Hepatic steroid sulfatase critically determines estrogenic activities of conjugated equine estrogens in human cells in vitro and in mice.

Conjugated equine estrogens (CEEs), whose brand name is Premarin, are widely used as a hormone-replacement therapy (HRT) drug to manage postmenopausal symptoms in women. Extracted from pregnant mare urine, CEEs are composed of nearly a dozen estrogens existing in an inactive sulfated form. To determine whether the hepatic steroid sulfatase (STS) is a key contributor to the efficacy of CEEs in HRT, we performed estrogen-responsive element (ERE) reporter gene assay, real-time PCR, and UPLC-MS/MS to assess the STS-dependent and inflammation-responsive estrogenic activity of CEEs in HepG2 cells and human primary hepatocytes. Using liver-specific STS-expressing transgenic mice, we also evaluated the effect of STS on the estrogenic activity of CEEs in vivo We observed that CEEs induce activity of the ERE reporter gene in an STS-dependent manner and that genetic or pharmacological inhibition of STS attenuates CEE estrogenic activity. In hepatocytes, inflammation enhanced CEE estrogenic activity by inducing STS gene expression. The inflammation-responsive estrogenic activity of CEEs, in turn, attenuated inflammation through the anti-inflammatory activity of the active estrogens. In vivo, transgenic mice with liver-specific STS expression exhibited markedly increased sensitivity to CEE-induced estrogenic activity in the uterus resulting from increased levels of liver-derived and circulating estrogens. Our results reveal a critical role of hepatic STS in mediating the hormone-replacing activity of CEEs. We propose that caution needs to be applied when Premarin is used in patients with chronic inflammatory liver diseases because such patients may have heightened sensitivity to CEEs due to the inflammatory induction of STS activity.

Adenosine Administration With a Stopcock Technique Delivers Lower-Than-Intended Drug Doses.

STUDY OBJECTIVE: Adenosine administration with a stopcock is the recommended treatment for pediatric patients with acute supraventricular tachycardia. Recent reports suggest that many infants do not respond to the first dose of adenosine administered. Our aim is to determine whether administration of adenosine with a stopcock delivers lower-than-expected drug doses in patients weighing less than 10 kg, corresponding to weights of infants. METHODS: We developed an in vitro model of adenosine delivery. Doses of adenosine corresponding to weights 2 to 25 kg were calculated, using a dose of 0.1 mg/kg, and administered through one port of a stopcock. Distilled water was administered through the second port. The adenosine concentration of the output was measured with mass spectrometry and results were confirmed with spectrophotometry of Evans blue. RESULTS: The mean doses of adenosine delivered through the stopcock increased as weight increased. The mean dose of adenosine delivered was 0.08 mg/kg for weights 2 to 9 kg and 0.1 mg/kg for weights 10 to 25 kg (95% confidence interval for difference of means -0.03 to -0.009). The median dose of adenosine delivered was 0.07 mg/kg (interquartile range [IQR] 0.06 to 0.07 mg/kg), 0.09 mg/kg (IQR 0.08 to 0.09 mg/kg), and 0.1 mg/kg (IQR 0.09 to 0.1 mg/kg) for weights 2 to 5, 6 to 9, and 10 to 25 kg, respectively (rank difference=100; P<.05 for 2 to 5 kg versus 10 to 25 kg). Similar results were obtained with spectrophotometry. CONCLUSION: Administration of adenosine through a stopcock delivers doses lower than intended in patients weighing less than 10 kg, which may account for the decreased response of infants to the first dose of adenosine.

Development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry method to quantify antiretroviral drug concentrations in human plasma for therapeutic monitoring.

