Emicizumab versus immunosuppressive therapy for the management of acquired hemophilia A.

BACKGROUND: Acquired hemophilia A (AHA) is an autoimmune bleeding disorder caused by neutralizing antibodies against coagulation factor VIII. Immunosuppressive therapy (IST) is standard of care to eradicate autoantibody production and protect from further bleeding but carries a risk of severe infection and mortality in frail patients with AHA. Recently, emicizumab has been studied for its potential to reduce the need for early and aggressive IST. OBJECTIVES: To compare outcomes of 2 studies that used either IST (GTH-AH 01/2010; N = 101) or prophylaxis with emicizumab (GTH-AHA-EMI; N = 47) early after diagnosis of AHA. METHODS: Baseline characteristics were balanced by propensity score matching. Primary endpoint was the rate of clinically relevant new bleeds during the first 12 weeks; secondary endpoints were adverse events and overall survival. RESULTS: The negative binominal model-based bleeding rate was 68% lower with emicizumab as compared with IST (incident rate ratio, 0.325; 95% CI, 0.182-0.581). No difference was apparent in the overall frequency of infections (emicizumab 21%, IST 29%) during the first 12 weeks, but infections were less often fatal in emicizumab-treated patients (0%) compared with IST-treated patients (11%). Thromboembolic events occurred less often with emicizumab (2%) than with IST (7%). Overall survival after 24 weeks was better with emicizumab (90% vs 76%; hazard ratio, 0.44; 95%, CI, 0.24-0.81). CONCLUSION: Using emicizumab instead of IST in the early phase after initial diagnosis of AHA reduced bleeding and fatal infections and improved overall survival.

Emicizumab prophylaxis in patients with acquired haemophilia A (GTH-AHA-EMI): an open-label, single-arm, multicentre, phase 2 study.

BACKGROUND: Acquired haemophilia A is caused by neutralising autoantibodies against coagulation factor VIII, leading to severe bleeding. Standard treatment involves immunosuppressive therapy, which is associated with adverse events and mortality in the frail population of patients with acquired haemophilia A. This study investigated whether emicizumab, a factor VIIIa mimetic antibody, protects patients with acquired haemophilia A from bleeding and allows deferral of immunosuppression during the first 12 weeks after diagnosis. METHODS: We report final results of an open-label, single-arm, phase 2 clinical trial. Adult patients with acquired haemophilia A from 16 haemophilia treatment centres in Germany and Austria were eligible if they had not previously received immunosuppression. Patients received emicizumab subcutaneously (6 and 3 mg/kg on days 1 and 2, 1·5 mg/kg weekly until week 12), but no immunosuppression. Follow-up was until week 24. The primary endpoint was the number of clinically relevant bleeds per patient-week until week 12. Emicizumab was considered effective if the mean bleeding rate was significantly below 0·15 bleeds per patient-week, the rate observed in a previous study of patients with acquired haemophilia A treated with bypassing agents and immunosuppression but no emicizumab. The study is registered with clinicaltrials.gov, NCT04188639 and is complete. FINDINGS: Of 49 patients screened from March 25, 2021, to June 10, 2022, 47 were enrolled (23 women, 24 men). Median age was 76 years (IQR 66-80), 46 (98%) of 47 patients were White, median factor VIII activity was 1·4 IU/dL (0·3-5·6), and median inhibitor concentration was 11·4 Bethesda units per mL (3·9-42·7). Mean breakthrough bleeding rate was 0·04 bleeds per patient-week (upper 97·5% CI 0·06). 33 (70%) of 47 patients had no bleeding events, seven patients (15%) had one bleed, six patients (13%) had two bleeds, and one patient (2%) had three bleeds. Adverse events of grade 3 or worse included COVID-19 (n=2), acute kidney injury (n=2), and stroke (n=1). Four of 47 patients died, including two deaths related to bleeding, one from COVID-19, and one from cardiac arrest (none were judged as related to emicizumab). INTERPRETATION: This study suggests that emicizumab prophylaxis prevents bleeding in patients with acquired haemophilia A and that immunosuppressive therapy can be deferred while patients are receiving this treatment. The low number of thromboembolic events, severe infections, and fatalities observed in this study are promising. FUNDING: This study was supported by funding from Hoffman-La Roche.

Impaired glucose and lipid metabolism in ageing aryl hydrocarbon receptor deficient mice.

