Potential of Powder Rheology for Detecting Unforeseen Cross-Contamination of Foreign Active Pharmaceutical Ingredients.

Unexpected cross-contamination by foreign components during the manufacturing and quality control of pharmaceutical products poses a serious threat to the stable supply of drugs and the safety of customers. In Japan, in 2020, a mix-up containing a sleeping drug went undetected by liquid chromatography during the final quality test because the test focused only on the main active pharmaceutical ingredient (API) and known impurities. In this study, we assessed the ability of a powder rheometer to analyze powder characteristics in detail to determine whether it can detect the influence of foreign APIs on powder flow. Aspirin, which was used as the host API, was combined with the guest APIs (acetaminophen from two manufacturers and albumin tannate) and subsequently subjected to shear and stability tests. The influence of known lubricants (magnesium stearate and leucine) on powder flow was also evaluated for standardized comparison. Using microscopic morphological analysis, the surface of the powder was observed to confirm physical interactions between the host and guest APIs. In most cases, the guest APIs were statistically detected due to characteristics such as their powder diameter, pre-milling, and cohesion properties. Furthermore, we evaluated the flowability of a formulation incorporating guest APIs for direct compression method along with additives such as microcrystalline cellulose, potato starch, and lactose. Even in the presence of several additives, the influence of the added guest APIs was successfully detected. In conclusion, powder rheometry is a promising method for ensuring stable product quality and reducing the risk of unforeseen cross-contamination by foreign APIs.

Cationic Ester Prodrugs of Curcumin with N,N-dimethyl Amino Acid Promoieties Improved Poor Water Solubility and Intestinal Absorption.

PURPOSE: Although curcumin (Cur) has powerful pharmacological effects, its use in medicine has not been established yet. The oral bioavailability (BA) of Cur is limited because of its poor water solubility. The purpose of this study was to confirm whether cationic N,N-dimethyl amino acid esters of Cur could act as prodrugs and improve its water solubility and oral bioavailability. METHODS: Two N,N-dimethyl amino acid esters of Cur were synthesized. The hydrolysis profile of the esters was evaluated using rat and human microsomes. A pharmacokinetic study after oral administration of the Cur ester derivatives was performed in rats and compared to the administration of suspended or dissolved Cur formulation. The anti-inflammatory effects of the Cur derivatives were evaluated using macrophage RAW 264.7 stimulated with lipopolysaccharide. RESULTS: Cur ester derivatives showed > 200 mM water solubility. The derivatives were reconverted to the parent compound (Cur) after cleavage of the ester bonds by microsomal esterase, indicating that the compounds could act as Cur prodrugs. The Cur prodrugs enhanced the absolute oral bioavailability of Cur by a 9- and threefold increase of suspended and dissolved Cur administration, respectively, thereby improving intestinal absorption. Cur prodrugs strongly attenuated COX2, iNOS, and ERK phosphorylation. CONCLUSIONS: The cationic N,N-dimethyl amino acid ester prodrugs of Cur improved the water solubility of Cur and enhanced oral bioavailability in rats. These Cur prodrugs may be good candidates for developing medicinal options previously unavailable due to the poor water solubility and oral BA of Cur.

Visualization of Absorbed Lipid in the Normal Duodenal Epithelium Using Magnifying Endoscopy with Narrow-Band Imaging.

BACKGROUND: Subepithelial microvascular pattern cannot be visualized on the surface of adenoma and carcinoma by magnifying endoscopy due to a white opaque substance (WOS), which consists of minute lipid droplets accumulated in the neoplastic epithelium. AIMS: We aimed to investigate whether the WOS is visualized in the duodenum after exogenous fat loading (FL) administration in an open-label, randomized, controlled study. METHODS: The patients scheduled to undergo endoscopic therapy for gastric epithelial neoplasms were enrolled in the study. They were randomly assigned to the FL or non-FL group. An initial (before FL administration) and follow-up (after two to three weeks) endoscopic examinations were conducted to observe the duodenal mucosa using magnifying narrow-band imaging. Each patient in the FL group consumed 250 ml of Ensure H® four hours before the follow-up examination. Two experienced endoscopists determined the grade of the WOS. FL test results were judged positive for patients who showed a higher grade at the follow-up examination than at the initial examination. The rate of positive test results was compared between the two groups. RESULTS: Twenty patients (10 in the FL and 10 in the non-FL groups) were included. FL test results were positive for all 10 patients in the FL group, while they were negative for all 10 patients in the non-FL group (P < 0.001 by Fisher's exact test). CONCLUSIONS: Lipids loaded onto normal duodenal epithelium were absorbed, and the absorbed lipid droplets appeared as WOS on magnifying narrow-band imaging.

