CLAIMS
`
`1.
`
`A composition comprising at least one C1 esterase inhibitor wl‘ierein the at leam one Cl
`
`
`i in?» nenent at 400U/i‘l‘il or more for use in Wtreafing, inhibiting, or
`
` preventing hereditary angioedema (HAE)
`
`
`
`and wherein Mid corn Mention is; mlittiflihlim‘ffid
`
`subeinniieowly.
`
`2.
`
`The composition for use according to claim 1, wherein the at least one C1 esterase
`
`inhibitor is present at up to—aheut 1000 U/ml,—ailie»ut 400 U/ml to—eheut 600 U/ml, or—aheut 500
`
`U/ml.
`
`3.
`
`The composition for use according to claim 1 or claim 2, wherein the at least one C1
`
`esterase inhibitor is administered at a dose ranging from 500 Unite to about 5.000 Mime—Wen?
`
`1,000 Units tonerth 3,500 Units, or anew 1,500 Units toeheut 2,500 Units.
`
`The composition for use according to any one of the preceding claims, wherein the at
`
`least one C1 esterase inhibitor is administered daily, every other day, every 3 days, once a week,
`
`twice a week or three times a week.
`
`$3.
`
`The composition for use according to any one of the preceding claims, wherein the
`
`subject is administered a high initial dose of the at least one C1 esterase inhibitor, followed by
`
`lower maintenance doses,
`
`optionally wherein the high initial dose is at least 1.5, 2, 3, 4, or 5 times the subsequent
`
`doses, and/or the high initial dose is administered intravenously and the at least one C1 esterase
`
`inhibitor is subsequently administered subcutaneously.
`
`if
`
`15
`
`Page 1 of 51
`
`CSL EXHIBIT 1076
`
`CSL v. Shire
`
`CSL EXHIBIT 1076
`CSL v. Shire
`
`Page 1 of 51
`
`

`

`“9
`
`t
`
`,1
`
`2
`
`,3 :,
`
`Eu?
`
`,3
`
`Eu?
`
`3
`
`a
`
`a
`
`a
`
`. The composition for use according to any preceding claim, wherein the administration of
`
`the at least one Cl esterase inhibitor leads to increased levels of the Cl esterase inhibitor in the
`
`blood of the subject, wherein the blood levels of the Cl esterase inhibitor are increased to at least
`
`0.3 U/mL, 0.4 U/mL, or up tom 1 U/mL.
`
`The composition for use according to any preceding claim, wherein the blood levels of
`
`the Cl esterase inhibitor are maintained at or above 0.4 U/mL for at least 50%, at least 75%, at
`
`least 90%, or at least 95% of the time.
`
`402%.
`
`The composition for use according to any preceding claim, wherein:
`
`(i) the hereditary angioedema is Type I or Type II HAE,
`
`(ii) the administration of the at least one Cl esterase inhibitor results in HAE prophylactic
`
`treatment,
`
`(iii) the treatment of HAE results in at least a reduction in the severity and/or number of
`
`HAE attacks, or
`
`(iv) the administration of the at least one Cl esterase inhibitor results in treatment of an
`
`HAE attack.
`
`The composition for use according to any preceding claim, wherein the at least one Cl
`
`esterase inhibitor is isolated or purified from human plasma or is recombinantly produced, and/or
`
`wherein the Cl esterase inhibitor has an amino acid sequence at least 90%, 95%, 98%,
`
`99% or 100% identical to the human Cl esterase inhibitor amino acid sequence of Figure l.
`
`The composition for use according to any preceding claim, wherein the composition
`
`comprises a buffer which comprises citrate or sodium citrate, optionally—alarm 5mM tom 50
`
`mM sodium citrate,—a¥wat 10 mM tom 30 mM sodium citrate, 09:5th 20 mM sodium
`
`citrate .
`
`Page 2 of51
`
`16
`
`Page 2 of 51
`
`

`

`«Kati. The composition for use according to any one of claims l-«l—lfl, com wisirw a buffer
`
`wherein the buffer does not contain citrate or citric acid.
`
`HQ. The composition for use according to any preceding claim, wherein the composition:
`
`(i) comprises at least one amino acid or salt thereof; and/or
`
`(ii) has a pH of between—aleu—t2 6.5 and 8.0.
`
`W
`c»
`
`‘m‘
`
`,
`
`,
`
`M
`i
`
`,
`
`‘
`
`A Mm“, m,
`a M.» .u I
`
`The composition for use according to any preceding claim wherein the composition is
`
`
`forrmd fi‘mn a ”‘ "
`" lyophilized form
`
`reconstitutmed in a buffer prior to
`
`administration.
`
`17
`
`Page 3 of51
`
`Page 3 of 51
`
`