Antiretroviral therapy (ART) is highly effective for the treatment of HIV-1 infection. ART previously consisted of concomitant administration of many drugs, multiple times per day. Currently, ART generally consists of two- or three-drug regimens once daily as fixed-dose combinations. Drug monitoring may be necessary to ensure adequate concentrations are achieved in the plasma over the dosing interval and prevent further HIV resistance formation. Additionally, nonadherence remains an issue, highlighting the need to ensure sufficient ART exposure. Towards this effort, we developed and validated a highly selective ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of a panel of nine antiretrovirals: abacavir, bictegravir, cabotegravir, dolutegravir, doravirine, emtricitabine, lamivudine, raltegravir, and tenofovir in human plasma. Using only 50 µL of plasma, a simple protein precipitation with acetonitrile with internal standards followed by reconstitution in 50 uL (high) or 400 uL (low) was performed. Analyte separation was achieved using a multistep UPLC gradient mixture of (A: 0.1% formic acid in water and B: acetonitrile) and a Waters CORTECS T3 (2.1 ×100 mm) column. The method was comprehensively validated according to the United States Food and Drug Administration Bioanalytical Guidelines over two clinically relevant ranges (1-250 ng/mL and 100-5000 ng/mL) with excellent linearity (R2 > 0.99 for all). The assay run time was 7.5 min. This method achieves acceptable performance of trueness (89.7-104.1%), repeatability, and precision (CV <15%), and allows for simultaneous quantification of guideline-recommended ART regimens. This method can be utilized for the therapeutic monitoring of antiretrovirals in human plasma.

Simultaneous quantification of five antiretrovirals in human tissues using ultra-high performance liquid chromatography-tandem mass spectrometry methods for therapeutic drug monitoring at the sites of action.

Although antiretroviral therapy (ART) is highly effective for the treatment of HIV-1 infection to suppress virus in the blood, HIV persists in tissues. HIV persistence in the tissues is due to numerous factors, and one of those factors are antiretroviral (ARV) concentrations. ARV concentrations in tissues must be adequate to suppress HIV at the sites of action. While therapeutic drug monitoring in the plasma is well-known, drug monitoring in the tissues provides local assessments of adequate ARV exposure to prevent localized HIV resistance formation. Towards these efforts, we validated an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method in human tissues (cervical, rectal, and vaginal tissues) for the simultaneous quantification of five ARVs: bictegravir, cabotegravir, dolutegravir, doravirine, and raltegravir. For this assay, protein precipitation with acetonitrile with stable, isotopically-labeled internal standards followed by supernatant pre-concentration was performed. Analyte separation was accomplished using a multistep UPLC gradient mixture of 0.1 % formic acid in water (A) and acetonitrile (B) with a Waters Cortecs T3 (2.1x100 mm) column. The assay was extensively validated as per the United States Food and Drug Administration Bioanalytical Method Validation Guidance over a clinically observed range (0.05-50 ng/mL) with superb linearity (R2 > 0.99 across all ARVs). The assay run time was 8.5 min. This analytical method achieves appropriate performance of trueness (85.5-107.4 %), repeatability, and precision (CV < 15 %). Our method will be employed for the therapeutic monitoring of guideline-recommended ARVs in human tissues for monitoring therapeutic efficacy in HIV treatment and prevention research efforts.

Systemic and local chronic inflammation and hormone disposition promote a tumor-permissive environment for breast cancer in older women.

Estrogen receptor positive (ER+) breast cancer is the most common subtype of breast cancer and is an age-related disease. The peak incidence of diagnosis occurs around age 70, even though these post-menopausal patients have low circulating levels of estradiol (E2). Despite the hormone sensitivity of age-related tumors, we have a limited understanding of the interplay between systemic and local hormones, chronic inflammation, and immune changes that contribute to the growth and development of these tumors. Here, we show that aged F344 rats treated with the dimethylbenz(a)anthracene / medroxyprogestrone acetate (DMBA/MPA) carcinogen develop more tumors at faster rates than their younger counterparts, suggesting that the aged environment promotes tumor initiation and impacts growth. Single-nuclei RNA-seq (snRNA-seq) of the tumors showed broad local immune dysfunction that was associated with circulating chronic inflammation. Across a broad cohort of specimens from patients with ER+ breast cancer and age-matched donors of normal breast tissue, we observe that even with an estrone (E1)-predominant estrogen disposition in the systemic circulation, tumors in older patients increase HSD17B7 expression to convert E1 to E2 in the tumor microenvironment (TME) and have local E2 levels similar to pre-menopausal patients. Concurrently, trackable increases in several chemokines, defined most notably by CCL2, promote a chronically inflamed but immune dysfunctional TME. This unique milieu in the aged TME, characterized by high local E2 and chemokine-enriched chronic inflammation, promotes both accumulation of tumor-associated macrophages (TAMs), which serve as signaling hubs, as well as polarization of TAMs towards a CD206+/PD-L1+, immunosuppressive phenotype. Pharmacologic targeting of estrogen signaling (either by HSD17B7 inhibition or with fulvestrant) and chemokine inflammation both decrease local E2 and prevent macrophage polarization. Overall, these findings suggest that chronic inflammation and hormonal disposition are critical contributors to the age-related nature of ER+ breast cancer development and growth and offer potential therapeutic insight to treat these patients. Translational Summary: We uncover the unique underpinnings establishing how the systemic host environment contributes to the aged breast tumor microenvironment, characterized by high local estradiol and chronic inflammation with immune dysregulation, and show that targeting avenues of estrogen conversion and chronic inflammation work to restore anti-tumor immunity.

