bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclin D2 and p27 Are Tissue-Specific Regulators of Tumorigenesis in Inhibin α Knockout Mice
Authors: Burns Kathleen H. , Agno Julio E. , Sicinski Piotr , Matzuk Martin M.
Published: July 2003

Inhibins are heterodimeric (α:βA and α:βB) endocrine, paracrine, and autocrine factors of the TGFβ superfamily that are produced predominantly by ovarian granulosa cells in females and testicular Sertoli cells in males. Control of granulosa and Sertoli cell proliferation is lost in the inhibin α (Inhα) knockout mouse model, leading to gonadotropin-dependent gonadal tumors of the granulosa/Sertoli cell lineage in both females and males. Castrate Inhα knockout mice develop sex steroidogenic tumors of the adrenal cortex. Physiological control of granulosa/Sertoli cell cycle progression depends on p27Kip1 and cyclin D2, which function in the G1 → S phase transition. To study the cell cycle-regulatory factors involved in ovarian, testicular, and adrenal tumor development in vivo, we have bred Inhα mutant mice to mice with targeted disruptions of the p27 and cyclin D2 genes. Our previous studies demonstrated that inhibins act cooperatively with p27 to negatively regulate granulosa cell proliferation, as double mutant mice lacking inhibins and p27 develop and succumb to ovarian tumors more rapidly than Inhα knockout mice. Here, we report that cyclin D2 antagonizes this inhibition and is key in promoting gonadal growth and tumor development, and tumor development is markedly suppressed in double-mutant mice. We found that double-knockout females lacking cyclin D2 and Inhα lived longer than mice lacking inhibins alone; the majority of these double-knockout mice lived longer than 17 wk, as opposed to inhibin α single-knockout females with 50% survival at between 12 and 13 wk of age. Moreover, 95% of inhibin α knockout males succumb to testicular tumor development by 12 wk of age, whereas double knockouts were protected from early signs of tumor development and had a 50% survival of 40 wk. Interestingly, the results of these studies reflect tissue-specific consequences of loss of these cell cycle regulators. In castrate mice, loss of p27 has little effect on adrenal cortical tumor progression in the absence of inhibins, whereas loss of cyclin D2 prolongs the lifespan of cyclin D2, Inhα double knockouts. After gonadectomy, 50% of cyclin D2, Inhα double-knockout males live to more than 46 wk of age, 10 wk longer than 50% of littermates lacking only inhibins. Similarly, 50% of female cyclin D2, inhibin α double knockouts live to 47 wk of age before succumbing to adrenal tumor development, in contrast to the 50% survival of Inhα single-knockout females at between 27 and 28 wk. Thus, identification of genetic modifiers of the Inhα knockout tumor phenotype has led us to a better appreciation of how specific components of the cell cycle machinery contribute to tumorigenesis in the ovary, testis, and adrenal gland.
Facile Synthesis of a Chitosan Hybrid of a Laminin-related Peptide and Its Antimetastatic Effect in Mice
Authors: Hojo Keiko , Maeda Mitsuko , Mu Yu , Kamada Haruhiko , Tsutsumi Yasuo , Ama Yasuhiro Nishiy , Yoshikawa Tomoko , Kurita Keisuke , Block Lawrence H , Mayumi Tadanori , Kawasaki Koichi
Published: July 2003

Laminin, a cell adhesion protein, consists of three peptide chains (α, β and γ-1). The β chain contains a Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence that has been found to inhibit experimental metastasis in mice. We have prepared a hybrid of a water-soluble chitosan and a laminin-related peptide, and have examined its inhibitory effect on experimental metastasis in mice.

A laminin-related peptide, acetyl-Tyr-Ile-Gly-Ser-Arg-βAla-OH (Ac-YIGSRβA-OH), was prepared by a solid-phase method. Ac-YIGSRβA-OH was then reacted with a watersoluble chitosan. βAla is a spacer and was placed to avoid racemization of the Arg residue when the peptide was coupled with chitosan. Although chitosan has amino groups, they did not react with the peptide. Four methods were tried to achieve a coupling reaction, the diphenylphosphoryl azide method, the diisopropylcarbodiimide/1-hydroxybenzotriazole method, the water-soluble carbodiimide (WSC), and the 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) method, but all four methods were unsuccessful. Therefore, a small spacer, tert-butyloxycarbonyl-Gly, was intercalated in chitosan, by the TBTU method, to facilitate its coupling with the peptide. After removal of the protecting group, the Gly-chitosan was coupled with Ac-YIGSRβA-OH by the watersoluble carbodiimide method to give Ac-YIGSRβAG-chitosan.

Conjugation of the peptide with the larger chitosan molecule did not reduce the inhibitory effect of the peptide on experimental metastasis in mice, it actually potentiated the antimetastatic effect, demonstrating that chitosan may be effective as a drug carrier for peptides.
Cyclic Dipeptides in the Induction of Maturation for Cancer Therapy
Authors: Graz C J M , Grant G D , Brauns S C , Hunt A , Jamie H , Milne P J
Published: July 2003

Studies have suggested a possible form of therapy based on the use of maturation-inducing compounds to induce differentiation of neoplastic cells and stimulate faster recovery of the normal cell population. The study of the effects of nine cyclic dipeptides on biochemical markers of differentiation implicated their potential to induce differentiation. Studies were undertaken to determine the specificity of these agents for HT-29 cell cultures as well as the identification of the signal transduction pathways affected by these agents inducing the differential gene expression observed in the cells.

