Patent Application
20090215675
The present invention is related to peptides, their use in the detection and inhibition of anti-β1-adrenergic receptor antibodies and diagnostic agents and pharmaceutical compositions containing the same.
Dilated cardiomyopathy (DCM) is a severe cardiac disease of young adults which will result in continuous decline of cardiac function if not treated. Said decline is based on a reduced cardiac function going along with dilatation of the heart muscle. In about 30% of the cases dilated cardiomyopathy is of genetic origin, about 10% of the cases are caused by toxic substances such as alcohol and chemotherapeutics and the remaining 60% of the cases are caused by acute or chronic infection of the heart muscle and concomitantly occurring immunological secondary reactions. Ultimately, heart transplantation is the means of choice for the treatment of dilated cardiomyopathy. Without treatment the patient is facing an untimely death (Richardson et al. (1996) Circulation, 93, 841-842).
In idiopathic dilated cardiomyopathy which is characterised by a loss of heart function without defined aetiology, a correlation of heart disease and autoimmune reactions against various myocardial antigens was found, such as autoantibodies against the heavy chain of myosin, laminin and the ADP/ATP transporter (Schulze et al. (1990) Circulation, 81, 859-869). However, there was no evidence that any of these antigens is relevant in terms of pathogenesis. In contrast thereto, functionally active antibodies against cardiac β1-receptors have been shown to play an important role in the pathogenesis of dilated cardiomyopathy. Immunological analysis have shown that the second out of a total of three extracellular domains of the β1-adrenergic receptor exhibits both a T-cell epitope and a B-cell epitope (Hoebecke et al. (1994), Methods Neuroscie. 25, 345-365) and meets thus the criteria for an auto-antigen in accordance with the first Witebsky's postulate.
In order to provide experimental evidence that this auto-antigen is relevant for the etiology of dilated cardiomyopathy, it was tried to induce immune cardiomyopathy in accordance with the second Witebsky's postulate. In a rat model experimental evidence was provided that stimulatory anti-β1-AR antibodies against the second extracellular domain of the β1-adrenergic receptor are involved in the etiology of dilated cardiomyopathy (β1-ECII) (Jahns et al. (2004) J. Clin. Invest. 113, 1419-1429). The β1-ECII-/GST fusion protein used showed 100% homology between human and rat. Each of the animals immunised against β1-ECII developed stimulatory anti-β1-antibodies and severe progressive dilatation and pump function failure of the left ventricle corresponding to immune cardiomyopathy which could be detected by echocardiography and was confirmed by invasive measurements and histologic analysis of the animals. In order to simulate β1-autoantibodies the serum of β1-antibody positive animals was transferred intravenously to genetically identical rats every four weeks. Again, a slowly progressive dilated immune cardiomyopathy was observed nine months after serum transfer as evidenced by echocardiography and as also confirmed by invasive measurements and morphological and histological studies of the hearts of the transfer animals. Using this approach, for the very first time it was shown that β1-antibody-induced immune cardiomyopathy can be transferred and meets thus the classical third Witebsky's postulate for an autoimmune pathogenesis of dilated cardiomyopathy (Freedman & Lefkowitz (2004) J. Clin. Invest. 113, 1378-1382).
In addition to the animal studies blood samples from patients were analysed for β1-receptor antibodies. Jahns et al. detected auto-antibodies against recombinant human β1-receptors in about 30% of all patients suffering from cardiac failure and idiopathic dilated cardiomyopathy. These patients exhibit a significantly decreased cardiac function compared to patients without such β1-receptor antibodies (Jahns et al. (1999) Circulation 99, 649-654). The stimulatory effect of the antibodies could be abolished by the cardioselective β1-receptor blocker bisoprolol. In view of this, Jahns et al. (Jahns et al. (1999), Circulation 99, 649-654; Jahns et al. (2000) JACC 36, 1280-1287; Jahns et al. (2004) JCI 113, 1419-1429) suggested to use β1-receptor antagonists such as bisoprolol as inhibitors of the interaction between anti-β1-adrenergic receptor antibodies and the native recombinant and non-recombinant β1-adrenergic receptors.
An alternative therapeutic approach for the treatment of dilated cardiomyopathy is the removal of the antibodies from the circulation of patients with functionally active and potentially harmful β1-receptor antibodies using immunoadsorption or immunoapharesis. In such a procedure, the blood of the patient is passed through columns removing the auto-antibody against β1-adrenergic receptors using a matrix coupled non-receptor homologous peptide or by inspecifically removing antibodies of the IgG subclass by binding the antibodies to protein A columns or to matrix coupled goat-anti-human IgG antibody. Such procedure, however, is both time and labour consuming. Additionally, due to the unspecific elimination of all immunoglobulins from the blood of the thus treated patients, numerous draw-backs arise such as an imbalanced immune system and immune response. Nevertheless it seems that antibody-positive DCM patients have at least a short term benefit from such treatment.
The problem underlying the present invention is thus to provide further means for the inhibition of anti-β1-adrenergic receptor antibodies. More particularly, the problem underlying the present invention is to provide means for the inhibition of anti-β1-adrenergic receptor antibodies.
