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`WO 2016/131958
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`PCT/EP2016/053559
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`5
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`Pharmaceutical formulations of C1 esterase inhibitor
`
`Field of invention
`
`The present invention relates to pharmaceutical formulations comprising the C1 esterase
`
`10
`
`inhibitor ("C1-I NH"), exhibiting a higher stability for prolonged storage and a reduced kinematic
`
`viscosity for ameliorated use in treating or preventing disorders related to kinin formation.
`
`Background
`
`C1-INH, a plasma glycoprotein with a molecular weight of 104 kDa, belongs to the protein family
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`15
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`of serine protease inhibitors (serpins), which regulate the activity of serine proteases by
`
`inhibiting their catalytic activity (Bock SC, et al., Biochemistry 1986, 25: 4292-4301). C1-INH
`
`inhibits the classical pathway of the complement system by inhibiting the activated serine
`
`proteases C1 s and C1 r. Furthermore, C1-INH is a major inhibitor of the contact activation
`
`system due to its ability to inhibit the activated serine proteases factor Xlla (FXlla), factor Xia
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`20
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`(FXla), and plasma kallikrein (Davis AE, Clin. lmmunol. 2005, 114: 3-9; Caliezi C et al.,
`
`Pharmacol. Rev. 2000, 52: 91-112). Deficiency in C1-INH leads to the clinical manifestation of
`
`hereditary angioedema (HAE), which is characterized by episodes of acute angioedema attacks
`
`in subcutaneous or submucosal tissues such as the skin, larynx, or visceral organs (Longhurst
`
`H, et al. Lancet 2012, 379: 474-481) which last between 1 and 7 days and occur at irregular
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`25
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`intervals. Abnormalities in C1-INH plasma content or in its functional activity (often referred to as
`
`a deficiency of functional C1-INH) result from various large and small mutations in the C1-INH
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`gene (vide supra) (Karnaukhova E, J. Hematol. Thromb. Dis., 2013, 1-7).
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`Two types of hereditary C1-INH deficiency generally exist. The more prevalent type I HAE is
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`30
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`characterized by low content (below 35% of normal) and low inhibitory activity of C1-INH in the
`
`circulation. Type II HAE is associated with normal or elevated antigenic levels of C1-INH of low
`
`functional activity. Recently, HAE with normal C1-INH (also known as type Ill HAE) has been
`
`described in two subcategories: (1) HAE due to mutation in the factor XII gene and, as a result,
`
`increased activity of factor XII leading to a high generation of bradykinin, and (2) HAE of
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`35
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`unknown genetic cause. HAE attacks can be treated effectively by administering C1-INH
`
`(Longhurst H, et al., Lancet 2012, 379: 474-481; Bork K, Allergy Asthma Clin. lmmunol. 2010, 6:
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`15). Moreover, administration of C1-INH has been shown to prevent edema formation in
`
`patients when given prophylactically. C1-INH is currently marketed e.g. as Berinert® (CSL
`Behring), Cetor® (Sanquin), Cinryze® (Shire), Ruconest® I Rhucin® (recombinant C1 inhibitor by
`
`Pharming). Due to its inhibitory effects on the complement and the contact activation systems,
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`5
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`C1-INH substitution restores normal homeostatic function and inhibits the excessive formation
`
`of vasoactive peptides such as bradykinin, which mediate the formation of angioedema.
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`C1-INH has been reported to reduce ischemia-reperfusion injury in rodent models for cerebral
`
`ischemia-reperfusion (De Simoni et al., J Cereb Blood Flow Metab. 2003, 23: 232-9; Akita et al.,
`2003, Neurosurgery 52: 395-400).
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`10
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`The C1-INH compositions commercially available for the treatment of C1-INH deficiency up to
`
`date are all large volume formulations, i.e., these formulations need to be administered by
`
`intravenous injection. In view of the fact that C1-INH has been shown to prevent edema
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`15
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`formation in patients with hereditary angioedema when given prophylactically (Cicardi M et al.,
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`Expert Opin. Pharmacother. 2007; 8: 3173-3181), there is a requirement for formulations that
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`can be easily self-administered by the affected patients at regular intervals.