Cationic N,N-Dimethylglycine Ester Prodrug of 2R-α-Tocotrienol Promotes Intestinal Absorption via Efficient Self-Micellization with Intrinsic Bile Acid Anion.

The intestinal absorption of hydrophobic compounds is severely influenced by their transportation rate through the unstirred water layer in the intestinal lumen. A member of the vitamin E family, α-Tocotrienol (α-T3) has remarkable pharmacological effects, but its intestinal absorption is hampered due to its hydrophobicity. Here, we prepared three ester derivatives of 2R-α-T3, and we selected a suitable prodrug compound using rat plasma and liver microsomes. The micellization profile of the selected compound in the presence of taurocholic acid (TCA) was evaluated. After gastrostomy administration of the prodrug candidate or α-T3 solution containing TCA, AUC values were determined for α-T3 in plasma obtained from bile duct-ligated rats. Among the three types in the efficiency of the reconversion to the parent drug, α-T3 N,N-dimethylglycinate (α-T3DMG) was the best prodrug; α-T3DMG formed mixed micelles via ion pairs with anionic TCA. The solubility of α-T3DMG in n-octanol/water depended on its ratio to TCA. The AUC after α-T3DMG administration to ligated rats was 2-fold higher than that after α-T3 administration, suggesting a smooth interaction with intrinsic bile acids. In conclusion, utilization of the prodrug synthesized using N,N-dimethylglycine ester may be a beneficial approach to promote intestinal absorption of α-T3 via self-micellization with intrinsic bile acid.

Novel Cationic Prodrug of Ubiquinol-10 Enhances Intestinal Absorption via Efficient Formation of Nanosized Mixed-Micelles with Bile Acid Anions.

The aim of this study was to develop a prodrug of ubiquinol-10 (UqH-10), the active form of ubiquinone-10 (Uq-10), for oral delivery. Bioavailability of UqH-10 is hampered by its high susceptibility to oxidation and water-insolubility. We prepared three novel N,N-dimethylglycine ester derivatives of UqH-10, including a 1-monoester (UqH-1-DMG), 4-monoester (UqH-4-DMG), and 1,4-bis-ester (UqH-DMG), and assessed their physicochemical properties in vitro and in vivo. UqH-DMG spontaneously formed an aqueous micelle solution comprising 20 nm particles at 36.5 °C. Cationic UqH-DMG formed nano-sized (5 nm) mixed-micelles with taurocholic acid. Reconversion of the derivatives to UqH-10 was accelerated in human liver microsomes. The oral bioavailability of UqH-10 after administration of UqH-derivatives or Uq-10 was determined in fasted and postprandial rats secreting normal and high levels of bile, respectively. In fasted rats, plasma UqH-10 after UqH-derivatives administration reached Cmax at 2-3 h and after Uq-10 administration, it remained low. The AUC0-24h of UqH-10 after UqH-derivatives administration was 2-3-fold higher than that after Uq-10 administration. In postprandial rats, the Tmax of UqH-10 after UqH-derivatives administration was an hour earlier than after Uq-10 administration. In conclusion, cationic UqH-derivatives are convenient prodrugs that enhance UqH-10 bioavailability by forming nanosized mixed-micelles with intestinal bile acids.

Atypical Antipsychotic Drug Ziprasidone Protects against Rotenone-Induced Neurotoxicity: An In Vitro Study.

Schizophrenia is a severe, chronic mental illness characterized by delusions, hallucinations, negative symptoms, and cognitive dysfunction. Recently, several studies have demonstrated that the pathogenesis of schizophrenia involves mitochondrial dysfunction and oxidative stress. However, the effect of antipsychotic drugs for these events has been poorly investigated. In the present study, we evaluated the neuroprotective effect of an atypical antipsychotic drug, ziprasidone (ZPD), on rotenone (ROT)-induced neurotoxicity involving oxidative stress in PC12 cells. Our data showed that ZPD treatment promoted the translocation of NF-E2-related factor-2 (Nrf2) from cytoplasm to nucleus and activated the expression of its target genes NAD(P)H quinone oxidoreductase (NQO-1), catalase (CAT), and heme oxygenase (HO-1). Additionally, ZPD prevented ROT-induced cell death and intracellular reactive oxygen species production. Interestingly, the use of serotonin 5-HT1A receptor antagonist 1-(2-methoxyphenyl)-4 (4-(2-phtalimido) butyl) piperazine (NAN-190) completely blocked the protective effect of ZPD against ROT-induced cell death. Our results demonstrate the neuroprotective effect of ZPD against ROT-induced neurotoxicity and suggest that ZPD may be a potential candidate for the prevention of mitochondrial dysfunction and oxidative stress in schizophrenia.

