Radioimmunoassay for plasma betamethasone 17-benzoate.

A sensitive radioimmunoassay for plasma betamethasone 17-benzoate has been developed. The antiserum used was obtained by immunizing rabbits with betamethasone 17-benzoate-21-hemisuccinate-bovine-serum-albumin conjugate. All of the endogenous steroids tested cross reacted less than 0.10%. A standard curve was established with a useful range from 0.05-5 ng. Reliability criteria were satisfactory. Measurement of plasma concentrations of betamethasone 17-benzoate was performed in patients and in rabbits following occlusive dressing of betamethasone 17-benzoate cream and gel base.     

Relative potency in acute and chronic suppressive effects of prednisolone and betamethasone on the hypothalamic-pituitary-adrenal axis in man

The relative potency in the hypothalamic-pituitary-adrenal (HPA) suppression of both prednisolone and betamethasone was examined in an acute study with normal volunteers and in a chronic study with glucocorticoid-treated patients. Circadian rhythm of plasma cortisol was studied after a single dose administration of 5 to 30 mg prednisolone or 0.5 to 3.0 mg betamethasone at 8:00 hr. Morning-rise of plasma cortisol occurred on the morning after the administration of 30 mg or less prednisolone but no morning rise was noted after the administration of 1.0 mg or more betamethasone. Plasma ACTH was slightly elevated on the morning after 30 mg prednisolone administration but showed low levels throughout the night after 3.0 mg betamethasone administration. Plasma cortisol responsiveness to ACTH was examined in patients before and during therapy with either prednisolone or betamethasone. The basal cortisol level was not suppressed and the responsiveness to ACTH remained nearly normal during long-term 5 mg prednisolone therapy, but these were completely suppressed during long-term 5 mg betamethasone therapy. The responsiveness to ACTH was nearly normal in patients receiving alternate-day therapy with prednisolone in such large doses as 50 or 60 mg every other day, but was completely suppressed in patients receiving 1.0 mg betamethasone every other day. The relative potency of betamethasone in acute and chronic suppressive effects on the HPA system seems to be much stronger than that of prednisolone in equivalent doses with comparable anti-inflammatory effects. It is also suggested that the alternate-day therapy with such long-acting steroids as betamethasone are useless in preventing HPA suppression.     

The endocrine disruption potential of betamethasone using Japanese medaka as a fish model

The presence of synthetic glucocorticosteroids (GCs) in surface water and their potential endocrine disruption (ED) activity at environmental concentrations has not been fully investigated. Synthetic GCs may interfere with endogenous receptors within the hypothalamic-pituitary-gonadal (HPG) axis and disruptions of this pathway can result in decreased reproduction and/or adverse developmental effects. Betamethasone, a synthetic GC, has been on the market in the United States since the 1980's. The Pharmaceutical Assessment and Transport Evaluation model estimated betamethasone concentrations to be <0.6 ng/L in 95% of all U.S. surface waters. Concentrations of 0.01, 0.1, and 1.0 µg betamethasone/L were used in a two-generation fish full life cycle study with Japanese medaka. Gross endpoints, secondary sexual characteristics, and vitellogenin expression were evaluated. The highest concentration at which ED outcomes are not anticipated was determined to be 0.1 µg/L. The ratio of the predicted environmental concentration to the no effect concentration for ED is less than one, indicating no risk to aquatic life from environmentally relevant concentrations of betamethasone.     

Late-gestation betamethasone enhances coronary artery responsiveness to angiotensin II in fetal sheep

