Chronic treatment of cycling rhesus monkeys with low doses of the antiprogestin ZK 137 316: morphometric assessment of the uterus and oviduct

The long-term effects of the antiprogestin ZK 137 316 on reproductive tract morphology in rhesus macaques were investigated. The monkeys were injected daily (i.m.) for five menstrual cycles with vehicle or 0.01, 0.03 or 0.1 mg ZK 137 316/kg body weight. Reproductive tracts (n = 3/ group) were collected during the mid-luteal phase (day 8) of the fifth cycle in the control, 0.01 and 0.03 mg/kg groups, or 6-7 days after the oestradiol peak in the 0.1 mg/kg group. ZK 137 316 treatment resulted in a dose-dependent atrophy of the endometrium, marked by reduced mitotic activity in the glands, compaction of the stroma, degradation of spiral arteries and dilation of veins. There was no effect of ZK 137 316 on myometrial or oviductal weight. Treatment with 0.1 and 0.03 mg/kg, but not 0.01 mg/kg resulted in fully ciliated and secretory oviducts, indicating a dose-dependent blockade of progesterone antagonism of oestrogen-dependent oviductal differentiation. In the endometrium, the suppressive action of progesterone on oestrogen and progestin receptors was also blocked by ZK 137 316 in a dose-dependent manner. However, endometrial atrophy appeared due to inhibition of progesterone action together with a blockade of oestrogen-dependent proliferation. The profoundly suppressed endometrium produced by chronic low-dose ZK 137 316 treatment is unlikely to support implantation. Such treatment may therefore provide a novel contraceptive modality.      

Reversible suppression of menstruation with progesterone antagonists in rhesus macaques

BACKGROUND: A reliable means of menstrual suppression would greatly improve the quality of life for women. Information is lacking on the direct endometrial effects and appropriate dosages of new antiprogestins that may be useful for this purpose. METHODS: The current work evaluated three different systems in macaque monkeys. First, the range of doses of two relatively new antiprogestins, ZK 137 316 and ZK 230 211, that would block progesterone action directly on the endometrium in artificially cycled, spayed rhesus macaques; second, the direct endometrial effects of ZK 230 211, a type III antiprogestin; and third, investigation of whether endometrial-suppressive doses administered chronically to intact, cycling monkeys could be used for reversible, menstrual suppression. RESULTS: The results in naturally cycling animals showed that ZK 137 316 blocked menstruation in all animals, but doses of 0.05 mg/kg blocked ovulation in 55.5% of animals and doses of 0.1 mg/kg blocked ovulation in 66.6% of the animals. However, all doses of ZK 230 211 that blocked menstruation also blocked ovulation. All progesterone antagonist (PA)-treated animals, regardless of dose, maintained normal follicular phase concentrations of oestradiol and returned to normal menstrual cyclicity within 15--41 days post-treatment.Therefore ZK 137 316, depending on dose, can allow ovulation but block menstruation, while ZK 230 211, a much more potent PA, blocks both ovulation and menstruation at all effective doses. Both PAs block unopposed oestrogenic action on the endometrium through their antiproliferative effects. CONCLUSIONS: Reversible amenorrhoea can be achieved with these two PAs, and they can protect the endometrium from the effects of unopposed oestrogen whether or not ovulation is blocked. Chronic, low dose PA treatment may provide a new option for women who wish to suppress their menstrual periods.     

Low-dose antiprogestin treatment prevents pregnancy in rhesus monkeys and is reversible after 1 year of treatment

BACKGROUND: Administration of low doses of an antiprogestin to rhesus monkeys permits ovarian/menstrual cyclicity, suppresses endometrial proliferation and prevents pregnancy without adverse or toxic side-effects after 5-6 months of daily treatment. The purpose of this study was to test the reversibility with respect to restoration of fertility after 1 year of low-dose antiprogestin treatment. METHODS: This experiment included a daily 1 year vehicle- or antiprogestin-treatment interval followed by a 9 month post-treatment interval for adult, female rhesus monkeys (n = 5/group) of proven fertility and exhibiting regular menstrual cycles. Co-habitation occurred with a male of proven fertility and vaginal swabs were taken to identify the presence of sperm during the treatment (antiprogestin females) and post-treatment intervals (vehicle and antiprogestin females). RESULTS: Mating and vaginal sperm were evident in all antiprogestin females during, and, in both groups, after treatment. Based on ultrasonography, none of the antiprogestin-treated females became pregnant during the treatment interval. However, upon cessation of treatment, pregnancy rates were similar between antiprogestin-treated (3/5) relative to vehicle-treated (4/5) females with live, healthy infants born in both groups. There were no differences between groups in fetal measurements, gestation lengths, live birth rates and infant weights. CONCLUSIONS: The reversal of the anti-fertility effects of chronic, low-dose antiprogestin treatment supports the clinical feasibility of potent and selective antiprogestins as potential contraceptives for women.     

