Description

Dihexa Nootropic Powder

 

CAS Number	1401708-83-5
Other Names	L-Isoleucinamide, N-(1-oxohexyl)-L-tyrosyl-N-(6-amino-6-oxohexyl)-; 9WYX65A5C2; N-hexanoic-Tyr-Ile-(6) aminohexanoic amide; PNB-0408
IUPAC Name	(2S,3S)-N-(6-amino-6-oxohexyl)-2-[[(2S)-2-(hexanoylamino)-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylpentanamide
Molecular Formula	C₂₇H₄₄N₄O₅
Molecular Weight	504.67
Purity	≥99% Pure (LC-MS)
Material Safety Data Sheet (MSDS)	View Material Safety Data Sheet
Liquid Availability	30mL liquid  (20mg/mL, 600mg bottle)
Powder Availability	10 milligrams (lyophilized/freeze-dried) 1 gram 60 capsules (5mg/capsule, 300mg bottle)
Gel Availability	N/A
Storage	Store in cool dry environment, away from direct sunlight.
Terms	All products are for laboratory developmental research USE ONLY. Products are not for human consumption.

 

What is Dihexa?

N-hexanoic-TyrIle-(6)-amino hexanoic amide, more commonly known as Dihexa, is a blood-brain barrier-permeable angiotensin IV analogue. Dihexa is categorized as a nootropic compound with a long cyclical half life and the potential to promote anti-dementia activity in cases of pharmacologically induced cognitive impairments [1]. Current research regarding Dihexa focuses on its ability to improve the recovery of peripheral nerve functioning, as well as how the compound interacts with the PI3K/AKT pathway to prompt the procognitive capacity of the nootropic.

 

Main Research Findings

1) The findings of the study suggest that Dihexa may have the potential to treat disorders, such as Alzheimer’s disease, that may benefit from the augmentation of synaptic connectivity.

2) Dihexa was shown to improve cognitive impairments and recover memory by inhibiting inflammation and decreasing neuronal loss by affecting the PI3K/AKT signaling pathways.

 

Selected Data

1) Previous research has determined the ability of AngIV analogs such as Dihexa, to increase cerebral blood flow, elicit neuroprotective effects, and promote long term potentiation, learning, and memory consolidation. That being said, the research team of McCoy et al examined the potential of the nootropic compound to induce spinogenesis and synaptogenesis. The study began by collecting blood samples from the jugular veins of fourth-month old, male Sprague-Dawley rats. The samples were incubated on ice and centrifuged to separate the serum, which was then collected, transferred to clean tubes, and stored until further experimentation took place [2].

10 ul of each drug solution was added to 90 ul of the rat blood serum samples and at specific time intervals the researchers terminated metabolism by adding 1 ml of ACN and acetic acid in order to precipitate the proteins which were then removed after overnight refrigeration followed by centrifugation. The supernatant was separated, dried, and rehydrated prior to HPLC separation and analysis. The degradation rate of the drugs was measured by observing the decrease in the area under the curve at the retention time of the drug. A plot of the concentration versus the time was then generated in able to find the degradation kinetic constant and calculate the half life

Microsomal metabolism was also examined by the research team using the liver microsomes from male rats and an NADPH-regenerating system prepared by adding 1.7 mg/ml NADP, 7.8 mg/ml glucose 6-phosphate, and 6 U/ml glucose-6-phosphate dehydrogenase to 10 ml of 2% sodium bicarbonate. A 500 um solution of Dihexa was prepared in acetonitrile and the collected liver microsomes were suspended in a solution of 0.5 mg/ml of 0.1 M Tris buffer at a pH 7.38. The suspended liver microsomes were placed on ice in prechilled microcentrifuge tubes, allowing for the addition of 640 ul of 0.1 M Tris buffer and 10 ul of 500 uM test compound to the sample. Following rotisserie hybridization and incubation for the appropriately assigned time, 500 ul of each sample was added to 500 ul of ice-cold acetonitrile. Samples were then analyzed by high-performance liquid chromatography/mass spectrometry in order to determine drug concentrations and calculate negative controls samples containing no liver microsomes [2].

Following the examination of microsomal metabolism, 24-month old male Sprague-Dawley rats weighing approximately 290-450 grams were utilized for the purpose of behavioral studies. Prior to behavioral studies, a guide cannula was positioned in the right hemisphere and was fitted with a beveled tip to stop the penetration depth at 2.5 mm. The cannula was secured to the skull and sealed with a thick stainless steel wire. After the surgery the animals were maintained under a 12 hour light/12 hour dark cycle and were closely monitored and gently handled for 5 minutes per day, 5-6 days after the surgery was performed [2].

