ARA-290 was built by scientists to keep only the "repair and calm inflammation" part of erythropoietin (EPO), the hormone your kidneys make to boost red blood cells, while stripping out the part that thickens your blood. It has been tested mainly as an experimental treatment for painful nerve damage (small-fiber neuropathy) in people with sarcoidosis and diabetes, with smaller side projects looking at diabetic eye disease, organ transplants, gut inflammation, and aging. It is not an approved medicine anywhere; it exists only as a research and clinical-trial drug developed under the name cibinetide. Because it is derived from EPO, it is also on the World Anti-Doping Agency's banned substance list for athletes.
How strong is the evidence?
Of the 34 papers reviewed, about a third are real human studies - five small phase 2 clinical trials (9 to 64 people each) in sarcoidosis nerve pain, diabetic neuropathy, and diabetic eye disease, plus a few reviews summarizing that same trial data. The rest are mouse, rat, and lab-dish studies exploring how it works and whether it could help with things like organ transplants, gut inflammation, heart aging, and bone loss. The human trials for nerve pain are consistent and encouraging, but every one of them is small and short (4 to 12 weeks), run mostly by the peptide's original developers, and no large or long-term trial has been done. One human trial (diabetic eye disease) largely failed to show benefit. That mix - real but limited human data, no approved use - is why this sits at "limited human evidence," not "clinical."
Uses
What people use it for
Nerve pain from small-fiber neuropathy (sarcoidosis)
Some human dataThe best-studied use. Several small trials in people with sarcoidosis-related nerve pain and damage found ARA-290 reduced pain scores and, in the better trials, actually helped small nerve fibers regrow.
Diabetic nerve pain and blood sugar control
Some human dataOne phase 2 trial in people with type 2 diabetes and painful neuropathy found improvements in blood sugar (HbA1c), cholesterol levels, and nerve pain scores over about two months.
Diabetic eye disease (macular edema)
Some human dataTested in a small trial as a way to protect the retina in diabetes-related swelling of the eye. It was safe, but it mostly failed to improve the actual eye measurements (vision, retinal thickness) researchers were tracking.
General anti-inflammatory / tissue-repair research
Animal / labExplored almost entirely in mice, rats, and cell dishes as a way to protect organs from inflammation and injury - gut inflammation, transplanted pancreatic islets, diabetic wounds, kidney damage from chemotherapy, and age-related heart decline. None of this has reached human testing yet.
Potential benefits
What it may help with
Less neuropathic pain and measurable nerve fiber regrowth
Some human dataIn sarcoidosis patients with small-fiber neuropathy, ARA-290 reduced pain scores versus placebo and, in a 64-person dose-finding trial, increased the density of tiny nerve fibers in the cornea (a proxy for nerve healing elsewhere in the body). The 4 mg/day dose showed the clearest effect.
Improved blood sugar and cholesterol in type 2 diabetes
Some human dataA month of daily dosing improved HbA1c (a 2-3 month average blood sugar marker) and lipid levels in diabetic patients with nerve pain, alongside improved pain scores.
Studies:25387363Better quality-of-life scores even when objective measures didn't move
Some human dataIn the diabetic eye disease trial, standard eye tests (vision, retinal thickness) didn't improve, but patients reported better scores on a validated quality-of-life questionnaire.
Studies:32674280Calms inflammation and protects tissue in animal and lab models
Animal / labAcross many disease models - colitis, transplanted pancreatic islets, diabetic wounds, kidney injury from chemotherapy drugs - ARA-290 consistently reduced inflammatory signaling and tissue damage. This is the core mechanism researchers keep testing in new organs and diseases.
May help nerves recover after injury or autoimmune attack
Animal / labIn rodent models of nerve injury and autoimmune nerve damage, ARA-290 reduced pain behavior and helped nerves regenerate, with effects lasting weeks after dosing stopped.
May protect the aging heart and reduce frailty
Animal / labIn a 15-month study of aged rats, long-term ARA-290 treatment reduced heart inflammation, preserved heart pumping function, and lowered markers of overall frailty compared to untreated rats.
Studies:36741836May increase bone density
Animal / labA month of treatment increased bone mineral density in mice by roughly 5%, and reduced the bone-eating cells (osteoclasts) that EPO itself tends to activate.
Studies:35008482
What to watch for
Side effects & risks
- Mild
- Moderate
Long-term and large-scale safety is unknown
Every human trial lasted 4 to 12 weeks with fewer than 65 people. Nobody has studied what happens with repeated cycles, higher doses, or use over years.
Dosing
Dosing — what studies used
There is no approved or standard human dose - ARA-290 is not a marketed drug. In the clinical trials that exist, researchers most commonly used 4 mg injected under the skin once a day for 4 to 12 weeks, though doses from 1 to 8 mg/day and an IV version (2 mg, three times a week) have also been tested. These were all medically supervised research doses using pharmaceutical-grade material, not real-world prescriptions, and nothing about repeat cycles or long-term use has been studied.
Sarcoidosis small-fiber neuropathy (early pilot trial)
Human trial2 mg
3 times per week · 4 weeks · Intravenous
First controlled human trial (22 patients); safe, reduced neuropathy symptom scores versus placebo.
Sarcoidosis small-fiber neuropathy (dose-finding trial)
Human trial1, 4, or 8 mg per day
Once daily · 28 days · Subcutaneous
64 patients; the 4 mg/day group showed the clearest improvement in corneal nerve fiber regrowth and pain.
Sarcoidosis small-fiber neuropathy (earlier daily-dosing trial)
Human trialNot stated in the published summary
Once daily · 28 days · Subcutaneous
Confirmed both pain relief and corneal nerve fiber regrowth; later replicated at 4 mg/day in a larger trial.
Type 2 diabetes with painful neuropathy
Human trial4 mg
Once daily · 28 days, with a 28-day drug-free follow-up · Subcutaneous
Self-administered by patients; improved blood sugar, cholesterol, and nerve pain scores.
Diabetic macular edema (eye disease)
Human trial4 mg
Once daily · 12 weeks · Subcutaneous
Self-administered; safe, but didn't meaningfully improve the eye measurements researchers were tracking.
Nerve injury pain (animal dose-response study)
Animal study3 to 60 micrograms per kg body weight
5 doses over 10 days · Effects tracked for up to 20 weeks · Subcutaneous
Rat study; 30 mcg/kg was the most effective dose for long-lasting pain relief without needing repeat dosing.
All dosing above came from medically supervised trials using pharmaceutical-grade peptide. There is no established at-home protocol, and unregulated "research chemical" versions carry unknown purity and contamination risk.
These figures describe what researchers used in studies. They are not a recommendation or a prescription.
Mechanism
How it works
ARA-290 is a tiny copy of one small piece of the erythropoietin (EPO) molecule - the hormone your kidneys make to tell your bone marrow to produce more red blood cells. Normal EPO does that by locking onto a receptor found on red blood cell precursors. But EPO also, separately, calms down injured or stressed tissue by locking onto a different receptor pairing (found on immune cells and damaged tissue) called the innate repair receptor. ARA-290 was designed to fit only that second receptor, so it can turn down inflammation and help cells survive and heal without telling the bone marrow to make more red blood cells. In practice, it seems to quiet down overactive immune cells (like macrophages), reduce inflammatory signaling in nerves and other tissues, and support nerve regrowth - all without the blood-thickening risk of real EPO.
Who should avoid it
- Not an approved medicine anywhere, so there's no official prescribing guidance or contraindication list.
- Anyone pregnant or breastfeeding, since it has not been studied in these groups.
- Competitive athletes - it is on the WADA banned substances list because it is derived from EPO.
- Anyone hoping to use it as a substitute for standard, proven treatment of diabetes or diagnosed neuropathy rather than alongside medical care.
Interactions to know
- No human drug-interaction studies exist.
- In animal organ-transplant studies it was combined with the immune-suppressing drug tacrolimus without problems, but this pairing hasn't been tested in people outside that specific research context.
The papers that matter most
Key studies
First controlled human trial of ARA-290; safe and reduced neuropathy symptom scores versus placebo over 4 weeks.
Safety and efficacy of ARA 290 in sarcoidosis patients with symptoms of small fiber neuropathy: a randomized, double-blind pilot study
Showed real nerve fiber regrowth, not just symptom relief - plus better exercise capacity and temperature sensation.