Effect of low daily doses of mifepristone on ovarian function and endometrial development

The effects of low daily doses of the antiprogestin mifepristone (RU 486) on ovarian and endometrial function were studied. The study included one control cycle, three treatment cycles and one follow-up cycle. During the treatment cycles, either 0.1 (n = 5) or 0.5 (n = 5) mg of mifepristone was administered once daily. Urine samples were collected three times weekly during the control and treatment cycles and pregnanediol glucuronide and oestrone glucuronide and luteinizing hormone (LH) were quantified by radioimmunoassay. Blood samples for cortisol measurement were collected once weekly and for serum glycodelin at the onset of menstruation. An endometrial biopsy was obtained in the mid-luteal phase in the control cycle and in the first and third treatment cycles and analysed by morphometric and histochemical methods. Binding of Dolichus biflorus agglutinin (DBA) lectin was measured and expression of progesterone and oestrogen receptors and glycodelin were analysed immunohistochemically. All cycles studied were ovulatory with an LH peak and elevated pregnanediol glucuronide concentrations. Follicular development seemed normal as judged by ultrasound examination. The length of the menstrual cycle and the menstrual bleeding were not significantly altered. Following administration of 0.5 mg mifepristone/day, endometrial development appeared to be slightly retarded and glandular diameter was significantly reduced. Furthermore, significant decreases in DBA lectin binding and endometrial expression of glycodelin were observed. Daily doses of 0.1 mg did not have any significant effect on the endometrium. No differences in oestrogen or progesterone receptor immunoactivity between control and treatment cycles were seen. This study provides further evidence that endometrial function is sensitive even to doses of antiprogestin that are low enough not to disturb ovulation. It remains to be established whether these effects are sufficient to prevent implantation.      

Respiratory mechanics in COPD patients who failed non-invasive ventilation: Role of intrinsic PEEP

The purpose was to determine if 2 weeks of buspirone suppressed post-hypoxic breathing instability and pauses in the C57BL/6J (B6) mouse. Study groups were vehicle (saline, n=8), low-dose (1.5 mg/kg, n=8), and high-dose buspirone (5.0 mg/kg, n=8). Frequency, measured by plethysmography, was the major metric, and a pause defined by breathing cessation >2.5 times the average frequency. Mice were tested after 16 days of ip injections of vehicle or drug. On day 17, 4 mice in each group were tested after buspirone and the 5-HT(1A) receptor antagonist, 4-iodo-N-{2-[4-(methoxyphenyl)-1-piperazinyl] ethyl}-N-2-pyridinyl-benzamide (p-MPPI, 5 mg/kg). A post-hypoxic pause was present in 6/8 animals given vehicle and 1/16 animals given buspirone at either dose, but always present (8/8) with p-MPPI, regardless of buspirone dose. Post-hypoxic frequency decline was blunted by buspirone (-10% vehicle vs. -5% at both doses) and restored by p-MPPI; ventilatory stability as described by the coefficient of variation which was reduced by buspirone (p<0.04) was increased by p-MPPI (0.01). In conclusion, buspirone administration after 2 weeks acts through the 5-HT(1A) receptor to reduce post-hypoxic ventilatory instability in the B6 strain.     