The cyclic dipeptides studied showed a high degree of specificity, having no significant effect on Caco-2 cells (P > 0.05), representing the normal gastrointestinal mucosa. All inducers administered were shown to affect the total energy state of HT-29 cells, an effect which increased the probability of HT-29 cell differentiation. Results indicated that those agents which induced differential gene expression acted at different steps in the isolated signal transduction pathway. Cyclo(Trp-Trp) and cyclo(Phe-Pro) induced a high degree of acetylation of histones (P < 0.05), while the remaining cyclic dipeptides induced a high degree of phosphorylation of histones (P < 0.05) (cyclo(Trp-Trp) induced a moderate degree of histone phosphorylation). The results from histone phosphorylation and acetylation and cyclic AMP responsive element binding protein phosphorylation studies suggest that the cyclic dipeptides activate a chromatin switch, which leads to the increase in accessibility of lineage-specific genes for transcription.
Investigation of Endothelial Hyperreactivity in the Obese Zucker Rat In-situ: Reversal by Vitamin E
Authors: Andrews Tara J , Laight David W , Änggård Erik E , Carrier Martin J
Published: July 2003

The obese Zucker rat, a popular model of insulin resistance allied with oxidant stress, is associated with either normal or paradoxically enhanced endothelial vasodilator function compared with its lean litter mate. We have investigated hindquarter endothelium-dependent vasodilation in the obese Zucker rat in-situ and have examined its relationship with oxidant stress.

In perfused hindquarter preparations equivalently preconstricted with phenylephrine, vasodilator responses to the endothelium-dependent agent acetylcholine (0.03–1000 pmol) were greater in obese (pD2 = 11.03±0.19) compared with lean (pD2 = 10.53±0.13) animals (P < 0.01, two-way analysis of variance). In contrast, maximal vasodilation to the nitric oxide (NO) donor sodium nitroprusside (100 nmol) was similar in obese (59.6±9.8%) and lean (51.9±2.6%) preparations (P > 0.05). However, this exaggerated vasodilator reactivity to acetylcholine in obese animals was abolished following four-week dietary supplementation with the lipophilic antioxidant vitamin E (obese pD2 = 10.74±0.18; lean pD2 = 10.74±0.08). This antioxidant-mediated effect was associated with a reduction (P < 0.02, two-way analysis of variance) and an enhancement (P < 0.01, two-way analysis of variance) in endothelium-dependent vasodilator responses in obese and lean hindlimb preparations, respectively.

Our data therefore now point to a differential modulation of hindquarter endothelium-dependent vasodilation in the obese and lean Zucker rat by the prevailing oxidant tone, resulting in an agonist-stimulated endothelial vasodilator hyperreactivity in obese animals.
Long-term Clomipramine Treatment Upregulates Forebrain Acetylcholine Muscarinic Receptors, and Reduces Behavioural Sensitivity to Scopolamine in Mice
Authors: Tsukagoshi Hiroshi , Morita Toshihiro , Hitomi Shinichiro , Saito Shigeru , Kadoi Yuji , Uchihashi Yoshitaka , Kuribara Hisashi , Goto Fumio
Published: July 2003

We have investigated the effects of long-term treatment with clomipramine, a tricyclic antidepressant, on central muscarinic acetylcholine receptors (mAChR) in mice.

Repeated clomipramine administration resulted in an increase in the forebrain receptor density value (Bmax) for [3H]quinuclidinyl benzilate, a muscarinic ligand (P < 0.05), that was dependent on dose per administration (saline or 5, 10, or 20 mg kg−1 once a day for 7 days) and number of days treated (20 mg kg−1 for 1, 3, 5, or 7 days). No change in apparent affinity (defined as the reciprocal of the dissociation constant) (KD) occurred. Seven daily treatments with clomipramine (saline or 5, 10, or 20 mg kg−1) reduced hyperlocomotion induced by scopolamine (0.5 mg kg−1, s.c.) dose-dependently, and the effect of 20 mg kg−1 clomipramine was significant (P < 0.05).

These results suggest that an upregulation of mAChR is produced by repeated clomipramine administration, and such a change is responsible for the decreased sensitivity to the muscarinic antagonist scopolamine.
Time Dependent Effects of Glucocorticoids on Adrenocorticotropin Secretion of Rat Pituitaries Ex-vivo
Authors: Bruns R , Rohdewald P
Published: July 2003

Different glucocorticoids have been compared with respect to the inhibition of corticotropin-releasing factor (CRF)-mediated adrenocorticotropin (ACTH) secretion from pituitary fragments of the rat. The influence of time of exposure to glucocorticoids and glucocorticoid concentration has been investigated.

CRF-stimulated ACTH secretion of perifused rat pituitary fragments was measured by a chemiluminescence immunoassay. ACTH secretion was monitored over three days. Inhibition of CRF-stimulated ACTH secretion by glucocorticoids was quantified by the area under the curve of CRF-stimulated ACTH secretion over baseline. Concentrations needed to inhibit ACTH secretion decreased with the receptor affinities of the glucocorticoids as follows: fluticasone propionate; receptor affinity 1800, concentration 10−8 M; budesonide, 935 and 3–2.5 times 10−8 M; flunisolide, 478 and 5 times 10−7 M; prednisolone, 10 and 10−6 M. CRF-stimulated secretion was inhibited by glucocorticoids after incubation for 1 min at concentrations between 10−8 and 10−6 M. The same absolute quantity of the glucocorticoids produced no inhibition when incubation was prolonged to 50 min or when a lower concentration was used. Immediately after the perifusion stimulation of ACTH secretion was observed.