Prodrugs for Skin Delivery of Menahydroquinone-4, an Active Form of Vitamin K2(20), Could Overcome the Photoinstability and Phototoxicity of Vitamin K2(20).

The effective delivery of menahydroquinone-4 (MKH), an active form of menaquinone-4 (MK-4, vitamin K2(20)), to the skin is beneficial in the treatment of various skin pathologies. However, its delivery through the application of MK-4 to the skin is hampered due to the photoinstability and phototoxicity of MK-4. This study aimed to evaluate the potential of ester prodrugs of MKH for its delivery into the skin to avoid the abovementioned issues. The ester prodrugs, MKH 1,4-bis-N,N-dimethylglycinate hydrochloride (MKH-DMG) and MKH 1,4-bis-hemisuccinate (MKH-SUC), were prepared using our previously reported methods. Photostability was determined under artificial sunlight and multi-wavelength light irradiation, phototoxicity was determined by intracellular ROS formation and cell viability of UVA-irradiated human epidermal keratinocyte cells (HaCaT), and delivery of MKH into HaCaT cells was assessed by measuring menaquinone-4 epoxide (MKO) levels. MKH prodrugs showed higher photostability than MK-4. Although MK-4 induced cellular ROS and reduced cell viability after UVA irradiation, MKH prodrugs did not affect either ROS generation or cell viability. MKH prodrugs enhanced intracellular MKO, indicating effective delivery of MKH and subsequent carboxylation activity. In conclusion, these MKH prodrugs show potential for the delivery of MKH into the skin without photoinstability and phototoxicity.

Antitumor Effects and Delivery Profiles of Menahydroquinone-4 Prodrugs with Ionic or Nonionic Promoiety to Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) shows poor prognosis owing to its very frequent recurrence even after curative treatment. Thus, an effective and safe long-term chemopreventive agent is strongly in demand. Menahydroquinone-4 (MKH) is an active form of menaquinone-4 (MK-4, vitamin K2) that is involved in the synthesis of vitamin K-dependent proteins in the liver. We hypothesized that efficient delivery of MKH might be critical to regulate HCC proliferation. The discovery of a suitable prodrug targeting HCC in terms of delivery and activation could reduce the clinical dose of MK-4 and maximize efficacy and safety. We previously showed that MKH dimethylglycinate (MKH-DMG) enables effective delivery of MKH into HCC cells and exhibits strong antitumor effects compared with MK-4. In this study, we prepared anionic MKH hemi-succinate (MKH-SUC) and non-ionic MKH acetate (MKH-ACT), in addition to cationic MKH-DMG, and evaluated MKH delivery profiles and antitumor effects in vitro. MKH-SUC showed the highest uptake and the most efficient release of MKH among the examined compounds and exhibited rapid and strong antitumor effects. These results indicate that MKH-SUC might have a good potential as an MKH delivery system for HCC that overcomes the limitations of MK-4 as a clinical chemopreventive agent.

Intracellular mechanisms underlying lipid accumulation (white opaque substance) in gastric epithelial neoplasms: A pilot study of expression profiles of lipid-metabolism-associated genes.