The problem underlying the present invention is solved in a first aspect by a peptide selected from the group comprising
cyclo(Ala-x-x-x-x-x-x-x-x-x-Cys-x-x-x-Pro-x-Cys-Cys-xk-Gln), (I)
Cyclo(Ala-x-x-Trp-x-x-Gly-x-Phe-x-Cys-xh-Gln), (II)
Cyclo(Ala-x-x-x-x-x-x-x-x-x-Cys-xj-Cys-x-x-x-Pro-x-Cys-Cys-xi-Gln); (III)
Ala-x1-Cys-xm-Cys-x-x-x-Pro-x-Cys-Cys-xn-Gln, (IV)
In an embodiment the peptide is a cyclic peptide of formula Ia:
cyclo(Ala-x2-x-x1-x-x1-x1-x-x2-x2-Cys-x-x-x1-Pro-x-Cys-Cys-xk-Gln), (Ia)
whereby k is any integer from 0 to 6, preferably any integer from 1 to 6, more preferably k=6;
In an embodiment the peptide is a cyclic peptide of formula Ib:
Cyclo(x4-x2-x4-x1-x4-x1x1-x4-x2-x2-Cys-x3-x5-x1-Pro-x2-Cys-Cys-x1-x3-x3-x4-x5-x2-x5) (Ib)
whereby
In an embodiment the peptide is a peptide of formula Ic:
cyclo(Ala-Arg-Ala-Glu-Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Thr-Asn-Arg-Gln). (Ic)
In a preferred embodiment at least one of acidic amino acid residues is replaced by a different amino acid selected from the group comprising acidic amino acids.
In a preferred embodiment at least one of the basic amino acid residues is replaced by a different amino acid selected from the group comprising basic amino acids.
In a preferred embodiment at least one of the aliphatic amino acid residues is replaced by a different amino acid selected from the group comprising aliphatic amino acids.
In a more preferred embodiment at least one Ala amino acid residue is replaced by Glu.
In an embodiment the peptide is a cyclic peptide of formula IIa:
Cyclo(Ala-x4-x2-Trp-x1-x3-Gly-x4-Phe-x3-Cys-xn-Gln), (IIa)
whereby n is any integer from 0 to 2, preferably 1 or 2;
whereby
In a preferred embodiment the peptide is a peptide of formula IIb:
cyclo(Ala-Gly-Arg-Trp-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Leu-Gln) (IIb)
or a peptide of formula IIc:
cyclo(Ala-Gly-Arg-Trp-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Gln) (IIc)
In a more preferred embodiment at least one of the acidic amino acid residues is replaced by a different amino acid selected from the group comprising acidic amino acids.
In another more preferred embodiment at least one of the basic amino acid residues is replaced by a different amino acid selected from the group comprising basic amino acids.
In another more preferred embodiment at least one of the aliphatic amino acid residues is replaced by a different amino acid selected from the group comprising aliphatic amino acids.
In another more preferred embodiment at least one Ala amino acid residue is replaced by Glu.
In preferred embodiment the peptide is a cyclic peptide of formula IIIa:
Cyclo(Ala-x4-x2-x3-x1-x3-x3-x4-x3-x3-Cys-xj-Cys-x-x-x-Pro-x-Cys-Cys-xi-Gln), (IIIa)
whereby i is any integer from 0 to 6, preferably any integer from 1 to 6, more preferably i=6;
whereby j is any integer from 0 to 4, preferably any integer from 1 to 4, more preferably j=4;
whereby
In a more preferred embodiment the peptide is a peptide of formula IIIb:
Cyclo(Ala-Gly-Arg-Trp-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Thr-Asn-Arg-Gln) (IIIb)
In more preferred embodiment at least one of the acidic amino acid residues is replaced by a different amino acid selected from the group comprising acidic amino acids.
In another more preferred embodiment at least one of the basic amino acid residues is replaced by a different amino acid selected from the group comprising basic amino acids.
In another more preferred embodiment at least one of the aliphatic amino acid residues is replaced by a different amino acid selected from the group comprising aliphatic amino acids.
In another more preferred embodiment at least one Ala amino acid residue is replaced by Glu.
In a preferred embodiment the peptide is a linear peptide of formula IVa:
Ala-x4-x2-x3-x1-x3-x3-x4-x3-x3-Cys-xm-Cys-x-x-x-Pro-x-Cys-Cys-xn-Gln), (IVa)
whereby n is any integer from 0 to 6, preferably any integer from 1 to 6, more preferably n=6;
whereby m is any integer from 0 to 4, preferably any integer from 1 to 4, more preferably m=4;
whereby
In a more preferred embodiment the peptide is a peptide of formula IVb:
Ala-Gly-Arg-Trp-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Thr-Asn-Arg-Gln (IVb)
In a further aspect the present invention is related to cyclic peptides having any of the two following formulae, whereby it is to be acknowledged that both peptides are covered by formula IV but are additional cyclic peptides:
In another more preferred embodiment at least one of the of acidic amino acid residues is replaced by a different amino acid selected from the group comprising acidic amino acids.
In another more preferred embodiment at least one of the basic amino acid residues is replaced by a different amino acid selected from the group comprising basic amino acids.
In another more preferred embodiment at least one of the aliphatic amino acid residues is replaced by a different amino acid selected from the group comprising aliphatic amino acids.
In another more preferred embodiment at least one Ala amino acid residue is replaced by Glu.
In an embodiment, in case the peptide is a cyclic peptide, the cyclization occurs by a linkage which is a covalent binding selected from the group comprising S—S linkages, peptide bonds, carbon bonds such as C—C or C═C, ester bonds, ether bonds, azo bonds, C—S—C linkages, C—N—C linkages and C═N—C linkages.
In a preferred embodiment the S—S linkage is formed by two Cys residues of the peptide.
In an alternative preferred embodiment the peptide bond is formed by the NH2 group of the N-terminal amino acid and the COOH group of the C-terminal amino acid.
In another alternative preferred embodiment additional bonds are formed by the side chain of NH2 groups and COOH groups of the constituent amino acids.
