NZ751555B2 - C1-inh compositions and methods for the prevention and treatment of disorders associated with c1 esterase inhibitor deficiency - Google Patents

Abstract

Lyophilised compositions and methods for the treatment and/or prevention of disorders associated with C1 esterase inhibitor deficiency, such as hereditary angioedema (HAE), via subcutaneous administration, wherein the lyophilised compositions when reconstituted comprise a C1 esterase inhibitor between 400 to 600 U/mL, a sodium citrate buffer, a pH in the range of 6.5 to 8.

Description

C1-INH COMPOSITIONS AND METHODS FOR THE PREVENTION AND TREATMENT OF DISORDERS ASSOCIATED WITH C1 ESTERASE INHIBITOR DEFICIENCY This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 61/791,399, filed March 15, 2013. The foregoing application is incorporated by reference herein.

FIELD OF THE INVENTION The present invention relates to the field of eutic agents and methods of use thereof. ically, the instant ion provides itions and methods for the treatment and/or prevention of disorders ated with C1 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 ion 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 C1 se 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 osal/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 of the C1 se 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 ting disorders associated with a deficiency of the C1 esterase inhibitor, such as HAE, are desired.

SUMMARY OF THE INVENTION In accordance with the instant invention, methods for ting, ng, 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 administering a composition comprising at least one Cl se inhibitor.

In accordance with the instant invention, therapeutic compositions are also ed. In a particular embodiment, the composition comprises at least one C1 esterase tor and, optionally, at least one pharmaceutically able carrier for delivery (e. g. intravenous or subcutaneous delivery). Kits comprising a composition 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 tor.

Figure 2 provides a graph of the effect of protein concentration on viscosity for initial spin tration samples.

DETAILED DESCRIPTION OF THE ION The restoration of active C1 esterase tor levels in patients having a disorder associated with nt or reduced levels of active C1 esterase inhibitor (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 Cl esterase inhibitor is sufficient to maintain the blood levels of the Cl esterase inhibitor. The SC administration of a Cl esterase tor 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 er associated with Cl esterase inhibitor deficiency in a subject are provided. In a particular embodiment, the methods comprise administering (e. g., subcutaneously or enously) to a subject in need thereof at least one Cl esterase inhibitor. In a particular ment, the C1 esterase inhibitor is administered subcutaneously after an initial administration of the Cl esterase inhibitor intravenously.

Cl esterase inhibitors are also known as C1 tors (C1 INH). C1 esterase inhibitors are tors of complement C1 and belong to the superfamily of serine nase 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, 173:163). In , the C1 se inhibitor is a heavily glycosylated rotein of approximately 76 kDa (Perkins et al. (1990) J. Mol. Biol., 2142751). The ty 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 C1 esterase inhibitor is human. An amino acid sequence of human C1 esterase inhibitor is provided in GenBank Accession No. CAA30314 (see also : 710, which also provides nucleotide sequences of the C1 esterase tor) 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 l. The C1 esterase inhibitor may be isolated or 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 se inhibitor is Cinryze®. In a particular embodiment, the C1 esterase inhibitor is present in the compositions of the instant invention at high concentration. Indeed, itions comprising very high levels of C1 esterase inhibitor have been determined to be surprisingly stable and active. In a ular 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 ular embodiment, the compositions of the instant invention do not contain citrate or citric acid. The compositions lacking citrate and citric acid are particularly useful for the aneous 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 administration), the buffer of the instant compositions comprises a carboxylic group.

For example, the buffer may be, without tion, 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 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 ation. 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 sing polysorbate 80 are also useful for lized ations.

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 se 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 ate. nine 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 es (e.g., 2 to about 5 amino acids, particularly di-peptides or tri-peptides).

As stated hereinabove, the instant invention asses 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 e, without limitation, acquired angioedema (AAE) and hereditary angioedema (HAE).

In a particular ment, the disorder is HAE and/or the attacks associated therewith. As stated hereinabove, HAE is a hreatening and debilitating disease that manifests as ent, submucosal/subcutaneous swelling attacks due to a deficiency of C1 esterase inhibitor , BL. (2008) N. Engl. J. Med, 35921027- 1036). In a particular embodiment, the hereditary angioedema is type I or type II.

Both type I and type II have a defective gene for the sis of Cl se 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. l. 98: 157—163).

