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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
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  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
  • C07K14/8121—Serpins
  • C07K14/8128—Antithrombin III
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a process for the preparation of latent antithrombin III.
• Antithrombin III is a plasma glycoprotein with a total molecular weight of 58.1 kDa (Lebing et al, Vox Sang. 67, 117-124, 1994), that inhibits serine proteases in the coagulation cascade and thus plays a major role in the regulation of blood clotting.
• Antithrombin III is an inhibitor of Factors IXa, Xa, XI and Xlla, as well as of thrombin.
• AT regulates clot formation in different stages of the coagulation cascade. A small decrease of AT content in the blood is associated with an increased risk of thromboembolism.
• Concentrates of AT are used in the prophylaxis and treatment of thromboembolic disorders in patients with acquired or hereditary antithrombin deficiency.
• AT has a function in many other processes of the human body, for example in angiogenesis and inflammatory responses.
• the function of AT in these physiological processes is not fully understood.
• L-AT and a selectively elastase cleaved variant have been shown to possess a strong antiangiogenic activity, and also to suppress tumor growth in mice that have been injected subcutaneously with a human neuroblastoma cell line (O'Reilly et al, Science 285, 1926-1928, 1999, and WO 00/20026) .
• L-AT must be considered a potential human anti-cancer drug.
• clinical evaluation of this potential drug remains to be performed. Purification of AT with affinity chromatography is done using purified heparin as solid phase bound ligand, as is known in the art.
• iller-Andersson et al discloses the use of heparin- Sepharose to purify human AT.
• This chromatographic system has also been useful for the separation between AT and L- AT, where the decreased affinity of heparin for L-AT relative to AT makes it possible to resolve the two components, as described by Chang and Harper (Thrombosis and Haemostasis 77, 323-328, 1997) .
• Hydrophobic interaction chromatography has been used for the separation of native and latent forms of AT (Karlsson, G & Winge, S. (2001) Protein Expr. Purif. 21:149-155)
• An object of the present invention is then to provide a process for the preparation of latent antithrombin III, L-AT, from a solution of native antithrombin III, AT, which process gives a higher yield of the desired product than the prior art process.
• a further object of the invention is to provide a process for the preparation of L-AT from AT, wherein the production of aggregates of AT polymers is kept to a minimum .
• Another object of the invention is to provide such a process for the conversion of AT to L-AT, in which commonly available reagents and buffer solutions are used, and which is performed in vitro.
• Still another object of the invention is to provide a process for the preparation of L-AT from AT, which is readily scaled up for industrial production of L-AT.
• a process which comprises incubation of a solution of native antithrombin III in the presence of sulfate ions and a buffer selected from Good's zwitterionic buffers. It has surprisingly been found that these incubation conditions makes possible the recovery of L-AT from the process in yields that are substantially higher than those obtained by methods of the prior art (notably the citrate conditions of Wardell et al) , while avoiding possible aggregation problems.
• FIG. 1 Heparin affinity chromatography of antithrombin using a sodium chloride gradient, 0-2 (5-60 min) .
• the injected amount of protein was 100 ⁇ g for sample A-B, and 150 ⁇ g for sample C-D. All samples were incubated at 60 °C for 16 h, except for the reference AT sample A, which was not heat-treated (sample 7 in the example) .
• Sample B (sample 6 in the example) was incubated according to Wardell, ie in 0.5 M citrate.
• Samples C (sample 2 in the example) and D (sample 1 in the example) were incubated in 5 mM HEPES, pH 7.4, with 0.9 and 0.8 M ammonium sulfate respectively. Integration of the low affinity heparin- binding peak, eluting at 22 min, gave 44%, 71% and 89% of the total integrated area for samples B, C, and D, respectively. Native AT eluted at 39 min.
• Figure 2 Native electrophoresis of antithrombin samples, using 12.5% polyacryl mide in a homogeneous gel. The amount of sample was 0.5 r ⁇ protein/lane, and the gels were silver-stained after running. All samples, except for lane 7, were incubated in 60 °C for 16 h.
• Lane 1 5 mM HEPES, 0.8 M ammonium sulfate, pH 7.4 Lane 2) 5 mM HEPES, 0.9 M ammonium sulfate, pH 7.4 Lane 3) 5 mM HEPES, 1.1 M ammonium sulfate, pH 7.4 Lane 4) 5 mM HEPES, 1.4 M ammonium sulfate, pH 7.4 Lane 5) 5 mM HEPES, 2.0 M ammonium sulfate, pH 7.4 Lane 6) 10 mol Tris/HCl, 0.5 M trisodium citrate, pH 7.4 (according to Wardell et al . 1997) Lane 7) Reference AT sample, not heat-treated
• Lane 8 25 mM sodium phosphate, 100 mM sodium chloride, pH 7.4 Lane 9) 25 mM HEPES, 0.8 M ammonium sulfate, pH 7.4 Lane 10) 5 mM HEPES, 0.5 M ammonium sulfate, pH 7.4 Lane 11) 5 mM HEPES, 2.0 M ammonium sulfate, pH 7.4 Lane 12) 5 mM HEPES, 0.8 M ammonium sulfate, pH 7.0 All lane numbers correspond to the sample numbers listed in the example below.
