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Discovery and Characterization of a Potent Interleukin-6 Binding Peptide with Neutralizing Activity In Vivo.

Ranganath S, Bhandari A, Avitahl-Curtis N, McMahon J, Wachtel D, Zhang J, Leitheiser C, Bernier SG, Liu G, Tran TT, Celino H, Tobin J, Jung J, Zhao H, Glen KE, Graul C, Griffin A, Schairer WC, Higgins C, Reza TL, Mowe E, Rivers S, Scott S, Monreal A, Shea C, Bourne G, Coons C, Smith A, Tang K, Mandyam RA, Masferrer J, Liu D, Patel DV, Fretzen A, Murphy CA, Milne GT, Smythe ML, Carlson KE - PLoS ONE (2015)

Bottom Line: Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys.Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP).This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.

View Article: PubMed Central - PubMed

Affiliation: Discovery Biology, Ironwood Pharmaceuticals, Cambridge, MA, United States of America.

ABSTRACT
Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman's Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.

No MeSH data available.


Related in: MedlinePlus

Determination of PEGylation site and effect of PEGylation on IC50 and plasma exposure.A. Ribbon diagram of ZDC (in cyan) bound to IL-6. Tolerated PEGylation sites on ZDC are indicated in green, and the final PEGylation site, position 29, is labeled. B. Five positions on the non-binding exoface of PN-2171 (sequence shown in S1 Table) were scanned with Lys, Nε-acetylated Lys (Lys-Ac), and LysPEG20L for their effect on IL-6 induced pSTAT3 in U937 cells. IC50 values are averages of n = 2 determinations. C. Effect of PEG molecular weight on the activity of peptides. PN-2519 is the non-PEGylated parent, PN-2520 contains a PEG40Br, PN-2566 contains PEG30L and PN-2567 contains a PEG20Br, all PEGylated at position 29. D. PK analysis in rats of peptide analogs with different molecular weight PEG moieties. In this experiment, the non-PEGylated comparator is PN-2365 (a PN-2519 analog with K26, K29). Peptides were administered by SC injection at 0.23 μmol/kg and plasma samples were taken at the indicated time points.
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pone.0141330.g002: Determination of PEGylation site and effect of PEGylation on IC50 and plasma exposure.A. Ribbon diagram of ZDC (in cyan) bound to IL-6. Tolerated PEGylation sites on ZDC are indicated in green, and the final PEGylation site, position 29, is labeled. B. Five positions on the non-binding exoface of PN-2171 (sequence shown in S1 Table) were scanned with Lys, Nε-acetylated Lys (Lys-Ac), and LysPEG20L for their effect on IL-6 induced pSTAT3 in U937 cells. IC50 values are averages of n = 2 determinations. C. Effect of PEG molecular weight on the activity of peptides. PN-2519 is the non-PEGylated parent, PN-2520 contains a PEG40Br, PN-2566 contains PEG30L and PN-2567 contains a PEG20Br, all PEGylated at position 29. D. PK analysis in rats of peptide analogs with different molecular weight PEG moieties. In this experiment, the non-PEGylated comparator is PN-2365 (a PN-2519 analog with K26, K29). Peptides were administered by SC injection at 0.23 μmol/kg and plasma samples were taken at the indicated time points.

Mentions: The remaining amino acids at positions 16, 26 and 29 of PN-2729 were optimized during evaluation of potential sites for PEGylation. It was anticipated, and indeed observed (Fig 2D), that an unmodified constrained peptide (PN-2365; S2 Table) would be cleared rapidly upon systemic administration. Since the intent was to develop an anti-IL-6 peptide that had prolonged PK, a PEGylation strategy was adopted. PEGylation of peptides and proteins has been extensively utilized to enhance PK properties [26]. To enable site-specific PEGylation, peptide analogs were required that had only one Lys (free amine) for acylation with activated PEG. Consequently Lys residues at position 26 and 29 were changed to Arg, and cell-based assays suggested that these changes were relatively well tolerated. R26 in PN-2519 was derived from this analysis (Table 1).


