Safety, pharmacokinetics and pharmacodynamics of multiple-ascending doses of the novel glucokinase activator AZD1656 in patients with type 2 diabetes mellitus.

AIMS: To assess the safety, pharmacokinetics and pharmacodynamics of multiple-ascending doses of the novel glucokinase activator AZD1656 in patients with type 2 diabetes mellitus (T2DM). METHODS: This randomized, single-blind, placebo-controlled, monotherapy study was carried out in two parts. In part A, 32 patients received AZD1656 (7, 20, 40 or 80 mg) twice daily or placebo for 8 days in hospital. In part B, another 20 patients received, as outpatients, individually titrated AZD1656 15-45 mg twice daily or placebo for 28 days. Safety, pharmacokinetics and pharmacodynamic variables were evaluated. RESULTS: AZD1656 was generally well tolerated. Pharmacokinetics of AZD1656 were virtually dose- and time-independent. AZD1656 was rapidly absorbed and eliminated. An active metabolite was formed which had a longer half-life than AZD1656, but showed ∼15% of the area under the plasma concentration versus time curve from 0 to 24 h compared with that of AZD1656. Renal excretion of AZD1656 and the metabolite was low. In part A, fasting plasma glucose (FPG) was reduced by up to 21% and mean 24-h plasma glucose was reduced by up to 24% with AZD1656 versus placebo, depending on dose. No dose-related changes in serum insulin or C-peptide were observed with AZD1656 at the end of treatment. Results in part B confirmed the glucose-lowering effect of AZD1656 versus placebo. CONCLUSIONS: AZD1656 was well tolerated with predictable pharmacokinetics in patients with T2DM. Dose-dependent reductions in plasma glucose were observed.

Higher than expected estradiol levels in aromatase inhibitor-treated, postmenopausal breast cancer patients.

OBJECTIVE: Vaginal estradiol is considered contraindicated in aromatase inhibitor (AI)-treated patients because of the risk of elevated estrogen levels. This leaves limited treatment options for patients experiencing gynecological symptoms. However, in clinical practice, no precise estimation has been performed of circulating estrogens and aromatase index in postmenopausal breast cancer patients on long-lasting AI or tamoxifen treatment. METHODS: Steroid hormones were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) and extraction radioimmunoassay (RIA). Postmenopausal AI-treated patients (n =33) were compared with tamoxifen-treated patients (n =34) and controls without vaginal treatment (n =56), with vaginal estradiol (n =25), or with estriol (n =11) treatment. RESULTS: By use of LC-MS/MS, median (range) estradiol plasma concentrations were 16.7 (2.4-162.6), 31.0 (13.4-77.1), 27.2 (7.8-115.8) and 33.3 (20.3-340.1) pmol/l in AI-treated breast cancer patients, tamoxifen-treated breast cancer patients, postmenopausal controls and postmenopausal controls on vaginal estradiol, respectively. The AI-treated group and subgroups had significantly lower estradiol and estrone concentrations than all other groups (p <0.05). There was extensive interindividual variation in estradiol concentration within the AI-treated group, measured using both LC-MS/MS (2.3-182.0 pmol/l) and extraction RIA (2.4-162.6 pmol/l). The AI-treated group had lower aromatase index compared to all other groups (p <0.05-0.001). CONCLUSION: Circulating estrogen levels may have been underestimated in previous longitudinal studies of AI-treated breast cancer patients. Additional studies are required to further evaluate the role of circulating estrogens in breast cancer patients suffering from gynecological symptoms.

Multiple pituitary hormone deficiency: management of puberty for optimal auxological results.

The overview in this paper focuses on ways of achieving optimal auxological results in puberty, principally in idiopathic and congenital multiple pituitary hormone deficiency (MPHD), suggested by the co-authors. We agreed that diagnosing gonadotrophin insufficiency/deficiency is difficult in young children and should be repeated in late prepuberty, but a firm diagnosis of MPHD helps avoid endocrine re-testing at the end of growth. The hypothalamic-pituitary axis must be reassessed periodically in evolving endocrinopathies, though current practice varies widely. Optimum age to induce puberty is 11-12 years in girls and 13-14 boys, and sex steroids are the preferred agents. Short-course testosterone to increase micropenis size is advantageous, but inducing early testicular maturation is not known to improve later fertility. There is also little evidence for increasing the dose of GH during puberty, though therapy should continue to final height, and possibly until peak bone mass is achieved. Delaying puberty is an option in septo-optic dysplasia, and minimising the dose of hydrocortisone is crucial in treating ACTH/cortisol insufficiency. Many unresolved questions remain in this difficult area.

