Main Research Findings

1) Dihexa has therapeutic potential to act as a treatment for various neurological disorders related to reduced synaptic connectivity, such as Alzheimer’s disease.

2) Treatment with Dihexa has been shown to improve memory and motor impairments by enhancing synaptic connectivity and facilitating the formation of new functional synapses.

 

Selected Data

1) This comprehensive study performed by the research team of McCoy et al aimed to develop metabolically stable AngIV analogs with enhanced blood-brain barrier (BBB) permeability and procognitive activity, specifically focusing on a molecule named Dihexa. The research utilized male Sprague-Dawley rats and various in vitro and ex vivo experimental models [1].

The peptides under investigation, include: Nle¹-AngIV (Nle-Tyr-Ile-His-Pro-Phe) and its analogs such as D-Nle-YIH, acetyl-NleYIH, γ-amino butyric acid-YIH, NleYI-amide, and the primary focus, N-hexanoic-Tyr-Ile-(6) aminohexanoic amide (Dihexa). These syntheses employed 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis methods, ensuring high purity and confirmed structure via liquid chromatography (LC) mass spectrometry (MS). Scopolamine hydrobromide (S-1875) from Sigma-Aldrich was used to induce cognitive deficits in behavioral studies.

To assess the metabolic stability of the synthesized peptides, serum metabolism studies were conducted using blood obtained from 4-month-old male Sprague-Dawley rats. Blood was collected via jugular vein catheters, centrifuged to obtain serum, and stored at -20°C. Drug solutions, typically 5 mg/ml in HPLC-grade water (except for Dihexa, prepared in DMSO), were added to rat serum, and the mixture was incubated at 37°C. Metabolism was halted at specific time intervals by precipitating proteins with acetonitrile and acetic acid. The supernatant was then analyzed by HPLC with a Rainin Econosphere ODS C18 column and an acetonitrile/water mobile phase containing 0.1% trifluoroacetic acid. The degradation rate and half-life (t1/2) of the drugs were determined by measuring the decrease in area under the curve (AUC) at their retention times [1].

For in vivo pharmacokinetic analysis, male Sprague-Dawley rats weighing ≥250 g were cannulated in the right jugular vein. After a 30-minute incubation on ice, blood samples were collected at various time points post-intravenous 10 mg/kg dose or intraperitoneal 20 mg/kg dose administration of Dihexa dissolved in 75% DMSO. The typical injection volume was 200 µl. Blood samples were immediately centrifuged, and plasma was transferred to pre-prepared tubes containing acetonitrile and an internal standard of Nle-YI-(6) aminohexanoic amide, 100 µg/ml in isotonic saline. These samples were then processed for HPLC/MS analysis, similar to the serum metabolism studies, using a Shimadzu HPLC/MS system. Pharmacokinetic parameters such as AUC, maximum plasma concentration (Cmax), terminal elimination t1/2, volume of distribution (Va), and clearance were calculated [1].

To evaluate Dihexa’s ability to cross the BBB and accumulate in brain regions, rats were fitted with carotid cannulas and infused with a mixture of [³H]Dihexa (10 µCi) and [¹⁴C]inulin (2 µCi), a vascular space marker, in 100 µl of isotonic saline. Thirty minutes post-infusion, brains were removed, dissected into specific regions, and blood samples were collected. After solubilization, the samples were analyzed for ³H and ¹⁴C content using dual window scintillation counting. The ratio of Dihexa to inulin in the blood was used to correct for any blood contamination in brain regions, while higher ratios in brain regions compared to blood indicated active concentration.

Next to assess Dihexa’s metabolic stability in the liver, pooled male rat liver microsomes were used. The microsomes were incubated with 500 µM solutions of Dihexa, piroxicam, verapamil, and 7-ethoxycoumarin, as controls for low, moderate, and highly metabolized compounds, respectively, in 0.1 M Tris buffer with an NADPH-regenerating system at 37°C. Metabolism was terminated by adding acetonitrile, and the samples were analyzed by HPLC/MS. Intrinsic clearance and half-life were calculated [1].

