Alginate Microsponges as a Scaffold for Delivery of a Therapeutic Peptide against Rheumatoid Arthritis.

The quest for biocompatible drug-delivery devices that could be able to open new administration routes is at the frontier of biomedical research. In this contribution, porous polysaccharide-based microsponges based on crosslinked alginate polymers were developed and characterized by optical spectroscopy and nanoscopic microscopy techniques. We show that macropores with a size distribution ranging from 50 to 120 nm enabled efficient loading and delivery of a therapeutic peptide (CIGB814), presently under a phase 3 clinical trial for the treatment of rheumatoid arthritis. Alginate microsponges showed 80% loading capacity and sustained peptide release over a few hours through a diffusional mechanism favored by partial erosion of the polymer scaffold. The edible and biocompatible nature of alginate polymers open promising perspectives for developing a new generation of polysaccharide-based carriers for the controlled delivery of peptide drugs, exploiting alternative routes with respect to intravenous administration.

Biodistribution and pharmacokinetic profiles of an altered peptide ligand derived from heat-shock proteins 60 in Lewis rats.

Human heat-shock protein 60 (HSP60) is an autoantigen involved in the pathogenesis of rheumatoid arthritis (RA). Epitopes derived from HSP60 can trigger activation of regulatory T cells (Treg). CIGB-814 is an altered peptide ligand (APL) derived from HSP60. In preclinical models, this peptide had anti-inflammatory effects and increased Treg. The results from phase I clinical trial indicated that CIGB-814 was safe and activated mechanisms associated with induction of tolerance. Biodistribution profile for inducers of tolerance is crucial for triggering its effects. The primary goal of this study in Lewis rats was to identify (1) the target organs of CIGB-814 and (2) the pharmacokinetics (PK) profile. 125I-CIGB-814 administered subcutaneously at three dose levels was distributed in the thyroid gland, but also at considerable levels to the stomach and small and large intestines. In addition, concentration of CIGB-814 was increased in lymph nodes (LNs) at 24 h, compared with 4-h post-administration. Small intestine and LNs are excellent sites for induction of tolerance, due to the characteristics of dendritic cells in these tissues. Maximum concentration of CIGB-814 in blood of Lewis rats at 0.5 to 1 h agrees with PK profile determined for patients. Altogether, these results support therapeutic possibilities of CIGB-814 for RA.

Development and validation of a bioanalytical method based on LC-MS/MS analysis for the quantitation of CIGB-814 peptide in plasma from Rheumatoid Arthritis patients.

CIGB-814, originally named as E18-3 APL1 or APL1 in preclinical experiments, is a novel therapeutic peptide candidate for Rheumatoid Arthritis (RA). It is an altered peptide ligand containing a novel CD4+ T-cell epitope of human heat shock protein 60 (83-109, MW 2988.38g/mol) with a mutation (D100→L) that increases its affinity for HLA-II type molecules associated to RA. A bioanalytical method, based on LC-MS/MS analysis, in the SRM mode was developed and fully validated to quantify this peptide in human plasma. An internal standard with the same amino acid sequence but labeled with three (13C615N2)-Lys residues was used for quantitation. The method provides a linear range from 1.5 to 48ng/mL (without matrix effect and carry over) and an accuracy and precision good enough for monitoring more than 80% of the AUC of the PK profile in a phase I clinical trial. The peptide was administered subcutaneously in three dose levels (1, 2.5 and 5mg) not normalized to the body weight of patients with RA. The low doses imposed an analytical challenge; however, a LLOQ of 1.5ng/mL enabled the PK analysis. The Cmax, reached at 0.5h, showed a great variability, that was most likely due to the non-normalized doses; the proposed mechanism for this peptide; and the variability between patients. A rapid clearance of this peptide (4-6h) is advantageous for an immunomodulatory drug, because the therapeutic schedule requires repeated dosages to restore peripheral tolerance.

APL-1, an altered peptide ligand derived from heat-shock protein, alone or combined with methotrexate attenuates murine collagen-induced arthritis.