The methods of the instant invention encompass the stration of at least one C1 esterase tor. Compositions comprising at least one C1 esterase inhibitor and, ally, at least one pharmaceutically acceptable carrier (e.g., one suitable for aneous or intravenous administration) are encompassed by the instant invention. Such itions 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 sing 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 titution buffer(s), syringes (e.g., able) for parenteral (e. g., subcutaneous) injection, and instruction material. In a particular embodiment, the kit comprises at least one pre-loaded e 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 ceutically 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 ction or protein aggregation).

The agents and compositions of the present ion can be administered by any suitable route, for example, by injection (e. g., for local (direct) or systemic stration. 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 additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), antioxidants (e.g., ascorbic acid, sodium sulfite), preservatives (e. g., Thimersol, benzyl alcohol) and bulking nces (e.g., lactose, mannitol). The pharmaceutical composition of the present invention can be prepared, for e, 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 itions 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 on, or a sterile solution comprising the appropriate pharmaceutically acceptable carriers (e. g., to reconstitute the compositions as bed hereinabove). The reconstitution buffer(s) may be provided in the kits of the instant invention or may be obtained or provided separately.

As used , aceutically 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 nces 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 sed 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 e (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. ceutical preparations for eral, 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. ceutical compositions containing a compound of the present invention as the active ingredient in intimate admixture with a pharmaceutical carrier can be prepared ing to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for stration, e.g., parenterally or subcutaneous. For parenterals, the carrier will usually comprise e water, though other ingredients, for example, to aid solubility or for preservative purposes, may be ed. Inj ectable suspensions may also be prepared, in which case appropriate liquid rs, suspending agents and the like may be employed.

A pharmaceutical preparation of the ion may be formulated in dosage unit form for ease of stration and uniformity of . 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 n a 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 als, 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 the dosage may be reduced to a nance level. The appropriate al 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 tor (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 Cl esterase inhibitor is present in the nance 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).

In a particular embodiment, the C1 esterase tor 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 (l Unit/ml is the mean quantity of C1 inhibitor present in 1 m1 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 ed by 1000U every 3 days results in the maintenance of about 0.4 U/ml in blood. Notably, for ease of use by the t, less frequent administrations may be red. The administration of a 2000U initial dose of Cl esterase inhibitor followed by 500U everyday with weekend ys from stration (i.e., 5 out of 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 C1 esterase inhibitor, but delayed compared to those receiving an initial high dose. ions The singular forms "a," "an," and "the" include plural nts unless the context clearly dictates otherwise.

As used herein, the term " may refer to i5%, i2%, or dzl%.

As used herein, the terms "host," "subject," and "patient" refer to any animal, 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 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 ent of HAE results in at least a reduction in the severity and/or number of HAE attacks.

WO 45519 The phrase "effective amount" refers to that amount of therapeutic agent that results in an ement 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 ment or listed in the US. copeia or other generally recognized pharmacopeia for use in animals, and more ularly in humans.

A "carrier" refers to, for e, a diluent, adjuvant, preservative (e. g., Thimersol, benzyl alcohol), xidant (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, ol), 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 bed in gton’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 ental 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 ation 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 (6.g. chromatographic methods, agarose or rylamide gel electrophoresis, HPLC analysis, and the like).

The following example is provided to illustrate various embodiments of the t invention. The example is illustrative and is not intended to limit the invention in any way.

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 s 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 n 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 g pipette tip. In order to calculate the sample ity, a standard curve was first ed using a set of rds with known viscosities. e (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 ce 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 sample during a measurement. For a 30 uL volume mark on the pipette tip, the micropipette was set to draw 42 uL.

Results The instant example ined the ability to develop a higher concentration liquid formulation of C1 INH as a monoformulation. The initial studies d on concentration of the stock on of C1 INH using a spin concentration .

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 s, there were measurable increases in viscosity as the concentrations exceeded 300 U/ml (Figure 2). At all pH values, the viscosity begins to increase ly 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 tration 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 inj ectable product (Table 2). The ity 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 of the same gelled, likely due to thermally induced aggregation. For the remaining formulations, the viscosity sed 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. [CIINHI —__-_ ate 13:33:06 "- - 17412.1 - ~ - -—_— -—-——_ - ——-—- - ~ ---_——400 . -———_—500 .

Table 2: Viscosity at t0 and after one week of storage at 40°C (t1). 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, ting that some degradation had already occurred just in the process of preparing the samples.

