Mari Okazaki

Background/Objectives: FAD012 (3,5-dimethyl-4-hydroxycinnamic acid), a ferulic acid (FA) derivative, exhibits greater cerebroprotective effects than FA in experimental cerebral ischemia. Prophylactic oral administration of FAD012 preserves cerebral blood flow and blood–brain barrier integrity, consistent with its protective effects on the cerebrovascular endothelium. Compared with FA, FAD012 has higher calculated lipophilicity and a lower topological polar surface area, suggesting altered membrane permeability and systemic disposition. However, its pharmacokinetic characteristics remain unclear. Methods: This study compared the pharmacokinetics and tissue distribution of FAD012 and FA in male Sprague–Dawley rats. After intravenous and oral administration (10 mg/kg), plasma concentration–time profiles were analyzed using compartmental pharmacokinetic models. Tissue distribution after 2 weeks of dietary administration and plasma protein binding were also evaluated. Results: After intravenous administration, FAD012 exhibited a longer elimination half-life and tended to have a larger volume of distribution than FA, without a significant difference in total clearance. After oral administration, FAD012 showed more rapid absorption, with a higher maximum plasma concentration and a shorter time to maximum plasma concentration, with no significant differences in the area under the concentration–time curve or absolute bioavailability. After chronic administration, unchanged FAD012 was present at a higher plasma concentration, alongside lower tissue-to-plasma concentration ratios. FAD012 also exhibited markedly higher plasma protein binding than FA. Conclusions: These findings demonstrate that structural modification of FA to FAD012 alters absorption and systemic disposition without increasing absolute bioavailability. The higher plasma concentration of unchanged FAD012 observed after chronic administration may contribute to its cerebrovascular protective effects and support its potential as a prophylactically administered cerebroprotective agent for ischemic stroke.

Ferulic acid (FA) is well known for its antioxidant properties and neuroprotective effects. To enhance these biological activities, we designed a novel series of FA derivatives by introducing a phenyl group at the α-position of the carboxyl moiety. Further structural modifications were achieved by incorporating hydroxy or alkoxy substituents at various positions on the two aromatic rings. A series of these derivatives were synthesized and evaluated for their antioxidant capacity using the DPPH radical scavenging assay, as well as their cytoprotective effects against oxidative stress in Neuro-2a cells. Among the synthesized compounds, one derivative exhibited significantly enhanced activity in both assays. Mechanistic studies indicated that this heightened efficacy is attributable to a unique reaction pathway involving dual antioxidant mechanisms.

Background/Objectives: The rapid progression of stroke often results in irreversible brain damage and poor outcomes when treatment is delayed. Prophylactic administration of FAD012 (3,5-dimethyl-4-hydroxycinnamic acid), a synthetic derivative of ferulic acid (FA), has demonstrated cerebroprotective effects in ischemic models through antioxidant and endothelial protective mechanisms. This study investigated the effects of FAD012 on cerebral infarction and blood–brain barrier (BBB) integrity using a photothrombotic stroke model in rats. Methods: Male Sprague Dawley rats received a single intraperitoneal injection of FAD012 or FA (100 or 300 mg/kg) 60 min prior to stroke induction. Under isoflurane anesthesia, the middle cerebral artery was exposed, and stroke was induced by intravenous administration of Rose Bengal followed by green laser irradiation. Cerebral blood flow (CBF) was monitored by laser Doppler flowmetry. BBB disruption was evaluated by Evans Blue extravasation and immunohistochemistry for tight junction (TJ) proteins. Results: Control rats exhibited extensive infarction, BBB disruption, and reduced expression of claudin-5, occludin, and ZO-1, along with fragmented collagen IV. In contrast, FAD012 (300 mg/kg) significantly attenuated CBF reduction, reduced infarct size, preserved BBB integrity, and maintained TJ protein expression, with greater efficacy than an equivalent dose of FA. FAD012 also preserved the expression and phosphorylation of endothelial nitric oxide synthase (eNOS), a key marker of vascular integrity. The CBF-preserving effect of FAD012 was completely abolished by N^G-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor. Conclusions: These findings suggest that FAD012 protects endothelial function, thereby contributing to the maintenance of CBF and BBB integrity, supporting its potential as a prophylactic therapeutic agent for ischemic stroke.