BACKGROUND AND AIM: White opaque substance (WOS) is a novel endoscopic finding in gastric neoplasms, indicating the intracellular accumulation of lipid droplets (LDs). However, gastric lipid metabolism has not been extensively investigated, even in normal mucosa. We investigated the expression profiles of lipid-metabolism-associated genes in gastric neoplasms. METHODS: Thirty-four patients with early gastric cancer or adenoma were enrolled in this study. Paired biopsy samples from tumor and adjacent non-tumor areas were obtained and analyzed by real-time polymerase chain reaction. Endoscopically resected specimens were evaluated histopathologically. RESULTS: Genes associated with ß-oxidation (peroxisome proliferator-activated receptor α, carnitine palmitoyltransferase 1A, and hydroxyacyl-CoA dehydrogenase), lipoprotein excretion (apolipoprotein B, microsomal triglyceride transfer protein, and acyl-CoA:cholesterol acyltransferase 2), fatty acid transport (fatty acid-binding protein), construction of triglycerides in the endoplasmic reticulum (acyl-CoA:diacylglycerol acyltransferase 1), and LD degradation/lipolysis (comparative gene identification-58, adipose triglyceride lipase) were significantly downregulated in neoplasms compared with non-tumor areas. Pyruvate dehydrogenase lipoamide kinase isozyme 4 (negative regulator of glycolysis) and adipophilin (LD surface component) were also repressed. Conversely, expression levels of genes associated with de novo lipogenesis (sterol regulatory element-binding protein 1c, acyl-CoA:diacylglycerol acyltransferase 2) were significantly enhanced in neoplasms. There was no significant difference in gene expression levels between carcinomas and adenomas, or between WOS-positive and WOS-negative neoplasms. CONCLUSION: Gene expression profiles in neoplasms suggest a predominance of lipid storage (lipogenesis/LD formation) over consumption (ß-oxidation/excretion/lipolysis). Lipid accumulation and WOS in gastric epithelial neoplasms may be caused by impaired mitochondrial oxidation, lipoprotein excretion, and LD degradation.

Effects of radical scavengers for reactive oxygen species on vitamin K-induced phototoxicity under UVA irradiation.

Vitamin K possesses efficacy as a topical dermatological agent. However, vitamin K is phototoxic and susceptible to photodegradation. Herein, we investigated the mechanisms underlying the phototoxicity of phylloquinone (PK, vitamin K1) and menaquinone-4 (MK-4, vitamin K2) under ultraviolet A (UVA) irradiation using various reactive oxygen species (ROS) scavengers. This resulted in the production of superoxide anion radicals via type I and singlet oxygen via type II photodynamic reactions, which were quenched by the ROS scavengers: superoxide dismutase and sodium azide (NaN3). In HaCaT cells, MK-4 and PK induced the production of intracellular ROS, particularly hydrogen peroxide, in response to UVA irradiation. Furthermore, the addition of catalase successfully decreased maximum ROS levels by approximately 30%. NaN3 and catalase decreased the maximum reduction in cell viability induced by UVA-irradiated PK and MK-4 in cell viability by approximately 2-7-fold. Additionally, ROS scavengers had no effect on the photodegradation of PK or MK-4 at 373 nm. Therefore, the phototoxicities of PK and MK-4 were attributed to the generation of singlet oxygen and hydrogen peroxide, underscoring the importance of photoshielding in circumventing phototoxicity.

Menahydroquinone-4 may play a key role in regulating CCL5 expression induced by epidermal growth factor receptor inhibitors.

Epidermal growth factor receptor (EGFR) inhibitors frequently cause severe skin rash as a side effect, which is a critical burden for patients who continuously receive drug treatments. Several recent clinical trials have shown that vitamin K is effective against these side effects; however, the underlying mechanisms remain unclear. EGFR inhibitors induce C-C motif chemokine ligand 5 (CCL5) in dermopathy. We hypothesized that menahydroquinone-4 (MKH), the active form of menaquinone-4 (MK-4, vitamin K2(20)), supplied by biosynthesis or external delivery, is essential for the suppressive effect on CCL5. The aim of this study was to explore the underlying mechanisms governing the relieving effects of MKH against skin rashes caused by EGFR inhibitors. The responses generated by EGFR inhibitors and the effect of MKH derivatives (two ester derivatives and MK-4) on them were evaluated using human skin cell lines (HaCaT and HSC-1). EGFR inhibitors downregulated UbiA prenyltransferase domain-containing protein-1 (UBIAD1, MKH synthetase) expression and MKH biosynthesis. Knockdown of UBIAD1 or γ-glutamyl carboxylase and treatment with warfarin upregulated CCL5 expression. MKH derivatives suppressed the CCL5 expression induced by EGFR inhibitors. Our data strongly suggest that MKH is involved in suppressing CCL5 expression and alleviating the skin damage caused by EGFR inhibitors.

Delivery of the reduced form of vitamin K2(20) to NIH/3T3 cells partially protects against rotenone induced cell death.