ARA 290 improves symptoms in patients with sarcoidosis-associated small nerve fiber loss and increases corneal nerve fiber density
Larger 64-patient dose-finding trial confirmed nerve fiber regrowth and pain relief, with 4 mg/day as the standout dose.
Cibinetide Improves Corneal Nerve Fiber Abundance in Patients With Sarcoidosis-Associated Small Nerve Fiber Loss and Neuropathic Pain
Extended the benefit beyond sarcoidosis - improved blood sugar control and diabetic nerve pain over 8 weeks.
ARA 290, a nonerythropoietic peptide engineered from erythropoietin, improves metabolic control and neuropathic symptoms in patients with type 2 diabetes
Important negative data point: safe, but failed to improve the core eye-health measurements it was designed to fix.
A Phase 2 Clinical Trial on the Use of Cibinetide for the Treatment of Diabetic Macular Edema
Explains the core mechanism - a 2-minute half-life peptide that flips a lasting cellular switch, and why that decouples tissue repair from blood-boosting effects.
Flipping the molecular switch for innate protection and repair of tissues: Long-lasting effects of a non-erythropoietic small peptide engineered from erythropoietin
Bottom line
ARA-290 (cibinetide) has real, repeated small-trial evidence for reducing nerve pain and helping small nerves regrow in sarcoidosis and diabetes, but it's not an approved drug, every trial so far has been small and short, and it flatly failed to help in the one eye-disease trial that tested it. Treat it as a promising research compound worth watching, not a proven treatment - and only under medical supervision if used at all.
Research papers
Studies we have on file for ARA-290. Tap a title to open it on PubMed. Labels like “animal” or “human trial” are rough guides.
34 papers
The time to develop treatments for diabetic neuropathy.
Diabetic neuropathy is a multifaceted condition affecting up to 50% of individuals with long standing diabetes. The most common presentation is peripheral diabetic sensory neuropathy (DPN). We carried out a systematic review of papers dealing with diabetic neuropathy on Pubmed in addition to a targeted Google search.Search terms included small fiber neuropathy,diffuse peripheral neuropathy, quantitative sensory testing, nerve conduction testing, intra-epidermal nerve fiber density, corneal confocal reflectance microscopy, aldose reductase inhbitors, nerve growth factor, alpha-lipoic acid, ruboxistaurin, nerve growth factor antibody, and cibinetide. Over the past half century, there have been a number of agents undergoing unsuccessful trials for treatment of DPN.There are several approved agents for relief of pain caused by diabetic neuropathy, but these do not affect the pathologic process. The failure to find treatments for diabetic neuropathy can be ascribed to (1) the complexity of design of studies and (2) the slow progression of the condition, necessitating long duration trials to prove efficacy.We propose a modification of the regulatory process to permit early introduction of agents with demonstrated safety and suggestion of benefit as well as prolongation of marketing exclusivity while long term trials are in progress to prove efficacy.
The Non-Erythropoietic EPO Analogue Cibinetide Inhibits Osteoclastogenesis In Vitro and Increases Bone Mineral Density in Mice.
The two erythropoietin (EPO) receptor forms mediate different cellular responses to erythropoietin. While hematopoiesis is mediated via the homodimeric EPO receptor (EPOR), tissue protection is conferred via a heteromer composed of EPOR and CD131. In the skeletal system, EPO stimulates osteoclast precursors and induces bone loss. However, the underlying molecular mechanisms are still elusive. Here, we evaluated the role of the heteromeric complex in bone metabolism in vivo and in vitro by using Cibinetide (CIB), a non-erythropoietic EPO analogue that exclusively binds the heteromeric receptor. CIB is administered either alone or in combination with EPO. One month of CIB treatment significantly increased the cortical (~5.8%) and trabecular (~5.2%) bone mineral density in C57BL/6J WT female mice. Similarly, administration of CIB for five consecutive days to female mice that concurrently received EPO on days one and four, reduced the number of osteoclast progenitors, defined by flow cytometry as Lin-CD11b-Ly6Chi CD115+, by 42.8% compared to treatment with EPO alone. In addition, CIB alone or in combination with EPO inhibited osteoclastogenesis in vitro. Our findings introduce CIB either as a stand-alone treatment, or in combination with EPO, as an appealing candidate for the treatment of the bone loss that accompanies EPO treatment.
A Phase 2 Clinical Trial on the Use of Cibinetide for the Treatment of Diabetic Macular Edema.
Evaluating the effects of cibinetide in diabetic macular edema (DME). Phase 2 trial. Naïve patients with >400 µm central retinal thickness (CRT) DME in one/both eyes were recruited (May 2016-April 2017) at the Belfast Health and Social Care Trust. The study eye was that with best vision and lowest CRT. Patients self-administered cibinetide 4 mg/day subcutaneously for 12 weeks. Primary and secondary outcomes: mean change from baseline to week 12 in best corrected visual acuity (BCVA), CRT, central retinal sensitivity, tear production, patient-reported outcomes, adverse events and antibodies to cibinetide. Descriptive statistics were used; exploratory analyses focused on non-study eyes, diabetic control, serum cytokines and albuminuria. Nine patients were recruited; eight completed the study. There was no improvement in mean change baseline-week 12 in BCVA (-2.9 + 5.0), CRT (10 + 94.6 microns), central retinal sensitivity (-0.53 + 1.9 dB) or tear production (-0.13 + 7.7 mm), but there was an improvement in National Eye Institute Visual Function Questionnaire (NEI VFQ-25) composite scores (2.7 + 3.1). Some participants experienced improvements in CRT, tear production, diabetic control and albuminuria. No serious adverse events/reactions or anti-cibinetide antibodies were seen. The cibinetide 12-week course was safe. Improvements in NEI VFQ-25 scores, CRT, tear production, diabetic control and albuminuria, observed in some participants, warrant further investigation. EudraCT number: 2015-001940-12. ISRCTN16962255-registration date 25.06.15.
Cibinetide dampens innate immune cell functions thus ameliorating the course of experimental colitis.
Two distinct forms of the erythropoietin receptor (EPOR) mediate the cellular responses to erythropoietin (EPO) in different tissues. EPOR homodimers signal to promote the maturation of erythroid progenitor cells. In other cell types, including immune cells, EPOR and the ß-common receptor (CD131) form heteromers (the innate repair receptor; IRR), and exert tissue protective effects. We used dextran sulphate sodium (DSS) to induce colitis in C57BL/6 N mice. Once colitis was established, mice were treated with solvent, EPO or the selective IRR agonist cibinetide. We found that both cibinetide and EPO ameliorated the clinical course of experimental colitis in mice, resulting in improved weight gain and survival. Correspondingly, DSS-exposed mice treated with cibinetide or EPO displayed preserved tissue integrity due to reduced infiltration of myeloid cells and diminished production of pro-inflammatory disease mediators including cytokines, chemokines and nitric oxide synthase-2. Experiments using LPS-activated primary macrophages revealed that the anti-inflammatory effects of cibinetide were dependent on CD131 and JAK2 functionality and were mediated via inhibition of NF-κB subunit p65 activity. Cibinetide activation of the IRR exerts potent anti-inflammatory effects, especially within the myeloid population, reduces disease activity and mortality in mice. Cibinetide thus holds promise as novel disease-modifying therapeutic of inflammatory bowel disease.
Early monocyte modulation by the non-erythropoietic peptide ARA 290 decelerates AD-like pathology progression.
Alzheimer's disease (AD) pathology is characterized by amyloid-β (Aβ) deposition and tau hyper-phosphorylation, accompanied by a progressive cognitive decline. Monocytes have been recently shown to play a major role in modulating Aβ pathology, and thereby have been pointed as potential therapeutic targets. However, the main challenge remains in identifying clinically relevant interventions that could modulate monocyte immune functions in absence of undesired off-target effects. Erythropoietin (EPO), a key regulator of erythrocyte production, has been shown to possess immunomodulatory potential and to provide beneficial effects in preclinical models of AD. However, the transition to use recombinant human EPO in clinical trials was hindered by unwanted erythropoietic effects that could lead to thrombosis. Here, we used a recently identified non-erythropoietic analogue of EPO, ARA 290, to evaluate its therapeutic potential in AD therapy. We first evaluated the effects of early systemic ARA 290 administration on AD-like pathology in an early-onset model, represented by young APP/PS1 mice. Our findings indicate that ARA 290 early treatment decelerated Aβ pathology progression in APP/PS1 mice while improving cognitive functions. ARA 290 potently increased the levels of total monocytes by specifically stimulating the generation of Ly6CLow patrolling subset, which are implicated in clearing Aβ from the cerebral vasculature, and subsequently reducing overall Aβ burden in the brain. Moreover, ARA 290 increased the levels of monocyte progenitors in the bone marrow. Using chimeric APP/PS1 mice in which Ly6CLow patrolling subset are selectively depleted, ARA 290 was inefficient in attenuating Aβ pathology and ameliorating cognitive functions in young animals. Interestingly, ARA 290 effects were compromised when delivered in a late-onset model, represented by aged APP1/PS1. In aged APP/PS1 mice in which AD-like pathology is at advanced stages, ARA 290 failed to reverse Aβ pathology and to increase the levels of circulating monocytes. Our study suggests that ARA 290 early systemic treatment could prevent AD-like progression via modulation of monocyte functions by specifically increasing the ratio of patrolling monocytes.
