Stability of Extruded 17β-Estradiol Solid Dispersions

Recrystallization is one of the main problems concerning the stability of solid dispersions. Different analytical methods were applied showing that no recrystallization occurred after treating melt extruded solid dispersions with 17β-Estradiol as the model drug with heat or water vapor. A skillful choice of excipients—a combination of polymers and additives—could be the reason for improving the stability. The requirements of the USP 23 for Estradiol tablets of 75% dissolved drug after 60 min were fulfilled after storing the tablets for 6 months at 40°C/75% RH. By observing the change in glass transition temperature, DSC analysis showed that the solid dispersions were stable against thermal stress. Isothermal microcalorimetry as well as moisture absorption gravimetry were methods to prove the stability of the solid dispersions against water vapor.     

Nasal Absorption Enhancement of 17β-Estradiol by Dimethyl-β-Cyclodextrin in Rabbits and Rats

A new formulation for nasal administration containing 17-estradiol (E₂) with dimethyl--cyclodextrin (DMC) as a solubilizer and absorption enhancer is described. Nasal administration of this E₂-DMC formulation gave a significantly higher E₂ absorption than an E₂ suspension in both rabbits and rats. Relative to an intravenous injection of the E₂-DMC formulation, absolute bioavailabilities of 94.6 and 67.2% were calculated for the nasal E₂-DMC formulation in rabbits and rats, respectively. Differences in bioavailability may have resulted from differences in experimental animal conditions. The effects on human nasal ciliary activity of the E₂-DMC formulation were studied with an in vitro method. The formulation was found to exert only a minor effect on ciliary beat frequency. Thus, nasal delivery of E₂, using a cyclodextrin inclusion formulation, may have potential for clinical application, e.g., in the therapy of postmenopausal disorders.     

Preparation and characterization of D,L-PLA loaded 17-β-Estradiol valerate by emulsion/evaporation methods

PLA microparticles containing 17-β-estradiol valerate were prepared by an emulsion/evaporation method in order to sustain drug release. This system was characterized concerning particle size, particle morphology and the influence of formulation and processing parameters on drug encapsulation and in vitro drug release. The biodegradation of the microparticles was observed by tissue histological analysis. Scanning electron microscopy and particle size analysis showed that the microparticles were spherical, presenting non-aggregated homogeneous surface and had diameters in the range of 718–880 nm (inert micro-particles) and 3–4 µm (drug loaded microparticles). The encapsulation efficiency was ～80%. Hormone released from microparticles was sustained. An in vivo degradation experiment confirmed that microparticles are biodegradable. The preparation method was shown to be suitable, since the morphological characteristics and efficiency yield were satisfactory. Thus, the method of developed microparticles seems to be a promising system for sustained release of 17-β-estradiol.     

Preparation and characterization of D, L-PLA loaded 17-β-Estradiol valerate by emulsion/evaporation methods

PLA microparticles containing 17-β-estradiol valerate were prepared by an emulsion/evaporation method in order to sustain drug release. This system was characterized concerning particle size, particle morphology and the influence of formulation and processing parameters on drug encapsulation and in vitro drug release. The biodegradation of the microparticles was observed by tissue histological analysis. Scanning electron microscopy and particle size analysis showed that the microparticles were spherical, presenting non-aggregated homogeneous surface and had diameters in the range of 718-880 nm (inert micro-particles) and 3-4 μm (drug loaded microparticles). The encapsulation efficiency was ～80%. Hormone released from microparticles was sustained. An in vivo degradation experiment confirmed that microparticles are biodegradable. The preparation method was shown to be suitable, since the morphological characteristics and efficiency yield were satisfactory. Thus, the method of developed microparticles seems to be a promising system for sustained release of 17-β-estradiol.     

17β-Estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway

We hypothesized that 17β-estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway. Rat aortic rings were contracted with cumulative addition of U46619, NaF, KCl or PDBu 30 min after pretreatment with 17β-estradiol (10, 30, and 100 μM) or vehicle. We measured the amount of GTP RhoA and the level of phosphorylation of the myosin light chain (MLC₂₀), myosin phosphatase targeting subunit 1 (MYPT1) and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI17). Pretreatment with 17β-estradiol dose-dependently inhibited the concentration-response curves in response to U46619, NaF or KCl, but not to PDBu. 17β-Estradiol decreased not only the level of phosphorylation of MYPT1^Thr855 and CPI17^Thr38 as well as MLC₂₀, but also the activity of RhoA induced by U46619 or NaF. However, 17β-estradiol did not affect the level of phosphorylation of CPI17 induced by PDBu. 17β-Estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway.     