Nocturnal application of transdermal estradiol patches produces levels of estradiol that mimic those seen at the onset of spontaneous puberty in girls.

The objective of pubertal induction in children with hypogonadism is to mimic spontaneous puberty in terms of physical and psychological development. In a clinical observation study, we induced puberty in 15 girls with hyper- or hypogonadotropic hypogonadism using low doses of transdermal estradiol patches attached only during the night and compared the estradiol concentrations obtained with those in healthy girls. Pubertal induction was started between the ages of 12.3 and 18.1 yr. A transdermal matrix patch of 17beta-estradiol (25 microg/24 h; Evorel, Janssen Pharmaceuticals-Cilag) was cut into pieces corresponding to 3.1, 4.2, or 6.2 microg/24 h initially and attached to the buttock. After 4-14 months, the dose was increased gradually. Serum 17beta-estradiol concentrations were measured every 2 h by RIA (detection limit, 6.0 pmol/L; 1.6 pg/mL). The results show that it is possible to mimic the spontaneous levels as well as the diurnal pattern of serum 17beta-estradiol in early puberty, by cutting a transdermal 17beta-estradiol matrix patch and attaching a part of it, corresponding to 0.08-0.12 microg estradiol/kg BW, to the buttock nocturnally. In most of the girls, breast development occurred within 3-6 months of the start of treatment.

Leptin levels show diurnal variation throughout puberty in healthy children, and follow a gender-specific pattern.

OBJECTIVE: To investigate the levels and diurnal rhythm of serum leptin in healthy children, and to investigate the association between leptin levels and sex steroids. METHODS: Four girls and four boys, all healthy volunteers, were followed longitudinally throughout puberty. Their chronological ages ranged from 8.7 to 19.5 years, and body composition, expressed as weight-for-height standard deviation scores (SDS), ranged between -1.7 and +2.4. Serum leptin, oestradiol and testosterone concentrations were measured by radioimmunoassay at 1000, 1400, 1800, 2200, 0200 and 0600 h. RESULTS: In all girls and boys, both prepubertally and during pubertal development, serum leptin levels increased during the night, with no difference in relative peak amplitude. In boys, the leptin concentrations increased until the initiation of puberty and then declined, whereas in girls, the concentrations increased throughout puberty. The inter-individual variation in mean leptin levels among girls decreased to 11% at the time of menarche. A positive correlation was found for both oestradiol and testosterone versus leptin in girls throughout puberty (r=0.64 and r=0.71 respectively, P<0.001). A negative correlation was found between leptin and testosterone in boys in mid- and late puberty (r=-0.66, P<0.01). No correlation was found between oestradiol and leptin in boys or between testosterone and leptin in pre- and early pubertal boys. CONCLUSION: Serum leptin concentrations show diurnal variation throughout pubertal development in both girls and boys. The changes in leptin levels during puberty follow a gender-specific pattern, probably due to an influence of sex steroids on leptin production.

Diurnal rhythm of testosterone secretion before and throughout puberty in healthy girls: correlation with 17beta-estradiol and dehydroepiandrosterone sulfate.

The regulation of androgen synthesis during puberty in females is complicated, with changes in steroidogenic and peripheral interconversion capacity. In the present study we have investigated the diurnal rhythm of testosterone secretion in 56 healthy girls before and during puberty, up to 2 yr postmenarche. The girls' ages ranged between 4.6-16.5 yr, and their height SD scores ranged between -3.6 and +3.7. One to 5 serum profiles (seven samples per 24 h) were taken from each girl for steroid measurements, and a total of 84 serum profiles were obtained. Serum testosterone concentrations were determined using a RIA with a detection limit of 30 pmol/L. The results demonstrate that there is a diurnal rhythm of testosterone secretion during both prepuberty and puberty in girls. The pattern has its nadir in the late evening or just after midnight, with the highest levels in the morning (0600-1000 h). Serum testosterone concentrations in prepubertal girls were significantly lower than those in pubertal girls and were significantly lower in early puberty than in girls in mid- or late puberty. No differences were found in levels between girls in midpuberty or late puberty. Before puberty, serum testosterone concentrations correlated with serum dehydroepiandrosterone sulfate, consistent with the adrenals being the major source of testosterone. After the onset of puberty, a correlation between testosterone and 17beta-estradiol was seen, consistent with the ovaries being the major source of testosterone during puberty. Furthermore, the present study showed that there is a relative hyperandrogenicity in early puberty, with high levels of androgens relative to estrogens.

Management of males with 45,X/46,XY gonadal dysgenesis.

