Preparation and characterization of a powder containing an oily liquid drug with Eudragit EPO or L100 copolymer.

Oily liquid drugs are not convenient for oral administration. We developed a powder containing clofibrate (CF), a model of an oily drug, using aminoalkyl methacrylate copolymer (EPO) or methacrylic acid copolymer (L100). CF or a mixture of CF and soybean oil was emulsified with EPO or L100 aqueous solution. Using a high-pressure homogenizer, a stable emulsion was obtained, and a powder was then obtained by lyophilization of the emulsion. The content of CF in the powder depended on the formulation, with the highest contents being 24.6% and 27.1% for EPO and L100, respectively. The incorporation ratio of CF was higher for L100 than for EPO. The powder using EPO was sticky because of leaked CF and the low glass transition temperature of EPO. The powder using L100 was a typical powder obtained by lyophilization. The leakage of CF from the powder was <2%, lower than for EPO powder. The dissolution of CF from powder using EPO was fast, regardless of the pH of the medium, but the powder using L100 showed enteric-soluble characteristics, indicating that CF is well incorporated in L100.

Novel chronotherapeutic rectal aminophylline delivery system for therapy of asthma.

The aim of this study was to develop a new chronotherapeutic pharmaceutical preparation as a sustained-release suppository for prevention and therapeutic use against bronchial asthma in the early morning. Sustained-release hollow-type (SR-HT) suppositories using sodium alginate (Alg-Na), sodium polyacrylate (PANa) or polyacrylate-PANa co-polymer (PA-PANa) as gelling polymers (gel agent) were prepared and pharmaceutical characteristics of these suppositories were investigated. Type A SR-HT suppositories comprised a suppository shell prepared with oleaginous base and containing aminophylline only or aminophylline with Alg-Na or PANa in the cavity (hollow space). Type B SR-HT suppositories comprised a suppository shell prepared with oleaginous base and gel agent (30%), with aminophylline in the hollow space. In drug-release studies, the acrylate polymer-containing suppositories showed linearity of delayed release rate, providing significantly decreased the highest concentration of theophylline in plasma (C(max)) and delayed the time required to reach C(max) (t(max)) and the mean residence time (MRT) after rectal administrated in rabbits. In particular, suppositories containing PA-PANa maintained significantly higher theophylline concentrations than control suppositories at 12h after rectal administration. Furthermore, histopathological examination indicated that these suppositories using acrylate polymers did not result in rectal lesions. The SR-HT suppository, particularly using PA-PANa as a gel agent, may thus be useful against nocturnal symptoms of asthma. In this study, we confirmed new formulation of sustained-release suppository for chronotherapy of theophylline using oily base material in combination with polymer such as PA-PANa. The hollow-type suppository containing oleaginous base and hydrophilic polymer in the shell could be useful device for rectal administration of various drugs with prolongation of plasma concentration.

Induction of apoptosis by new ent-kaurene-type diterpenoids isolated from the New Zealand liverwort Jungermannia species.

Some diterpenoids show various biological activities, including anti-inflammatory, anti-HIV and anti-tumor activity. Previously, we have focused our research on the apoptosis-inducing properties of diterpenoids and found that some ent-kaurene-type diterpenoids induced apoptosis in human leukemia HL-60 cells. In this study, we have investigated the induction of apoptosis in HL-60 cells by the novel ent-kaurene-type diterpenoids, jungermannenones A (JA), B (JB), C (JC) and D (JD), isolated from the New Zealand liverwort Jungermannia species. Treatment of the cells with each compound for 12 h resulted in cytotoxicity (IC (50) values: A, 1.3; B, 5.3; C, 7.8; D, 2.7 microM) and caused DNA fragmentation and nuclear condensation, both biochemical markers of the induction of apoptosis. Treatment with the compounds resulted in activation of caspases, including caspase-3 and caspase-8. A broad-spectrum inhibitor of caspases, Z-Asp-CH (2)-DCB, attenuated the cytotoxicity induced by these compounds, suggesting that JA, JB, JC and JD induced apoptosis through a caspase-dependent pathway. JA and JD inhibited the activity of nuclear factor-kappaB, which is a transcriptional factor of anti-apoptotic factors. Thus, some of these new ent-kaurene-type diterpenoids may be promising candidates for anti-tumor agents.

Activation of p38 mitogen-activated protein kinase during ent-11alpha-hydroxy-16-kauren-15-one-induced apoptosis in human leukemia HL-60 cells.