Antenatal glucocorticoids are used to promote the maturation of fetuses at risk for preterm delivery. While perinatal glucocorticoid exposure has clear immediate benefits to cardiorespiratory function, there is emerging evidence of adverse long-term effects. To determine if antenatal betamethasone alters vascular reactivity, we examined isometric contraction of endothelium-intact coronary and mesenteric arteries isolated from twin fetal sheep at 121-124 days gestation (term being 145 days). One twin received betamethasone (10 microg/h iv) while the second twin received vehicle (0.9% NaCl) for 48 h immediately before the final physiological measurements and tissue harvesting. Fetuses that received betamethasone had higher mean arterial blood pressures than the saline-treated twin controls (53 +/- 1 vs. 48 +/- 1 mmHg, P < 0.05). Coronary vessels from betamethasone-treated fetuses exhibited enhanced peak responses to ANG II (72 +/- 17 vs. 23 +/- 6% of the maximal response to 120 mM KCl, P < 0.05). There was no significant difference in response of the coronary arteries to other vasoactive compounds [KCl, U-46619, sodium nitroprusside, 8-bromo-cGMP (8-BrcGMP), isoproterenol, and forskolin]. Contractile responses to ANG II were similar in betamethasone and control mesenteric arteries (48 +/- 17 vs. 36 +/- 12% of the maximal response to 10-6 M U-46619). Western blot analysis revealed AT1 receptor protein expression was increased by betamethasone in coronary but not in mesenteric arteries. These findings demonstrate that antenatal betamethasone exposure enhances coronary but not mesenteric artery vasoconstriction to ANG II by selectively upregulating coronary artery AT1 receptor protein expression.     

Analysis of betamethasone in rat plasma using automated solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry. Determination of plasma concentrations in rat following oral and intravenous administration

A method is described for the determination of betamethasone in rat plasma by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The analyte was recovered from plasma by solid-phase extraction and subsequently analyzed by LC-MS-MS. A Packard Multiprobe II, an automated liquid handling system, was employed for the preparation and extraction of a 96-well plate containing unknown plasma samples, standards and quality control samples in an automated fashion. Prednisolone, a structurally related steroid, was used as an internal standard. Using the described approach, a limit of quantitation of 2 ng/ml was achieved with a 50 microl aliquot of rat plasma. The described level of sensitivity allowed the determination of betamethasone concentrations and subsequent measurement of kinetic parameters of betamethasone in rat. Combination of automated plasma extraction and the sensitivity and selectivity of LC-MS-MS offers a valuable alternative to the methodologies currently used for the quantitation of steroids in biological fluids.     

Serum free thyroid hormones are decreased by betamethasone treatment in Graves' disease

38 patients with Graves' disease were treated at random with the glucocorticosteroid betamethasone or with placebo. The daily oral dose was 6.0 mg for the first 5 days, 4.5 mg for the following week, and then 3.0 mg. The serum free triiodothyronine (FT3) concentration decreased within 5 days, while the free thyroxine (FT4) level was reduced first after 3 weeks of betamethasone treatment. The suppressed serum thyrotropin concentration did not change. In the placebo group no significant constant alterations were found in any of the variables studied. The results corroborate that betamethasone decreases FT3 and to a less degree also FT4, which earlier has been indicated by indirect methods, although the mechanisms behind the changes remain to be clarified. Since FT3 is more readily available for the metabolic effects in tissues the rapid striking fall in its concentration is an argument for glucocorticoid treatment in selected patients with severe hyperthyroidism of Graves' disease.     

Determination of betamethasone and betamethasone 17-monopropionate in human plasma by liquid chromatography-positive/negative electrospray ionization tandem mass spectrometry

This study presents a high-performance liquid chromatography-positive/negative electrospray ionization tandem mass spectrometric (LC-ESI(+/-)-MS-MS) method for the determination of betamethasone (BOH) and betamethasone 17-monopropionate (B17P) in human plasma using beclomethasone dipropionate as the internal standard (I.S.). Both compounds were extracted from human plasma with ether-cyclohexane (4:1, v/v) and were separated by HPLC on a Hanbon Lichrospher C(18) column with a mobile phase of methanol-water (85:15, v/v) at a flow rate of 0.7ml/min. Calibration curves were linear over the range of 0.10-50ng/ml for BOH and 0.050-50ng/ml for B17P. The inter-run relative standard deviations were less than 14.4% for BOH and 12.3% for B17P. The intra-run relative standard deviations were less than 9.3% for BOH and 7.9% for B17P. The mean plasma extraction recovery for BOH and B17P were in the ranges of 82.7-85.9% and 83.6-85.3%, respectively. The method was successfully applied to study the pharmacokinetics of a new formulation of betamethasone phosphate/betamethasone dipropionate injection in healthy Chinese volunteers.     

Fetal and neonatal breathing movements in man after betamethasone.