Mitochondria generate energy through the action of the electron transport chain (ETC) and ATP synthase. Mitochondrial malfunction can lead to various disorders, including neurodegenerative diseases. Several reports have shown that menaquinone-4 (MK-4, vitamin K2(20)), a safe drug for osteoporosis, may improve mitochondrial function. Here, we hypothesized that the efficient delivery of menahydroquinone-4 (MKH), an active form of MK-4, could exert a supporting effect. We verified the effects of MKH delivery on mitochondrial dysfunction by using MK-4 and MKH ester derivatives in NIH/3T3 mouse fibroblast cells treated with mitochondrial inhibitors. MK-4 and MKH derivatives suppressed cell death, the decline in mitochondrial membrane potential (MMP), excessive reactive oxygen species (ROS) production, and a decrease in intrinsic coenzyme Q9 (CoQ9) induced by rotenone (ROT, complex I inhibitor). MK-4 and MKH derivatives delivered MKH to NIH/3T3 cells, acting as an effective MKH prodrug, proving that the delivered MKH may reflect the mitigation effects on ROT-induced mitochondrial dysfunction. MKH prodrugs are also effective against 3-nitropropionic acid (3-NP, complex II inhibitor) and carbonyl cyanide-m-chlorophenylhydrazone (CCCP, uncoupler)-induced cell death. In conclusion, MKH delivery may mitigate mitochondrial dysfunction by maintaining MMP, ROS, and CoQ9, indicating that MKH prodrugs may be good candidates for treating mitochondrial disorders.

Changes in the Penetration Rate of Biosimilar Infliximab Within Japan Using a Japanese Claims Database.

PURPOSE: Infliximab, which was approved in 2002, had its first biosimilar launched in 2014 across Japan. However, the penetration rate of this biosimilar remains unclear given the limited data regarding its current clinical use throughout Japan. This study was conducted to describe the current clinical characteristics of patients receiving infliximab and the penetration rate of the reference infliximab and/or biosimilar infliximab using a Japanese administrative claims database. PATIENTS AND METHODS: This retrospective, descriptive study utilized the Japan Medical Data Vision database, a nationwide hospital-based database. Data on patients receiving infliximab recorded from April 2008 to March 2019 were extracted from the database. Patient characteristics of the reference and biosimilar infliximab groups and penetration rates according to fiscal year, target diseases diagnosis, and subsidy for intractable diseases were examined. RESULTS: A total of 9735 patients were extracted for analysis, among whom 92% (n=8950) and 8% (n=785) received only reference infliximab and its biosimilar, respectively. Both groups exhibited similar clinical characteristics. The biosimilar penetration rate increased from 0.8% in 2014 to 22.5% in 2018, with overall penetration rates throughout the period according to diagnosis (with or without subsidy) being 14.4% (with, 4.1%; without, 16.4%), 4.7% (with, 3.7%; without, 10.6%), 5.7% (with, 4.5%; without, 13.5%), and 7.5% (with, 4.4%; without, 8.2%) for rheumatoid arthritis, Crohn's disease, ulcerative colitis, and psoriasis, respectively. CONCLUSION: Biosimilar infliximab is prescribed for patients with similar characteristics to reference infliximab. Despite the increasing penetration rates according to target disease, they remain much lower among patients receiving subsidy for intractable disease than among those who do not.

Dermoscopic diagnostic performance of Japanese dermatologists for skin tumors differs by patient origin: A deep learning convolutional neural network closes the gap.

In the dermoscopic diagnosis of skin tumors, it remains unclear whether a deep neural network (DNN) trained with images from fair-skinned-predominant archives is helpful when applied for patients with darker skin. This study compared the performance of 30 Japanese dermatologists with that of a DNN for the dermoscopic diagnosis of International Skin Imaging Collaboration (ISIC) and Shinshu (Japanese only) datasets to classify malignant melanoma, melanocytic nevus, basal cell carcinoma and benign keratosis on the non-volar skin. The DNN was trained using 12 254 images from the ISIC set and 594 images from the Shinshu set. The sensitivity for malignancy prediction by the dermatologists was significantly higher for the Shinshu set than for the ISIC set (0.853 [95% confidence interval, 0.820-0.885] vs 0.608 [0.553-0.664], P < 0.001). The specificity of the DNN at the dermatologists' mean sensitivity value was 0.962 for the Shinshu set and 1.00 for the ISIC set and significantly higher than that for the human readers (both P < 0.001). The dermoscopic diagnostic performance of dermatologists for skin tumors tended to be less accurate for patients of non-local populations, particularly in relation to the dominant skin type. A DNN may help close this gap in the clinical setting.