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`Long-term prophylaxis of HAE aims to prevent or to minimize the number and severity of
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`20
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`angioedema attacks. However, the medications currently available for long-term prophylaxis are
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`in many cases not optimal. Oral antifibrinolytics requiring multiple daily doses are relatively
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`ineffective and frequently associated with significant side effects. Anabolic androgens are
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`convenient to take and usually effective at doses <200 mg/day but can be associated with
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`significant risk of serious side effects. Currently available formulations of C1-INH require
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`25
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`intravenous access, imposing a burden on the patient, the healthcare provider, or both.
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`Maintenance of intravenous access has required many patients to have venous ports implanted,
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`which are associated with increased risks of infection and thrombosis. Plasma levels of
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`functional C1-I NH fall rapidly following intravenous administration of therapeutic dosages of C1-
`
`l NH concentrates, reaching near basal levels within 3 days.
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`30
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`When manufacturing protein therapeutics, such as the C1-INH, regulatory authorities strongly
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`recommend manufacturers of therapeutic protein products to minimize protein multimerisation
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`and aggregation as much as possible. Moreover, an increased stability of protein therapeutics is
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`highly desired for prolonged storage of such therapeutics. Conditions, which increase the
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`35
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`protein's stability are also the best conditions to prevent denaturation and formation of high
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`molecular weight components (HMWC) by multimerisation and aggregation, in particular of the
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`therapeutic protein.
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`Therefore, strategies that minimize HMWC formation are highly desired to be developed as
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`5
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`early as feasible in product development. This can be achieved by e.g. using an appropriate cell
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`substrate, selecting manufacturing conditions that minimize HMWC formation, employing a
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`purification scheme that removes HMWC to the greatest extent possible, choosing a container
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`system, which minimizes HMWC formation of the protein, and most notably, choosing a
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`formulation that minimizes HMWC formation, degradation and denaturation during storage.
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`10
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`Hence, formulation components are principally chosen based on their ability to preserve the
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`native conformation of the therapeutic protein by preventing denaturation due to hydrophobic
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`interactions that may lead to HMWC formation, as well as by preventing chemical degradation,
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`including truncation, oxidation, and deamidation (Cleland et al., Crit. Rev. Ther. Drug Carrier
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`15
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`Syst. 1993, 10(4): 307-377; Shire et al., J. Pharm. Sci. 2004, 93(6): 1390-1402; Wakankarand
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`Borchardt, J. Pharm. Sci. 2006, 95(11 ): 2321-2336).
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`The potential clinical consequences of immune responses induced by protein HMWC may
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`depend on the loss or preservation of native epitopes in the HMWC: (a) some antibodies
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`20
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`generated by the human subject against HMWC containing native protein may bind to
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`monomeric protein as well as to the HMWC and may inhibit or neutralize product activity. (b),
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`other antibodies to denatured/degraded and hence aggregated protein bind uniquely to the
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`HMWC material, but not to native protein monomers (Guidance for Industry lmmunogenicity
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`Assessment for Therapeutic Protein Products, U.S. Department of Health and Human Services,
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`25
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`Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for
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`Biologics Evaluation and Research (CBER), August 2014).
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`The development of a concentrate comprising C1-INH (333
`
`IU/ml) and hyaluronidase
`
`(rHuPH20) by Viropharma (now: Shire) in subcutaneous administration was stopped during
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`30
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`clinical development: in particular almost all participants of the clinical trial suffered from
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`adverse events at the injection site.
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`WO 2014/145519 discloses C1-INH compositions having about 400 or 500 U/ml C1-INH. It is
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`suggested not to use citrate or citric acid as a buffer substance for subcutaneous administration.
