Enhanced Intracellular Delivery and Improved Antitumor Efficacy of Menaquinone-4

Hepatocellular carcinoma (HCC) is a major malignant tumor type that occurs globally. HCC incidence is increasing, especially in Asian countries. Despite many therapeutic approaches, the long-term prognosis of HCC remains poor because of frequent recurrence due to intrahepatic metastasis or multicentric carcinogenesis. Therefore, it is necessary to develop effective and safe chemopreventive agents to improve the prognosis of HCC. Menaquinone-4 (MK-4) has a suppressive effect on HCC, but cellular delivery is poor. We hypothesized that effective cellular delivery of menahydroquinone-4 (MKH), a fully reduced form of MK-4, would regulate HCC growth and metastasis. We developed a bioreductive activation-independent delivery system with the N,N-dimethylglycine ester of MKH (MKH-bis-DMG) to deliver MKH to HCC cells without any bioreductive processing of MK-4. MKH-bis-DMG inhibited the proliferation of both DCP-positive and DCP-negative HCC cell lines in a time- and dose-dependent manner via G1/S cell-cycle arrest. We assessed the effect of MKH-derivatives on HCC metastasis using a mouse model of spleen-liver metastasis. The mean tumor hepatic replacement area of MKH-bis-DMG treated mice was significantly less than that of untreated mice. In conclusion, MKH-bis-DMG may be beneficial as a chemopreventive agent for recurrent HCC

Inhibition of nerve growth factor-induced neurite outgrowth from PC12 cells by dexamethasone: signaling pathways through the glucocorticoid receptor and phosphorylated Akt and ERK1/2.

Glucocorticoids are important mediators of the stress response and are commonly employed as drugs for the suppression of immune rejection after organ transplantation. Previous investigations uncovered the possibility of mood depression in patients undergoing long-term treatment with synthetic glucocorticoids, including dexamethasone (DEX). Exogenous glucocorticoids and their synthetic derivatives can also adversely affect the development of the central nervous system. Although neurite extension from rat pheochromocytoma-derived PC12 cells and a variety of primary neurons is stimulated by nerve growth factor (NGF), and signaling pathways triggered by the binding of NGF to tyrosine kinase receptor type 1 (TrkA) function in both neurite outgrowth and neuronal survival, the effect of DEX on the activation of regulatory proteins and pathways downstream of TrkA has not been well characterized. To analyze the influence of DEX on NGF-induced neurite outgrowth and signaling, PC12 cells, a widely utilized model of neuronal differentiation, were pretreated with the glucocorticoid prior to NGF induction. NGF-induced neurite outgrowth was attenuated by pretreatment with DEX, even in the absence of DEX after the addition of NGF. Moreover, DEX suppressed the phosphorylation of Akt and extracellular-regulated kinase 1/2 (ERK1/2) in the neurite outgrowth signaling cascade initiated by NGF. Finally, the glucocorticoid receptor (GR) antagonist, RU38486, counteracted the inhibitory effect of DEX pretreatment, not only on the phosphorylation of Akt and ERK1/2, but also on neurite extension from PC12 cells. These results suggest that DEX binding to the GR impairs NGF-promoted neurite outgrowth by interfering with the activation/phosphorylation of Akt and ERK1/2. These novel findings are likely to be useful for elucidating the central nervous system depressive mechanism(s) of action of DEX and other glucocorticoids

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

Menahydroquinone-4 Prodrug: A Promising Candidate Anti-Hepatocellular Carcinoma Agent

Recently, new therapeutics have been developed for hepatocellular carcinoma (HCC). However, the overall survival rate of HCC patients is still unsatisfactory; one of the reasons for this is the high frequency of recurrence after radical treatment. Consequently, to improve prognosis, it will be important to develop a novel anti-tumor agent that is especially effective against HCC recurrence. For clinical application, long-term safety, together with high anti-tumor efficacy, is desirable. Recent studies have proposed menahydroquinone-4 1,4-bis-N,N-dimethylglycinate hydrochloride (MKH-DMG), a prodrug of menahydroquinone-4 (MKH), as a promising candidate for HCC treatment including the inhibition of recurrence; MKH-DMG has been shown to achieve good selective accumulation of MKH in tumor cells, resulting in satisfactory inhibition of cell proliferation in des-γ-carboxyl prothrombin (DCP)-positive and DCP-negative HCC cell lines. In a spleen-liver metastasis mouse model, MKH-DMG has been demonstrated to have anti-proliferation and anti-metastatic effects in vivo. The characteristics of MKH-DMG as a novel anti-HCC agent are presented in this review article

Evaluation of pharmacokinetics and the stability of daptomycin in serum at various temperatures

Background: Daptomycin exhibits concentration-dependent antibacterial activity. By monitoring daptomycin serum concentrations, clinicians may be able to predict the effectiveness of treatments for infections more accurately. However, it has been reported that daptomycin concentrations in plasma samples stored at −20 °C decrease approximately 25% after 4 weeks. The aim of this study was to evaluate the stability of daptomycin in serum at various temperatures. Methods: Daptomycin serum samples were prepared and stored at different temperatures. The stability of daptomycin under various conditions was evaluated by sequential measurements of concentration. Results: Although the loss of concentration of daptomycin in serum samples stored in freezers (−80 °C and −20 °C) was less than 10% after 168 days (6 months), the concentrations in samples stored in a refrigerator (4 °C) decreased by more than 70% over the same period. Furthermore, daptomycin concentrations in serum samples stored at close to body temperature (35 °C, 37 °C, and 39 °C) decreased by more than 50% after only 24 h. Conclusions: The results of the present study demonstrate that the measurement of serum concentrations of daptomycin needs to be performed rapidly. Furthermore, the degradation of daptomycin in serum may be involved in its elimination from the living body

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

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