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.

 

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