(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(10) International Publication Number
`
`WO 2014/145519 A2
`
`\ g9\
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`
`18 September 2014 (18.09.2014) WI P O I P C T
`
`(51) International Patent Classification:
`A6IK 38/I7(2006.01)
`_
`_
`_
`(21) InternatlonalAppllcatlon Number:
`
`(22) International Filing Date:
`
`PCTMS2014/O30309
`
`(25) Filing Language:
`(26) Publication Language:
`
`17 March 2014 (17.03.2014)
`.
`Enghsh
`English
`
`(30) Priority Data:
`61/791,399
`
`15 March 2013 (15.03.2013)
`
`US
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`Ao, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ. LA. LC. LK. LR. LS. LT, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG,N1, NO, NZ,
`OM, PA, PE, PG, PH, PL, PT, QA, Ro, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,
`ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`(71) Applicant (for all designated States except US)3 VIRO‘
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
`PHARMA HOLDINGS LIMITED; Canon's Court, 22
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, T],
`Victoria Street, Hamilton, HM 12 (BM).
`TM), Emma“ (AL, AT’ BE’ BG’ CH’ CY’ CZ’ DE’ DK’
`Inventors afar US only): GALLAGHER, Cynthia; 730
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`.
`.
`Stockton Dr1ve, Exton, PA 19341 (US). RUDDY, Steven,
`MC’ MK’ MT’ NL’ NO’ PL’ PT’ RO’ RS’ SE’ SI’ SK’ SM’
`730 Stockton Drive Exton PA 19341 (US)
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`'
`’
`’
`KM, ML, MR, NE, SN, TD, TG).
`Inventor: MANNING, Mark, Cornell; 4630 Sorrel Lane,
`Published:
`Johnstown, CO 80534-6404 (US).
`(74) Agents: NETTER, Robert, C., Jr. et a1.; Dann, Dorfman, _
`Herrell & Skillman, 1601 Market Street, Suite 2400, Phil-
`adelphia, PA 19103-2307 (US).
`
`(72)
`
`(72)
`
`without international search report and to be republished
`upon receipt ofthat report (Rule 48.2(g))
`
`(54) Title: Cl-INH COMPOSITIONS AND METHODS FOR THE PREVENTION AND TREATMENT OF DISORDERS ASSO-
`CIATED WITH C1 ESTERASE INHIBITOR DEFICENCY
`
`(57) Abstract: Compositions and methods for the treatment and/or prevention of disorders associated with C1 esterase inhibitor de -
`ficiency are disclosed.
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`CSL v. Shire, |PR2017-01512
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`WO 2014/145519
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`PCT/US2014/030309
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`C1-INH COMPOSITIONS AND METHODS FOR THE PREVENTION AND
`
`TREATMENT OF DISORDERS ASSOCIATED WITH C1 ESTERASE
`
`INHIBITOR DEFICENCY
`
`
`
`This application claims priority under 35 U.S.C. §119(e) to US. Provisional
`
`Patent Application No. 61/791,399, filed March 15, 2013. The foregoing application
`
`10
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`is incorporated by reference herein.
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`FIELD OF THE INVENTION
`
`The present invention relates to the field of therapeutic agents and methods of
`
`use thereof. Specifically, the instant invention provides compositions and methods
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`15
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`for the treatment and/or prevention of disorders associated with C1 esterase inhibitor
`
`deficiency.
`
`BACKGROUND OF THE INVENTION
`
`Several publications and patent documents are cited throughout the
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`20
`
`specification in order to describe the state of the art to which this invention pertains.
`
`Full citations of these references can be found throughout the specification. Each of
`
`these citations is incorporated herein by reference as though set forth in full.