Kaurene-type diterpenes possess various biological activities including antitumor and anti-inflammatory effects. Indeed, we have found that an ent-kaurene diterpene, ent-11alpha-hydroxy-16-kauren-15-one (KD), induced apoptosis via caspase-8 activation in human promyelocytic leukemia HL-60 cells. However, the mechanism of caspase-8 activation by KD is not clear. In this study, we investigated the involvement of p38 mitogen-activated protein kinase (p38 (MAPK)) in KD-induced apoptosis. p38 (MAPK) was activated by treatment with KD parallel to DNA ladder formation. Pretreatment with SB203580, a specific inhibitor of p38 (MAPK), attenuated induction of apoptosis by KD and inhibited activation of caspase-8. Cleavage of Bid, a typical substrate of caspase-8, was also inhibited by treatment with SB203580, suggesting that activation of p38 (MAPK) occurs upstream of caspase-8 during KD-induced apoptosis.

An ent-kaurene diterpene enhances apoptosis induced by tumor necrosis factor in human leukemia cells.

Some antitumor agents, including tumor necrosis factor-alpha (TNF-alpha) and camptothecin (CPT), often cause resistance of tumor cells to antitumor agents through activation of the nuclear factor-kappa B (NF-kappa B) pathway that leads to up-regulation of anti-apoptotic proteins. Therefore, co-treatment of an inhibitor of the NF-kappa B pathway with antitumor agents is a useful strategy for chemotherapy. Here we report that ent-11 alpha-hydroxy-16-kauren-15-one (KD) selectively inhibits NF-kappa B-dependent gene expression due to treatment with TNF-alpha. KD in combination with TNF-alpha caused a dramatic increase in apoptosis in human leukemia cells accompanied by activation of caspases. A broad-spectrum inhibitor of caspases decreased the apoptosis induced by treatment with KD and TNF-alpha. KD in combination with CPT also caused an increase in apoptosis. These results suggest that the apoptotic potency of co-treatment of KD with TNF-alpha or CPT is elicited through selective inhibition of NF-kappa B-dependent anti-apoptotic proteins and thus may provide a basis for the development of useful approaches to the treatment of leukemia.

A comparison of apoptosis and necrosis induced by ent-kaurene-type diterpenoids in HL-60 cells.

We previously reported that ent-11alpha-hydroxy-16-kauren-15-one (KD) induces apoptosis through a caspase-dependent pathway and the induction of apoptosis is dependent on its enone group in human leukemia cells. Here we investigated the abilities of some KD-related compounds with enone group (Fig. 1) to induce apoptosis and to activate some caspases. The IC50 values of ent-kaurene-related compounds possessing the enone group, ent-1beta-hydroxy-9(11),16-kauradien-15-one (1), ent-9(11),16-kauradiene-12,15-dione (2) and the rearranged ent-kaurane-type diterpene (3), against HL-60 cells after 12 h of treatment were 40 microM, 1.8 microM and 5.5 microM, respectively. Although treatment with 3 induced apoptosis, DNA ladder formation was not observed after treatment with 1 or 2. Induction of necrosis, as assayed by trypan blue staining, was observed after treatment with 1 or 2. Treatment with compound 1, 2 or 3 induced proteolysis of poly(ADP-ribose) polymerase (PARP), a substrate of caspase-3, and processing of caspase-3. Activation of caspase-8 and processing of Bid, a typical substrate of caspase-8, were also observed on treatment with these compounds. Pretreatment with a broad-spectrum inhibitor of caspases attenuated apoptosis induced by 3 but not necrosis induced by 1 and 2. In summary, KD-related compounds are a unique family of diterpenes that cause either caspase-dependent apoptotic or necrotic cell death.

Adipocyte-derived leucine aminopeptidase suppresses angiogenesis in human endometrial carcinoma via renin-angiotensin system.

