EA042954B1 - COMPOSITION CONTAINING C1-INH AND THEIR USE FOR THE TREATMENT, INHIBITION OR PREVENTION OF HEREDITARY ANGIOEDEMA - Google Patents

Description

This application claims, pursuant to Section 35, §119(e), USC, priority of U.S. Provisional Application No. 61/791399, filed March 15, 2013. The above application is incorporated here by reference.

Field of invention

The present invention relates to the field of therapeutic agents and methods of their use. In particular, the present invention provides a composition containing C1-INH and its use for the treatment, inhibition or prevention of hereditary angioedema (HAE).

Prior Art

Several publications and patents are cited in the present description of the invention in order to describe the state of the art to which this invention pertains. Full citations of these references can be found in this specification. Each of these quotations is incorporated here by reference in its entirety.

Hereditary angioedema (HAE) is a rare, life-threatening genetic disorder caused by deficiency of the C1 esterase inhibitor (see www.haei.org and www.haea.org for general information). At least 6,500 people in the United States of America and at least 10,000 people in Europe suffer from HAE. Patients suffering from HAE experience recurrent, unpredictable, debilitating, life-threatening bouts of inflammation and submucosal/subcutaneous edema. Inflammation usually affects the larynx, abdomen, face, limbs, and genitourinary tract. This genetic disorder is the result of a defect in the gene that controls the synthesis of the C1 esterase inhibitor. Accordingly, restoring active C1 esterase inhibitor levels in these patients to normal or near normal is an effective measure in the treatment of HAE. However, new and improved methods for the treatment and prevention of C1 esterase inhibitor deficiency disorders such as HAE are desired.

Brief summary of the invention

The present invention provides a composition for inhibiting, treating and/or preventing hereditary angioedema (HAE) in a subject. In a specific embodiment, the composition contains from 400 U/ml to 600 U/ml of a C1 esterase inhibitor, a citrate buffer or a phosphate buffer, wherein the pH of said composition is between 6.5 and 8.0, and said composition is a subcutaneous composition. introductions. Also provided herein is the use of a composition according to the invention for the treatment, inhibition or prevention of hereditary angioedema (HAE).

Brief description of graphic materials

In FIG. 1 shows the amino acid sequence of a human C1 esterase inhibitor.

In FIG. 2 is a plot of the effect of protein concentration on viscosity for samples concentrated by initial centrifugation.

Detailed description of the invention

Restoration of the levels of active C1 esterase inhibitors in patients suffering from a disorder associated with insufficient or reduced levels of an active C1 esterase inhibitor (eg, HAO) is an effective measure in the treatment of such disorders. Currently, a C1 esterase inhibitor (such as Cinryze® (ViroPharma, Inc.; Exton, PA)) is intravenously administered to a patient by healthcare professionals. The present invention provides C1 esterase inhibitor compositions (such as Cinryze®) that are also effective when administered subcutaneously (SC). Surprisingly, subcutaneous administration of the C1 esterase inhibitor was sufficient to maintain blood levels of the C1 esterase inhibitor. SC administration of a C1 esterase inhibitor satisfies a previously unfulfilled need that has existed in medicine due to the limitations associated with intravenous administration in patients suffering from HAE.

The present invention provides a composition for the treatment, inhibition and/or prevention of hereditary angioedema in a subject.

C1 esterase inhibitors are also known as C1 (C1-INH) inhibitors. C1 esterase inhibitors are complement C1 inhibitors and belong to the superfamily of serine protease inhibitors. The human C1 esterase inhibitor is a 500 amino acid protein including a 22 amino acid signal sequence (Carter et al. (1988) Eur. J. Biochem., 173:163). In plasma, the C1 esterase inhibitor is present as a highly glycosylated glycoprotein of approximately 76 kDa (Perkins et al. (1990) J. Mol. Biol., 214:751). C1 esterase inhibitor activity can be assayed using known methods (see, for example, Drouet et al. (1988) Clin. Chim. Acta., 174:121-30). In a specific embodiment, the C1 esterase inhibitor is human. The amino acid sequence of the human C1 esterase inhibitor is available from GenBank under the catalog number CAA30314 (see also GeneID: 710, which also lists the nucleotide sequences of the C1 esterase inhibitor), and is shown in FIG. 1. The C1 esterase inhibitor for use in the methods of the present invention may have an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence on fig. 1. The C1 esterase inhibitor can be isolated or purified from blood plasma (eg, human blood plasma) or recombinantly produced. If purified from blood plasma, the C1 esterase inhibitor can be nanofiltered and pasteurized. In a specific embodiment, the plasma-derived C1 esterase inhibitor is Cinryze®. In a specific embodiment, the C1 esterase inhibitor is present in the compositions of the present invention at a high concentration. Indeed, compositions containing very high levels of C1 esterase inhibitor have been found to be surprisingly stable and active. In a specific embodiment, the C1 esterase inhibitor is present at a concentration of 400 U/mL to 600 U/mL, or about 500 U/mL.