The results suggest the possibility of minimizing the side effects of glucocorticoids by prolonging drug release.
Influence of Extracellular K+ Concentrations on Quinidine-induced K+ Current Inhibition in Rat Ventricular Myocytes
Authors: Hirota Michiko , Ohtani Hisakazu , Hanada Erika , Sato Hitoshi , Kotaki Hajime , Uemura Hiroko , Nakaya Haruaki , Iga Tatsuji
Published: July 2003

Hypokalaemia is one of the important risk factors for development of torsades de pointes. We recently reported that hypokalaemia increased the electrocardiographic QT interval in rats treated with quinidine, but did not alter the arrhythmogenic potency of quinidine. In this study, we have investigated the influence of extracellular potassium concentration ([K+]o) on the inhibition of several types of cardiac potassium currents by quinidine. Such types of currents include the delayed rectifier potassium current (IK), the transient outward current (Ito), and the inward rectifier potassium current (IK1), as measured in isolated rat ventricular cells using patch-clamp techniques.

Concentration-dependent effects of quinidine on IK, Ito, and IK1 were evaluated under both normal ([K+]o = 5.4 mM) and hypokalaemic ([K+]o = 3.5 mM) conditions. In contrast to both IK and Ito, which were barely influenced by changes in [K+]o, IK1 was significantly inhibited by hypokalaemia. Furthermore, while quinidine suppressed both IK and Ito in a concentration-dependent manner, the inhibitory potency of quinidine on these currents was not influenced by changes in [K+]o. The respective normal and hypokalaemic IC50 values for quinidine were 11.4 and 10.0 μM (IK), and 17.6 and 17.3 μM (Ito). Although higher concentrations of quinidine were required to inhibit IK1, the inhibitory potency of quinidine was also found to be insensitive to changes in [K+]o. Thus, in rats, the inhibitory potency of quinidine for the K+ current-types IK, Ito and IK1 is barely influenced by changes in [K+]o.

These findings are consistent with our previous report showing that the QT-prolonging potency of quinidine was not altered under hypokalaemic conditions. However, whilst hypokalaemia does not affect IK or Ito, it can inhibit IK1 and can result in QT prolongation in-vivo.
Comparison of Analgesic Effects of Khat (Catha edulis Forsk) Extract, D-Amphetamine and Ibuprofen in Mice
Authors: Connor John , Makonnen Eyasu , Rostom Amr
Published: July 2003

We have compared the analgesic properties of khat (Catha edulis Forsk) extract, amphetamine and ibuprofen in mice. After intragastric administration of the drugs analgesia was measured relative to water-injected controls using the hot-plate, the tail-flick, and abdominal-constriction tests.

At the highest doses examined (amphetamine 1.8 mg kg−1, ibuprofen 90 mg kg−1, khat extract 1800 mg kg−1), all three substances produced analgesia, but the order of efficacy varied with the test. Khat and ibuprofen were significantly different from the control in the hot-plate assay at three or more time points post-injection. In the tail-flick test, khat and amphetamine were efficacious; ibuprofen means were somewhat lower but still significantly different from control. Higher doses of the drugs decreased the number of responses in the acetic acid-induced abdominal-constriction assay.

We conclude that khat, like amphetamine and ibuprofen, can relieve pain. Differences in assay results may reflect differences in modes and sites of action, as well as in the type of pain generated by the chemical and thermal stimuli for nociception.
Effect of Sho-saiko-to Extract on HGF and TGF-β Levels of Intraorgans in Liver-injured Rats after Partial Hepatectomy
Authors: Ono Masahide , Miyamura Mitsuhiko , Kyotani Shojiro , Saibara Toshiji , Ohnishi Saburo , Nishioka Yutaka
Published: July 2003

To examine the effects of Sho-saiko-to extract on liver regeneration, Sho-saiko-to extract (0.75%, 1.5% or 3.) was administered to 70% partial hepatectomized rats with dimethyl-nitrosamine-induced liver-injury.

S phase cell number, liver retinoid levels, hepatocyte growth factor (HGF) and transforming growth factor-beta (TGF-β) levels in each intraorgan were measured as indicators of liver regeneration. Three to seven days after hepatectomy, HGF and TGF-β levels of the liver and spleen of the Sho-saiko-to extract groups were significantly different from the levels of the ordinary food group (P < 0.05–0.1). HGF levels in the Sho-saiko-to extract groups were approximately 1.3–1.8 times higher in the liver and approximately 1.8–2.1 times higher in the spleen compared with the levels found in the ordinary food group. TGF-β levels in the Sho-saiko-to extract groups were approximately 0.38–0.47 times the level in the liver and 0.58–0.77 times the level in the spleen of the ordinary food group. There was no difference in HGF and TGF-β levels of the kidney and lung between the Sho-saiko-to extract group and the ordinary food group. There was a significant and positive correlation between HGF level and S phase cell number in the liver (r = 0.826, P < 0.01). There was a significant and negative correlation between TGF-β level and the retinoid level in the liver (r = −0.696, P < 0.01). In addition, the levels of the active constituents of Sho-saiko-to extract (glycyrrhetic acid, baicalin and baicalein) showed high values in the liver and spleen of partial hepatectomized rats, and increased from the third day after partial hepatectomy.

These results show that Sho-saiko-to extract induces liver regeneration by increasing the production of HGF and suppressing the production of TGF-β in the liver and spleen of partial hepatectomized rats. It was considered that the increase in the Sho-saiko-to extract active constituent levels in the liver and spleen greatly influences this action.
Inhibitory Effect of Euphol, a Triterpene Alcohol from the Roots of Euphorbia kansui, on Tumour Promotion by 12-O-Tetradecanoylphorbol-13-acetate in Two-stage Carcinogenesis in Mouse Skin
Authors: Yasukawa Ken , Akihisa Toshihiro , Yoshida Zen-Ya , Takido Michio
Published: July 2003

The anti-inflammatory activity of euphol, twelve other triterpene alcohols and sitosterol-β-D-glucopyranoside, isolated from the dichloromethane extract of the roots of Euphorbia kansui, has been evaluated in mice with inflammation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA).