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`35
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`The disclosed formulations contain only particular buffer substances in low concentrations with
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`no other excipient added. All disclosed C1-INH formulations have a relatively low overall purity
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`of about 67% monomer content at t0 . Although the initial viscosity levels are within the limits
`usually set for use of an injectable product, there is a need for products with an even better
`
`viscosity. With regard to stability the WO 2014/145519 discloses only data after one week at
`
`40°C and after two weeks at 25°C, i.e. no long-term stability data are shown and thus long-term
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`5
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`stability is unproven. In the present invention it has been found, that the long-term stability of the
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`C1-INH formulations which are disclosed in WO 2014/145519 can be considerably improved.
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`In summary, there is a need for a C1-INH formulation that has a proven long-term storage
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`stability by being less prone to HMWC formation as well as to denaturation and degradation,
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`10
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`does not cause serious drug-related adverse events, cause only acceptable treatment(cid:173)
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`emergent adverse events at the injection site and has a low viscosity. Furthermore, a
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`formulation, which is easily administered in high concentrations at low volumes, is urgently
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`needed. In addition, it would be desirable that such a formulation can be used for prophylactic
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`therapy as well as for acute therapy of patients suffering from hereditary angioedema.
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`15
`
`Summary
`
`The present invention provides low volume formulations comprising high concentrations of C1-
`
`INH and having an increased C1-INH stability, which makes said formulations well-suited for
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`20
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`prolonged storage. Moreover, said formulations exhibit a reduced viscosity, which simplifies the
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`subcutaneous and
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`intravenous application of such compounds,
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`in particular, as said
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`formulations may be applied by the patients themselves.
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`The present invention further relates to use of such formulations in the acute and/or prophylactic
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`25
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`treatment of disorders related to kinin formation.
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`In particular, the present invention relates to stable pharmaceutical formulations comprising
`
`(a)
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`C1-INH at a concentration of about 400 IU/ml - 2,000 IU/ml; and
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`sodium citrate having a calculated osmolarity of 20-120 mOsm/L or sodium di-
`(b)
`hydrogen phosphate I di-sodium hydrogen phosphate having a calculated osmolarity of
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`60-120 mOsm/L; and
`
`(c)
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`one or more physiologically acceptable salt(s), other than the substances in (b),
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`having a calculated osmolarity of 150-600 mOsm/L; or
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`one or more amino acid(s) selected from glycine and/or one or more basic and/or
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`one or more acidic L-amino acid(s) or a salt/salts thereof having a calculated osmolarity
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`30
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`35
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`of 50-500 mOsm/L; or
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`one or more physiologically acceptable salt(s), other than the substances in (b),
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`and one or more amino acid(s) selected from glycine and/or one or more basic and/or
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`one or more acidic L-amino acid(s) or a salt/salts thereof having together a calculated
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`osmolarity of 80-740 mOsm/L;
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`5
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`wherein
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`the
`
`overall
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`calculated
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`osmolarity
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`of
`
`the
`
`formulation
`
`is
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`170-800 mOsm/L.
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`In various embodiments, the physiologically acceptable salt of the above formulation is a
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`physiologically acceptable sodium salt, preferably selected from sodium chloride, disodium
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`10
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`EDTA, sodium acetate, sodium succinate and sodium sulphate.
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`In various embodiments, the basic L-amino acid is arginine, lysine and/or histidine or a salt/salts
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`thereof, preferably hydrochloride(s).
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`15
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`In various embodiments, the acidic L-amino acid is L-glutamic acid and/or L-aspartic acid or a
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`salt/salts thereof, preferably sodium salt(s).
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`In various embodiments the pH of the pharmaceutical formulations referred to above is between
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`about 6. 7 and about 7 .5.