In a specific embodiment, the compositions of the present invention do not contain citrate or citric acid. Compositions free of citrate and citric acid are particularly useful for subcutaneous administration of a C1 esterase inhibitor because citrate or citric acid can cause a reaction at the injection site. In a particular embodiment, the buffer in the compositions of the present invention is sodium phosphate (eg, 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 specific embodiment (for example, for intravenous administration), the buffer in the compositions in accordance with the present invention contains a carboxyl group. For example, the buffer may, without limitation, be citrate, succinate, tartrate, maleate, acetate, and salts thereof. In a specific embodiment, the buffer in the compositions of the present invention is sodium citrate or citrate (eg, 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 present invention may have a pH range of 6.5 to 8.0, in particular from about 6.5 to about 7.5, and in particular from about 6.5 to about 7.0.

The compositions of the present invention may also contain polysorbate 80 (TWEEN). Compositions containing polysorbate 80 are particularly useful as they reduce/limit protein aggregation. Polysorbate 80 can also limit protein interactions when the composition is brought into contact with silicone-containing lubricants/oils, such as lubricants/oils used in syringes and other injection devices. Compositions containing polysorbate 80 are also useful for lyophilized preparations. In a specific embodiment, polysorbate 80 is present at a concentration of from about 0.01% to about 0.1%, in particular from about 0.025% to about 0.075%, in particular about 0.05%.

Compositions of the present invention may also contain sucrose. Sucrose can be added as a bulking agent as well as a lyophilization protective agent. In a specific embodiment, sucrose is added to the compositions to be lyophilized. In a particular embodiment, the compositions contain from about 25 mM to about 125 mM sucrose, in particular from about 50 mM to about 100 mM sucrose.

The compositions of the present invention may also contain at least one amino acid or a salt thereof, in particular methionine and/or arginine. Arginine, which carries a positive charge on its side chain, can be used to buffer phosphate solutions. Methionine acts as a stabilizer (for example, limiting oxidation). The amino acids may be present in the composition as single amino acids or as short peptides (eg, 2 to about 5 amino acids, such as dipeptides or tripeptides).

As stated above, the present invention encompasses use in the treatment, inhibition and/or prevention of hereditary angioedema. As stated above, HAE is a life-threatening and severe disease that manifests as recurrent bouts of submucosal/subcutaneous swelling due to deficiency of the C1 esterase inhibitor (Zuraw, B.L. (2008) N. Engl. J. Med., 359: 1027-1036 ). In a specific embodiment, hereditary angioedema is type I or type II edema.

Both type I and type II are associated with a defect in the gene responsible for the synthesis of the C1 esterase inhibitor, as a result of which the C1 inhibitor is not produced (HAO type I) or a non-functional C1 inhibitor (HAO type II) is produced (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 present invention encompasses compositions containing at least one C1 esterase inhibitor and a citrate buffer or a phosphate buffer. Such compositions may be administered in a therapeutically effective amount to a patient in need of such administration for the treatment of a C1 esterase inhibitor deficiency disorder.

