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  Process for preparing cardiodilatin fragments; highly purified cardiodilatin fragments and intermediate products for the preparation of sameUSPTO Application #: 20060122371Title: Process for preparing cardiodilatin fragments; highly purified cardiodilatin fragments and intermediate products for the preparation of same Abstract: The invention relates to a process for the preparation of cardiodilatin fragments, to highly purified cardiodilatin fragments, and to appropriate intermediates for the preparation of said fragments. Furthermore, the invention relates to highly purified cardiodilatin fragments which are free of peptide impurities and exhibit a single migration peak in capillary electrophoresis, as well as to appropriate processes for the preparation of same. (end of abstract) Agent: Arent Fox PLLC - Washington, DC, US Inventors: Hansueli Immer, Wolf-Georg Forssmann, Knut Adermann, Christian Klessen USPTO Applicaton #: 20060122371 - Class: 530327000 (USPTO) Related Patent Categories: Chemistry: Natural Resins Or Derivatives; Peptides Or Proteins; Lignins Or Reaction Products Thereof, Peptides Of 3 To 100 Amino Acid Residues, 11 To 14 Amino Acid Residues In Defined Sequence The Patent Description & Claims data below is from USPTO Patent Application 20060122371. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The invention relates to a process for the preparation of cardiodilatin fragments, to highly purified cardiodilatin fragments, and to appropriate intermediates for the preparation of said fragments. [0002] The present invention is directed to a process for the preparation of cardiodilatin fragments of formula I R.sup.1-ANP(105-121)-R.sup.2 (I), [0003] having a chain length of 17-37 amino acids in total, wherein ANP(105-121) represents the amino acid sequence [SEQ ID NO. 1], [0004] R.sup.1 represents an amino acid chain of sequence ANP(90-104) [SEQ ID NO. 2] or fragments thereof having a chain length of 0-15 amino acids, and [0005] R.sup.2 represents an amino acid chain of sequence ANP(122-126) [SEQ ID NO. 3] or fragments thereof having a chain length of 0-5 amino acids, [0006] wherein synthesis is effected via condensation of at least three partial fragments, and condensation of the partial fragments to give the cardiodilatin fragments of formula I is carried out between the amino acid positions Gly.sup.108 and Arg.sup.109 and the amino acid positions Gly.sup.120 and Cys.sup.121. [0007] Cardiodilatin is a peptide of the class of natriuretic peptides. These peptides play an important role in regulating the balance of salts and water in the body. The prototype of natriuretic hormones is cardiodilatin, also referred to in literature as atrial natriuretic peptide (CDD/ANP). The isolation of cardiodilatin and the preparation of biologically active fragments of cardiodilatin are known from U.S. Pat. No. 4,751,284 (cf., W. G. Forssmann et al., Klin. Wochenschr. 1986, 64 (Suppl. VI), 4-12). A review on isolation and characterization of cardiodilatin and fragments thereof, as well as their physiological properties has been published in Eur. J. Clin. Invest. 1986, 16; 439-451 (W. G. Forssmann). From EP 0,349,545, a specific cardiodilatin fragment having a chain length of 32 amino acids is known. Meanwhile, this fragment is also referred to in literature as urodilatin (INN: ularitide). Furthermore, U.S. Pat. No. 5,354,900 (Suntory) describes a biologically active fragment having a chain length of 28 amino acids, known as .alpha.-hANP. Further biologically active cardiodilatin fragments or derivatives thereof have been described in EP 0,180,615. Therein, in particular, cardiodilatin fragments are described which begin with the amino acid position Arg.sup.102 at the N-terminus and end with the amino acid position Arg.sup.125 or Arg.sup.126 at the C-terminus. Instead of the designation cardiodilatin, the literature frequently uses the designation "atrial natriuretic peptide" (ANP). In the numbering of the sequences of the cardiodilatin amino acids used in the following, reference is made to the nomenclature used for the ANF/CDD (1-126) peptide (=ANP) in EP 0,349,545. [0008] A common structural feature of all hitherto known biologically active cardiodilatin fragments is the formation of a disulfide bridge between the amino acids Cys.sup.105 and Cys.sup.121, resulting in a stable ring of 17 amino acids. It is believed that the formation of this ring is substantially responsible for the biological activity of the cardiodilatin