TPA (1.7 nmol; 1.0 μg/ear) was dissolved in acetone and 10 μL delivered to the inner and outer surfaces of the right ear of ICR mice. A triterpene alcohol, sterol glucoside or vehicle (20 μL; chloroform—methanol 1: 1), was applied topically approximately 30 min before each TPA treatment. The ear thickness was measured before treatment and then oedema was measured 6 h after TPA treatment.

For the two-stage carcinogenesis experiment, initiation was accomplished by administration of a single topical application of 7,12-dimethylbenz[a]anthracene (DMBA; 195 nmol; 50 μg/mouse) to the shaved backs of mice. Promotion was with 1.7 nmol (1.0 μg) TPA, applied twice weekly to the same shaved area, begun one week after the initiation. Euphol (2.0 μmol; 853 μg), or its vehicle (acetone-dimethylsulphoxide, 9:1; 100 μL), was applied topically 30 min before each TPA treatment. The number and diameter of skin tumours were measured every other week for 20 weeks.

All the compounds were found to possess marked inhibitory activity and their 50% inhibitory dose for TPA-induced inflammation was 0.2–1.0 mg/ear. Topical application of euphol (2.0 μmol; 853 μg/mouse) markedly suppressed the tumour-promoting effect of TPA (1.7 nmol; 1.0 μg/mouse) in mouse skin initiated with DMBA.
Analysis of the Stability and Degradation Products of Triptolide
Authors: Mao Yan Ping , Tao Xue Lian , Lipsky Peter E
Published: July 2003

Triptolide is the major active ingredient of the Chinese herbal remedy Tripterygium wilfordii Hook F. (TwHF). As triptolide content is used to estimate the potency of preparations of TwHF, assessment of its stability is warranted.

The accelerated stability of triptolide was investigated in 5% ethanol solution in a light-protected environment at pH 6.9, within a temperature range of 60–90°C. The observed degradation rate followed first-order kinetics. The degradation rate constant (K25°C) obtained by trending line analysis of Arrhenius plots of triptolide was 1.4125 times 10−4 h−1. The times to degrade 10% (t1/10) and 50% (t1/2) at 25°C were 31 and 204 days, respectively. Stability tests of triptolide in different solvents and different pH conditions (pH 4–10) in a light-protected environment at room temperature demonstrated that basic medium and a hydrophilic solvent were the major factors that accelerated the degradation of triptolide. Triptolide exhibited the fastest degradation rate at pH 10 and the slowest rate at pH 6. In a solvent comparison, triptolide was found to be very stable in chloroform. The stability of triptolide in organic polar solvents tested at both 100% and 90% concentration was greater in ethanol than in methanol than in dimethylsulphoxide. Stability was also greater in a mixture of solvent: pH 6 buffer (9:1) than in 100% solvent alone. An exception was ethyl acetate, which is less polar than the other solvents tested, but permitted more rapid degradation of triptolide. Two of the degradation products of triptolide were isolated and identified by HPLC and mass spectroscopy as triptriolide and triptonide. This suggested that the decomposition of triptolide occurred at the C12 and C13 epoxy group and the C14 hydroxyl. The opening of the C12 and C13 epoxy is an irreversible reaction, but the reaction occurring on the C14 hydroxyl is reversible.

These results show that the major degradation pathway of triptolide involves decomposition of the C12 and C13 epoxy group. Since this reaction is very slow at 4°C at pH 6, stability is enhanced under these conditions.
Triboelectrification of Spray-dried Lactose Prepared from Different Feedstock Concentrations
Authors: Cassidy Orla E , Carter Paul A , Rowley Geoff , Merrifield David R
Published: July 2003

Powder systems may acquire electrostatic charge during various pharmaceutical processing operations and may give rise to difficulties in handling and powder flow, mainly due to adhesion/cohesion effects. We have investigated the electrostatic charging of spray-dried lactose prepared from different feedstock concentrations using a laboratory spray-dryer.

Triboelectrification of the spray-dried lactose samples was effected through contact with the stainless steel surface of either a mixing vessel or a cyclone separator. Results from both techniques showed differences in charge accumulation and particle-steel adhesion between the spray-dried lactose samples. As the feedstock concentration used to produce the spray-dried lactose was increased in the range 10–50% w/v, the mean charge on the lactose decreased from −20.8 to −1.3 nC g−1 and −54.9 to −4.1 nC g−1 for the mixing vessel and cyclone separator, respectively, with a corresponding decrease in adhesion. In addition, as the feedstock concentration was increased from 10 to 50% w/v, decreases were obtained in surface area values (1.06 to 0.56 m2 g−1), pore diameter (198.7 to 83.5 μm) and pore volume (1.09 to 0.75 cm3 g−1), and together with differences in crystal form correlated with the charge and adhesion results.

The results suggested that the feedstock concentration could have a considerable influence on the charging and adhesional properties of spray-dried lactose. This may have relevance during pharmaceutical processing and manufacturing operations.
Development of a Lyophilization Formulation that Preserves the Biological Activity of the Platelet-inducing Cytokine Interleukin-11 at Low Concentrations
Authors: Page Christopher , Dawson Peter , Woollacott David , Thorpe Robin , Mire-Sluis Anthony
Published: July 2003

Recombinant human interleukin-11 (rhIL-11) is a licensed biological therapeutic product in at least one country and is used to combat thrombocytopenia during chemotherapeutic regimens, as well as undergoing clinical trials for a range of other disorders. Following attempts to lyophilize IL-11 at low concentrations, it was clear that a significant loss of recoverable biological activity occurred.

Investigation of a variety of factors, including the type of container in which the rhIL-11 was lyophilized, revealed that surface adsorption to glass was a major factor resulting in loss of activity of rhIL-11 in solution (> 40% reduction after 3 h at room temperature), in addition to losses of activity post-lyophilization. To overcome this problem, different formulations containing combinations of human serum albumin (HSA), trehalose and Tween-20 have been investigated.