The agents and compositions of the present invention may be administered by any suitable route, such as by injection (eg, for local (direct) or systemic administration). In a specific embodiment, the composition is administered subcutaneously or intravenously. As a rule, a pharmaceutically acceptable carrier in the composition is selected from the group of diluents, preservatives, solubility

- 2 042954 lysers, emulsifiers, adjuvants and/or carriers. The compositions may include diluents with different buffering contents (eg Tris HCl, acetate, phosphate), pH and ionic strength, and additives such as detergents and solubilizing agents (eg Tween 80, polysorbate 80), antioxidants (eg ascorbic acid, metabisulphite sodium), preservatives (eg thimerosal, benzyl alcohol) and bulking agents (eg lactose, mannitol). The pharmaceutical composition of the present invention may be prepared, for example, in liquid form, or may be presented in dried powder form (eg, lyophilized for subsequent dilution).

In a specific embodiment, the compositions are prepared in lyophilized form. When the compositions are presented in lyophilized form, they are reconstituted before use (for example, within one hour, several hours, or a day or more before use) with a suitable buffer (for example, sterile water, sterile saline, or a sterile solution containing the appropriate pharmaceutically acceptable carriers (eg for reconstitution of the compositions described above) Reconstitution buffer(s) may be present in the kits of the present invention or may be obtained or supplied separately.

As used herein, the term pharmaceutically acceptable carrier includes any and all solvents, dispersion media, and the like, which may be suitable for the desired route of administration of the pharmaceutical preparation, examples of which are given in the previous paragraph. The use of such media for pharmaceutically active substances is known in the art. Any conventional media or agents may be considered for use in a pharmaceutical formulation, provided they are not incompatible with the molecules being administered.

The choice of a suitable pharmaceutical preparation depends on the chosen route of administration. In this case, the pharmaceutical preparation contains molecules dispersed in a medium compatible with the tissue into which they are administered. Methods for preparing compositions for parenteral or subcutaneous administration are well known in the art (see, for example, Remington's Pharmaceutical Science (E.W. Martin, Mack Publishing Co., Easton, PA)).

As indicated above, the agents of the present invention are administered parenterally - for example by intravenous injection into the bloodstream, and/or by subcutaneous injection. Pharmaceutical preparations for parenteral, intravenous and subcutaneous injection are known in the art. If parenteral injection is chosen as the route of administration of the molecules, the steps required to ensure that sufficient molecules to achieve a biological effect reach the target cells must be taken.

Pharmaceutical compositions containing a compound of the present invention as an active ingredient in intimate admixture with a pharmaceutical carrier may be prepared in accordance with conventional methods for the preparation of pharmaceutical mixtures. The carrier may be presented in a wide variety of forms depending on the form of the drug desired for administration, such as parenteral or subcutaneous. In the case of preparations for parenteral administration, the carrier will usually contain sterile water, although other ingredients may be included, for example to aid solubility or preservation objectives. Injectable suspensions may also be prepared using suitable liquid carriers, suspending agents, and the like.

The pharmaceutical preparation of the invention can be formulated into unit dosage form for ease of administration and dose uniformity. The term unit dosage form, as used herein, refers to a physically discrete unit of a pharmaceutical product that is acceptable to the patient being treated. Each dose should contain an amount of the active ingredient calculated to achieve the desired effect, in association with the selected pharmaceutical carrier. Standard doses may be proportionally increased or decreased based on the weight of the patient. Suitable concentrations to alleviate a particular pathological condition can be determined by calculations using a concentration versus dose curve. Suitable unit doses can also be determined by evaluating the effectiveness of the treatment.

The pharmaceutical preparation containing the molecules of the present invention may be administered at suitable time intervals, for example, daily, every other day, every three days, five days out of every seven days, or at least one, two or three times a week or more up to alleviate or reduce the intensity of pathological symptoms, after which the dose can be reduced to a maintenance level. A suitable interval in a particular case may usually depend on the condition of the patient.

In a specific embodiment, the C1 esterase inhibitor is present in the composition or is administered in the range of from about 100 units to about 10,000 units; from about 500 units to about 5000 units; from about 1000 units to about 3500 units, or from about 1500 units to about 2500 units. In a specific embodiment, at least about 2000 units are used. In a specific embodiment, a high initial dose of C1 esterase inhibitor (as listed above) is used (may be administered intravenously

- 3 042954 but)) and then smaller maintenance doses. For example, a high initial dose may be at least 1.5; 2; 3; 4 or 5 times higher than subsequent doses. In a specific embodiment, the C1 esterase inhibitor is present in the maintenance composition, or administered for maintenance, in the range of about 100 units to about 5000 units; from about 250 units to about 2000 units; from about 250 units to about 1000 units; or approximately 500 units. In the methods according to the present invention, high initial doses of the C1 esterase inhibitor are possible (for example, they may be possible in prophylactic methods).

