EP0939770A1 - Peptide natriuretique cerebral (bnp) specifique pour un recepteur - Google Patents

Peptide natriuretique cerebral (bnp) specifique pour un recepteur

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Publication number
EP0939770A1
EP0939770A1 EP97910899A EP97910899A EP0939770A1 EP 0939770 A1 EP0939770 A1 EP 0939770A1 EP 97910899 A EP97910899 A EP 97910899A EP 97910899 A EP97910899 A EP 97910899A EP 0939770 A1 EP0939770 A1 EP 0939770A1
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EP
European Patent Office
Prior art keywords
arg
ser
gly
lys
seq
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EP97910899A
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German (de)
English (en)
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David G. Lowe
Jill R. Schoenfeld
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Genentech Inc
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Genentech Inc
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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • 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
    • A61P7/10—Antioedematous agents; Diuretics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • 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 compounds having natriuretic, diuretic, and/or vasodilatory activity in mammals
  • the compounds of this invention are related to B-type natriuretic peptide (BNP)
  • BNP B-type natriuretic peptide
  • the compounds exhibit decreased binding affinity to the natriuretic peptide clearance receptor (NPR-C) compared to wild-type BNP or the wild-type type A natriuretic peptide (ANP) and advantageously have enhanced potency
  • NPR-C natriuretic peptide clearance receptor
  • ANP wild-type type A natriuretic peptide
  • the invention also relates to pharmaceutical compositions comprising the novel compounds as well as their use in diagnostic, therapeutic, and prophylactic methods Description of Related Disclosures Three natriuretic peptides, type A, type B and type C, have been identified which play a role in hemodynamics (Rosenzweig, A , and Seid
  • Both ANP and BNP are primarily cardiac hormones that are released by the heart in its role as an endocrine organ regulating fluid and electrolyte hemostasis
  • Human BNP is stored in the atrium and ventricle (Hosodo et al , (1991 ) Hypertension 17 1 152-1 156) as the mature hormone whereas ANP is stored in cardiocytes as the precursor BNP was first isolated from porcine brain (Sudoh et al , (1988) Nature 332 78- 81) although it is synthesized m and secreted into the circulation from the porcine heart (Aburaya et al , (1989) Biochem Biophys Res Commun 158 360-368) Porcine BNP circulates as a 26 or 32 amino acid polypeptide and exhibits structural homology with ANP (Sudoh et al , (1988) Nature 332 78-81) The cDNA sequence encoding human BNP precursor, hBNP[l-108] has been determined (S
  • natriuresis sodium (natriuresis) and water (diuresis) by the kidneys
  • GFR glomerular filtration rate
  • Sodium reabsorption is regulated, in part, by the adrenal steroid hormone aldosterone, in part by blood pressure, hematoc ⁇ t and plasma viscosity and in part by the various natriuretic factors or hormones (deBold, A J et al , (1981) Lilt Sciences 28 89-94, Garcia R , (1982) Expe ⁇ entia 38 1071-73, Currie, M S et al , (1983) Science 221 71 -73, Flynr T G et al , (1983) Biochem Biophys Res Commun 1 17 8
  • the present invention provides compounds which have natriuretic, diuretic, and/or vasodilatory activity in mammals
  • the compounds of this invention are related to the B-type natriuretic peptide (BNP) and exhibit decreased binding affinity to the natriuretic peptide clearance receptor (NPR-C) compared to BNP
  • BNP B-type natriuretic peptide
  • NPR-C natriuretic peptide clearance receptor
  • the compounds and related compositions allow for a potent NPR-A receptor stimulation providing for low dose pharmaceutical formulations
  • the compositions of the present invention are useful in therapeutic and prophylactic methods for inducing natruresis, diuresis or vasodilation or associated processes
  • the compounds of the present invention have a decreased binding affinity for the human clearance receptor (hNPR-C) compared to wild-type BNP
  • the compounds exhibit the same or an increased affinity for the type- A natriuretic peptide receptor
  • at least one of amino acid residues Xaal9, Xaa23, Xaa24 or Xaa25 of wild-type BNP is selected according to the following scheme in order to produce a BNP variant or related compound of the invention
  • Xaal9 is selected from the group consisting of Ser. Arg, Ala. Asn, Gly and conservative substitutions thereof;
  • Xaa23 is selected from the group Gly, Met, Phe, Leu and conservative substitutions thereof;
  • Xaa24 is selected from the group Tip, Tyr, Phe, and conservative substitutions thereof; and Xaa25 is selected from the group Gly, Arg, and conservative substitutions thereof.
  • A is selected from the group consisting of H, C j -C ⁇ alkanoyl and Thr-Ala-Pro-Arg (SEQ ID NO: 30);
  • is absent or selected from the group consisting of a peptide from between 1 and 10 amino acids, Gly-Ser-Gly-, Val-Gln-Gly-Ser-Gly (SEQ ID NO: 31 ) and Ser-Pro-Lys-Met- Val-Gln-Gly-Ser-Gly (SEQ ID NO: 32);
  • Xaa j c is selected from the group consisting of Arg, Ala, Asn, Gly and Ser;
  • Xaa23 is selected from the group Gly, Met, Phe, Leu;
  • Xaa2 is selected from the group Tip, Tyr, Phe; ⁇ aa 25 ' s se ' ecte d fr° m tne group Gly, and Arg;
  • R2 is selected from the group consisting of a 1 to 6 amino acid peptide and Lys-Val-Leu-Arg-Arg-His- (SEQ ID NO: 33);
  • the composition of the present invention is a polypeptide and the invention encompasses a composition of matter comprising a nucleic acid, preferably DNA, encoding the polypeptide of the invention.
  • the invention further comprises an expression control sequence operably linked to the DNA molecule, an expression vector, preferably a plasmid, comprising the DNA molecule, where the control sequence is recognized by a host cell transformed with the vector, and a host cell transformed with the vector.
  • the polypeptide compositions of the present invention may be made by a process which includes the steps of synthesizing nucleic acid sequences encoding any of the amino acid sequences of the invention, ligating the nucleic acid sequence into a suitable expression vector capable of expressing the nucleic acid sequence in a suitable host, transforming the host with the expression vector into which the nucleic acid sequence has been ligated, and culturing the host under conditions suitable for expression of the nucleic acid sequence, whereby the protein encoded by the selected nucleic acid sequence is expressed by the host.
  • the polypeptide is then recovered from the host cell culture.
  • the ligating step may further contemplate ligating the nucleic acid into a suitable expression vector such that the nucleic acid is operably linked to a suitable secretory signal, whereby the amino acid sequence is secreted by the host.