The problem underlying the present invention is solved in a second aspect by a composition comprising at least one of the peptides according to the first aspect of the present invention and a carrier.
The problem underlying the present invention is solved in a third aspect by a diagnostic agent comprising at least one of the peptides according to the first aspect of the present invention.
In an embodiment the diagnostic agent is for the detection of anti-β-adrenergic receptor antibodies.
In a preferred embodiment the diagnostic agent comprises at least one further biologically active compound.
The problem underlying the present invention is solved in a fourth aspect by a diagnostic kit for the detection of anti-β1-adrenergic receptor antibodies comprising a peptide according to the first aspect of the present invention or a diagnostic agent according to the third aspect of the present invention.
The problem underlying the present invention is solved in a fifth aspect by a pharmaceutical composition comprising at least one of the peptides according to the first aspect of the present invention and a pharmaceutically acceptable carrier.
In an embodiment the pharmaceutical composition additionally comprises a further pharmaceutically active agent.
In a preferred embodiment the further pharmaceutically active agent is selected from the group comprising a beta receptor blocker, preferably selective β1-adrenergic receptor blockers.
In a more preferred embodiment the further pharmaceutically active agent is selected from the group of selective β1-adrenergic receptor blockers comprising atenolol, metoprolol, nebivolol, and bisoprolol or the non-selective beta blocker carvedilol.
The problem underlying the present invention is solved in a sixth aspect by the use of a peptide according to the first aspect of the present invention, for the manufacture of a medicament.
In an embodiment the medicament is for the treatment and/or prevention of a disease, whereby such disease is selected from the group of heart diseases, comprising infectious and non-infectious heart disease, ischemic and non-ischemic heart disease, inflammatory heart disease and myocarditis, cardiac dilatation, idiopathic cardiomyopathy, idiopathic dilated cardiomyopathy, immune-cardiomyopathy, heart failure, and any cardiac arrhythmia including ventricular and supraventricular premature capture beats.
In a preferred embodiment the disease is idiopathic dilated cardiomyopathy and preferably anti-β-AR antibody-induced dilated immune-cardiomyopathy.
In an embodiment the medicament comprises at least one further pharmaceutically active compound.
In an embodiment the medicament is for the treatment and/or prevention of patients having antibodies against β-adrenergic receptors, preferably β1-adrenergic receptors.
In an embodiment the medicament is for inducing immune tolerance.
The problem underlying the present invention is solved in a seventh aspect by the use of a peptide according to the first aspect of the present invention for the manufacture of a medicament, whereby the medicament is for inducing immune tolerance, preferably by suppression of the production of anti-β-adrenergic receptor antibodies and more preferably by suppression of the production of anti-β1-adrenergic receptor antibodies.
The problem underlying the present invention is solved in an eighth aspect by the use of a peptide according to the first aspect of the present invention for inducing immune tolerance by suppression of the production of anti-β1-adrenergic receptor antibodies through blockade of the antigen-recognition sites of the antibody-producing prae B-cells.
In a still further aspect the problem underlying the present invention is solved by the use of a peptide according to the present invention in a method of diagnosis.
In an embodiment the method is for the diagnosis of idiopathic dilated cardiomyopathy, preferably anti-β-AR antibody-induced dilated immune-cardiomyopathy.
In preferred embodiment the method is a FRET-based method.
Without wishing to be bound by any theory in the following, the present inventors have surprisingly found that a number of both cyclic as well as linear peptides are, in principle, active as inhibitors of anti-β-adrenergic receptor antibodies, more particularly as inhibitors of anti-β1-adrenergic receptor antibodies.
It has to be acknowledged that, as also shown in
Also, the present inventors have surprisingly found that the peptides according to the present invention obviously act through an additional mechanism different from a capturing of the autoantibodies which activate the β1-adrenergic receptors resulting in, among others, dilated immune-cardiomyopathy and heart failure. Rather, the peptides according to the present invention seem to be suitable to induce immune tolerance since endogeneous production of anti-β1-adrenergic receptor antibodies rapidly decreased and finally stopped within 5-6 months in both prophylactically (
It will be understood that for the various peptides of the present invention, a certain flexibility and variability in the primary sequence, i.e. the amino acid sequence is possible as long as the overall secondary and tertiary structure of the respective peptides which is defined by at least some fixed amino acid residues and by their spatial arrangement, is ensured. Moreover the number of amino acids and thus the length of the primary structure appears to be crucial for the biological effects of the various peptides of the present invention. A peptide-length equal or above 26 amino acids (primary structure) is thought to be capable of stimulating directly (that is, without the use of carrier proteins) immunocompetent T-cells and thus may provoke a paradoxal increase in anti-β1-receptor antibody production through T-cell mediated B-cell stimulation (data not shown). Therefore, in the subsequent therapy study using a limited number of supple-mentary pilot animals we tested shorter peptid-variants of the 25 amino-acid β1-ECII-peptide, i.e. cyclic peptide-variants comprising either 18 or 16 amino-acids (AA). The generated constructs were: ECII-18AA, cyclo(Ala-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Gln); and ECII-16AA, cyclo(Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Tyr-Gln)). Wheras the circulating anti-β1-ECII antibody titers were reduced by about a same extent with either the 25AA cylic peptide Ic or the 18AA construct (see
Insofar, the specific peptides in terms of defined amino acid sequences are particularly preferred embodiments of the more general peptides, i.e the peptides represented by the generic formulae herein.