Endothelial dysfunction is closely linked to inflammation and oxidative stress and ultimately contributes to the development of cardiovascular diseases. Lipopolysaccharide (LPS), a major component of Gram-negative bacteria, induces vascular inflammation and oxidative damage in experimental models. Curcumin (Cur), a polyphenol from Curcuma longa, is well known for its anti-inflammatory and antioxidant properties. In this study, we examined the protective effects of CUD003, a novel synthetic Cur derivative, on the LPS-induced impairment of endothelium-dependent relaxation in the thoracic aorta of mice. Male ICR mice were pretreated with CUD003 or Cur (3 or 10 mg/kg, p.o.) 30 min prior to LPS injection (10 mg/kg, i.p.). Twenty-four hours after LPS injection, vascular reactivity was assessed in isolated aortic rings by evaluating vasorelaxation and vasoconstriction responses. LPS markedly impaired acetylcholine-induced vasorelaxation in the phenylephrine (PE)-precontracted aortic rings, while PE-induced contraction and sodium nitroprusside-induced relaxation were preserved, indicating that LPS impaired endothelium-dependent relaxation without affecting smooth muscle function. Immunohistochemical analysis revealed a reduction in eNOS expression and elevated levels of TNF-α, COX-2, O₂⁻, and malondialdehyde, indicating enhanced inflammation and oxidative stress in the aorta. Pretreatment with CUD003 (10 mg/kg) significantly ameliorated these changes and showed superior protective effects compared to the same dose of Cur. These findings suggest that CUD003 protects against LPS-induced vascular dysfunction and suppresses inflammation and oxidative stress, supporting its potential as a preventive candidate against vascular inflammation and dysfunction.

Reactive oxygen species (ROS) contribute to cerebral damage in transient cerebral ischemia, making their elimination a key therapeutic target. Osteogenic disorder Shionogi (ODS) rats, which lack endogenous L-ascorbic acid (AA) synthesis, serve as a useful model for investigating AA’s protective effects against ischemic brain injury. ODS rats were given an AA-free diet (0% AA), 0.1% AA, or 1% AA in drinking water for two weeks before undergoing middle cerebral artery occlusion and reperfusion (MCAO/Re). The 0% AA group exhibited pronounced damage following MCAO/Re, characterized by the induction of lipid peroxidation, O₂⁻ production, inflammation-related gene expression, and extensive infarct formation. In contrast, the 1% AA group showed reductions in these markers, along with fewer TUNEL-positive cells and a smaller infarct volume. Notably, sodium-dependent vitamin C transporter 2 (SVCT2) expression increased in both two AA-supplemented groups, although the 0.1% AA group did not exhibit sufficient improvement in post-ischemic damage. A two-week intake of AA significantly alleviated MCAO/Re-mediated injuries associated with oxidative stress and inflammation in ODS rats. Sufficient AA intake is thus supposed to mitigate ischemic damage, possibly through SVCT2 upregulation and enhanced AA availability, leading to the suppression of oxidative stress and inflammation.

