(12) United States Patent
`Savarese et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 9.220,700 B2
`Dec. 29, 2015
`
`US009220700B2
`
`(54) CYSTEINE FOR PHYSIOLOGICAL
`NJECTION
`(75) Inventors: John J. Savarese, Southbury, CT (US);
`Paul M. Heerdt, Greenwich, CT (US)
`(73) Assignee: Cornell University, Ithaca, NY (US)
`
`*) Notice:
`
`Subject to any disclaimer, the term of this
`y
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 771 days.
`13/391,154
`Aug. 18, 2010
`PCT/US2010/045907
`
`(21) Appl. No.:
`(22) PCT Filed:
`(86). PCT No.:
`S371 (c)(1),
`(2), (4) Date: May 7, 2012
`(87) PCT Pub. No.: WO2011/022491
`PCT Pub. Date: Feb. 24, 2011
`
`(65)
`
`Prior Publication Data
`US 2012/O214873 A1
`Aug. 23, 2012
`
`Related U.S. Application Data
`(60) Provisional application No. 61/235,191, filed on Aug.
`19, 2009.
`Foreign Application Priority Data
`
`(30)
`
`Mar. 17, 2010 (WO)................ PCT/US2O10(OOO796
`
`(51) Int. Cl.
`A 6LX3L/95
`A6 IK3I/98
`A6 IK9/00
`A6 IK9/06
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(Continued)
`
`(52) U.S. Cl.
`CPC ............. A6 IK3I/198 (2013.01); A61 K9/0019
`(2013.01); A61 K9/06 (2013.01); A61 K3I/375
`(2013.01); A61 K47/22 (2013.01)
`(58) Field of Classification Search
`CPC ........................... A61K 31/195; A61 K31/198
`USPC .......................................................... 514/562
`See application file for complete search history.
`
`(56)
`
`References Cited
`
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`
`FOREIGN PATENT DOCUMENTS
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`OTHER PUBLICATIONS
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`“U.S. Appl. No. 13/257.214. Notice of Allowance mailed Jul. 19.
`2013, 19 pgs.
`
`(Continued)
`
`Primary Examiner — San-Ming Hui
`(74) Attorney, Agent, or Firm — Schwegman Lundberg &
`Woessner, P.A.
`
`ABSTRACT
`(57)
`This application describes methods of making and using
`physiological cysteine solutions useful for reversing a neuro
`muscular blockade caused by a cysteine-reversible neuro
`muscular blockade agent, that overcomes problems of cys
`teine precipitation and dimerization.
`6 Claims, 5 Drawing Sheets
`
`750
`
`7.00
`
`6.50
`
`6.00
`
`SSO
`2 500
`4SO
`
`4.00
`
`3SO
`
`3.00
`150
`
`1.70
`
`2.10
`190
`STARTING pH
`
`230
`
`2SO
`
`Eton Ex. 1120
`1 of 55
`
`

`

`US 9.220,700 B2
`Page 2
`
`(51) Int. Cl.
`A 6LX3L/375
`A6 IK 47/22
`
`(2006.01)
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`
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`

`

`U.S. Patent
`
`Dec. 29, 2015
`
`Sheet 1 of 5
`
`US 9.220,700 B2
`
`750
`
`7.00
`
`6.50
`
`6.00
`
`5.50
`
`s 500
`
`450
`
`4.00
`
`3.50
`
`3.00
`1.50
`
`1.70
`
`2.10
`1.90
`STARTING pH
`FIG, 1
`
`2.30
`
`2SO
`
`Eton Ex. 1120
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`

`

`U.S. Patent
`
`Dec. 29, 2015
`
`Sheet 2 of 5
`
`US 9.220,700 B2
`
`1.90
`
`18O
`
`pH 170
`
`160
`
`150
`10
`
`pH
`
`255
`
`250
`
`2.45
`
`240
`
`2.35
`
`2.30
`
`10
`
`15
`
`20
`
`25
`
`TEMP DEGREESC
`
`FIG, 2A
`
`30
`
`35
`
`15
`
`20
`
`25
`
`30
`
`35
`
`TEMP DEGREESC
`
`FIG. 2B
`
`Eton Ex. 1120
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`U.S. Patent
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`Dec. 29, 2015
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`Sheet 3 of 5
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`US 9.220,700 B2
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`
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`-O-FORMULATION 1
`-- FORMULATION2
`-A-FORMULATION3
`-O-FORMULATIONS
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`U.S. Patent
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`Dec. 29, 2015
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`Sheet 4 of 5
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`US 9.220,700 B2
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`5
`
`s t 5
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`2 ra
`35
`&
`
`
`
`5
`a
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`2. 2
`
`s
`
`TIME (MIN)
`FIG, 4A
`
`TIME (MIN)
`FIG, 4B
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`-O-CONTROL
`-O-10 mg/kg
`-V-20 mg/kg
`-0-50 mg/kg
`-H 100 mg/kg
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`U.S. Patent
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`Dec. 29, 2015
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`Sheet 5 of 5
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`US 9.220,700 B2
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`
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`90S.O
`43 MNE
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`P
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`COMPOSITE OF5 SEC. AWERAGES
`-100 mg/kg.L-CYCTEINE, n=3
`100 mg/kg D-CYCTEINE, n=3
`
`------
`
`u- -
`83-24
`P = 0.38
`
`6.OMINUTES
`
`---
`
`S
`
`Ye^
`
`3
`
`9 O
`
`
`
`
`
`85
`
`8
`
`d
`s 75
`5
`e
`
`O
`
`20
`
`40
`
`60
`
`8O
`
`100
`
`120
`
`145
`
`2
`is 140
`2
`
`s
`as
`
`135
`
`
`
`130
`
`\
`?