Two formulations were successful in entirely preserving the biological activity of rhIL-11 through lyophilization and subsequent reconstitution (potency estimates of formulated relative to original material being ≥0.97). Accelerated degradation studies, performed at intervals over a six-month period, demonstrated the stability of freeze-dried rhIL-11 using these formulations (predicted annual reduction in potency after storage at −20°C ≤1.4%).

In conclusion, we have developed a working combination of excipients (0.5% HSA, 0.1% trehalose and 0.02% Tween-20 in potassium phosphate buffer (pH 7.4)) to formulate a stable rhIL-11 freeze-dried product in glass containers, with no loss in potency. These findings should facilitate development of low dose rhIL-11 products and be an indicator of caution to those using this and other material with similar physical properties, without taking appropriate precautions to avoid losses through adsorption.
Tissue Distribution and Pharmacological Potential of SM-16896, a Novel Oestrogen-bisphosphonate Hybrid Compound
Authors: Tsushima Naomi , Yabuki Masashi , Harada Hideyuki , Katsumata Takashi , Kanamaru Hiroshi , Nakatsuka Iwao , Yamamoto Michihiro , Nakatsuka Masashi
Published: July 2003

Postmenopausal osteoporosis is caused mainly by a deficiency of oestrogen with rapid bone loss. To target oestrogen to the bone effectively, we have synthesized and evaluated the effects of a novel hybrid compound of oestrogen and bisphosphonate, SM-16896. The tissue distribution pattern and pharmacological potential are reported.

Although the affinity for calf uterine oestrogen receptor was very low (IC50: 73.3 μM; 1/25000 of that of 17β-oestradiol (2.84 nM)), SM-16896 showed oestrogenic activity. SM-16896 (1 μM) induced a 4.5-fold transcriptional activity in rat osteosarcoma UMR-106 cells compared with vehicle-treated control, when we used the expression vector for human oestrogen receptor and a CAT reporter plasmid containing an oestrogen-responsive element. The distribution of SM-16896 after a subcutaneous administration to 7-week-old female rats was examined by radioluminography using 3H-labelled SM-16896. At 30 min after the administration, significant radioactivity was detected in the bone. At 24 h after administration, a high level of radioactivity was detected in the bone, but in the uterus it was only at a background level. Daily subcutaneous administration of 0.5 mg kg−1 SM-16896 for 12 weeks (five times per week) to 13-week-old ovariectomized rats suppressed the ovariectomized-induced reduction in bone mineral density. A bone mineral density ratio of 120% was maintained compared with sham-operated rats, whereas a relatively low suppression of uterine weight was observed (about 50% loss compared with sham-operated rats). In the same experiment, the implantation of a 17β-oestradiol time-release pellet (0.25 mg/pellet/90 days) almost completely suppressed the reduction of both the bone mineral density and uterine tissue weight. It is likely that the effect of SM-16896 on bone was due to its oestrogenic activity, since 1.0 mg kg−1 SM-18108, the bisphosphonate moiety of this compound, had no effect on bone in 7-week-old ovariectomized rats.

The results suggest that SM-16896, a bisphosphonate-conjugated oestrogen, showed a preference profile in the uterus and bone due to its characteristic distribution pattern compared with the natural oestrogen analogue 17β-oestradiol. Thus, bisphosphonate-conjugated oestrogens have the potential to improve patient compliance in oestrogen therapy by minimizing adverse effects and reducing the frequency of medication.
The Effect of Divalent Cations on the Membrane Properties and Pharmacokinetics in Rat of the Lipid A Analogue E5531
Authors: Asai Yasuyuki , Sano Yoshihisa , Kikuchi Kiyomi , Iwamoto Kiyoshi , Watanabe Sumio
Published: July 2003

To obtain information on the effects of Ca2+ on the membrane properties of the lipid A analogue E5531, we have determined the aggregate size, zeta potential, membrane fluidity, micropolarity and permeability of the E5531 membrane as a function of Ca2+ levels.

Within the molar ratios of [Ca2+]/[E5531] = 1 and 3, Ca2+ increased the zeta potential of the E5531 membrane but had no effect on aggregate size (approximately 20 nm). Within the above ratios, Ca2+ decreased the membrane fluidity, as measured by micropolarity of E5531 and increased the phase transition temperature.

The pharmacokinetics in rats for these samples with different membrane fluidity, prepared by changing the pre-dose formulation concentration of Ca2+, was determined and a correlation between membrane fluidity and pharmacokinetics was clearly observed.

It thus appears that Ca2+ effects the membrane fluidity of E5531 as well as its pharmacokinetics in rats.
Oral, Intraperitoneal and Intravenous Pharmacokinetics of Deramciclane and its N-desmethyl Metabolite in the Rat
Authors: Nemes Katalin Balogh , Abermann Miklós , Bojti Erzsébet , Grézal Gyula , Al-Behaisi Samar , Klebovich Imre
Published: July 2003

The pharmacokinetic properties of deramciclane fumarate (EGIS-3886), a new potential anxiolitic agent, and its N-desmethyl metabolite have been investigated in Wistar rats after 10 mg kg−1 deramciclane fumarate was administered orally, intraperitoneally or intravenously.

A highly sensitive, validated and optimized gas chromatographic method with nitrogen selective detection (GC-NPD) using a solid-phase extraction technique was used to determine plasma levels of the parent compound and its N-desmethyl metabolite.

