Main Research Findings

1) Early modulation of monocytes initiated by administration of the peptide ARA 290, was found to decelerate the progression of pathology similar to Alzheimer’s disease.

2) The peptide ARA 290 has been shown to relieve pathophysiological pain by inhibiting activity of the TRPV1 channel.

3) By suppressing the spinal microglia response, the peptide ARA 290 has the potential to produce long term relief of various types of neuropathic pain.

 

Selected Data

1) Development of Alzheimer’s disease is associated with the accumulation of amyloid-beta plaque and tau hyperphosphorylation. Recent studies have found that monocyte activation plays a crucial role in regulating pathology related to amyloid-beta deposition. APA 290 is a non-erythropoietic peptide that has been shown to potentially address progression of Alzheimer’s-like pathology. The research team of Al-Onaizi et al examined the effect of systemic administration of the peptide on disease progression.

For the purpose of this experiment, 4-9 month old male APP/PS1 and APP/PS1/CX3GR1 triple transgenic chimeric mice with GFP-tagged peripheral patrolling monocytes were used to conduct 2-photon intravital imaging. 4 month old APP/PS1 mice were used to represent early onset of Alzheimer’s disease while 8 month old APP/PS1 mice were used to represent late onset of Alzheimer’s disease. They were treated with either a vehicle or active doses of ARA 290 and euthanized at 5 and 9 months, respectively. Similarly, 5 month old chimeric mice were treated following the same protocol and euthanized at 6 months. Furthermore, to test the early benefits of peptide treatment, 4 month old mice were treated with either a vehicle or ARA 290 for 5 days followed by euthanasia. All animals were housed under standard laboratory conditions with ad libitum access to food and water and were randomized into experimental treatment groups in order to undergo neurobehavioral testing [1].

The neurobehavioral tests performed in this study were the water T-Maze test and the novel object recognition test. Following treatment with ARA 290, the water T-maze test was used to assess differences in right/left discriminatory learning based on the mices’ ability to remember the spatial location of a submerged platform. The mice were placed in the testing apparatus and allowed to swim freely until they could locate and escape through the submerged platform in the right or left arm of the maze. During the initial learning and acquisition phase, the mice underwent a maximum of 24 trials and were given a 10 minute rest period after every block of 10 trials; criterion was considered achieved if the mice were able to complete 5 consecutive trials without error. The acquisition and retention trialing took place two days later and the mice were expected to find the submerged platform on the other side of the maze. Average swimming speed and the amount of time it took to find the platform was recorded by the research team [1].

The novel object recognition test was used to assess the mices’ learning and recognition memory based on their innate trait to explore the environment for novel objects. The test took place in a fiberglass open rectangular field; during the learning phase the mice were placed in the apparatus with two identical objects and allowed to explore freely for 5 minutes. The animals were returned to their home cages and reintroduced to the apparatus an hour later for the retention phase of the study. The researchers placed the mice into the apparatus with one of the familiar objects and a similar novel object and monitored their activity and movements via a video-tracking system [1].

2) The research team of Zhang et al examined the ability of the peptide ARA 290 to relieve pathophysiological pain by targeting TRPV1 channels and integrating the immune system and nociceptive signals. For the purpose of this study, behavioral and physiological experiments were carried out using 2 month old C57/BL6 mice. The physiological examination included dissociated neurons that were transferred and plated in combination with HEK cells that overexpress TRPV1 channels. The samples were stained, incubated, and imaged at 1 second intervals followed by delivery of drugs and stimulation to the cells through a gravity driven perfusion system and suction pipette. The samples were pre-incubated with ARA 290 and baseline fluorescence levels were collected for 20 seconds prior to KCl stimulus application for 15 seconds, ending with a 2 minute washout. Calcium activity was calculated by the research team using the observed changes in fluorescence [2].

In terms of behavioral testing, three groups of 8 mice were subcutaneously injected in the left hind paw with either 1 nM ARA 290, 10 nM ARA 290 in 10 uL of PBS, or PBS only. 15 minutes after the peptide injection the mice were injected with a 10 uM capsaicin solution in the same place. After 30 minutes of resting the mice were subjected to the paw withdrawal threshold assay. Beginning procedures of the examination include placing the mice into separate chambers with mesh floor stands and allow them to habituate for 15 minutes before noxious stimulus was applied. After the incubation period the mice were placed in a plastic chamber to rest while the paw withdrawal frequencies were measured at 1, 2, 4,8, and 24 hours post-capsaicin injection. A positive response to the test was a quick withdrawal of the paw during or after the stimulation, all tests were repeated 10 times for the purpose of statistical analysis. It is important to note that after all behavioral and physiological experiments, the dorsal root ganglion and the trigeminal ganglion were dissected for further evaluation [2].