17β-Estradiol/levonorgestrel transdermal system for the management of the symptomatic menopausal woman

Recent publications of the initial outcomes from the Women’s Health Initiative (WHI) study of menopausal management have raised concerns over the safety of hormone therapy [1,2]. Every study, no matter how large or well conducted, has biases and limitations that preclude the ability to apply the outcomes to a larger group of individuals not specifically evaluated in the analysis. In particular, the hormonal arms of the WHI evaluated only a single dose of a daily oral regimen of conjugated equine oestrogen 0.625 mg [1,2], combined with medroxyprogesterone acetate 2.5 mg if the subject had a uterus [1]. The failure to evaluate non-oral regimens prevented the evaluation of hormone delivery systems that have been shown to provide similar symptom relief to oral regimens, but with a considerably different physiological impact. The once-weekly transdermal patch (Climara Pro^?) releasing 17β-estradiol 0.045 mg/day and levonorgestrel 0.015 mg/day has been shown to be highly effective in rapidly reducing the frequency and intensity of vasomotor symptoms and to significantly improve all categories in the quality of life Women’s Health Questionnaire [3]. In addition, this transdermal combination system was not associated with any cases of endometrial hyperplasia, adverse impacts on cholesterol or lipid values and was associated with an increasing rate of amenorrhoea over time [3]. The 17β-estradiol/levonorgestrel transdermal system is approved in the US for the treatment of moderate-to-severe vasomotor symptoms and the prevention and treatment of urogenital atrophy.     

Pulmonary Absorption of Insulin Mediated by Tetradecyl-β-Maltoside and Dimethyl-β-Cyclodextrin

Purpose. To determine if tetradecyl--maltoside (TDM) and dimethyl--cyclodextrin (DMCD) enhance pulmonary absorption of insulin and to investigate if they do so by a reversible action on respiratory epithelium. Methods. Insulin formulated with saline, TDM, or DMCD was administered intratracheally, after laryngoscopic visualization, as a spray to anesthetized rats. Reversibility studies were conducted in intact rats by administering insulin at different time points after administration of TDM or DMCD. The pharmacodynamics and pharmacokinetics of insulin formulations were assessed by measuring plasma glucose and plasma insulin concentrations. Results. When insulin formulated with increasing concentrations (0.06-0.25%) of TDM or DMCD were administered to anesthetized rats, there was a concentration-dependent decrease in plasma glucose and increase in plasma insulin concentrations. The relative bioavailability of insulin formulations containing TDM was higher (0.34-0.84%) than that of formulations containing DMCD (0.19-0.48%). When insulin was administered 120 min after an agent was administered, in the reversibility study, no significant change in plasma glucose and insulin levels occurred compared to control. Conclusions. Both TDM and DMBCD enhance pulmonary absorption of insulin, with TDM being more efficacious than DMCD in enhancing insulin absorption via pulmonary administration. The effects of TDM and DMCD on respiratory epithelium are reversible, and the epithelium reestablishes its normal physiologic barrier 120 min after exposure to these agents.     

Effect of 17-β-Estradiol and Ginsenoside Rg1 on Reactive Microglia Induced by β-Amyloid Peptides

Abstract

The reactive microglias induced by 25 μmol of β-amyloid peptides (Aβ25–35) and/or IFN-γ can initiate the microglial respiratory burst and release NO, respectively. Oxidative stress and inflammatory function have been implicated in Alzheimer's disease (AD). We showed that 10 μmol 17-β-estradiol (E₂) and 1–10 μmol ginsenoside Rg₁ (Rg₁) could prevent the toxicity of Aβ25–35 and/or IFN-γ to microglias, inhibit the microglial respiratory burst activity and decrease the accumulation of NO. These results demonstrated the protectional effect of E₂ or Rg₁ on neuron from damaging by reactive microglias in AD.     

17β-Estradiol inhibition of PPARγ-induced adipogenesis and adipocyte-specific gene expression

Aim:

To investigate the molecular interaction of peroxisome proliferator-activated receptor γ (PPARγ) with 17β-estradiol (E) in the regulation of adipogenesis.

Methods:

Female ovariectomized (OVX) mice and differentiated 3T3-L1 adipocytes were treated with combinations of the PPARγ agonist troglitazone or E, and the variables and determinants of adipogenesis were measured using in vivo and in vitro approaches.