ARA 290 for treatment of small fiber neuropathy in sarcoidosis.
Painful peripheral neuropathy is a common, difficult-to-treat complication associated with a variety of diseases, including diabetes mellitus and sarcoidosis. It is caused by damage of small and autonomic nerve fibers, resulting in potentially debilitating symptoms of neuropathic pain and autonomic dysfunction. The limited efficacy of current treatment options dictates a rationalized design of novel compounds. The authors present the recent data from two Phase II clinical trials on ARA290, an erythropoietin derivative with tissue protective and healing properties that does not stimulate erythropoiesis. ARA 290 treatment was consistently associated with a significant improvement of neuropathic pain symptoms in sarcoidosis patients, evidenced by a decrease in pain scores on validated questionnaires. Moreover, ARA 290 treatment resulted in significant increases in corneal nerve fibers, improved sensory pain thresholds, improved quality of life and physical functioning. Current treatment modalities of neuropathy are based on a trial-and-error approach, have limited efficacy and come with significant side effects. Given the excellent safety profile while reducing neuropathy symptoms, the prospects of ARA 290 treatment in sarcoid neuropathy seem promising. The long-lasting beneficial effects of ARA 290 on both pain-related and non-pain-related symptoms in sarcoidosis patients prompt additional studies on potential disease-modifying properties of ARA 290.
Cibinetide Protects Isolated Human Islets in a Stressful Environment and Improves Engraftment in the Perspective of Intra Portal Islet Transplantation.
During intra-portal pancreatic islet transplantation (PITx), innate immune reactions such as the instant blood mediated inflammatory reaction (IBMIR) cause an immediate loss of islets. The non-hematopoietic erythropoietin analogue cibinetide has previously shown islet-protective effects in mouse PITx. Herein, we aimed to confirm cibinetide's efficacy on human islets, and to characterize its effect on IBMIR. We cultured human islets with pro-inflammatory cytokines for 18 hours with or without cibinetide. ATP content and caspase 3/7 activity were measured. Dynamic glucose perfusion assay was used to evaluate islet function. To evaluate cibinetides effect on IBMIR, human islets were incubated in heparinized polyvinyl chloride tubing system with ABO compatible blood and rotated for 60 minutes to mimic the portal vein system. Moreover, human islets were transplanted into athymic mice livers via the portal vein with or without perioperative cibinetide treatment. The mice were sacrificed six days following transplantation and the livers were analyzed for human insulin and serum for human C-peptide levels. Histological examination of recipient livers to evaluate islet graft infiltration by CD11b+ cells was performed. Our results show that cibinetide maintained human islet ATP levels and reduced the caspase 3/7 activity during culture with pro-inflammatory cytokines and improved their insulin secreting capacity. In the PVC loop system, administration of cibinetide reduced the IBMIR-induced platelet consumption. In human islet to athymic mice PITx, cibinetide treatment showed an increased amount of human insulin in the livers and higher serum human C-peptide, while histological examination of the livers showed reduced infiltration of pro-inflammatory CD11b+ cells around islets grafts compared to the controls. In summary, Cibinetide protected isolated human islets in a pro-inflammatory milieu and reduced IBMIR related platelet consumption. It improved engraftment of human islets in athymic mice. The study confirms that cibinetide is a promising agent to be used in clinical PITx.
Improvement of Islet Allograft Function Using Cibinetide, an Innate Repair Receptor Ligand.
During intraportal pancreatic islet transplantation (PITx), early inflammatory reactions cause an immediate loss of more than half of the transplanted graft and potentiate subsequent allograft rejection. Previous findings suggest that cibinetide, a selective innate repair receptor agonist, exerts islet protective and antiinflammatory properties and improved transplant efficacy in syngeneic mouse PITx model. In a stepwise approach toward a clinical application, we have here investigated the short- and long-term effects of cibinetide in an allogeneic mouse PITx model. Streptozotocin-induced diabetic C57BL/6N (H-2) mice were transplanted with 320 (marginal) or 450 (standard) islets from BALB/c (H-2) mice via the portal vein. Recipients were treated perioperative and thereafter daily during 14 d with cibinetide (120 µg/kg), with or without tacrolimus injection (0.4 mg/kg/d) during days 4-14 after transplantation. Graft function was assessed using nonfasting glucose measurements. Relative gene expressions of proinflammatory cytokines and proinsulin of the graft-bearing liver were assessed by quantitative polymerase chain reaction. Cibinetide's effects on dendritic cell maturation were investigated in vitro. Cibinetide ameliorated the local inflammatory responses in the liver and improved glycemic control immediately after allogeneic PITx and significantly delayed the onset of allograft loss. Combination treatment with cibinetide and low-dose tacrolimus significantly improved long-term graft survival following allogeneic PITx. In vitro experiments indicated that cibinetide lowered bone-marrow-derived-immature-dendritic cell maturation and subsequently reduced allogeneic T-cell response. Cibinetide reduced the initial transplantation-related severe inflammation and delayed the subsequent alloreactivity. Cibinetide, in combination with low-dose tacrolimus, could significantly improve long-term graft survival in allogeneic PITx.
Activation of the EPOR-β common receptor complex by cibinetide ameliorates impaired wound healing in mice with genetic diabetes.
Diabetes is characterized by poor wound healing which currently lacks an efficacious treatment. The innate repair receptor (IRR) is a master regulator of tissue protection and repair which is expressed as a response injury or metabolic stress, including in diabetes. Activation of the IRR might provide benefit for diabetic wound healing. A specific IRR agonist cibinetide was administered in an incisional wound healing model performed mice with genetic diabetes (db+/db+) and compared to the normal wild-type. Animals were treated daily with cibinetide (30μg/kg/s.c.) or vehicle and euthanized 3, 7, and 14days after the injury to quantitate vascular endothelial growth factor (VEGF), malondialdehyde (MAL), phospho-Akt (pAkt), phospho e-NOS (p-eNOS), and nitrite/nitrate content within the wound. Additional evaluations included quantification of skin histological change, angiogenesis, scar strength, and time to complete wound closure. Throughout the wound healing process diabetic animals treated with vehicle exhibited increased wound MAL with reduced VEGF, pAkt, peNOS and nitrite/nitrate, all associated with poor re-epitheliziation, angiogenesis, and wound breaking strength. Cibenitide administration significantly improved these abnormalities. The results suggest that cibinetide-mediated IRR activation may represent an interesting strategy to treat diabetes-associated wound healing.
Phase-targeted erythropoietin derivatives for traumatic brain injury: bridging mechanisms to precision therapy.
Traumatic brain injury (TBI) unfolds through a well-defined chronology-hyperacute excitotoxic and inflammasome bursts, acute apoptotic and blood-brain-barrier failure, and subacute neurovascular remodeling-that no single-pathway drug can adequately cover. Recombinant erythropoietin (EPO) limits secondary damage in animals, yet its erythropoietic drive and thrombotic liability have stalled clinical adoption. This review integrates structural biology, pharmacology and translational data on four engineered EPO derivatives-carbamylated EPO, asialo-EPO, darbepoetin alfa and the helix-B surface peptide (HBSP/cibinetide)-that decouple cytoprotection from red-cell stimulation. We first outline how specific modifications (carbamylation, desialylation, hyper-glycosylation or helix truncation) bias EPOR signaling toward PI3K-AKT and away from JAK2-STAT5. We then match each derivative to its optimal injury window. Meta-analyses of randomized trials suggest a possible trend toward lower short-term mortality without a consistent functional benefit or thrombotic signal. By integrating molecular mechanisms, experimental findings, and early clinical observations, this review outlines hypotheses and future trial frameworks for phase-targeted, erythropoietin-based neuroprotection. Further controlled studies are required to establish safety, efficacy, and optimal therapeutic timing before translation to routine clinical use.