3) In addition to pathophysiological pain related to TRPV1 receptors, the research team of Swartjes et al examined the ability of ARA 290 to provide long-term relief of neuropathic pain with suppression of the spinal microglia response. This is a relevant topic in research considering that neuropathic pain is considered a debilitating condition characterized by lesions in the nervous system either centrally or peripherally, resulting in hyperalgesia and allodynia to mechanical and thermal stimuli. Current research shows that inflammation both in the peripheral and central nervous system plays a crucial role in the development of neuropathic pain [3].

Additionally, evidence found that mechanisms underlying allodynia and hyperalgesia are related to the communication between neurons and glial cells as the glia become more abundant and activated in response to the release of proliferative molecules. The activated glial cells regulate and maintain the inflammatory response by releasing mediators such as TNF-alpha and interleukin 1-beta. Based on the previous research findings, the team of Swartjes et al sought to clarify the involvement of astrocytes and microglia in neuropathic pain and how they may be targeted for treatment purposes.

ARA 290 was found to be a neuroprotective synthetic amino acid erythropoietin derivative that had the ability to produce long-term pain relief in cases of peripheral nerve damage. The pain relieving effects of the peptide are produced by the activation of beta-common receptors that form a heterocomplex with EPO receptors. The heterocomplex is referred to as the innate repair receptor that becomes naturally upregulated in cases of tissue injury.

Activation of the IRR promotes tissue repair and neurite outgrowth, and initiates an anti-inflammatory response that results in the inhibition of cell death signaling and apoptosis. Based on this information regarding the mechanism of action of ARA 290, the research team investigated the dose-dependent changes in mechanical and thermal allodynia in response to the peptide. Additionally, the anti-inflammatory effects of the peptide at the spinal cord level were further assessed by visualizing astrocytes and microglia via immunohistochemistry [3].

 

Discussion

1) The research team of Al-Onaizi et al induced Alzheimer’s-like pathology in APP/PS1 mice in order to evaluate the effects of the peptide ARA 290 on the accumulation and deposition of amyloid beta plaques in the brain. Pathology in the mice began to develop around 3-4 months and treatment started either at 4 months to represent early onset Alzheimer’s or 8 months to represent late onset Alzheimer’s. The mice underwent neurobehavioral testing in the form of the water T-maze test and a novel object recognition test. Once treatment and testing protocols were fulfilled the mice were euthanized and brains were dissected in order to immunolabel sections with 6E10 antibody in order to visualize amyloid beta deposits in the hippocampus and the cerebral cortex. That being said, observation of the brain sections showed a clear reduction in the amount of plaque in mice treated with ARA 290 compared to those treated with a vehicle [1].

Figure 1: Changes in and amount of plaque in the (A, B) cerebral cortex and (C, D) the hippocampus

As it was previously mentioned, the water T-maze test was used to assess left/right discrimination learning while the novel object recognition test was used to assess long-term memory. During the initial acquisition stage the results reported that there was no difference between the number of trials it took to reach criterion between the APP/PS1 mice treated with a vehicle versus those treated with ARA 290. However, when observing the behavior of the mice in the retention phase, the researchers found that it took the mice treated with ARA 290 fewer numbers of trials to reach criterion in comparison to the mice treated with a vehicle [1].

Figure 2: Trials to criterion during A) the acquisition phase and B) retention phase of the water T-maze test.

In terms of the novel object recognition test, it was found that the APP/PS1 mice treated with the peptide did not exhibit any significant differences in object exploration from the mice treated with a vehicle during the acquisition phase. This was compared to the retention phase where exploration of the novel object dramatically increased in the test subjects treated with ARA 290 compared to those treated with a vehicle. These findings indicated that treatment with ARA 290 early on in the disease progression has the potential to combat cognitive degeneration [1].

Figure 3: Exploration time of the novel object during E) the acquisition phase and F) the retention phase

2) As it was previously mentioned, test subjects underwent a paw withdrawal test in order to evaluate the ability of ARA 20 to relieve capsaicin-induced hypersensitivity in an animal model using live mice. Capsaicin was injected into the hindpaw of the test subjects followed by an injection of either 1 nM ARA 290, 10 nM ARA 290, or 10 uL of a vehicle compound. Hypersensitivity of the TRPV1 channels was measured by assessing the threshold of pressure that caused paw withdrawal as well as the frequency of spontaneous paw withdrawal. The results showed that administration of 10 uL PBS did not increase the paw threshold withdrawal, however both 1 nM and 10 nM of ARA 290 was found to relieve mechanical hypersensitivity induced by capsaicin. The attenuation occurred in a dose-dependent manner where 1 nM partially relieved the mechanical hypersensitivity while it was almost completely gone with 10 nM of ARA 290 [2].

Figure 4: Changes in A) paw withdrawal threshold and B) paw withdrawal frequency in mice treated with ARA 290 after mechanical hypersensitivity was induced through capsaicin injection.