Nineteen women in the last trimester of pregnancy were treated with betamethasone 12 mg daily for three consecutive days. The fetal breathing movements were monitored by an ultrasonic method before and after the treatment and the patterns of movements were compared to those in controls. In seven treated cases the recording of breathing was performed also in the neonatal period. Betamethasone in the given dose, which causes pronounced alterations in the fetal corticosteroid balance, does not influence the pattern of breathing movements in the fetus and newborn. Although corticosteroids are known to affect several functions of CNS, the maturation of mechanisms regulating breathing movements in the perinatal period is apparently not accelerated by betamethasone.     

Prenatal betamethasone exposure results in pituitary-adrenal hyporesponsiveness in adult sheep

Fetal exposure to synthetic glucocorticoids in sheep results in increased fetal hypothalamic-pituitary-adrenal (HPA) activity persisting to one year of age. We aimed to determine the effects of single or repeated maternal or fetal betamethasone injections on offspring HPA activity at 2 and 3 yr of age and whether changes in adrenal mediators of steroidogenesis contribute to changes in pituitary-adrenal function. Pregnant ewes or their fetuses received either repeated intramuscular saline or betamethasone injections (0.5 mg/kg) at 104, 111, 118, and 124 days of gestation (dG) or a single betamethasone injection at 104 dG followed by saline at 111, 118, and 124 dG. Offspring were catheterized at 2 and 3 yr of age and given corticotrophin-releasing hormone + arginine vasopressin challenges. Adrenal tissue was collected for quantitative RT-PCR mRNA determination at 3.5 yr of age. In 2-yr-old offspring, maternal betamethasone injections did not alter basal ACTH or cortisol levels, but repeated injections elevated ACTH responses. At 3 yr of age, basal ACTH was elevated, and both basal and stimulated cortisol levels were suppressed by repeated maternal injections. Basal and stimulated cortisol-to-ACTH ratios and basal cortisol-to-cytochrome P-450 17alpha-hydroxylase (P450c17) mRNA ratios were suppressed by repeated injections. Repeated fetal betamethasone injections attenuated basal ACTH and cortisol levels in offspring at 2 but not 3 yr of age. Plasma changes were not associated with altered adrenal P450c17, ACTH receptor, beta-hydroxysteroid dehydrogenase, or glucocorticoid receptor mRNA levels. These data suggest that maternal, but not fetal, betamethasone administration results in adrenal suppression in adulthood.     

Repeated betamethasone treatment of pregnant sheep programs persistent reductions in circulating IGF-I and IGF-binding proteins in progeny

Exposure to synthetic glucocorticoids in utero markedly improves survival after preterm birth, but repeated exposures impair fetal and postnatal growth and are associated with evidence of insulin resistance in later life. The insulin-like growth factor (IGF) axis is an important regulator of growth and metabolism before and after birth. We have therefore investigated the effects of repeated maternal betamethasone injections on plasma IGF-I, IGF-II, and IGF-binding proteins (IGFBP) in fetal and postnatal progeny in the sheep. Pregnant sheep carrying male fetuses were injected with saline or betamethasone at 104, 111, and 118 days of gestation (dG, term approximately 150 dG). Plasma samples were collected postmortem from fetuses before (75, 84, 101 dG) or after one (109 dG), two (116 dG), or three (121-122, 132-133, 145-147 dG) doses of saline or betamethasone and from progeny at 42 and 84 days of age. Fetal weight was reduced after two or more maternal betamethasone injections, and this effect persisted to term. Repeated betamethasone exposures reduced plasma IGF-I and total IGFBP in fetuses at 133 dG and progeny at 84 days, and reduced plasma IGFBP-3 at 84 days. Fetal plasma IGF-II tended to increase transiently at 109 dG following the first betamethasone injection. Fetal, placental, and/or postnatal weights correlated positively with concomitant plasma IGF-I, IGF-II, and total IGFBP. We conclude that repeated exposure to synthetic glucocorticoids in utero programs the IGF axis before and after birth, which may contribute to the adverse effects of betamethasone exposure on growth and metabolism.     