derivatives. At position Cys.sup.105, the cardiodilatin fragments are substituted by an amino acid chain R.sup.1 having a chain length of 0-15 amino acids, and at position Cys.sup.121 by a chain R.sup.2 having a chain length of 0-5 amino acids. In the [SEQ ID NO. 1], the central region ANP(105-121) is presented in linearized form. [0009] The cardiodilatin fragment ANP(95-126), with the INN designation ularitide, is a particularly stable and biologically active human peptide, having diuretic activity and a relaxing effect on the smooth vascular muscles, which is formed of 32 amino acids and has the following sequence, wherein both the cysteine amino acids at positions 11 and 27 in the peptide are forming a disulfide bridge: [0010] Urodilatin is found in human urine. EP 0,349,545 describes a process for recovering urodilatin from urine using alginic acid, wherein the peptides adsorbed to alginic acid are eluted, the eluate is fractionated according to conventional purification methods, and the active fraction is recovered using a test based on the examination of the relaxing effect of urodilatin on the smooth muscles. [0011] Furthermore, EP 0,349,545 describes a stepwise chemical synthesis of urodilatin using the Merrifield process (J. Am. Chem. Soc. 1963, 85; 2149-2156), at a solid phase according to the ABI standard program following the Boc strategy. In addition, this patent specification describes the preparation of urodilatin from the partial fragment ANP(99-126). This fragment is bound to a solid phase, and is reacted with a second partial fragment, the tetrapeptide Boc-Thr(But)-Ala-Pro-Arg(Tos). The peptide ANP(95-126) obtained from the condensation is removed from the support, subjected to cyclization after removal of the protecting groups and subsequently, is processed and purified in a per se known manner. [0012] Similarly, EP 0,180,615 describes the chemical synthesis using a solid support, wherein formation of the cardiodilatin fragments described therein is effected successively, starting from the C-terminus in direction of the N-terminus. Here, condensation via partial fragments is not described. [0013] However, the cardiodilatin fragments prepared according to the procedures described in literature did not have the purity necessary for clinical studies and for the authorization as medicinal product because, due to the synthesis, peptide impurities had been introduced into the final product which could not be removed even by subsequent purification processes. Due to their immunogenic properties, the impurities may give rise to undesirable side-effects when administered to the patient, so that therapeutic application involved risk. Moreover, the synthesis could be accomplished at only a small scale under reasonable technical input and was not economically suitable for a larger production scale. Furthermore, another drawback of known processes for synthesis was the existing potential risk of racemization due to which the urodilatin was obtained with lower purity, lower biological activity and in insufficient yield. Racemization of the product which frequently occurs with existing syntheses often resulted in insufficient optical purity of the final product, and these impurities frequently cannot be removed or only with exceedingly high technical input. [0014] Thus, it is an object of the invention to develop an improved process for the chemical synthesis of cardiodilatin fragments which does not involve the above-mentioned drawbacks. [0015] The object of the invention is attained by performing the synthesis of cardiodilatin fragments on the basis of the Merrifield process using a specific selection of peptide fragments. [0016] Surprisingly, the course of synthesis has been found to be optimal when the cardiodilatin fragments are formed using three partial fragments, with the condensation of the partial fragments to give the cardiodilatin fragment of formula I being performed in such fashion that the formation is effected via condensation of partial fragments and bond formation between the amino acid positions Gly.sup.108 and Arg.sup.109 and the amino acid positions Gly.sup.120 and Cys.sup.121. This process is advantageous in that the cardiodilatin fragments of formula I can be obtained in higher yields and in higher purity as compared to the synthetic processes known from prior art. [0017] The synthesis of the cardiodilatin fragments of formula I is effected in such way that initially, the three partial fragments having the sequences R.sup.1-ANP(105-108), ANP(109-120) and