  • the present invention further extends to therapeutic applications for the compounds described herein.
  • the invention includes a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the compound of the invention.
  • Pharmaceutical compositions comprising these compounds can be used in the treatment or prophylaxis of various pathological conditions associated with water or electrolyte imbalance and hypertension, especially renovascular hypertension.
  • pathological conditions include, for example, congestive heart failure (CHF), nephrotic syndrome and hepatic cirrhosis, pulmonary disease, and renal failure due to ineffective renal perfusion or reduced glomerular filtration rate.
  • CHF congestive heart failure
  • nephrotic syndrome and hepatic cirrhosis pulmonary disease
  • renal failure due to ineffective renal perfusion or reduced glomerular filtration rate.
  • the results show that the peptides are full agonists on this receptor, with potency similar to BNP.
  • Figure 2 Stimulation of cGMP second messenger production on human natriuretic peptide receptor-A expressed in 293 cells. cGMP values for natriuretic peptide stimulation of natriuretic peptide receptor-A at o
  • ANP refers to the 28 amino acid residue peptide reported by Kangawa et al., Biochem. Biophys. Res. Comm. (1984) 118(1): 131-139; BNP refers to SEQ ID NO: 1 ; BNP02 refers to SEQ ID NO: 2; BNP04 refers to SEQ ID NO: 7; BNP05 refers to SEQ ID NO: 7; ANF 131 refers to SEQ ID NO: 50; BNP06 refers to SEQ ID NO: 48; and BNP07 refers to SEQ ID NO: 49.
  • the results show that the peptides are full agonists on this receptor, with potency similar to BNP.
  • Figure 3 Alignment of the amino acid sequence of BNP from mammalian species; human (Sudoh, et al., (1989) Biochem Biophy. Res. Commun. 159: 1427-1434) (SEQ ID NO 1), canine (Seilhamer et al., (1989) Biochem. Biophys. Res. Commun 165:650-658) (SEQ ID NO 44), porcine (Sudoh, T., et al., (1988) Nature 332:78-81) (SEQ ID NO 45), rat (Kojima et al., (1989) Biophys. Biochem. Res. Commun.
  • amino acid or amino acid residue refers to naturally occurring L amino acids or to D amino acids as described further below with respect to variants.
  • the commonly used one- and three- letter abbreviations for amino acids are used herein (Bruce Alberts et al., Molecular Biology of the Cell, Garland Publishing, Inc., New York (3d ed. 1994)).
  • BNP type-B natriuretic peptide
  • wild-type BNP wild-type BNP
  • sequence of BNP from mammalian species is known, for example, human (Sudoh, et al., (1989) Biochem Biophy. Res. Commun. 159:1427-1434) (SEQ ID NO 1), canine (Seilhamer et al., (1989) Biochem. Biophys. Res.
  • human BNP means the 32 amino acid residue peptide reported by Sudoh, T., et al., (1988) Nature 332:78-81 having the following primary amino acid sequence: 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 Ser-Pro-Lys-Met- Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Glu- 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 1)
  • residue position number is used in conjunction with the three letter amino acid nomenclature to designate the residue at which a substitution is made in the BNP related compounds of this invention.
  • the nomenclature "BNP Serl9Arg” or the like is used in a BNP related compound in which arginine (Arg) replaces serine (Ser) at residue position number 19 of wild-type BNP.
  • BNP Serl9Arg or the like is used in a BNP related compound in which arginine (Arg) replaces serine (Ser) at residue position number 19 of wild-type BNP.
  • Multiple substitutions are designated in the same manner with a comma separating each substitution.
  • BNP variant and "BNP related compound” are defined as a compound having at least qualitative biological activity in common with the polypeptide of SEQ ID NO: 1 and having at least one amino acid substitution as defined herein for conferring receptor specificity. Preferred amino acid substitutions are outlined in Table I of Example I.
  • the qualitating biological activity referred to is the capacity to stimulate cyclic GMP production in cells expressing the guanylate cyclase-linked natriuretic peptide receptor NPR-A.
  • the NPR-A receptor amino acid sequence deduced from cDNAs encoding the receptor is known (Chinkers et al., (1989) Nature 338:78-83; Lowe et al. (1989) EMBO J.
  • NPR-A receptor cDNA Functional expression of the NPR-A receptor cDNA in cultured mammalian cells which confers both ANP and BNP binding and guanylate cyclase activity is also known (Chinkers, et al., (1989) Nature 338:78-83). Assay systems for measurement of binding affinities for NPR receptors as well as cGMP accumulation are known (see, International Application No. PCT/US94/12591). Additionally, in preferred embodiments, the BNP variant or BNP related compound of the present invention is believed to compete with any wild-type ANP (Kangawa et al., (1984) Biochem. Biophys. Res. Commun.