Effect of simultaneous administration of betamethasone and triiodothyronine (T3) on the development of functional pulmonary maturation in fetal rabbit

Recent experimental evidence suggests that a combination of glucocorticoid and thyroid hormone may be more effective than either hormone alone in accelerating morphologic as well as biochemical mammalian fetal lung maturation. We have demonstrated that IM administration of T3 to the rabbit doe is associated with enhanced functional fetal lung maturation. We investigated the effect of simultaneous administration of T3 and betamethasone on the development of functional fetal lung maturation and the duration of survival after premature delivery. On day 25 and 26 of pregnancy, T3 (175 micrograms/kg/dose) betamethasone (85 micrograms/kg/dose), T3 plus betamethasone or the appropriate amount of the vehicles were injected. The functional fetal pulmonary maturity and the duration of survival after premature delivery were assessed on day 27 of gestation. Although enhanced functional fetal lung maturation was observed after T3 or betamethasone administration, there was no additive effect after simultaneous administration of both. The duration of survival on premature delivery was enhanced in betamethasone but not T3 or T3 plus betamethasone group when compared to control. Further animal experimentation seems necessary before a clinical trial of T3 plus betamethasone therapy is considered.     

Long-term effects of betamethasone on blood pressure and hypothalamo-pituitary-adrenocortical function in spontaneously hypertensive and normotensive rats

An enhanced hypothalamo-pituitary-adrenocortical (HPA) activity has been described during onset of elevated blood pressure in spontaneously hypertensive rats (SHR). An instability of the HPA axis could thus contribute to the development of hypertension in these animals. Glucocorticoid effects on blood pressure and HPA function were studied therefore in SHR and normotensive Wistar-Kyoto (WKY) and Wistar rats. Beginning at 4 weeks of age, the rats were treated with 0.1 and 0.5 microgram betamethasone per milliliter drinking water for 7 weeks. SHR and WKY responded with a significant elevation in average blood pressure. In SHR, mean blood pressure rose from 181.4 +/- 3.9 (mean +/- SEM) to 203.1 +/- 2.8 mm Hg in response to the lower dose of betamethasone and to 209.2 +/- 4.0 mm Hg in response to 0.5 microgram betamethasone per milliliter drinking water. In WKY, blood pressure increased from 134.4 +/- 3.3 to 148.2 +/- 3.0 and 157.9 +/- 4.5 mm Hg in response to the lower and higher dose of betamethasone, respectively. No significant effect was seen in Wistar rats, where the mean blood pressure values changed insignificantly from 133.8 +/- 2.1 to 136.3 +/- 3.2 and 135.6 +/- 2.4 mm Hg. Stress-induced secretion of corticosterone was significantly suppressed in a dose-dependent manner in all three strains. Stress-induced secretion of adrenocorticotropin was markedly reduced by 0.5 microgram betamethasone per milliliter in SHR and by both doses in WKY. No significant effect, however, was seen in Wistar rats. A predisposition to the hypertensiogenic actions of glucocorticoids was found therefore in SHR and WKY, but not in Wistar rats.     

Discovery of betamethasone 17alpha-carbamates as dissociated glucocorticoid receptor modulators in the rat

A series of betamethasone 17alpha-carbamates were designed, synthesized, and evaluated for their ability to dissociate the two main functions of the glucocorticoid receptor, that is, transactivation and transrepression, in rat cell lines. A number of alkyl substituted betamethasone 17alpha-carbamates were identified with excellent affinity for the glucocorticoid receptor (e.g., 7, GR IC(50) 5.1 nM) and indicated dissociated profiles in functional assays of transactivation (rat tyrosine aminotransferase, TAT, and rat glutamine synthetase, GS) and transrepression (human A549 cells, MMP-1 assay). Gratifyingly, the in-vivo profile of these compounds, for example, 7, also indicated potent anti-inflammatory activity with impaired effects on glucose, insulin, triglycerides, and body weight. Taken together, these results indicate that dissociated glucocorticoid receptor modulators can be identified in rodents.     