Effects of luteal phase administration of mifepristone (RU486) and prostaglandin analogue or inhibitor on endometrium in the rhesus monkey

Early luteal phase administration of a potent anti-progestin like mifepristone (RU486) inhibits blastocyst implantation and the establishment of pregnancy without marked changes in menstrual cyclicity and ovarian steroid hormone profiles; however, the underlying mechanism is not very clear. In the present study, a hypothesis that prostaglandins (PG) are involved in the anti-gestatory action of luteal phase mifepristone was tested. Endometrial changes in rhesus monkeys were examined following luteal phase administration of mifepristone, a prostaglandin synthesis inhibitor (diclofenac) and a prostaglandin analogue (misoprostol) either alone or in combination. Twenty-five monkeys were randomly assigned to six groups: group 1 (n = 4), normal control group; group 2 (n = 4), mifepristone (2 mg, daily, s.c.) treated group; group 3 (n = 4), diclofenac (25 mg, daily, i.m.) treated group; group 4 (n = 4), misoprostol (100 microg, daily, oral) treated group; group 5 (n = 5), mifepristone and diclofenac (same dosages as for groups 2 and 3) treated group; group 6 (n = 4), mifepristone and misoprostol (same dosages as for groups 2 and 4) treated group. All treatments were given to monkeys on days 16-18 of mated cycles and endometrial tissue samples were collected on day 20. With diclofenac alone (group 3), marginal changes were observed in glandular, stromal and vascular compartments, and there were few apoptotic bodies in gland cells; partial inhibition and delay in implantation was earlier reported. Significantly higher oestrogen receptor expression in glandular epithelial cells as compared with all other treatment groups was found after treatment with misoprostol alone (group 4) and was associated with normal fecundity. The anti-nidatory action of luteal phase antiprogestin treatment alone or in combination with diclofenac or misoprostol was associated with altered endometrial histometric features characterized by glandular apoptosis, regression in secretory functions, decreased oedema, extravasation and a higher degree of stromal leukocytic infiltration. In these three groups (groups 2, 5 and 6) receptors for oestrogen and progesterone receptors were significantly higher in stromal cells, and lower in vascular cells, while glandular cells showed significantly higher progesterone receptors compared with the control group. The anti-nidatory activity of mifepristone and associated endometrial changes could not be accentuated or attenuated with co-administration of PGE or diclofenac, nor could these be mimicked by these agents alone.      

Effects of ciclesonide and fluticasone propionate on allergen-induced airway inflammation and remodeling features

BACKGROUND: Several topical corticosteroids are available as anti-inflammatory treatment for asthma. Their comparative effects on allergic inflammation and airway remodeling are unclear. OBJECTIVE: We compared the effects of ciclesonide with those of fluticasone propionate in a Brown Norway rat model of chronic allergic asthma. METHODS: Rats sensitized and exposed to ovalbumin (OVA) were treated with dry powder vehicle, ciclesonide, or fluticasone (0.01, 0.03, and 0.1 mg/kg administered intratracheally) 24 hours and 1 hour before each of 6 OVA exposures. In a second protocol we administered 0.1 mg/kg ciclesonide or fluticasone only after the third OVA exposure. RESULTS: Ciclesonide at all doses inhibited the allergen-induced increase in airway eosinophils and T cells, reduced goblet cell hyperplasia, and decreased 5-bromo-2'-deoxyuridine-immunoreactive airway smooth muscle (ASM) and epithelial cells. At 0.03 and 0.1 mg/kg ciclesonide, bronchial hyperresponsiveness (BHR) was also inhibited. Fluticasone did not attenuate allergen-induced BHR, despite inhibiting airway eosinophils and T cells, goblet cell hyperplasia, and 5-bromo-2'-deoxyuridine-immunoreactive ASM and epithelial cells. Fluticasone (0.1 mg/kg) caused a significant reduction in body weight (9%) compared with ciclesonide (0.1 mg/kg). Ciclesonide did not change plasma corticosterone levels, whereas fluticasone (0.1 mg/kg) reduced them. In the second protocol both fluticasone and ciclesonide inhibited BHR, bronchial inflammation, goblet cell hyperplasia, and ASM proliferation. CONCLUSION: Ciclesonide potently inhibited chronic allergic inflammation, remodeling, and BHR without having an effect on body weight and the hypothalamic-pituitary-adrenal axis. Fluticasone prevented airway inflammation but not BHR, but both fluticasone and ciclesonide are effective at reversal of BHR, inflammation, and remodeling features.     