Male Sprague-Dawley rats were used for behavioral testing, with 24-month-old rats of mixed sex also included for aged rat models. Scopolamine hydrobromide at a dose of 70 nmol was administered to induce memory deficits reminiscent of Alzheimer’s disease. Dihexa or its analogs were administered 5 minutes after scopolamine, either intracerebroventricularly at doses of 0.1 or 1 nmol), intraperitoneally at doses of 0.05, 0.25, or 0.50 mg/kg, or orally at doses of 1.25 or 2.0 mg/kg. The Morris water maze task, a spatial memory test, involved an 8-day acquisition phase and a Day 9 probe trial. Swim latency, swim distance, time spent in the target quadrant, and number of quadrant crossings were recorded using a computerized video tracking system [1].

Hippocampal neurons from P1 Sprague-Dawley rats were cultured and transfected with monomeric red fluorescent protein to visualize dendritic arbors. Neurons were treated with a vehicle, Nle¹-AngIV, or Dihexa for 5 days or acutely for 30 minutes. Cells were then fixed and imaged using an inverted confocal microscope. Dendritic spine density was measured on primary and secondary dendrites, and spine-head width was also assessed. Hippocampal slices from P4 Sprague-Dawley rats were also cultured and biolistically transfected with tomato fluorescent protein to visualize dendritic arbors. Slices were stimulated with a vehicle, Nle¹-AngIV, or Dihexa for 2 days. Spinosogenesis was assessed by measuring spine numbers per 50-µm dendritic length.

Finally, transfected neurons were immunostained for presynaptic markers, VGLUT1 and synapsin, and postsynaptic marker, PSD-95, to assess functional synapse formation. Microscopic images were analyzed for percent correlation between spines and markers. Whole-cell patch-clamp recordings were performed on mRFP-β-actin-transfected hippocampal neurons to measure miniature excitatory postsynaptic currents (mEPSCs), reflecting synaptic activity [1].

2) This review article completed by the research team of Wright et al summarizes the methods and materials employed in numerous studies concerning the development and testing of small molecule AngIV analogs, particularly Dihexa, for Alzheimer’s (AD) and Parkinson’s (PD) diseases. The primary “materials” discussed are various angiotensin peptides, their synthetic analogs, and specific receptor antagonists, while the “methods” encompass biochemical assays, animal models of neurodegenerative diseases, and behavioral assessments [2].

The overarching material developed and investigated comprises AngIV-based compounds, designed to overcome the limitations of native peptides like AngIV, which suffer from poor metabolic stability and inability to cross the BBB due to their size and hydrophilicity. Initial efforts involved synthesizing AngIV analogs with extended half-lives by incorporating reduced peptide bonds between residues, as exemplified by Divalinal-AngIV and Norleural-AngIV. Other researchers explored modifying the peptide backbone, such as replacing amino acids with a styrene moiety or reducing amide bonds.

A significant focus was on developing inhibitors of insulin-regulated aminopeptidase (IRAP), a protein identified as the AT4 receptor, with the goal of enhancing metabolic stability and BBB permeability. This line of research directly informed the development of Dihexa. Dihexa, chemically known as N-hexanoic-Tyr-Ile-(6)-aminohexanoic amide, emerged from efforts to create a small molecule that retains the memory-enhancing properties of Nle¹-AngIV and its N-terminal fragments. The design rationale prioritized oral efficacy, increased metabolic stability, and improved BBB penetrability. Key molecular modifications aimed at increasing hydrophobicity and decreasing hydrogen-bonding potential. Dihexa was synthesized as a small molecule compound that, in in vitro studies, demonstrated its ability to induce spinogenesis and synaptogenesis at picomolar concentrations. Pharmacokinetic analysis revealed it to be orally active with significantly increased metabolic stability, exhibiting a plasma half-life of 335.5 minutes, allowing for prolonged systemic availability [2].