  • qualitating biological activity may be defined as the ability to compete with any wild-type BNP or ANP for binding to NPR-A receptor thereby inducing cGMP accumulation
  • the term "compete” and "ability to compete” are relative terms
  • the terms, when used to describe the activity of the BNP variant mean a BNP variant that when added in a 10-fold molar excess to wild-type BNP or wild-type ANP in a standard receptor binding assay produces at least a 50% inhibition of binding of the wild-type BNP or wild-type ANP
  • the BNP variant will produce at least a 50% inhibition of binding in a 5-fold molar excess and most preferably at least a 2-fold molar excess
  • a most preferred BNP variant or BNP related compound of the present invention will produce at least a 50% inhibition of binding when present a l
  • a characteristic of the BNP related compound or BNP variant of the present invention is a decreased affinity for the natriuretic clearance receptor NPR-C Therefore variants or compounds of the present invention have at least quali t ative biological activity in common with wild-type BNP but have a decreased affinity for the clearance receptor NPR-C when compared to BNP in a standard receptor binding assay as described herein (see Bennett et al , (l 991 ) J Biol Chem 266 23060-23067)
  • the BNP variants and compounds of the present invention are, in general, homologous ammo acid sequences of the rat, porcine, canine or other mammalian BNP's or homologous amino acid sequences of the sequence of SEQ ID NO 1 includmg homologous in vitro generated variants having the qualitative biological activity defined above
  • Homology with respect to the BNP variants of the present invention is defined as the percentage of ammo acid residues in a candidate sequence that are identical with either the amino acid residues in SEQ ID NO 1, the ammo acid sequence of a mammalian BNP or a composite sequence as defined herein after aligning the sequences and introducing gaps if necessary to achieve the maximum identity (Figure 3)
  • No N- or C- terminal extension or deletion in the candidate sequence shall be construed as reducing identity
  • Composite ammo acid within the present invention refers to an alternate ammo acid having the same position in the 32 amino acid residue structure as human BNP from other mammalian vertebrate species Therefore,
  • the invention contemplates a BNP variant having at least the qualitative biological activity as defined above and having, for example, at least about 75% ammo acid homology with the polypeptide of SEQ ID NO 1 or the polypeptide of SEQ ID NO 1 lacking the 5 carboxyl terminal ammo acid residues and/or the 9 amino terminal residues
  • the BNP variant amino acid sequence preferably will share at least 80%, more preferably, greater than 85% sequence homology with the sequence of SEQ ID NO 1
  • a BNP variant or related compound may exhibit less than 50% sequence homology with the sequence of SEQ ID NO 1 and still retain the characteristics of the BNP variant or BNP related compound as defined above
  • Included m the definition of BNP variant or BNP related compound of the present mvention are ammo acid sequence variants of the SEQ ID NO 1 wherein an ammo acid m addition to those described herein which confer receptor specificity has been substituted by another residue, including predetermined mutations (e g site directed PCR mutagenesis), other composite amino acid substitutions
  • amino acid sequence variants can be generated by substitutions which utilize the D rather than L form of an ammo acid This is especially useful in stabilizing the BNP variant of the present invention against enzymatic degradation
  • stabilizing amino acid substitutions have been described for ANP (Nutt and Veber, (1987) Endoc ⁇ n and Metab Clin N Amer 16(1) 19-41 )
  • ANP analogues includedmg the introduction of D-amino acids where appropriate can be conducted for BNP and are included the definition of BNP variant or BNP related compound
  • the term variant is meant to include, in addition to those substitutions described herein for receptor specificity, substitutions that confer increased stability of BNP molecules such as chiral ammo acid replacements which may offer resistance to metabolic degradation
  • the present invention is meant to include BNP variants and related compounds that have decreased susceptibility to hydrolysis by neutral endopeptidase 24 1 1 (EC3 4 24 1 1) (NEP) and angiotensin converting enzyme (ACE) as well as improved potency or duration of action
  • BNP variants wherein an ammo acid has been added to or deleted from the N-terminal or C-terminal 17 member ring structure of wild- type BNP
  • Ammo acid sequence insertions include carboxyl-terminal fusions ranging in length from one residue to polypeptides contaming a hundred or more residues mcludmg pre and pro ammo acid sequences as described (see for example Ogawa et al , (1994) J Clin Invest 93 191 1-1921)
  • Other examples of terminal insertions include BNP variants with a composite N-terminal or C-terminal sequence from another mammalian species or from another natriuretic peptide (see Figure 3) Such amino terminal substitutions have been described (Shimekake et al , (1992) FEBS, 309(2) 185-189) Suitable N-terminal sequences are described in International Publication No WO 89/12060 and include Gly, Ser Gly, Asp/
  • Exemplary C-terminal insertions are described in International Publication No 89/12069 and mcluded (OH), NH, orNR'R" where R' and R" are independently H, lower alkyl, or Asn/Lys, Asn/Lys-Val, Asn/Lys- Val-Leu, Asn/Lys- Val-Leu-Arg (SEQ ID NO 40), Asn/Lys- Val-Leu-Arg-Arg/Lys (SEQ ID NO 41 ), Asn/Lys- Val-Leu-Arg-Arg/Lys-Tyr-His (SEQ ID NO 42), or the amides (NH or NR'R”) thereof
  • amino acid substitution variants have at least one amino acid residue in addition to those described herem for conferring receptor specificity m the BNP variant molecule removed and a different residue inserted m its place
  • the sites for substitutional mutagenesis include sites where ammo acids found in the BNP variant from various species are substantially different in terms of side chain bulk, charge and or hydrophobicity These ammo acids are substituted with the exemplary conservative substitutions as described herein below including the exemplary non-naturally occurring amino acids
  • the residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous solution
  • the residue has a positive charge due to association with H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH Neutral/non-polar
  • the residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contamed when the peptide is in aqueous medium
  • Ammo acid residues can be further classified as cyclic or noncyclic, aromatic or non aromatic with respect to their side chain groups these designations being commonplace to the skilled artisan Original Exemplary Conservative Preferred Conservative
  • Peptides synthesized by the standard solid phase synthesis techniques described here, for example, are not limited to amino acids encoded by genes for substitutions involving the amino acids. Commonly encountered amino acids which are not encoded by the genetic code, include, for example, those described in International Publication No.
  • WO 90/01940 as well as 2-amino adipic acid (Aad) for Glu and Asp; 2- aminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric (Abu) acid for Met, Leu, and other aliphatic amino acids; 2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids; 2-aminoisobutyric acid (Aib) for Gly; cyclohexylalanine (Cha) for Val, and Leu and He; homoarginine (Har) for Arg and Lys; 2,3- diaminopropionic acid (Dpr) for Lys, Arg and His; N-ethylglycine (EtGly) for Gly, Pro, and Ala; N- ethylglycine (EtGly) for Gly, Pro, and Ala; N-ethylasparigine (EtAsn) for Asn, and
  • Leu, and Val p-amidinophenylalanine for Ala; N-methylglycine (MeGly, sarcosine) for Gly, Pro, and Ala; N-methylisoleucine (Melle) for He; Norvaline (Nva) for Met and other aliphatic amino acids; Norleucine (Nle) for Met and other aliphatic amino acids; Ornithine (Orn) for Lys, Arg and His; Citrulline (Cit) and methionine sulfoxide (MSO) for Thr, Asn and Gin; N-methylphenylalanine (MePhe), trimethylphenylalanine, halo (F, Cl, Br, and I)phenylalanine, triflourylphenylalanine, for Phe.
  • a useful method for identification of certain residues or regions of the BNP variant for amino acid substitution other than those described herein for receptor specificity is called alanine scanning mutagenesis as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • a residue or group of target residues are identified (e.g. charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
  • Those domains demonstrating functional sensitivity to the substitution then are refined by introducing further or other variations at or for the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined the nature of the mutation per se need not be predetermined.
  • insertional BNP variants or BNP related compounds include the fusion to the N- or C- terminus of the BNP molecule of immunogenic polypeptides, e.g., bacterial polypeptides such as beta lactamase or an enzyme encoded by E coli Tip locus or yeast protein, and C-terminal fusion with proteins having a long half- life such a.', immunoglobulin constant region or other immunoglobulin regions, albumin, or ferritin as described in WO 89/02922 published 6 April 1989.
