PCT
`WORLD INTELLECTUAL PROPERIT ORGANIZATION
`International Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION 1REATY (PCT)
`WO 95/22560
`
`(11) International Publication Number:
`
`(51) International Patent Classification 6 :
`C07K 14/475, A61K 38/18
`
`Al
`
`(43) International Publication Date:
`
`24 August 1995 (24.08.95)
`
`·' I
`
`(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
`
`(81) Designated States: AM, AT, AU, BB, BG, BR, BY, CA, CH,
`CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, JP, KE, KG,
`KP, KR, KZ, LK, LR, LT, LU, LV, MD, MG, MN, MW,
`MX, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SI, SK, TJ,
`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).
`
`(71) Applicant: THE SYNTEX-SYNERGEN NEUROSCIENCE
`JOINT VENTURE [US/US); 1885 33rd Street, Boulder, CO Published
`With international search report.
`80301-2546 (US).
`
`(72) Inventors: DIX, Daniel, B.; 1183 Twin Peaks Circle, Long(cid:173)
`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 A venue, Englewood,
`CO 80111 (US).
`
`(54) Title: PHARMACEUTICAL FORMULATIONS OF CNTF
`
`(57) Abstract
`
`Pharmaceutical formulations of ciliary 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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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify Stales party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AT
`AU
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF .
`CG
`CH
`Cl
`CM
`CN
`cs
`CZ
`DE
`DK
`ES
`Fl
`FR
`GA
`
`Auslria
`Australia
`Barbados
`Belgium
`Bwitina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switwland
`COCe d'Ivoire
`Cameroon
`China
`Czechoslovakia
`Czech Republic
`Germany
`Dcnmarlc
`Spain
`Finland
`France
`Gabon
`
`GB
`GE
`GN
`GR
`HU
`IE
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LI
`LK
`LU
`LV
`MC
`MD
`MG
`ML
`MN
`
`United Kingdom
`Georgia
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Kenya
`Kyrgystan
`Democratic People's Republic
`of Korea
`Republic of Korea
`Kazakhstan
`Liechtenstein
`Sri Lanka
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`Mali
`Mongolia
`
`MR
`Mauritania
`MW Malawi
`NE
`Niger
`Nelherlands
`NL
`NO
`Norway
`NZ
`New Zealand
`PL
`Poland
`Fr
`Ponugal
`RO
`Romania
`RU
`Russian Federation
`SD
`Sudan
`Sweden
`SE
`SI
`Slovenia
`SK
`Slovakia
`SN
`Senegal
`TD
`Chad
`Togo
`TG
`TJ
`Tajikistan
`1T
`Trinidad and Tobago
`Ukraine
`UA
`us
`United Stales of America
`uz
`Uzbelclstan
`Viet Nam
`VN
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`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.
`
`BACKGROUND OF THE INVENTION
`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 oc·cur 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 a~
`Parkinson's disease, Alzheimer's disease, and
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`WO95/22560
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`- 2-
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`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 cel ls. 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 f or 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
`fac tor 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 t he 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
`i ncorporated herein by reference, shows the surprising
`effectiveness of systemically administered CNTF to
`accelerate l ocal recovery at the site of peripheral
`nerve damage.
`The ab i lity of CNTF to protect motor neur ons from
`lesion - induced death may also mak~ it e f fective in
`preventing nerve cell degeneration associated with such
`neurodegenerative diseases as Parkinson's d~sease,
`Alzheimer's disease, Amyotrophic Lateral Scl erosis
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`-3-
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`(ALS ) , and Spinal Muscular Atrophy (SMA). U.S. patent
`application se r i al numbe r 08/015,218 filed February 8,
`1993 and U.S. patent application serial number
`08/116,440 filed September 3, 1993, bot h of which are
`entitled Methods for Treating Amyotrophic Lateral
`Scl eros i s With CNTF, are specifically incorporated
`herein by reference. These patents present evidence
`demonstrating the effecti veness of treat i ng amyotrophic
`lateral sclerosis i n huma ns wi th CNTF.