Studies on the AD model primarily utilized rats in behavioral experiments designed to mimic cognitive impairment. Amnesia was induced via intracerebroventricular (icv) administration of scopolamine at a dose of 70 nmol in 2 µl artificial cerebrospinal fluid, with control animals receiving either aCSF or distilled water. Dihexa was administered orally via gavage at doses of 1.25 mg/kg or 2.0 mg/kg, 25 minutes before behavioral testing. The Morris Water Maze task served as the primary behavioral assessment, involving 8 days of training with 5 trials per day. On day 9, probe trials were conducted by removing the hidden platform, allowing animals to search for 120 seconds. Key outcome measures included the latency to find the platform during training and the time spent in the target quadrant during probe trials, which are indicators of spatial memory retention [2].

For the PD model, rats with unilateral 6-hydroxydopamine (6-OHDA) lesions in the substantia nigra were used to replicate PD pathology. Dihexa was administered intraperitoneally (i.p.) at 0.5 mg/kg in phosphate buffered saline (PBS) on alternate days, commencing 15 days post-surgery and continuing through day 48, with control animals receiving PBS alone. Behavioral assessments focused on motor functions, specifically walking stride length analysis and forepaw rope hang time. Additionally, histological assessment involved Tyrosine Hydroxylase (TH) staining in the substantia nigra to evaluate the integrity and potential restoration of dopaminergic neurons, with evaluations performed at 1 month and 3 months post-lesion.

The investigation into the AT4/HGF/c-Met system link revealed distinct roles for different compounds. Norleual-AngIV was used to inhibit HGF binding to the c-Met receptor, consequently blocking HGF-dependent signaling pathways involved in proliferation, invasion, and scattering. This inhibitory action was attributed to Norleual-AngIV’s capacity to prevent HGF dimerization, a crucial step for c-Met activation, while Dihexa was subsequently confirmed to act as an agonist within this system. Binding studies employed tritiated small molecule HGF analogs to map the distribution of HGF/c-Met receptors in the brain, establishing a correlation with known AT4 receptor densities. Furthermore, in vitro cellular and molecular methods demonstrated that Nle¹-AngIV stimulated dendritic spine numbers, increased spine size, and enhanced miniature excitatory postsynaptic currents in hippocampal neurons. Similar in vitro work showed that HGF promoted dendritic arborization in hippocampal neurons, an effect that could be blocked by NMDA receptor antagonists and was dependent on intracellular calcium and CaMKII activity [2].

 

Discussion

1) The results of this study completed by McCoy et al systematically evaluated the metabolic stability, BBB permeability, procognitive activity, and synaptogenic potential of Dihexa and other Nle¹-AngIV analogs, ultimately aiming to identify a therapeutic agent for dementia.

Initial investigations focused on the metabolic stability of various Nle¹-AngIV-derived peptides in rat serum. The parent compound, Nle¹-AngIV, demonstrated an exceedingly short half-life of less than 2 minutes. However, N-terminal modifications, such as N-acetylation, D-norleucine substitution, or replacement with γ-aminobutyric acid (GABA), dramatically elongated the peptides’ half-lives. For instance, N-Acetyl-Nle-Tyr-Ile-His had a half-life of 115 ± 7.6 min, D-Nle-Tyr-Ile showed 225 ± 23.7 min, and GABA-Tyr-Ile exhibited a remarkable 946 ± 234 min. C-terminal amidation, as seen in Nle-Tyr-Ile-His-NH₂, also provided a more modest increase in stability. Dihexa, with its combined N-hexanoic-Tyr-Ile-(6) aminohexanoic amide structure, exhibited a significantly extended half-life of 335.5 ± 9.5 min, confirming that both N- and C-terminal modifications are effective strategies for improving metabolic stability [1].