Males with the 45,X/46,XY karyotype and malformations of the external genitalia carry an increased risk of developing germ cell neoplasia of the gonads. We have studied gonadal tissue from 10 individuals, 0.3-17 years of age, with a male phenotype and either hypospadias and/or cryptorchidism. Four patients, 0.3-15 years of age, had carcinoma in situ, 1 boy had Sertoli-cell-only pattern and the remainder prepubertal histology. Gonadoblastoma or invasive carcinoma was not found. On the basis of our current knowledge we propose a strategy for management and follow-up of these boys in order to detect possible premalignant histological changes early and prevent development of a gonadal tumour.

Twenty-four-hour profiles of luteinizing hormone, follicle-stimulating hormone, testosterone, and estradiol levels: a semilongitudinal study throughout puberty in healthy boys.

To follow and correlate gonadotropin and sex steroid changes throughout puberty, 24-h profiles of LH, FSH, testosterone, and estradiol were taken on several occasions for between 2-9.5 yr in 12 healthy boys, aged 8.7-18.2 yr. Serum concentrations of LH and FSH were measured every 20 min, whereas testosterone and estradiol were measured every 2-4 h during the 24-h period. The prepubertal boys (Tanner stage 1) were subdivided into two groups: Pre 1, with a testicular volume of 1-2 mL, and Pre 2, with a testicular volume of 3 mL. Pubertal stages were classified, according to testicular volume, as early puberty (pubertal stage 2; 4-9 mL), midpuberty (pubertal stages 3-4; 10-15 mL), and late puberty (pubertal stage 5; > or = 16 mL). Mean levels of LH and FSH increased with pubertal development, although the increase in LH was greater than that in FSH. These increases were due to elevated basal levels of LH and FSH as well as to increases in the number of peaks and the peak amplitudes of LH. No diurnal rhythm was found in boys at stage Pre 1. Thereafter, a clear diurnal rhythm appeared for LH, and later in puberty, an ultradian rhythm was superimposed, as shown by time-sequence analyses. A diurnal rhythm also existed for FSH, but was much less marked than that for LH despite a clear covariation between LH and FSH, as shown from cross-correlation studies. Testosterone also showed diurnal variations from the late prepubertal stage, followed by increasing levels during both day and night in puberty. We conclude that during puberty, gonadotropin levels rise differently for LH and FSH, which may be due to the development of differences in feedback mechanisms. Despite covariation between LH and FSH, only LH showed a clear diurnal variation. In parallel, nocturnal variations in testosterone and estradiol were found. Changes in mean levels of LH, testosterone, and estradiol as well as their mean daytime and nighttime levels follow each other from the prepubertal stages to late puberty.

Diurnal rhythm of 17 beta-estradiol secretion throughout pubertal development in healthy girls: evaluation by a sensitive radioimmunoassay.

Puberty is initiated by a nocturnal rise in gonadotropin secretion, which, in boys, results in an increased nocturnal secretion of testosterone. To characterize any similar diurnal rhythm of 17 beta-estradiol in healthy girls, we determined the secretion of 17 beta-estradiol before and during puberty. The study group consisted of 45 healthy girls whose height SD scores ranged from -3.7 to +4.9 compared with Swedish growth reference values. One to 6 profiles of 17 beta-estradiol (7 samples/24 h) were obtained from each girl during puberty and from 21 of the girls before clinical signs of puberty (a total of 76 serum profiles). Serum 17 beta-estradiol concentrations were determined using a modified RIA. The detection limit for the RIA was 1.8 fmol/tube, which corresponded to a serum level of 7.8 pmol/L in extracted serum. It was considered that levels above 50 pmol/L could be determined accurately without extraction. The serum levels of 17 beta-estradiol in prepubertal girls were, in most cases, below the detection limit, except in the morning, when in 17 of the 21 prepubertal girls, serum 17 beta-estradiol levels were just above the detection limit. All girls in early puberty (Tanner breast stage 2) had measurable serum levels of 17 beta-estradiol in the morning, whereas 10 of these 15 girls had levels below the detection limit around midnight. Later in puberty (Tanner breast stages 3 and 4), but before menarche, the diurnal rhythm was more obvious, with high levels of 17 beta-estradiol during the latter part of the night and in the morning. This diurnal rhythm was lost by 1 yr after menarche. There was a high degree of correlation between serum concentrations of 17 beta-estradiol and bone age, whereas there was much less, if any, correlation between 17 beta-estradiol and levels of sex hormone-binding globulin or dehydroepiandrosterone sulfate during puberty. We conclude that the nocturnal rise in gonadotropin secretion during puberty in girls is accompanied by an increased secretion of 17 beta-estradiol in the morning. This diurnal rhythm is lost 1 yr after menarche. Determination of 17 beta-estradiol levels in the morning could be useful in determining the initiation of puberty, whereas determinations in the late evening could provide information on the tempo of puberty.