`In addition,
`Sendtner et al . (1990 ) Nature 345:440 - 441, showed that
`local application of CNTF prevents lesion-induced dea th
`o f 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 damag~
`is the instability of CNTF i n soluti on. CNTF i n
`solution rapidly precipit ates when agitated or
`s ubjected to thermal incubation. To the extent that
`any of the protein is denatured, the eff ective amount
`of biologically active CNTF is diminished. Protein
`i ntegrity must
`t herefo re be maintained during
`manufacture and storage as well as during
`administration. Proteins are particul arly prone to
`degradation at elevated temperatures. Excipi ent s known
`t o physical ly stabilize proteins in solutions appear to
`neg atively affect the thermal stability of CNTF .
`In addition, CNTF is subj ect to loss from solut ion
`by nonspecific adsorption to the s urface areas o f
`storage containers and di s pensing dev ices. Such
`nonspecific bi ndi ng may occur to a variety of materials
`including glass and plastics, for example, polyethylene
`or pol ypropylene . These materials may be in the f orm
`of vials, tubing, syringes, i nplantabl e infusi on
`devices', o r any other surf ace which may come in contact
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`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
`temperatures.
`In certain such administrations the CNTF
`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 f or 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 formul ation
`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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`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 greatl y 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 l yophil ization
`and subsequent reformulations. Also included wi thin
`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 reconstitut ion .
`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:
`(a) ciliary neurotrophic factor;
`(b)
`two or more stabilizing agents;
`a sufficient amount of a biologically
`(c)
`acceptable salt to maintain isotonicity; and
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`-6-
`
`a buffer i n an amount sufficient to maintain
`(d)
`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
`a patient in need t hereof.
`In particul ar, the
`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 ~y 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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`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 CNTF 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 40c . 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
`suitable for certain situations. The aqueous CNTF
`formulations described herein are suitable f or
`maintaining the stability and bioactivity of CNTF in
`aqueous solution. The aqueous formulations sui table
`for intrathecal administration are suitable for
`maintaining the stability and bioactivity of CNTF in
`situations where the for mulation is maintai~ed at
`elevated temperatures for long periods of time and is
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`-8-
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`contained in high surface area containers. The
`lyophilization formulations of CNTF are suitable for
`maintaining the stability and bioactivity of CNTF when
`subjected to lyophilization and subsequent
`reconst itution. The most preferred embodiments of the
`present invention are aqueous formulations that are
`stable prior to lyophilizat ion and that ' have retained
`their bioactivity following l yophilization , 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 precipi tation from
`solution, the rmal degradation and adsorption. Such
`agents include non- ionic surfactants such a
`polysorbate-80, as well as propylene glycol,
`polyethyl ene g l ycol, 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 a lbumin, sodium phosphate, and t ris
`(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 prevent ion and treatment of peripheral
`nerve damage.
`"Biologically acceptable" applies to
`materials characterized by the absence of significant
`adverse biological e ffects i n vivo.
`"Room temperature"
`is between about 22 0c to about 25 oC .
`"Body
`tempera t ure" is between about 36oc to about 400c .
`"Lyophilizable f ormulation" refers to an aqueous
`formulat ion of CNTF which may be f reez e dried to a
`moisture content of less than about 2% and which
`retains a t least about 70% of the initial CNTF
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`"Isotonic" refers to
`bioactivity upon reconstitution.
`a solution having approximately the same osmotic
`pressure as blood serum, about 300 mM per liter. A
`"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
`{rhCNTF). 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 l east 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(cid:173)
`PAGE or other means known to those skilled in the art.
`
`A. Aqueous CNTF formulations.
`In the aqueous CNTF formulat ions 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 t he
`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
`anot her embodiment of the invention, Tween® is
`preferred.