`
`Hereditary angioedema (HAE) is a rare, life-threatening, genetic disorder
`
`caused by a deficiency of the Clesterase inhibitor (see generally www.haei.org and
`
`25 www.haea.org). At least 6,500 people in the United States and at least 10,000 people
`
`in Europe have HAE. HAE patients experience recurrent, unpredictable, debilitating,
`
`life-threatening attacks of inflammation and submucosal/subcutaneous swelling. The
`
`inflammation is typically of the larynx, abdomen, face, extremities, and urogenital
`
`tract. This genetic disorder is a result of a defect in the gene controlling the synthesis
`
`30
`
`of the C1 esterase inhibitor. Accordingly, restoring the levels of active C1 esterase
`
`inhibitor in these patients to or near normal levels is an effective measure for treating
`
`HAE. Still, new and improved methods of treating and preventing disorders
`
`associated with a deficiency of the Clesterase inhibitor, such as HAE, are desired.
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`
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`SUMMARY OF THE INVENTION
`
`In accordance with the instant invention, methods for inhibiting, treating,
`
`and/or preventing a disorder associated with a deficiency in C1 esterase inhibitor in a
`
`subject are provided. In a particular embodiment, the method comprises
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`5
`
`administering a composition comprising at least one C1 esterase inhibitor.
`
`In accordance with the instant invention, therapeutic compositions are also
`
`provided. In a particular embodiment, the composition comprises at least one C1
`
`esterase inhibitor and, optionally, at least one pharmaceutically acceptable carrier for
`
`delivery (6. g. intravenous or subcutaneous delivery). Kits comprising a composition
`
`10
`
`comprising at least one C1 esterase inhibitor are also provided herein.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Figure 1 provides an amino acid sequence of human C1 esterase inhibitor.
`
`Figure 2 provides a graph of the effect of protein concentration on viscosity
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`15
`
`for initial spin concentration samples.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`The restoration of active C1 esterase inhibitor levels in patients having a
`
`disorder associated with deficient or reduced levels of active C1 esterase inhibitor
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`20
`
`(e. g., HAE) is an effective measure for treating such disorders. Currently, C1 esterase
`
`inhibitor (such as Cinryze® (ViroPharma, Inc.; Exton, PA)) is administered to a
`
`patient intravenously by a medical professional. Herein, formulations of a C1 esterase
`
`inhibitor (such as Cinryze®) are provided which are also effective for subcutaneous
`
`(SC) administration. Surprisingly, the subcutaneous administration of the C1 esterase
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`25
`
`inhibitor is sufficient to maintain the blood levels of the C1 esterase inhibitor. The SC
`
`administration of a Cl esterase inhibitor fulfills an unmet medical need due to the
`
`limitations of intravenous administration in HAE patients.
`
`In accordance with the instant invention, compositions and methods for
`
`inhibiting (e. g., reducing or slowing), treating, and/or preventing a disorder associated
`
`30 with Cl esterase inhibitor deficiency in a subject are provided. In a particular
`
`embodiment, the methods comprise administering (e.g., subcutaneously or
`
`intravenously) to a subject in need thereof at least one C1 esterase inhibitor. In a
`
`particular embodiment, the C1 esterase inhibitor is administered subcutaneously after
`
`an initial administration of the C1 esterase inhibitor intravenously.
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`C1 esterase inhibitors are also known as C1 inhibitors (C1 INH). C1 esterase
`
`inhibitors are inhibitors of complement C1 and belong to the superfamily of serine
`
`proteinase inhibitors. Human C1 esterase inhibitor is a protein of 500 amino acids,
`
`including a 22 amino acid signal sequence (Carter et a1. (1988) Eur. J. Biochem.,
`
`5
`
`173:163). In plasma, the C1 esterase inhibitor is a heavily glycosylated glycoprotein
`
`of approximately 76 kDa (Perkins et al. (1990) J. Mol. Biol., 2142751). The activity
`
`of a C1 esterase inhibitor may be assayed by known methods (see, e.g., Drouet et al.