After oral administration the absorption of the parent compound was very fast (tmax 0.5 h). The maximum plasma concentration (Cmax) was detected at 44.9, ≥177.8 and ≥2643.0 ng mL−1 after oral, intraperitoneal and intravenous administration of deramciclane, respectively. For the metabolite the respective Cmax values were 32.0, ≥25.4 and 51.0 ng mL−1. The pharmacokinetic curves of both the parent compound and its metabolite showed enterohepatic recirculation for all administration routes. The biological half-life (tβ1/2) for deramciclane ranged from 3.42 to 5.44 h and for the N-desmethyl metabolite the range was 2.90–5.44 h, after administration of the drug by the three different routes. After intravenous administration AUC0-∞ of deramciclane was 29.2- and 5.4-times higher than that observed after oral and intraperitoneal treatment, respectively. These AUC0-∞ ratios were only 2.1- and 1.5-times higher for the metabolite. The absolute bioavailability of deramciclane in rats was 3.42% after oral and 18.49% after intraperitoneal administration.

The comparative pharmacokinetic study of deramciclane in rat after the different administration routes showed fast absorption. Furthermore, plasma levels were found to be administration route-dependent, low bioavailability of the parent compound indicated an extremely fast and strong first-pass metabolism. The apparent volume of distribution suggested strong tissue binding after administration of the drug by any of the three routes studied.
The Effect of Fenfluramine on the Pulmonary Disposition of 5-Hydroxytryptamine in the Isolated Perfused Rat Lung: a Comparison with Chlorphentermine
Authors: Valodia P , Syce J A
Published: July 2003

A possible mechanism for fenfluramine-induced pulmonary hypertension has been investigated. Fenfluramine, like chlorphentermine, may inhibit the pulmonary uptake and/or metabolism of 5-hydroxytryptamine (5-HT). This allows more 5-HT to remain in the pulmonary circulation, where it may exert a greater vasoconstrictor action resulting in pulmonary hypertension. Chlorphentermine has been shown to inhibit the uptake and metabolism of 5-HT.

The effect of fenfluramine on the pulmonary disposition of [14C]5-HT has been investigated, in comparison with chlorphentermine, using a recirculating isolated perfused rat lung system. The pulmonary disposition of [14C]5-HT was assessed by measuring the change in [14C]5-HT concentration in the perfusion medium during the experiment and at the end, and the concentration in the lung at the end of the experiment. The concentration of 5-hydroxyindoleacetic acid, a metabolite of 5-HT, was measured in perfusate and lung samples. Mean pulmonary clearance of 5-HT for the control lung and lungs challenged with either fenfluramine (2.5 μM) or chlorphentermine (2.5 μM) was 4.514, 1.316 and 1.007 mL min−1, respectively (n = 5). The concentration of 5-HT found in the lungs at the end of the experiment for the control and the lungs preloaded with fenfluramine or chlorphentermine was 695.05 ± 9.69, 638.65 ± 10.27 and 617.3 ± 14.38 ng g−1, respectively.

Fenfluramine, like chlorphentermine, inhibited the pulmonary disposition of 5-HT resulting in an elevated perfusate level of 5-HT. This is a possible contributing mechanism for fenfluramine-induced pulmonary hypertension. The effect of fenfluramine was less pronounced than chlorphentermine.
Glutamate and Kynurenate in the Rat Central Nervous System Following Treatments with Tail Ischaemia or Diclofenac
Authors: Edwards Stephen R , Mather Laurence E , Lin Yiguang , Power Ian , Cousins Michael J
Published: July 2003

Kynurenate is an endogenous antagonist at the allosteric glycine site on the N-methyl-D-aspartate (NMDA) receptor, and may have a role in ameliorating nociceptive processes through modulation of NMDA receptor function. While antinociceptive effects of non-steroidal anti-inflammatory drugs (NSAIDs) are mediated peripherally and possibly centrally through inhibition of prostaglandin synthesis, there is also evidence for centrally mediated prostaglandin-independent antinociceptive effects that may result from increased central nervous system (CNS) concentrations of kynurenate. We have investigated the effects of the NSAID diclofenac, (40 mg kg−1, s.c.; administered to rats 1 h before killing) or the exposure of rats to noxious stimulation (tail ischaemia for 20 min before killing), on the concentrations of glutamate and kynurenate in discrete CNS regions.

Regional CNS tissue concentrations of diclofenac were between 3.0–3.8 nmol g−1. The corresponding regional glutamate concentrations ranged between 4.8–10.6 μmol g−1, and were significantly lower in the ischaemia group when compared with both control (15%, P < 0.05) and diclofenac-treated (19%, P < 0.002) groups. Kynurenate concentrations in these CNS regions ranged between 3.3–45.8 pmol g−1. Pairwise comparisons between the control and diclofenac-treated groups found significant increases in kynurenate concentrations in the diencephalon and lumbo-sacral regions of the CNS (P = 0.05).

Noxious stimulation from tail ischaemia appeared to be associated with increased release of glutamate. Additionally, NSAIDs appeared to increase kynurenate concentrations in the spinal cord and diencephalon. Antagonism by kynurenate of glutamate effects at NMDA receptors may contribute to the antinociceptive effects of NSAIDs.
Fluvastatin, a New Inhibitor of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase, Resists Hydroxyl Radical Generation in the Rat Myocardium
Authors: Obata Toshio , Ebihara Akio , Yamanaka Yasumitsu
Published: July 2003

The aim of this study was to determine whether fluvastatin, an inhibitor of low-density lipoprotein (LDL) oxidation, can resist Cu (II)-induced hydroxyl radical generation (.OH) in the extracellular fluid of rat myocardium.