  • immunogenic polypeptides e.g., bacterial polypeptides such as beta lactamase or an enzyme encoded by E coli Tip locus or yeast protein
  • C-terminal fusion with proteins having a long half- life such a.', immunoglobulin constant region or other immunoglobulin regions, albumin, or ferritin as described in WO 89/02922 published 6 April 1989.
  • C j -C ⁇ alkyl when used to describe a BNP variant means a branched, unbranched or cyclic, saturated aliphatic hydrocarbon radical, having the number of carbon atoms specified.
  • Representative examples of these alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n- pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 2,2-dimethylbutyl, cycolhexyl and the like.
  • the terms “lower alkyl” and"C j -Cgalkyl” are synonymous and used interchangeably.
  • a preferred "C, - Cgalkyl” group is methyl.
  • C j -C ⁇ alkanoyl when used to describe a BNP variant encompasses groups such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, caproyl, and the like.
  • C ⁇ -C ⁇ aryl when used to describe a BNP variant means a homocyclic hydrocarbon aromatic radical, whether or not fused, having the number of carbon atoms designated.
  • Preferred aryl groups include phenyl, napthyl, biphenyl, phenanthrenyl, naphthacenyl, and the like (see e.g. Lang's Handbook of Chemistry (Dean, J. A., ed) 13 th ed. Table 7-2 [1985]).
  • BNP variant means one, two, or three aryl groups having the number of carbon atoms designated, appended to an alkyl radical having the number of carbon atoms designated including but not limited to; benzyl, napthylmethyl, phenethyl, benzyhydryl
  • a preferred arylalkyl group is the benzyl group.
  • “Pharmaceutically acceptable salts” include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicyclic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid, propionic acid, glycolic acid, pyru
  • “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts.
  • Salts derived from pharmaceutically acceptable organic nontoxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine resins and the like.
  • Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline, and caffeine.
  • the BNP variants and related compounds of this invention have at least one amino acid of wild-type BNP substituted as described herein for conferring NPR-A receptor specificity. Therefore, preferred compounds of this invention are synthetic hormone agonists of the human natriuretic peptide receptor-A (hNPR-A). This receptor is found in kidney, smooth muscle, adrenal and other tissues (Napier M.A. et al, (1984) Proc. Nat. Acad. Sci. USA, 81 :5946-5950; Hori et al., (1985) Biochem. Biophys. Res. Commun. 129:773-779; Quirion et al., (1986) Proc. Natl. Acad. Sci.
  • hNPR-A human natriuretic peptide receptor-A
  • hNPR-A contains an intracellular guanyl cyclase domain.
  • the action of hNPR-A is mediated by hydrolysis of GTP to produce the second messenger cGMP. Accordingly, preferred compounds of this invention stimulate hNPR-A to produce cGMP to at least the same extent as wild-type BNP.
  • the preferred compounds of this invention do not however bind to the human natriuretic peptide clearance receptor-C (hNPR-C) to the same extent as wild-type BNP.
  • hNPR-C human natriuretic peptide clearance receptor-C
  • Both in vitro and m vivo systems can be used to identify the compounds of the present invention. Since binding of BNP to the NPR-A cellular receptor is presumptively a prerequisite to qualitative biological activity the BNP related compounds of the present invention are identified by screening for binding of the candidate compound to the NPR-A and NPR-C receptors.
  • Primary screens for the compounds of the present invention therefore include vasorelaxant assays in rat and rabbit aorta tissues, relaxation of chick rectum intestinal smooth muscle tissue, natriuresis in rats, as well as the receptor specific binding assays and cGMP formation in cell culture assays described herein above and in the following Example sections.
  • assays have been developed for the screening of BNP which evaluate the ability of the BNP to compete with a labeled native BNP for binding to intact cultured cells or membrane preparations derived from cultured cells expressing the cellular receptors.
  • NPR-A expressing 293 cells produced as described by Lowe and Fendly (1992) J. Biol. Chem. 267:21691-21697 may be utilized.
  • the cDNA for NPR-C (Lowe et al., (1990) Nucleic acids Res. 18:3412) may be transfected into suitable host cells for continuous expression.
  • radiolabeled or florescent labeled BNP is incubated with immobilized NPR-A or NPR-C receptor-immunoglobulin chimeras in varying concentration of unlabeled candidate compound.
  • concentrations of successful candidate compound effectively prevent binding of labeled BNP to immobilized receptor chimeras.
  • the concentration of unlabeled peptide at which 50% maximal BNP is displaced is referred to as the EC50 and reflects the receptor binding affinity. Therefore a candidate compound with an EC50 of lOOnM displays a substantially weaker interaction with a receptor than peptide with an EC50 of 10 nM.
  • BNP related compounds or variants within the present invention have a decreased binding affinity for NPR-C and corresponding NPR-A receptor specificity. Therefore, according to the present invention a BNP variant with an EC50 greater than wild type BNP for the NPR-C is said to display a weaker affinity for the NPR-C receptor and therefore a decreased affinity for the clearance receptor.
  • receptor specificity is reflected in the ratio of binding affinity of the candidate compound for NPR-A and NPR-C.
  • a change in the ratio of binding affinity for the candidate compound to NPR-A compared to NPR-C is referred to as a change in receptor specificity.
  • it is a characteristic of the BNP variant or BNP related compound to exhibit a decrease in the ratio of binding affinity for NPR-A compared to NPR-C as compared to wild-type BNP in the same assay.
  • Preferred compounds and variants according to this aspect of the invention exhibit at least a 10 fold decrease in the ratio of the binding affinities compared to wild-type BNP and more preferably greater that a 100 fold decrease in the ratio.
  • substitutions can occur individually or in combination.
  • Preferred among the individual substitutions conferring receptor specificity is a BNP variant or related compound which has a substitution for amino acid residue 19 of wild-type BNP as provided in the preceding paragraph.
  • Preferred among combination substitutions conferring receptor specificity is a BNP variant or related compound where each of amino acids 23, 24, and 25 of wild type BNP are substituted according to the scheme presented in the preceding paragraph.
  • Such preferred combination substitutions may be accompanied by a substitution of residue 19 according to the scheme provided in the preceding paragraph.