`

`
`
`15
`
`20
`
`25
`
`30
`
`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
`
`for the treatment and/or prevention of disorders associated with Cl esterase inhibitor
`
`deficiency.
`
`BACKGROUND OF THE INVENTION
`
`Several publications and patent documents are cited throughout the
`
`specification in order to describe the state of the art to which this invention pertains.
`
`Full citations ofthese references can be found throughout the specification.%%hef
`
`
`
`Hereditary angioedema (HAE) is a rare, life-threatening, genetic disorder
`
`caused by a deficiency ofthe Cl esterase inhibitor (see generally www.haei.org and
`
`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 ofa defect in the gene controlling the synthesis
`
`of the C1 esterase inhibitor. Accordingly, restoring the levels of active Cl 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 Cl esterase inhibitor, such as HAE, are desired.
`
`Page 4 of51
`
`Page 4 of 51
`
`

`

`SUMMARY OF THE INVENTION
`
`
`
`The invention rovides a, com ositien com . risin I at least one C l esterase
`
`inhibitor wherein the at least one C l esterase inhibitor is
`resent at 400U/ml or more for
`
`
`
`
`
`reventin T heredita
`
`use in treatin r
`inhibitin 7 or
`com osition is administered subcutaneousl
`.
`
`
`
`
`an rioedema HAE‘ and wherein said
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Figure 1 provides an amino acid sequence of human Cl esterase inhibitor.
`
`Figure 2 provides a graph of the effect of protein concentration on viscosity
`
`for initial spin concentration samples.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`The restoration of active Cl esterase inhibitor levels in patients having a
`
`disorder associated with deficient or reduced levels of active C1 esterase inhibitor
`
`(e. g., HAE) is an effective measure for treating such disorders. Currently, Cl esterase
`
`inhibitor (such as Cinryze® (ViroPharma, Inc.; Exton, PA)) is administered to a
`
`patient intravenously by a medical professional. Herein, formulations of a Cl esterase
`
`inhibitor (such as Cinryze®) are provided which are also effective for subcutaneous
`
`(SC) administration. Surprisingly, the subcutaneous administration of the Cl esterase
`
`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 andfor use in methods
`
`for inhibiting (e. g., reducing or slowing), treating, and/or preventing a disorder
`
`associated with C1 esterase inhibitor deficiency in a subject are provided. In a
`
`particular embodiment, the methods comprise administering (egrsubcutaneouslyer—
`
`to a subject in need thereof at least one C1 esterase inhibitor. In a
`
`particular embodiment, the Cl esterase inhibitor is administered subcutaneously after
`
`an initial administration of the Cl esterase inhibitor intravenously.
`2
`
`Page 5 of51
`
`Page 5 of 51
`
`

`

`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 al. (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., 214:751). The activity
`
`of a C1 esterase inhibitor may be assayed by known methods (see, e.g., Drouet et a1.
`
`(1988) Clin. Chim. Acta., 174: 121-30). In a particular embodiment, the C1 esterase
`
`inhibitor is human. An amino acid sequence of human C1 esterase inhibitor is
`
`10
`
`provided in GenBank Accession No. CAA30314 (see also GeneID: 710, which also
`
`provides nucleotide sequences of the C1 esterase inhibitor) and Figure 1. 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
`
`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
`
`20
`
`high levels ofCl esterase inhibitor have been determined to be surprisingly stable and
`
`active. In a particular embodiment, the C1 esterase inhibitor is present at 313%
`
`Ic171mlto«abeut—1%‘(991—17innr1,—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
`
`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 ofthe 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 embodimentWHntravenew
`
`30
`
`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
`
`Page 6 of51
`
`Page 6 of 51
`
`

