Atrial natriuretic peptide-Fc, ANP-Fc, fusion proteins: semisynthesis, in vitro activity and pharmacokinetics in rats.

Atrial natriuretic peptide (ANP) may be a useful molecule for the treatment of cardiovascular diseases due to its potent natriuretic effects. In an effort to prolong the short in vivo half-life of ANP, fusions of the peptide to the Fc domain of IgG were generated using a semisynthetic methodology. Synthetic ANP peptides were synthesized with thioesters at either the N- or C-termini of the peptide and subsequently linked to the N-terminus of recombinantly expressed Fc using native chemical ligation. The linker length between the ANP and Fc moieties was varied among 2, 11, or 16 amino acids. In addition, either one ("monomeric") or two ("dimeric") ANP peptides were linked to Fc to study whether this modification had an effect on in vitro activity and/or in vivo half-life. The various constructs were studied for in vitro activity using a cell-based cGMP assay. The ANP-Fc fusion constructs were between 16- and ∼375-fold weaker than unconjugated ANP in this assay, and a trend was observed where the most potent conjugates were those with longer linkers and in the dimeric configuration. The pharmacokinetics of several constructs were assessed in rats, and the half-life of the ANP-Fc's were found to be approximately 2 orders of magnitude longer than that of the unconjugated peptide. There was no significant difference in terminal half-life between the monomeric and dimeric constructs (2.8-5.5 h), but a trend was observed where the C(max) of the monomeric constructs was approximately 3-fold higher than that of the dimeric constructs, although the origin of this effect is not understood. These novel ANP-Fc fusion constructs hold promise for future therapeutic application in the treatment of cardiovascular diseases.

PEGylation enhances the therapeutic potential of peptide antagonists of the neonatal Fc receptor, FcRn.

Peptides targeting the human neonatal Fc receptor (FcRn) were conjugated to poly(ethylene glycol) (PEG) polymers to study their effect on inhibition of the IgG:FcRn protein-protein interaction both in vitro and in mice. Both linear (5-40kDa) and branched (20, 40kDa) PEG aldehydes were conjugated to an amine-containing linker of a homodimeric anti-FcRn peptide using reductive alkylation chemistry. It was found that conjugation of PEG to the peptide compromised the in vitro activity, with larger and branched PEGs causing the most dramatic losses in activity. The conjugates were evaluated in transgenic mice for their ability to accelerate the catabolism of human IgG. Optimal pharmacodynamic properties were observed with PEG-peptide conjugates that contained 20-40kDa linear PEGs and a 20kDa branched PEG. The optimal PEG-peptide conjugates were more effective in vivo than the unconjugated peptide control on a mole:mole and mg/kg basis, and represent potential new longer-acting peptide therapeutics for the treatment of humorally-mediated autoimmune disease.

Synthesis and structure-activity relationships of dimeric peptide antagonists of the human immunoglobulin G-human neonatal Fc receptor (IgG-FcRn) interaction.

The neonatal Fc receptor, FcRn, regulates the half-life of IgG in vivo and may be a target in the treatment of autoimmune disease. Monomeric peptide antagonists of the human IgG-human FcRn interaction were dimerized using three different synthetic methodologies: thiol/alkyl halide coupling of unprotected peptides, reductive alkylation of unprotected peptides, and on-resin amide bond formation with protected peptides. It was found that dimerization of monomeric peptides increased the in vitro activity of the peptide monomers more than 200-fold. Human IgG catabolism experiments in human FcRn transgenic mice were used to assess the in vivo activity of peptide dimers that possessed different linkers, cyclizations, and affinities for FcRn. Overall, it was found that the linker joining two monomeric peptides had only a minor effect on the in vitro potency but that in vitro potency was predictive of in vivo activity.

Aspirin at low-intermediate concentrations protects retinal vessels in experimental diabetic retinopathy through non-platelet-mediated effects.

The prevention of diabetic retinopathy requires drugs that leverage the benefits of glycemic control without adding the burden of side effects. Aspirin at dosages of 1-1.5 g/day has prevented manifestations of diabetic retinal microangiopathy in a clinical trial as well as in studies with dogs. Because lower and safer doses of aspirin could be used if its beneficial effects on retinopathy were due to antithrombotic effects, we compared the effects of a selective antiplatelet drug (clopidogrel) to those of aspirin in streptozotocin-induced diabetic rats. Clopidogrel did not prevent neuronal apoptosis, glial reactivity, capillary cell apoptosis, or acellular capillaries in the retina of diabetic rats. Aspirin, at doses yielding serum levels (<0.6 mmol/l) well below the anti-inflammatory range for humans, prevented apoptosis of capillary cells and the development of acellular capillaries but did not prevent neuroglial abnormalities. The aldose reductase inhibitor sorbinil, used as the benchmark for the effect of the other drugs, prevented all abnormalities. The diabetic rat retina showed increased expression of the transcription factor CCAAT/enhancer-binding protein-beta, one of the known targets of low-intermediate concentrations of aspirin. Thus we found a spectrum of drug efficacy on the prevention of experimental diabetic retinopathy, ranging from the absent effect of a selective antiplatelet drug to the prevention of all abnormalities by an aldose reductase inhibitor. Aspirin at low-intermediate concentrations selectively prevented microangiopathy. The minimal effective dose of aspirin should now be sought.