  • Exemplary peptides are given below:
  • hBNP Arg 13 Ser, Lysl4Pro, Ser 19 Arg (SEQ ID NO: 1 1) hBNP LysHThr, Serl9Arg, Ser21Gln; (SEQ ID NO: 12) hBNP Lysl4Asn, Serl9Arg, Ser20His, Ser21Leu; (SEQ ID NO: 13) hBNP Lys l4Asn, Serl9Ala, Ser20Arg, Ser21Glu; (SEQ ID NO: 14) hBNP Lysl4Asn, Serl9Arg, Ser20Lys, Ser21Thr; (SEQ ID NO: 15) hBNP Lys l4Asn, Serl9Arg, Ser20Gly; (SEQ ID NO: 16) hBNP Lysl4Asn, Serl9Gly, Ser20Arg, Ser21Ala; (SEQ ID NO: 17) hBNP Lysl4Asn, Serl9Arg, Serl9Arg,
  • One method of producing compounds of the present invention involves chemical synthesis of the polypeptide, followed by treatment under oxidizing conditions appropriate to obtain the native conformation, that is, the correct disulfide bond linkages This can be accomplished usmg methodologies well known to those skilled in the art (see Atherton, E , and Sheppard R C , Solid Phase Peptide Synthesis A Practical Approach, IRL Press, Oxford England (1989), Kelley, R F & Winkler, M E in Genetic Engineering Principles and Methods, Setlow, J K , ed , Plenum Press, N Y , vol 12, pp 1-19 (1990), Stewart, J M & Young, J D Solid Phase Peptide Synthesis, Pierce Chemical Co Rockford, IL(1984))
  • Polypeptides of the invention may be conveniently prepared using solid phase peptide synthesis (Mer ⁇ field, (1964) J Am Chem Soc , 85 2149, Houghten, (1985) Proc Natl Acad Sci USA 82 5132)
  • Solid phase synthesis begins at the carboxy-terminus of the putative peptide by coupling a protected ammo acid to a suitable resin (e g chloromethylated polystyrene resin) as shown in Figures 1-1 and 1-2, on pages 2 and 4 of Stewart and Young supra
  • a suitable resin e g chloromethylated polystyrene resin
  • a suitable resin e g chloromethylated polystyrene resin
  • the next ⁇ -ammo- and side-chain protec t ed ammo acid in the synthesis is added
  • the remaining ⁇ -amino- and, if necessary, side- chain-protected amino acids are then coupled sequentially in the desired order by condensation to obtain an intermediate compound connected to the
  • condensation between two amino acids, or an ammo acid and a peptide, or a peptide and a peptide can be carried out according to the usual condensation methods such as the azide method, mixed acid anhydride method, DCC (N,N'-d ⁇ cyclohexylcarbodum ⁇ de) or DIC (N,N'-d ⁇ sopropylcarbodum ⁇ de)methods, active ester method (p-nitrophenyl ester method, BOP [benzot ⁇ azole-1-yl-oxy-t ⁇ s (dimethylammo) phosphonium hexafluorophosphate] method, N-hydroxysuccinic acid lmido ester method, etc, and Woodward reagent K method
  • Common to chemical synthesis of peptides is the protection of any reactive side-chain groups of the amino acids with suitable protecting groups Ultimately these protecting groups are removed after the desired polypeptide chain has been sequentially assembled Also common is the protection of the ⁇ -amino group on an amino acid or
  • Suitable protective groups for protecting the ⁇ -and e- amino side chain groups are exemplified by benzyloxycarbonyl (abbreviated Z), isonicotinyloxycarbonyl (iNOC), O-chlorobenzyloxycarbonyl [Z(2C1)], p-nitrobenzyloxycarbonyl p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl, (Boc), t- amyloxycarbonyl (Aoc), isoboronyloxycarbonyl, adamatyloxycarbonyl, 2-(4-b ⁇ henyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonylethoxycarbonyl (Msc), trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulphenyl (NPS), diphenylpho
  • Protective groups for the carboxy functional group are exemplified by; benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (Obut), 4-pyridylmethyl ester (OPic), and the like. It is often desirable that specific amino acids such as arginine, cysteine, and serine possessing a functional group other than amino and carboxyl groups are protected by a suitable protective group.
  • the guanidino group of arginine may be protected with nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzenesulfonyl, 4-methoxy-2, 6-dimethylbenzenesulfonyl (Mds), 1,3,5- trimethylphenysulfonyl (Mts), and the like.
  • the thiol group of cysteine may be protected with p- methoxybenzyl, friphenylmethyl, acetylaminomethyl ethylcarbamoyle, 4-methylbenzyl, 2, 4, 6-trimethy-benzyl (Tmb) etc, and the hydroxyl group of serine can be protected with benzyl, t-butyl, acetyl, tetrahydropyranyl and the like.
  • the intermediate peptide is removed from the resin support by treatment with a reagent, such as liquid HF and one or more thio-containing scavengers, which not only cleaves the peptide from the resin, but also cleaves all the remaining side-chain protecting groups.
  • a reagent such as liquid HF and one or more thio-containing scavengers, which not only cleaves the peptide from the resin, but also cleaves all the remaining side-chain protecting groups.
  • a reagent such as liquid HF and one or more thio-containing scavengers
  • a thio-cresol and cresol scavenger mixture is used.
  • the resin is washed with ether, and immediately transferred to a large volume of dilute acetic acid to solubilize and minimize intermolecular cross-linking.
  • a 250 ⁇ M polypeptide concentration is diluted in about 2 liters of 0.1 M acetic acid solution. The solution is then stirred and its pH adjusted to about 8.0 using ammonium hydroxide. Upon pH adjustment, the polypeptide takes its desired conformational arrangement.
  • the present invention encompasses a nucleic acid, preferably DNA, encodmg the protein component of a BNP variant that contain the amino acid substitutions described herein
  • the invention further comprises an expression control sequence operably linked to the DNA molecule, an expression vector, preferably a plasmid, comprising the DNA molecule, where the control sequence is recognized by a host cell transformed with the vector
  • the compounds of the present invention may be made by a process which includes the steps of synthesizing (by art standard techniques) nucleic acid sequences encoding any of the amino acid sequences described here , ligating the nucleic acid sequence into a suitable expression vector capable of expressing the nucleic acid sequence in a suitable host, transforming the host with the expression vector to which the nucleic acid sequence has been ligated, culturing the host under conditions suitable for expression of the nucleic acid sequence, whereby the protein encoded by the selected nucleic acid sequence is expressed by the host
  • the ligating step may further contemplate ligating the nucleic acid into a suitable expression vector such that the nucleic acid is operably linked to a suitable secretory signal, whereby the amino acid sequence is secreted by the host
  • the secretory signal may be selected, for example, from the group consisting of the leader sequence of stll, ecotm, lamB, he ⁇ es gD, lpp, alkaline phosphatas
  • the host cell may be prokaryotic or eukaryotic Prokaryotes are preferred for cloning and expressing DNA sequences to produce parent polypeptides, segment substituted polypeptides, residue-substituted polypeptides and polypeptide variants
  • E coli K12 strain 294 ATCC No 31446
  • E coli B E coli.