`
`(1988) Clin. Chim. Acta., 174:121-30). In a particular embodiment, the Cl esterase
`
`inhibitor is human. An amino acid sequence of human C1 esterase inhibitor is
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`10
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`provided in GenBank Accession No. CAA30314 (see also GeneID: 710, which also
`
`provides nucleotide sequences of the C1 esterase inhibitor) and Figure l. A C1
`
`esterase inhibitor for use in the methods of the instant invention may have an amino
`
`acid sequence that has at least 65, 70, 75, 80, 85, 90, 95, 98, 99, or 100% identity with
`
`the amino acid sequence of Figure 1. The C1 esterase inhibitor may be isolated or
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`15
`
`purified from plasma (e. g., human plasma) or recombinantly produced. When
`
`purified from plasma, the C1 esterase inhibitor may be nanofiltered and pasteurized.
`
`In a particular embodiment, the plasma-derived C1 esterase inhibitor is Cinryze®. In
`
`a particular embodiment, the C1 esterase inhibitor is present in the compositions of
`
`the instant invention at high concentration. Indeed, compositions comprising very
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`20
`
`high levels of C1 esterase inhibitor have been determined to be surprisingly stable and
`
`active. In a particular embodiment, the C1 esterase inhibitor is present at about 250
`
`U/ml to about 1000 U/ml, about 400 U/ml to about 600 U/ml, or about 500 U/ml.
`
`In a particular embodiment, the compositions of the instant invention do not
`
`contain citrate or citric acid. The compositions lacking citrate and citric acid are
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`25
`
`particularly useful for the subcutaneous administration of the C1 esterase inhibitor as
`
`citrate/citric acid can cause an injection site reaction. In a particular embodiment, the
`
`buffer of the instant compositions is sodium phosphate (e.g., about 5 mM to about 50
`
`mM sodium phosphate, about 10 mM to about 30 mM sodium phosphate, or about 20
`
`mM sodium phosphate). In a particular embodiment (e. g., for intravenous
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`30
`
`administration), the buffer of the instant compositions comprises a carboxylic group.
`
`For example, the buffer may be, without limitation, citrate, succinate, tartarate,
`
`maleate, acetate, and salts thereof. In a particular embodiment, the buffer of the
`
`instant composition is citrate or sodium citrate (e. g., about 5 mM to about 50 mM
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`sodium citrate, about 10 mM to about 30 mM sodium citrate, or about 20 mM sodium
`
`citrate).
`
`The compositions of the instant invention may have a pH range of about 6.5 or
`
`higher, particularly about 6.5 to about 8.0, particularly about 6.5 to about 7.5, and
`
`more particularly about 6.5 to about 7.0.
`
`The compositions of the instant invention may also comprise polysorbate 80
`
`(TWEEN). Compositions comprising polysorbate 80 are particularly useful as they
`
`reduce/mitigate protein aggregation. Polysorbate 80 can also limit protein
`
`interactions when the composition comes into contact with silicon containing
`
`lubricants/oils such as those used in syringes and other administration devices.
`
`Compositions comprising polysorbate 80 are also useful for lyophilized preparations.
`
`In a particular embodiment, the polysorbate 80 is present at a concentration of about
`
`0.01% to about 0.1%, particularly about 0.025% to about 0.075%, particularly about
`
`0.05%.
`
`The compositions of the instant invention may also comprise sucrose. Sucrose
`
`can be added as a “bulking” agent as well as a lyo-protectant.
`
`In a particular
`
`embodiment, sucrose is added to compositions to be lyophilized. In a particular
`
`embodiment, the compositions comprise about 25 mM to about 125 mM sucrose,
`
`particularly about 50 mM to about 100 mM sucrose.
`
`The compositions of the instant invention may also comprise at least one
`
`amino acid or salt thereof, particularly methionine and/or arginine. Arginine carries a
`
`positive charge on its side chain can be used to buffer solutions with phosphate.
`
`Methionine acts as a stabilizer (e. g., by limiting oxidation). The amino acids may be
`
`present in the composition as individual amino acids or present as short peptides (e. g.,
`
`2 to about 5 amino acids, particularly di-peptides or tri-peptides).