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`20
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`25
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`30
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`35
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`In some embodiments, the pharmaceutical formulation comprises
`
`(a)
`
`(b)
`
`(c)
`
`(d)
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`about 400-625 IU/ml C1-INH;
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`about 20-120 mOsm/L sodium citrate;
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`about 50-300 mOsm/L glycine; and
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`about 190-400 mOsm/L sodium chloride,
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`wherein the overall calculated osmolarity of the formulation is 260-600 mOsm/L.
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`In various embodiments, the melting temperature of C1-INH measured by DSF in the above
`
`formulations is about 55°C or higher, preferably about 55-60°C.
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`In various embodiments, the formulations above may further comprise
`
`(a)
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`a detergent selected from the group consisting of PS80 (polysorbate 80) and
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`PS20 (polysorbate 20); and/or
`
`(b)
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`a preservative and/or antioxidant selected
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`from
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`the group consisting of
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`benzylalcohol, cresol, phenol, methionine and glutathione.
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`In various embodiments, the C1-INH is human C1-INH. In preferred embodiments, the human
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`C1-INH is derived from human plasma.
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`In various embodiments, the formulations referred to above comprise an absolute amount of
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`5
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`C1-INH of at least 1,200 IU, at least 1,500 IU or at least 1,800 IU per finished dosage form.
`
`In various embodiments, the above formulations are
`
`(a)
`
`(b)
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`10
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`obtainable by reconstitution of a lyophilized powder with a suitable liquid, or
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`provided as a liquid formulation.
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`In all of the above referenced embodiments, the formulation can be administered via
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`subcutaneous administration or via
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`intravenous administration, whereby optionally said
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`formulation may be self-administered by the patient.
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`15
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`In various embodiments, the kinematic viscosity of the formulation is below 10 mm2/s, below 8
`mm2/s, below 6 mm2/s or below 5 mm2/s.
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`In various embodiments, the formulation according to the invention comprises less than 10% of
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`high molecular weight components (HMWC), less than 8% of HMWC, less than 5% of HMWC or
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`20
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`less than 3% of HMWC.
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`Another aspect of the invention refers to pharmaceutical formulations for use
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`in the acute and/or prophylactic treatment of a disorder related to kinin formation, in
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`particular hereditary angioedema (HAE), preferably HAE type I, HAE type II or HAE type
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`Ill, secondary brain edema, edema of the central nervous system, hypotensive shock, or
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`edema during or after contacting blood with an artificial surface;
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`in the acute and/or prophylactic treatment of a disorder related to an
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`ischemia(cid:173)
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`reperfusion injury (IRI), in particular wherein the IRI is due to surgical intervention, in
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`particular vascular surgery, cardiac surgery, neurosurgery, trauma surgery, cancer
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`surgery, orthopedic surgery, transplantation, minimally invasive surgery, or insertion of a
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`device for delivery of a pharmacologically active substance or for mechanical removal of
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`complete or partial obstructions;
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`in the acute and/or prophylactic treatment of retinopathy; or
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`in preventing rejection of transplanted tissue in a patient.
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`30
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`35
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`Another aspect of the invention refers to kits comprising the pharmaceutical formulation of the
`
`invention as a lyophilized powder and a respective volume of a suitable liquid for reconstitution.
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`Yet another aspect of the invention refers to kits comprising the pharmaceutical formulation of
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`the invention and at least one syringe and/or one needle. And yet another aspect refers to a
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`5
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`syringe prefilled with a liquid pharmaceutical formulation of the invention.