  • E coli c600 and c600hfl E coli W3110 (F-, gamma-, prototrophic /ATCC No 27325), bacilli such as Bacillus subtilis, and other enterobacte ⁇ aceae such as Salmonella -typhimurium or Serratia marcesans, and various pseudomonas species
  • the preferred prokaryote is E coli W31 10 (ATCC 27325)
  • the polypeptides When expressed by prokaryotes the polypeptides typically contain an N- terminal methionine or a formyl methionine and are not glycosylated In the case of fusion proteins, the N- terminal methionine or formyl methionine resides on the ammo terminus of the fusion protein or the signal sequence of the fusion protein
  • eukaryotic organisms such as yeast cultures, or cells derived from multicellular organisms may be used In principle, any such cell culture is workable
  • any such cell culture is workable
  • useful host cell lines are VERO and HeLa cells, Chinese Hamster Ovary (CHO) cell lines, W138, 293, BHK, COS-7 and MDCK cell lines
  • Preferred expression vectors of the present invention may be selected from, for example, pBR322, phGHl, pB0475, pB1537, pRIT5, pRIT2T, pKK233-2, pDR540, pPL-lambda and pB1537, with the most preferred vector being pB1537
  • a preferred vector for direct expression of the BNP variants of the present invention is pB1537 (Cunningham et al (1994) EMBO J 13 2508-2515) and contains o ⁇ gms of replication for E coli, the alkaline phosphatase promoter, the stll signal sequence, the BNP variant gene, and the ampicillm resistance gene
  • Other preferred vectors are pRlT5 and pRlT2T (Pharmacia Biotechnology) These vectors contain appropriate promoters followed by the Z domain of protein A. allowing genes inserted into the vectors to be expressed as fusion proteins
  • vectors can be constructed us g standard techniques by combining the relevant traits of the vectors described herein
  • a vector contaming the origins of replication for phage and E coli, which allow it to be shuttled between such hosts, is used thereby facilitating both mutagenesis and expression
  • Relevant traits of the vector include the promoter, the ⁇ bosome bind g site, the variant gene or gene fusion (the Z domain of protein A and a BNP variant and its linker), the signal sequence, the antibiotic resistance markers, the copy number, and the appropriate origins of replication
  • a variation on the above procedures contemplates the use of gene fusions, wherem the gene encoding a BNP variant is associated, in the vector, with a gene encoding another protein or a fragment of another protein
  • the "other" protein is often a protein or peptide which can be secreted by the cell, making >t possible to isolate and purify the desired protein from the culture medium and eliminating the necessity of destroying the host cells which arises when the desired protein remains inside the cell
  • the fusion protein can be expressed mtracellularly It is useful to use fusion proteins that are highly expressed
  • BNP variants expressed as fusion proteins may be properly folded or may require folding to obtain the native structure
  • the properly folded fusion protein may be active and useful as pharmaceutical drug
  • a chaotrope such as a guanidine HCl
  • a redox buffer containing, for example, reduced and oxidized dithiothreitol or glutathione at the appropriate ratios, pH, and temperature, such that the protein of interest is refolded to its native structure.
  • a chaotrope such as guanidine HCl
  • a redox buffer containing, for example, reduced and oxidized dithiothreitol or glutathione at the appropriate ratios, pH, and temperature, such that the protein of interest is refolded to its native structure.
  • More preferred would be the correctly folded native protein that is obtained from the fusion protein by methods known in the art.
  • Fusion proteins can be cleaved using chemicals, such as cyanogen bromide, which cleaves at a methionine, or hydroxylamine, which cleaves between an Asn and Gly.
  • chemicals such as cyanogen bromide, which cleaves at a methionine, or hydroxylamine, which cleaves between an Asn and Gly.
  • the nucleotide base pairs encoding these amino acids may be inserted just prior to the 5' end of the BNP variant gene.
  • proteolytic cleavage of fusion proteins which has been recently reviewed (Carter, P. (1990) in Protein Purification: From Molecular Mechanisms to Large-Scale Processes, Ladisch, M. R, Willson, R. C, Painton, C. C, and Builder, S. E, eds, American Chemical Society Symposium Series No. 427, Ch 13, 181-193).
  • Proteases such as Factor Xa, thrombin, subtilisin and mutants thereof, have been successfully used to cleave fusion nroteins.
  • a peptide linker that is amenable to cleavage by the protease used is inserted between the "other" protein (e.g. the Z domain of protein A) and the protein of interest, such as a BNP variant.
  • the nucleotide base pairs encoding the linker are inserted between the genes or gene fragments coding for the other proteins.
  • Proteolytic cleavage of the partially purified fusion protein containing the correct linker can then be carried out on either the native fusion protein, or the reduced or denatured fusion protein.
  • BNP variants may be purified from the broths of E. coli cultures grown in 10 liter fermentors by batch reverse phase chromatography, cation exchange chromatography and C18 reverse phase HPLC.
  • Compounds of the present invention have natriuretic, diuretic and vasorelaxant activity and may inhibit the release of aldosterone and renin. Thus, these compounds find use as therapeutic agents in the treatment of various pathological conditions associated with water or electrolyte imbalance and hypertension, especially renovascular hypertension.
  • the compounds of the present invention can be used analogous to ANP peptides in therapeutic and prophylactic measures.
  • BNP has potent diuretic and systemic vasorelaxant properties (Kambayashi et al, (1990) Biochem. Biophys. Res. Commun. 173:599-605).
  • BNP BNP-like protein
  • Metabolism 41 1273-1275
  • Mukoyama M et al, (1991) J. Clin. Invest. 87:1402-14112.
  • Both BNP and ANP are unregulated proportionally during chronic hypoxia (Am. J. Physiol. (1994) 266:L308-L315). Therefore any condition resulting in blood volume or blood pressure overload is a candidate.
  • Such conditions include, for example, cardiac diseases such as congestive heart failure (CHF), cardiomyopathies, myocarditis, valve diseases such as rheumatic heart disease, coronary heart disease such as myocardial ischemia and infarction, arrhythmias such as paroxysmal atrial tachycardia and atrial fibrillation and flutter, congenital heart defects, restrictive disorders such as pericarditis and cardiac tamponade.
  • CHF congestive heart failure
  • cardiomyopathies myocarditis
  • myocarditis valve diseases such as rheumatic heart disease
  • coronary heart disease such as myocardial ischemia and infarction
  • arrhythmias such as paroxysmal atrial tachycardia and atrial fibrillation and flutter
  • congenital heart defects congenital heart defects
  • restrictive disorders such as pericarditis and cardiac tamponade.