ANP(121)-R.sup.2 are prepared according to the Merrifield process. Then, preferably, condensation of the three partial fragments to give the cardiodilatin fragment of formula I is effected in two partial steps, whereby in a first step, condensation between the amino acid positions Gly.sup.120 and Cys.sup.121 of the partial fragments ANP(109-120) and Cys.sup.121-R.sup.2 is effected, with the intermediate fragment ANP(109-121)-R.sup.2 being formed. Then, in a subsequent second step, condensation of the thus obtained fragment ANP(109-121)-R.sup.2 with the third partial fragment R.sup.1-ANP(105-108) is effected, forming the desired cardiodilatin fragment of formula I. Using the process according to the invention, the yield of cardiodilatin fragments is between 15 and 20%, based on the amount of each cardiodilatin partial fragment used as starting material. [0018] The three partial fragments having the sequences R.sup.1-ANP(105-108), ANP(109-120) and ANP(121)-R.sup.2 are prepared according to the Merrifield process, wherein the amino acids with functional groups (hydroxy, carboxy, amino, or mercapto groups) present in the sequence are substituted by appropriate protecting groups. For example, as suitable protecting groups the following groups are possible: [0019] protecting groups for hydroxy groups: Boc (t-butyloxycarbonyl), tBu (t-butyl ether); [0020] protecting groups for amino functions: Fmoc (9-fluorenylmethoxycarbonyl), Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl), Trt (trityl); [0021] protecting groups for carboxy groups: OtBu (t-butyl ester); [0022] protecting groups for mercapto groups: Acm (acetamidomethyl) or Trt. [0023] Here, the following protecting groups are preferred for the following amino acids: tBu for the amino acids Thr, Asn, Tyr or Ser; Pbf or Pmc for the amino acid Arg; Acm for the amino acid Cys; OtBu for the amino acid Asp; Trt for the amino acids Gln, Asn or Cys. [0024] Using the Fmoc strategy (B. Riniker et al., Tetrahedron 1993, 49; 9307-9320), the protected partial fragments ANP(109-120), R.sup.1-ANP(105-108) and ANP(121)-R.sup.2 are formed on a solid support material. All the materials generally used in the Merrifield synthesis may serve as solid support materials. Preferred as support material is polystyrene functionalized as aminomethyl or benzhydrylamino compound. The superacid-sensitive bonding of the peptide fragments to the resin by means of the 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid linker allows their removal without impeding the side-chain protection. The fragments are purified by digestion with various solvents. Thus, the three starting fragments ANP(109-120), R.sup.1-ANP(105-108) and ANP(121)-R.sup.2 are obtained with a C-terminal free carboxyl group and in good purity. When forming the peptides on the support resin, the yield in every single step of addition of one amino acid is nearly quantitative and is about 97-99% [0025] The flow diagram in FIG. 1 illustrates the principle of synthesis, with urodilatin ANP(95-126) as an example. Here, condensation of the fragment Boc-1-14-OH (1) [this nomenclature corresponds to the general designation of fragment R.sup.1-ANP(105-108), wherein R.sup.1=ANP(95-104)] with the fragment H-15-32-OtBu (5) [corresponding to an ANP nomenclature of ANP(109-121)-R.sup.2, wherein R.sup.2=ANP(122-126)] is effected. This fragment (5) is synthesized from the fragments Fmoc-15-26-OH (2) [corresponding to an ANP nomenclature of ANP(109-120)] and H-27-32-OtBu (3c) [corresponding to an ANP nomenclature of ANP(121)-R.sup.2]. FIG. 2 represents the fragments synthesized and modified with protecting groups. [0026] In the next step, the carboxyl group of fragment (3a) is converted to the t-butyl ester (3b) (cf., Riniker et al., 22nd Europ. Peptide Symposium Interlaken, September 1992 (L7)). Subsequent removal of the Fmoc group from fragment (3b) leads to the product (3c). This is fused with fragment (2), resulting in fragment (4). Removal of the Fmoc protecting group and condensation of the obtained fragment (5) with fragment (1) leads to the fully protected urodilatin (6). Removal of the protecting groups by treatment with trifluoroacetic acid and 1,3-propanedithiol as a scavenger provides the linear peptide (7) which is cyclized to crude urodilatin (8) by oxidation with iodine solution. This is desalted, purified and may be lyophilized subsequently. The synthesis of other cardiodilatin fragments is conducted in an analogous fashion. Continue reading... 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