Expression of acute-phase response proteins in retinal Müller cells in diabetes.

PURPOSE: To characterize the whole spectrum of gene expression changes induced by diabetes in retinal Müller glial cells. METHODS: Müller cells were isolated from the retina of streptozotocin-diabetic and age-matched control rats by gradient centrifugation and immediately processed for RNA isolation. The gene expression profile of Müller cells was studied with the GeneChip Rat Genome oligonucleotide array (Affymetrix, Santa Clara, CA). The upregulation of acute-phase proteins in the retina of diabetic rats was confirmed by Northern and Western blot analyses. Real-time-RT-PCR was used to study the retinal expression of inflammatory cytokines. RESULTS: Gene expression profiling identified 78 genes as differentially expressed in diabetic Müller cells. One third of these genes were associated with inflammation, including a large cluster (18% of the differentially expressed genes) of acute-phase response proteins: alpha2-macroglobulin, ceruloplasmin, complement components, lipocalin-2, metallothionein, serine protease inhibitor-2, transferrin, tissue inhibitor of metalloproteases-1, transthyretin, and the transcription factor C/EBPdelta. Northern and Western blot analyses confirmed the upregulation of alpha2-macroglobulin and ceruloplasmin in the diabetic retina, but not in the cerebral cortex and liver of the same animals. The acute-phase response of Müller cells in diabetes was associated with upregulation of interleukin (IL)-1beta in the retina. CONCLUSIONS: Müller cells acquire a complex and specific reactive phenotype in diabetes characterized by the induction of acute-phase response proteins and other inflammation-related genes. The concomitant upregulation of IL-1beta in the retina of diabetic rats points to this cytokine as a possible mediator of the acute-phase response mounted by Müller cells in diabetes.

Studies of rat and human retinas predict a role for the polyol pathway in human diabetic retinopathy.

The polyol (sorbitol) pathway of glucose metabolism is activated in many cell types when intracellular glucose concentrations are high, and it can generate cellular stress through several mechanisms. The role of the polyol pathway in the pathogenesis of diabetic retinopathy has remained uncertain, in part because it has been examined preferentially in galactose-induced retinopathy and in part because inhibition studies may not have achieved full blockade of the pathway. Having observed that the streptozotocin-induced diabetic rat accurately models many cellular processes characteristic of human diabetic retinopathy, we tested in the diabetic rat if documented inhibition of the polyol pathway prevents a sequence of retinal vascular abnormalities also present in human diabetes. An inhibitor of aldose reductase, the rate-limiting enzyme in the pathway, prevented the early activation of complement in the wall of retinal vessels and the decreased levels of complement inhibitors in diabetic rats, as well as the later apoptosis of vascular pericytes and endothelial cells and the development of acellular capillaries. Both rat and human retinal endothelial cells showed aldose reductase immunoreactivity, and human retinas exposed to high glucose in organ culture increased the production of sorbitol by a degree similar to that observed in the rat. Excess aldose reductase activity can be a mechanism for human diabetic retinopathy.

A role for the polyol pathway in the early neuroretinal apoptosis and glial changes induced by diabetes in the rat.

We tested the hypothesis that the apoptosis of inner retina neurons and increased expression of glial fibrillary acidic protein (GFAP) observed in the rat after a short duration of diabetes are mediated by polyol pathway activity. Rats with 10 weeks of streptozotocin-induced diabetes and GHb levels of 16 +/- 2% (mean +/- SD) showed increased retinal levels of sorbitol and fructose, attenuation of GFAP immunostaining in astrocytes, appearance of prominent GFAP expression in Müller glial cells, and a fourfold increase in the number of apoptotic neurons when compared with nondiabetic rats. The cells undergoing apoptosis were immunoreactive for aldose reductase. Sorbinil, an inhibitor of aldose reductase, prevented all abnormalities. Intensive insulin treatment also prevented most abnormalities, despite reducing GHb only to 12 +/- 1%. Diabetic mice, known to have much lower aldose reductase activity in other tissues when compared with rats, did not accumulate sorbitol and fructose in the retina and were protected from neuronal apoptosis and GFAP changes in the presence of GHb levels of 14 +/- 2%. This work documents discrete cellular consequences of polyol pathway activity in the retina, and it suggests that activation of the pathway and "retinal neuropathy" require severe hyperglycemia and/or high activity of aldose reductase. These findings have implications for how to evaluate the role of the polyol pathway in diabetic retinopathy.