Hypothermia decreases cerebrospinal fluid asymmetric dimethylarginine levels in children with traumatic brain injury.

OBJECTIVES: Pathological increases in asymmetric dimethylarginine, an endogenous nitric oxide synthase inhibitor, have been implicated in endothelial dysfunction and vascular diseases. Reduced nitric oxide early after traumatic brain injury may contribute to hypoperfusion. Currently, methods to quantify asymmetric dimethylarginine in the cerebrospinal fluid have not been fully explored. We aimed to develop and validate a method to determine asymmetric dimethylarginine in the cerebrospinal fluid of a pediatric traumatic brain injury population and to use this method to assess the effects of 1) traumatic brain injury and 2) therapeutic hypothermia on this mediator. DESIGN, SETTING, AND PATIENTS: An ancillary study to a prospective, phase II randomized clinical trial of early hypothermia in a tertiary care pediatric intensive care unit for children with Traumatic brain injury admitted to Children's Hospital of Pittsburgh. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A UPLC-MS/MS method was developed and validated to quantitate asymmetric dimethylarginine. A total of 56 samples collected over 3 days with injury onset were analyzed from the cerebrospinal fluid of consented therapeutic hypothermia (n = 9) and normothermia (n = 10) children. Children undergoing diagnostic lumbar puncture (n = 5) were enrolled as controls. Asymmetric dimethylarginine was present at a quantifiable level in all samples. Mean asymmetric dimethylarginine levels were significantly increased in normothermic Traumatic brain injury children compared with that in control (0.19 ± 0.08 µmol/L and 0.11 ± 0.02 µmol/L, respectively, p = 0.01), and hypothermic children had significantly reduced mean asymmetric dimethylarginine levels (0.11 ± 0.05 µmol/L) vs. normothermic (p = 0.03) measured on day 3. Patient demographics including age, gender, and nitric oxide levels (measured as nitrite and nitrate using liquid chromatography coupled with Griess reaction) did not significantly differ between normothermia and hypothermia groups. Also, nitric oxide levels did not correlate with asymmetric dimethylarginine concentrations. CONCLUSIONS: Asymmetric dimethylarginine levels were significantly increased in the cerebrospinal fluid of traumatic brain injury children. Early hypothermia attenuated this increase. The implications of attenuated asymmetric dimethylarginine on nitric oxide synthases activity and regional cerebral blood flow after traumatic brain injury by therapeutic hypothermia deserve future study.

Development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry method for quantifying lenacapavir plasma concentrations: Application to therapeutic monitoring.