`
`As stated hereinabove, the instant invention encompasses methods of treating,
`
`inhibiting, and or preventing any condition or disease associated with an absolute or
`
`relative deficiency of functional Cl esterase inhibitor. Such disorders include,
`
`without limitation, acquired angioedema (AAE) and hereditary angioedema (HAE).
`
`In a particular embodiment, the disorder is HAE and/or the attacks associated
`
`therewith. As stated hereinabove, HAE is a life-threatening and debilitating disease
`
`that manifests as recurrent, submucosal/subcutaneous swelling attacks due to a
`
`deficiency of Cl esterase inhibitor (Zuraw, BL. (2008) N. Engl. J. Med., 35921027-
`
`1036). In a particular embodiment, the hereditary angioedema is type I or type II.
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`Both type I and type II have a defective gene for the synthesis of Cl esterase inhibitor
`
`that produce either no Cl inhibitor (HAE type I) or a dysfunctional Cl inhibitor (HAE
`
`type II) (Rosen et al. (1965) Science 148: 957-958; Bissler et al. (1997) Proc. Assoc.
`
`Am. Physicians 109: 164-173; Zuraw et al. (2000) J. Allergy Clin. Immunol. 105:
`
`541-546; Bowen et al. (2001) Clin. Immunol. 98: 157—163).
`
`The methods of the instant invention encompass the administration of at least
`
`one C1 esterase inhibitor. Compositions comprising at least one C1 esterase inhibitor
`
`and, optionally, at least one pharmaceutically acceptable carrier (e.g., one suitable for
`
`subcutaneous or intravenous administration) are encompassed by the instant
`
`invention. Such compositions may be administered, in a therapeutically effective
`
`amount, to a patient in need thereof for the treatment of a disorder associated with C1
`
`esterase inhibitor deficiency. The instant invention also encompasses kits comprising
`
`at least one composition of the instant invention, e. g., a composition comprising at
`
`least one C1 esterase inhibitor and, optionally, at least one pharmaceutically
`
`acceptable carrier (e. g., one suitable for intravenous or subcutaneous administration).
`
`The kits may further comprise at least one of reconstitution buffer(s), syringes (e. g.,
`
`disposable) for parenteral (e. g., subcutaneous) injection, and instruction material. In a
`
`particular embodiment, the kit comprises at least one pre-loaded syringe comprising
`
`the C1 esterase inhibitor and at least one pharmaceutically acceptable carrier. For
`
`example, a syringe may be loaded with at least one Cl esterase inhibitor with at least
`
`one pharmaceutically acceptable carrier for administration (e.g., intravenous or
`
`subcutaneous administration). Alternatively, a single syringe may be loaded with
`
`lyophilized C1 esterase inhibitor. In a particular embodiment, the preloaded syringes
`
`have a pharmaceutical composition that contains polysorbate 80 as a component (e. g.,
`
`in an amount that prevents protein-silicone interaction or protein aggregation).
`
`The agents and compositions of the present invention can be administered by
`
`any suitable route, for example, by injection (e. g., for local (direct) or systemic
`
`administration. In a particular embodiment, the composition is administered
`
`subcutaneously or intravenously. In general, the pharmaceutically acceptable carrier
`
`of the composition is selected from the group of diluents, preservatives, solubilizers,
`
`emulsifiers, adjuvants and/or carriers. The compositions can include diluents of
`
`various buffer content (e.g., Tris HCl, acetate, phosphate), pH and ionic strength; and
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`additives such as detergents and solubilizing agents (e. g., Tween 80, Polysorbate 80),
`
`antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,
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`10
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`20
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`25
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`30
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`Thimersol, benzyl alcohol) and bulking substances (e. g., lactose, mannitol). The
`
`pharmaceutical composition of the present invention can be prepared, for example, in
`
`liquid form, or can be in dried powder form (e. g., lyophilized for later reconstitution).