Induction of tolerance to autoantigens in vivo is a complex process that involves several mechanisms such as the induction of regulatory T cells and changes in the cytokine and chemokine profiles. This approach represents an attractive alternative for treatment of autoimmune diseases. APL-1 is an altered peptide ligand derived from a novel CD4 + T cell epitope of human heat-shock protein of 60 kDa (HSP60), an autoantigen involved in the pathogenesis of rheumatoid arthritis (RA). We have shown previously that this peptide efficiently inhibited the course of adjuvant-induced arthritis in Lewis rats and induced regulatory T cell (Treg) in ex vivo assay with PBMC isolated from RA patients. This study was undertaken to evaluate the therapeutic effect of APL-1 and its combination with methotrexate (MTX) in collagen-induced arthritis (CIA). CIA was induced in male DBA/1 mice at 8 weeks of age by immunization with chicken collagen. APL, MTX or both were administrated beginning from arthritis onset. Therapeutic effect was evaluated by arthritis and joint pathologic scores. In addition, TNFα and IL-10 in sera were measured by ELISA. Treg induction was assessed by FACS analysis. APL-1 inhibits efficiently the course of arthritis in CIA, similar to MTX. In addition, therapy with APL-1 plus MTX reduced CIA in mice, associated with an increase in Treg. These facts reinforce the therapeutic possibilities of APL-1 as a candidate drug for treatment of RA.

APL1, an altered peptide ligand derived from human heat-shock protein 60, increases the frequency of Tregs and its suppressive capacity against antigen responding effector CD4 + T cells from rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by a chronic relapsing-remitting joint inflammation. Perturbations in the balance between CD4 + T cells producing IL-17 and CD4 + CD25(high)FoxP3 + Tregs correlate with irreversible bone and cartilage destruction in RA. APL1 is an altered peptide ligand derived from a CD4+ T-cell epitope of human HSP60, an autoantigen expressed in the inflamed synovium, which increases the frequency of CD4 + CD25(high)FoxP3+ Tregs in peripheral blood mononuclear cells from RA patients. The aim of this study was to evaluate the suppressive capacity of Tregs induced by APL1 on proliferation of effector CD4+ T cells using co-culture experiments. Enhanced Treg-mediated suppression was observed in APL1-treated cultures compared with cells cultured only with media. Subsequent analyses using autologous cross-over experiments showed that the enhanced Treg suppression in APL1-treated cultures could reflect increased suppressive function of Tregs against APL1-responsive T cells. On the other hand, APL1-treatment had a significant effect reducing IL-17 levels produced by effector CD4+ T cells. Hence, this peptide has the ability to increase the frequency of Tregs and their suppressive properties whereas effector T cells produce less IL-17. Thus, we propose that APL1 therapy could help to ameliorate the pathogenic Th17/Treg balance in RA patients.

APL-2, an altered peptide ligand derived from heat-shock protein 60, induces interleukin-10 in peripheral blood mononuclear cell derived from juvenile idiopathic arthritis patients and downregulates the inflammatory response in collagen-induced arthritis model.

Juvenile idiopathic arthritis (JIA) is a heterogeneous group of diseases characterized by autoimmune arthritis of unknown cause with onset before age of 16 years. Methotrexate provides clinical benefits in JIA. For children who do not respond to methotrexate, treatment with anti-tumor necrosis factor (TNF)-α is an option. However, some patients do not respond or are intolerant to anti-TNF therapy. Induction of peripheral tolerance has long been considered a promising approach to the treatment of chronic autoimmune diseases. We aimed to evaluate the potentialities of two altered peptide ligands (APLs) derived from human heat-shock protein 60, an autoantigen involved in the pathogenesis of autoimmune arthritis, in JIA patients. Interferon (IFN)-γ, TNF-α and interleukin (IL)-10 levels were determined in ex vivo assays using peripheral blood mononuclear cells (PBMC) from these patients. Wild-type peptide and one of these APLs increased IFN-γ and TNF-α levels. Unlike, the other APLs (called APL2) increased the IL-10 level without affecting IFN-γ and TNF-α levels. On the other hand, APL2 induces a marked activation of T cells since it transforms cell cycle phase's distribution of CD4+ T cells from these patients. In addition, we evaluated the therapeutic effect of APL2 in collagen-induced arthritis model. Therapy with APL2 reduced arthritis scores and histological lesions in mice. This effect was associated to a decrease in TNF-α and IL-17 levels. These results indicate a therapeutic potentiality of APL2 for JIA.