Effect of an Na+/H+ exchange inhibitor,SM-20550, on ischemic preconditioning in rabbits

The effects of SM-20550 [N-(aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonic acid], an Na+/H+ exchange inhibitor, on ischemic preconditioning (IPC) were studied in a rabbit model of myocardial ischemia and reperfusion injury. Anesthetized rabbits underwent occlusion of the coronary artery (30 min) followed by reperfusion (5 h). In SM-20550-treated animals, SM-20550 was intravenously administered at 0.03 mg/kg or 0.1 mg/kg before ischemia (30 min). Treatment with SM-20550 at 0.03 mg/kg had a nonsignificant tendency to reduce infarct size (18%). In contrast, 0.1 mg/kg of SM-20550 significantly reduced infarct size by 62%. In animals with IPC, the condition was induced by 2 or 5 min of ischemia and 10 min of reperfusion prior to sustained ischemia (30 min). Although 5 min of IPC significantly reduced infarct size by 72%, 2 min of IPC reduced infarct size by only 27%, which was not significant. The combination of 5 min of IPC and 0.1 mg/kg of SM-20550 significantly reduced infarct size by 78%. This reduction in infarct size was similar to that produced by 0.1 mg/kg SM-20550 or 5 min of IPC alone. Moreover, the combination of 2 min of IPC and 0.03 mg/kg of SM-20550 significantly reduced infarct size by 64%, although neither 0.03 mg/kg SM-20550 nor 2 min of IPC alone reduced infarct size significantly. These results indicate that an Na+/H+ exchange inhibitor SM-20550, does not antagonize the cardioprotective effect of IPC. SM-20550 and IPC appeared to act synergistically to exert a combined cardioprotective effect.     

Peptide leukotriene involvement in pulmonary eosinophil migration upon antigen challenge in the actively sensitized guinea pig.

Male Dunkin Hartley guinea pigs, actively sensitized to ovalbumin (OA) and pretreated with mepyramine (1 mg/kg i.p.), were challenged with OA as an aerosol. Histological analysis of the lung for eosinophils (EOs) showed significant accumulations in the submucosa of the airways (8 h: 1.477.3 +/- 43 EOs/mm2 airway, n = 3, p less than 0.01), after antigen challenge compared to saline control (478.6 +/- 104 EOs/mm2 airway, n = 4). Pretreatment with both mepyramine and cimetidine (10 mg/kg i.p.) did not affect this response. Aerosolization of OA to unsensitized guinea pigs resulted in no significant accumulation of EOs (360.2 +/- 49 EOs/mm2 airway, n = 4) when compared with the saline-challenged sensitized control group. Pretreatment with the leukotriene (LT)D4 receptor antagonist MK-571 (1 mg/kg p.o.) significantly inhibited the OA-induced EO migration (95 +/- 5% inhibition, n = 5, p less than 0.01) compared to vehicle in the presence of mepyramine and cimetidine, while indomethacin (3 mg/kg p.o., n = 4) had no effect. Aerosolization of synthetic LTC4 (0.3-30 micrograms/ml) resulted in a dose-dependent migration of EOs into the submucosal layer over 8 h, reaching significance at the 10-micrograms/ml dose, with comparable results obtained with LTD4. Pretreatment of animals with MK-571 (1 mg/kg p.o.) before LTC4 and LTD4 aerosol results in inhibition of the response in both cases, suggesting that this effect may be mediated through the LTD4 receptor. We conclude that peptide LTs produce eosinophilia in the airways of the guinea pig.     

Anti-nidatory effect of a single, early post-ovulatory administration of mifepristone (RU 486) in the rhesus monkey