`
`In a particular embodiment, the compositions are formulated in lyophilized
`
`form. Where the compositions are provided in lyophilized form, the compositions are
`
`reconstituted prior to use (e. g., within an hour, hours, or day or more of use) by an
`
`appropriate buffer (e. g., sterile water, a sterile saline solution, or a sterile solution
`
`comprising the appropriate pharmaceutically acceptable carriers (e.g., to reconstitute
`
`the compositions as described hereinabove). The reconstitution buffer(s) may be
`
`provided in the kits of the instant invention or may be obtained or provided
`
`separately.
`
`As used herein, “pharmaceutically acceptable carrier” includes any and all
`
`solvents, dispersion media and the like which may be appropriate for the desired route
`
`of administration of the pharmaceutical preparation, as exemplified in the preceding
`
`paragraph. The use of such media for pharmaceutically active substances is known in
`
`the art. Except insofar as any conventional media or agent is incompatible with the
`
`molecules to be administered, its use in the pharmaceutical preparation is
`
`contemplated.
`
`Selection of a suitable pharmaceutical preparation depends upon the method of
`
`administration chosen. In this instance, a pharmaceutical preparation comprises the
`
`molecules dispersed in a medium that is compatible with the tissue to which it is
`
`being administered. Methods for preparing parenterally or subcutaneously
`
`administrable compositions are well known in the art (see, e.g., Remington’s
`
`Pharmaceutical Science (E.W. Martin, Mack Publishing Co., Easton, PA)).
`
`As stated hereinabove, agents of the instant invention are administered
`
`parenterally — for example by intravenous injection into the blood stream and/or by
`
`subcutaneous injection. Pharmaceutical preparations for parenteral, intravenous, and
`
`subcutaneous injection are known in the art. If parenteral injection is selected as a
`
`method for administering the molecules, steps should be taken to ensure that
`
`sufficient amounts of the molecules reach their target cells to exert a biological effect.
`
`Pharmaceutical compositions containing a compound of the present invention
`
`as the active ingredient in intimate admixture with a pharmaceutical carrier can be
`
`prepared according to conventional pharmaceutical compounding techniques. The
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`carrier may take a wide variety of forms depending on the form of preparation desired
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`for administration, e. g., parenterally or subcutaneous. For parenterals, the carrier will
`
`usually comprise sterile water, though other ingredients, for example, to aid solubility
`
`or for preservative purposes, may be included.
`
`Inj ectable suspensions may also be
`
`prepared, in which case appropriate liquid carriers, suspending agents and the like
`
`may be employed.
`
`A pharmaceutical preparation of the invention may be formulated in dosage
`
`unit form for ease of administration and uniformity of dosage. Dosage unit form, as
`
`used herein, refers to a physically discrete unit of the pharmaceutical preparation
`
`appropriate for the patient undergoing treatment. Each dosage should contain a
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`10
`
`quantity of active ingredient calculated to produce the desired effect in association
`
`with the selected pharmaceutical carrier. Dosage units may be proportionately
`
`increased or decreased based on the weight of the patient. Appropriate concentrations
`
`for alleviation of a particular pathological condition may be determined by dosage
`
`concentration curve calculations. Appropriate dosage unit may also be determined by
`
`assessing the efficacy of the treatment.
`
`The pharmaceutical preparation comprising the molecules of the instant
`
`invention may be administered at appropriate intervals, for example, daily, every
`
`other day, every three days, five out of every 7 days, or at least one, two or three times
`
`a week or more until the pathological symptoms are reduced or alleviated, after which
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`20
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`the dosage may be reduced to a maintenance level. The appropriate interval in a
`
`particular case would normally depend on the condition of the patient.
