Perioperative Marinobufagenin (MBG) Measurement May Improve Acute Kidney Injury Risk Assessment in Patients Undergoing Major Cardiac Surgery: A Proof-of-Concept Study.

Background and Objectives: Acute kidney injury (AKI) remains a significant complication following major cardiac surgery. Marinobufagenin (MBG), a cardiotonic steroid involved in sodium balance and blood pressure regulation, has been linked to organ damage after ischemia-reperfusion events. This pilot, prospective study investigates the utility of circulating MBG to improve AKI risk assessment in cardiac surgery patients as a stand-alone biomarker and after inclusion in a validated risk model (STS-AKI score). Materials and Methods: We included 45 patients undergoing elective cardiac surgery. The MBG levels were measured preoperatively and at 4, 8, and 12 h post-surgery. The AKI was defined according to the KDIGO guidelines. Statistical analyses assessed the diagnostic and prognostic utility of MBG and its integration with the STS-AKI score. Results: An AKI occurred in 26.7% of the patients. The STS-AKI score performed well in this cohort (AUC: 0.736). The MBG levels displayed a decreasing trend in the whole population after surgery (p = 0.02). However, in the AKI patients, MBG increased at 4 and 8 h before decreasing at 12 h post-surgery. The MBG changes from the baseline to 8 h and from 8 to 12 h post-surgery showed a remarkable diagnostic accuracy for an AKI (AUCs: 0.917 and 0.843, respectively). Integrating these MBG changes with the STS-AKI score significantly improved the model performance, including discrimination, calibration, and risk reclassification. Conclusions: The MBG measurement, particularly any dynamic changes post-surgery, enhances AKI risk stratification in cardiac surgery patients. Integrating MBG with the STS-AKI score offers more accurate risk predictions, potentially leading to better patient management and outcomes.

The Effects of Short-Term Changes in Sodium Intake on Plasma Marinobufagenin Levels in Patients with Primary Salt-Sensitive and Salt-Insensitive Hypertension.

Increased marinobufagenin (MBG) synthesis has been suggested in response to high dietary salt intake. The aim of this study was to determine the effects of short-term changes in sodium intake on plasma MBG levels in patients with primary salt-sensitive and salt-insensitive hypertension. In total, 51 patients with primary hypertension were evaluated during acute sodium restriction and sodium loading. Plasma or serum concentrations of MBG, natriuretic pro-peptides, aldosterone, sodium, potassium, as well as hematocrit (Hct) value, plasma renin activity (PRA) and urinary sodium and potassium excretion were measured. Ambulatory blood pressure monitoring (ABPM) and echocardiography were performed at baseline. In salt-sensitive patients with primary hypertension plasma MBG correlated positively with diastolic blood pressure (ABPM) and serum NT-proANP concentration at baseline and with serum NT-proANP concentration after dietary sodium restriction. In this subgroup plasma MBG concentration decreased during sodium restriction, and a parallel increase of PRA was observed. Acute salt loading further decreased plasma MBG concentration in salt-sensitive subjects in contrast to salt insensitive patients. No correlation was found between plasma MBG concentration and left ventricular mass index. In conclusion, in salt-sensitive hypertensive patients plasma MBG concentration correlates with 24-h diastolic blood pressure and dietary sodium restriction reduces plasma MBG levels. Decreased MBG secretion in response to acute salt loading may play an important role in the pathogenesis of salt sensitivity.

The endogenous cardiotonic steroid Marinobufagenin and decline in estimated glomerular filtration rate at follow-up in patients with arterial hypertension.

BACKGROUND: Marinobufagenin (MBG) is an endogenous cardiotonic steroid (CTS) that inhibits the Na+/K+-ATPase. Human MBG is significantly increased in end-stage renal disease and immunization against MBG attenuates cardiovascular fibrosis in a rat model of uremic cardiomyopathy. Mineralocorticoid antagonists (MRA) block MBG binding sites and decrease proteinuria in chronic kidney disease (CKD) patients. We therefore aimed to investigate the association of MBG and albuminuria, as a marker of renal damage, as well as MBG and decline of glomerular filtration rate (GFR). METHODS: The Graz endocrine causes of hypertension (GECOH) study is a single center study of adults routinely referred for screening of endocrine hypertension. Plasma MBG was measured by an enzyme-linked immunoassay, and in a post-hoc analysis, follow-up creatinine levels were obtained. Patients with proteinuria >3.5g/day at baseline were excluded from further evaluation. RESULTS: We measured MBG concentrations in 40 hypertensive subjects and excluded one patient due to pre-existing proteinuria. Plasma MBG was significantly correlated with albuminuria (Spearman ρ = .357; p = .028) and proteinuria (ρ = .336; p = .039). In linear regression analysis, the association remained significant after adjustment for age, sex, and BMI (ß = .306; p = .036), and for mean systolic blood pressure (ß = .352; p = .034). In follow-up analyses (N = 30), MBG was significantly associated with decline in GFR after adjustment for time-to-follow-up (ß = -.374; p = .042). CONCLUSION: The findings suggest that MBG plasma concentrations were associated with albuminuria as well as decline in kidney function. Whether MBG predicts hard renal endpoints warrants further investigations.