`

`sodium citrate, about 10 mM to about 30 mM sodium citrate, or about 20 mM sodium
`
`citrate).
`
`The compositions ofthe 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
`
`5 more particularly about 6.5 to about7.0.
`
`The compositions ofthe 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
`
`10
`
`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%.
`
`15
`
`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.
`
`20
`
`The compositions ofthe 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.,
`
`25
`
`2 to about 5 amino acids, particularly di-peptides or tri-peptides).
`
`As stated hereinabove, the instant isclosure encompasses
`
`compositions for use in methods of treating, inhibiting, and or preventing any
`
`condition or disease associated with an absolute or relative deficiency of functional
`
`C1 esterase inhibitor. Such disorders include, without limitation, acquired
`
`30
`
`angioedema (AAE) and hereditary angioedema (HAE). In
`
`embedimentthe invention, 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 ofCl esterase inhibitor (Zuraw, BL (2008) N. Engl. J. Med., 359:1027-
`
`1036). In a particular embodiment, the hereditary angioedema is type I or type II.
`
`Page 7 of51
`
`4
`
`Page 7 of 51
`
`

`

`Both type I and type II have a defective gene for the synthesis of C1 esterase inhibitor
`
`that produce either no C1 inhibitor (HAE type I) or a dysfunctional C1 inhibitor (HAE
`
`type II) (Rosen et a1. (1965) Science 148: 957-958; Bissler et a1. (1997) Proc. Assoc.
`
`Am. Physicians 109: 164-173; Zuraw et al. (2000) J. Allergy Clin. Immunol. 105:
`
`541-546; Bowen et a1. (2001) Clin. Immunol. 98: 157-163).
`
`The comp ositions for use according to methedsetlthe instant invention
`
`encompass the administration of at least one C1 esterase inhibitor, as defined in the
`
`claims. 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 as defined in
`
`the claims. 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 disclosure also encompasses kits
`
`comprising at least one composition ofthe 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) as defined in the claims. 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 embedimentaspect,
`
`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 C1 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 embodimentaspgct, 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 ofthe present Wdisclosure can be
`
`administered by any suitable route, for example, by injection (e. g., for local (direct)
`
`or systemic administration. In apartieulaeembedimentthe invention, the composition
`
`is administered subcutaneouslyWin accordance with the invention. 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 additives such as detergents and
`
`solubilizing agents (e.g., Tween 80, Polysorbate 80), antioxidants (e.g., ascorbic acid,
`
`sodium metabisulflte), preservatives (e. g.,
`
`Page 8 of51
`
`Page 8 of 51
`
`

`

`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
`
`5
`
`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
`
`10
`
`provided in the kits of the instant Wdisclosure 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
`
`15
`
`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
`
`20
`
`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)).
`
`25
`
`As stated hereinabove, agents of the instant mention—disclosure 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
`
`30
`
`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
`
`carrier may take a wide variety of forms depending on the form ofpreparation desired
`
`Page 9 of51
`
`6
`
`Page 9 of 51
`
`

`

`for administrationyeagrrparenterallw (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
`
`5
`
`like may be employed.
`
`A pharmaceutical preparation ofthe 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
`
`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 ofa particular pathological condition may be determined by dosage
`
`concentration curve calculations. Appropriate dosage unit may also be determined by
`
`15
`
`assessing the efficacy ofthe 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
`
`20
`
`the dosage may be reduced to a maintenance level. The appropriate interval in a
`
`particular case would normally depend on the condition ofthe 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
`
`25
`
`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 ofthe 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).
`
`Page 10 of51
`
`Page 10 of 51
`
`

`

`In a particular embodiment, the Cl 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
`
`5
`
`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 oftime or all
`
`ofthe 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 of just below 0.4 U/ml
`
`10
`
`in blood. Further, the administration of a 2000U initial dose of Cl 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 Cl esterase inhibitor
`
`followed by 500U everyday with weekend holidays from administration (i.e., 5 out of
`
`15
`
`7 days) also results in the maintenance of about 0.4 U/ml or higher in blood. Notably,
`
`the administration of only the maintenance doses leads to increased and
`
`physiologically relevant blood levels of the Cl esterase inhibitor, but delayed
`
`compared to those receiving an initial high dose.
`
`20 Definitions
`
`H H
`
`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 5%, 2%, or ::l%.
`
`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
`
`ofthe patient (e.g., in one or more symptoms), delay in the progression ofthe
`
`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.
`
`Page 11 of51
`
`Page 11 of 51
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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
`
`5
`
`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.
`
`10
`
`A "carrier" refers to, for example, a diluent, adjuvant, preservative (e. g.,
`
`Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulflte),
`
`solubilizer (e.g., TWEEN 80, Polysorbate 80), emulsifler, 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
`
`15
`
`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,
`
`20
`
`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
`
`25
`
`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-
`
`30
`
`60% by weight ofa 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).
`
`Page 12 of51
`
`Page 12 of 51
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`