Dysphagia is a serious complication of stroke, yet effective pharmacological treatments remain limited. This study investigated the effects of FAD012 (3,5-dimethyl-4-hydroxy cinnamic acid), a synthetic derivative of ferulic acid (FA), on cerebral damage and swallowing dysfunction in a rat model of bilateral common carotid artery occlusion (2VO). Sprague–Dawley rats were orally administered FAD012 (3 or 10 mg/kg), FA (10 mg/kg), or 0.5% carboxymethyl cellulose (CMC, suspension vehicle) starting one week before 2VO. Two weeks after 2VO surgery, which was performed under isoflurane anesthesia, reflex swallowing was assessed by electromyographic recordings of the mylohyoid muscle under urethane anesthesia. Two weeks after 2VO, cerebral blood flow (CBF) declined to approximately 40% of baseline, and the number of reflex swallowing responses was significantly reduced in the CMC group. Additionally, 2VO induced O₂⁻ production, apoptotic cell death in the striatum, and a reduction in tyrosine hydroxylase expression. Substance P (SP) levels in the laryngopharyngeal mucosa, positively regulated by dopaminergic signaling in the basal ganglia, also decreased. FAD012 (10 mg/kg) effectively prevented the 2VO-induced reduction in CBF, enhanced the reflex swallowing, and preserved the dopamine-SP system. Notably, FAD012 exerted significantly stronger effects than FA at the same dose. These findings suggest that FAD012 maintains CBF under cerebral hypoperfusion and enhances the swallowing reflex by maintaining neuronal function in the striatal and laryngopharyngeal regions of 2VO rats.

Tissue plasminogen activator, aiming to restore cerebral blood flow (CBF), has been used for acute ischemic strokes in clinics; however, its narrow therapeutic time window remains a serious concern. To develop novel prophylactic drugs to alleviate cerebral ischemia/reperfusion injuries, ferulic acid derivative 012 (FAD012) was synthesized and showed comparable antioxidant properties to ferulic acid (FA) and probably possesses the potent ability to cross the blood–brain barrier. A more potent cytoprotective effect of FAD012 against H₂O₂-induced cytotoxicity in PC12 cells was also observed. In vivo toxicity was not observed in rats given a long-term oral administration of FAD012, indicating its good tolerability. A one-week-course oral administration of FAD012 significantly alleviated middle cerebral artery occlusion (MCAO)-induced cerebral ischemia/reperfusion injuries in rats, accompanied by the restoration of CBF and endothelial nitrogen oxide synthetase (eNOS) expression. Treatment with FAD012 significantly restored the cell viability and eNOS expression damaged by H₂O₂, used to mimic MCAO-triggered oxidative stress, in rat brain microvascular endothelial cells. Our findings suggested that FAD012 protected the viability of vascular endothelium and maintained eNOS expression, ultimately contributing to the restoration of CBF, and may provide a rationale for the development of FAD012 into an effective prophylactic drug for patients at high risk of stroke.

To explore the molecular mechanisms of action underlying the antileukemia activities of darinaparsin, an organic arsenical approved for the treatment of peripheral T–cell lymphoma in Japan, cytotoxicity of darinaparsin was evaluated in leukemia cell lines NB4, U-937, MOLT-4 and HL-60. Darinaparsin was a more potent cytotoxic than sodium arsenite, and induced apoptosis/necrosis in NB4 and HL-60 cells. In NB4 cells exhibiting the highest susceptibility to darinaparsin, apoptosis induction was accompanied by the activation of caspase-8/-9/-3, a substantial decrease in Bid expression, and was suppressed by Boc-D-FMK, a pancaspase inhibitor, suggesting that darinaparsin triggered a convergence of the extrinsic and intrinsic pathways of apoptosis via Bid truncation. A dramatic increase in the expression level of γH2AX, a DNA damage marker, occurred in parallel with G₂/M arrest. Activation of p53 and the inhibition of cdc25C/cyclin B1/cdc2 were concomitantly observed in treated cells. Downregulation of c-Myc, along with inactivation of E2F1 associated with the activation of Rb, was observed, suggesting the critical roles of p53 and c-Myc in darinaparsin-mediated G₂/M arrest. Trolox, an antioxidative reagent, suppressed the apoptosis induction but failed to correct G₂/M arrest, suggesting that oxidative stress primarily contributed to apoptosis induction. Suppression of Notch1 signaling was also confirmed. Our findings provide novel insights into molecular mechanisms underlying the cytotoxicity of darinaparsin and strong rationale for its new clinical application for patients with different types of cancer.