Although current antiretroviral therapy (ART) effectively suppresses HIV in the blood, regimens may fail due to suboptimal treatment history and non-adherence to ART. In these scenarios, accumulation of viral resistance mutations to ART drug classes may occur. For these treatment-experienced people living with HIV (PLWH), activity against resistant viral strains is required; lack of therapeutic efficacy will result in continued viral replication and progression to acquired immunodeficiency syndrome. New treatment options have emerged. Lenacapavir is a first-in-class long-acting HIV-1 capsid inhibitor approved for the treatment of HIV in treatment-experienced patients. Lenacapavir is approved with an initiation regimen of oral and subcutaneous injection dosing followed by subcutaneous self-injection every 6 months. With infrequent dosing, therapeutic drug monitoring may be necessary to ensure adequate concentrations are consistently achieved in the plasma to assure treatment adherence and prevent further HIV resistance formation. To this end, we developed and validated a highly selective ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify lenacapavir concentrations in human plasma. A simple protein precipitation with acetonitrile followed by supernatant dilution was performed. Lenacapavir and its stable labeled internal standard were separated at 1.90 min using a multi-step UPLC gradient. The assay for lenacapavir quantification was extensively validated according to the United States Food and Drug Administration Bioanalytical Guidelines over a clinically relevant range of 0.1 to 500 ng/mL with excellent linearity (R2 ≥ 0.9960). This analytical method achieves acceptable performance of trueness (89.7-104.1 %), repeatability, and precision (CV < 15 %). We applied this method to quantify a clinical sample and to determine the percent protein-unbound. This method can be utilized for the therapeutic monitoring of lenacapavir in human plasma for monitoring HIV treatment efficacy.

Clinical rel mutations in Staphylococcus aureus prime pathogen expansion under nutrient stress.

Persistent infection by Staphylococcus aureus has been linked to the bacterial stringent response (SR), a conserved stress response pathway regulated by the Rel protein. Rel synthesizes (p)ppGpp "alarmones" in response to amino acid starvation, which enables adaptation to stress by modulating bacterial growth and virulence. We previously identified five novel protein-altering mutations in rel that arose in patients with persistent methicillin-resistant S. aureus bacteremia. The mutations mapped to both the enzymatic and regulatory protein domains of Rel. Here, we set out to characterize the phenotype of these mutations to understand how they may have been selected in vivo. After introducing each mutation into S. aureus strain JE2, we analyzed growth, fitness, and antibiotic profiles. Despite being located in different protein domains, we found that all of the mutations converged on the same phenotype. Each shortened the time of lag phase growth and imparted a fitness advantage in nutritionally depleted conditions. Through quantification of intracellular (p)ppGpp, we link this phenotype to increased SR activation, specifically during the stationary phase of growth. In contrast to two previously identified clinical rel mutations, we find that our rel mutations do not cause antibiotic tolerance. Instead, our findings suggest that in vivo selection was due to an augmented SR that primes cells for growth in nutrient-poor conditions, which may be a strategy for evading host-imposed nutritional immunity. Importance Host and pathogen compete for available nutrition during infection. For bacteria, the stringent response (SR) regulator Rel responds to amino acid deprivation by signaling the cell to modulate its growth rate, metabolism, and virulence. In this report, we characterize five rel mutations that arose during cases of persistent methicillin-resistant Staphylococcus aureus bacteremia. We find that all of the mutations augmented SR signaling specifically under nutrient-poor conditions, enabling the cell to more readily grow and survive. Our findings reveal a strategy used by bacterial pathogens to evade the nutritional immunity imposed by host tissues during infection.

Hepatic Estrogen Sulfotransferase Distantly Sensitizes Mice to Hemorrhagic Shock-Induced Acute Lung Injury.

Hemorrhagic shock (HS) is a potential life-threatening condition that may lead to injury to multiple organs, including the lung. The estrogen sulfotransferase (EST, or SULT1E1) is a conjugating enzyme that sulfonates and deactivates estrogens. In this report, we showed that the expression of Est was markedly induced in the liver but not in the lung of female mice subject to HS and resuscitation. Genetic ablation or pharmacological inhibition of Est effectively protected female mice from HS-induced acute lung injury (ALI), including interstitial edema, neutrophil mobilization and infiltration, and inflammation. The pulmonoprotective effect of Est ablation or inhibition was sex-specific, because the HS-induced ALI was not affected in male Est-/- mice. Mechanistically, the pulmonoprotective phenotype in female Est-/- mice was accompanied by increased lung and circulating levels of estrogens, attenuated pulmonary inflammation, and inhibition of neutrophil mobilization from the bone marrow and neutrophil infiltration to the lung, whereas the pulmonoprotective effect was abolished upon ovariectomy, suggesting that the protection was estrogen dependent. The pulmonoprotective effect of Est ablation was also tissue specific, as loss of Est had little effect on HS-induced liver injury. Moreover, transgenic reconstitution of human EST in the liver of global Est-/- mice abolished the pulmonoprotective effect, suggesting that it is the EST in the liver that sensitizes mice to HS-induced ALI. Taken together, our results revealed a sex- and tissue-specific role of EST in HS-induced ALI. Pharmacological inhibition of EST may represent an effective approach to manage HS-induced ALI.