To address the critical issue of BBB permeability, studies using [³H]Dihexa and [¹⁴C]inulin in rats revealed that Dihexa avidly concentrated in all examined brain regions including the prefrontal cortex, hippocampus, hypothalamus, striatum, thalamus, midbrain, brain stem, and cerebellum, compared to blood. The ratios of [³H]/[¹⁴C] CPM/Gram consistently exceeded that observed in blood, confirming Dihexa’s ability to cross the BBB. Pharmacokinetic parameters after intravenous administration in rats showed Dihexa possessed a long half-life of 12.68 days and was extensively distributed, indicated by a large volume of distribution (Va). Moreover, microsomal metabolism studies demonstrated that Dihexa had very low phase I metabolism, with an average intrinsic clearance of 2.72 µl/min/mg and a half-life of 509.4 minutes. Predicted physicochemical properties supported its hydrophobic character and oral bioavailability, suggesting it is a metabolically stable and BBB-permeable molecule [1].

The procognitive activity of Dihexa was evaluated in the Morris water maze task using the scopolamine-induced cognitive deficit model in young rats and also in aged rats. In young rats with scopolamine-induced deficits, all tested Dihexa treatment groups significantly improved water maze performance. For intracerebroventricular administration, both low doses of 0.1 nmol and high doses of 1.0 nmol of Dihexa significantly reduced escape latency compared to the scopolamine group from day 2 onwards. The high-dose Dihexa group was indistinguishable from vehicle controls across all testing days. Similarly, intraperitoneal administration of 0.25 mg/kg and 0.50 mg/kg and oral administration of 2.0 mg/kg of Dihexa significantly improved performance to levels comparable with vehicle controls. Probe trials on day 9 consistently showed that high doses of Dihexa, regardless of administration method, significantly increased time spent in the target quadrant compared to scopolamine-impaired groups, indicating preserved learned task memory. In the aged rat model, oral administration of Dihexa also significantly improved water maze performance on most test days.

 

Figure 1: Changes in A) escape latency and B) time spent in quadrants across all treatment groups.

Beyond behavioral improvements, Dihexa demonstrated potent synaptogenic activity. In cultured hippocampal neurons, Dihexa treatment for 5 days induced a near 3-fold increase in the number of actin-enriched spines by 41 spines/50µm for Dihexa vs. 15 spines for the vehicle. Nle¹-AngIV also increased spine numbers to 32 spines/50µm, but to a lesser extent than Dihexa. Acute 30-minute applications of Dihexa or Nle¹-AngIV also significantly increased spine numbers compared to vehicles by 23.9 and 22.6 vs. 17.4 spines/50µm, respectively.

Immunocytochemical analysis confirmed that these newly formed spines were functional, showing similar percent correlations with presynaptic markers VGLUT1 and synapsin, and postsynaptic marker, PSD-95, as control-treated neurons. Electrophysiological recordings further supported this, revealing that Dihexa caused a 1.6-fold increase and Nle¹-AngIV caused a 1.7-fold increase in the frequency of AMPA-mediated mEPSCs compared to vehicle-treated neurons, indicating an expansion of functional synapses without altering individual synapse properties such as amplitude, rise, or decay times. In organotypic hippocampal slice cultures, Dihexa and Nle¹-AngIV similarly augmented spinogenesis, with control slices showing 7 spines per 50-µm dendrite length, compared to 11 for both Dihexa and Nle¹-AngIV treated slices [1].

In summary, the results demonstrate that Dihexa is a metabolically stable and BBB-permeable AngIV analog that effectively reverses scopolamine-induced cognitive deficits and improves spatial learning in aged rats. These behavioral benefits are underpinned by its marked ability to induce spinogenesis and promote functional synaptogenesis in hippocampal neurons, highlighting its potential as a robust procognitive and antidementia agent [1].

2) This review completed by Wright et al investigates the development of small molecule AngIV analogs as potential treatments for AD and PD, two debilitating neurodegenerative conditions currently lacking effective disease-modifying therapies. Traditional treatments for AD, such as cholinesterase inhibitors and NMDA receptor antagonists, offer only modest symptomatic relief, while L-DOPA for PD primarily addresses motor symptoms, with limited efficacy against non-motor dysfunctions and progression. This research explores a novel therapeutic avenue by targeting the brain’s renin-angiotensin system (RAS) and its interaction with the hepatocyte growth factor (HGF)/c-Met receptor system [2].