  • kidney diseases such as chronic renal failure and nephrosis, cirrhosis, primary aldosteronism, Cushing's disease, preeclampsia, toxemia of pregnancy, premenstrual syndrome, nephrotic syndrome and hepatic cirrhosis, pulmonary disease, and renal failure due to ineffective renal perfusion or reduced glomerular filtration rate.
  • the compounds and compositions can be administered to humans in a manner similar to ANP and other similar therapeutic agents.
  • the dosage to be administered will depend on the usual factors including; age, weight, sex, condition of the patient, specific disorder being treated, and route of administration. In general, the dosage required for therapeutic efficacy will range from about 0.01 to 1000 mg/kg, more usually 0.1 to 25 mg/kg of the host body weight. Alternatively, dosages within these ranges can be administered by constant infusion over an extended period of time until the desired therapeutic benefits have been obtained.
  • the present invention also provides compositions containing an effective amount of compounds of the present invention, including the nontoxic addition salts, amides and esters thereof, which may, alone, serve to provide the above-recited therapeutic benefits.
  • the peptide compounds may be formulated into the compositions as neutral or salt forms.
  • Pharmaceutically acceptable nontoxic salts include the acid addition salts (formed with the free amino groups) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyl amine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyl amine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • Such compositions can be provided together with physiologically tolerable liquid, gel or solid diluents, adjuvants and excipients.
  • compositions are prepared as sterile, injectable liquid solutions or suspensions.
  • Compositions may also be emulsified.
  • the active ingredient is often mixed with diluents or excipients which are physiologically tolerable and compatible with the active ingredient. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof.
  • diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof.
  • the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH-buffering agents, and the like.
  • auxiliary substances such as wetting or emulsifying agents, stabilizing or pH-buffering agents, and the like.
  • compounds of the present invention can also be employed as intermediates in the synthesis of such useful compounds.
  • compounds of the present invention whose activity levels are reduced or eliminated entirely can serve to modulate the activity of other diuretic, natriuretic or vasorelaxant compounds, including compounds outside the scope of the present invention, by, for example, binding to alternate receptors, stimulating receptor turnover, or providing alternate substrates for degradative enzyme of receptor activity and thus inhibiting these enzymes or receptors.
  • such compounds can be delivered as admixtures with other active compounds or can be delivered separately, for example, in their own carriers.
  • compositions may be prepared alone or in combination with a C receptor ligand such as, for example SC-46542, or an inhibitor of the enzyme primarily responsible for the degradation of BNP such as SCH 32615, SCH 344826, SCH 39370, SQ 2900072, thiorfan and UK 69578.
  • a C receptor ligand such as, for example SC-46542
  • an inhibitor of the enzyme primarily responsible for the degradation of BNP such as SCH 32615, SCH 344826, SCH 39370, SQ 2900072, thiorfan and UK 69578.
  • Sythetic compounds which occupy the C-receptor increase the concentrations of circulating BNP and may be used in combination with the peptides of the present invention.
  • U.S. Patent No 4,740,499 describes a method of prolonging or enhancing the bioactivity of an atrial peptide using two specific inhibitor of endopeptidase 24.11, thio ⁇ han or keloto ⁇ han administered simultaneous with the atrial
  • Monovalent phagemid libraries (Lowman, H. B. et al, (1991) Methods: Companion Methods Enzymol. 3:205-216) of BNP were generated by site directed mutagenesis (Kunkel, T. A, et al, (1991) Methods Enzymol. 204:125-139).
  • Each library contained three, four or five codons fully randomized for all 20 amino acids (as shown in Table 1 below) using oligonucleotides that mutated target codons to NNS sequences (where N represents a mixture of all four bases and S is a mixture of both C and G).
  • Stocks of approximately 10 phagemid per mL were prepared from PEG precipitates of culture broths from XL-1 Blue cells containing the plasmid and superinfected with K07 helper phage.
  • the coat solution was discarded and the wells were blocked for one hour at room temperature with 5% skim milk in 25mM sodium carbonate (pH9.6). After washing with 0.01% Tween® in PBS, NPR-A IgG was added at 1 ⁇ g/ml in binding buffer (2% skim milk in PBS) for 1 hour at room temperature. The wells were washed again. 10 ⁇ l of phage stock (250 fold concentrated from growth media, stored in PBS) was brought up to 100 ⁇ l with binding buffer and incubated in the wells for 2 hours at room temperature.
  • the phage were eluted with 100 ⁇ l 0.2M glycine (pH 2.0), then neutralized with 13 ⁇ l 1M Tris Base and titered by infecting E. coli (XLl-blue Stratagene, Inc., San Diego, CA). The remaining selected phagemid particles were propagated for the use in the next selection cycle.
  • Receptor specific selection was done by adding competing NPRC IgG (Bennett et al. (1991) supra) during the phage binding step in the NPR-A IgG coated wells. This was done in seven rounds of selection with the amount of competing CIgG increasing from 20nM to 200nM.
  • tert-butyloxycarbonyl Boc
  • p-toluenesulfonyl Tos
  • 4-methylbenzyl MeBzl
  • benzyl Bzl
  • 2- bromobenzyloxycarbonyl Br-Z
  • cyclohexyl ester Ochex
  • 4-methoxylbenzyl MeOBzl
  • 2-chlorobenzyloxycarbonyl Cl-Z
  • hydrogen fluoride HF
  • benzotriazolyloxytris(dimethylamino)phosphoniumhexafluorophosphate BOP
  • methylene chloride DCM
  • trifluoroacetic acid TMA
  • DMA dimethylacetamide
  • Peptides were assembled using an Applied Biosystems 430A automated peptide synthesizer. Protected amino acids were obtained from various vendors. Side chain protection was Asp (Ochex), Cys (MeOBzl), Arg (Tos), Ser(Bzl), Thr (Bzl), Lys (Cl-Z), and Tyr (Br-Z).
  • the peptides were assembled on 0.7 g (1.0 mmol) of Boc-Tyr(Br-Z) PAM resin (Applied Biosystems). The coupling protocols were those recommended by Applied Biosystems and DMA was used instead of DMF as solvent. After chain assembly the N-terminal Boc group was removed using TFA. The peptide resin was then washed with dichloromethane followed by methanol and then dried under vacuum. The peptide was then deprotected and removed from the resin by stirring the peptide-resin in 20 ml of a mixture of HF (92.5%), anisole (5%) and ethylmethylsulfide (2.5%) for 1 hr at 0 degrees.