Discovery and Characterization of a Potent Interleukin-6 Binding Peptide with Neutralizing Activity In Vivo.

Ranganath S, Bhandari A, Avitahl-Curtis N, McMahon J, Wachtel D, Zhang J, Leitheiser C, Bernier SG, Liu G, Tran TT, Celino H, Tobin J, Jung J, Zhao H, Glen KE, Graul C, Griffin A, Schairer WC, Higgins C, Reza TL, Mowe E, Rivers S, Scott S, Monreal A, Shea C, Bourne G, Coons C, Smith A, Tang K, Mandyam RA, Masferrer J, Liu D, Patel DV, Fretzen A, Murphy CA, Milne GT, Smythe ML, Carlson KE - PLoS ONE (2015)

Determination of PEGylation site and effect of PEGylation on IC50 and plasma exposure.A. Ribbon diagram of ZDC (in cyan) bound to IL-6. Tolerated PEGylation sites on ZDC are indicated in green, and the final PEGylation site, position 29, is labeled. B. Five positions on the non-binding exoface of PN-2171 (sequence shown in S1 Table) were scanned with Lys, Nε-acetylated Lys (Lys-Ac), and LysPEG20L for their effect on IL-6 induced pSTAT3 in U937 cells. IC50 values are averages of n = 2 determinations. C. Effect of PEG molecular weight on the activity of peptides. PN-2519 is the non-PEGylated parent, PN-2520 contains a PEG40Br, PN-2566 contains PEG30L and PN-2567 contains a PEG20Br, all PEGylated at position 29. D. PK analysis in rats of peptide analogs with different molecular weight PEG moieties. In this experiment, the non-PEGylated comparator is PN-2365 (a PN-2519 analog with K26, K29). Peptides were administered by SC injection at 0.23 μmol/kg and plasma samples were taken at the indicated time points.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4640888&req=5

pone.0141330.g002: Determination of PEGylation site and effect of PEGylation on IC50 and plasma exposure.A. Ribbon diagram of ZDC (in cyan) bound to IL-6. Tolerated PEGylation sites on ZDC are indicated in green, and the final PEGylation site, position 29, is labeled. B. Five positions on the non-binding exoface of PN-2171 (sequence shown in S1 Table) were scanned with Lys, Nε-acetylated Lys (Lys-Ac), and LysPEG20L for their effect on IL-6 induced pSTAT3 in U937 cells. IC50 values are averages of n = 2 determinations. C. Effect of PEG molecular weight on the activity of peptides. PN-2519 is the non-PEGylated parent, PN-2520 contains a PEG40Br, PN-2566 contains PEG30L and PN-2567 contains a PEG20Br, all PEGylated at position 29. D. PK analysis in rats of peptide analogs with different molecular weight PEG moieties. In this experiment, the non-PEGylated comparator is PN-2365 (a PN-2519 analog with K26, K29). Peptides were administered by SC injection at 0.23 μmol/kg and plasma samples were taken at the indicated time points.
Mentions: The remaining amino acids at positions 16, 26 and 29 of PN-2729 were optimized during evaluation of potential sites for PEGylation. It was anticipated, and indeed observed (Fig 2D), that an unmodified constrained peptide (PN-2365; S2 Table) would be cleared rapidly upon systemic administration. Since the intent was to develop an anti-IL-6 peptide that had prolonged PK, a PEGylation strategy was adopted. PEGylation of peptides and proteins has been extensively utilized to enhance PK properties [26]. To enable site-specific PEGylation, peptide analogs were required that had only one Lys (free amine) for acylation with activated PEG. Consequently Lys residues at position 26 and 29 were changed to Arg, and cell-based assays suggested that these changes were relatively well tolerated. R26 in PN-2519 was derived from this analysis (Table 1).

Bottom Line: Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys.Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP).This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.

View Article: PubMed Central - PubMed

Affiliation: Discovery Biology, Ironwood Pharmaceuticals, Cambridge, MA, United States of America.

ABSTRACT
Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman's Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.

No MeSH data available.


Related in: MedlinePlus