The vasoreparative potential of endothelial colony-forming cells in the ischemic retina is enhanced by cibinetide, a non-hematopoietic erythropoietin mimetic.
Retinal ischemia remains a common sight threatening end-point in blinding diseases such as diabetic retinopathy and retinopathy of prematurity. Endothelial colony forming cells (ECFCs) represent a subpopulation of endothelial progenitors with therapeutic utility for promoting reparative angiogenesis in the ischaemic retina. The current study has investigated the potential of enhancing this cell therapy approach by the dampening of the pro-inflammatory milieu typical of ischemic retina. Based on recent findings that ARA290 (cibinetide), a peptide based on the Helix-B domain of erythropoietin (EPO), is anti-inflammatory and tissue-protective, the effect of this peptide on ECFC-mediated vascular regeneration was studied in the ischemic retina. The effects of ARA290 on pro-survival signaling and function were assessed in ECFC cultures in vitro. Efficacy of ECFC transplantation therapy to promote retinal vascular repair in the presence and absence of ARA290 was studied in the oxygen induced retinopathy (OIR) model of retinal ischemia. The inflammatory cytokine profile and microglial activation were studied as readouts of inflammation. ARA290 activated pro-survival signaling and enhanced cell viability in response to H2O2-mediated oxidative stress in ECFCs in vitro. Preconditioning of ECFCs with EPO or ARA290 prior to delivery to the ischemic retina did not enhance vasoreparative function. ARA290 delivered systemically to OIR mice reduced pro-inflammatory expression of IL-1β and TNF-α in the mouse retina. Following intravitreal transplantation, ECFCs incorporated into the damaged retinal vasculature and significantly reduced avascular area. The vasoreparative function of ECFCs was enhanced in the presence of ARA290 but not EPO. Regulation of the pro-inflammatory milieu of the ischemic retina can be enhanced by ARA290 and may be a useful adjunct to ECFC-based cell therapy for ischemic retinopathies.
Flipping the molecular switch for innate protection and repair of tissues: Long-lasting effects of a non-erythropoietic small peptide engineered from erythropoietin.
Many disease processes activate a cellular stress response that initiates a cascade of inflammation and damage. However, this process also triggers a tissue protection and repair system mediated by locally-produced hyposialated erythropoietin (hsEPO). Although recombinant EPO is used widely for treating anemia, potential use of recombinant EPO for tissue-protection is limited by rises in hematocrit, platelet activation, and selectin expression resulting in a high risk of thrombosis. Importantly, the erythropoietic and tissue-protective effects of EPO are mediated by different receptors. Whereas EPO stimulates red cell progenitors by binding to an EPO receptor (EPOR) homodimer, a heterodimer receptor complex composed of EPOR and β common receptor (βcR) subunits, termed the innate repair receptor (IRR), activates tissue protection and repair. The IRR is typically not expressed by normal tissues, but instead is rapidly induced by injury or inflammation. Based on this understanding, EPO derivatives have been developed which selectively activate the IRR without interacting with the EPOR homodimer. The latest generation of specific ligands of the IRR includes an 11 amino acid peptide modeled from the three dimensional structure of the EPO in the region of helix B called pyroglutamate helix B surface peptide (pHBSP; ARA-290). Despite a short plasma half-life (~2min), pHBSP activates a molecular switch that triggers sustained biological effects that have been observed in a number of experimental animal models of disease and in clinical trials. This review summarizes pharmacokinetic and pharmacodynamic data and discusses the molecular mechanisms underlying the long-lasting effects of this short-lived peptide.
Cibinetide Improves Corneal Nerve Fiber Abundance in Patients With Sarcoidosis-Associated Small Nerve Fiber Loss and Neuropathic Pain.
Sarcoidosis frequently is complicated by small nerve fiber loss (SNFL), which can be quantified using corneal confocal microscopy (CCM). Prior studies suggest that the innate repair receptor agonist cibinetide reverses corneal nerve loss. This phase 2b, 28-day, randomized trial of 64 subjects with sarcoid-associated SNFL and neuropathic pain assessed the effect of cibinetide on corneal nerve fiber area (CNFA) and regenerating intraepidermal fibers (GAP-43+) as surrogate endpoints for disease modification, pain severity, and functional capacity (6-minute walk test [6MWT]). Cibinetide (1, 4, or 8 mg/day) was compared to placebo. The primary study endpoint was a change in CNFA at 28 days. The placebo-corrected mean change from baseline CNFA (μm2) at day 28 was 109 (95% confidence interval [CI], -429, 647), 697 (159, 1236; P = 0.012), and 431 (-130, 992) in the 1, 4, and 8 mg groups, respectively. Intraepidermal GAP-43+ fibers increased in the 4 mg group (P = 0.035). Further, changes in CNFA correlated with changes in GAP-43+ (ρ = 0.575; P = 0.025) and 6MWT (ρ = 0.645; P = 0.009). Pain improved significantly in all groups, with subjects having moderate-severe pain reporting a clinically meaningful placebo-corrected decrease in pain intensity in the 4 mg group (P = 0.157). Cibinetide significantly increased small nerve fiber abundance in the cornea and skin, consistent with a disease modifying effect. The relationships between CNFA and other clinical measures of disease support its use as a surrogate endpoint to assess potential disease modifying therapies for neuropathy.
Managing fatigue in sarcoidosis - A systematic review of the evidence.
Fatigue is a common manifestation of sarcoidosis, often persisting without evidence of disease activity. First-line therapies for sarcoidosis have limited effect on fatigue. This review aimed to assess the treatment options targeting sarcoidosis-associated fatigue. Medline and Web of Science were searched in November 2015; the bibliographies of these papers, and relevant review papers, were also searched. Studies were included if they reported on the efficacy of interventions (both pharmacological and non-pharmacological) on fatigue scores in sarcoidosis patients. Eight studies were identified that fulfilled the inclusion criteria. These studies evaluated six different interventions (infliximab, adalimumab, ARA 290, methylphenidate, armodafinil and exercise programmes). There is evidence to support a treatment effect of anti-tumour necrosis factor (TNF)-αtherapies (adalimumab and infliximab) and neurostimulants (methylphenidate and armodafinil), but within five of the studies, the risk of bias was high within most domains and the remaining three studies included only small numbers of participants and were short in duration. Trial evidence for treating fatigue as a manifestation of sarcoidosis is limited and requires further investigation. Anti-TNF-α therapies may be beneficial in patients with organ-threatening disease. Neurostimulants have some trial evidence supporting improvements in fatigue but further investigation is needed before they can be recommended.
ARA 290, a nonerythropoietic peptide engineered from erythropoietin, improves metabolic control and neuropathic symptoms in patients with type 2 diabetes.
Although erythropoietin ameliorates experimental type 2 diabetes with neuropathy, serious side effects limit its potential clinical use. ARA 290, a nonhematopoietic peptide designed from the structure of erythropoietin, interacts selectively with the innate repair receptor that mediates tissue protection. ARA 290 has shown efficacy in preclinical and clinical studies of metabolic control and neuropathy. To evaluate the potential activity of ARA 290 in type 2 diabetes and painful neuropathy, subjects were enrolled in this phase 2 study. ARA 290 (4 mg) or placebo were self-administered subcutaneously daily for 28 d and the subjects followed for an additional month without further treatment. No potential safety issues were identified. Subjects receiving ARA 290 exhibited an improvement in hemoglobin A(1c) (Hb A(1c)) and lipid profiles throughout the 56 d observation period. Neuropathic symptoms as assessed by the PainDetect questionnaire improved significantly in the ARA 290 group. Mean corneal nerve fiber density (CNFD) was reduced significantly compared with normal controls and subjects with a mean CNFD >1 standard deviation from normal showed a significant increase in CNFD compared with no change in the placebo group. These observations suggest that ARA 290 may benefit both metabolic control and neuropathy in subjects with type 2 diabetes and deserves continued clinical evaluation.
ARA 290 relieves pathophysiological pain by targeting TRPV1 channel: Integration between immune system and nociception.
