healthy, and 0.002 for HF + 1 mg/kg COR-1 vs. months at doses of 1 1 to 4 mg/kg body weight. Administration of COR-1 resulted in markedly reduced anti-?1EC2-expressing memory B lymphocytes in the spleen despite continued antigenic boosts, but did not significantly decrease overall peripheral anti-?1EC2 titers. COR-1 did not induce any anti-?1EC2 or other immune response in na?ve rats (corresponding to findings in healthy human volunteers). It did not cause any toxic side effects in GLP studies in dogs, rats or mice, and the no observed adverse effect level (NOAEL) exceeded the therapeutic doses by 100-fold. Conclusion The second generation immunomodulating epitope-mimicking cyclopeptide COR-1 (also termed JNJ-5442840) offers promise to treat immune-mediated cardiac diseases. Introduction Heart failure (HF) is a life-threatening syndrome characterized by shortness of breath, fluid retention, and reduced cardiac function. Despite recent advances in pharmacotherapy, about 50% of patients die within four years[1]. One key player in the regulation of cardiac function is the beta1-adrenergic receptor (?1-AR) situated in the membrane of cardiomyocytes. Upon physical or psychical stress ?1-AR transmit some of the effects of catecholamines to the heart[2C4]. Whereas short-term adrenergic stimulation serves to temporarily improve cardiac performance on demand, chronic activation of the sympathetic nervous system has the opposite effect, and over time leads to progressive deterioration of cardiac structure and function[5]. Several studies have shown that many heart failure patients exhibit catecholamine-like acting autoantibodies directed against the cardiac ?1-AR (anti-?1Cabs)[6C9]. Such receptor-stimulating anti-?1Cabs are particularly found in patients with idiopathic dilated cardiomyopathy (DCM), a non-ischemic heart muscle disease of unknown etiology characterized by dilatation and impaired contraction of the left ventricle[10]. Clinically, the Purvalanol A presence of stimulating anti-?1Cabs has been associated with a more severely reduced cardiac function[11], a higher incidence of life-threatening ventricular arrhythmias and sudden cardiac death[12], and an increased cardiovascular mortality risk[13]. However, efficient and specific therapeutic strategies to combat these harmful receptor-antibodies are still lacking. Most functional anti-?1Cabs were shown to target the second extracellular loop of the ?1-AR protein (?1EC2), representing the largest of in total three EC-loops and, thus, a readily accessible Purvalanol A target on the cell surface[7,14]. Moreover, ?1EC2 contains T- and B-cell epitopes[15] turning it into a potent self-antigen. The receptors crystal structure suggests that ?1EC2 is essential for the stabilization and locking of the receptors catecholamine-binding pocket[14,16]. Thus, it seems conceivable that conformational anti-?1EC2Cabs may allosterically increase ?1-receptor activity[7,17]. Monthly immunization of Lewis rats with fusion proteins containing ?1EC2 gives rise to stimulating anti-?1EC2Cabs. Within 9 months anti-?1EC2Cpositive rats develop progressive left ventricular dilatation, wall thinning, and downregulation Purvalanol A of cardiac ?1-AR,a feature typical for human DCM [6,18,19]. We found that ?1EC2Cmimicking cyclopeptides given either (a) shortly after the induction Purvalanol A of Mdk stimulating anti-?1EC2Cabs or (b) in overt heart failure strongly improved the development and/or course of heart failure[20]. They were more efficient than the clinically used ?1-AR receptor blocker bisoprolol[20]. In this follow-up study, we investigated whether the novel cyclic peptide COR-1 (also termed JNJ-5442840) also improves important functional and immunological parameters which characterise autoimmune heart failure. We also tested COR-1 effects on na?ve animals, and potential side effects in comprehensive toxicological and pharmacokinetic studies. Materials and methods Generation and characterization of ?1-EC2-homologous cyclopeptides Cyclic peptides (CP) were synthesized by Polypeptide, Strasbourg, France according to described protocols of fluorenylmethoxycarbonyl (FMOC) resin-based amino acid chain elongation, and subsequent head-to-tail cyclisation. Fmoc-Asp(OBut)-(Dmb)Gly-OH was attached to a 2-chlorotrityl chloride resin (MERCK/NOVA BIOCHEM) yielding a resin of 0,30 mmol/g. Peptide synthesis was done by a standard cycle of deblocking with 30% piperidine/ N,N-dimethylformamide (DMF) (5+12 min) and coupling with 3 eq. Fmoc-amino acid/TBTU/6 eq. N-methylmorpholine (NMM) in DMF (double coupling, 2 x 30 min). After cleavage from the resin by 20% hexafluoroisopropanol (HFIP)/DCM (2 x 20 min), the isolated crude peptides were cyclized by 3 eq 7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP)/ 5 eq. diisopropylethylamine (DIEA) in DMF overnight, the solvent was evaporated and the crude peptides were deblocked by trifluoroacetic acid (TFA)/water/ thioanisol Purvalanol A (TIS) (95:5: 3) in 2h. Then, the peptides were purified up to 95% by means of HPLC and analyzed by MALDI-TOF mass spectrometry. Intramolecular disulphide bridges between cysteins form and reproducibly at these circumstances spontaneously. The produced cyclopeptide ?1EC2-CP was biochemically analyzed by ruthless water chromatography (HPLC), and by mass spectroscopy (MALDI). HPLC was completed within a Waters Parting Modul 2690 using a Waters Dual Lambda absorbance detector jointly; absorbance was read at 220 nm. After cyclization and peptide-synthesis, the samples had been dissolved in.