In a specific embodiment, the C1 esterase inhibitor is administered at such a frequency and dose as to increase the level of C1 esterase inhibitor to at least about 0.3, and in particular 0.4 U/mL, or even up to about 1 U/mL (1 U/mL). ml is the average amount of C1 inhibitor present in 1 ml of normal human plasma) in the subject's blood. For example, the C1 esterase inhibitor level may be maintained at or above 0.4 U/mL for at least 50%, at least 75%, at least 90%, at least 95% or more of the time, or the entire time period. (for example, the period of time during which the drug is administered). For example, administering an initial dose of a C1 esterase inhibitor of 2000 IU followed by 250 IU every day or 500 IU every other day results in blood levels just below 0.4 U/mL. In addition, administration of an initial dose of 2000 U C1 esterase inhibitor followed by 1000 U every 3 days results in a blood level of approximately 0.4 U/mL. In particular, less frequent administrations may be preferred for ease of use by the patient. Administration of an initial dose of C1 esterase inhibitor of 2000 U followed by 500 U every day with no administration on weekends (i.e. 5 out of 7 days) also results in a blood level of approximately 0.4 U/day. ml or higher. Notably, administration of only maintenance doses results in increased and physiologically relevant levels of the C1 esterase inhibitor in the blood, which is however delayed in time compared to when a high initial dose is administered.

Definitions

Unless otherwise clearly stated, singular nouns include plural objects.

As used herein, the term approximately may refer to ±5%, ±2%, or ±1%.

As used herein, the terms host, subject, and patient refer to any animal, including humans.

The term prevent as used herein refers to the prophylactic treatment of a subject at risk of developing a condition (eg, HAE or HAE attack), resulting in a decrease in the likelihood that the subject will develop the condition.

As used herein, treat refers to any type of treatment that benefits a patient suffering from a disorder, including improving the patient's condition (eg, one or more symptoms), delaying the progress of the condition, and the like. In a specific embodiment, treatment of HAE results in at least a reduction in the severity and/or number of attacks of HAE.

The phrase effective amount refers to the amount of a therapeutic agent that results in an improvement in the patient's condition. A therapeutically effective amount of a compound or pharmaceutical composition refers to an amount effective to prevent, inhibit, treat, or reduce the symptoms of a particular disorder or disease.

Pharmaceutically acceptable indicates approval by a regulatory agency of the federal or state government, or indication in the USP or other generally accepted pharmacopoeia for use in animals, and in particular humans.

The carrier refers, for example, to a diluent, adjuvant, preservative (eg thimerosal, benzyl alcohol), antioxidant (eg ascorbic acid, sodium metabisulphite), solubilizer (eg TWEEN 80, polysorbate 80), emulsifier, buffer (eg Tris HCl, acetate, phosphate), water, aqueous solutions, oils, bulking agent (eg lactose, mannitol), cryo-/lyo-protective agents, tonicity regulator, excipient, auxiliary agent or diluent with which the 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 a protein, nucleic acid, compound, or cell that is sufficiently separated from the environment with which it can naturally be associated (eg, such that it is present in an essentially pure form). Selected does not necessarily mean the exclusion of artificial or synthetic mixtures with other compounds or materials, or the presence of impurities which do not affect the fundamental activity and which may be present, for example, due to incomplete purification.

The term substantially pure refers to a preparation containing at least 50-60% by weight,

- 4 042954 of this material (eg nucleic acid, oligonucleotide, protein, etc.). In some embodiments, the preparation contains at least 75 wt.%, in particular 90-95% or more by weight of this compound. Purity is measured by methods appropriate to the compound (eg, chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC (high performance liquid chromatography) analysis, and the like).