The hypothesis that post-coital administration of mifepristone(RU 486) as a single dose in the early luteal phase can be aneffective anti-nidatory strategy was tested using the rhesusmonkey as the experimental model. Incidence of pregnancy, vaginalbleeding patterns, profiles of menstrual cyclicity and of serumlevels of progesterone and oestrogen were examined followingadministration of RU 486 as a single dose of 10 mg/kg and 2mg/kg body weight on the second day after ovulation. In controlmonkeys (group 1; n = 5) receiving the vehicle alone (benzylbenzoate: olive oil, 1: 4, v/v) there was a 60% pregnancy rate.Following s.c. administration of RU 486 at both doses, no pregnancywas recorded in a total of 33 treatment cycles in 12 monkeys.Five monkeys received RU 486 at 10 mg/kg s.c. (group 2) in threeconsecutive cycles. All animals had complete inhibition of implantation;in addition, the treatment cycle length was prolonged (P <0.001) due to an extension of the luteal phase. The subsequentfollicular phase was unaffected. Mild, premature vaginal bleedingduring the luteal phase was recorded in five treatment cycles,3–5 days after drug application. Though the serum profilesof progesterone and oestrogen in these monkeys showed markedindividual variations, there was a characteristic progesteronerebound about 18–20 days after drug administration. Monkeysin group 3 were given RU 486 at 2 mg/kg, s.c. either for threeconsecutive cycles (group 3a; n = 4) or for two consecutivecycles (group 3b; n = 3). Premature luteal phase vaginal bleedingoccurred only in four treatment cycles, within 2–6 dayspost-treatment. An increase in both the duration (P < 0.001)and degree (P < 0.001) of menstrual flow as compared withthe pre-treatment cycles was recorded in six treatment cyclesof three monkeys in group 3. These animals did not have prematureluteal phase vaginal bleeding. Collectively, 100% protectionagainst pregnancy with no change in the cycle length was obtainedin all seven monkeys in 18 treatment cycles. Analysis of pooleddata revealed that the subsequent follicular phase, as wellas the ovarian steroid hormone profiles of treatment cycleswere unaffected. Thus, a single application of RU 486 in theearly secretory phase appears to be a potential anti-implantationstrategy for intercepting pregnancy in the primate.     

The role of low progesterone and tension as triggers of perimenstrual chocolate and sweets craving: some negative experimental evidence.

Approximately half of the 40-50% of North American women who crave chocolate or sweets do so principally in the perimenstrum, the part of the menstrual cycle surrounding the onset of menstruation. We test two hypotheses about the events that trigger these cravings: 1) the premenstrual drop in progesterone levels; or 2) dysphoria or tension in the perimenstruum. Chocolate craving, sweets craving, and other perimenstrual symptoms were rated daily for six menstrual cycles by a sample of women with severe premenstrual syndrome (PMS). Forty-four women satisfied criteria for cyclicity in chocolate craving, and 44 for sweet craving, determined during the first two cycles. Thirty-four subjects satisfied criteria for craving of both chocolate and sweets. After placebo treatments during the third cycle, subjects were randomly assigned, double blind, to administration of placebo, oral micronized progesterone, or alprazolam (a tranquillizer). Treatments were administered from the beginning of the third week to the second day postonset of menstruation during the fourth to sixth months of study. Neither progesterone nor alprazolam decreased chocolate or sweets craving.     

Discovery of LH-regulated genes in the primate corpus luteum

Circulating LH is essential for the development and function of the primate corpus luteum (CL) during the menstrual cycle. However, the cellular and molecular processes whereby LH controls luteal structure and function are poorly understood. Therefore, studies were initiated to identify gene products that are regulated by gonadotrophin in the monkey CL. Rhesus monkeys either were untreated (controls, CTRL; n = 3) or received the GnRH antagonist Antide (ANT; 3 mg/kg body weight, n = 3) to inhibit pituitary LH secretion on day 6 of the luteal phase in spontaneous menstrual cycles. The CL was removed 24 h later. RNA was extracted and converted to cDNA. The CTRL and ANT cDNA were differentially labelled with fluorescent dyes (Cy3-CTRL and Cy5-ANT) and hybridized onto microarrays containing 11,600 human cDNA. The selected cDNA were analysed further via semi-quantitative RT-PCR (a) to validate the microarray results and (b) to determine if their expression varies in the CL (n = 3/stage) between the mid (day 6-8), late (day 14-16), or very late (day 18-19, menses) luteal phase of the natural cycle. After normalization of the fluorescence data, 206 cDNA (1.8% of the total) exhibited > or = 2-fold change in expression after ANT. Of the 25 cDNA exhibiting a > or = 6-fold change, 6 were up-regulated and 19 were down-regulated. Twenty-two of these 25 cDNA were validated by RT-PCR as differentially expressed in the ANT group, relative to the CTRL group, and 11 of 25 changed (P < 0.05) correspondingly in the late-to-very late luteal phase. Thus, we have identified gene products that are regulated by gonadotrophin in the primate CL that may be important in luteal regression during the menstrual cycle.     