An enzyme immunoassay for plasma betamethasone

A sensitive enzyme immunoassay for plasma betamethasone was developed using betamethasone-3-(O-carboxymethyl)oxime-β-D-galactosidase conjugate as a labelled antigen and 4-methylumbelliferyl-β-D-galactoside as a fluorescence substrate. The performances of the enzyme immunoassay were compared with that of a radioimmunoassay using ³H-betamethasone and the same antiserum. The minimal detectable level for the enzyme immunoassay was 0.15 pg/tube or 0.15 ng/ml of plasma, which was remarkably more sensitive than the radioimmunoassay level of 10 pg/tube or 2 ng/ml of plasma. The specificity was sufficient, in particular, the cross reactivity of cortisol was 0.008%. However, the precision of the enzyme immunoassay was inferior to that of the radioimmunoassay.     

Intra-amniotic LPS and antenatal betamethasone: inflammation and maturation in preterm lamb lungs

The proinflammatory stimulus of chorioamnionitis is commonly associated with preterm delivery. Women at risk of preterm delivery receive antenatal glucocorticoids to functionally mature the fetal lung. However, the effects of the combined exposures of chorioamnionitis and antenatal glucocorticoids on the fetus are poorly understood. Time-mated ewes with singleton fetuses received an intra-amniotic injection of lipopolysaccharide (LPS) either preceding or following maternal intramuscular betamethasone 7 or 14 days before delivery, and the fetuses were delivered at 120 days gestational age (GA) (term = 150 days GA). Gestation matched controls received intra-amniotic and maternal intramuscular saline. Compared with saline controls, intra-amniotic LPS increased inflammatory cells in the bronchoalveolar lavage and myeloperoxidase, Toll-like receptor 2 and 4 mRNA, PU.1, CD3, and Foxp3-positive cells in the fetal lung. LPS-induced lung maturation measured as increased airway surfactant and improved lung gas volumes. Intra-amniotic LPS-induced inflammation persisted until 14 days after exposure. Betamethasone treatment alone induced modest lung maturation but, when administered before intra-amniotic LPS, suppressed lung inflammation. Interestingly, betamethasone treatment after LPS did not counteract inflammation but enhanced lung maturation. We conclude that the order of exposures of intra-amniotic LPS or maternal betamethasone had large effects on fetal lung inflammation and maturation.     

Maternal and feto-placental prostanoid responses to a single course of antenatal betamethasone

We tested the hypothesis that antenatal betamethasone alters prostanoid levels in the maternal and feto-placental compartments. Forty-three singleton pregnancies were studied. Group I were women treated with a single course of antenatal betamethasone and who delivered <37 weeks gestation; Group II were untreated women who delivered <37 weeks; and Group III were untreated women who delivered >38 weeks. Maternal and mixed cord blood; and placental samples were collected at delivery and analyzed for PGE2, PGF(2alpha), 6-ketoPGF(1alpha), and TxB2 levels. Antenatal betamethasone decreased maternal PGE2 levels with concomitant increases in the feto-placental compartment. Umbilical cord TxB2 levels in the treated group were significantly lower than the non-treated pre-term and term groups resulting in a higher 6-ketoPGF(1alpha):TxB2 ratio. Considering the regulatory role of PGE2 and PGI2 in fetal lung development and neonatal transition homeostasis, these results suggest a mechanism, at least in part, for the beneficial effects of antenatal steroids on fetal lung maturation and neonatal cardio-pulmonary homeostasis at birth.     

Effects of betamethasone,sulindac and quinacrine drugs on the inflammatory neoangiogenesis response induced by polyurethane sponge implanted in mouse

In this study, we showed the effect of the betamethasone, sulindac and quinacrine alone or combined, on the inflammatory angiogenesis promoted by polyurethane sponge on mice. The main finding reported here is that the formation of new blood vessels was strongly inhibited by low concentration of betamethasone, sulindac or quinacrine, whether alone or in combination. It is known that steroidal anti-inflammatory drugs inhibit the enzymes required for the production of prostaglandins through a nuclear glucocorticoid receptor (GR) mediated mechanism. This mechanism may occur in endothelial cells as well. Considering that activity of cyclo-oxigenases 1 and 2 is inhibited by sulindac, and that these enzymes are located in the stromal tissue, we propose that the anti-angiogenic effect of these agents may occur via inhibition of both COX isoforms. On the other hand, quinacrine inhibited PLA2 activity, and we propose here that the anti-angiogenic effect occurs via inhibition of the enzyme PLA2. The potentiated effect of the association of betamethasone, sulindac and quinacrine may have some therapeutic benefit in the control of pathological angiogenesis. Further studies are required to validate these propositions.     