In situ phase transition of microemulsions for parenteral injection yielding lyotropic liquid crystalline carriers of the antitumor drug bufalin.

In this work, we used the small angle X-ray scattering (SAXS) method for controlled preparation of in situ forming sustained-release carriers for the antitumor drug bufalin (BUF), which has very poor solubility and a considerable cardiotoxicity in a non-encapsulated state. To that aim, we exploited the pseudo-ternary phase diagram of an oil(O)/surfactant(S)/water(W) system containing medium chain capric/caprylic triglycerides (MCT) and a co-surfactant blend of Macrogol (15)-hydroxystearate (Solutol HS 15) and sorbitan monooleate (Span 80). Two compositions with different oil contents (sample B and C) were selected from the microemulsion region of the phase diagram in order to study the effect of the aqueous environment on their structural behavior. A phase transition from a microemulsion (ME) to a liquid crystalline phase (LC) was established by SAXS upon progressive dilution. The drug bufalin (BUF) was encapsulated in the microemulsions with low viscosity, whereas the release of the drug occurred from the in situ generated lamellar liquid crystalline structures. The formulations were characterized by SAXS, dynamic light scattering (DLS), cryogenic transmission electron microscopy (Cryo-TEM), rheology, drug loading and encapsulation efficiency, and in vitro release profiles. A correlation was suggested between the structures of the in situ phase-transition formed LCME formulations, the differences in their viscosities and drug release profiles. The performed cytotoxicity, cell apoptosis and pharmacokinetic experiments showed an enhanced bioavailability of BUF after encapsulation. These results suggest potential clinical applications for the obtained safe in situ phase-transition sustained-release formulations of BUF.

Antibody to Marinobufagenin Reverses Placenta-Induced Fibrosis of Umbilical Arteries in Preeclampsia.

BACKGROUND: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Immunoneutralization of heightened MBG by Digibind, a digoxin antibody, reduces blood pressure (BP) in patients with PE, and anti-MBG monoclonal antibody lessens BP in a rat model of PE. Recently, we demonstrated that MBG induces fibrosis in cardiovascular tissues via a mechanism involving inhibition of Fli-1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. OBJECTIVES AND METHODS: We hypothesized that in PE, elevated placental MBG levels are associated with development of fibrosis in umbilical arteries. Eleven patients with PE (mean BP 124 ± 4 mmHg; age 29 ± 2 years; 39 weeks gest. age) and 10 gestational age-matched normal pregnant subjects (mean BP 92 ± 2 mmHg; controls) were enrolled in the clinical study. RESULTS: PE was associated with a higher placental (0.04 ± 0.01 vs. 0.49 ± 0.11 pmol/g; p < 0.01) and plasma MBG (0.5 ± 0.1 vs. 1.6 ± 0.5 nmol/L; p < 0.01), lower Na/K-ATPase activity in erythrocytes (2.7 ± 0.2 vs. 1.5 ± 0.2 µmol Pi/mL/hr; p < 0.01), 9-fold decrease of Fli-1 level and 2.5-fold increase of collagen-1 in placentae (p < 0.01) vs. control. Incubation of umbilical arteries from control patients with 1 nmol/L MBG was associated with four-fold decrease in Fli-1 level and two-fold increase in collagen-1 level vs. those incubated with placebo (p < 0.01), i.e., physiological concentration of MBG mimicked effect of PE in vitro. Collagen-1 abundance in umbilical arteries from PE patients was 4-fold higher than in control arteries, and this PE-associated fibrosis was reversed by monoclonal anti-MBG antibody ex vivo. CONCLUSION: These results demonstrate that elevated placental MBG level is implicated in the development of fibrosis of the placenta and umbilical arteries in PE.

Plasma marinobufagenin immunoreactivity in patients with chronic kidney disease: a case control study.