Growth Inhibitory Effects of Ester Derivatives of Menahydroquinone-4, the Reduced Form of Vitamin K2(20), on All-Trans Retinoic Acid-Resistant HL60 Cell Line.

The first-choice drug for acute promyelocytic leukemia (APL), all-trans retinoic acid (ATRA), frequently causes drug-resistance and some adverse effects. Thus, an effective and safe agent for ATRA-resistant APL is needed. Menaquinone-4 (MK-4, vitamin K2(20)), used for osteoporosis treatment, does not have serious adverse effects. It has been reported that MK-4 has growth-inhibitory effects on HL60 cells by inducing apoptosis via the activation of Bcl-2 antagonist killer 1 (BAK). However, the effect of MK-4 on ATRA-resistant APL has not been reported. Here, we show that ester derivatives of menahydroquinone-4 (MKH; a reduced form of MK-4), MKH 1,4-bis-N,N-dimethylglycinate (MKH-DMG) and MKH 1,4-bis-hemi-succinate (MKH-SUC), exerted strong growth-inhibitory effects even on ATRA-resistant HL60 (HL-60R) cells compared with ATRA and MK-4. MKH delivery after MKH-SUC treatment was higher than that after MK-4 treatment, and the results indicated apoptosis induced by BAK activation. In contrast, for MKH-DMG, reconversion to MKH was slow and apoptosis was not observed. We suggest that the ester forms, including monoesters of MKH-DMG, exhibit another mechanism independent of apoptosis. In conclusion, the MKH derivatives (MKH-SUC and MKH-DMG) inhibited not only HL60 cells but also HL-60R cells, indicating a potential to overcome ATRA resistance.

NPC1L1 inhibitor ezetimibe is a reliable therapeutic agent for non-obese patients with nonalcoholic fatty liver disease.

BACKGROUND: We recently examined the distribution of abdominal fat, dietary intake and biochemical data in patients with nonalcoholic fatty liver disease (NAFLD) and found that non-obese NAFLD patients did not necessarily exhibit insulin resistance and/or dysregulated secretion of adipocytokines. However, dietary cholesterol intake was superabundant in non-obese patients compared with obese patients, although total energy and carbohydrate intake was not excessive. Therefore, excess cholesterol intake appears to be one of the main factors associated with NAFLD development and liver injury. METHODS: We reviewed a year of follow-up data of non-obese NAFLD patients treated with Niemann-Pick C1 like 1 inhibitor ezetimibe to evaluate its therapeutic effect on clinical parameters related to NAFLD. Without any dietary or exercise modification, 10 mg/day of ezetimibe was given to 8 patients. In 4 of 8 patients, ezetimibe was administered initially. In the remaining 4 patients, medication was switched from ursodeoxycholic acid to ezetimibe. RESULTS: In each patient, body mass index was maintained under 25 kg/m2 during the observation period. Serum ALT levels significantly decreased within 6 months and in 4 patients levels reached the normal range (<30 U/L), which was accompanied with at least a 10% decrease in serum total cholesterol and LDL-cholesterol. However, ultrasonographic findings of fatty liver did not show obvious improvement for a year. CONCLUSION: We conclude that the cholesterol absorption inhibitor ezetimibe can suppress hepatic injury in non-obese patients with NAFLD and that ezetimibe may offer a novel treatment for NAFLD.

Evaluation of photostability and phototoxicity of esterified derivatives of ubiquinol-10 and their application as prodrugs of reduced coenzyme Q10 for topical administration.

