Main Research Findings

1) Treatment with a combination of Dihexa, Vitamin C, and Forskolin was found to substitute growth factors to induce hepatic specification.

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

 

Selected Data

1) The study conducted by the research team of Pan et al aimed to develop an efficient and cost-effective small-molecule strategy for generating functional hepatic cells from human pluripotent stem cells (hPSCs). This involved several distinct phases, from initial cell culture and direct differentiation to functional assays and in vivo transplantation. Human PSC lines, including the UC15 iPSC line and the H1 embryonic stem cell (ESC) line, were maintained under standard conditions. Cells were cultured in mTeSR1 medium on 100-fold-diluted Matrigel matrix and routinely passaged using Accutase [1].

For hepatic differentiation, a stepwise approach was employed. Definitive Endoderm (DE) induction was the first critical step. When hPSCs reached approximately 70% confluency, the culture medium was switched to RPMI1640 supplemented with 1× B27. This basal medium was then augmented with a specific cocktail of small molecules: 3 µM or 1 µM CHIR99021 (CHIR), IDE1, 2 µM Ly294002 (Ly), and 0.75 µM PD0332991 (PD). The cells were treated with these cocktails for 3 days, with variations in CHIR concentration and the presence of Ly or PD to optimize DE induction efficiency [1].

Following DE induction, the cells underwent hepatic specification to produce hepatoblasts (HBs). This stage involved culturing DE cells in RPMI1640 medium supplemented with 1× B27. Initially, growth factors such as 20 ng/mL BMP2 and 30 ng/mL FGF4 were used as a control. Crucially, the researchers explored substituting these expensive growth factors with a small-molecule cocktail, specifically 10 µg/mL Vitamin C, 0.1 µM Dihexa, and 10 µM Forskolin (VDF cocktail), to assess its efficacy in inducing hepatic specification.

Once generated, HBs were maintained in an expansion medium. This medium consisted of RPMI1640, 1× B27 supplement, and 1× ITS, further supplemented with a chemical cocktail named ACDF SV. This cocktail included 5 µM A8301, 3 µM CHIR, 0.1 μΜ Dihexa, 10 µM Forskolin, 0.5 µM SAG, and 10 µg/mL Vitamin C. For replating, HBs were dissociated using Accutase and reseeded onto Matrigel-coated plates. For the final stage of maturation into functional hepatocyte-like cells (HLCs), the proliferative HBs were cultured in hepatoZYME-SFM medium supplemented with 1X GlutaMAX. This maturation medium contained a specific small-molecule cocktail: 5 μΜ A8301, 0.1 μΜ dexamethasone (Dex), 0.1 µM Dihexa, and 0.5 mM NH4Cl. The medium was refreshed daily throughout the 5-day differentiation period. All chemical compounds were sourced from Selleck unless otherwise noted, and growth factors from PeproTech [1].

The functionality of the differentiated HLCs was rigorously assessed using several in vitro assays. Culture media from 24-hour incubated cells were collected, stored at -80 °C, and analyzed for urea concentration using LC/MS/MS API3000, normalized to total cell protein. HLC cultures were incubated with conventional probe substrates for CYP3A4 (6 µM midazolam), CYP2C9 (10 µM diclofenac), and CYP2D6 (10 µM dextromethorphan) for 2 hours. Metabolite production was quantified by LC/MS/MS API3000 and normalized to total cell protein. Periodic acid-Schiff (PAS) staining was performed. Cells were fixed with 4% PFA for 30 minutes, and intracellular glycogen was stained according to the manufacturer’s instructions. Differentiated cells were incubated in media with 1 mg/ml ICG for 1 hour at 37 °C, washed with PBS, and imaged using a phase-contrast microscope. HLCs were cultured with Alexa-Flour 488-ac-LDL for 1 hour, followed by immunohistochemistry and DAPI counterstaining [1].

Immune-deficient NOD-SCID-IL2RG-/- mice (NSI mice, GIBH) were used as recipients for in vivo transplantation experiments. To induce acute liver injury, 8-week-old NSI mice received two consecutive intraperitoneal injections of DMN (7 mg/kg, Sigma, 1.0% dissolved in saline). Two days post-DMN treatment, 1 × 10^6 hepatic cells were intrasplenically transplanted into the injured mice. Recipient mice livers were harvested at various time points to monitor transplantation and repopulation.

Total RNA was extracted using TRIzol reagent and quantified with NanoDrop 2000. cDNA was reverse-transcribed using ReverTra Ace and oligo-dT. Quantitative RT-PCR was performed on a CFX96 machine using SYBR Green Premix. GAPDH was used for normalization, and experiments were repeated a minimum of three times. Cells were fixed with 4% PFA for 30 minutes, permeabilized with 0.1% Triton X-100, and blocked with 5% normal goat or donkey serum. Primary antibodies were incubated overnight at 4 °C, followed by Alexa Fluor-conjugated secondary antibodies. Nuclei were stained with 5 µg/mL DAPI.