  • the cyclization was begun immediately by adding 0.003 M K ⁇ fFe ⁇ N) ⁇ ] (1 g dissolved in 100 mL of distilled water) dropwise (ca. 1 drop/ 10 sec.) with vigorous stirring. Addition of the iron solution was halted when a bright yellow color persisted, and the reaction mixture was stirred for an additional 4 to 5 h. The pH of the cyclized solution was adjusted to 4.5 with HOAc.
  • Bio Rex 70 cation exchange resin 200-400 mesh in the Na + form was washed with 1 N HCl and then with distilled water. Approximately 1 10 g of this resin was added to the cyclized peptide solution and stirred overnight. The resin was filtered, washed exhaustively with distilled water, and slurry packed into a column. The peptide was eluted using 70% aqueous HOAc, identified by TLC (ninhydrin visualization) and lyophilized. The peptide was dissolved in 0.1% TFA in water, loaded onto a 2.5 cm x 50 cm C- 18 reversed phase column (15 micron, 300A) and eluted with a shallow gradient of increasing acetonitrile.
  • the elution conditions consisted of a 10% to 50% acetonitrile (containing 0.1% TFA) gradient at 0.5% min. .
  • the aqueous phase was 0.1% TFA in water.
  • Product containing fractions were then lyophilized to afford the pure title peptide.
  • the compounds of Examples 3 - 6 were analogously prepared.
  • Membrane Preparation Membranes from stable 293 cell l es expressing NPR-A (Lowe and Fendly (1992) J Biol Chem ) or NPR-C (Cunnmgham et al ( ⁇ 99 ⁇ ) supra) were prepared by removing the cells from tissue culture platrs with 0 5mM EDTA in PBS, cent ⁇ fuging at 225xg, 4° C, and resuspending m 50mM Hepes Ph7 4, ImM EDTA, ImM DTT, 250mM Sucrose, 0 7ug/ml Pepstatin A, 0 5ug/ml Leupeptin, ImM PMSF, luM Phosphoramidon The resuspended cells were homogenized for 30 seconds with a B ⁇ nkman polytron on setting 5 The homogenate was centrifuged in 30ml corex tubes in Sorval HB-4 rotor at 400xg ( 1500rpm) for 10 minutes at 4°C The super
  • Example 7 The peptides were assayed for stimulation CGMP production.
  • Cell Culture Cells were maintained in F12 (w/o GHT)/Low Glucose DMEM (50/50), 25 mM Hepes pH 7.2, 2mM Glutamine, 400 micrograms/ml G418 and 10% dialyzed calf serum. Log phase cells were removed from plates with 0.5 mM EDTA in PBS, taken up in media and cell concentration measured with a coulter counter. Cells were plated to 12 well dishes at a density of 300,000 cells/well, 18 to 24 hours prior to stimulation.
  • Figures 1 and 2 demonstrate the measurement of cGMP production in 293 cells expressing hNPR-A. Wild-type and variants stimulate guanyl cyclase activity in the membrane preparations as measured by cGMP production.
  • Glu Arg lie Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Ser Ser Ser Ser Phe Trp Arg Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Ser Ser Ser Ser Met Trp Arg Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Arg His Ser Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Arg His Leu Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Arg Ser Ser Ser Phe Trp Arg Cys Lys Val Leu Arg 20 25 30 Arg His 32
  • Glu Arg lie Arg His Ser Ser Met Trp Arg Cys Lys Val Leu Arg 20 25 30 Arg His 32
  • Glu Arg lie Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Arg His Leu Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Ala Arg Glu Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Arg Lys Thr Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30 Arg His 32
  • Glu Arg lie Gly Arg Ala Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Arg Trp Ser Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Arg Asn Thr Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Arg His Ser Thr Gly Leu Gly Cys Lys Val Leu Arg 20 25 30 Arg His 32
  • Glu Arg lie Arg Thr Lys Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30 Arg His 32
  • Glu Arg lie Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Arg Gly Met Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Ser Ser Ser Ser Met Tyr Arg Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Ser Ser Ser Ser Leu Trp Arg Cys Lys Val Leu Arg 20 25 30
  • Glu Arg lie Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30
  • Asp Arg lie Gly Ser Leu Ser Gly Leu Gly Cys Asn Val Leu Arg 20 25 30
  • Asp Arg lie Gly Ser Leu Ser Gly Leu Gly Cys Asn Val Leu Arg 20 25 30
  • Lys Val Thr His lie Ser Ser Cys Phe Gly His Lys lie Asp Arg 20 25 30 lie Gly Ser Val Ser Arg Leu Gly Cys Asn Ala Leu Lys Leu Leu
  • Asp Arg lie Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg 20 25 30 Arg His 32

Abstract

Composés apparentés à BNP présentant une affinité limitée de fixation au récepteur C du peptide natriurétique, mais possédant une affinité améliorée ou équivalent par rapport au BNP de type sauvage pour le récepteur A du peptide natriurétique. Ces composés peuvent être utilisés avantageusement pour le traitement ou la prophylaxie de différents états pathologiques associés au déséquilibre aqueux ou électrolytique.
EP97910899A 1996-10-22 1997-10-09 Peptide natriuretique cerebral (bnp) specifique pour un recepteur Withdrawn EP0939770A1 (fr)

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US10052366B2 (en) 2012-05-21 2018-08-21 Alexion Pharmaceuticsl, Inc. Compositions comprising alkaline phosphatase and/or natriuretic peptide and methods of use thereof
WO2016007873A1 (fr) 2014-07-11 2016-01-14 The Regents Of The University Of Michigan Compositions et méthodes de traitement de la craniosynostose
BR112017011900A2 (pt) 2014-12-05 2018-02-27 Alexion Pharma Inc tratamento de ataques com fosfatase alcalina recombinante
JP6868561B2 (ja) 2015-01-28 2021-05-12 アレクシオン ファーマシューティカルズ， インコーポレイテッド アルカリホスファターゼ欠損を有する被験者を治療する方法
AU2016308624B2 (en) 2015-08-17 2022-06-23 Alexion Pharmaceuticals, Inc. Manufacturing of alkaline phosphatases
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EP3368062A4 (fr) 2015-10-30 2019-07-03 Alexion Pharmaceuticals, Inc. Méthodes de traitement de la craniosynostose chez un patient
EP3426286A4 (fr) 2016-03-08 2019-12-04 Alexion Pharmaceuticals, Inc. Méthodes de traitement de l'hypophosphatasie chez l'enfant
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AU4816497A (en) 1998-05-15

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