PURPOSE: Angiotensin (Ang) II was reported to induce vascular endothelial growth factor (VEGF) expression in various cells. Adipocyte-derived leucine aminopeptidase (A-LAP) is a novel member of the M1 family of zinc metallopeptidases. Enzymatic characterization demonstrated that A-LAP hydrolyzes Ang II. This study examined the role of A-LAP in angiogenesis of human endometrial carcinoma. EXPERIMENTAL DESIGN: We investigated whether Ang II induces VEGF expression in human endometrial carcinoma cells. To investigate the possible function of A-LAP in angiogenesis of endometrial carcinoma, we transfected A-LAP cDNA into HEC-1A cells, showing the lowest expression of A-LAP. RESULTS: In the present study, we showed that Ang II enhanced VEGF expression in a dose-dependent manner in endometrial carcinoma cells (HEC-1A cells). Overexpression of A-LAP attenuated Ang II-induced VEGF expression in HEC-1A cells. In addition, Human umbilical vascular endothelial cell migration was increased in conditioned media from Ang II-treated wild-type cells, but not in conditioned media from Ang II-treated A-LAP-overexpressing cells (HEC-1A-A-LAP cells). In an in vivo study, we showed that A-LAP overexpression in endometrial carcinoma cells results in a reduction of VEGF immunoreactivity and the number of blood vessels within tumors. CONCLUSIONS: Our study demonstrates that it is feasible to overexpress Ang II-degrading enzymes in cultured cells and that this overexpression attenuated some effects of exogenous and endogenous Ang II. These experiments are a first step toward the development of novel strategies to selectively antagonize locally generated Ang II and suppress VEGF-induced angiogenesis in endometrial carcinoma.

New ent-kaurene-type diterpenoids possessing cytotoxicity from the New Zealand liverwort Jungermannia species.

Two new ent-kaurene-type and a new rearranged ent-kaurene-type diterpenoids possessing cytotoxicity against a human leukemia cell line have been isolated from the New Zealand liverwort Jungermannia species, together with previously known ent-kaurene-type diterpenoids. Their structures were established based on extensive NMR techniques.

[Apoptosis-inducing properties of ent-kaurene-type diterpenoids from the liverwort Jungermannia truncata].

Ent-11alpha-hydroxy-16-kauren-15-one (1) induced apoptosis in a human leukemia cell line (HL-60 cells), however, the apoptosis-inducing properties of 1 and its related compounds remain to be proved. We examined the involvement of caspases, a family of cysteine aspartic proteases, which play a central role in induction of apoptosis, in apoptosis induced by the compounds in HL-60 cells. Treatment of the cells with compounds 1, 2 and 3 with the enone group at C-15/C-16 caused DNA fragmentation, a sign of induction of apoptosis, and proteolysis of poly(ADP-ribose) polymerase (PARP), a hallmark of caspase activation. Z-Asp-CH2-DCB, abroad spectrum inhibitor of caspases, abolished the appearance of DNA fragmentation and also significantly attenuated the cytotoxic effects. These data suggest that induction of apoptosis by 1 and some of its related compounds are dependent on caspases activation and might be partly involved in the cytotoxicity in HL-60 cells.

[Preparation and evaluation of press-coated aminophylline tablet using crystalline cellulose and polyethylene glycol in the outer shell for timed-release dosage forms].

In an attempt to achieve chronopharmacotherapy for asthma, press-coated tablets (250 mg), which contained aminophylline in the core tablet in the form of low-substituted hydroxypropylcellulose (L-HPC) and coated with crystalline cellulose (PH-102) and polyethylene glycol (PEG) at various molecular weights and mixing ratios in the amounts of PH-102 and PEG as the outer shell (press-coating material), were prepared (chronopharmaceutics). Their applicability as timed-release (delayed-release) tablets with a lag time of disintegration and a subsequent rapid drug release phase was investigated. Various types of press-coated tablets were prepared using a tableting machine, and their aminophylline dissolution profiles were evaluated by the JP paddle method. Tablets with the timed-release characteristics could be prepared, and the lag time of disintegration was prolonged as the molecular weight and the amount of PEG, for example PEG 500,000, in the outer shell were increased. The lag time of disintegration could be controlled by the above-mentioned method, however, the pH of the medium had no effect on disintegration of the tablet and dissolution behavior of theophylline. The press-coated tablet (core tablet:aminophylline 50 mg, L-HPC and PEG 6000; outer shell:PH-102:PEG = 8:2 200 mg) with the timed-release characteristics was administered orally to rabbits for an in vivo test. Theophylline was first detected in plasma more than 2 h after administration; thus, this tablet showed a timed-release characteristics in the gastrointestinal tract. The time (tmax) required to reach the maximum plasma theophylline concentration (Cmax) observed after administration of the press-coated tablet was significantly (p < 0.05) delayed compared with that observed after administration of aminophylline solution in the control experiment. However, there was no difference in Cmax and area under the plasma theophylline concentration-time curve (AUC0-->24) between the press-coated tablet and aminophylline solution. These results suggest that the press-coated aminophylline tablet (with the timed-release characteristic) offers a promising forms of theophylline chronotherapy for asthma.