Disturbed homeostasis of glucose and lipid metabolism are dominant features of the so-called metabolic syndrome (MetS) and can increase the risk for the development of type 2 diabetes (T2D), a severe metabolic disease. T2D prevalence increases with age. The aryl hydrocarbon receptor (AHR) is a sensor of small molecules including dietary components. AHR has been identified as potential regulator of glucose homeostasis and lipid metabolism. Epidemiologically, exposure to xenobiotic AHR ligands such as polycyclic aromatic hydrocarbons is linked to T2D. We assess here the potential role of the AHR in disturbances of glucose and lipid metabolism in young (age 2-5 months) and old (age > 1,5 years) AHR-deficient (AHR KO) mice. Fasted young wildtype (WT) and AHR-KO mice displayed similar blood glucose kinetics after challenge with intra-peritoneal glucose injection. However, old AHR-KO mice showed lower tolerance than WT to i.p. administered glucose, i.e. glucose levels rose higher and returned more slowly to normal levels. Old mice had overall higher insulin levels than young mice, and old AHR-KO had a somewhat disturbed insulin kinetic in the serum after glucose challenge. Surprisingly, young AHR-KO mice had significantly lower triglycerides, cholesterol, high density lipoprotein values than WT, i.e., a dyslipidemic profile. With ageing, AHR-KO and WT mice did not differ in these lipid levels, except for slightly reduced levels of triglycerides and cholesterol. In conclusion, our findings in AHR KO mice suggest that AHR expression is relevant for the maintenance of glucose and lipid homeostasis in old mice.

Effects of estradiol, estrogen receptor subtype-selective agonists and genistein on glucose metabolism in leptin resistant female Zucker diabetic fatty (ZDF) rats.

The leptin resistant Zucker diabetic fatty (ZDF) rats are hyperphagic and become obese, but whereas the males develop type 2 diabetes mellitus (T2DM), the females remain euglycaemic. As estrogen deficiency is known to increase the risk of developing T2DM, we evaluated the role of ER subtypes alpha and beta in the development of glucose tolerance in leptin resistant ovariectomized (OVX) ZDF rats. At least six rats per group were treated with either vehicle (OVX), 17ß-estradiol (E2), ER subtype-selective agonists (Alpha and Beta), or genistein (Gen) for 17 weeks. At the end of the treatment period a glucose tolerance assay was performed and the metabolic flux of (13)C-glucose for the E2 group was investigated. OVX ZDF rats treated with E2, Alpha, Beta, and Gen tolerated the glucose significantly better than untreated controls. E2 treatment increased absorbance/flux of (13)C-glucose to metabolic relevant tissues such liver, adipose tissue, gastrocnemius, and soleus muscle. Moreover, whereas Alpha treatment markedly increased mRNA expression of GLUT4 in gastrocnemius muscle, Beta treatment resulted in the largest fiber sizes of the soleus muscle. Treatment with Gen increased both the mRNA expression of GLUT 4 and the fiber sizes in the skeletal muscle. In addition, E2 and Alpha treatment decreased food intake and body weight gain. In summary, estrogen-improved glucose absorption is mediated via different molecular mechanisms: while activation of ER alpha seems to stimulate muscular GLUT4 functionality, activation of ER beta results in a hypertrophy of muscle fibers. In addition, selective activation of ER alpha decreased food intake and body weight gain. Our data further indicate that ER subtype-selective agonists and genistein improve systemic glucose tolerance also in the absence of a functional leptin signaling pathway.

Molecular effects of ER alpha- and beta-selective agonists on regulation of energy homeostasis in obese female Wistar rats.

The molecular mechanisms underlying the effects of selective ER subtype activation on lipogenesis, adipogenesis, lipid utilization and storage as well as glucose metabolism are currently largely unknown and were analyzed in female OVX Wistar rats on a high-fat diet. Rats received estradiol (E2), ER subtype-selective agonists (Alpha and Beta), and genistein (Gen) for 10 weeks. In adipose tissue, treatment with E2, Alpha, and Beta significantly decreased lipogenic (SREBP-1c, FAS) and adipogenic genes (LPL, PPAR gamma). In liver and skeletal muscle of E2-, Alpha-, Beta-, and Gen-treated animals, lipogenesis and triglyceride accumulation were significantly reduced. Increased hepatic and muscular PPAR gamma mRNA expression was observed in untreated, Beta- and Gen-treated animals, which correlates with increased hepatic glucose uptake. However, only untreated animals showed impaired insulin sensitivity compared to all other groups. Therefore, PPAR gamma up-regulation in untreated animals suggests a compensatory mechanism, probably due to increased triglyceride accumulation in non-adipose tissues. Beta- and Gen-treated animals may benefit from the anabolic potency of ER beta that ameliorates lipid and glucose utilization in muscle. Activation of either ER subtype reduces fat enrichment and improves insulin sensitivity. Depending on the investigated tissue, different molecular pathways seem to be involved.

Metabolic effects of estrogen substitution in combination with targeted exercise training on the therapy of obesity in ovariectomized Wistar rats.