`/ 137+1} \
`/ 3.6MINUTES
`PXOOS
`-
`
`COMPOSITE OFSSEC. AWERAGES
`-100 mg/kg.L-CYCTEINE, n=3
`100 mg/kg D-CYCTEINE, n=3
`
`O
`
`20
`
`40
`
`8O
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`100
`
`120
`
`60
`FIG, 5B
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`US 9.220,700 B2
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`1.
`CYSTEINE FOR PHYSIOLOGICAL
`INJECTION
`
`This application is a U.S. National Stage Filing under 35
`U.S.C. 371 from International Application No. PCT/US2010/
`045907, filed Aug. 18, 2010, and published as WO 2011/
`022491 A1 on Feb. 24, 2011, which application claims prior
`ity to U.S. Provisional Patent Application Ser. No. 61/235,
`191, filed Aug. 19, 2009 and to PCT Application Ser. No.
`PCT/US2010/000796, filed Mar. 17, 2010, the contents of
`each of which applications and publication are specifically
`incorporated herein by reference in their entireties.
`This application is also related to U.S. Ser. No. 11/951,114,
`filed Dec. 5, 2007; U.S. application Ser. No. 10/975,197, filed
`Oct. 28, 2004: PCT Application Ser. No. PCT/US2004/
`035869, filed Oct. 28, 2004; U.S. Application Ser. No.
`60/515,048, filed Oct. 28, 2004; and U.S. Provisional Appli
`cation Ser. No. 61/160,915, filed Mar. 19, 2009, the contents
`of each of which applications are specifically incorporated
`herein by reference in their entireties.
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`BACKGROUND OF THE INVENTION
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`2
`wherein oxygen is Substantially removed from the aque
`ous solution of cysteine, the buffering Solution and/or
`cysteine Solution for physiological administration.
`In some embodiments, the aqueous solution of cysteine has a
`pH of about 1.90 to about 2.00. The aqueous solution of
`cysteine can be made from physiologically acceptable sol
`vents, for example, water, physiological saline, Sugar Solu
`tions and combinations thereof.
`As used herein, the term "cysteine' includes both L-cys
`teine and D-cysteine, or any mixture thereof, e.g., a racemate,
`unless other specified. As described below, use of the L- or
`D-isomer of cysteine can be beneficial in certain situations.
`For example, L-cysteine is typically cheaper and more readily
`available. However, when administering large amounts of
`cysteine, the D-isomer of cysteine may be preferred because
`it is also active and tends to minimize side effects (e.g.,
`adverse cardiovascular effects).
`The buffering solution used solution to generate a cysteine
`Solution for physiological administration can include a weak
`base. Examples of compounds that can be present in the
`buffering solution include tris(hydroxymethyl)methylamine
`(Tris), N.N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hy
`droxymethyl)methylglycine (Tricine), 4-2-hydroxyethyl-1-
`piperazineethanesulfonate (HEPES), tris(hydroxymethyl)
`methylaminoethanesulfonate
`(TES).3-(N-morpholino)
`propanesulfonate
`(MOPS),
`piperazine-N,N'-bis(2-
`ethanesulfonate (PIPES).2-(N-morpholino)ethanesulfonate
`(MES) or combinations thereof.
`As described herein, cysteine solutions with more than 50
`mg/ml cysteine are difficult to make, however the cysteine
`Solution for physiological administration described herein
`can have a cysteine concentration of about 100 to about 300
`mg/ml, or about 150-250 mg/ml.