The primary behavioral study performed was the water maze test that consisted of a circular apparatus painted black and filled with water to a depth of 26 cm. The tank was sectioned into four equal quadrants that were defined as southeast, southwest, northwest, and northeast. The hidden platform was randomly placed in one of the four quadrants and submerged 2 cm below the surface of the water. Entry points to the tank were randomly assigned to one of four quadrant corners situated north, south, east, and west, while the walls of the tank were covered with spatial cues of different shapes and colors. Each test subject received an injection of 70 nmol of scopolamine hydrobromide in 2 ul of artificial cerebrospinal fluid that was delivered over a 20 second time period, 20 minutes prior to the water maze testing. This was followed by administration of Dihexa mixed with 2 ul of artificial cerebrospinal fluid delivered 5 minutes before water maze testing began [2].

Acquisition trials were conducted over the course of 8 consecutive days with 5 trials conducted each day. On day 1 before the first trial, the test subjects were placed on the platform for 30 seconds in order to familiarize themselves. The first trial began by facing the rat towards the wall of the maze at one of the entry points. The test subjects were then allowed to swim around the tank for 120 seconds in order to locate the hidden platform. Once the platform was located the animal was allowed a 30 second rest period before the next trial started. If the rat could not locate the platform the researchers placed the rat onto the platform and allowed them a 30 second rest period before the next trial started. On day 9 following acquisition training, an additional trial was conducted where the platform was removed and the rat was required to swim for the entire 120 second duration. This allowed the research team to determine the animals’ learned responses by counting how many times each subject crossed the quadrant where the platform was located. Swim path was recorded using a computerized tracking system that displayed swim latency and swim distance [2].

2) The research team of Sun et al examined the potential of Dihexa to improve cognitive functioning related to Alzheimer’s disease by targeting the brain AngIV/PI3K/AKT axis. 6 month old male APP/PS1 mice and wild-type C57 mice were used for the purpose of this study. The test subjects were housed in a standard animal room under a 12 hour light/12 hour dark day/night cycle with ad libitum access to food and water. The first part of the study began by randomly dividing the test subjects in four groups including: wild type, APP/PS1, APP/PS1 + Dihexa administered at a dose of 1.44 mg/kg, and APP/PS1 + Dihexa administered at a dose of 2.88 mg/kg [1].

The second part of the study began by randomly dividing the test subjects in three groups including: APP/PS1, Dihexa administered at a dose of 2.88 mg/kg, and 2.88 mg/kg of Dihexa + 0.5 mg/kg of wortmannin. Both Dihexa and wortmannin were prepared by dissolving the compounds in 10% DMSA, 40% PEG 300, 5% Tween 80, and 45% saline. Dihexa was administered to the APP/PS1 mice intraperitoneally from 6 to 9 months of age and 0.9% saline was administered to the wild type group once per day over a three month time period.

After the drugs were administered to the test subjects for 3 months they underwent the Morris water maze test. A round black tub was filled with water and divided into four equal regions labeled as north (N), south (S), east (E), and west (W). A small platform was submerged 1 cm below the water surface, in the center of the northeast quadrant of the tank. Each mouse underwent 4 trials per day for 5 consecutive days. They were allowed 60 seconds to search the tank for the platform and at the end of each session they were placed on the platform and remained there for 30 seconds. On the 6th day the platform was removed and the researchers recorded the number of times the individual mice crossed the quadrant where the platform was previously located, over the course of 60 seconds [1].

After all necessary data was collected for the Morris water maze test, the mice were euthanized and the brain tissue was dissected. The brain tissue obtained was weighed and PBS was added in order to ensure a weight (g) to volume (mL) ratio of 1:9. The samples were homogenized to allow for centrifugation of the supernatant, followed by measurement of AngIV, TNF-alpha, IL-10, and IL-1-beta levels through the use of a sensitive and specific ELISA assay. Following perfusion, the brain was fixed for 48 hours and embedded in paraffin. The 4 um thick paraffin sections were then dehydrated, stained with methylene blue, and washed with distilled water, followed by a repeat of dehydrating and washing the sections in order to prepare for a Nissl staining trial. All positively stained cells were counted and the “% Nissl positive neuron” was calculated by dividing the number of positive neurons by the total number of neurons in each cell [1].