Upon storage for one week at 40° C, the pH 5.9 samples gelled, making analysis by RP HPLC impossible. For all of the other samples, the t purity was essentially unchanged, indicating that little, if any, chemical degradation occurs for e under these conditions. m [c1 INH]_" phosphate 82.87 i 0.75 . . _84 74 i- 1 244 . . _—gel 82.99 :t 0.64 ——-— —_-— —-_— ———— 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 t1. er, the RP HPLC data indicate that there are small losses due to chemical degradation. Higher pH seems to diminish the rate of degradation and there may be some ivity to buffer composition.

While the chemical stability of C1 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 C1 INH mixture, leading to an overall ‘purity’ of about ~67% at t0. Upon storage at 40° C for one week (t1), the overall monomer content of the samples decreased to 54—56 % for the s with pH 6.9 and higher. There was little difference n the two different pH conditions, the different buffers and the two protein concentrations. When stored for two weeks at 25° C (t2), the pH 5.9 samples did not gel, as they did at the higher storage temperature. However, there was appreciably higher degradation, especially with histidine buffer. For these at pH 6.9 or 7.9, the loss as measured by SEC was about 2% or so, compared to the 10-12 % loss at the higher temperature for half of the time.

Buffer _—a _——" ——_—" ——_-_ _—67.41 :t 0 36 54.79 i 0.14 65.45 i 0.23 WO 45519 -—citrate Table 4: Monomer content by SEC upon storage at 25° C (t2) or 40° C (t1).

The data indicate that the rate of degradation will be about 13—fold to 35-fold slower at 4° C than at 25° C. The higher estimate comes from using an Arrhenius plot.

The lower estimate comes from determine the average loss as the temperature is decreased by 5° C and extrapolating to a e temperature of 4° C. Using the current data as an indicator, this predicts a loss of about 3 to 10 % loss after two years at refrigerated temperatures. In other words, a liquid ation s to be quite stable based on these data. Furthermore, the degradation rates are roughly comparable between the 400 U/mL and 500 U/mL samples, suggesting that developing the higher concentration ation is just as viable.

The degradation rate is much faster at pH 5.9, leading to gelation at 40° C and greater losses at 25° C. Thus, further pH/buffer screening will focus on the pH 6.5 to 8.0 range. There is a clear buffer effect on viscosity and possibly also on stability.

The studies demonstrated that there is not a solubility limit to preparing C1 TNH at concentrations up to 500 U/ml. There is an increase in viscosity once the concentrations reach the 400-500 U/ml range (which is buffer dependent with citrate being better than phosphate which is better than Tris), but they are manageable and still allow facile delivery by injection for standard syringe systems. In general, C1 INH is relatively stable to chemical degradation, as determined by RP HPLC.

While certain of the preferred ments of the present invention have been described and specifically exemplified above, it is not ed that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the ing claims.

Claims (39)

What is claimed is:

1. A lyophilized pharmaceutical composition comprising sodium citrate, and at least 2000 U of C1 se tor and less than 5000 U of C1 esterase inhibitor, wherein following reconstitution, said lyophilized composition achieves a solution comprising: (i) a concentration of about 400 to about 600 U/mL of C1 esterase inhibitor, (ii) citrate at a concentration of about 5mM to about 15mM, and (iii) a pH ranging from about 6.5 to about 8.0, wherein the solution is for subcutaneous stration to treat HAE and wherein the C1 esterase tor comprises the amino acid sequence of residues 23 to 500 of SEQ ID NO: 1.

2. The lyophilized pharmaceutical composition of claim 1, wherein following reconstitution said lized composition achieves a solution comprising a concentration of 500 U/mL of C1 esterase inhibitor.

3. The lyophilized pharmaceutical composition of claim 1 or 2, wherein the pH of the solution ranges from about 6.5 to about 7.5.

4. The lyophilized pharmaceutical composition of claim 1 or 2, wherein the pH of the solution ranges from about 6.5 to about 7.

5. The lized ceutical composition of any one of claims 1 to 4, wherein the concentration of citrate in the solution is about 7mM to about 13mM.

6. The lyophilized pharmaceutical composition of claim 5, wherein the concentration of citrate in the solution is about 10mM.

7. The lyophilized pharmaceutical composition of any one of claims 1 to 6, wherein the composition is for HAE prophylactic treatment.