Results:

Troglitazone (250 mg·kg⁻¹·d⁻¹ for 13 weeks) decreased the size of adipocytes without the change in white adipose tissue (WAT) mass and increased the expression of adipocyte-specific genes, such as PPARγ, adipocyte fatty acid binding protein, and lipoprotein lipase, compared with OVX control mice. E (0.05 mg/pellet, sc implanted) significantly reduced WAT mass, adipocyte size, and adipose marker gene expression. When mice were concomitantly treated with troglitazone and E, E blunted the effects of troglitazone on WAT mass, adipocyte size, and adipose PPARγ target gene expression. Consistent with the in vivo data, E (10 μmol/L) treatment inhibited lipid accumulation and the expression of adipocyte-specific genes caused by troglitazone (10 μmol/L) in 3T3-L1 cells. E (10 μmol/L) also decreased troglitazone-induced PPARγ reporter activity through both estrogen receptor (ER) α and ERβ. Mechanistic studies indicated that E (0.1 μmol/L) decreased the DNA binding of PPARγ induced by troglitazone (1 μmol/L) and inhibited the recruitment of the PPARγ coactivator CREB-binding protein.

Conclusion:

These results suggest that in vivo and in vitro treatment of E interferes with the actions of PPARγ on adipogenesis by down-regulating adipogenesis-related genes, which are mediated through the inhibition of PPARγ coactivator recruitment. In addition, it is likely that the activities of PPARγ activators may be enhanced in estrogen-deficient states.     

Low Doses of 17��-Estradiol and 17��-Estradiol Facilitate, Whereas Higher Doses of Estrone and 17��- and 17��-Estradiol Impair, Contextual Fear Conditioning in Adult Female Rats

Estrogens are known to exert significant structural and functional effects in the hippocampus of adult rodents. In particular, 17β-estradiol can improve, impair, or have no effect on hippocampus-dependent learning and memory depending on dose and time of administration. The effects of other forms of estrogen, such as estrone and 17α-estradiol, on hippocampus-dependent learning have not been as thoroughly investigated. Therefore, the purpose of this study was to investigate the effects of 17β-estradiol, estrone, and 17α-estradiol at three different doses on two different tasks: hippocampus-dependent contextual fear conditioning and hippocampus-independent cued fear conditioning. Adult ovariectomized female rats were injected with one of the estrogens at one of the three doses 30 mins before conditioning to assess the rapid effects of these estrogens on acquisition. Twenty-four hours later memory for the context was examined and 1 h later memory for the cue (tone) was assessed. Levels of synaptophysin were examined in the dorsal hippocampus of rats to identify a potential synaptic correlate of hormonal effects on contextual fear conditioning. Low 17β-estradiol and 17α-estradiol enhanced, whereas high 17β-estradiol and 17α-estradiol impaired, contextual fear conditioning. Only the middle dose of estrone severely impaired contextual fear conditioning. Estrogens did not alter performance in the hippocampus-independent cued task. Synaptophysin expression was increased by estrone (at a middle and high dose) and 17β-estradiol (at a middle dose) in the CA3 region of the hippocampus and was not correlated with cognition. The results of this study indicate that estradiol can positively or negatively influence hippocampus-dependent learning and memory, whereas estrone impairs hippocampus-dependent learning and memory in a dose-dependent manner. These results have important therapeutic implications, as estrone, a main component of a widely used hormone replacement therapy, was shown to have either a negative effect or no effect on learning and memory. It may be possible to use 17α-estradiol and lower doses of estrogens as potential alternatives in hormone replacement therapies.     

Pharmacokinetics and Tolerability of a Novel 17β-Estradiol and Progesterone Intravaginal Ring in Sheep

This study reports the preparation, in vitro release, pharmacokinetics, and local tolerability of novel ethylene-vinyl acetate intravaginal rings (IVRs) delivering 17β-estradiol (E₂) and progesterone (P), in drug-naïve ovariectomized female Dorset crossbred sheep. After preparation and assessment of in vitro release of E₂ and P, animals were randomized to treatment groups 1 or 2 (comparator rings releasing 50 or 100 μg/d E₂, respectively), groups 3 or 4 (ethylene-vinyl acetate IVRs, 160 μg/d E₂ with 4 [160/4 IVR] or 8 mg/d P [160/8 IVR], respectively), or group 5 (160 μg E₂ and 10 mg P administered intravenously). IVRs were placed on day 1 and remained in place through day 29. Animals underwent daily examinations to confirm ring placement, and vaginal irritation was scored from 0 (none) to 4 (severe). Blood samples were taken at scheduled times for pharmacokinetic analysis. Postmortem examinations performed on groups 1-4 were macroscopic and microscopic evaluations, including irritation scoring and histopathology. IVRs were retained over 28 days in all but 1 animal (group 4). In all animal groups, clinical observations showed no significant abnormal findings. Pharmacokinetic analysis in the animals showed sustained release of E₂ and P over a 28-day period. Irritation scores and microscopic assessments were consistent with foreign object placement. A novel 2-drug IVR delivery system was well tolerated in a sheep model and pharmacokinetic release was as expected over a 28-day release period. These results will guide future human clinical studies.     