Termination of early pregnancy with ZK 98, 734: pharmacokinetic behaviour and clinical effect

The antiprogestin RU 486 (mifepristone) is highly effectivein inducing early abortion in women only if the compound iscombined with a prostaglandin analogue. A new related antiprogestin,ZK 98, 734, has been reported in animal studies to be much morepotent as an abortifacient than mifepristone, concomitant withless antiglucocorticoid activity. The aim of the present two-centrestudy was to explore the abortifacient efficacy and plasma concentrationsof ZK 98, 734 in women seeking abortion. A total of 96 pregnantwomen with amenorrhoea of <49 days were treated with oraldoses of 12.5, 25, 50 or 100 mg ZK 98, 734 twice daily for 4days. The overall rate of complete abortion and continuing livepregnancies was 68 and 20% respectively, i.e. results comparablewith treatment with mifepristone alone. No dose-response relationshipwas noted. In patients with complete abortion, signs of lutealsysfunction in terms of oestradiol and progesterone productionwere evident on the fourth treatment day, in contrast to patientswith failures. Increased amounts of cortisol and prolactin werefound during treatment both in successfully treated patientsand failures, whereas aldosterone values remained unaffected.The effect on cortisol may indicate some antiglucocorticoidactivity in the human. The concentrations of ZK 98, 734 in peripheralblood after 25, 50 and 100 mg twice daily for 4 days were similar.The values were slightly above 0.5µmol/l on the secondday of treatment. Maximal concentrations of 0.7 µmol/lwere seen on treatment day 4. Plasma concentrations of ZK 98,734 did not differ in cases of complete abortion and failures.In conclusion, the abortifacient efficacy in relation to doseof the new antigestaged ZK 98, 734 does not essentially differfrom that of mifepristone treatment alone.     

The role of pre-ovulatory progesterone in the midcycle gonadotrophin surge, ovulation and subsequent luteal phase: studies with RU486 in rhesus monkeys

Conflicting evidence exists on the possible physiological roleof progesterone in the regulation of the midcycle surge of gonadotrophinsduring the normal primate menstrual cycle. We designed the presentstudy based on the availability of a potent antiprogesterone,RU486, that acts by binding to the progesterone receptor withoutinducing progestational activity. Regularly cycling rhesus monkeysreceived daily administration of RU486,10 mg orally (n = 8)or vehicle (n = 5) from the day of the menstrual cycle in whichserum oestradiol was 130 pg/ml or more, and a laparoscopy revealedthe presence of a dominant follicle. While vehicle administrationdid not affect the normal ovulatory pattern nor the hormonalmilieu of the menstrual cycles, RU486 induced marked aberrationsduring the treated cycles. Delay of ovulation with a normalsubsequent luteal phase was observed in three animals. Threeanimals remained anovulatory until the following cycle and twoanimals that ovulated on days 14 and 16 of the treated cycleshad short luteal phases. Analysis of daily FSH, LH, oestradioland progesterone revealed that the administration of RU486 disruptedthe midcycle pattern of gonadotrophins by disrupting them afterthe surge was initiated. Oestradiol surges were not differentfrom controls and in all animals the ascendant levels of progesteronewere interrupted by the administration of the antiprogesterone.This study clearly shows that the pre-ovulatory administrationof RU486, a potent antiprogesterone, alters pre-ovulatory gonadotrophinsecretion, inducing different degrees of menstrual irregularitiessuch as anovulation, delayed ovulations and short luteal phases.Based on these results it is possible to speculate that progesteroneexerts a facilitatory effect (positive feed-back) on the midcyclegonadotrophin peak that induces ovulation in primates.     

Analysis of gene mutations and clastogenicity following short-term treatment with azathioprine in MutaMouse.