The brain RAS, particularly AngIV, has been implicated in learning, memory, neuroprotection, and cerebral blood flow regulation. Initial research suggested that the memory-enhancing effects often attributed to AngII were, in fact, mediated by its conversion to AngIV, acting at the AT4 receptor subtype. There has been ongoing debate regarding the precise identity of the AT4 receptor, with some evidence suggesting it is the insulin-regulated aminopeptidase (IRAP). However, a central hypothesis presented in this study is that the AT4 receptor system coincides with the HGF/c-Met receptor system. The HGF/c-Met system is critical for neurogenesis, neuronal survival, neurite outgrowth, synaptic plasticity, and cerebroprotection, functions that significantly overlap with those attributed to AngIV. This convergence of function prompted the investigation into AngIV analogs as potential modulators of the HGF/c-Met pathway.

Previous AngIV analogs, while showing promise in animal models of dementia, were hindered by poor metabolic stability indicated by a short half-life, and an inability to cross the BBB due to their peptide nature. To overcome these limitations, the researchers designed and synthesized novel small molecules with improved metabolic stability, BBB penetrability, and oral activity. This effort led to the development of Dihexa (N-hexanoic-Tyr-Ile-(6)-aminohexanoic amide), a key focus of this study [2].

The results for Dihexa were highly promising. In preclinical models, Dihexa demonstrated superior pharmacokinetic properties, including good oral activity and effective BBB penetration, along with enhanced metabolic stability with a half-life of 335.5 minutes. Functionally, Dihexa was found to induce spinogenesis and synaptogenesis in hippocampal neurons at picomolar concentrations, indicating its ability to augment synaptic connectivity [2].

In an Alzheimer’s disease model using scopolamine-induced amnesia in rats, Dihexa significantly improved spatial memory. Rats treated with Dihexa showed acquisition curves in the Morris water maze task that were comparable to control animals, and significantly better than those receiving scopolamine alone. Subsequent probe trials confirmed that Dihexa-treated rats exhibited persistent memory for the target quadrant, suggesting effective cognitive rescue.

For Parkinson’s disease, the study utilized a unilateral 6-OHDA lesioned rat model. Treatment with Dihexa led to a notable recovery in motor function, as evidenced by improved rope hang times. Dihexa-treated rats quickly regained motor performance equivalent to sham-operated controls, and significantly surpassed those receiving a placebo. Histological analysis further supported these behavioral findings, showing partial restoration of tyrosine hydroxylase staining in the substantia nigra of Dihexa-treated animals, indicative of neuronal protection and recovery [2].

The mechanism underlying Dihexa’s effects appears to involve its action as an HGF/c-Met receptor agonist. The authors confirmed that AngIV analogs can inhibit HGF binding to c-Met and HGF-dependent signaling by interfering with HGF dimerization, a crucial step for c-Met activation. Dihexa, by acting as an HGF/c-Met receptor agonist, facilitates this dimerization, leading to the beneficial neurotrophic and synaptogenic effects observed.

In conclusion, this study highlights Dihexa as a novel small molecule AngIV analog that can overcome the limitations of previous peptide-based compounds. Its ability to penetrate the BBB, its metabolic stability, and its significant efficacy in restoring cognitive and motor functions in animal models of AD and PD, likely through modulating the HGF/c-Met system, position it as a highly promising therapeutic candidate for these debilitating neurodegenerative diseases [2].

 

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] McCoy AT, Benoist CC, Wright JW, et al. Evaluation of metabolically stabilized angiotensin IV analogs as procognitive/antidementia agents. J Pharmacol Exp Ther. 2013;344(1):141-154. doi:10.1124/jpet.112.199497.

[2] Wright JW, Kawas LH, Harding JW. The development of small molecule angiotensin IV analogs to treat Alzheimer’s and Parkinson’s diseases. Prog Neurobiol. 2015;125:26-46. doi:10.1016/j.pneurobio.2014.11.004

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

2) Dihexa was found to have the potential to facilitate functional recovery in cases of peripheral nerve injury.

 

Selected Data

1) 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].