Studies of the luteinization process in the rat: development of catecholamine response on progesterone production during the peri-ovulatory period.

The present study was undertaken to investigate whether pre-ovulatory follicles have an adrenergic response in terms of progesterone production. Extirpated pre-ovulatory follicles obtained both before and after the endogenous gonadotropin surge and newly formed corpora lutea were obtained from the PMSG rat ovulatory model. Follicles and corpora lutea were incubated for 120 min in MEM with Earle's salt and 10 mM Hepes, 37 degrees C, pH 7.4 and 100% oxygen, with 30 microM noradrenaline or 10 micrograms ml-1 LH-B9). Pre-ovulatory follicles were barely stimulable by noradrenaline, while newly formed corpora lutea responded markedly. Luteinizing hormone (LH) levels significantly increased progesterone accumulation in all groups. In order to determine whether preovulatory follicles need intact surrounding tissue for an adrenergic response on progesterone production, pieces of ovaries containing pre-ovulatory follicles were incubated. No significant effect of noradrenaline or adrenaline was seen, while LH had a substantial effect. The results show that catecholamines acutely exert a selective effect on steroidogenesis in the ovary with a marked stimulatory effect on corpora lutea and a marginal effect on the pre-ovulatory follicle.

Redistribution of ovarian blood flow after injection of human chorionic gonadotropin and luteinizing hormone in the adult pseudopregnant rat.

It is well known that LH and human CG (hCG) induce an increase in total ovarian blood flow. The effect of LH/hCG on luteal blood flow, however, is unknown. This work studies the effect of hCG on both luteal and ovarian blood flows at different stages of pseudopregnancy in adult female rats. Pseudopregnancy was induced by mating with sterile male rats. The length of pseudopregnancy was 13 +/- 1 days and, during this time, blood flow was measured by the injection of radioactive microspheres during anesthesia. At autopsy, the corpora lutea were identified and extirpated under a stereomicroscope. These, and the remaining ovary, were then counted for radioactivity and the blood flow was calculated. Progesterone levels were determined in plasma and ovarian tissues. Furthermore, the responsiveness of adenylate cyclase was tested in ovarian tissues at day 6 of pseudopregnancy. An intraarterial injection of hCG (50 IU) or vehicle (saline) was given 20 min before the blood flow determinations in anesthetized rats. The luteal blood flow was not changed by hCG on days 2, 6, and 11 of pseudopregnancy, whereas in the remaining ovary the blood flow increased more than 2-fold, thereby resulting in redistribution of the blood flow. Ten micrograms of NIH-LH-B9, tested at day 6 of pseudopregnancy, mimicked the effect of hCG. At day 6 of pseudopregnancy, hCG (50 IU) was given ip to conscious rats 200 min and 24 h before blood flow determinations. At 200 min after hCG there was a more pronounced redistribution of ovarian blood flow with a 45% reduction in luteal blood flow and a 4-fold increase in flow through the remaining ovary. LH as well as hCG doubled the progesterone content of the remaining ovary. In the corpora lutea an increased progesterone content was seen after 200 min of hCG exposure. At 24 h after hCG injection, all parameters had returned to control levels except that adenylate cyclase was nonresponsive. The increase in the total ovarian blood flow coincides with the increased steroidogenesis and these effects are likely due to release of metabolites and/or vasoactive substances. Despite this increase, the blood flow of the corpus luteum was not increased rendering vascular mechanisms unlikely as a part of the acute LH/hCG effects on corpus luteum of pseudopregnancy.

Beta-adrenergic receptor concentration in corpora lutea of different ages obtained from pregnant mare serum gonadotropin-treated rats.

We have measured the beta-adrenergic receptor content in 1- to 10-day-old corpora lutea. Corpora lutea of defined ages were obtained by sc injection of 8 IU PMSG to 26-day-old rats, leading to ovulation and formation of corpora lutea in the early morning of day 29. Membrane fractions of isolated corpora lutea were incubated with various concentrations of the beta-adrenergic antagonist [125I]iodohydroxybenzylpindolol [125I]iodo-HYP, 12.5-2500 pM) for 60 min at 22 C. Alprenolol (10(-5) M) was used to determine nonspecific binding. Bound and free [125I]iodo-HYP was separated by filtration and washing on Whatman GF/C filters under vacuum. There was a 3-fold increase in beta-adrenergic receptor concentration during the first 2-3 days of corpus luteum formation, followed by a decline in the beta-adrenergic receptor content with luteal age. The rat luteal beta-adrenergic receptor seems to be of the beta 2-subtype as determined from adenylate cyclase stimulation as well as from displacement of [125I]iodo-HYP binding by various beta-adrenergic agonists.