ARA 290 is an erythropoietin-derived polypeptide that possesses analgesic and tissue protective effect in many diseases such as diabetes and cancer. The analgesic effect of ARA 290 is mediated by its anti-inflammatory and immunomodulatory functions, or more specifically, by targeting the innate repair receptor (IRR) to down-regulate inflammation to alleviate neuropathic pain. However, whether other mechanisms or pathways are involved in ARA 290-mediated analgesic effect remains elusive. In this study, we are particularly interested in whether ARA 290 could directly target peripheral nociceptors by blocking or influencing receptors in pain sensation. Using calcium imaging, cell culture and behavioral tests, we demonstrated that ARA 290 was able to specifically inhibit TRPV1 channel activity, and relieve the mechanical hypersensitivity induced by capsaicin. Our study suggested that ARA 290 could potentially function as a novel antagonist for TRPV1 channel. This finding would not only contribute to the development of new pain treatment using ARA 290, but also help to improve our understanding of the integration between the immune system and the peripheral nervous system.
A small erythropoietin derived non-hematopoietic peptide reduces cardiac inflammation, attenuates age associated declines in heart function and prolongs healthspan.
Aging is associated with increased levels of reactive oxygen species and inflammation that disrupt proteostasis and mitochondrial function and leads to organism-wide frailty later in life. ARA290 (cibinetide), an 11-aa non-hematopoietic peptide sequence within the cardioprotective domain of erythropoietin, mediates tissue protection by reducing inflammation and fibrosis. Age-associated cardiac inflammation is linked to structural and functional changes in the heart, including mitochondrial dysfunction, impaired proteostasis, hypertrophic cardiac remodeling, and contractile dysfunction. Can ARA290 ameliorate these age-associated cardiac changes and the severity of frailty in advanced age? We conducted an integrated longitudinal (n = 48) and cross-sectional (n = 144) 15 months randomized controlled trial in which 18-month-old Fischer 344 x Brown Norway rats were randomly assigned to either receive chronic ARA290 treatment or saline. Serial echocardiography, tail blood pressure and body weight were evaluated repeatedly at 4-month intervals. A frailty index was calculated at the final timepoint (33 months of age). Tissues were harvested at 4-month intervals to define inflammatory markers and left ventricular tissue remodeling. Mitochondrial and myocardial cell health was assessed in isolated left ventricular myocytes. Kaplan-Meier survival curves were established. Mixed ANOVA tests and linear mixed regression analysis were employed to determine the effects of age, treatment, and age-treatment interactions. Chronic ARA290 treatment mitigated age-related increases in the cardiac non-myocyte to myocyte ratio, infiltrating leukocytes and monocytes, pro-inflammatory cytokines, total NF-κB, and p-NF-κB. Additionally, ARA290 treatment enhanced cardiomyocyte autophagy flux and reduced cellular accumulation of lipofuscin. The cardiomyocyte mitochondrial permeability transition pore response to oxidant stress was desensitized following chronic ARA290 treatment. Concurrently, ARA290 significantly blunted the age-associated elevation in blood pressure and preserved the LV ejection fraction. Finally, ARA290 preserved body weight and significantly reduced other markers of organism-wide frailty at the end of life. Administration of ARA290 reduces cell and tissue inflammation, mitigates structural and functional changes within the cardiovascular system leading to amelioration of frailty and preserved healthspan.
Erythropoietin-derived nonerythropoietic peptide ameliorates experimental autoimmune neuritis by inflammation suppression and tissue protection.
Experimental autoimmune neuritis (EAN) is an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system. Erythropoietin (EPO) has been known to promote EAN recovery but its haematopoiesis stimulating effects may limit its clinic application. Here we investigated the effects and potential mechanisms of an EPO-derived nonerythropoietic peptide, ARA 290, in EAN. Exogenous ARA 290 intervention greatly improved EAN recovery, improved nerve regeneration and remyelination, and suppressed nerve inflammation. Furthermore, haematopoiesis was not induced by ARA 290 during EAN treatment. ARA 290 intervention suppressed lymphocyte proliferation and altered helper T cell differentiation by inducing increase of Foxp3+/CD4+ regulatory T cells and IL-4+/CD4+ Th2 cells and decrease of IFN-γ+/CD4+ Th1 cells in EAN. In addition, ARA 290 inhibited inflammatory macrophage activation and promoted its phagocytic activity. In vitro, ARA 290 was shown to promote Schwann cell proliferation and inhibit its inflammatory activation. In summary, our data demonstrated that ARA 290 could effectively suppress EAN by attenuating inflammation and exerting direct cell protection, indicating that ARA 290 could be a potent candidate for treatment of autoimmune neuropathies.
Synthesis and evaluation of 99mTc-DOTA-ARA-290 as potential SPECT tracer for targeting cardiac ischemic region.
Myocardial infarction caused by ischemia of heart tissue is the main reason for death worldwide; therefore, early detection can reduce mortality and treatment costs. Erythropoietin (EPO) has protection effects on ischemic tissue due to nonhematopoietic peptide (pHBSP; ARA-290) which is derived from the B-subunit of EPO. We designed and synthesized a modified DOTA-(Lys-Dabcyl6, Phe7)-ARA-290 using Fmoc solid-phase peptide synthesis strategies. To improve serum stability, Fmoc-Lys-(Dabcyl)-OH as lipophilic amino acid was synthesized along with Fmoc-Phe-OH which then were substituted with Arg6 and Ala7, respectively; they were then investigated for the ability to detect ischemic cardiac imaging. DOTA-(Lys-Dabcyl6,Phe7)-ARA-290 was labeled with technetium 99m, and its radiochemical purity (RCP), stability in the presence of human serum and, specific bind to hypoxic H9c2 cells were evaluated. In vivo studies for biodistribution and SPECT scintigraphy were checked in a normal and cardiac ischemia rat model. Radiolabeling purity was obtained more than 96% by ITLC, and in vitro stability of the radiopeptide up to 6 hr was 85%. The binding of 99mTc-ARA-290 to hypoxic cells was remarkably higher than normoxic cells (3 times higher than normoxic cells at 1 hr). Biodistribution and SPECT imaging on the cardiac ischemic model showed that radiopeptide considerably accumulated in the ischemic region (cardiac ischemic-to-lung rate = 3.65 ID/g % at 0.5 hr). The results of studies, in vitro and in vivo, indicated that 99mTc-DOTA-(Lys-Dabcyl6,Phe7)-ARA-290 could be an appropriate candidate for early diagnosis of cardiac ischemia.
Corneal nerve fiber size adds utility to the diagnosis and assessment of therapeutic response in patients with small fiber neuropathy.
Small fiber neuropathy (SFN) is a common feature of many inflammatory diseases, often presenting with pain and disability. SFN is diagnosed using symptoms, thermal threshold testing, and intra-epidermal nerve fiber quantification. Corneal confocal microscopy (CCM) is an ophthalmic imaging technique which non-invasively quantifies corneal nerve fiber (CNF) density, branch density and length, and has comparable diagnostic and superior ability to identify nerve regeneration compared to skin biopsy. CNF size (width and area) depends upon the number of fibers within each nerve, as well as pathology (e.g., swelling), and may provide additional sensitivity to diagnose SFN and identify nerve repair. We have compared the utility of the standard CCM variables employed to CNF size in patients with diabetic sensorimotor polyneuropathy or sarcoidosis-associated SFN, and in patients with SFN following cibinetide administration, an agent which promotes nerve repair. The results show that: 1) CNF width distribution and area depend upon neuropathy severity; 2) CNF area, density, branch density and length possess comparable discriminatory power for diagnosing neuropathy; 3) CNF area is related to length by a quadratic function which is predictive for both healthy subjects and those with SFN; 4) CNF area is a useful variable for quantifying change in CNF morphology.
A Nonhematopoietic Erythropoietin Analogue, ARA 290, Inhibits Macrophage Activation and Prevents Damage to Transplanted Islets.