When assessing the effects of ARA 290 on the activity levels of TRPV1 channels, the research team of Zhang et al first hypothesized that the peptide was able to target the TRPV1 channels to increase their thresholds. This was tested by applying various doses of a capsaicin solution to dissociated dorsal root and trigeminal ganglions treated with fluo-4 AM. Calcium responses of lower concentrations of capsaicin were normalized to the response of the 10 uM dose. The same gradient of capsaicin solutions were then combined with 0.3 nM, 1 nM, and 10 nM concentrations of ARA 290 and applied to the dissociated dorsal root and trigeminal ganglions. After generating the concentration curve of capsaicin mixed with ARA 290, the results of this portion of the study found that ARA 290 was able to shift the concentration curve of capsaicin to the right in both sets of ganglions. This was interpreted by the research team as an increase in the TRPV1 channel threshold to capsaicin, with the 10 nM concentration being the most effective [2].

Figure 5: Changes in the capsaicin response in A) dorsal root ganglions and B) trigeminal ganglions in response to ARA 290 administered in concentrations of 0.3 nM, 1 nM, and 10 nM.

The researchers also assessed the effects of the peptide on TRPV1 channels in over-expressed HEK293 cells in order to further confirm that ARA 290 specifically targets the TRPV1 channels. When adding ARA 290 to 10 uM of capsaicin, the peptide was able to inhibit calcium responses invoked by the capsaicin in a concentration dependent manner. Calcium responses in the TRPV1 over-expression HEK293 cells were found to reduce by 90% with an application of 0.1 nM of ARA 290 and reach 100% with 3 nM and 10 nM concentrations of the peptide [2].

Figure 6: Changes in capsaicin response in TRPV1 over-expressed HEK293 cells in response to administration of ARA 290.

3) In order to assess the ability of ARA 290 to reduce mechanical allodynia and cold allodynia, the research team of Swartjes et al treated rats with either a vehicle or an active dose of the peptide following the induction of spinal nerve injury. ARA 290 was administered in doses of 3, 10, 30, and 60, ug/kg on days 1, 3, 6, 8, and 10 and was found to significantly reduce mechanical and tactile allodynia for up to 20 weeks in comparison to treatment with a vehicle compound. Analysis of the collected data found that administration of 30 ug/kg or 60 ug/kg of ARA 290 elicited the most significant relief of allodynia while concentrations of 1, 3, and 10 ug/kg did not result in any remarkable changes in levels of mechanical allodynia. That being said, higher doses of the peptide were associated with greater relief periods from mechanical allodynia due to a higher toleration of filament stimulation at a greater force [3].

Figure 7: (A) The effect of treatment with a vehicle (red), 3 ug/kg ARA 290 (yellow), 10 ug/kg ARA 290 (green), 30 ug/kg ARA 290 (light blue), or 60 ug/kg ARA 290 (dark blue) administered at days 1, 3, 6, 8, and 10 post-surgery. (B) Correlation of the relief of mechanical allodynia and the treatment dosage of ARA 290

In addition to mechanical allodynia the research team also assessed the effects of the peptide on cold allodynia following induction of a spared nerve injury. Cold allodynia responses typically developed within 7-14 days and were found to be relieved by treatment with ARA 290 in a dose dependent manner. While a linear ARA 290 dose-response was observed, all treatment dosages of the peptide were found to be more effective at attenuating the cold allodynia response in comparison to administration of the treatment compound. Higher treatment doses of the peptide were also found to correspond to the test subjects’ experiencing less cold allodynia due to the slower response they had to the application of acetone [3].

Figure 8: (A) The effect of treatment with a vehicle (red), 3 ug/kg ARA 290 (yellow), 10 ug/kg ARA 290 (green), 30 ug/kg ARA 290 (light blue), or 60 ug/kg ARA 290 (dark blue) administered at days 1, 3, 6, 8, and 10 post-surgery. (B) Correlation of the relief of cold allodynia and the treatment dosage of ARA 290

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Al-Onaizi MA, Thériault P, Lecordier S, Prefontaine P, Rivest S, ElAli A. Early monocyte modulation by the non-erythropoietic peptide ARA 290 decelerates AD-like pathology progression. Brain Behav Immun. 2022 Jan;99:363-382. doi: 10.1016/j.bbi.2021.07.016. Epub 2021 Jul 31. PMID: 34343617.

[2] Zhang W, Yu G, Zhang M. ARA 290 relieves pathophysiological pain by targeting TRPV1 channel: Integration between immune system and nociception. Peptides. 2016 Feb;76:73-9. doi: 10.1016/j.peptides.2016.01.003. Epub 2016 Jan 13. PMID: 26774587.

[3] Swartjes M, van Velzen M, Niesters M, Aarts L, Brines M, Dunne A, Cerami A, Dahan A. 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. Mol Pain. 2014 Feb 16;10:13. doi: 10.1186/1744-8069-10-13. PMID: 24529189; PMCID: PMC3928087.

 

 

 

PEPTIDES PREFER THE COLD
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