Experimental data have shown increased plasma levels of marinobufagenin in kidney failure. In this case-controlled retrospective analysis, we evaluated plasma marinobufagenin immunoreactivity in hemodialysis patients compared with subjects with normal kidney function. Sixty-eight adult hemodialysis patients with chronic kidney disease (34 females and 34 males) as well as 68 age-, gender-, and blood pressure-matched subjects without chronic kidney disease were enrolled. Patients on stable hemodialysis regimen for at least 3 mo before the study were included. Exclusion criteria were: age <18 yr, severe liver or heart insufficiency, and overhydration. Subjects without chronic kidney disease must have had an estimated glomerular filtration rate ≥60 ml·min-1·1.72 m-2 according to the Modification of Diet in Renal Disease formula. Plasma marinobufagenin immunoreactivity was significantly ( P < 0.001) higher in hemodialysis patients (1.66 ± 1.13 nmol/l) compared with subjects with normal kidney function (0.46 ± 0.23). In hemodialysis patients, plasma marinobufagenin immunoreactivity was higher in men compared with women. A significant positive correlation has been found between plasma marinobufagenin immunoreactivity and serum NT-proBNP, NT-proANP, or aldosterone concentrations in all analyzed subjects. In hemodialyzed patients with plasma marinobufagenin immunoreactivity above median value 5-yr, all-cause mortality was higher compared with those with plasma marinobufagenin concentration below median. We have shown that plasma marinobufagenin immunoreactivity is increased in patients with end-stage kidney failure treated with hemodialysis parallel to the increase in serum NT-proBNP, NT-proANP, and aldosterone concentrations. Higher marinobufagenin immunoreactivity has been associated with worse survival in hemodialyzed patients.

Revealing of endogenous Marinobufagin by an ultra-specific and sensitive UHPLC-MS/MS assay in pregnant women.

Marinobufagenin (MBG) is a bufadienolide cardiac inotrope implicated in volume expansion-mediated hypertensive states including essential hypertension and preeclampsia (PE). Endogenous MBG is an inhibitor of the α1-isoform of Na+,K+-ATPase with vasoconstrictive and cardiotonic properties, causing hypertension and natriuresis. Elevated endogenous MBG-like material levels have been described by immunoassays in salt-sensitive pregnant and preeclamptic rats as well as in preeclamptic human patients. The rise of endogenous MBG-like material appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential biomarkers for PE. The weak specificity and the high variability of the published immunoassays gives no certification about endogenous MBG existence. This led us to set-up a highly specific and sensitive analytical method to detect MBG in plasma at low levels relying on liquid chromatography combined to mass spectrometry (UHPLC-MS/MS) with recording of 7 highly specific MRM transitions for MBG. Pure MBG standard used in the method development was obtained by purification from the Bufo marinus toad venom. d3-25-hydroxyvitamin D3 was used as internal standard. An increasing organic gradient with mobile phase A and B composed of 97:3 (v/v) H2O: MeOH and 50:45:5 (v/v/v) MeOH:IPA:H2O at pH 4.5 respectively was used on a Pursuit 3 PFP column (100 mm × 3 mm; 3 µm) to allow elution and separation of the plasmatic compounds. Chromatographic analyses of plasma samples were preceded by a precipitation of proteins pretreatment. The developed UHPLC-MS/MS assay has been applied to early-pregnant women plasma samples allowing us to investigate MBG plasma levels. Thanks to the high specificity of the assay we were able to authenticate and certify the presence of endogenous MBG in early-pregnant women plasma with the use of the 7 selected specific mass transitions. These pioneering preliminary results are giving a promising perspective for early preeclampsia risk assessment in pregnant women.

Genetic Control of Serum Marinobufagenin in the Spontaneously Hypertensive Rat and the Relationship to Blood Pressure.