Erythropoietin exerts anti-inflammatory, antiapoptotic, and cytoprotective effects in addition to its hematopoietic action. A nonhematopoietic erythropoietin analogue, ARA 290, has similar properties. The efficacy of pancreatic islet transplantation (PITx) is reduced due to islet damage that occurs during isolation and from the severe inflammatory reactions caused by the transplantation procedure. We investigated whether ARA 290 protects islets and ameliorates inflammatory responses following PITx thus improving engraftment. The effects of ARA 290 on pancreatic islets of C57BL/6J (H-2) mice and on murine macrophages were investigated using an in vitro culture model. As a marginal PITx, 185 islets were transplanted into the liver of streptozotocin-induced diabetic mice (H-2) via the portal vein. Recipients were given ARA 290 (120 μg/kg) intraperitoneally just before and at 0, 6, and 24 hours after PITx. Liver samples were obtained at 12 hours after PITx, and expression levels of proinflammatory cytokines were assessed. ARA 290 protected islets from cytokine-induced damage and apoptosis. Secretion of pro-inflammatory cytokines (IL-6, IL-12, and TNF-α) from macrophages was significantly inhibited by ARA 290. After the marginal PITx, ARA 290 treatment significantly improved the blood glucose levels when compared to those of control animals (P < 0.001). Upregulation of monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, IL-1β, and IL-6 messenger RNA expression within the liver was suppressed by ARA 290 treatment. ARA 290 protected pancreatic islets from cytokine-induced damage and apoptosis and ameliorated the inflammatory response after PITx. ARA 290 appears to be a promising candidate for improvement of PITx.
ARA 290, a peptide derived from the tertiary structure of erythropoietin, produces long-term relief of neuropathic pain coupled with suppression of the spinal microglia response.
Neuropathic pain is a difficult to treat disorder arising from central or peripheral nervous system lesions. The etiology of neuropathic pain consists of several overlapping pathways converging into an exaggerated pain state with symptoms such as allodynia and hyperalgesia. One of these pathways involves activation of spinal cord microglia and astrocytes, which drive and maintain the inflammatory response following the lesion. These cells are a potential target for drugs for neuropathic pain relief. In this current study, we investigated the dose-effect relationship of the tissue protective peptide ARA 290, derived from the tertiary structure of erythropoietin, on allodynia and concurrent spinal cord microglia and astrocytes. Following a spared nerve injury in rats, vehicle or ARA290 (administered in either one of 4 doses: 3, 10, 30 and 60 μg/kg) was administered on days 1, 3, 6, 8 and 10. ARA290 exerted a dose-response effect by significantly reducing mechanical allodynia up to 20 weeks when compared to vehicle. The reduction of cold allodynia was significant up to 20 weeks for the doses 3, 10, 30 and 60 μg/kg when compared to vehicle. The effect 10 and 30 μg/kg ARA290 and vehicle on the microglia response (iba-1-immunoreactivity, iba-1-IR) and astrocyte reaction (GFAP-immunoreactivity, GFAP-IR) was investigated in animals surviving 2 (group 1) or 20 (group 2) weeks following lesion or sham surgery. In group 1, significant microglia reactivity was observed in the L5 segment of the spinal cord of animals treated with vehicle when compared to sham operated, while animals treated with 10 or 30 μg/kg did not show a increase. In group 2, a more widespread and increased microglia reactivity was observed for animals treated with 0 and 10 μg/kg when compared to sham operated animals, indicated by involvement of more spinal cord segments and higher iba-1-IR. Animals treated with 30 μg/kg did not show increased microglia reactivity. No difference in astrocyte reaction was observed. The erythropoietin-analogue ARA290 dose-dependently reduced allodynia coupled to suppression of the spinal microglia response, suggestive of a mechanistic link between ARA290-induced suppression of central inflammation and relief of neuropathic pain symptoms.
Sarcoidosis and pain caused by small-fiber neuropathy.
Sarcoidosis is a chronic inflammatory illness and small-fiber neuropathy (SFN) is one of the disabling and often chronic manifestations of the disease. SFN presents with peripheral pain and symptoms of autonomic dysfunction. The character of the pain can be burning or shooting. Besides, allodynia and hyperesthesia can exist. Diagnosis is usually made on the basis of clinical features, in combination with abnormal specialized tests. The aim of treatment is often to reduce pain; however, total pain relieve is seldom achieved. The role of TNF-α in the pathogenesis of SFN in sarcoidosis appears interesting to explore. Novel therapeutic agents such as ARA 290, a nonhematopoietic erythropoietin analogue with potent anti-inflammatory and tissue protective properties, are interesting to explore in the treatment of SFN in sarcoidosis.
An engineered non-erythropoietic erythropoietin-derived peptide, ARA290, attenuates doxorubicin induced genotoxicity and oxidative stress.
Erythropoietin (EPO) applies anti-inflammatory, anti-apoptotic, anti-oxidant and cytoprotective effects besides its hematopoietic action. A nonhematopoietic peptide engineered from EPO, ARA 290, interacts selectively with the innate repair receptor and has similar possessions. ARA290 mediates tissue protection without hematopoietic side-effects of EPO which limit its clinical application. Doxorubicin (DOX) is the broad-spectrum chemotherapeutic agent, but its use is limited by the development of nonspecific toxicity on noncancerous tissues especially in cardiac cells. Mechanisms behind the DOX-induced toxicities are enhanced level of oxidative damage, inflammation and apoptosis. In the present study, we have investigated whether ARA290 acts as a chemoprotective agent modulating the cytotoxicity, genotoxicity and oxidative stress induced in vitro by DOX. The genoprotective effect of ARA290 on DOX-induced toxicity in three cell line (HepG2, HGF & Stem cell) were assessed. Cells were treated with ARA290 (50-400 nM) and DOX (1 μM) in pretreatment condition. Cytotoxicity was evaluated using the MTT assay, genoprotective effect of ARA290 were evaluated using the micronucleus test and comet assay. AR290 significantly reduced the percentage of DNA in tail and the frequency of micronuclei induced by DOX. Besides, DOX impaired anti-oxidant defense enzyme activities and induced inflammation and apoptotic cell death. ARA290 markedly attenuated DOX induced oxidative stress and protected against DOX induced inflammation and apoptotic cell death. This result proposes that ARA290 can act as a protective agent, reducing DOX-induced DNA damage and oxidative stress, and it is possible that this protection could also extend to cardiac cells.
Safety and efficacy of ARA 290 in sarcoidosis patients with symptoms of small fiber neuropathy: a randomized, double-blind pilot study.
ARA 290 (a peptide designed to activate the innate repair receptor that arrests injury and initiates cytoprotection, antiinflammation and healing) reduces allodynia in preclinical neuropathy models. We studied the safety and efficacy of ARA 290 to reduce symptoms of small fiber neuropathy (SFN) in patients with sarcoidosis. A total of 22 patients diagnosed with sarcoidosis and symptoms of SFN were enrolled in a double-blind, placebo-controlled exploratory trial consisting of three times weekly intravenous dosing of ARA 290 (2 mg; n = 12) or placebo (n = 10) for 4 wks. Inclusion criteria were a diagnosis of neuropathy and a spontaneous pain score of ≥5 (Brief Pain Inventory [BPI]). Endpoints assessed were changes in pain intensity and the small fiber neuropathy screening list (SFNSL) score, quality of life (SF-36), depressive symptoms (Inventory of Depressive Symptomatology [IDS]) and fatigue (Fatigue Assessment Scale [FAS]). No safety concerns were raised by clinical or laboratory assessments. The ARA 290 group showed significant (p < 0.05) improvement at wk 4 in SFNSL score compared with placebo (Δ -11.5 ± 3.04 versus Δ -2.9 ± 3.34 [standard error of the mean]). Additionally, the ARA 290 group showed a significant change from baseline in the pain and physical functioning dimensions of the SF-36 (Δ -23.4 ± 5.5 and Δ -14.6 ± 3.9, respectively). The mean BPI and FAS scores improved significantly but equivalently in both patient groups. No change was observed in the IDS. ARA 290 appears to be safe in patients with sarcoidosis and can reduce neuropathic symptoms.
Potential role of erythropoietin receptors and ligands in attenuating apoptosis and inflammation in critical limb ischemia.