In accordance therewith, the various generic formulae refer to a basic peptide structure as reflected by formulae I, II, III and IV, whereby more specific formulae and thus peptides being further defined in a less generic manner but still being covered by formulae I, II, III and IV being referred to herein as Ia, Ib, Ic, IIa, IIb, IIIa, IIIb, IVa and IVb, respectively. It will also be understood by the ones skilled in the art that the individual amino acid may be replaced by another naturally occurring or synthetic amino acid, preferably if both amino acids belong to the same category of amino acids. In accordance therewith, for example, an acidic amino acid can be replaced by another acidic amino acid, a basic amino acid may be replaced by another basic amino acid and so on. It will also be acknowledged by the ones skilled in the art that one or several of the amino acids forming the peptide of the present invention may be modified. In accordance therewith any amino acid as used herein preferably also represents its modified form. For example, an alanine residue as used herein also comprises modified alanine. Such modifications may, among others, be a methylation or acylation or the like, whereby such modification or modified amino acid is preferably comprised by the present invention as long as the thus modified amino acid and more particularly the peptide containing said thus modified amino acid is still functionally active as defined herein, more particularly functionally active as an inhibitor of β1-adrenergic receptors and even more preferably active in inhibiting the interaction between β1-adrenergic receptors and antibodies, more preferably auto-antibodies directed against β1-adrenergic receptors. Respective assays for determining whether such a peptide, i.e. a peptide comprising one or several modified amino acids, fulfils this requirement, are known to the one skilled in the art and, among others, also described herein, particularly in the example part hereof.
The invention comprises also derivatives of the peptides such as salts with physiologic organic and anorganic acids like HCl, H2SO4, H3PO4, malic acid, fumaric acid, citronic acid, tatratic acid, acetic acid.
As used herein, the sequences of the various peptides are indicated from the N-terminus to the C-terminus, whereby the N-terminus is at the left side and the C-terminus is at the right side of the respective depicted amino acid sequence.
Preferably an acidic amino acid is an amino acid selected from the group comprising Asp, Asn, Glu, and Gln; preferably a basic amino acid is an amino acid selected from the group comprising Arg and Lys; preferably a neutral amino acid is an amino acid selected from the group comprising Gly, Ala, Ser, Thr, Val, Leu, Ile; preferably an aliphatic amino acid is an amino acid which is selected from the group comprising Gly, Ala, Ser, Thr, Val, Leu, Ile, Asp, Asn, Glu, Gln, Arg, Lys, Cys and Met.
As used herein, the expression that one particular amino acid, such as, e.g., a basic amino acid, is replaced by a different amino acid which is selected from a respective particular group of amino acids, such as, e.g., the group comprising basic amino acids, preferably means that the particular amino acid is replaced by another, i.e. different amino acid under the proviso that such different amino acid is part of the respective particular group of amino acids. To the extent indicated herein, this is applicable to each of the particular amino acid and, in principle, each such replacement is independent of any other replacement optionally made in relation to other amino acids forming the respective peptide.
The peptides according to the present invention may be used as a diagnostic agent and for the manufacture of a medicament for the treatment of diseases or may be used in a composition, preferably a pharmaceutical composition, a diagnostic composition and a diagnostic kit, preferably for the detection of anti-β-adrenergic receptor antibodies, more preferably for the detection of anti-β1-adrenergic receptor antibodies. Such diseases are preferably those, where the β1-adrenergic receptor is activated in a non-physiological manner, more particularly is activated by antibodies, more preferably by auto-antibodies which are directed against the β1-adrenergic receptor. More specifically, such diseases comprise, however, are not limited thereto, the group of heart diseases, comprising infectious and non-infectious heart disease, ischemic and non-ischemic heart disease, inflammatory heart disease and myocarditis, cardiac dilatation, idiopathic cardio-myopathy, idiopathic dilated cardiomyopathy, immune-cardiomyopathy, heart failure, and any cardiac arrhythmia including ventricular and supraventricular premature capture. Such pharmaceutical composition may additionally or alternatively also be used for the treatment of patients having antibodies against β-adrenergic receptors, preferably β1-adrenergic receptors. A further subgroup of patients which may be treated by the pharmaceutical composition according to the present invention are those patients suffering from any of the diseases described herein, more particularly the group of heart diseases, comprising infectious and non-infectious heart disease, ischemic and non-ischemic heart disease, inflammatory heart disease and myocarditis, cardiac dilatation, idiopathic cardiomyopathy, idiopathic dilated cardiomyopathy, immune-cardiomyopathy, heart failure, and any cardiac arrhythmia including ventricular and supraventricular premature capture and having at the same time the antibodies directed against β-adrenergic receptors, more preferably antibodies against the β1-adrenergic receptor, whereby in a preferred embodiment the antibodies are auto-antibodies. What is said herein for the pharmaceutical composition applies also to the medicament for the manufacture of which the peptides of the present invention are used. As used herein, compounds and peptides are used in an interchangeable manner herein.
Apart from containing at least one peptide of the present invention, the composition may either comprise two or a plurality of cyclic peptides of the present invention and/or other β-receptor blockers, more particularly β1-adrenergic receptor blockers. Examples therefore are, among others, bisoprolol, metoprolol, atenolol, nebivolol, and carvedilol. This kind of combination provides for protection from antibody-induced selective β1-receptor-downregulation by the peptides (see
The pharmaceutical composition typically comprises a carrier, more preferably a pharmaceutically acceptable carrier, excipient or diluent.