17β-Estradiol attenuates the activity of the glutamate transporter type 3 expressed in Xenopus oocytes

Estrogen, a neuroactive sex hormone in the brain, enhances neuronal excitability and increases seizures. Glutamate transporters help in limiting the excitatory neurotransmission by uptaking glutamate from the synapses. We investigated the effects of 17β-estradiol on the activity of a glutamate transporter, excitatory amino acid transporter 3 (EAAT3), in Xenopus oocytes. EAAT3 was expressed in Xenopus oocytes by injection of rat EAAT3 mRNA. l-Glutamate (30 μM)-induced membrane currents mediated by EAAT3 were measured using the two-electrode voltage clamp technique. 17β-Estradiol reduced EAAT3 activity in a concentration- and time-dependent manner. 17β-Estradiol (10nM for 72h) significantly decreased V(max) but had no effect on K(m) of EAAT3 for glutamate. When 17β-estradiol treated oocytes were incubated with phorbol-12-myrisate-13-acetate, a protein kinase C (PKC) activator, 17β-estradiol-induced decrease in EAAT3 activity was abolished. Furthermore, in pretreatment of oocytes with chelerythrine or staurosporine, two PKC inhibitors, EAAT3 activity was significantly decreased. However, there was no statistical difference among the 17β-estradiol, PKC inhibitor, or 17β-estradiol plus PKC inhibitor groups. Likewise, wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, significantly reduced basal EAAT3 activity, but the activity did not differ among the 17β-estradiol, wortmannin, or 17β-estradiol plus wortmannin groups. Estradiol receptor inhibitor, fulvestrant, did not change the reduced EAAT3 activity by 17β-estradiol. Our results suggest that 17β-estradiol decreases EAAT3 activity. PKC and PI3K seem to be involved in this effect, possibly not via estradiol receptors.     

Prediction of Biliary Excretion in Rats and Humans Using Molecular Weight and Quantitative Structure–Pharmacokinetic Relationships

The aims were (1) to evaluate the molecular weight (MW) dependence of biliary excretion and (2) to develop quantitative structure–pharmacokinetic relationships (QSPKR) to predict biliary clearance (CL_b) and percentage of administered dose excreted in bile as parent drug (PD_b) in rats and humans. CL_b and PD_b data were collected from the literature for rats and humans. Receiver operating characteristic curve analysis was utilized to determine whether a MW threshold exists for PD_b. Stepwise multiple linear regression (MLR) was used to derive QSPKR models. The predictive performance of the models was evaluated by internal validation using the leave-one-out method and external test groups. A MW threshold of 400 Da was determined for PD_b for anions in rats, while 475 Da was the cutoff for anions in humans. MW thresholds were not present for cations or cations/neutral compounds in either rats or humans. The QSPKR model for human CL_b showed a significant correlation (R² = 0.819) with good prediction performance (Q² = 0.722). The model was further assessed using a test group, yielding a geometric mean fold-error of 2.68. QSPKR models with significant correlation and good predictability were also developed for CL_b in rats and PD_b data for anions or cation/neutral compounds in rats and humans. Both CL_b and PD_b data were further evaluated for subsets of MRP2 or P-glycoprotein substrates, and significant relationships were derived. QSPKR models were successfully developed for biliary excretion of non-congeneric compounds in rats and humans, providing a quantitative prediction of biliary clearance of compounds.Key words: biliary clearance, humans, in silico, molecular weight cutoff, molecular volume, QSPKR, rats     

Leflunomide increased the renal exposure of acyclovir by inhibiting OAT1/3 and MRP2

Rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide(LEF)and the antiviral drug acyclovir to reduce the high risk of infection.Acyclovir is a substrate of organic anion transporter(OAT)1/3 and multidrug resistance-associated protein(MRP)2.Considering the extraordinarily long half-life of LEF’s active metabolite teriflunomide(TER)and the kidney injury risk of acyclovir,it is necessary to elucidate the potential impact of LEF on the disposition of acyclovir.Here we used a specific MRP inhibitor MK571 and probenecid(OAT1/3 and MRP2 inhibitor)to assess the effects of MRP2 and OAT1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats.We showed that LEF and probenecid,but not MK571 significantly increased the plasma concentration of acyclovir.However,kidney and liver exposures of acyclovir were increased when coadministered with LEF,probenecid or MK571.The kidney/plasma ratio of acyclovir was increased to approximately 2-fold by LEF or probenecid,whereas it was increased to as much as 14.5-fold by MK571.Consistently,these drugs markedly decreased the urinary excretion of acyclovir.TER(0.5−100μmol/L)dose-dependently increased the accumulation of acyclovir in MRP2-MDCK cells with an IC50 value of 4.91μmol/L.TER(5μmol/L)significantly inhibited the uptake of acyclovir in hOAT1/3-HEK293 cells.These results suggest that LEF/TER increased the kidney accumulation of acyclovir by inhibiting the efflux transporter MRP2,which increased its kidney/plasma ratio and renal injury risk.However,the inhibitory effects of LEF/TER on OAT1/3 reduced the tubular cells’uptake of acyclovir and increased the plasma concentration.     

Ursodeoxycholyl lysophosphatidylethanolamide attenuates hepatofibrogenesis by impairment of TGF-β1/Smad2/3 signalling

Background and Purpose

Chronic hepatic inflammation results in liver fibrosis. As effective anti-fibrogenic agents are lacking, we investigated ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE), a synthetic bile acid–phospholipid conjugate with anti-inflammatory and anti-apoptotic properties for tis effects on hepatic fibrogenesis.

Experimental Approach

To stimulate fibrogenesis, LX2 hepatic stellate cells were cultured with conditioned medium from CL48 liver cells after exposure to stress-inducing conditions – methionine–choline-deficient (MCD) medium or TNFα/cycloheximide (CHX) – with or without UDCA-LPE preincubation. Anti-fibrogenic effects of UDCA-LPE were further studied in CL48 and LX2 cells and in primary human hepatic stellate cells (HHStec) directly exposed to TGF-β1. To test UDCA-LPE in vivo, C57BL/6 mice were fed a MCD diet for 11 weeks followed by 30 mg·kg⁻¹ UDCA-LPE 3× per week for 2.5 weeks.

Key Results

Expression of α-smooth muscle actin (α-SMA), α1-collagen, vimentin and TGF-β1 was down-regulated by up to 93% by UDCA-LPE in LX-2 cells cultured with conditioned medium. Also, UDCA-LPE inhibited Smad3 phosphorylation in CL48 cells incubated with MCD medium or TNFα/CHX and in LX2 cells exposed to conditioned medium. UDCA-LPE also decreased phosphorylated Smad3 and Smad2 directly induced by TGF-β1. Inhibition of TGF-β1/Smad2/3 signalling with down-regulation of target genes was confirmed in HHStec. In vivo, UDCA-LPE decreased hepatic α-SMA, α1-collagen and TGF-β1 expression and markedly lowered α-SMA protein and collagen deposition in MCD mice.

Conclusions and Implications

By blocking TGF-β1/Smad2/3 signalling, UDCA-LPE suppressed key mediators of hepatic fibrogenesis. Thus, UDCA-LPE could be suitable for prevention of fibrotic progression of chronic liver disease.     

Introduction of an electron withdrawing group on the hydroxyphenylnaphthol scaffold improves the potency of 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2) inhibitors

Estrogen deficiency in postmenopausal women or elderly men is often associated with the skeletal disease osteoporosis. The supplementation of estradiol (E2) in osteoporotic patients is known to prevent bone fracture but cannot be administered because of adverse effect. As 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2) oxidizes E2 to its inactive form estrone (E1) and has been found in osteoblastic cells, it is an attractive target for the treatment of osteoporosis. Twenty-one novel, naphthalene-derived compounds have been synthesized and evaluated for their 17β-HSD2 inhibition and their selectivity toward 17β-HSD1 and the estrogen receptors (ERs) α and β. Compound 19 turned out to be the most potent and selective inhibitor of 17β-HSD2 in cell-free assays and had a very good cellular activity in MDA-MB-231 cells, expressing naturally 17β-HSD2. It also showed marked inhibition of the E1-formation by the rat and mouse orthologous enzymes and strong inhibition of monkey 17β-HSD2. It is thus an appropriate candidate to be further evaluated in a disease-oriented model.