The mutagenicity and clastogenicity of the immunosuppressive drug azathioprine (AZA), a multitissue rodent carcinogen and IARC-classified human carcinogen, was investigated using transgenic lacZ mice (MutaMouse). Male animals (n = 5 per group) were dosed with AZA (10, 50, 100 mg/kg p.o. daily for 5 days), vehicle (n = 10), or the positive control, chlorambucil (15 mg/kg i.p., n = 3), and killed 24 hr or 25 days after the last treatment. Micronucleus assays were performed with bone marrow (24-hr samples) or peripheral blood (24-hr and 25-day samples) and DNA was extracted from bone marrow and liver for gene mutation analysis at the transgenic lacZ locus. AZA induced 5.3-111.3-fold increases in %MNPCE (P < 0.01) in bone marrow compared with vehicle control, accompanied by 4.4-5. 6-fold increases in %MNRETs (P < 0.01) in peripheral blood. Chlorambucil caused a 14.5-fold increase in %MNRET and there was evidence of significant stem cell toxicity in both positive control and AZA treatment groups. By day 25, however, there was evidence of substantial recovery of the bone marrow as determined by the frequency of RET, and the %MNRET in all treatment groups was the same as the vehicle control. Analysis of lacZ MF showed 1.4-1.6-fold increases in AZA 24-hr bone marrow samples, increasing to approximately 2.0-fold above concurrent controls by day 25 (medium dose P < 0.05, high dose P < 0.01). For liver, there was a 2-fold increase in MF (P < 0.05) in the 24-hr sample at the highest dose only, and increases of 1.3-1.5-fold by day 25 in the medium (P < 0. 05) and high (P = 0.055) dose groups, respectively. The positive control, chlorambucil, induced 2-3-fold increases (P < 0.01) in mean MF in both bone marrow (25-day sample) and liver (24-hr and 25-day samples). These data confirm the clastogenicity of AZA in the mouse, and show that this compound induces gene mutations in bone marrow and liver, in vivo, at the highest dose and supports the view that AZA is a genotoxic carcinogen.     

A single mid-follicular dose of CDB-2914, a new antiprogestin, inhibits folliculogenesis and endometrial differentiation in normally cycling women

Previous studies in women have shown that the antiprogestin mifepristone delays or inhibits folliculogenesis. The purpose of this study was to explore whether a new analogue, CDB-2914, has similar effects on folliculogenesis, ovulation, or on subsequent luteal phase endometrial maturation. Forty-four normally cycling, healthy women recorded urine LH and vaginal bleeding during pre-treatment, treatment, and post-treatment cycles. At a lead follicle diameter of 14-16 mm, a single oral dose (10, 50, 100 mg) of CDB-2914 or placebo was given, and daily ultrasound, oestradiol and progesterone were obtained until follicular collapse; an endometrial biopsy was obtained 5-7 days later. Single doses of CDB-2914 were well tolerated. Mid-follicular CDB-2914 suppressed lead follicle growth, causing a dose-dependent delay in folliculogenesis and suppression of plasma oestradiol. At higher doses, a new lead follicle was often recruited. Although luteinized unruptured follicles were observed at the 100 mg dose, all women had follicular collapse. There was a significant delay in endometrial maturation after CDB-2914 at all doses. The treatment cycle was lengthened by 1-2 weeks in 30% at 100, 27% at 50 and 9% at 10 mg. CDB-2914 altered ovarian and endometrial physiology without major effects on menstrual cyclicity and may have therapeutic utility.     

Effects of bisphosphonates on joint damage and bone loss in rat adjuvant-induced arthritis

Objective and design: Examination of the effects of bisphosphonates on joint damage and generalized bone loss.Materials: Adjuvant-arthritis was induced by injection of Mycobacterium butyricum into the footpad of the right hind paw of Lewis rats (8 animals/group) on day 0.Treatment: Arthritic rats were treated with the vehicle (saline), etidronate or alendronate (subcutaneously, daily 5 times a week for 3 weeks from day 1 to day 21). Experiment-1: Etidronate (0.1, 0.5, 2.5, 12.5 mg/kg) or alendronate (0.02, 0.1, 0.5, 2.5 mg/kg), Experiment-2: Etidronate (2.5, 5, 10mg/ kg) or alendronate (0.001, 0.01, 0.1 mg/kg).Methods: In the adjuvant-injected side of the hind limbs, paw swelling was evaluated at 1-week intervals, and bone mineral density (BMD) in the proximal tibia, histopathology and radiographical findings in the tibio-tarsal region were evaluated at the time of sacrifice (on day 21).Results: In all treatment schedules, both bisphosphonates significantly prevented paw swelling and bone loss. Alendronate reduced paw swelling at higher doses (over 0.1 mg/ kg) compared with its effect on BMD decrease (over 0.001 mg/kg). In contrast, etidronate reduced paw swelling and joint damage at doses similar to those (over 2.5 mg/kg) prevented BMD decrease.Conclusions: Both etidronate and alendronate are effective in reducing arthritic damage, but their effective dose ranges for inflammatory responses and BMD decrease clearly differ; i.e., the etidronate dose ranges for anti-inflammatory and anti-resorptive effects are similar, whereas the dose range for anti-inflammatory effects of alendronate is 100-fold higher than that for its anti-resorptive effects.Received 21 January 2003; returned for revision 14 July 2003; accepted by M. Katori 15 August 2003     