`
`Detailed Description
`
`Definitions
`
`10
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`According to the present invention, the term "C1 esterase inhibitor" or "C1 inhibitor" ("C1-INH")
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`refers to the proteins or fragments thereof that function as serine protease inhibitors and inhibit
`
`proteases associated with the complement system, preferably proteases C1 r and C1 s as well
`
`as MASP-1 and MASP-2, with the kallikrein-kinin system, preferably plasma kallikrein and factor
`
`Xlla, and with the coagulation system, preferably factor Xia and factor Xlla. In addition, the C1-
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`15
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`INH can serve as an anti-inflammatory molecule that reduces the selectin-mediated leukocyte
`
`adhesion to endothelial cells. C1-INH as used herein can be the native serine protease inhibitor
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`or an active fragment thereof, or it can comprise a recombinant peptide, a synthetic peptide,
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`peptide mimetic, or peptide fragment that provides similar functional properties, such as the
`
`inhibition of proteases C1 r and C1 s, and/or MASP-1 and MASP-2, and/or plasma kallikrein,
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`20
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`and/or factor Xlla, and/or factor Xia. The term C1-INH shall also encompass all natural occurring
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`alleles, splice variants and isoforms which have the same or similar functions as the C1-INH.
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`For further disclosure regarding the structure and function of C1-INH, see US 4,915,945,
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`US 5,939,389, US 6,248,365, US 7,053,176 and WO 2007/073186.
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`25 One "unit" ("U") of C1-INH is equivalent to the C1-INH activity in 1 ml of fresh citrated plasma of
`
`healthy donors. The C1-INH may also be determined in "international units" ("IU"). These units
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`are based on the current World Health Organization (WHO) standard for C1-I NH concentrates
`
`(08/256) which was calibrated in an international collaborative study using normal local human
`
`plasma pools. In general, U and IU are equivalent.
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`30
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`The term "hereditary angioedema" ("HAE") as used herein relates to angioedema caused by a
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`low content and low inhibitory activity of C1-INH in the circulation (HAE type I) or by the
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`presence of normal or elevated antigenic levels of C1-INH of low functional activity (HAE type
`
`II). The term "HAE" as used herein also encompasses HAE with normal C1-INH (also known as
`
`35 HAE type Ill) which has been described recently in two subcategories: (1) HAE due to mutation
`
`in the factor XII gene and, as a result, increased activity of factor XII leading to a high
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`generation of bradykinin, and (2) HAE of unknown genetic cause. In patients suffering from
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`hereditary angioedema, edema attacks can occur in various intervals, including a daily, weekly,
`
`monthly, or even yearly basis. Furthermore, there are affected patients wherein no edema
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`occurs.
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`5
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`The term "angioedema" ("edema") as used herein relates to swelling of tissue, for example
`
`swelling of skin or mucosa. The swelling can occur, for example, in the face, at hands or feet or
`
`on the genitals. Furthermore, swelling can occur in the gastro-intestinal tract or in the respiratory
`
`tract. Other organs can also be affected. Swelling persists usually between one and three days.
`
`10 However, remission can already occur after hours or not until weeks.
`
`The term "ischemia-reperfusion injury" ("IRI") is an injury caused by the return of blood into
`
`tissue ("reperfusion") after an ischemia or a lack of oxygen. Direct damage to the tissue is
`
`caused by the interruption of the blood flow, mainly due to loss of oxygenation to the viable
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`15
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`tissue, ultimately leading to infarction if not reversed. However, if the insult is reversed, the
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`reperfusion of the ischemic tissue may paradoxically cause further "indirect" damage. Upon long
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`duration of ischemia, the "direct" damage resulting from hypoxia alone is the predominant
`
`mechanism. For shorter durations of ischemia, the "indirect" reperfusion mediated damage
`
`increasingly contributes to the damage caused.
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`20
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`The term "retinopathy" as used herein relates to acute or persistent damage of the eye.
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`Retinopathy can be caused by diabetes mellitus (leading to diabetic retinopathy), arterial
`
`hypertension (leading to hypertensive retinopathy), prematurity of the newborn (leading to
`
`retinopathy of prematurity), exposure to ionizing radiation (radiation retinopathy), direct sunlight
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`25
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`exposure (solar retinopathy), sickle cell disease, retinal vascular disease such as retinal vein or
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`artery occlusion, trauma, especially to the head and other diseases or conditions. Many types of
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`retinopathy are proliferative resulting, most often, from neovascularization or the overgrowth of
`
`blood vessels. Angiogenesis, the sprouting of new vessels is the hallmark precursor that may
`
`result in blindness or severe vision loss particularly if the macula becomes affected. In rare
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`30
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`cases, retinopathy is caused by genetic diseases.