For s.c. or i.v.-injection, compounds of the invention may be formulated in aqueous solution, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically saline buffer. For transmucosal and transpulmonal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
The use of pharmaceutical acceptable carriers to formulate the compounds according to the present invention into dosages or pharmaceutical compositions suitable for systemic, i.e. intravenous/intraarterial, or subcutaneous administration is within the scope of the present invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be readily formulated using pharmaceutically acceptable carriers well known in the art into dosages suitable for subcutaneous or oral administration. Such carriers enable the compounds according to the present invention to be formulated as tablets, pills, capsules, dragees, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
Compounds according to the present invention or medicaments comprising them, intended to be administered intracorporally/intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered near the cell surface. Delivery systems involving liposomes are disclosed in U.S. Pat. No. 4,880,635 to Janoff et al. The publications and patents provide useful descriptions of techniques for liposome drug delivery and are incorporated by reference herein in their entirety. Pharmaceutical compositions comprising a compound according to the present invention for parenteral and/or subcutaneous administration include aqueous solutions of the active compound(s) in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or castor oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injections suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions and to allow for a constantly slow release of the substance in the organism.
Pharmaceutical compositions comprising a compound according to the present invention for oral use can be obtained by combining the active compound(s) with solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, sorbitol, and the like; cellulose preparations, such as, for example, maize starch wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone (PVP) and the like, as well as mixtures of any two or more thereof. If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate, and the like.
Dragee cores as a pharmaceutical composition comprising a compound according to the present invention are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, suitable organic solvents or solvent mixtures, and the like. Preferably a gastric juice resistant coating such as derivatives of cellulose Aquateric®, HP50® or HP55®, polymer of methacrylic acid and methacrylic acid esters (Eutragid® L, Eutragid® S; retard forms Eutragid® RL und Eutragid® RS or derivatives of polyvinyl are used. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations comprising a compound according to the present invention which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
The pharmaceutical composition may be present in the range of 10 μg/kg to 100 mg/kg depending on the application form, preferably s.c. or i.v. application every two or four weeks. In the rat 1 mg/kg s.c. or i.v. every other month were sufficient to obtain therapeutic levels of the compounds according to the present invention, with the respective dosage for human preferably being about 1-10 mg/kg i.v. or s.c.
It is within the present invention that the pharmaceutical composition is used for the treatment of any of the diseases and patient groups as defined above including the detection of anti-β-receptor antibodies in these patients by using the aforementioned compounds. Also, the peptides according to the present invention may be used for the preparation of a medicament for the treatment and/or prevention of any of the diseases and patient groups as defined above in connection with the pharmaceutical composition.
Finally, the present invention is related to a method for the treatment of patients suffering from or being at risk to develop a disease as disclosed herein, more particularly heart diseases, comprising infectious and non-infectious heart disease, ischemic and non-ischemic heart disease, inflammatory heart disease and myocarditis, cardiac dilatation, idiopathic cardio-myopathy, idiopathic dilated cardiomyopathy, immune-cardiomyopathy, heart failure, and any cardiac arrhythmia including ventricular and supraventricular premature capture beats, whereby the patient is in need of such treatment and whereby the method comprises administering to said patient a pharmaceutically effective amount of the peptide of the present invention, or the pharmaceutical composition or the medicament disclosed herein. Preferably, a therapeutically effective dose refers to that amount of the active ingredient that produces amelioration of symptoms or a prolongation of survival of a subject which can be determined by the one skilled in the art doing routine testing.
A “patient” for the purposes of the present invention, i.e. to whom a compound according to the present invention or a pharmaceutical composition according to the present invention is administered, includes both humans and other animals and organisms. Thus the compounds, pharmaceutical compositions and methods are applicable to or in connection with both human therapy and veterinary applications including diagnostic(s), diagnostic procedures and methods as well as staging procedures and methods. For example, the veterinary applications include, but are not limited to, canine, bovine, feline, porcine, caprine, equine, and ovine animals, as well as other domesticated animals including reptiles, such as iguanas, turtles and snakes, birds such as finches and members of the parrot family, lagomorphs such as rabbits, rodents such as rats, mice, guinea pigs and hamsters, amphibians, fish, and arthropods. Valuable non-domesticated animals, such as zoo animals, may also be treated. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human.
In a further aspect, the present invention is related to a method for diagnosing a patient which can be treated using the peptide, pharmaceutical composition and medicament according to the present invention. Preferably, such method comprises the steps as described in the example part hereof aspect the present invention is thus related to the use of peptides according to the present invention as a diagnostic or for the manufacture of a diagnostic test. The rational behind this diagnostic use of the peptides according to the present invention is their interaction with the structures described above and in particular with the anti-β-adrenergic receptor antibodies.
So far, the definition of antibody-positivity depends on highly divergent screening methods such as, e.g., ELISA with receptor peptides, Western blotting of heart tissues, functional assays with neonatal rat cardiomyocytes, or detection by surface plasmon resonance. Until now, this issue has not been solved satisfactorily.