2) The research team of Weiss et al examined the potential of Dihexa, mesenchymal stem cells (MSC), and Granulocyte-Colony Stimulating Factor (G-CSF) to promote the recovery of limb damage as a result of nerve damage. For the purpose of this study, 10 to 12 week old inbred male Lewis rats weighing approximately 300 grams each were used. Male rats were used over female rats in order to eliminate any skewed data that may result from hormonal fluctuations related to the rats’ reproductive cycles. All housing, handling, and experimentation that involved the rats were conducted following the procedures set in place by the National Institutes of Health guide for the care and usage of laboratory animals [2].

All of the test subjects included in the study were randomly assigned to 1 of 10 different experimental treatment groups. Group 1 was a vehicle control that received an intravenous injection of saline. Group 2 was a vehicle control that received local Hydrogel and an intravenous injection of saline. Group 3 received local Hydrogel and an intravenous injection of MSC. Group 4 received local MSC via Hydrogel and an intravenous injection of MSC. Group 5 received local MSC and G-CSF via Hydrogel and an intravenous injection of MSC. Group 6 received local MSC and Dihexa via Hydrogel and an intravenous injection of MSC. Group 7 received local MSC and G-CSF via Hydrogel, an intravenous injection of MSC, and an injection into the gastrocnemius muscle with MSC and G-CSF dissolved in saline. Group 8 received local MSC and Dihexa via Hydrogel, an intravenous injection of MSC, and an injection into the gastrocnemius muscle with MSC dissolved in saline and Dihexa dissolved in DMSO. Group 9 acted as a vehicle control for Dihexa in group 6 and received local MSC and DMSO via hydrogel and an intravenous injection of MSC. Finally, group 10 acted as a vehicle control for Dihexa in group 8 and received local MSC and DMSO via hydrogel, and intravenous injection of MSC, and an injection into the gastrocnemius muscle with MSC and DMSO [2].

The animals underwent a sciatic nerve repair model that began with the transection of the main sciatic nerve above the trifurcation point into the tibial, fibular, and sural branches. Following the nerve transection, heparin was delivered through the tail vein and the animal was monitored under anesthesia for an hour to simulate the transport time it takes to get treatment in cases of traumatic peripheral nerve damage. The surgery was performed on the right hind limb and the left limb served as a control. 1 week after the surgery was performed the rats were administered manual physiotherapy to the right hind limb 1-2 times per week for 5 minutes at a time. Primary outcome measures assessed in this study included limb sensory and motor functioning at various time points until 16 weeks post-surgery. Secondary outcome measures assessed in this included gastrocnemius mass and the presence of a foot-flexion contracture [2].

Sensory functioning was examined using the flexor withdrawal spinal reflex. Testing began 1 week after the sciatic nerve repair surgery was conducted and occurred at weekly intervals. Using forceps, the stimulus was applied to the rats’ hind limb by momentarily pinching the areas innervated by the tibial, fibular, and sural nerves. A normal response to the stimulus was first determined in the unoperated left hind limb and was defined as an immediate withdrawal of the limb with or without a vocalization. The same stimulus was then applied to the operated right hind limb and was graded on a scale of 0-3 in comparison to the left hind limb response. A score of 0 was defined as no response; 1, mild response; 2, moderate response; and 3, normal strong response.

Motor functioning was evaluated starting at 2 weeks post-surgery and testing was conducted on a biweekly basis using a walking track analysis where the rats were made to walk in a confined walkway lined with white paper and led into a dark shelter. The researchers applied water soluble black ink to the plantar surfaces of the rats’ hindpaws prior to walking down the walkway from its entrance and into the shelter. The typical outcome measure used by researchers is the Sciatic Function Index that assesses hind limb motor function based on toe to toe and toe to heel distance. Unfortunately the SFI could not be calculated by the research team as there was poor toe to toe print separation. That being said, an alternative measure was used by the research team that assessed motor function by grading the toe and heel foot print characteristic on a scale from 0-4, with 0 defined as no print, non functional, and 4 defined as complete print, near normal functioning [2].