`The aqueous solution of cysteine and/or the stable solution
`of cysteine can include other ingredients, for example, glu
`tathione. When glutathione is present it can be included at a
`concentration of 100 to 200 mg/ml. Mixtures of L-cysteine
`and D-cysteine can also be included in the aqueous Solution of
`cysteine and/or the stable solution of cysteine. Other ingre
`dients that can be included in the aqueous solution of cysteine
`and/or the stable solution of cysteine are, for example, a
`bacteriostatic agent, a chelating agent, an antioxidant or a
`combination thereof. Examples of antioxidants that can be
`present in the aqueous solutions of cysteine and/or the stable
`Solutions of cysteine include Vitamins, cofactors and combi
`nations thereof. For example, the antioxidant can be ascorbic
`acid, vitaminA, vitamin E. coenzyme Q10, a flavonoid and/or
`combinations thereof. Examples of chelating agents that can
`be present in the aqueous solutions of cysteine and/or the
`stable solutions of cysteine include citric acid, dicarboxym
`ethyl-glutamic
`acid,
`ethylenediaminedisuccinic
`acid
`(EDDS), ethylenediaminetetraacetic acid (EDTA), hepta
`Sodium salt of diethylene triamine penta (methylene phos
`phonic acid) (DTPMPNa), malic acid, nitrilotriacetic acid
`(NTA), methionine, oxalic acid, phosphoric acid, polar amino
`acids (e.g., arginine, asparagine, aspartic acid, glutamic acid,
`glutamine, lysine, and ornithine), siderophores (e.g., Desfer
`rioxamine B). Succinic acid and combinations thereof.
`Another aspect of the invention is a method of making a
`stable cysteine solution that can be stored prior to physiologi
`cal administration, where the method involves:
`(a) removing oxygen from a Volume of physiological saline
`or water,
`(b) adding Sufficient cysteine to the physiological saline or
`water to generate a 150-250 mg/ml cysteine solution;
`
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`During certain Surgical procedures, neuromuscular block
`ing agents must be employed. However, neuromuscular
`blocking agents literally paralyze a patient for the time during
`which they are active. Hence, the use of neuromuscular
`blocking agents is restricted to situations where muscle relax
`ation is essential for effective treatment of a patient, for
`example, selected Surgical procedures involving intubation of
`the trachea. Because paralysis can interfere with essential
`body functions (e.g. breathing) the physician selects a neuro
`muscular blocking agent that will be active for as long as
`needed but no more than is needed. For example, when a
`breathing tube must be inserted into the trachea of a patient, a
`neuromuscular blocking agent is used to relax the tracheal
`muscles and permit intubation. However, the neuromuscular
`blocking agent also relaxes the muscles of the chest, thereby
`causing the patient to stop breathing. The anesthesiologist
`must quickly insert the breathing tube into the patient's tra
`chea and begin ventilation of the lungs. If the tube cannot be
`inserted quickly enough, the physician must intervene with
`some form of artificial resuscitation or the patient may suffer
`45
`oxygen deprivation, and the associated tissue and brain dam
`age that may result from lack of oxygen. Fast reversal of the
`neuromuscular blocking agent by a rapidly acting antagonist
`can remove the patient from danger and avoid, or minimize,
`the duration of artificial resuscitation.
`The inventors have developed neuromuscular blocking
`agents that, Surprisingly, are quickly reversed by injection of
`a thiol Such as the amino acid, cysteine. However, physiologi
`cal cysteine Solutions at useful concentrations are unstable
`and easily oxidize or dimerize.
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`SUMMARY OF THE INVENTION
`
`Thus, one aspect of the invention is a method of making a
`cysteine Solution for physiological administration that
`includes:
`(a) obtaining an aqueous Solution of cysteine with a pH of
`about 1.80 to about 2.20; and
`(b) mixing the aqueous Solution of cysteine with a buffer
`ing Solution to generate a cysteine solution for physi
`ological administration that has a pH of about 4.5 to
`about 5.5,
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`(c) adjusting the pH of the 150-250 mg/ml cysteine solu
`tion to a pH of about 1.90 to about 2.00 to generate a
`cysteine solution with a pH of about 1.90 to about 2.00:
`(d) sparging the cysteine solution with the pH of about 1.90
`to about 2.00 with non-oxygen containing gas to gener
`ate a deoxygenated cysteine solution; and
`(e) dispensing the deoxygenated cysteine Solution into a
`container and sealing the container,
`to thereby make the stable cysteine solution that can be stored
`prior to physiological administration.