RDP58, a locally active TNF inhibitor, is effective in the dextran sulphate mouse model of chronic colitis

OBJECTIVE AND DESIGN: RDP58 is a novel anti-inflammatory peptide that inhibits TNF synthesis and upregulates heme oxygenase-1. RDP58 therapy was evaluated in the dextran sodium sulphate (DSS) model of chronic colitis. MATERIAL: Colitis was induced by giving DSS to mice (n = 8 animals/group). Toxicity studies were done in Rhesus monkeys (n = 5), dogs (n = 3) and mice (n = 10). TREATMENT: In colitis, mice were treated with p.o. vehicle (saline), RDP58 (5 and 10 mg/kg/day) or 5-ASA (50 mg/kg/day). METHODS: Disease activity index (DAI) was used as the endpoint of efficacy. RESULTS: RDP58 therapy significantly reduced DAI and histological scores in all animals. DAI scores in RDP58 treated animals declined faster than 5-ASA. RDP58 at 5 or 10 mg/ kg/day significantly reduced DAI compared to 5-ASA. RDP58 significantly reduced acute, chronic and total inflammation scores. It enhanced re-epithelialization by reducing crypt scores. RDP58 was not bioavailable and was well tolerated. CONCLUSIONS: Therapeutic efficacy of RDP58 combined with a lack of bioavailibility and toxicity suggest that RDP58 may be a promising new therapeutic for IBD.     

牛磺酸联合安定治疗脑缺血再灌注损伤的实验研究

目的:研究牛磺酸联合安定对实验性脑缺血再灌注损伤大鼠的神经保护作用。方法:SD雄性大鼠随机分为假手术组、脑缺血再灌注损伤组、牛磺酸治疗组(200mg·kg-1)、安定治疗组(10mg·kg-1)、联合治疗组(牛磺酸100mg·kg-1+安定5mg·kg-1),每组12只。采用大脑中动脉栓塞法(MCAO)建立大鼠局灶性脑缺血模型,2h后拔出栓线形成再灌注,再灌注时各组分别给药,脑缺血再灌注损伤组注射等剂量的生理盐水,12h后各组重复注射1次。另分批实验同样5组动物,每组16只,分别于再灌注后10h给药,12h后重复治疗1次。各组中12只动物同先前5组于再灌注后48h观测神经行为学评分、脑梗死体积以及脑含水量测定。每组中余下4只大鼠,2周后行尼氏染色观察脑组织病理学改变。结果:与脑缺血再灌注损伤组相比,缺血后2h、12h联合治疗均能显著降低大鼠神经行为学评分、减少脑含水量、缩小脑梗死体积,同时能明显减轻海马神经元变性坏死(P0.01或P0.05),且其保护作用优于牛磺酸或安定单用组。结论:缺血性脑损害所致急、慢性损伤时牛磺酸联合安定具有明显的神经保护作用。     

Activation of mitochondrial ATP-sensitive potassium channels prevents neuronal cell death after ischemia in neonatal rats

Activation of mitochondrial ATP-sensitive potassium channels (mK(ATP)) has been shown to protect against cell death following ischemia/reperfusion in the heart but not in brain. We examined whether mK(ATP) activation with diazoxide (DIZ) prevents neuronal cell death following hypoxia-ischemia (HI) in 7-day-old rat pups. Rat pups were subjected to HI (left carotid ligation; 8% O(2); 2.5 h), following administration of vehicle, 1.9 mg/kg DIZ, 3.8 mg/kg DIZ or DIZ plus 10 mg/kg 5-hydroxydecanoic acid (mK(ATP) antagonist). Total infarct volume was reduced from 99.8+/-2.7% in vehicle animals to 80.6+/-4.2% in 3.8 mg/kg DIZ treated animals (n=85, P<0.05). Western blotting showed K(ATP) subunits concentrated in mitochondria. Fluorescent studies indicated DIZ directly depolarized the mitochondria. In conclusion, selective opening of mK(ATP) prior to HI results in neuroprotection in immature rats.