BACKGROUND: We have investigated serum levels of immunoreactive marinobufagenin (MBG) in 16- to 20-week-old spontaneously hypertensive rats (SHRs)-A3 and in the normotensive Wistar-Kyoto (WKY) rat strain in the absence of salt loading, and we have investigated the genetic control of serum MBG. METHODS AND RESULTS: We genotyped the F2 progeny of an SHR-A3×WKY intercross using a genome-wide panel of 253 single-nucleotide polymorphism markers that were dimorphic between SHR-A3 and WKY and measured serum MBG by ELISA. Serum MBG levels were lower in SHR-A3 than WKY rats (0.39±0.07 and 1.27±0.40 nmol/L, respectively), suggesting that MBG may not play a role in the markedly divergent blood pressure measured by telemetry in rats of these 2 strains (SHR-A3 and WKY, 198.3±4.43 and 116.8±1.51 mm Hg, respectively). The strain difference in serum MBG was investigated to determine whether genomic regions influencing MBG might be identified by genetic mapping. Quantitative trait locus mapping indicated a single locus influencing serum MBG in the region of chromosome 6q12. Homozygosity of WKY alleles at this locus was associated with increased serum MBG levels. We surveyed whole genome sequences from our SHR-A3 and WKY lines, seeking coding sequence variation between SHR-A3 and WKY within the mapped locus that might explain the inherited strain difference in serum MBG. CONCLUSIONS: We identified amino acid substitution in the sterol transport protein Abcg5, present in SHR-A3, but absent in WKY, that is a potential mechanism influencing MBG levels.

Involvement of the Bufadienolides in the Detection and Therapy of the Acute Respiratory Distress Syndrome.

PURPOSE: The acute respiratory distress syndrome (ARDS) represents a major challenge for clinicians as well as basic scientists. The mortality rate for ARDS has been maintained within the range of 40-52%. The authors have examined the involvement of the "cardiotonic steroids" in the pathogenesis and therapy of ARDS. We have studied the possible role of the bufadienolide, marinobufagenin (MBG), in the pathogenesis of ARDS in both a rat model of ARDS and in patients afflicted with that disorder. In addition, the potential therapeutic benefit of an antagonist of MBG, resibufogenin (RBG), in an animal model has been evaluated. METHOD: A syndrome resembling human ARDS was produced in the rat by exposing the animals to 100% oxygen for 48 h. In other animals, RBG was administered to these "hyperoxic" rats, and the serum MBG was measured. In human ICU patients, urinary samples were examined for levels of MBG, and the values were compared to those obtained from other ICU patients admitted with diagnoses other than ARDS. RESULTS: (1) Exposure of rats to hyperoxia produced a histologic picture which resembled that of human ARDS. (2) Serum levels of MBG in the "hyperoxic" rats substantially exceeded those obtained in animals exposed to ambient oxygen levels and were reduced to normal by RBG. (3) In ARDS patients, substantial elevations in urinary MBG were obtained compared to those in non-ARDS ICU patients. CONCLUSIONS: MBG may serve as an important biomarker for the development of ARDS, and RBG may represent a preventative/therapy in this disorder.

Simultaneous determination of ginsenosides and bufadienolides in rat plasma after the oral administration of Shexiang Baoxin Pill for pharmacokinetic study by liquid chromatography tandem mass spectrometry following solid-phase extraction.

A sensitive and reliable bioanalytical method was established for quantitati\ve and pharmacokinetic investigation of nine ginsenosides and seven bufadienolides in rat plasma after the oral administration of Shexiang Baoxin Pill by liquid chromatography-electrospray ionization tandem mass spectrometry, using tinidazole and digoxin as internal standards (ISTDs). All of the analytes and ISTDs obtained satisfactory recoveries by solid-phase extraction using an Oasis HLB µElution Plate, which was eluted with methanol and ethyl acetate successively, and chromatographic separation was achieved on a Shim-pack XR-ODSIIcolumn (75 × 2.0 mm, 2.2 µm) with gradient elution using a mixture of acetonitrile-0.1% formic acid solution (v/v) as the mobile phase at a flow rate of 0.3 mL/min. Detection was carried out by a triple-quadrupole tandem mass spectrometry with positive/negative ion switching multiple reaction monitoring mode. All analytes showed good linearity over a wide concentration range (r2 > 0.99). The lower limit of quantification was in the range 0.625-12.5 ng/mL for bufadienolides and 2-5.5 ng/mL for ginsenosides, and the mean recoveries of all analytes were in the range 78.29-99.35%. The intra- and inter-day precisions (RSD) were in the range 0.08-12.38% with the accuracies between 86.09 and 99.40%. The validated method was then successfully applied to pharmacokinetic study of the above 16 compounds in rat plasma. Pharmacokinetic results indicated that the developed extraction and analytical method could be employed as a rapid, effective technique for pharmacokinetic study of multiple components, especially various polarity that are difficult to extract simultaneously.

ASSOSIATION OF ENDOGENOUS CARDIOTONIC STEROIDS WITH SALT-SENSITIVITY OF BLOOD PRESSURE IN GEORGIAN POPULATION.