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

Example

Research on a centrifugal concentrator

Protein was applied to centrifuge concentrators and centrifuged at 10,500 rpm for 5 to 10 minutes. After stopping the centrifugation of the samples, the final volumes in centrifuge concentrators were recorded and the approximate protein concentration was calculated. Additional protein was added to centrifuge concentrators and centrifuged until the desired protein concentration was reached, at which point a UV measurement was taken. For each protein concentration, a UV measurement and a viscosity measurement were performed. The above procedure was continued until the viscosity of the protein began to prevent further concentration of the sample.

Viscosity measurements

Viscosity was determined by measuring the time it takes a sample to travel a predetermined distance in the gel filling a pipette tip. To calculate the viscosity of the sample, a standard curve was first built using a set of standards with known viscosities. Sucrose (or Briquet) solutions are suitable for constructing such a curve, but any material that has a known viscosity at a certain temperature should also be suitable.

To make measurements, the pipette plunger is squeezed, the pipette tip is immersed in the sample vial, the plunger is released, and the time it takes the liquid to travel a predetermined distance in the pipette tip is measured with a stopwatch. The distance used for these experiments was 30 µl of water. It is important to note that the pipette tip is only suitable for a single experiment, so multiple tips are used for parallel sample studies. In addition, the volume aspirated into the pipette tip must be larger than the volume marked on the tip to ensure that the sample is aspirated uniformly during measurement. In the case where the volume marking on the pipette tip was 30 µl, the micropipette was adjusted to aspirate 42 µl.

results

In the present example, it has been found that a higher concentration of C1-INH liquid composition can be obtained as a mono-composition. Initial studies were focused on the concentration of the stock solution of C1-INH using the method of centrifugal concentration. The pH of the solutions was initially adjusted, but no other excipient was added. Three pH values were studied (pH 5.9, 6.9 and 7.9). Upon centrifugal concentration, all solutions remained clear to concentrations of approximately 500 U/mL (approximately 100 mg/mL) at all pH values tested (Table 1). Although the dissolution limit was not reached in these studies, measurable increases in viscosity were found at concentrations in excess of 300 U/ml (FIG. 2). At all pH values, the viscosity begins to increase markedly when the concentration of C1-INH exceeds 400 U/ml.

7.9 6.9 5.9

U/ml Viscosity U/ml Viscosity U/ml Viscosity

93.12 0.99 182.4 415.18 3.95 289.4 454.81 13.74 378.6 501.17 30.43 479.0

4.23 187.2 2.36 4.90 296.9 7.71 12.08 396.7 5.46 14.67 478.8 24.09

Tab. 1: final concentrations (in units/ml) and viscosities of samples prepared in centrifuge concentration experiments. These values are based on the initial concentration of 160 U/mL of the original drug lot.

A larger feasibility study was conducted in which various buffers (20 mM phosphate, 20 mM citrate, and 20 mM Tris) were tested at each of the three pH targets. Samples of 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. Initial viscosities exceeded pure water values (~1 mPa-s) but were within the range normally specified for use as an injectable product (Table 2). Viscosities for samples at 400 U/mL were less than at 500 U/mL, typically by 7-10 mPa-s. When stored at 40°C for one week, the viscosity of all samples increased. At pH 5.9, all samples gelled, probably due to thermally induced aggregation. In the remaining compositions, the viscosity increased to some extent. In some cases, these values exceeded 30 mPa-s. The increase in viscosity was less when stored at 25°C than at 40°C. There was only little, if any, change for the pH 6.9 samples, indicating that pH 6.9 may be more favorable in terms of long-term storage stability.

pH [C1 INH] Buffer to t1 t2 5.9 400 phosphate 13.3+0.6 gel 17.4+2.1 500 24.6+1.5 gel 36.9+7.3 400 histidine 14.7+0.8 gel 19.1+2.5 500 27.7+3.8 gel 27.7+3.8 6.9 400 phosphate 12.2+1.5 16.1+0.6 11.9+3.0 500 20.8±2.0 35.3±2.1 32.1±7.7 400 citrate 7.4+0.8 9.2+0.7 7.1+0.6 500 14.4+3.2 19.8+1.1 12.6+0.5 7.9 400 phosphate 8.2+1.2 12.8+0.7 22.0+3.5 500 16.2+1.4 23.1+2.1 25.5+7.5 400 tris 14.1+0.7 18.7+0.7 30.0+3.8 500 20.5+0.9 33.3+6.2 31.0+1.8

Tab. 2: Viscosity at t0 and after one week of storage at 40°C (t1). Viscosity is expressed in mPa-s.