One approach for using the peptides according to the present invention as a diagnostic and in a diagnostic method, respectively, is a a three-step screening procedure comprising performing an ELISA with the peptides according to the present invention as well as determining immunofluorescence and determining cAMP responses in cells expressing native human beta-AR. It is to be acknowledged that each and any of the aforementioned steps can as such be preformed for the detection of said antibodies using the peptides according to the present invention. A large number of heart failure patients may thus be screened for functionally active anti-beta1-Abs. In connection with such diagnostic method the definition of functionally active anti-beta1-Abs is preferably based on their effects on receptor-mediated signalling, that is, their effects on cellular cAMP levels and on the activity of the cAMP-dependent protein kinase (PKA). Cyclic AMP is an universal second messenger of many G protein-coupled receptors including the beta-adrenergic receptor family. It exerts its effects via PKA, cAMP-gated ion channels, phosphodiesterases, and exchange proteins directly activated by cAMP, known as Epac1 and 2. The prior art describes several fluorescence methods for measuring cAMP in intact cells which can all be used in connection with the diagnostic method of the present invention. Fluorescence resonance energy transfer (FRET) between green fluorescent protein (GFP) variants fused to the regulatory and catalytic subunits of PKA has been described to study the spatio-temporal dynamics of cAMP in neurons (Hempel C M, Vincent P, Adams S R, Tsien R Y, Selverston A I. Nature. 1996; 384:113-114) or cardiac myocytes. (Zaccolo M, Pozzan T., Science. 2002; 295:1711-1715).
More recently, single chain fluorescence indicators have been described in the art which are characterized by having an enhanced cyan (CFP) or yellow fluorescent protein (YFP) directly fused to the cAMP-binding domain of Epac-proteins, which allowed to achieve a higher sensitivity and better temporal resolution of the cAMP measurements. Such system is, among others described in WO2005/052186 the disclosure of which is incorporated herein by reference in its entirety. Such system can be used in connection with any diagnostic procedure using the peptides according to the present invention. Also such system can be used for, however is not limited thereto, analyzing the prevalence of functionally active anti-beta1-Abs. Preferably such diagnostic method is applied to a cohort of previously antibody-typed DCM patients or any individual to be assessed insofar or any individual suspected of suffering from any of the diseases described herein or being at risk to suffer therefrom. In a further step of the diagnostic method and screening method, the ability of beta-blockers to inhibit anti-beta1-Ab-induced receptor activation can be assessed and determined, respectively.
The afore described assay which is a FRET-based method as described in WO 2005/052186 making use of the peptides according to the present invention is advantageous insofar as it is simpler, less time consuming, and at the same time discloses or identifies all DCM patients previously considered anti-beta1-ECII-positive. This embodiment of a FRET based method of diagnosing making use of one or several of the peptides according to the present invention is based on detecting antibody-induced increases in cAMP. This second messenger most likely accounts for the harmful effects provoked by activating anti-beta1-Abs. Therefore, it is advantageous that this method, which is preferably used as a diagnostic method, and which quantifies antibody-induced cAMP signals allows to differentiate between functional anti-beta1-Ab-classes. In fact, by applying this method a previously undetected patient population characterized by the presence of “low” activating autoantibodies presumably targeting a different receptor domain, has been identified. Additionally, this method suggests that these classes are preferably directed against different epitopes of the receptor. Taken together, screening by Epac-FRET appears to represent a very sensitive single step approach, allowing to detect different kinds of activating antibodies directed against the human beta1-AR. Therefore, the present invention is also related to the use of one or several of the peptides according to the present invention for use in an Epac-FRET assay. More preferably such Epac-FRET assay is used for diagnosis, even more preferably for the diagnosis of patients suffering from or suspected of suffering from any of the disease described herein.
A further finding underlying the present invention is that functional anti-beta 1-Abs were detected in almost two thirds of patients with DCM—about half of them “high” activator IgG, it means directed against beta 1-ECII, and about another half “low” activator IgG, it means directed against beta 1-ECI. Insofar, these patients define groups of patients which can be diagnosed and treated, respectively, with the peptides according to the present invention. Mortality curves for the two FRET-classified populations demonstrated that “low” activator IgG patients do not significantly differ in mortality from DCM patients without activating auto-antibodies, whereas “high” activator IgG patients have a significantly higher mortality. Thus, anti-beta 1-Abs directed against beta 1-ECII are clinically particularly relevant and are, therefore, a particularly preferred marker and target, respectivela, in or subject to the diagnostic methods disclosed herein. The capability of the FRET-based approach which is a preferred method of diagnosis according to the present invention to differentiate between prognostically distinct anti-beta1-Abs in heart failure, preferably due to DCM, provides for the clinical relevance of this method.
In a still further aspect the present invention is related to a diagnostic agent. Such diagnostic agent consists of or comprises at least one peptide of the present invention. Preferably the diagnostic agent consists of a peptide of the present invention, whereby the peptide preferably comprises a label. Such label may be selected from the group comprising radioactive labels and fluorescent labels. Respective labels are known to the ones skilled in the art. Typically, the peptide is the part of the diagnostic agent conferring specific binding characteristics to the diagnostic agent, preferably binding to anti-β1-adrenergic receptor antibodies, whereas the label confers the signalling characteristics to the diagnostic agent.
The diagnostic agent may comprise, apart from the labelled or unlabelled peptide of the present invention, a further biologically active compound. Such further biologically active compound, in a preferred embodiment, is a means to confer signalling characteristics to the diagnostic agent, particularly in case the peptide of the present invention is unlabelled. For example, the further biologically active compound can be an antibody, preferably a monoclonal antibody, and more preferably a labelled antibody specifically binding to a peptide of the present invention or to a complex consisting of a peptide of the present invention and an anti-β-adrenergic receptor antibody, preferably an anti-β1-adrenergic receptor antibody.
The kit in accordance with the present invention comprises at least a feature which is selected from the group comprising a peptide of the present invention and a diagnostic agent according to the present invention. In an embodiment the kit further comprises an instruction leaflet, and/or a buffer for use in the application of the kit, and/or at least one reaction vessel for carrying out the detection reaction for which the kit is or is to be used. In a further embodiment, some or all of the reagents used in connection with the application of said kit are present as portions useful in carrying out the reaction(s) for which the kit is to be used.