Managing critical limb ischemia (CLI) is challenging. Furthermore, ischemic myopathy prevents good functional outcome after revascularization. Hence, we have focused on limiting the tissue damage rather than angiogenesis, which has traditionally been the motivation to develop nonsurgical treatments for CLI. Erythropoietin (EPO) protects ischemic tissue, and this property may also benefit CLI. The objective of this study was to examine the expression of the tissue-protective EPO receptor complex (EPOR-CD131 [β-chain of interleukin (IL)-3/IL-5/granulocyte macrophage colony-stimulating factor receptor]) in skeletal muscle obtained from humans with CLI. Because native EPO is thrombogenic, the antiapoptotic and anti-inflammatory effects of a nonhematopoietic helix-B peptide of EPO (ARA 290) were investigated on ischemic myotubes in vitro. Tissue was obtained from gastrocnemius muscle of 12 patients undergoing amputation for CLI and from 12 patients without limb ischemia. The expression of EPOR and CD131 was demonstrated by immunohistochemistry and Western blot. A validated in vitro model of myotube ischemia was used in which mature C2C12 myotubes were cultured 6 to 12 hours in a depleted media and gas mixture (20% CO2 and 80% N2). The myotubes were pretreated with EPO or ARA 290 before exposure to simulated ischemia. Apoptosis and cell death were determined by cleaved caspase-3 assay and lactate dehydrogenase release assay. Enzyme-linked immunosorbent assay measured the inflammatory cytokines. EPOR and CD131 were expressed and significantly upregulated in CLI (average optical density [OD] in Western blot [control vs CLI] EPOR, 0.05 U vs 0.1 U; CD131, 0.10 U vs 0.22 U; P < .01). There was colocalization of EPOR and CD131 in the sarcolemma (cell membrane) of the skeletal myofiber. There was no difference in the distribution of colocalization between the CLI and the normal muscle. The ischemic myotubes treated by ARA 290 in vitro had a significantly decreased number of apoptotic cells (ischemia vs ischemia plus ARA 290: 71.1% vs 55.1%; P < .01), cleaved caspase-3 (OD of ischemia vs ischemia plus ARA 290: 0.15 U vs 0.02 U; P < .01), lactate dehydrogenase release (ischemia vs ischemia plus ARA 290: 32.5 U/L vs 21.3 U/L; P < .01), and IL-6 release (OD at 450 nm, ischemia vs ischemia plus ARA 290: 0.18 vs 0.13; P < .01). This study demonstrates the expression and the upregulation of EPOR and CD131 in CLI and also shows that EPOR and CDI are colocalized in the cell membrane of both ischemic and control muscle fiber. The in vitro experiments demonstrate that ARA 290 decreases inflammation and apoptosis of ischemic myotubes. ARA 290 may potentially be used as adjunctive treatment for CLI.
ARA 290 improves symptoms in patients with sarcoidosis-associated small nerve fiber loss and increases corneal nerve fiber density.
Small nerve fiber loss and damage (SNFLD) is a frequent complication of sarcoidosis that is associated with autonomic dysfunction and sensory abnormalities, including pain syndromes that severely degrade the quality of life. SNFLD is hypothesized to arise from the effects of immune dysregulation, an essential feature of sarcoidosis, on the peripheral and central nervous systems. Current therapy of sarcoidosis-associated SNFLD consists primarily of immune suppression and symptomatic treatment; however, this treatment is typically unsatisfactory. ARA 290 is a small peptide engineered to activate the innate repair receptor that antagonizes inflammatory processes and stimulates tissue repair. Here we show in a blinded, placebo-controlled trial that 28 d of daily subcutaneous administration of ARA 290 in a group of patients with documented SNFLD significantly improves neuropathic symptoms. In addition to improved patient-reported symptom-based outcomes, ARA 290 administration was also associated with a significant increase in corneal small nerve fiber density, changes in cutaneous temperature sensitivity, and an increased exercise capacity as assessed by the 6-minute walk test. On the basis of these results and of prior studies, ARA 290 is a potential disease-modifying agent for treatment of sarcoidosis-associated SNFLD.
Characterization of in vitro generated metabolites of selected peptides <2 kDa prohibited in sports.
With an increasing number of prohibited substances in doping controls, knowledge about their metabolism is crucial for efficient analysis. While for low molecular mass molecules, standard protocols for in vitro metabolism experiments are well established, the situation with peptidic drugs has been shown to be substantially more heterogeneous and complex. Two principle strategies aiming at simulating the metabolism of lower molecular mass peptides in vitro are presented within this study. The prohibited peptides ARA-290, GHRP-3, and Peforelin, with a to-date unknown metabolism, were chosen as model compounds for these experiments and metabolism after incubation with different blood specimens (EDTA-, heparin-, citrate-plasma, and serum) and exposure to recombinant amidase were investigated. The characterization of in vitro generated drug-derived peptidic analytes was accomplished by means of liquid chromatography coupled to high resolution mass spectrometry. Identification of the generated metabolites was ensured by dedicated high resolution product ion experiments after liquid chromatographic separation. While extensive exopeptidase-driven metabolism was observed for ARA-290 (with one main metabolite PyrEQLERALN), GHRP-3 and Peforelin were found to exhibit a considerable metabolic stability with a low tendency for deamidation only. Copyright © 2017 John Wiley & Sons, Ltd.
Immunometabolic dysregulation drives selective executive cognitive dysfunction in male db/db mice.
Type 2 diabetes (T2D) is associated with cognitive impairment, with executive functions such as cognitive flexibility being particularly vulnerable. Growing evidence suggests that chronic inflammatory and metabolic stress contributes to diabetes -related brain dysfunction, yet behavioral assessment in animal models is often confounded by anxiety, altered motivation, and reduced response vigor. In this study, we used a translational touchscreen-based operant platform to distinguish associative learning from executive cognitive flexibility in db/db mice, a well-established genetic model of T2D, and to evaluate the effects of the non-erythropoietic erythropoietin derived peptide ARA 290. Male db/db mice and age matched db/m heterozygote lean controls were tested using pairwise visual discrimination to assess associative learning and reversal learning to probe cognitive flexibility. Metabolic function was evaluated using glucose and insulin tolerance tests, while immune and metabolic effects of ARA 290 were evaluated by flow cytometry and RNA-seq. Db/db mice displayed delayed task engagement and longer response latencies during pretraining and acquisition, yet maintained intact associative learning accuracy. In contrast, they exhibited pronounced impairments in cognitive flexibility during reversal learning, characterized by increased perseveration and reduced adaptation to changed reward contingencies. Treatment with ARA 290 improved insulin sensitivity and altered circulating monocyte proportions but did not rescue deficits in executive cognitive flexibility. RNA-seq of the hippocampus revealed enrichment of immune pathways consistent with chronic low-grade inflammation, providing molecular context for the observed behavioral phenotype. Together, these findings demonstrate that T2D selectively impairs executive cognitive flexibility while sparing basic associative learning, and that improvement in peripheral metabolic function and altered monocyte proportions are insufficient to restore executive cognition. This work highlights the value of touchscreen-based paradigms for resolving distinct cognitive domains in metabolic disease and highlights the need to target brain specific immunometabolic mechanisms to address diabetes-associated cognitive dysfunction.
Mechanistic Approach for Protective Effect of ARA290, a Specific Ligand for the Erythropoietin/CD131 Heteroreceptor, against Cisplatin-Induced Nephrotoxicity, the Involvement of Apoptosis and Inflammation Pathways.
ARA 290, an 11-amino acid linear nonhematopoietic peptide derived from the three-dimensional structure of helix B of the erythropoietin (EPO), interacts selectively with the innate repair receptor (IRR) that arbitrates tissue protection. The aim of this study was to investigate the protective effects of ARA290 against cisplatin-induced nephrotoxicity. For this purpose, HEK-293 and ACHN cells were treated with ARA290 (50-400 nM) and cisplatin (2.5 μM) in pretreatment condition. Then, cytotoxicity, genotoxicity, oxidative stress parameters (ROS, GPx, SOD, and MDA), and inflammatory markers (TNFα, IL6, and IL1β) were evaluated. Furthermore, apoptotic cell death was assessed via caspase-3 activity and tunnel assay. To determine the molecular mechanisms of the possible nephroprotective effects of ARA290, gene and protein expressions of TNFα, IL1β, IL6, Caspase-3, Bax, and Bcl2 were evaluated by real-time PCR and western blot assay, respectively. The findings indicated that ARA290 significantly reduced the DNA damage parameters of comet assay and the frequency of micronuclei induced by cisplatin. Besides, ARA290 improved cisplatin-induced oxidative stress by reducing MDA/ROS levels and enhancing antioxidant enzyme levels. In addition, reduced levels of pro-inflammatory cytokines indicated that cisplatin-induced renal inflammation was mitigated upon the treatment with ARA290. Besides, ARA290 ameliorates cisplatin-induced cell injury by antagonizing apoptosis. Furthermore, the molecular findings indicated that gene and protein levels of TNFα, IL1β, IL6, Caspase-3, and Bax were significantly decreased and gene and protein levels of Bcl2 significantly increased in the ARA290 plus cisplatin group compared with the cisplatin group. These findings revealed that ARA290 as a potent chemo-preventive agent exerted a protective effect on cisplatin-induced nephrotoxicity mostly through its anti-apoptotic, anti-inflammatory, and antioxidant potentials and also suggested that ARA290 might be a new therapeutic approach for patients with acute kidney injury.