Notably, at pH 6.9, citrate formulations have lower viscosities than phosphate ones, although at pH 7.9, phosphate buffer provided lower viscosity than tris buffer. Higher viscosity implies that more force is required to deliver a given volume of drug within a given time frame.

Purity according to reverse phase HPLC was initially about 86-87% for formulations at pH 6.9 and above (Table 3). Initial levels were lower at pH 5.9, indicating that some degree of degradation had already occurred during sample preparation. When stored for one week at 40°C, samples with pH 5.9 turned into a gel, which led to the fact that analysis using reversed-phase HPLC became impossible. For all other samples, the percentage purity did not change substantially, indicating that there would be little, if any, chemical degradation when stored under these conditions.

pH [C1 INH] Buffer to t1 t2 5.9 400 phosphate 82.87+0.75 gel 81.10+2.11 500 84.74+1.24 gel 83.61+1.02 400 histidine 84.11+1.53 gel 85.34+1.55 500 86.36+0.76 gel 82.99+0.64 6.9 400 phosphate 87.14+0.67 88.59+0.29 85.19+2.00 500 86.44+1.49 85.65+1.32 84.07+1.24 400 citrate 86.67+1.36 82.92+1.48 86.03+0.87 500 86.89+1.24 86.74+0.88 84.42+1.19 7.9 400 phosphate 86.09+1.14 85.29+0.84 85.98+0.90 500 86.47±1.15 83.57±1.33 84.00±0.97 400 tris 87.14+0.98 81.74+7.89 86.14+0.81 500 88.74+0.82 87.24+1.47 87.30+0.95

Tab. 3: percent purity according to reverse phase HPLC when stored at 25°C (t2) or 40°C (t1).

In the case of samples that were stored for two weeks at 25°C, there were small losses compared to those observed at time t1. Taken together, the reverse phase HPLC data indicate that there is little loss due to chemical degradation. A higher pH appears to result in a reduced rate of degradation and some sensitivity to the buffer composition may occur.

Although the chemical stability of C1-INH does not appear to change on storage, there is some physical instability as detected by gel permeation chromatography (Table 4). Other proteins are also present in the C1-INH mixture, resulting in an overall 'purity' at time t0 of approximately 67%. When stored at 40°C for one week (P), the total content of monomers in the samples is reduced to 54-56% for samples with a pH of 6.9 and above. There was little difference between the two different pH conditions, different buffers, and two protein concentrations. When stored for two weeks at 25°C (t2) at pH 5.9, the samples did not gel as they did at the higher storage temperature. However, markedly higher degradation was found, especially with histidine buffer. For these cases at pH 6.9 or 7.9, the loss measured by gel permeation chromatography was approximately 2% or so, compared to a loss of 10-12% at higher temperature for half the time.________________

pH [C1 INH] Buffer to t1 t2 5.9 400 phosphate 68.32+1.04 gel 62.56+0.94 500 67.19+0.14 gel 61.46+0.14 400 histidine 64.68+0.42 gel 46.58+1.09 500 66.60+0.08 gel 44.48+1.04 6.9 400 phosphate 67.85+0.22 55.29+0.36 500 67.41±0.36 54.79±0.14 65.45±0.23 400 citrate 67.82+0.07 56.14+0.41 65.49+0.16 500 67.43+0.30 56.59+0.33 65.03+0.36 7.9 400 phosphate 67.85+0.09 54.96+0.52 61.31+0.25 500 67.58+0.40 55.57+0.56 64.98+0.50 400 Tris 67.63+0.27 55.40+0.30 65.70+0.56 500 67.67+0.47 56.18+0.64 66.19+0.84

Tab. 4: monomer content according to gel permeation chromatography when stored at 25°C (t2) or 40°C (tl)

The data indicate that the rate of degradation is about 13-35 times slower at 4°C than at 25°C. A higher estimate is obtained using the Arrhenius plot. A lower estimate is obtained by determining the average loss per 5°C decrease in temperature and extrapolating to a storage temperature of 4°C. Using this data as an indicator, a loss of approximately 3-10% over two years at refrigerated storage temperature can be predicted. In other words, as follows from these data, the liquid composition appears to be very stable. In addition, the degradation rates of the 400 and 500 U/mL samples are approximately comparable, indicating that the development of a higher concentration formulation is feasible.