`
`The term "acute treatment" or "treatment" as used herein relates to the treatment of a patient
`
`displaying acute symptoms. Acute treatment can occur from the appearance of the symptom
`
`until the full remission of the symptom. An acute treatment can occur once or several times until
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`35
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`the desired therapeutic effect is achieved.
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`The term "prophylactic treatment" or "prophylaxis" or "prevention" as used herein relates to the
`
`treatment of a patient in order to prevent the occurrence of symptoms. Prophylactic treatment
`
`can occur at regular intervals of days, weeks or months. Prophylactic treatment can also
`
`occasionally occur.
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`5
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`The term "about" means within an acceptable error range for a particular value which partially
`
`depends on the limitations of the measurement system.
`
`The term "HMWC" or "high molecular weight components" as used herein refers to any self-
`
`10
`
`associated, i.e. multimerised or aggregated protein species, in particular of C1-INH, with
`
`monomer defined as the smallest functional subunit. HMWC are further classified based on five
`
`characteristics: size,
`
`reversibility/dissociation, conformation, chemical modification, and
`
`morphology (Narhi et al., J. Pharm. Sci. 2012, 101 (2): 493-498).
`
`15 HMWC, in particular multimers and aggregates, have been recognized for their potential to elicit
`
`immune responses to therapeutic protein products for over a half-century (Gamble, Int. Arch.
`
`Allergy Appl. lmmunol. 1966, 30(5): 446-455). The underlying mechanisms by which protein
`
`HMWC may elicit or enhance immune responses include inter alia the following: extensive
`
`cross-linking of B-cell receptors, causing efficient B-cell activation (Dintzis et al., J. lmmunol.
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`20
`
`1989, 143(4): 1239-1244; Bachmann et al., Science 1993, 262(5138): 1448-1451); enhancing
`
`antigen uptake, processing, and presentation; and triggering immunostimulatory danger signals
`
`(Seong and Matzinger, Nat. Rev. lmmunol. 2004, 4(6): 469-478). Such mechanisms may
`
`enhance recruitment of T-cell help needed for generation of high-affinity, isotype-switched lgG
`
`antibody, whereby the antibody response is most often associated with neutralization of product
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`25
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`efficacy (Bachmann and Zinkernagel, Annu. Rev. lmmunol. 1997, 15:235-70).
`
`The term "finished dosage form (FDF)" of a drug is a dosage form of the drug which has
`
`undergone all stages of manufacture, including packaging in its final container and labelling.
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`30
`
`The term "physiologically acceptable salt" of this invention refers to salts in formulations that are
`
`mainly used in treating medical conditions in humans, in particular to treating or preventing
`
`disorders related to kinin formation. Further, a physiological acceptable salt refers to ionic
`
`substances which are soluble, i.e. in the liquid, preferably aqueous, state a physiological
`
`acceptable salt will be present in form of its dissolved cation(s) and anion(s), and which will not
`
`35
`
`cause serious adverse side events after administration to the human body. In this sense, the
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`formulations or their finished dosage forms are appropriate for physiological practice together
`with other excipients.
`
`The term "osmotic concentration", or "osmolarity", is the measure of solute concentration,
`
`5
`
`defined as
`
`number
`
`osmoles (Osm)
`
`per litre
`
`of solution (osmol/L or
`
`Osmolarity measures the number of osmoles of solute
`
`per unit volume of solution,
`
`whereas molarity measures the number moles
`
`unit volume of solution.