Sensitizing Triple Negative Breast Cancer to Tamoxifen Chemotherapy via a Redox-Responsive Vorinostat-containing Polymeric Prodrug Nanocarrier.

There is an urgent and unmet need to develop effective therapies for triple negative breast cancers (TNBCs) which are much more aggressive and have poor prognosis due to lack of receptor targets for Her2-targeted and endocrine therapy. In this study we systematically evaluated the effect of Vorinostat (SAHA, a pan-HDAC inhibitor) in reactivating the expression of functional estrogen receptor α (ERα) and synergizing with tamoxifen (TAM, a selective estrogen-receptor modulator) in antitumor activity. In addition, a SAHA prodrug-based dual functional nanocarrier was developed for codelivery of SAHA and TAM for effective combination therapy. Methods: A SAHA-containing polymeric nanocarrier, POEG-co-PVDSAHA was developed via reversible addition-fragmentation transfer (RAFT) polymerization with SAHA incorporated into the polymer through a redox-responsive disulfide linkage. The effect of both free SAHA and POEG-co-PVDSAHA on reactivating the expression of functional ERα was investigated in several human and murine TNBC cell lines via examining the mRNA and protein expression of ERα target genes. The cytotoxicity of free SAHA and TAM combination and TAM-loaded POEG-co-PVDSAHA micelles was examined via MTT assay. The in vivo antitumor activity of TAM-loaded POEG-co-PVDSAHA was investigated in a murine breast cancer model (4T1.2). Results: Both free SAHA and POEG-co-PVDSAHA were effective in inducing the reexpression of functional estrogen receptor α (ERα), which may have helped to sensitize TNBCs to TAM. More importantly, POEG-co-PVDSAHA self-assembled to form small-sized micellar carrier that is effective in formulating and codelivery of TAM. TAM-loaded POEG-co-PVDSAHA micelles exhibited enhanced and synergistic cytotoxicity against TNBC cell lines compared with free SAHA, free TAM and TAM loaded into a pharmacologically inert control carrier (POEG-co-PVMA). In addition, codelivery of TAM via POEG-co-PVDSAHA micelles led to significantly improved antitumor efficacy in 4T1.2 tumor model compared with other groups such as combination of free SAHA and TAM and TAM-loaded POEG-co-PVMA micelles. Conclusion: Our prodrug-based co-delivery system may provide an effective and simple strategy to re-sensitize TNBCs to TAM-based hormone therapy.

Melatonin inhibits cytosolic mitochondrial DNA-induced neuroinflammatory signaling in accelerated aging and neurodegeneration.

Chronic inflammation is a pathologic feature of neurodegeneration and aging; however, the mechanism regulating this process is not understood. Melatonin, an endogenous free radical scavenger synthesized by neuronal mitochondria, decreases with aging and neurodegeneration. We proposed that insufficient melatonin levels impair mitochondrial homeostasis, resulting in mitochondrial DNA (mtDNA) release and activation of cytosolic DNA-mediated inflammatory response in neurons. We found increased mitochondrial oxidative stress and decreased mitochondrial membrane potential, with higher mtDNA release in brain and primary cerebro-cortical neurons of melatonin-deficient aralkylamine N-acetyltransferase (AANAT) knockout mice. Cytosolic mtDNA activated the cGAS/STING/IRF3 pathway, stimulating inflammatory cytokine generation. We found that Huntington's disease mice had increased mtDNA release, cGAS activation, and inflammation, all inhibited by exogenous melatonin. Thus, we demonstrated that cytosolic mtDNA activated the inflammatory response in aging and neurodegeneration, a process modulated by melatonin. Furthermore, our data suggest that AANAT knockout mice are a model of accelerated aging.