`
`In a particular embodiment, the C1 esterase inhibitor is present in the
`
`composition or is administered in the range of about 100 Units to about 10,000 Units;
`
`about 500 Units to about 5,000 Units; about 1,000 Units to about 3,500 Units, or
`
`about 1,500 Units to about 2,500 Units. In a particular embodiment, at least about
`
`2,000 Units is used. In a particular embodiment, a high initial dose of the C1 esterase
`
`inhibitor (as listed above (may be administered intravenously)) is used, followed by
`
`lower maintenance doses. For example, the high initial dose may be at least 1.5, 2, 3,
`
`4, or 5 times the subsequent doses. In a particular embodiment, the C1 esterase
`
`30
`
`inhibitor is present in the maintenance composition or is administered for
`
`maintenance in the range of about 100 Units to about 5,000 Units; about .250 Units to
`
`about 2,000 Units; about 250 Units to about 1,000 Units; or about 500 Units. The
`
`high initial does of the C1 esterase inhibitor is optional in the methods of the instantly
`
`claimed invention (e. g., may be optional with prophylactic methods).
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`In a particular embodiment, the C1 esterase inhibitor is administered with a
`
`frequency and dosage so as to increase the C1 esterase inhibitor level to at least about
`
`0.3 or, more particularly, 0.4 U/ml or more up to about 1 U/ml (1 Unit/ml is the mean
`
`quantity of Cl inhibitor present in 1 ml of normal human plasma) in the blood of the
`
`subject. For example, the C1 esterase inhibitor level may be kept at or above 0.4
`
`U/ml for at least 50%, at least 75%, at least 90%, at least 95% or more of time or all
`
`of the time (e. g., the time during which drug is being administered). For example, the
`
`administration of a 2000U initial dose of C1 esterase inhibitor followed by 250U
`
`everyday or 500U every other day results in the maintenance ofjust below 0.4 U/ml
`
`in blood. Further, the administration of a 2000U initial dose of C1 esterase inhibitor
`
`followed by 1000U every 3 days results in the maintenance of about 0.4 U/ml in
`
`blood. Notably, for ease of use by the patient, less frequent administrations may be
`
`preferred. The administration of a 2000U initial dose of C1 esterase inhibitor
`
`followed by 500U everyday with weekend holidays from administration (i.e., 5 out of
`
`7 days) also results in the maintenance of about 0.4 U/ml or higher in blood. Notably,
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`10
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`I5
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`the administration of only the maintenance doses leads to increased and
`
`physiologically relevant blood levels of the C1 esterase inhibitor, but delayed
`
`compared to those receiving an initial high dose.
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`20
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`Definitions
`
`The singular forms “a,” “an,” and “the” include plural referents unless the
`
`context clearly dictates otherwise.
`
`As used herein, the term “about” may refer to i5%, i2%, or i1%.
`
`As used herein, the terms “host,” “subject,” and “patient” refer to any animal,
`
`25
`
`including humans.
`
`As used herein, the term “prevent” refers to the prophylactic treatment of a
`
`subject who is at risk of developing a condition (e.g., HAE or HAE attack) resulting
`
`in a decrease in the probability that the subject will develop the condition.
`
`The term “treat” as used herein refers to any type of treatment that imparts a
`
`30
`
`benefit to a patient afflicted with a disorder, including improvement in the condition
`
`of the patient (e. g., in one or more symptoms), delay in the progression of the
`
`condition, etc. In a particular embodiment, the treatment of HAE results in at least a
`
`reduction in the severity and/or number of HAE attacks.
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`CSL v. Shire, IPR2017-01512
`Shire Ex. 2004, Page 9
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`Shire Ex. 2004, Page 9
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`PCT/US2014/030309
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`The phrase “effective amount” refers to that amount of therapeutic agent that
`
`results in an improvement in the patient’s condition. A “therapeutically effective
`
`amount” of a compound or a pharmaceutical composition refers to an amount
`
`effective to prevent, inhibit, treat, or lessen the symptoms of a particular disorder or
`
`disease.