`Such a stable cysteine solution, for example, can be stored
`for up to about 1 year without significant precipitation or
`dimerization of the cysteine. In some embodiments, the stable
`cysteine is stored frozen.
`When administration of the cysteine is desired, the stable
`cysteine Solution can be adjusted to a pH to about 4.4 to about
`5.5, to thereby generate a physiological Solution of cysteine.
`Thus, to adjust the pH of the stable cysteine solution, the
`following steps can be followed:
`f) adding a buffering Solution to the stable cysteine Solution
`to adjust the pH to about 4.4 to about 5.5 without expos
`ing the stable cysteine solution to significant oxygen;
`g) mixing the buffering solution into the stable cysteine
`Solution to generate a cysteine solution for physiological
`administration that has a pH of about 4.5 to about 5.5,
`wherein the mixing is performed without substantial
`exposure of the stable cysteine solution or the buffering
`Solution to oxygen.
`In many embodiments, it is advisable to Substantially remove
`oxygen from the buffering solution before adding the buffer
`ing solution to the stable cysteine Solution.
`Another aspect of the invention is a kit that includes:
`(a) a first container with a stable solution of cysteine that
`has a pH of about 1.8 to about 2.1;
`(b) a second container with a buffering Solution for raising
`the pH of the stable solution of cysteine; and
`(c) instructions for storing the kit, for raising the pH of the
`stable solution of cysteine to an appropriate pH and/or
`for administering the physiological Solution of cysteine
`to reverse a neuromuscular blockade.
`The kit can also include other components. For example, the
`kit can include one or more Syringes and needles for transfer
`ring an appropriate amount of the buffering solution to the
`stable solution of cysteine and/or administering the physi
`ological Solution of cysteine to a patient. Moreover, the kit
`can also include a third container that contains a neuromus
`cular blocking agent, whereina neuromuscular blockade gen
`erated in a Subject by the neuromuscular blocking agent can
`be reversed by cysteine. In many embodiments, it is advisable
`to Substantially remove oxygen from the first container and/or
`the second container. The stable solution of cysteine can be
`made by any of the methods and procedures described herein.
`Another aspect of the invention is a method of reversing a
`neuromuscular blockade in a patient comprising administer
`ing an effective amount of a physiological solution of cysteine
`to the patient, wherein the physiological Solution of cysteine
`is made by a method comprising:
`(a) removing oxygen from a Volume of physiological saline
`or water,
`(b) adding sufficient cysteine to the physiological saline or
`water to generate a 150-250 mg/ml cysteine solution;
`(c) adjusting the pH of the 150-250 mg/ml cysteine solu
`tion to a pH of about 1.90 to about 2.00 to generate a
`cysteine solution with a pH of about 1.90 to about 2.00:
`(d) sparging the cysteine solution with the pH of about 1.90
`to about 2.00 with non-oxygen containing gas to gener
`ate a deoxygenated cysteine solution;
`
`US 9.220,700 B2
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`4
`(e) dispensing the deoxygenated cysteine Solution into a
`container and sealing the container to thereby make the
`stable cysteine solution that can be stored prior to physi
`ological administration;
`(f) adding a buffering Solution to the stable cysteine solu
`tion to adjust the pH to about 4.4 to about 5.5 without
`exposing the stable cysteine solution to significant oxy
`gen; and
`(g) mixing the buffering solution into the stable cysteine
`Solution to generate a physiological Solution of cysteine
`that has a pH of about 4.5 to about 5.5, wherein the
`mixing is performed without Substantial exposure of the
`stable cysteine solution or the buffering solution to oxy
`gen.
`In many embodiments, it is advisable to Substantially remove
`oxygen from the buffering Solution.
`
`DESCRIPTION OF THE FIGURES
`
`FIG.1 graphically illustrates the final pH of L-Cys-HCl pH
`1.95 Solutions after mixing with 1.0 mL 3.6M Tris pH 8.0, for
`cysteine sample WFJ-00017.
`FIGS. 2A and 2B illustrate the effect of temperature on the
`pH of standard and L-Cys-HCl solutions. The effect oftem
`perature on a pH standard (1.679) is shown in FIG. 2A while
`the effect of temperature on an L-Cys-HCl solution is shown
`in FIG. 2B.
`FIG. 3 shows the time in hours when precipitation was
`observed in various L-Cys-HCl solutions (formulations 1, 2,
`3 and 5) as a function of pH. Formulations 1, 3 and 5 were
`particle free fo