Finally, the samples were prepared for Western blotting analysis by lysing the brain homogenates on ice for 300 minutes in 100 mL of a lysis buffer composed of 120 mM NaCL, 40 mM Tris (pH 8), and 0.1% NP40, followed by centrifugation. Additionally, a bicinchoninic acid (BCA) assay was used to determine the protein concentration; 30 ug of the protein was the separated using 10% SDS-PAGE and electroblotting onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% non-fat milk and incubated overnight at 4 degrees Celsius, with primary antibodies. This procedure was followed by incubation of the membranes with secondary antibodies at room temperature for 2 hours [1].

 

Discussion

1) The goal of the research team of McCoy et al was to find an AngIV analog capable of permeating the blood brain barrier and eliciting procognitive activity by improving the metabolic stability of the tri- and tetrapeptide N-terminals of AngIV analogs. Various structural alterations were made at the N-terminal such as substitution of D-norleucine for L-norleucine, N-acetylation of norleucine, and replacement of norleucine with GABA in order to improve overall stability by reducing susceptibility to aminopeptidases.

The compounds were incubated in the presence of rat blood serum samples and all of the resulting incubates were analyzed by HPLC in order to determine their rate of metabolic activity. The results reported that AngIV had a very short half life of less than 2 minutes, however, each of the N-terminal modified compounds, such as Dihexa, exhibited elongated half-lives in comparison to the parent compound. These findings suggest that reduced degradation of the N-terminal leads to an improvement in metabolic stability [2].

Dihexa also helps contribute to the improvement of metabolic stability through its high level of bioavailability for distribution into tissues. In addition to its bioavailability and extended half life, rat liver microsomes were examined as an additional measurement of the effects of Dihexa on metabolic rate. Initial results reported that phase I metabolism of the nootropic was very low with an intrinsic clearance of 2.72 ul/min/mg, as well as an average half life of approximately 510 minutes. The clearance rate of Dihexa was compared to those of similar nootropic compounds such as piroxicam, verapamil, and 7-ethoxycoumarin [2].

In terms of behavioral testing, Dihexa was assessed for its ability to reverse deficits induced by the administration of scopolamine, seen in the subjects’ performance in the water maze test. The initial trial was run to verify the neuroprotective and cognitive effects of Dihexa; the collected results reported that both high and low doses of the nootropic significantly increase performance on the water maze test in comparison to the rats that were only administered scopolamine. It is important to mention that on all testing days, the group receiving the higher dose of Dihexa experienced performance that was indistinguishable from the control group administered only vehicle compounds [2].

Additionally, the research team sought to determine if there were any significant changes in results based on whether the nootropic was delivered via a cannula, an intraperitoneal injection, or oral administration. For all three methods of delivery there was a clear dose-response relationship between performance on the water maze test and administration of Dihexa. That being said, the high doses of the nootropic measured at 0.5 mg/kg/day for intraperitoneal delivery and 2.0 mg/kg/day for oral delivery, and resulted in a performance that was indistinguishable from the control group and significantly better than the animals treated with scopolamine alone [2].

Figure 1: Changes in scopolamine-dependent learning deficits in response to treatment with Dihexa. A) rats pretreated with 70 nmol of scopolamine and administered Dihexa via cannula. B) rats pretreated with 70 nmol of scopolamine and administered Dihexa via intraperitoneal injection. C) rats pretreated with 70 nmol of scopolamine and administered Dihexa via oral delivery methods.

After 8 days of acquisition training, a 9th day of trials commenced where the platform was taken away from the apparatus and the rats had to swim for a full 120 seconds while the research team recorded how many times each rat passed into the quadrant where the platform was previously located. The test subjects administered the highest dose of Dihexa spent the most amount of time in the target quadrant compared to the groups of animals treated with scopolamine alone. That being said, the findings indicated that there was a dose-dependent relationship between administration of the nootropic and the recorded escape latencies [2].

Figure 2: Time spent in the target quadrant by test subjects in each experimental treatment group. A) mice administered Dihexa through a cannula, B) mice administered Dihexa via intraperitoneal injection, and C) mice administered Dihexa via oral delivery methods

2) In order to determine whether AngIV is involved in the development of Alzheimer’s, the research team of Sun et al detected the baseline levels of AngIV in both wild type and APP/PS1 mice. In comparison to the wild type mice, the APP/PS1 mice were found to have significantly lower levels of AngIV in the brain. That being said, Dihexa was administered to the rats in doses of 1.44 mg/kg of 2.88 mg/kg in order to see how levels of AngIV changed in response to the nootropic compound. The results reported that both doses of Dihexa increased levels of AngIV in the brains of APP/PS1 mice, with the 2.88 mg/kg dose increasing these levels to almost the same amount in the wild type. These findings suggest that levels of AngIV in the brain potentially plays a role in the development of Alzheimer’s disease [1].