The effect of betamethasone and fetal sex on the synthesis and maturation of lung surfactant phospholipids in rabbits

In the present study we investigated the maturation of the surfactant phospholipids and the role of fetal sex on the effect of betamethasone in male and female rabbit fetuses. Betamethasone was administered to the doe (0.2 mg/kg intramuscularly) 42 and 18 h prior to killing. The fetuses were studied at 27 and 28 days from conception. Results from the alveolar lavage show that male fetuses tended to have a lower disaturated phosphatidylcholine/sphingomyelin ratio and lower levels of phosphatidylinositol. Phosphatidylglycerol was detected in trace amounts. This was apparently due to the high extracellular levels of myo-inositol inhibiting the synthesis of surfactant phosphatidylglycerol while increasing the synthesis of surfactant phosphatidylinositol. Betamethasone increased the recovery of disaturated phosphatidylcholine and phosphatidylinositol from the lung lavage in both sexes. As studied in lung slices in vitro, the betamethasone treatment decreased the incorporation of glucose into phospholipids, including into the fatty acid moiety of disaturated phosphatidylcholine, although it had no significant effect on the incorporation of glucose into the glycerol moiety of disaturated phosphatidylcholine. However, the addition of palmitate increased the incorporation of glucose into the glycerol moiety of disaturated phosphatidylcholine. The betamethasone treatment did not increase the incorporation of [1-14C]pyruvate into disaturated phosphatidylcholine. Following betamethasone administration, the availability of fatty acids may become rate-limiting for the synthesis of surfactant phospholipids. Betamethasone increased the activities of phosphatidic acid phosphohydrolase and phosphatidate cytidyltransferase in a fraction of microsomal membranes. The present evidence suggests that the glucocorticoid-induced lung maturation and the maturation of the normal lung are associated with an increase in the activity of the enzymes which are involved in metabolizing phosphatidic acid to neutral and acidic surfactant secretion of the male fetus was not explained by possible sex-related differences in the biosynthesis of the phospholipids.     

Exaggerated sympathetic mediated responses to behavioral or pharmacological challenges following antenatal betamethasone exposure

Glucocorticoid administration to women at risk for preterm delivery is standard practice to enhance neonatal survival. However, antenatal betamethasone exposure (β-exposure) increases mean arterial pressure (MAP) in adult sheep (1.8 yr old) and results in impaired baroreflex sensitivity (BRS) for control of heart rate (HR). In the current studies we tested the hypothesis that enhanced sympathetic nervous system and hypothalamo-pituitary-adrenal (HPA) axis-mediated responses are evident at an early age in β-exposed sheep. Pregnant ewes were administered betamethasone (0.17 mg/kg twice over 24 h) or vehicle (Veh-control) on the 80th day of gestation, and offspring were delivered at full term. Female β-exposed and control offspring instrumented at age 42 ± 3 days for conscious continuous recording of MAP and HR had similar resting values at baseline. However, BRS was ~45% lower in β-exposed offspring. β-Exposed lambs allowed to suckle for 10 min had a greater elevation in MAP than Veh-control lambs (19 ± 1 vs 12 ± 2 mmHg; n = 4-5, P < 0.05). MAP was reduced by 20% from baseline via sodium nitroprusside infusion (SNP) over 10 min, which triggered a rebound increase in MAP only in β-exposed lambs. HR increased with the reduction in MAP during SNP infusion in Veh-control lambs, whereas there was no change in HR with the reduction in MAP in β-exposed lambs. Combined vasopressin-CRF injection caused greater increases in MAP in the β-exposed lambs. Cortisol and ACTH responses were higher in response to SNP hypotension in the β-exposed lambs. The data reveal enhanced sympathetic and HPA axis responses associated with impaired BRS preceding differences in resting MAP in preweanling female lambs exposed in utero to glucocorticoids. The consequences of these alterations at an early age include eventual development of higher blood pressure in this ovine model of fetal programming.