A sensitive and robust UPLC-MS/MS method for quantitation of estrogens and progestogens in human serum.

OBJECTIVE: With the widespread use of sex-steroid hormones in contraceptives and hormone replacement therapy, there is an increasing need for reliable analytical methods. We report the development of a sensitive and robust UPLC-MS/MS method for quantitation of both endogenous and synthetic sex-steroid hormones in human serum. STUDY DESIGN: We developed and validated a UPLC-MS/MS method to quantify progestogens (etonogestrel, levonorgestrel, medroxyprogesterone acetate, norethindrone, progesterone) and estrogens (estradiol and ethinyl estradiol) with good accuracy, high sensitivity, and excellent robustness. We then applied the method to the analysis of sex-steroid hormones in serum from 451 clinical research participants. RESULTS: Each UPLC-MS/MS analysis was 6.5 min. The lower limits of quantitation (LLOQs) were 25 pg/ml for the progestogens, and 2.5 and 5.0 pg/ml for estradiol and ethinyl estradiol, respectively. When estradiol was analyzed without assessment of progestogens, the LLOQ was reduced to 1 pg/ml. The calibration curves were linear from 25-50,000, 2.5-2000 (1-2000 for estrogens-only analysis) and 5-2000 pg/ml, respectively. Both the accuracy and precision were below±15% not only for routine validation (intraday and interday), but for long-term (>2 years) assay robustness with external controls, thereby, demonstrating the utility of this method for multi-year clinical trial assessments of progestogens and estrogens. We applied the method to quantify sex-steroid levels in 1804 clinical samples. CONCLUSIONS: We successfully developed a UPLC-MS/MS method, and overcame the matrix suppression to allow sensitive quantitation of both synthetic and endogenous sex-steroid hormones in human serum. IMPLICATIONS: We developed a sensitive and robust UPLC-MS/MS method to accurately measure the levels of sex-steroid hormones in serum. The method overcame matrix interference barriers and achieved excellent long-term stability and reproducibility (≥96.9% accuracy; ≤13.0% relative variability measured with external controls over 2 years), demonstrating its utility in clinical sample analysis.

A microsomal based method to detect aromatase activity in different brain regions of the rat using ultra performance liquid chromatography-mass spectrometry.

Aromatase (ARO) is a cytochrome P450 enzyme that accounts for local estrogen production in the brain. The goal of this study was to develop a microsomal based assay to sensitively and reliably detect the low levels of ARO activity in different brain regions. Enzyme activity was detected based on the conversion of testosterone to estradiol. Quantity of estradiol was measured using ultra performance liquid chromatography-mass spectrometry. Detection was linear over a range of 2.5-200pg/ml estradiol, and was reproducible with intra- and inter-assay coefficients of variation (CV) <15%. Estradiol production using isolated microsomes was linear with time up to 30min as well as linearly related to amount of microsome. Substrate concentration curves revealed enzymatic kinetics (hippocampus: Vmax and Km: 0.57pmol estradiol/h per mg microsome and 48.58nM; amygdala: Vmax and Km: 1.69pmol estradiol/h per mg microsome and 48.4nM; preoptic area: Vmax and Km: 0.96pmol estradiol/h per mg microsome and 44.31nM) with testosterone used at a saturating concentration of 400nM. Anastrozole treatment blocked ARO activity in hippocampal and ovarian microsomes, indicating that the assay is specific for ARO. Also, we showed that the distribution of the long form ARO mRNA (CYP19A1) in different regions of the brain is correlated with ARO activity, with highest levels in the amygdala, followed by preoptic area and hippocampus. In the frontal cortex, very little long form ARO mRNA, and little to no ARO activity, were detected. These findings demonstrate that the microsomal incubation (MIB) assay is a sensitive and reliable method for quantifying ARO activity in discrete brain regions.