Cells were dissociated with Accutase and resuspended in PBS with 3% BSA. For cell sorting, cell suspensions were stained with FITC-conjugated human Ep-CAM antibody and Alexa Fluor APC-conjugated human C-Kit for 30 minutes on ice. EpCAM+/C-Kit+ cells were sorted using a MoFloTM fluorescence-activated cell sorter. Sorted cells were reseeded on Matrigel-pre-coated plates and expanded. For proliferation analysis, expanded HBs were fixed with 4% PFA, incubated in blocking and permeabilizing buffers (0.1% Triton X-100, 5% normal donkey serum), and then incubated with APC-conjugated Ki67 and other indicated antibodies [1].

2) 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. 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 [2] .

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 [2].

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 [2].

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 [2].

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 [2].

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 [2].

 

Discussion

1) This study performed by researchers Pan et al developed an efficient and cost-effective small-molecule strategy for generating functional hepatic cells from hPSCs, demonstrating its potential for cell-based therapy and drug discovery. The initial attempts to induce DE differentiation using small-molecule cocktails containing CHIR and IDE1 (CI-I and CI) proved inefficient. Immunostaining revealed mixed cell populations with many undifferentiated hPSCs and mesoderm cells, and DE markers SOX17 and FOXA2 were expressed at lower levels compared to the Activin A-based control (CA). Gene expression analysis indicated that these suboptimal results were likely due to lower endogenous Nodal signaling and elevated BMP signal activities [1].

To improve DE differentiation, the researchers incorporated PI3K inhibitor LY294002 (Ly) or CDK4/6 inhibitor PD into the small-molecule cocktails. The enhanced cocktails, CILy (CHIR+IDE1+Ly) and CIP (CHIR+IDE1+PD), significantly increased the expression of DE markers SOX17 and FOXA2, with CIP showing the highest efficiency, comparable to the traditional CA method. Further analysis of signaling pathways confirmed that PD effectively upregulated endogenous TGF-β/Nodal signaling and downstream Smad2/3 transduction, while concurrently downregulating BMP signaling, leading to robust DE formation. In contrast, while Ly boosted WNT3A expression and accelerated primitive streak/MD induction, it resulted in a mixed population that included unwanted mesoderm [1].

For hepatic specification, the study identified a novel small-molecule cocktail, VDF (Vitamin C, Dihexa, and Forskolin), that could effectively substitute for expensive growth factors like BMPs, FGFs, and HGF. This VDF cocktail induced high expression of key hepatic markers such as AFP, HNF4α, and Albumin (ALB), with an efficiency of 95.5% (HNF4a+/AFP+ positive cells), closely matching that of the growth factor-based control. This successful substitution was further validated using an independent iPSC line.

The HBs generated using this small-molecule protocol were successfully purified by EpCAM antibody sorting, achieving 95.2% purity. These purified HBs demonstrated robust self-renewal capacity, expanding in a small-molecule-defined medium (ACDFSV) for over 20 passages without losing their proliferative capacity or characteristic morphology. FACS analysis confirmed that at passage 10, 54.7% of EpCAM+ HBs co-expressed the proliferative marker Ki67, and immunostaining showed sustained co-expression of AFP, HNF4α, and Ki67. Importantly, these expanded HBs retained bipotency, co-expressing both hepatic and early cholangiocyte markers [1].

Upon maturation, the proliferative HBs differentiated into functional HLCs exhibiting typical polygonal morphology and distinct round nuclei. In vitro functional analyses confirmed their mature hepatocyte characteristics: they showed high transcription and protein expression of ALB, A1AT, CYP3A4, and CYP2C9, comparable to primary human hepatocytes (PHs). Moreover, these HLCs displayed significant CYP450 metabolic activities for CYP3A4, CYP2C9, and CYP2D6, similar to PHs. They also demonstrated a high urea secretion pattern of approximately 50% of PHs and possessed the abilities for cytoplasmic glycogen storage, indocyanine green (ICG) uptake, and LDL uptake.

Finally, the in vivo efficacy of these small-molecule-derived HBs was demonstrated through transplantation into DMN-induced acute liver failure NSI mice. The transplanted HBs significantly improved the survival rate of recipient mice to nearly 80% survival compared to the sham control group, which experienced high mortality. Histological examination showed reduced liver necrosis and restored tissue morphology in transplanted mice. Tracing experiments with Dil-labeled HBs and human ALB (hALB) confirmed that the HBs successfully homed to the injured liver and differentiated into mature hepatocytes. Levels of AST and ALT were significantly decreased, indicating functional recovery of the damaged liver. Four weeks post-transplantation, widespread hALB-positive cells were observed, and FACS analysis showed that approximately 15% of the total hepatocyte mass was derived from the transplanted human HBs, with increasing hALB secretion in the mouse serum over time [1].

2) 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 [2].

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 [2].

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 [2].

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 [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] Pan T, Wang N, Zhang J, et al. Efficiently generate functional hepatic cells from human pluripotent stem cells by complete small-molecule strategy. Stem Cell Res Ther. 2022;13(1):159. Published 2022 Apr 11. doi:10.1186/s13287-022-02831-1

[2] 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.

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.

 

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