`
`can be measured e.g. by measuring the freezing point
`
`by methods
`
`10
`
`known to the person skilled in the
`
`Methods for measuring the
`
`of a solution by
`
`freezing point depression are described, for
`
`in the European
`
`the U.S. Pharmacopeia chapter 785. For example, for water, 1 Osmol of a solute added to
`
`1 kg of water lowers the freezing point by 1.86°C.
`
`15
`
`theoretical calculation of osmolarity is
`
`known to the person skilled in the
`
`Briefly,
`
`one
`
`for
`
`component of the solution the product of the osmotic coefficient f, the
`
`number
`
`particles n into which the molecule
`
`in
`
`and the molar concentration
`
`that component, and sums the result over all components. Hence, the osmolarity (in Osm/L)
`
`20
`
`25
`
`30
`
`35
`
`a solution can be calculated as follows:
`
`= l':ti ni Ci
`
`index i represents the identity
`
`a particular component, such as a salt
`
`f is the
`
`coefficient for that particular component;
`
`n is the number of
`
`into which the molecule of that particular component
`
`in water;
`
`C is the molar concentration of that particular component.
`
`The molar concentration
`
`slight temperature dependence; for the present purpose it is
`
`referred
`
`to the concentrations at
`
`preferably 10-30°C, more preferably
`
`at
`
`atmospheric
`
`At concentrations typically used in the claimed
`
`for
`
`an
`
`coefficient of 1
`
`particular component gives a sufficient approximation for calculating the
`
`of a solution.
`
`example, sodium chloride is a particular component which
`
`dissociates in
`
`into two particles; the osmolarity of sodium chloride is twofold of its
`Sodium di-hydrogen phosphate I di-sodium hydrogen phosphate is in
`
`applicable pH range approximately an equimolar mixture of both components, so that it will
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`
`dissociate in water into two or three particles, respectively; the osmolarity of phosphate is 2.5-
`
`fold
`
`its molar concentration. As used herein the molar concentrations of C1-INH in the
`
`claimed
`
`are very
`
`and can
`
`disregarded while calculating the osmolarity.
`
`It is well known that deviations between the measured osmolarity and the calculated osmolarity
`
`5 may occur. In particular in solutions where highly charged proteins, such as C1-INH, are present
`
`there might be a strong influence of that protein to the measured osmolarity due to the Donan
`
`potential. Hence, the term "osmolarity" or "calculated osmolarity" of this invention refers to the
`
`calculated osmolarity.
`
`10
`
`The term "DSF" refers to "differential scanning fluorimetry", which is a thermal shift assay or
`
`thermal denaturation assay that measures the stability of a target protein and a subsequent
`
`increase in the melting temperature of a protein upon binding of a ligand to the protein. The
`
`binding of low molecular weight ligands can increase the thermal stability of a protein. This
`
`stability change is measured by performing a thermal denaturation curve in the presence of a
`
`15
`
`fluorescent dye, such as Sypro Orange. When the protein unfolds, the exposed hydrophobic
`
`surfaces bind the dye, resulting in an increase in fluorescence. The stability curve and its
`
`midpoint value (melting temperature mt) are obtained by gradually increasing the temperature to
`
`unfold the protein and measuring the fluorescence at each point. In other words, the DSF
`
`monitors thermal unfolding of proteins in the presence of a fluorescent dye and the temperature
`
`20
`
`at which a protein unfolds is measured by an increase in the fluorescence of that dye with
`
`affinity for hydrophobic parts of protein, which are exposed as the protein unfolds. The
`
`difference in melting temperature can be used to rank buffer conditions, additives, according to
`
`their enhancement of protein stability.
`
`25
`
`The melting temperature (mt) is defined by the Gibbs free energy of unfolding (~Gu). An
`
`increase of temperature leads to a decrease of the portion of folded protein, and a decrease of
`
`~Gu. ~Gu equals zero at a state, where the ratio of folded and unfolded proteins is equal. The
`
`corresponding temperature is the melting temperature of a protein. Hence, the melting
`
`temperature is a measure for assessing the thermal stability of a protein. The formulations of
`
`30
`
`this invention offer an increase of melting temperatures as compared to prior art C1-INH
`
`formulations, as can be seen in Tables 2-3. A higher stability of such formulations makes them
`
`more suited for longer C1-INH storage.