This investigation differentiates types of essential hypertension in a Georgian population as well as describes endogenous cardiotonic steroids in salt-sensitive and salt-resistant subjects. This case control study included 185 subjects: 94 cases with stage 1 essential hypertension (JNC7) naïve to antihypertensive treatment, and 91 controls. A salt-sensitivity test was used to dichotomize case and control groups into salt-sensitive and salt-resistant subgroups. Blood and urine samples were obtained to categorize participants as consuming high and low salt diets. Endogenous cardiotonic steroids, sodium and plasma-renin activity (PRA) were measured in both samples at the different sodium conditions. Determinants of circulating levels of endogenous sodium pump inhibitors were carried out using the ELISA and RIA methods; PRA was assessed by radioimmunoassay. Descriptive statistics were used to analyze the data. Differences in variables between sodium conditions were assessed using paired t-tests. Salt-sensitivity was found in 60.5% of the total population investigated, with a higher proportion in females. A statistically significant positive correlation was found between salt-sensitivity and age in females (r=0.262, p<0.01), and with 24-hour urine sodium concentration changes (r=0.334, p<0.01). A significant negative correlation was found between salt-sensitivity and PRA. At the high sodium condition, endogenous MBG and OU were high in salt-sensitive subjects compared to those who were salt-resistant. These compounds decreased with a low-salt diet in both salt-sensitive cases and controls but remained the same in salt-resistant individuals. The MBG and OU levels positively correlated with systolic blood pressure in salt-sensitive individuals but no variability was evident among salt-resistant subjects. Our results show that MBG and OU levels start to increase at the normotensive stage and sustained high concentrations can lead to elevated systolic blood pressure, a risk factor for arterial hypertension in salt-sensitive subjects.

The role of endogenous cardiotonic steroids in pathogenesis of cardiovascular and renal complications of arterial hypertension.

Endogenous cardiotonic steroids (CTS), also called digitalis-like factors, are a group of steroid hormones linking high salt intake and elevated blood pressure and in part responsible for target organ damage in arterial hypertension. CTS act primarily through their ability to inhibit the ubiquitous transport enzyme sodium-potassium adenosine triphosphatase (Na⁺/K⁺-ATPase). A portion of Na⁺/K⁺-ATPase does not seem to actively "pump" sodium and potassium but is closely associated with other key signaling proteins. Plasma concentration and urine excretion of CTS are increased in experimental models with volume expansion and on a high salt diet. Elevated plasma concentration of marinobufagenin has been shown in volume-expanded states such as essential hypertension, primary aldosteronism, chronic renal failure, congestive heart failure and pregnancy. In experimental models marinobufagenin induces heart and kidney fibrosis to the same extent as observed in uremia. Neutralization of marinobufagenin with antibodies prevents such heart remodeling. Expanding our understanding of this new class of hormones may lead to development of novel and effective therapeutic strategies in hypertensive patients with renal and cardiovascular complications.

Elevated Plasma Marinobufagenin, An Endogenous Cardiotonic Steroid, Is Associated With Right Ventricular Dysfunction and Nitrative Stress in Heart Failure.

BACKGROUND: Plasma levels of cardiotonic steroids are elevated in volume-expanded states, such as chronic kidney disease, but the role of these natriuretic hormones in subjects with heart failure (HF) is unclear. We sought to determine the prognostic role of the cardiotonic steroids marinobufagenin (MBG) in HF, particularly in relation to long-term outcomes. METHODS AND RESULTS: We first measured plasma MBG levels and performed comprehensive clinical, laboratory, and echocardiographic assessment in 245 patients with HF. All-cause mortality, cardiac transplantation, and HF hospitalization were tracked for 5 years. In our study cohort, median (interquartile range) MBG was 583 (383-812) pM. Higher MBG was associated with higher myeloperoxidase (r=0.42, P<0.0001), B-type natriuretic peptide (r=0.25, P=0.001), and asymmetrical dimethylarginine (r=0.32, P<0.001). Elevated levels of MBG were associated with measures of worse right ventricular function (RV s', r=-0.39, P<0.0001) and predicted increased risk of adverse clinical outcomes (MBG≥574 pmol/L: hazard ratio 1.58 [1.10-2.31], P=0.014) even after adjustment for age, sex, diabetes mellitus, and ischemic pathogenesis. In mice, a left anterior descending coronary artery ligation model of HF lead to increases in MBG, whereas infusion of MBG into mice for 4 weeks lead to significant increases in myeloperoxidase, asymmetrical dimethylarginine, and cardiac fibrosis. CONCLUSIONS: In the setting of HF, elevated plasma levels of MBG are associated with right ventricular dysfunction and predict worse long-term clinical outcomes in multivariable models adjusting for established clinical and biochemical risk factors. Infusion of MBG seems to directly contribute to increased nitrative stress and cardiac fibrosis.