Postmenopausal women tend to have a higher risk in developing obesity and thus metabolic syndrome. Recently we could demonstrate that physical activity and estrogen replacement are effective strategies to prevent the development of nutritional induced obesity in an animal model. The aim of this study was to determine the combined effects of estrogen treatment and exercise training on already established obesity. Therefore ovariectomized (OVX) and sham-operated (SHAM) female Wistar rats were exposed to a high fat diet for ten months. After this induction period obese SHAM and OVX rats either remained sedentary or performed treadmill training for six weeks. In addition OVX rats were treated with 17ß-Estradiol (E(2)) alone, or in combination with training. Before and after intervention effects on lipid and glucose metabolism were investigated. Training resulted in SHAM and OVX rats in a significant decrease of body weight, subcutaneous and visceral body fat, size of adipocytes and the serum levels of leptin, cholesterol, low-density lipoprotein and triglycerides. In OVX animals E(2) treatment resulted in similar effects. Often the combination of E(2) treatment and training was most effective. Analysis of the respiratory quotient indicates that SHAM animals had a better fat burning capacity than OVX rats. There was a tendency that training in SHAM animals and E(2) treatment in OVX animals could improve this capacity. Analysis of glucose metabolism revealed that obese SHAM animals had higher glucose tolerance than OVX animals. Training improved glucose tolerance in SHAM and OVX rats, E(2) treatment in OVX rats. The combination of both was most effective. Our results indicate that even after a short intervention period of six weeks E(2) treatment and exercise training improve parameters related to lipid as well as glucose metabolism and energy expenditure in a model of already established obesity. In conclusion a combination of hormone replacement therapy and exercise training could be a very effective strategy to encourage the therapy of diet-induced obesity and its metabolic consequences in postmenopausal women.

Impact of estradiol, ER subtype specific agonists and genistein on energy homeostasis in a rat model of nutrition induced obesity.

Estrogens are known to be involved in the control of energy homeostasis. Here we investigated the role of ER alpha and ER beta in a model of nutrition induced obesity. Ovariectomized Wistar rats were fed a high fat diet and received either vehicle, E2, ER subtype selective agonists (Alpha and Beta) or genistein. After 10 weeks, body weight, visceral fat, serum leptin, blood lipids, and in the soleus muscle anabolic markers were determined. Treatment with E2 and Alpha decreased body weight, total cholesterol and VLDL. Visceral fat mass, adipocyte size, and serum leptin were reduced by E2, Alpha and Beta. In the soleus muscle, treatment with E2 and Beta modulated Igf1 and Pax7 gene expression and resulted in larger muscle fibers. Our data indicate that blood lipids are affected via ER alpha, whereas activation of ER beta results in an increase of soleus muscle mass. Adipose tissue homeostasis seems to be affected via both ERs.

Effect of 17ß-estradiol and flavonoids on the regulation of expression of newly identified oestrogen responsive genes in a rat raphe nuclei-derived cell line.

Due to the health risks attributed to perimenopausal hormone therapy, phytoestrogens such as flavonoids are receiving widespread attention to help alleviate menopausal symptoms, including hormone-driven mood disorders. Based on our previous reporter gene study regarding their transactivational activity in raphe nuclei cells from a brain region involved in regulation of mood disturbances, we herein study their effects on the regulation of expression of 17ß-estradiol (E2)-regulated genes. DNA microarray was used to globally assess E2-induced gene expression in RNDA cells, a rat raphe nuclei-derived cellular model expressing oestrogen receptor ß. Out of 212 regulated genes, six were selected for verification and as endpoints for the effect of flavonoids on the regulation of mRNA expression in proliferating as well as differentiating RNDA cells. Under proliferative conditions, E2 up-regulated mRNA expression of Cml-5, Sox-18 and Krt-19. Similar effects were observed in response to 8-prenylnaringenin (8-PN), genistein (GEN), daidzein (DAI) and equol (EQ). In line with E2, mRNA expression of Nefm and Zdhhc-2 was down-regulated following 8-PN, GEN, DAI, EQ and naringenin treatment. No regulation was observed on Slc6a4 mRNA expression in response to E2 or the flavonoids in proliferating RNDA cells. When cells were shifted to conditions promoting differentiation, changes in cell morphology, in mRNA expression levels and in responsiveness towards E2 and the tested flavonoids were noticed. These expression studies additionally highlighted some of the genes as markers for RNDA cellular differentiation. RNDA cells should prove useful to elucidate molecular and cellular mechanisms of exogenous oestrogen receptor ligands with neural cell populations.

Physical activity and estrogen treatment reduce visceral body fat and serum levels of leptin in an additive manner in a diet induced animal model of obesity.