`In yet another embodiment of the invention,
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`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
`{37oC or above).
`In the absence of a physically
`stabilizing agent such as glycer?l, CNTF in solution
`must be stored at
`-2ooc 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
`formul ation 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
`
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`the present invention, the formula tion further
`comprises glycerol in the concent ration range between 4
`- 20%.
`In a further preferred embodiment of the
`present invention, the formulation comprises 0.01 - 8.0
`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), 1-5 0 rnM sodium
`phosphate, pH 6-8, 1 - 50 rnM tris (hydroxyrnethyl )
`aminomethane, and 100 - 400 rnM sodium chloride.
`In a second embodiment of the aqueous CNTF
`formulation of the present i nvention, 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%
`In a more preferred embodiment of the present
`(w/ v ).
`invention, the formulation is further comprised of
`glycerol in the concentration range between 4 - 20%
`(w/v ).
`In a further preferred embodiment of t he
`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 rnM sodium phosphate, pH 6-8, 1
`- 50 rnM tris (hydroxyrnethyl ) aminomethane, and 100 -
`400 rnM sodium chloride.
`In a third embodiment of the aqueous CNTF
`formulation o f the present invention , an alcohol is
`added to s tabilize 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 concentr.ation range of O .1 -
`20% (v/v).
`In a more preferred embodiment of the
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`present invention, the formulation further comprises
`glycerol in the concentration range between 4 - 20%
`(w/v).
`In a furt her 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 of the
`present invention are useful for intrathecal d~livery.
`The intrathecal CNTF formulations of the present
`invention represent a major advance in solving the
`problems of CNTF 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 inpl antable
`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 chlor.ide (KCl}, calcium chloride
`(CaC1 2 }, and magnesium chloride (MgC1 2 ) . The salt may
`be one salt or a combination of salts. A preferred
`formulation comprises 100 to 400 mM salt of the aqueous
`formulation.
`One intrathecal formulat ion of t he present
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`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 formulat ion 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 .
`In a preferred
`embodiment, the formulation is further comprised of 5%
`l ysine.
`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.
`As described above, the therapeut ic compositions
`of this invention may be administered intrathecally by
`continuous infusion from an implanted or an external
`pump. Other effective administration forms, such a s
`parenterally slow-release fo rmulations, parentally by
`injection, inhalant mists, orally active formulations,
`or suppositories, are also envisioned.
`
`C.
`
`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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`lyophilized material. The aqueous lyophilized
`formulations of this invention are particularly useful
`for providing long term storage of CNTF . The
`lyophilizable formulations of this i nvention 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.
`CNTF is present in the lyophilizable formulations
`over t he 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.
`I deally, the choice of buffer takes i nto account
`potential pH shifts during lyophilization caused by
`sequential crystallization of buffer components. For
`example, wit h phosphate buffers, the basic component
`has a -higher eutectic point than the acidic component,
`hence i t crystallizes out first and t he 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 t he same salts useful in the aqueous
`formu l ations described above, is present in the
`lyophilizable f ormulations at the same or reduced
`concentrations as that in the aqueous formulations.
`Because t he salt concentration may increase during
`lyophilization, it may be desirable to reduce the
`concentration of salt present in the l yophilizable
`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.
`Optionally, the lyophilizable formulations
`comprise a biologically acceptable water soluble
`carrier. The carriers and the concentration of
`carriers which may be used in the lyophilizable
`formulations of this inven tion are the same as those
`that are suitable for use i n the aqueous fo rmulat i ons .
`The lyophilizable formulations of t his invention
`are generally lyophilized to a residual moisture
`content of less than about 2%; however , formulations
`which retain CNTF biological activity at higher or
`l ower amounts of moisture content are also
`contemplated.
`An example of a formulation of CNTF which retains
`bioact ivity after lyophilization and r econstitution is
`comprised of 0.5 mg/ml CNTF, 85 m~/ml s uc rose, 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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`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