In preferred embodiments, the following abbreviations have shall have the following meanings: Ab or ab: antibody, Abs os abs: antibodies, AR: adrenergic receptor, EC extra cellular and AA amino acid.
The present invention will now be further illustrated by the following figures and examples, from which further advantages, features and embodiments may be taken, whereby
a is a diagram indicating the titer course of specific anti-ECII antibodies in the therapy arm of the study, given as % of the respective titers at the time point treatment was initiated (“starting value”), whereby “Beta1 untreated” means immunized anti-1-positive cardio-myo-pathic not treated animals, “Beta1/bisop.” means immunized anti-1-positive cardio-myo-pathic animals treated with bisoprolol (15 mg/kg, orally), “Beta1/pept. ECII-25AA” means immunized anti-1-positive cardio-myo-pathic animals treated with the 25AA peptide Ic in a dosage of 1 mg/kg intravenously, and “Beta1/bisop.+pept.25AA” means treatment with a combination of bisoprolol and the 25AA peptide Ic. Therapy was started after 8 months of regular (1× monthly) immunization. Monthly immunization was continued under therapy.
b is a diagram comparing the titer course of specific anti-ECII antibodies dependent on the different peptide variants used in the therapy arm of the study, whereby 25AA means the cyclic peptide Ic, 18AA means the shorter 18 amino-acid variant, and 16AA the shorter 16 amino-acid variant of the peptide. “Beta1 untreated” means immunized anti-1-positive cardio-myo-pathic not treated animals, “Beta1/pept. ECI” means immunized anti-1-positive cardio-myo-pathic animals treated with a peptide corresponding to the first extracellular receptor loop (1 mg/kg, intravenously, negative control—supposed and shown to not affect the anti-ECII antibody-titers), “Beta1/pept. ECII-25AA, ECII-18AA, ECII-16AA” means immunized anti-1-positive cardio-myo-pathic animals treated with the 25AA-, 18AA-, or 16AA-peptide, respectively.
Linear peptides are synthesised using the solid phase Fmoc protocol on a Multiple Peptide Synthesizer (SYROII, MultiSynTech GmbH, Witten). The synthesis was performed using side chain protected Fmoc amino acid derivatives on Rink Amide or Rink Amide MBHA resins (Novabiochem-Merk Biosciences GmbH, Bad Soden). The Fmoc amino acids are activated through diisopropylcarbodiimide/N-hydroxybenzotriazole or benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate.
For the synthesis of cyclic peptides on the solid phase Fmoc-Glu-ODmab or another Fmoc amino acid having a side chain protecting group which can be selectively cleaved off in an orthogonal manner, is incorporated at the C-terminal end of the linear peptide. After selective removal of the Dmab side chain using 2% hydrazine monohydrate in N,N′-dimethylformamide the resin-bound linear peptide is treated in the presence of diisopropylcarbodiimide and N-hydroxy-9-azabenzotriazole in N,N′-dimethylformamide for several hours. The cyclisation was monitored by taking samples and performing the Kaiser-Test and, if necessary, repeated. The cleaving off of the cyclic peptide from the synthesis resin generates a peptide amide and the removal of the protective groups of the side chain is done by treating the resin with trifluoro acetic acid/triisopropylsilane/ethandithiole/water for 2 hours.
The animal model used in this example and any other example described herein if not indicated to the contrary, is the human analogue rat model. Prior to evaluating and testing, respectively, this human analogue rat model using bisoprolol and the various compounds of the present invention, more particularly compounds of formula I c the animals were treated as follows.
In order to generate anti-β1-receptor antibodies the animals were immunized against the second β1-receptor loop. In animals neither receiving bisoprolol nor any peptide according to the present invention, progressive dilated immune cardiomyopathy was observed after 8 months of immunization (
Prophylactic treatment of the animals by administering 1 mg/kg of the cyclic peptide of formula I c either subcutaneously (n=4) or intravenously (n=4) every 4 weeks prevented a further increase of the specific anti-β1-ECII antibody-titer with the titer even continuously declining in the further course of the study (
To the big surprise of the present inventors, from the fifth month on no more immune response was observed upon antigen boost every 4 weeks as shown in
As shown in
Upon administration of the peptide of the present invention peptide Ic every 4 weeks, a surprisingly rapid decrease in the specific anti-beta 1-ECII titer was observed as shown in
Both in the rat model as well as in DCM patients the stimulatory, i.e. allosteric effects of β1-ECII-antibodies can be abolished with a cardioselective beta blocker (used in accordance with standard medication). From this it is concluded that β1-(auto)antibody positive patients will profit more than β1-(auto)antibody negative patients from the present invention. This suggests an even higher efficiency of a combination therapy consisting of bisoprolol and a peptide of the present invention for anti-beta-1-ECII-antibody positive patients. Supposed additive mechanisms could be, within others, protection from antibody-induced receptor-downregulation by the peptide together with a synergistic β1-receptor upregulation by the beta-blockers (like bisoprolol or metoprolol, see
In general, the number of amino acids and thus the length of the primary structure appears to be crucial for the biological effects of the peptides of the present invention. A peptide-length equal or above 26 amino acids (primary structure) is thought to be capable of stimulating directly (that is, without the use of carrier proteins) immunocompetent T-cells and thus provoke a paradoxal increase in anti-β1-receptor antibody production through T-cell mediated B-cell stimulation. Therefore, in the frame of the therapy study a limited number of pilot animals was treated with shorter peptid-variants of the 25 amino-acid peptide Ic, i.e. variants comprising either 18 or 16 amino-acids of the cyclic peptide. The used constructs were: ECII-18AA, cyclo(Ala-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Gln); and ECII-16AA, cyclo(Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Tyr-Gln)). Upon administration of shorter peptide-variants of the present invention peptide every 4 weeks, a rapid decrease in the specific anti-beta 1-ECII titer was equally observed within the first 6 months of therapy. Wheras in the following months the circulating anti-β1-ECII antibody titers were reduced by about a same extent with either the 25 AA cylic peptide Ic or the 18 AA construct (see
This detection of β-receptor antibodies (anti-β1-Abs) in body fluids is based on the measurement of the impact of antibodies on the β receptor mediated signal transduction by optical detection of changes in the concentration of the intracellular messenger cyclic adenosine monophosphate (cAMP). Such a cAMP test system is, in principle, described in PCT-application WO 2005/052186.