Estrogen replacement and physical activity have been demonstrated to reduce the risk to develop a metabolic syndrome in postmenopausal women. In this study we investigate the combined effects of endurance training and estrogen substitution in a rat animal model of diet induced obesity. Effects on lipid and glucose metabolism were evaluated. Ovariectomized (OVX) or sham-operated (SHAM) female Wistar rats were fed with a high fat diet (HF) for 9 weeks. After 3 weeks of overnutrition the OVX rats either remained sedentary, performed treadmill training, received 17ß-Estradiol (E(2)), or combined treatment. The OVX rats had a greater increase in body weight and serum levels of cholesterol, triglyceride and low-density lipoprotein cholesterol (LDL). These parameters could be reduced by E(2) and more effectively E(2) in combination with exercise. Also the increase of visceral body fat and leptin could be improved by E(2) and exercise. This combination showed synergistic effects. Serum levels of insulin could be reduced by exercise training, E(2) substitution revealed no significant changes. Our results indicate that ovariectomy increases the susceptibility to develop obesity. In addition they show that the combination of hormone replacement therapy (HRT) and physical activity may influence parameters related to lipid metabolism positively in an additive manner. The results of this study provide evidence that the combination of HRT with physical activity could be a very effective strategy to prevent the development of a metabolic syndrome induced by overnutrition.

Time dependency of uterine effects of naringenin type phytoestrogens in vivo.

Phytoestrogens exhibit significant estrogen agonistic/antagonistic properties in animals and humans. Naturally occurring flavonoids with a naringenin backbone like 8-prenylnaringenin (8-PN) and 6-(1,1-dimethylallyl)naringenin (6-DMAN) are considered to be some of the most potent phytochemicals activating nuclear receptors. 8-PN is a more potent estrogenic substance while 6-DMAN appears to have a higher antiandrogenic potency, however these are less well characterized compared to other phytoestrogens such as genistein. The aim of this study was to assess the estrogenic properties of 8-PN and 6-DMAN in an ovariectomized in vivo rat model. 8-PN and 6-DMAN were applied at concentrations of 15mg/kgBW. We assessed the uterotrophic response after 7h, 24h and 72h of treatment. In contrast to 8-PN, 6-DMAN did not alter uterine wet weight or the level of expression of proliferation markers at any time point. In contrast to the uterotrophic response, 6-DMAN stimulated uterine mRNA expression of estrogen responsive genes carrying an estrogen response element (ERE) in the ovariectomized rats, but to a lesser extent than E2 and 8-PN. In all treatment regimens, the mRNA expression of estrogen receptors alpha and beta mRNA was measured. In summary, we assessed the time dependent uterine responses and estrogenic activities of 6-DMAN and 8-PN. In contrast to 8-PN which mimicked the E2 induced responses on uterine wet weight and gene expression, 6-DMAN has no uterotrophic effect and only regulated the mRNA expression of genes carrying an ERE. Therefore, 6-DMAN is an exciting candidate molecule for future investigations and potentially a natural occurring selective estrogen receptor modulator.

Activation of estrogen receptor-beta by a special extract of Rheum rhaponticum (ERr 731), its aglycones and structurally related compounds.

The special extract ERr 731 from the roots of Rheum rhaponticum is the major constituent of Phytoestrol N which is used for the treatment of climacteric symptoms in menopausal women. However, the molecular mode of action of ERr 731 was unknown. For the first time, ERr 731 and its aglycones trans-rhapontigenin and desoxyrhapontigenin were investigated with regard to the activation of the estrogen receptor-alpha or estrogen receptor-beta (ERalpha, ERbeta). The related hydroxystilbenes cis-rhapontigenin, resveratrol and piceatannol were studied as comparators. As controls, 17beta-estradiol or the selective ERalpha-(propylpyrazoltriol) or ERbeta-agonists (diarylpropionitril) were used. Neither in ERalpha-expressing yeast cells, in the ERalpha-responsive Ishikawa cells, nor in human endometrial HEC-1B cells transiently transfected with the ERalpha an activation of ERalpha by ERr 731 or the other single compounds was detected. Furthermore, an antiestrogenic effect was not observed. In contrast in human endometrial HEC-1B cells transiently transfected with the ERbeta, 100 ng/ml ERr 731 and the single compounds significantly induced the ERbeta-coupled luciferase activity in a range comparable to 10(-8)M 17beta-estradiol. All effects were abolished with the pure ER antagonist ICI 182780, indicating an ER-specific effect. The ERbeta agonistic activity by ERr 731 could be of importance for its clinical use, as central functions relevant to climacteric complaints are proposed to be mediated via ERbeta activation.