Ubiquinol-10 (UqH-10), the fully reduced form of ubiquinone-10 (Uq-10, coenzyme Q10 ), is an antioxidant and is involved in energy production. However, physicochemical disadvantages, such as rapid oxidation, water-insolubility, photoinstability, and phototoxicity, limit its application. We previously reported that UqH-10 1,4-bis-N,N-dimethylglycinate improved the oxidation susceptibility and poor bioavailability of UqH-10 in rats. Herein, we evaluated the photochemical properties of UqH-esterified derivatives (N,N-dimethylglycinate, hemi-succinate, ethylsuccinate, and hemi-glutarate). Photostability was examined by irradiation using artificial sunlight and monochromatic light. The concentration of each compound was determined using LC-MS/MS. Phototoxicity was assessed by singlet oxygen and superoxide assays. Delivery of UqH-10 via UqH-esters to the HaCaT human keratinocyte cell line was determined using LC-MS/MS. UqH-esters showed higher photostability to artificial sunlight than Uq-10 and UqH-10. Uq-10 and UqH-10 were rapidly degraded by monochromatic light at 279 nm, whereas UqH-esters were more stable. UVA and/or UVB irradiation generated high levels of singlet oxygen and superoxide in Uq-10, whereas UqH-esters were unreactive. Additionally, UqH-esters effectively delivered UqH-10 to HaCaT cells following efficient uptake in their ester forms and ester bond hydrolysis in the cells. In conclusion, UqH-ester derivatives exhibit higher photostability and lower phototoxicity compared with Uq-10 and UqH-10.

Ester derivatives of phyllohydroquinone effectively deliver the active form of vitamin K1 topically, owing to their non-photosensitivity.

Topical application of phylloquinone (PK) is beneficial to the skin; however, its topical use is limited in Europe owing to its photosensitive properties such as photodegradation and phototoxicity. We evaluated the suitability of ester derivatives of phyllohydroquinone (PKH), the active form of PK, for topical application to overcome the abovementioned problems of PK. We used the PKH derivatives PKH-1,4-bis-N,N-dimethylglycinate hydrochloride (PKH-DMG) and PKH-1,4-bis-hemisuccinate (PKH-SUC) for our studies. Photostability was determined by measuring the residual concentration after irradiation with artificial sunlight and multi-wavelength light. Phototoxicity after ultraviolet A (UVA) irradiation was assessed by measuring drug-induced singlet oxygen and intracellular reactive oxygen species (ROS) generation, and cell viability of a human epidermal keratinocyte cell line (HaCaT). Delivery of PKH into HaCaT cells was assessed by measuring PK epoxide (PKO) levels. The PKH derivatives showed higher photostability than PK. After UVA irradiation, PK induced high singlet oxygen levels and intracellular ROS generation, and reduced cell viability, whereas the PKH derivatives showed no effects. The PKH derivatives increased intracellular PKO levels. AUCPKO(0-72 h) values after PKH-DMG and PKH-SUC treatments were 0.741- and 22.9-fold higher than that after PK treatment, respectively. In conclusion, PKH derivatives act as PKH prodrugs and are suitable for topical application without the need for special protection from light.

The Kampo medicine Yokukansan (YKS) enhances nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells.

Accumulating evidence indicates that neurotrophic factor-like substances involved in the induction of neurotrophic factor synthesis may aid in the treatment of neurological disorders, such as Alzheimer's disease. Yokukansan (YKS), a traditional Kampo medicine, has been used for the treatment of anxiety and mood disorders. In the present study, we aimed to identify the signaling pathways associated with YKS-mediated enhancement of nerve growth factor (NGF)-induced neurite extension in rat pheochromocytoma (PC12) cells. Akt and extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation levels were assessed by western blot analysis, in the presence of YKS and following the treatment with TrkA inhibitor, K252a. YKS treatment (NGF+YKS 0.5 group) enhanced NGF-induced neurite outgrowth and phosphorylation/activation of Akt and ERK1/2 in PC12 cells. Moreover, YKS-induced effects were inhibited by the treatment with the TrkA receptor antagonist K252a (NGF+YKS 0.5+K252a group); no significant difference in neurite outgrowth was observed between K252a-treated (NGF+YKS 0.5+K252a group) and NGF-K252a-treated cells (NGF+K252a group). However, neurite outgrowth in K252a-treated cells (NGF+K252a and NGF+YKS 0.5+K252a group) reached only one-third of the level in NGF-treated cells (NGF group). NGF-mediated Akt phosphorylation increased by YKS was also inhibited by K252a treatment (NGF+YKS 0.5+K252a group), but no significant difference in ERK1/2 phosphorylation was observed between NGF-YKS-K252a- and NGF-treated cells (NGF group). Our results indicate that YKS treatment enhanced NGF-induced neurite outgrowth via induction of Akt and ERK1/2 phosphorylation, following the binding of NGF to the TrkA receptor. These findings may be useful in the development of novel therapeutic strategies for the treatment of Alzheimer's disease.