Rats were anaesthetized and sodium salicylate in Ringer's solution (O.5 nmol μL−1 min−1) was infused through a microdialysis probe to detect the generation of .OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) in the myocardium. When Cu (II) (0, 10, 25 or 50 μm) was administered to 1 mm tyramine-pretreated rats, a marked elevation in the levels of 2,3-DHBA was found, indicating a positive linear correlation between Cu (II) and the increase in .OH formation trapped as 2,3-DHBA in the dialysate (r2 = 0.977). In the presence of fluvastatin (100 μm), a marked decrease in the levels of 2,3-DHBA was found. Corresponding experiments performed with iron (II) (0, 10, 25 or 50 μm), showed a marked elevation in the levels of 2,3-DHBA, indicating a positive linear correlation between iron (II) and the increase in .OH formation trapped as 2,3-DHBA in the dialysate (r2 = 0.986). However, in the presence of fluvastatin (100 μm) a small decrease in the level of 2,3-DHBA was found.

The results show that iron (II) against LDL oxidation may be insensitive compared with Cu (II). Cu (II)-induced .OH formation may be reduced by inhibiting LDL with fluvastatin.
The Role of Nitric Oxide and Prostaglandin E2 on the Hyperalgesia Induced by Excitatory Amino Acids in Rats
Authors: Park Yang Hae , Shin Chang Yell , Lee Tai Sang , Huh In Hoi , Sohn Uy Dong
Published: July 2003

The present study was designed to investigate the role of nitric oxide (NO), N-methyl-D-aspartate (NMDA) receptor and prostaglandins on hyperalgesia induced in rats by excitatory amino acids and the possibility that prostaglandins may act as the retrograde messenger in the spinal cord like NO.

Nω-nitro-L-arginine methyl ester (L-NAME; 500 μg/paw, intraplantarly (i.pl.)), MK-801 (10 μg/paw, i.pl.) or indomethacin (300 μg/paw, i.pl.) reduced the duration of phase 2 of the biting/licking and scratching (B/L + S) response induced by formalin injection from 255.6±16.7 s to 155.6 ± 16.9, 172.2 ± 33.3 or 205.6 ± 16.7 s, respectively. L-NAME (0–3 mg, i.th.), MK-801 (8 μg, i.th.) or indomethacin (20 μg, i.th) reduced the duration of phase 2 of the B/L + S response induced by saline injection from 288.5 ± 7.7 s to 207.7 ± 19.2, 184.6 ± 7.7 or 192.3 ± 38.5 s, respectively.

L-NAME or indomethacin injected into the spinal cord of the rat significantly reduced the hyperalgesia induced by NMDA (1 μg, i.th.) from 43.8 ± 4.6% to 12.3 ± 3.1 and 19.2 ± 2.3%, respectively. It is assumed that NO produced by excitatory amino acids may increase prostaglandin production by cyclooxygenase activation. L-NAME, MK-801 or indomethacin injected into the rat spinal cord significantly reduced the hyperalgesia induced by prostaglandin E2 (PGE2, 25 ng, i.th.) in the tail-flick test from 40.6 ± 3.5% to 18.2 ± 3.2, 18.8 ± 1.8 or 17.6 ± 4.1%, respectively, but had little effect on hyperalgesia in the paw pressure test (except for indomethacin).

In conclusion, NO and PGE2 affect the hyperalgesia induced by excitatory amino acids. It is suggested that PGE2, like NO, may act as a retrograde messenger in the spinal cord.
Protective Effect of Curcumin in Rat Liver Injury Induced by Carbon Tetrachloride
Authors: Park Eun-Jeon , Jeon Chul Hyun , Ko Geonil , Kim Jaebaek , Sohn Dong Hwan
Published: July 2003

This study was carried out to investigate the protective effects of curcumin on acute or subacute carbon tetrachloride-induced liver damage in rats.

Acute hepatotoxicity was induced by intraperitoneal injection of carbon tetrachloride after 4 consecutive days of curcumin treatment. Subacute hepatotoxicity was induced by oral administration of carbon tetrachloride twice a week during 4 weeks of curcumin treatment. In rats with acute liver injury, curcumin (100 and 200 mg kg−1) lowered the activity of serum alanine aminotransferase to 52–53% (P < 0.05) and aspartate aminotransferase to about 62% (P < 0.05) those of control rats. In rats with subacute liver injury, curcumin (100 mg kg−1) lowered the activity of serum alanine aminotransferase to 34% (P < 0.01) and alkaline phosphatase to 53% (P < 0.05) of control rats. The liver hydroxyproline content in the curcumin (100 mg kg−1)-treated group was reduced to 48% of the carbon tetrachloride control group (P < 0.01). Malondialdehyde levels in curcumin (100 mg kg−1) treated rat liver was decreased to 67% of the control rat liver (P < 0.01) in subacute injury.

It was concluded that curcumin improved both acute and subacute liver injury induced by carbon tetrachloride in rats.
SM-20220, a Potent Na+/H+ Exchange Inhibitor, Improves Consciousness Recovery and Neurological Outcome Following Transient Cerebral Ischaemia in Gerbils
Authors: Kuribayashi Yoshikazu , Itoh Natsuko , Horikawa Naotsugu , Ohashi Naohito
Published: July 2003

We studied the cerebroprotective effect of SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulphonate), a newly synthesized Na+/H+ exchanger (NHE) inhibitor, in Mongolian gerbil global ischaemia.

Transient cerebral ischaemia was induced by clipping both common carotid arteries for 30 min followed by 24 h reperfusion. Intravenous administration of SM-20220 (0.3 or 1.O mg kg−1) immediately after reperfusion significantly shortened the consciousness recovery time (P < 0.01). SM-20220 also improved the neurological outcome (McGraw's scale) after reperfusion. At the dose of 1.0 mg kg−1, the mortality rate was significantly reduced at 24 h after reperfusion (P < 0.01).