Prostaglandin F2 alpha inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages.

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F2 alpha (PGF2 alpha). The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF2 alpha. A dose-dependent inhibition by PGF2 alpha (0.5-50 microM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165-165 microM). The stimulation by epinephrine on progesterone production was inhibited by PGF2 alpha (5 microM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF2 alpha can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF2 alpha shows in this respect the same age dependent inhibitory pattern as in relation to LH stimulation.

Development of catecholamine responsiveness in granulosa cells from preovulatory rat follicles--dependence on preovulatory luteinizing hormone surge.

Factors responsible for development of catecholamine (CA) responsiveness in granulosa cells (Gc) of preovulatory follicles from immature rats injected with 10 IU of pregnant mare's serum gonadotropin (PMSG) on Day 26 were studied. CA did not stimulate cyclic AMP (cAMP) production in whole follicles isolated before (morning) or after (evening) the preovulatory gonadotropin surge on Day 28, while newly formed corpora lutea found on Day 29 responded to CA. Gc from the preovulatory follicles did not respond to CA when tested immediately after isolation. Gc were cultured for various periods in Eagle's MEM without serum and subsequently tested for a possible stimulation of cAMP and steroidogenic responses by CA. In Gc from follicles isolated in the morning (AM-Gc) and cultured for 12 or 24 h, no response to CA was found, while the Gc isolated in the evening (PM-Gc) and cultured similarly for 12 h showed a marked response to CA both in stimulation of cAMP and progesterone production. By a total or partial elimination of the gonadotropin surge, using pentobarbital in combination with luteinizing hormone (LH) or using specific antisera to LH or follicle-stimulating hormone (FSH), it was found that previous exposure to LH was necessary for the PM-Gc to develop CA responsiveness during culture. Further, it was possible to induce CA responsiveness in AM-Gc by treatment of rats with LH for a short period in vivo followed by a period of culture. The appearance of CA responsiveness in PM-Gc cultured for 12 h was abolished when cycloheximide (5 micrograms/ml) was present during culture. It appears that the following three conditions have to be satisfied for the isolated Gc to develop CA responsiveness: 1) exposure to LH in vivo, 2) culture for a short period, and 3) an active protein synthesis. It is concluded that under physiological conditions the process of luteinization is associated with acquisition of responsiveness to CA and this process depends on the LH component of the preovulatory gonadotropin surge.

In vivo effect of noradrenaline on the cyclic AMP level in rat corpora lutea.

Under in vitro conditions adrenaline and noradrenaline can stimulate the production of cyclic AMP in rat corpora lutea to a similar extent as a maximal dose of LH. The present study was undertaken to compare the effects of noradrenaline and LH under in vivo conditions. Anaesthetized rats bearing 2 days old corpora lutea were either infused intraarterially with noradrenaline (0.4 microgram/min) or given a single intraarterial injection of 5 micrograms LH. The cAMP levels in whole ovaries had increased already after 30 sec of noradrenaline infusion. These levels remained high for a few minutes and, thereafter the effect decreased with time. No effect was seen after 20 min of infusion. Infusion of noradrenaline for 20 min increased cAMP in corpora lutea, but not in non-luteal tissue. LH was effective both in luteal and non-luteal tissue. The results show that noradrenaline in vivo can increase the cAMP content only in corpus luteum and within a rather short time period (5 min). This effect is different from that seen with LH which, instead, acts on both corpus luteum and stromal tissue.

Catecholamine stimulation of cyclic AMP and progesterone production in rat corpora lutea of different ages.

In vitro effects of catecholamines (adrenaline and noradrenaline) and adrenergic antagonists on adenosine 3',5'-cyclic monophosphate (cAMP) and progesterone production by rat corpora lutea (CL) of different ages (1-8 days old) were studied. To obtain defined ages of CL a pregnant mare's serum gonadotrophin (PMSG) model was used. The effect of catecholamines on cAMP decreased with luteal age while the effect on progesterone production was maximal on 5 day old CL. The beta-blocker propranolol inhibited the effects of catecholamines in concentrations around 10-(5) M. The effects of LH could only be inhibited with higher doses of propranolol known to exert unspecific effects. These results support the theory that LH and catecholamine effects on rat corpora lutea are mediated through different receptors.