Ketamine does not produce relief of neuropathic pain in mice lacking the β-common receptor (CD131).
Neuropathic pain (NP) is a debilitating condition associated with traumatic, metabolic, autoimmune and neurological etiologies. Although the triggers for NP are diverse, there are common underlying pathways, including activation of immune cells in the spinal cord and up-regulation of the N-methyl-D-aspartate receptor (NMDAR). Ketamine, a well-known NDMAR antagonist, reduces neuropathic pain in a sustained manner. Recent study has shown that the novel 11-amino acid peptide erythropoietin derivative ARA290 produces a similar, long-lasting relief of NP. Here, we show that both drugs also have similar effects on the expression of mRNA of the NMDAR, as well as that of microglia, astrocytes and chemokine (C-C motif) ligand 2, all-important contributors to the development of NP. Although the effects of ketamine and ARA 290 on NP and its molecular mediators suggest a common mechanism of action, ARA 290 has no affinity for the NMDAR and acts specifically via the innate repair receptor (IRR) involved in tissue protection. We speculated therefore, that the IRR might be critically involved in the action of ketamine on neuropathic pain. To evaluate this, we studied the effects of ketamine and ARA 290 on acute pain, side effects, and allodynia following a spared nerve injury model in mice lacking the β-common receptor (βcR), a structural component of the IRR. Ketamine (50 mg/kg) and ARA 290 (30 µg/kg) produced divergent effects on acute pain: ketamine produced profound antinociception accompanied with psychomotor side effects, but ARA290 did not, in both normal and knock out mice. In contrast, while both drugs were antiallodynic in WT mice, they had no effect on NP in mice lacking the βcR. Together, these results show that an intact IRR is required for the effective treatment of NP with either ketamine or ARA 290, but is not involved in ketamine's analgesic and side effects.
Detecting peptidic drugs, drug candidates and analogs in sports doping: current status and future directions.
With the growing availability of mature systems and strategies in biotechnology and the continuously expanding knowledge of cellular processes and involved biomolecules, human sports drug testing has become a considerably complex field in the arena of analytical chemistry. Proving the exogenous origin of peptidic drugs and respective analogs at lowest concentration levels in biological specimens (commonly blood, serum and urine) of rather limited volume is required to pursue an action against cheating athletes. Therefore, approaches employing chromatographic-mass spectrometric, electrophoretic, immunological and combined test methods have been required and developed. These allow detecting the misuse of peptidic compounds of lower (such as growth hormone-releasing peptides, ARA-290, TB-500, AOD-9604, CJC-1295, desmopressin, luteinizing hormone-releasing hormones, synacthen, etc.), intermediate (e.g., insulins, IGF-1 and analogs, 'full-length' mechano growth factor, growth hormone, chorionic gonadotropin, erythropoietin, etc.) and higher (e.g., stamulumab) molecular mass with desired specificity and sensitivity. A gap between the technically possible detection and the day-to-day analytical practice, however, still needs to be closed.
Simplifying and expanding the screening for peptides <2 kDa by direct urine injection, liquid chromatography, and ion mobility mass spectrometry.
The analysis of low-molecular-mass peptides in doping controls has become a mandatory aspect in sports drug testing and, thus, the number of samples that has to be tested for these analytes has been steadily increasing. Several peptides <2 kDa with performance-enhancing properties are covered by the list of prohibited substances of the World Anti-Doping Agency including Desmopressin, LH-RH, Buserelin, Triptorelin, Leuprolide, GHRP-1, GHRP-2, GHRP-3, GHRP-4, GHRP-5,GHRP-6, Alexamorelin, Ipamorelin, Hexarelin, ARA-290, AOD-9604, TB-500 and Anamorelin. With the presented method employing direct urine injection into a liquid chromatograph followed by ion-mobility time-of-flight mass spectrometry, a facile, specific and sensitive assay for the aforementioned peptidic compounds is provided. The accomplished sensitivity allows for limits of detection between 50 and 500 pg/mL and thus covers the minimum required performance level of 2 ng/mL accordingly. The method is precise (imprecision <20%) and linear in the estimated working range between 0 and 10 ng/mL. The stability of the peptides in urine was tested, and -20°C was found to be the appropriate storage temperature for sports drug testing. Finally, proof-of-concept was shown by analysing elimination study urine samples collected from individuals having administered GHRP-6, GHRP-2, or LHRH.
Erythropoietin receptor (EpoR) agonism is used to treat a wide range of disease.
The erythropoietin receptor (EpoR) was discovered and described in red blood cells (RBCs), stimulating its proliferation and survival. The target in humans for EpoR agonists drugs appears clear-to treat anemia. However, there is evidence of the pleitropic actions of erythropoietin (Epo). For that reason, rhEpo therapy was suggested as a reliable approach for treating a broad range of pathologies, including heart and cardiovascular diseases, neurodegenerative disorders (Parkinson's and Alzheimer's disease), spinal cord injury, stroke, diabetic retinopathy and rare diseases (Friedreich ataxia). Unfortunately, the side effects of rhEpo are also evident. A new generation of nonhematopoietic EpoR agonists drugs (asialoEpo, Cepo and ARA 290) have been investigated and further developed. These EpoR agonists, without the erythropoietic activity of Epo, while preserving its tissue-protective properties, will provide better outcomes in ongoing clinical trials. Nonhematopoietic EpoR agonists represent safer and more effective surrogates for the treatment of several diseases such as brain and peripheral nerve injury, diabetic complications, renal ischemia, rare diseases, myocardial infarction, chronic heart disease and others.
Quick links (PubMed)
- PMID 33423557 — 2021 · The time to develop treatments for diabetic neuropathy.
- PMID 35008482 — 2021 · The Non-Erythropoietic EPO Analogue Cibinetide Inhibits Osteoclastogenes…
- PMID 32674280 — 2020 · A Phase 2 Clinical Trial on the Use of Cibinetide for the Treatment of D…
- PMID 29026145 — 2017 · Cibinetide dampens innate immune cell functions thus ameliorating the co…
- PMID 34343617 — 2022 · Early monocyte modulation by the non-erythropoietic peptide ARA 290 dece…
- PMID 24555851 — 2014 · ARA 290 for treatment of small fiber neuropathy in sarcoidosis.
- PMID 34498509 — 2021 · Cibinetide Protects Isolated Human Islets in a Stressful Environment and…
- PMID 32345869 — 2020 · Improvement of Islet Allograft Function Using Cibinetide, an Innate Repa…
- PMID 29223734 — 2018 · Activation of the EPOR-β common receptor complex by cibinetide ame…
- PMID 41659975 — 2025 · Phase-targeted erythropoietin derivatives for traumatic brain injury: br…
- PMID 30876881 — 2019 · The vasoreparative potential of endothelial colony-forming cells in the …
- PMID 25728128 — 2015 · Flipping the molecular switch for innate protection and repair of tissue…
- PMID 28475703 — 2017 · Cibinetide Improves Corneal Nerve Fiber Abundance in Patients With Sarco…
- PMID 27507833 — 2017 · Managing fatigue in sarcoidosis - A systematic review of the evidence.
- PMID 25387363 — 2015 · ARA 290, a nonerythropoietic peptide engineered from erythropoietin, imp…
- PMID 26774587 — 2016 · ARA 290 relieves pathophysiological pain by targeting TRPV1 channel: Int…
- PMID 36741836 — 2022 · A small erythropoietin derived non-hematopoietic peptide reduces cardiac…
- PMID 24603865 — 2014 · Erythropoietin-derived nonerythropoietic peptide ameliorates experimenta…
- PMID 35317117 — 2021 · Synthesis and evaluation of 99mTc-DOTA-ARA-290 as potential SPECT tracer…
- PMID 29549285 — 2018 · Corneal nerve fiber size adds utility to the diagnosis and assessment of…
- PMID 26683514 — 2016 · A Nonhematopoietic Erythropoietin Analogue, ARA 290, Inhibits Macrophage…
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