As it was previously mentioned the secondary outcome measures observed by the researchers included gastrocnemius muscle mass and foot flexion contractures. Foot flexion contracture in the rats were evaluated and graded on a scale of 0-4 with 0 being no contracture; 1, 0-30 degrees; 2, 31-60 degrees; 3, 61-90 degrees; and 4, >90 degrees. Following the end of the sensation and motor studies, the rats were euthanized and both the left and right gastrocnemius muscles were dissected and weighed in order to compare the muscle mass of the operated limb (right) versus the non-operated limb (left) [2].

 

Discussion

1) 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 1: 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 2: Changes in escape latency in the four different experimental groups included in the first part of the study.

Figure 3: 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 4: 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 5: 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].

2) The results of the sensory functioning reported that the fibular nerve boundary was recovered by approximately the first week, followed by recovery of the tibial and sural nerve boundaries. Similar to the fibular nerve the saphenous nerve boundary was recovered earlier, however, the saphenous nerve does not branch off of the sciatic nerve so sciatic nerve transection was not expected to affect the innervation of the saphenous nerve and all response to stimulus of the saphenous nerve was considered normal. Two weeks after the sciatic nerve repair all experimental groups ranged between a 1 and a 2 on the 0-3 graded functioning scale. In groups 7 and 8 including animals receiving injections in the gastrocnemius muscle of G-CSF and Dihexa, the sensory function was more pronounced by week 2 post-surgery. By week 10 sensory function improved in all of the experimental groups with grades on the 0-3 scale ranging from 2.6-3.0. Overall, results of the study found that total sensory function recovery improved early in group 7: mice administered G-CSF; and group 8: mice administered Dihexa [2].

Figure 6: Changes in sensory function grade in experimental groups 1-10 over the course of a 16 week time period following sciatic nerve repair surgery.

In terms of motor functioning, walking track footprints were graded on a scale of 0-4. At two weeks post-surgery all 10 of the experimental groups had grades ranging from 2.3 to 4.00, however, motor function ended up deteriorating by week 16 post-surgery. The exceptions were group 7: mice administered G-CSF; and group 8: mice administered Dihexa, both through injection to the gastrocnemius muscle. These two groups experienced a significant improvement in motor functioning by 16 weeks after the sciatic nerve repair. Additionally, the research team noted that these improvements were seen when the nootropic was administered in the gastrocnemius in combination with mesenchymal stem cells. When administered locally and intraperitoneally there were no remarkable changes in motor functioning observed by the researchers [2].

Figure 7: Recovery of motor functioning in groups of mice administered MSC, G-CSK, and Dihexa, determined by walking track foot prints of normal and sciatic nerve resected limbs.

As for the secondary outcome measures of gastrocnemius muscle mass and flexion foot contracture, there was a significant decrease in muscle mass seen in the right hind limb that underwent sciatic nerve transection, in comparison to the left hind limb that was not operated on. Overall loss of muscle mass ranged from 32-45% and none of the 10 experimental treatment groups experienced any significant changes in muscle mass when administered treatment. Additionally, flexion foot contractures were graded on a scale of 0-4. When compared to the control group, the foot flexion contracture angle was found to be reduced most significantly in group 7: mice administered MSC and G-CSF, with a grade of 1.2; and group 8: mice administered MSC and Dihexa, with a grade of 1.8 [2].

Figure 8: Changes in gastrocnemius muscle atrophy between left and right hind limbs, across the 10 experimental treatment groups.

Figure 9: Foot flexion contracture grade in mice treated with Dihexa and G-CSF, in comparison to the control group of mice.

 

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] Weiss JB, Phillips CJ, Malin EW, Gorantla VS, Harding JW, Salgar SK. Stem cell, Granulocyte-Colony Stimulating Factor and/or Dihexa to promote limb function recovery in a rat sciatic nerve damage-repair model: Experimental animal studies. Ann Med Surg (Lond). 2021 Oct 8;71:102917. doi: 10.1016/j.amsu.2021.102917. PMID: 34703584; PMCID: PMC8524106.

 

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