`
`The term "WFI" refers to "water for injection". It is water intended for use in the manufacture of
`
`35 medicines for parenteral administration, the solvent of which is water. Alternatively, it refers to
`
`water that is used to dissolve or dilute substances or preparations for parenteral administration.
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`It is purified by distillation or a purification process, which is equivalent or superior to distillation
`
`in the removal of chemicals and microorganisms.
`
`The kinematic viscosity (m2/s) is the ratio between the dynamic viscosity [Pa*s = kg/m·s] and
`the density of a fluid [kg/m3].
`
`5
`
`The terms "sodium citrate" or "Na-citrate" or "Na3-citrate" of this invention refers to Tri-sodium(cid:173)
`citrate, i.e. Na3C(OH)(COO-)(CH2C00-)2.
`
`10
`
`C1 esterase inhibitor
`
`In certain embodiments of the invention, the C1-INH is a plasma-derived or a recombinant C1-
`
`INH. In further embodiments said inhibitor is identical to the naturally occurring human protein or
`
`a variant thereof. In other embodiments, said inhibitor is human C1-INH. In other embodiments,
`
`said inhibitor is a recombinant analogue of human C1-INH protein.
`
`15
`
`According to the present invention, the C1-INH may be modified to improve its bioavailability
`
`and/or half-life, to improve its efficacy and/or to reduce its potential side effects. The
`
`modification can be introduced during recombinant synthesis or otherwise. Examples for such
`
`modifications are glycosylation, PEGylation and HESylation of the C1-INH or an albumin fusion
`
`20
`
`of the described C1-INH. In some embodiments, C1-INH comprises a fusion construct between
`
`C1-INH and albumin, in particular human albumin. In some embodiments, the albumin is a
`
`recombinant protein. In certain embodiments, the C1-INH and albumin proteins are joined
`
`directly, or via a linker polypeptide. For further disclosure regarding glycosylation and albumin
`
`fusion of proteins, see WO 01/79271.
`
`25
`
`Preparation of C1-INH
`
`For the purpose of this invention, the C1-INH can be produced according to methods known to
`
`the skilled person. For example, plasma-derived C1-INH can be prepared by collecting blood
`
`plasma from several donors. Donors of plasma should be healthy as defined in the art.
`
`30
`
`Preferably, the plasma of several (1000 or more) healthy donors is pooled and optionally further
`
`processed. An exemplary process for preparing C1-INH for therapeutic purposes is disclosed in
`
`US 4,915,945. Alternatively, in other embodiments, C1-INH can be collected and concentrated
`
`from natural tissue sources using techniques known in the art. Recombinant C1-INH can be
`
`prepared by known methods.
`
`35
`
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`
`In certain embodiments, C1-INH is derived from human plasma. In further embodiments, C1-
`
`INH is prepared by recombinant expression.
`
`A commercially available product comprising C1-INH is, e.g., plasma-derived Berinert® (CSL
`
`5
`
`Behring). Berinert® is manufactured according to A. Feussner et al. (Transfusion 2014, 54:
`
`2566-73) and is indicated for treatment of hereditary angioedema and congenital deficiencies.
`
`Alternative commercially available products comprising C1-INH are plasma-derived Cetor®
`(Sanquin), Cinryze® (Shire), and recombinant Ruconest® I Rhucin® (Pharming).
`
`10 C1-INH Formulations
`The present invention relates to stable pharmaceutical formulations comprising (a) C1-INH.
`
`These highly concentrated formulations of the invention are provided in low volume formulations
`
`having a low viscosity. The formulations are well-tolerated and suitable for intravenous and in
`
`particular subcutaneous administ