8. A kit comprising the lyophilized pharmaceutical composition as d in any one of claims 1 to 7 and at least one syringe and/or a reconstitution buffer.

9. A kit comprising at least one syringe pre-loaded with the lyophilized pharmaceutical composition of any one of claims 1 to 7, and instructions for using said kit.

10. The kit of claim 9, wherein the instructions are for abdominal subcutaneous administration.

11. A syringe pre-loaded with the ition of any one of claims 1 to 7, wherein the syringe is adapted for aneous administration of the composition.

12. The syringe of claim 11, wherein said syringe is adapted for abdominal subcutaneous administration of the composition.

13. Use of the lyophilized pharmaceutical composition as defined in any one of claims 1 to 7 in the manufacture of a medicament for increasing the level of Cl se inhibitor in the blood of a subject in need thereof.

14. The use according to claim 13, wherein the composition is for increasing the level of C1 esterase inhibitor in the blood of the t to at least 0.3 U/mL.

15. The use of claim 13, wherein the composition is for increasing the level of Cl esterase inhibitor in the blood of the subject to at least 0.4 U/mL.

16. The use of claim 13, wherein the composition is for sing the level of Cl esterase inhibitor in the blood of the subject to at least 1 U/mL.

17. The use of any one of claims 13 to 16, wherein the composition is for maintaining increased blood levels of Cl esterase inhibitor at least 50% of the time between uses of the composition.

18. The use of any one of claims 13 to 16, wherein the composition is for maintaining increased blood levels of Cl esterase inhibitor at about 95% of the time between uses of the composition.

19. The use of any one of claims 13 to 16, wherein the composition is for ining increased blood levels of Cl esterase inhibitor 100% of the time between uses of the composition.

20. The use of claim 13, n the medicament is for the treatment, inhibition and/or prevention of tary angioedema (HAE) in a subject in need thereof, n the medicament is for administration subcutaneously following titution.

21. The use of any one of claims 13 to 20, wherein the composition is for administration in a high initial dose of Cl esterase inhibitor, followed by at least one subsequent lower maintenance dose.

22. The use according to claim 21, wherein the high l dose is at least 1.5 times the at least one subsequent lower maintenance dose.

23. The use according to claim 21, wherein the high initial dose is at least 2 times the at least one subsequent lower maintenance dose.

24. The use according to claim 21, wherein the high initial dose is at least 3 times the at least one subsequent lower maintenance dose.

25. The use according to claim 21, wherein the high initial dose is at least 4 times the at least one subsequent lower maintenance dose.

26. The use according to claim 21, wherein the high initial dose is at least 5 times the at least one subsequent lower maintenance dose.

27. The use according to claim 21, wherein the at least one subsequent lower maintenance dose is for subsequent administration subcutaneously.

28. The use according to any one of claims 21 to 27, wherein the high initial dose ses about 2,000 U of Cl esterase.

29. The use according to any one of claims 21 to 27, wherein the high initial dose comprises about 3,000 U of Cl esterase.

30. The use of any one of claims 20 to 29, wherein: (i) the hereditary angioedema is Type I or Type II HAE, (ii) the administration of the Cl esterase inhibitor results in HAE lactic 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 C1 esterase inhibitor results in treatment of an HAE attack.

31. The use according to any one of claims 13 to 30, wherein the C1 esterase inhibitor is isolated or purified from human plasma or is inantly ed.

32. The use of any one of claims 13 to 31, wherein the composition is for administration daily, every other day, or every three days.

33. The use of any one of claims 13 to 31, n the composition is for administration one, two, or three times a week.

34. The use of any one of claims 13 to 33, wherein the pH ranges from about 6.5 to about 7.5.

35. The use of any one of claims 13 to 33, wherein the pH ranges from about 6.5 to about

36. The use of any one of claims 34 to 35, wherein the composition further comprises at least one amino acid or salt f.

37. The use of any one of claims 13 to 36, wherein the composition is for administration which results in HAE prophylactic treatment.

38. Use of C1 esterase inhibitor in the manufacture of a composition as defined in any one of claims 1 to 7, which is formulated for subcutaneous administration of C1 esterase inhibitor in a human patient in need thereof.

39. The use of any one of claims 34-37, wherein the composition comprises 400 U/mL to 500 U/mL of C1 esterase inhibitor. VV()