`
`“Pharmaceutically acceptable” indicates approval by a regulatory agency of
`
`the Federal or a state government or listed in the US. Pharmacopeia or other
`
`generally recognized pharmacopeia for use in animals, and more particularly in
`
`humans.
`
`A “carrier” refers to, for example, a diluent, adjuvant, preservative (e. g.,
`
`Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite),
`
`solubilizer (e.g., TWEEN 80, Polysorbate 80), emulsifier, buffer (e.g., Tris HCl,
`
`acetate, phosphate), water, aqueous solutions, oils, bulking substance (e.g., lactose,
`
`mannitol), cryo-/lyo— protectants, tonicity modifier, excipient, auxilliary agent or
`
`vehicle with which an active agent of the present invention is administered. Suitable
`
`pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by
`
`E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The
`
`Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et
`
`al., Eds, Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe,
`
`et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical
`
`Association, Washington.
`
`The term “isolated” may refer to protein, nucleic acid, compound, or cell that
`
`has been sufficiently separated from the environment with which it would naturally be
`
`associated (e.g., so as to exist in “substantially pure” form). “Isolated” does not
`
`necessarily mean the exclusion of artificial or synthetic mixtures with other
`
`compounds or materials, or the presence of impurities that do not interfere with the
`
`fundamental activity, and that may be present, for example, due to incomplete
`
`purification.
`
`The term “substantially pure” refers to a preparation comprising at least 50—
`
`60% by weight of a given material (e. g., nucleic acid, oligonucleotide, protein, etc.).
`
`In certain embodiments, the preparation comprises at least 75% by weight,
`
`particularly 90-95% or more by weight of the given compound. Purity is measured by
`
`methods appropriate for the given compound (e.g. chromatographic methods, agarose
`
`or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
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`Shire Ex. 2004, Page 10
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`The following example is provided to illustrate various embodiments of the
`
`present invention. The example is illustrative and is not intended to limit the
`
`5
`
`invention in any way.
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`
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`10
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`15
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`20
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`25
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`30
`
`Spin Concentration Studies
`
`EXAMPLE
`
`The protein was loaded into the spin concentrators and rotated at 10,500 rpms
`
`for 5 to 10 minutes. When the samples stopped rotating, the final volumes in the spin
`
`concentrators were recorded and a rough protein concentration was calculated for
`
`each one. Additional protein was added to the spin concentrators and rotated until the
`
`desired protein concentration was reached, at which point a UV measurement was
`
`made. At each target protein concentration a UV and viscosity measurement was
`
`performed. The above procedure continued until the viscosity of the protein prevented
`
`the sample from being further concentrated.
`
`Viscosity Measurements
`
`Viscosity was determined by measuring the amount of time the sample took to
`
`be drawn to a predetermined distance in a gel loading pipette tip. In order to calculate
`
`the sample viscosity, a standard curve was first prepared using a set of standards with
`
`known viscosities. Sucrose (or Brix) solutions are suitable for preparing such a curve,
`
`but any material with known viscosity at a defined temperature should be appropriate.
`
`In order to make a measurement, the pipette plunger is depressed, the pipette
`
`tip is inserted into the sample vial, the plunger is released, and the time for the fluid to
`
`travel a predetermined distance in the pipette tip was measured with a stop watch. The
`
`distance used for these experiments was 30 uL of water. In important note, a pipette
`
`tip is only reliable for a single measurement, so multiple tips are used to make
`
`replicate measurements of a sample. Also, the volume to be drawn into the pipette tip
`
`should be larger than the volume marked on the tip to ensure a uniform pull on the
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`Shire Ex. 2004, Page 11
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`PCT/US2014/030309
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`sample during a measurement. For a 30 uL volume mark on the pipette tip, the
`
`micropipette was set to draw 42 uL.
`
`
`
`10
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`15
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`20
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`25
`
`Results
`
`The instant example determined the ability to develop a higher concentration
`
`liquid formulation of C1 INH as a monoforrnulation. The initial studies focused on
`
`concentration of the stock solution of C1 INH using a spin concentration method.