To detect anti-β1-Abs by their ability to induce β1-receptor-mediated increases in cAMP, a new highly sensitive cAMP sensor (Epac1-camps) using fluorescence resonance energy transfer is employed. With this technology, a total of 56 patient sera and 40 control sera of a collective were analysed using this method, whereby the patient collective is the same as already published by Jahns et al. in 1999 and assayed using peptide immuno assay (ELISA) and 125iodine labelled cAMP assays for β-receptor antibodies (Jahns et al., Circulation 1999, supra). All those patients which had been β-receptor antibody-positive (n=17) in this earlier study, also showed a significant Epac-1 signal (49±4%). IgG from controls and from about half (17/39) of DCM patients previously judged anti-β1-ECII-negative did not significantly affect cellular cAMP.
Surprisingly, the new technology detected anti-β1-Abs in more than half (22/39) of DCM patients formerly judged antibody-negative, but the cAMP signals induced by these antibodies were generally lower (31±5%) than in the previous group.
Betablockers failed to fully prevent the antibody-induced β1-receptor activation, with carvedilol being more effective than other β-blockers. Blocking experiments demonstrated that “high” or “low” activator IgG were blocked better by peptides corresponding to the second or first extra-cellular β1-receptor-loop, respectively.
Taken together, the analysis of Epac-FRET signals from all 56 patients revealed an anti-beta 1-Ab prevalence of almost 70% (n=39/56) in DCM, which is much higher than detected by ELISA, immunofluorescence, and cAMP-RIA which proves the suitability of the peptides according to the present invention for the diagnosis of DCM. The Abs+ group can be separated into two subgroups on the basis of the FRET-data, which were classified as “low” (FRET amplitude 20-40% of isomax) and “high” activators (FRET amplitude ≧40% of isomax).
The two newly identified FRET-positive populations were studied in more detail. Analyzing the concentration-response relation we found that “low” activator IgG, even at higher concentra-tions, did not achieve cAMP levels of a similar order of magnitude as induced by “high” activator IgG (
In this assay for antibodies directed against β-adrenergic receptors the antibody induced cAMP production is measured in single cells such as, for example, HEK 293 cells or CHW cells which express the β-adrenergic receptor preferably in a concentration of 0.15 bis 0.3 pmol/mg membrane protein and are transiently transfected with an Epac-1 sensor.
The Epac-1 sensor as used is a fusion protein of Epac (exchange protein-1 directly activated by cAMP) and the binding domain E157-E316 coupled to a yellow fluorescent protein (YFP) and the cyan fluorescent protein (CFP) as described in the paper of Nikolaev et al. (JBC, 2004, 279 (36), 37215-8).
These cells are transfected preferably with 10 μg Epac-1 sensor/10 cm dish (diameter) using preferably calcium phosphate precipitation. The medium is preferably changed 12 to 18 hours after transfection. Preferably, 24 to 48 hours after transfection the antibody-induced receptor-mediated signal transduction is determined by measuring the changes in the cellular cAMP titre using the fluorescence resonance energy transfer described in Nikoaev et al. (supra).
The assay is preferably performed using serum samples of patients suffering from dilated cardiomyopathy and healthy controls. The IgG antibodies are preferably isolated from the serum using caprylic acid method, as described in the paper of Jahns et al. (Eur. J. Pharmacol., 1996, 113, 1419-1429. Prior to use the samples are preferably diluted with PBS buffer 1:6. Upon addition of the antibodies an increase of the intracellular cAMP level can be detected after about 100 to about 150 seconds, as expressed by a decrease in the fluorescence signal generated by the binding of cAMP to the Epac-1 sensor (corresponding to activation of the receptor). The antibody-induced value is calculated as percentage of the value obtained upon addition of 5 μM of the β-adrenergic agonist isoproterenol.
Apart from that, using this particular assay two further antibody positive patient groups were identified: three patients had a receptor antibody having a similar effect on intracellular cAMP-increase (44±2%) and another 19 patients had Epac-1-FRET-positive antibodies which had a significantly lower effect on cAMP-increase (31±5%). Blocking experiments using epitope-homologous peptides showed that the antibodies of the first group are directed against the second extracellular β1-receptor loop, whereas the antibodies of the second group are directed against the first extracellular β1-receptor loop.
This novel method of detecting anti-beta1-Abs proved to be much faster and more sensitive than previous methods and is additionally suitable to identify functionally active β-receptor antibodies against various receptor domains. It also revealed an insufficient ability of β-blockers to prevent the anti-beta1-Ab-induced receptor activation. It opens new venues for the research on anti-beta1-Abs in heart failure and eventual treatment options.
The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 05007056.4 | Mar 2005 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP06/02977 | 3/31/2006 | WO | 00 | 10/1/2008 |