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

Studies have shown that there is no dissolution limit for the preparation of C1 INH at concentrations up to 500 U/ml. There is an increase in viscosity after the concentration reaches the range of 400-500 U/mL (buffer dependent, with citrate being better than phosphate, which in turn is better than Tris), but this is manageable and still allows easy delivery by injection in the case of standard syringe systems. Typically, C1-INH is relatively resistant to chemical degradation as determined by reverse phase HPLC.

While some of the preferred embodiments of the present invention have been specifically described above by way of example, the invention is not intended to be limited to such embodiments. Various modifications can be made without departing from the scope and spirit of the present invention, which are set forth in the following claims.

Claims (14)

1. Composition for the treatment, inhibition or prevention of hereditary angioedema (HAE), containing from 400 to 600 IU / ml of a C1 esterase inhibitor, citrate buffer or phosphate buffer, while the pH of this composition is from 6.5 to 8.0, where said composition is a composition for subcutaneous administration. 2. Composition according to claim 1, characterized in that said C1 esterase inhibitor is present in an amount of 400 to 500 units/ml. 3. Composition according to any one of claims 1 or 2, characterized in that said C1 esterase inhibitor is isolated or purified from human plasma or obtained by a recombinant method, and/or -7 042954 said C1 esterase inhibitor has an amino acid sequence that is at least 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of the human C1 esterase inhibitor shown in FIG. 1. 4. Composition according to any one of claims 1 to 3, characterized in that said composition contains a citrate buffer containing sodium citrate or citrate, possibly from 5 to 50 mM sodium citrate, from 10 to 30 mM sodium citrate, or 20 mM sodium citrate . 5. Composition according to any one of claims 1 to 3, characterized in that said phosphate buffer contains sodium phosphate, possibly 5 to 50 mM sodium phosphate, 10 to 30 mM sodium phosphate, or 20 mM sodium phosphate. 6. Composition according to any one of claims 1 to 5, characterized in that said composition contains at least one amino acid or a salt thereof. 7. Composition according to any one of claims 1 to 6, characterized in that said composition is obtained from a lyophilized form by reconstitution with a buffer before administration. 8. The use of a composition according to any one of claims 1 to 7 for the treatment, inhibition or prevention of hereditary angioedema (HAE). 9. Use according to claim 8, characterized in that said C1 esterase inhibitor is administered daily, every other day, every three days, once a week, twice a week or three times a week. 10. The use according to claim 8 or 9, characterized in that said C1 esterase inhibitor is administered to the subject at a high initial dose, and then subsequent lower maintenance doses are administered, optionally said high initial dose of at least 1.5, 2, 3, 4 or 5 times the subsequent doses and/or said high initial dose is administered intravenously and then said C1 esterase inhibitor is administered subcutaneously. 11. The use according to any one of claims 8 to 10, characterized in that said administration of the C1 esterase inhibitor increases blood levels of the C1 esterase inhibitor in the subject, wherein blood levels of said C1 esterase inhibitor increase to at least 0.3 U/ ml, 0.4 U/ml or up to 1 U/ml. 12. Use according to any one of claims 8 to 11, characterized in that said levels of the C1 esterase inhibitor in the blood remain equal to or greater than 0.4 U/ml for at least 50%, at least 75%, at least 90% or at least 95% of the time. 13. Use according to any one of claims 8-12, characterized in that: (i) hereditary angioedema (HAE) is type I or II edema; (ii) administration of said C1 esterase inhibitor provides prophylactic treatment for HAE; (iii) said treatment for HAE provides at least a reduction in the severity and/or number of attacks of HAE; or (iv) administering said C1 esterase inhibitor provides treatment for an attack of HAE. 14. Use according to any one of claims 8 to 13, characterized in that said C1 esterase inhibitor is administered at a dose of 500 to 5000 units, 1000 to 3500 units or 1500 to 2500 units.