polysorbate 80 (Tween® 80), 4.0 mg/ml cysteine,
`0.1 mg/ml disodium EDTA, and 2.0 mg/ml citric acid (pH
`5.0).
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`
`-16-
`
`A further example of a formulation of CNTF which
`retains bioactivity after lyophilization and
`reconstitution is comprised of 0.5 mg/ml CNTF, 80 mg/ml
`polyethylene glycol 3350 (PEG 3350), 50 mg/ml lysine,
`20 mg/ml arginine,
`4 mg/ml cysteine, 0.58 mg/ml sodium
`chloride, and 1.6 mg/ml histidine (pH 7.5). Any higher
`molecular weight polyethylene glycol that is a solid at
`room temperature may be used in this formulation.
`In
`addition, glutathione may be used in place of cysteine,
`which may provide improved solution state stability.
`The most preferred embodiment of the inve