This study shows that NHE is involved in the aggravation of cerebral function, represented by consciousness recovery, and neurological outcome following transient forebrain ischaemia, and that its inhibitor may exert protective effects on post-ischaemic brain damage.
Estimation of Individual Sennosides in Plant Materials and Marketed Formulations by an HPTLC Method
Authors: Shah Shailesh A , Ravishankara M N , Nirmal Anjoo , Shishoo Chamanlal J , Rathod Ishwarsinh S , Suhagia Bhanubhai N
Published: July 2003

Senna is a well-known drug, used in the Ayurvedic and Allopathic systems of medicine, and is a treatment for constipation. The purgative action of senna and its formulations is due to the presence of sennosides A and B. An HPTLC method has been developed for the determination of individual sennosides (A, B, C, D) without any derivatization in marketed formulations (three tablet formulations, two granule formulations and one liquid formulation) and plant materials (senna leaf and pod).

The methanolic solution of a sample was applied on a pre-coated silica gel G60 F254 TLC plate (E. Merck.) and was developed using n-propanol: ethyl acetate: water: glacial acetic acid (3:3:2:0.1 v/v) as the mobile phase. The relative band speeds (Rf values) obtained were 0.35, 0.25, 0.61, 0.46 for sennosides A, B, C and D, respectively. The densitometric response was monitored at 366 nm. Calibration curves were found to be linear in the concentration ranges 193–1356, 402–2817, 71–497 and 132–927 ng per spot for sennosides A, B, C, and D, respectively. The correlation coefficients were found to be 0.9978, 0.9987, 0.9939 and 0.9983 respectively for sennosides A, B, C and D. The result obtained with the HPTLC method for total sennoside content was compared with the results using the pharmacopoeial methods (spectrophotometric (British Pharmacopoeia) and spectrofluorimetric (United States Pharmacopeia) using the ‘F’ test). The results revealed no significant difference in the three different methods for estimation of total sennoside.

The proposed HPTLC method was found to be simple, specific, precise, accurate and rapid. It can be used for routine quality control of sennosides or senna-containing formulations for individual sennosides.
Identification of Kaempferol as a Monoamine Oxidase Inhibitor and Potential Neuroprotectant in Extracts of Ginkgo Biloba Leaves
Authors: Sloley B D , Urichuk L J , Morley P , Durkin J , Shan J J , Pang P K T , Coutts R T
Published: July 2003

The effects of Ginkgo biloba leaf extract on rat brain or livermonoamine oxidase (MAO)-A and -B activity, biogenic amine concentration in nervous tissue, N-methyl-d-aspartate (NMDA)- and N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)-induced neurotoxicity and antioxidant activity was investigated to determine the effects of the extract on monoamine catabolism and neuroprotection.

Ginkgo biloba leaf extract was shown to produce in-vitro inhibition of rat brain MAO-A and -B. The Ginkgo biloba extract was chromatographed on a reverse-phase HPLC system and two of the components isolated were shown to be MAO inhibitors (MAOIs). These MAOIs were identified by high-resolution mass spectrometry as kaempferol and isorhamnetin. Pure kaempferol and a number of related flavonoids were examined as MAOIs in-vitro. Kaempferol, apigenin and chrysin proved to be potent MAOIs, but produced more pronounced inhibition of MAO-A than MAO-B. IC50 (50% inhibition concentration) values for the ability of these three flavones to inhibit MAO-A were 7 times 10−7, 1 times 10−6 and 2 times 10−6  m, respectively. Ginkgo biloba leaf extract and kaempferol were found to have no effect ex-vivo on rat or mouse brain MAO or on concentrations of dopamine, noradrenaline, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid. Kaempferol was shown to protect against NMDA-induced neuronal toxicity in-vitro in rat cortical cultures, but did not prevent DSP-4-induced noradrenergic neurotoxicity in an in-vivo model. Both Ginkgo biloba extract and kaempferol were demonstrated to be antioxidants in a lipid-peroxidation assay.

This data indicates that the MAO-inhibiting activity of Ginkgo biloba extract is primarily due to the presence of kaempferol. Ginkgo biloba extract has properties indicative of potential neuroprotective ability.
Protection by Osbeckia aspera Against Carbon Tetrachloride-mediated Alterations in Microsomal Drug Metabolizing Enzyme Activity
Authors: Jayatilaka K A P W , Thabrew M I
Published: July 2003

Previous investigations have confirmed the protective effect of Osbeckia aspera leaf extract on carbon tetrachloride-mediated liver injury in rat models. It is well known that the earliest alterations in liver cell structure and function following carbon tetrachloride poisoning involve the endoplasmic reticulum and its drug metabolizing enzymes. Therefore, we investigated whether an aqueous leaf extract of O. aspera could offer protection against carbon tetrachloride-induced changes in the microsomal drug metabolizing enzymes aniline hydroxylase and p-aminopyrine N-demethylase. This enzyme activity was compared with phenobarbital-induced righting reflex and lipid peroxidation.

Treatment of rats with the aqueous leaf extract of O. aspera (before or after the administration of carbon tetrachloride) resulted in a marked decrease in carbon tetrachloride-mediated alterations in aniline hydroxylase and p-aminopyrine N-demethylase activity, phenobarbital-induced loss of righting reflex and malondialdehyde formation due to lipid peroxidation. The Km value of these enzymes in control and Osbeckia-treated rats were the same.

These results show that the plant extract can markedly decrease the carbon tetrachloride-mediated reduction in aniline hydroxylase and p-aminopyrine N-demethylase activity and inhibit peroxidative damage to the cell membrane. Phenobarbital-induced sleeping time in rats and kinetic enzyme studies suggested that the effects of the plant extract was neither due to an induction of the drug-metabolizing enzymes under investigation, nor due to an alteration in the Km values of these enzymes.