`
`The solutions were initially adjusted for pH but no other excipient was added. Three
`
`pH values were investigated (pH 5.9, 6.9, and 7.9). Upon spin concentration, all of
`
`the solutions remained clear up to concentrations up ~500 U/ml (approximately 100
`
`mg/ml) for all pH values tested (Table 1). While the solubility limit was not reached
`
`in these studies, there were measurable increases in viscosity as the concentrations
`
`exceeded 300 U/ml (Figure 2). At all pH values, the viscosity begins to increase
`
`markedly when the C1 INH concentration goes above 400 U/ml.
`
`7.9
`
`6.9
`
`5 .9
`
`U/mL
`
`viscosity
`
`U/mL
`
`viscosity
`
`U/mL
`
`viscosity
`
`
`
`
`
`
`
`
`
`
`
`
`415.18
`3.95
`289.4
`4.90
`296.9
`7.71
`——_———
`
`454.81
`13.74
`378.6
`12.08
`396.7
`5.46
`——————
`501.17
`30.43
`479.0
`14.67
`478.8
`24.09
`
`Table 1: Final concentrations (in U/mL) and viscosities for samples prepared during
`
`the spin concentration experiments. These values were based on the initial 160 U/mL
`
`concentration of the initial bulk drug.
`
`A larger feasibility study was performed examining different buffers (20 mM
`
`phosphate, 20 mM citrate, and 20 mM Tris) at each of the three target pH values.
`
`Samples of both 400 U/ml and 500 U/ml were prepared and evaluated for stability
`
`after one week at 40° C and after two weeks at 25° C. The initial viscosity levels were
`
`well above the values for pure water (~1 mPa-s), but well within the limits usually set
`
`for use as an injectable product (Table 2). The viscosity values for the 400 U/ml
`
`samples were less than at 500 U/ml, usually by 7 to 10 mPa-s. Upon storage at 400 C
`
`for one week, the viscosity of all of the samples increased. At pH 5.9, all of the same
`
`gelled, likely due to thermally induced aggregation. For the remaining formulations,
`
`ll
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`CSL v. Shire, |PR2017-01512
`Shire Ex. 2004, Page 12
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`PCT/US2014/030309
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`the viscosity increased to some degree. In some cases these values exceeded 30 mPa-
`
`s. The increase in viscosity was less upon 25° C storage than at 40° C. There was
`
`little, if any change, for the samples at pH 6.9, indicating that pH 6.9 may be more
`
`favorable for long-term storage stability.
`
`
`
`
`
`
`
`
`
`
`IE.
`
`
`
`[C1 INH]
`00
`4
`
`
`
`.
`.
`l33:l:06
`phosphate “ l7.4i:2.1
`.
`.
`
`
`
`.
`
`.
`
`500
`
`4
`
`00
`
`500
`
`00
`
`500
`
`.
`— l44:l:32
`4
`00
`- phosphate
`8.2 :l: 1.2
`500
`.
`— 16.2114
`.
`
`4
`
`00
`
`198i11
`12 8 :l: 0.7
`231:21
`
`12640.5
`22.0 :l: 3.5
`25.5:75
`
`
`
`---m--- ---
`
`500
`31011.8
`33.3 :I: 6 2
`_— 20.5 i 0.9
`
`
`
`Table 2: Viscosity at t0 and after one week of storage at 40°C (tl). Viscosity is
`
`reported in mPa-s.
`
`Notably, at pH 6.9, citrate formulations had lower viscosity values than for
`
`phosphate, while at pH 7.9, phosphate buffer produced lower viscosities than tris
`
`buffer. Higher viscosities will mean greater force will be required to deliver a
`
`specified volume of the drug within a certain time frame.
`
`The purity by RP HPLC was initially near 86 to 87% for the formulations at
`
`pH 6.9 and above (Table 3). The initial levels were lower at pH 5.9, suggesting that
`
`some degradation had