`

`The following example is provided to illustrate various embodiments ofthe
`
`present invention. The example is illustrative and is not intended to limit the
`
`5
`
`invention in any way.
`
`1 0
`
`EXAMPLE
`
`Spin Concentration Studies
`
`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
`
`15
`
`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.
`
`20
`
`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
`
`25
`
`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
`
`30
`
`distance used for these experiments was 30 uL ofwater. 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
`
`Page 13 ofSl
`
`10
`
`Page 13 of 51
`
`

`

`sample during a measurement. For a 30 uL volume mark on the pipette tip, the
`
`micropipette was set to draw 42 uL.
`
`Results
`
`5
`
`The instant example determined the ability to develop a higher concentration
`
`liquid formulation ofCl INH as a monoformulation. The initial studies focused on
`
`concentration ofthe stock solution of Cl 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
`
`10
`
`the solutions remained clear up to concentrations up N500 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.
`
`15
`
`7.9
`
`6.9
`
`5.9
`
`U/mL
`
`viscosity
`
`U/mL
`
`viscosity
`
`/mL
`
`viscosity
`
`m
`
`
`
`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.
`
`20
`
`A larger feasibility study was performed examining different buffers (20 mM
`
`phosphate, 20 mM citrate, and 20 mM Tris) at each ofthe 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
`
`25
`
`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 40° C
`
`for one week, the viscosity of all of the samples increased. At pH 5.9, all ofthe same
`
`gelled, likely due to thermally induced aggregation. For the remaining formulations,
`
`Page 14 of51
`
`11
`
`Page 14 of 51
`
`

`

`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.
`
`H
`
`— g6
`
`
`
`—
`
`7.4
`0.8
`9.2
`0.7
`
`
`
`
`
`
`
`
`12.8 :: 0.7
`23.1221
`18.7::0.7
`
`Table 2: Viscosity at t0 and after one week ofstorage at 40°C (tl). Viscosity is
`
`reported in mPa-s.
`
`otably, at pH 6.9, citrate formulations had lower viscosity values than for
`
`10
`
`phosphate, while at pH 7.9, phosphate buffer produced lower viscositiesthan 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
`
`15
`
`some degradation had already occurred just in the process ofpreparing the samples.
`
`Upon storage for one week at 40° C, the pH 5.9 samples gelled, making analysis by
`
`RP HPLC impossible. For all ofthe other samples, the percent purity was essentially
`
`unchanged, indicating that little, if any, chemical degradation occurs for storage under
`
`these conditions.
`
`20
`
`
`
`[c1 INH]
`
`pH
`5.9
`
`phosphate
`
`82.87::0.75
`
`t2
`
`:2.11
`
`.
`
`Page 15 of51
`
`12
`
`---n------- N
`
`
`
`
`
`7.9
`
`00
`
`5
`
`phosphate
`
`8.2 :: 1.2
`
`16.2:
`.
`00
`4
`_ .
`.
`500
`
`
`
`Page 15 of 51
`
`

`

`87.14
`phosphate
`_ 86.44
`86.67
`
`0.67
`1.49
`1.36
`
`88.59
`85.65
`82.92
`
`0.29
`1.32
`1.48
`
`
`
`
`
`
`
`85.19
`84.07
`86.03
`
`2.00
`1.24
`0.87
`
`Table 3: Percent purity by RP HPLC upon storage at 25° C (t2) or 40° C (t1).
`
`For samples stored for two weeks at 25° C, there were small losses,
`
`comparable to what was seen at tl. Together, the RP HPLC data indicate that there
`
`5
`
`are small losses due to chemical degradation. Higher pH seems to diminish the rate of
`
`degradation and there may be some sensitivity to buffer composition.
`
`While the chemical stability ofCl INH seems to be unchanged upon storage,
`
`there is come physical instability observed as indicated by SEC (Table 4). There are
`
`other proteins present in the Cl INH mixture, leading to an overall 'purity' of about
`
`10
`
`~67% at t0. Up