Figure 3: The average levels of AngIV in the brain in the four different experimental groups included in the first part of the study.

In addition to levels of AngIV in the brain, the test subjects underwent the Morris water maze test in order to measure the cognitive ability of APP/PS1 mice when administered Dihexa. From day 1 of the experiment to day 5 the escape latency was found to remarkably decrease, however, escape latency in the APP/PS1 mice was higher than that of the wild type mice. That being said, both the 1.44 mg/kg and 2.88 mg/kg doses of the nootropic were successful at decreasing the escape latency to various degrees, with this effect being most prominent on the 4th and 5th days of the experiment. Overall, the researchers found that the APP/PS1 mice treated with Dihexa exhibited a significantly better performance in comparison to the control mice when assessing the number of platform crossings. These findings indicate that treatment with Dihexa improves cognition in APP/PS1 mice [1].

Figure 4: Changes in escape latency in the four different experimental groups included in the first part of the study.

Figure 5: The average number of crossing into the target quadrant in each of the four different experimental groups included in the first part of the study.

Following the Morris water maze test, Nissl staining was used to observe the amount of positive neuronal cells present. In comparison to the wild type mice, APP/PS1 mice experienced significant synaptic loss as well as a reduction in the number of neuronal cells in the cerebral cortex. When treated with both the 1.44 mg/kg dose and the 2.88 mg/kg dose of Dihexa, the APP/PS1 mice experienced an increase in the number of neuronal cells present in the cerebral cortex. Based on the results on the Nissl staining, the research team was able to conclude that treatment with Dihexa attenuated the rate of neuronal loss in the brains of APP/PS1 mice [1].

Figure 6: Percentage of Nissl-positive neurons in each of the four different experimental groups included in the first part of the study.

Additionally, in order to explore the mechanism of action being neuronal apoptosis, the research team assessed levels of neuroinflammation and glial activation by detecting levels of IL-1-beta, IL-10, and TNF-alpha in the brain. Baseline measurements found that TNF-alpha and IL-1-beta levels in the APP/PS1 group of mice were much higher than the wild type group of mice. However, when the mice were treated with Dihexa, levels of both IL-1-beta and TNF-alpha were found to significantly decrease. On the other hand, baseline measurements found that levels of IL-10 in APP/PS1 mice were significantly reduced in comparison to wild type mice, and when treated with Dihexa these mice experienced an increase in IL-10 levels. These findings suggest that the nootropic compound elicits neuroprotective effects on nerve cells in the brain damaged by inflammatory factors [1].

Figure 7: Changes in the levels of A) IL-1-beta, B) TNF-alpha, and C) IL-10 in each of the four different experimental groups included in the first part of the study.

It is important to mention that the research team took their experimental procedures a step further in order to define the relationship between Dihexa and the PI3K/AKT signaling pathway, wortmannin, a PI3K inhibitor, was administered to the mice intragastrically to detect the number of neuronal cells and inflammatory factors present. The introduction of wortmannin was found to significantly reverse the expression of PI3K and AKT, as well as the anti-apoptotic and anti-inflammatory effects of Dihexa, resulting in a decrease in the number of neuronal cells present in the cortex and the levels of IL-10, and an increase in the levels of TNF-alpha and IL-1-beta [1].

 

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] Sun X, Deng Y, Fu X, Wang S, Duan R, Zhang Y. AngIV-Analog Dihexa Rescues Cognitive Impairment and Recovers Memory in the APP/PS1 Mouse via the PI3K/AKT Signaling Pathway. Brain Sci. 2021 Nov 11;11(11):1487. doi: 10.3390/brainsci11111487. PMID: 34827486; PMCID: PMC8615599.

[2] McCoy AT, Benoist CC, Wright JW, Kawas LH, Bule-Ghogare JM, Zhu M, Appleyard SM, Wayman GA, Harding JW. Evaluation of metabolically stabilized angiotensin IV analogs as procognitive/antidementia agents. J Pharmacol Exp Ther. 2013 Jan;344(1):141-54. doi: 10.1124/jpet.112.199497. Epub 2012 Oct 10. PMID: 23055539; PMCID: PMC3533412.

 

Dihexa is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.

 

 

 

 

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2024-10-30-Umbrella-Labs-Dihexa-Certificate-Of-Analysis-COA.pdf

 

 

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