• the invention provides a process for the preparation of latent antithrombin III (referred to as L-AT) , starting from a solution of antithrombin III in its native form (referred to as AT) .
• L-AT latent antithrombin III
• AT can be isolated from blood plasma by heparin-Sepharose chromatography as has been described.
• the AT is then incubated in the presence of sulfate ions and a buffer.
• the incubation temperature and duration can be readily determined by the skilled person, but normal pasteurization conditions, such as a temperature of about 60 °C during about 16 hours, have been found to work well.
• the sulfate ions are preferably provided in the form of a sulfate salt.
• a sulfate salt is preferred.
• ammonium sulfate is preferred.
• a suitable concentration of sulfate ions in the process according to the invention lies in the range from 0.5 to 2.0 M, preferably from 0.7 to 1 M, a concentration between 0.8 and 0.9 M being most preferred.
• Another component of the incubation mixture is a buffer selected from Good's zwitterionic buffers (Good et al, Biochemistry 5, 467-477, 1966) .
• the buffer should fulfil most or all of the following requirements: it should exhibit a pK a value of between about 6 and about 9, a maximum solubility in water and a minimum solubility in other solvents, produce a minimum of salt effects, be stable at the experimental conditions used, and not absorb light in the visible or ultraviolet spectral regions (so as not to interfere with spectrophotometric measurements).
• Good's zwitterionic buffers including buffers such as HEPES, MES and PIPES, typically present the desired characteristics. The use of HEPES is particularly preferred in the process according to the invention.
• the widely used Tris buffer is unsuitable for the purposes of the invention.
• Preferred buffer concentrations are somewhat dependent on the buffer chosen, but typically lie in the range from 1 to 25 mM, more preferably from 2.5 to 10 mM, most preferably from 4 to 6 mM.
• the pH of the incubation reaction should lie between pH 6 and pH 9, preferably between pH 7 and pH 8, most preferably between pH 7.4 and pH 7.6.
• separation of the L-AT thus obtained from remaining AT is preferably performed using heparin affinity chromatography.
• the L-AT exhibits a lower binding affinity to heparin than AT, eluting substantially faster and enabling easy separation of the two forms of antithrombin III.
• the preparation of L-AT thus obtained is advantageously subjected to treatment for the inactivation or removal of pathogens, particularly in the form of viruses and prions .
• This can be done in any stage of the process using one of several methods for inactivation or removal known in the art, or combinations of such methods. Examples of such methods include chemical inactivation, heat inactivation, light inactivation, microwave inactivation and nano-filtration removal.
• a dead-end filtration procedure with a high salt content, like that described in WO96/00237, is particularly preferred, alone or in combination with other procedures.
• the removal and inactivation of pathogens can also be performed when the antithrombin III molecules are in the native state, before conversion to L-AT.
• the invention is further illustrated by the following, non-limiting example.
• EXAMPLE A laboratory sample of AT, > 95% pure, was obtained from Plasma Products, Pharmacia, Sweden. This sample was prepared according to known methods (Miller- Andersson et al, supra) and used for induction of the latent form of antithrombin.
• Sample 9 25 mM HEPES, 0.8 M ammonium sulfate, pH 7.4
• Sample 10 5 mM HEPES, 0.5 M ammonium sulfate, pH 7.4
• L-AT The formation of L-AT in the samples was analyzed by heparin affinity chromatography, and the presence of aggregates was analyzed by native electrophoresis.
• Electrophoresis was performed using a 12.5% polyacrylamide homogeneous Phast ® gel (Amersham Pharmacia).
• Sample 2 (incubation in 0.9 M ammonium sulfate) was analyzed regarding biological AT activity with the thrombin chro ogenic peptide substrate (S-2238) (Chromogenix, Molndal, Sweden), according to Handeland et al . (Scand J. Haematol . 31, 427-436, 1983).
• the assay solution consisted of thrombin, heparin, chromogenic substrate and sample, and the response after incubation was recorded as a change in absorbance at 405 nm.
• Heparin affinity chromatography gave elution of native AT at 39 min (about 0.9 M sodium chloride) and the main latent peak eluted at 22 min (about 0.3 M sodium chloride) (figure 1A-1B) . Integration of the low heparin-binding peak indicated a yield of 44% (figure IB) for the sample prepared according to Wardell' s method (sample 6), while incubation in 0.9 and 0.8 M ammonium sulfate (samples 2 and 1, respectively) yielded 71% and 89% respectively of the total integrated area (figure 1C-1D) . Table 1 shows that the percentage of formed L-AT decreases at increased concentration of ammonium sulfate/HEPES or at a higher pH value.
• Table 1 Heparin affinity chromatography. Formation of 5 L-AT in various sample buffers after 16 h incubation in 60 °C.
• the percentage of L-AT formed will decrease at a higher concentration of ammonium sulfate/HEPES or at a higher pH value. In addition, for the prevention of formation of aggregates, it is necessary not to use too low an ammonium sulfate concentration or too low a pH value .

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