Marinobufagenin-induced vascular fibrosis is a likely target for mineralocorticoid antagonists.

OBJECTIVE: Endogenous cardiotonic steroids, including marinobufagenin (MBG), stimulate vascular synthesis of collagen. Because mineralocorticoid antagonists competitively antagonize effect of cardiotonic steroids on the Na/K-ATPase, we hypothesized that spironolactone would reverse the profibrotic effects of MBG. METHODS: Experiment 1: Explants of thoracic aortae and aortic vascular smooth muscle cells from Wistar rats were cultured for 24 h in the presence of vehicle or MBG (100 nmol/l) with or without canrenone (10 µmol/l), an active metabolite of spironolactone. Experiment 2: In 16 patients (56 ±â€Š2 years) with resistant hypertension on a combined (lisinopril/amlodipine/hydrochlorothiazide) therapy, we determined arterial pressure, pulse wave velocity, plasma MBG, and erythrocyte Na/K-ATPase before and 6 months after addition of placebo (n = 8) or spironolactone (50 mg/day; n = 8) to the therapy. RESULTS: In rat aortic explants and in vascular smooth muscle cells, pretreatment with MBG resulted in a two-fold rise in collagen-1, and a marked reduction in the sensitivity of the aortic rings to the vasorelaxant effect of sodium nitroprusside following endothelin-1-induced constriction (EC50 = 480 ±â€Š67 vs. 23 ±â€Š3 nmol/l in vehicle-treated rings; P < 0.01). Canrenone blocked effects of MBG on collagen synthesis and restored sensitivity of vascular rings to sodium nitroprusside (EC50 = 17 ±â€Š1 nmol/l). Resistant hypertension patients exhibited elevated plasma MBG (0.42 ±â€Š0.07 vs. 0.24 ±â€Š0.03 nmol/l; P = 0.01) and reduced Na/K-ATPase activity (1.9 ±â€Š0.15 vs. 2.8 ±â€Š0.2 µmol Pi/ml per h, P < 0.01) vs. seven healthy individuals. Six-month administration of spironolactone, unlike placebo treatment, was associated with a decrease in pulse wave velocity and arterial pressure, and with restoration of Na/K-ATPase activity in the presence of unchanged MBG levels. CONCLUSION: MBG-induced vascular fibrosis is a likely target for spironolactone.

Pharmacokinetics and tissue distribution of five bufadienolides from the Shexiang Baoxin Pill following oral administration to mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Shexiang Baoxin Pill (SBP) is a well-known composite formula of traditional Chinese medicine (TCM), widely used to treat cardiovascular diseases such as angina pectoris and myocardial infarction. Bufadienolides are major active compounds of Venenum Bufonis, which is one of the seven materiamedicas that comprise the Shexiang Baoxin Pill. Previous pharmacokinetics studies of bufadienolides have typically used a single medicinal material delivered to rats. In this study, we have chosen the mouse, a more proper animal model than the rat, to investigate the in vivo pharmacokinetics and tissue distribution of bufadienolides from the Shexiang Baoxin Pill. MATERIALS AND METHODS: The concentrations of bufadienolides in plasma and tissues were identified using high performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS/MS). The samples were prepared by liquid-liquid extraction with ethyl acetate, and the separation of bufadienolides was achieved using an ACQUITY HSS T3 column by gradient elution using water (containing 0.1% formic acid) and acetonitrile as the mobile phase at a flow rate of 0.3 mL/min. The pharmacokinetic parameters were determined using non-compartmental analysis. RESULTS: The results showed that the five bufadienolides were rapidly absorbed and distributed into the body. The pharmacokinetic curve showed double peaks after oral administration. The major tissue depots for resibufogenin, bufalin, and bufotalin in mice were the intestines, lung and kidney, whereas the major tissue depots of gamabufotalin and arenobufagin were the intestines, liver and kidney. CONCLUSION: The information gained from this research provides a meaningful insight for the clinical applications of the Shexiang Baoxin Pill.

Plasma level of the endogenous sodium pump ligand marinobufagenin is related to the salt-sensitivity in men.

OBJECTIVE: Salt-induced elevation of the endogenous digitalis like sodium pump ligand marinobufagenin (MBG) in the Dahl salt-sensitive rats resulted in elevated blood pressure (BP). Here, we tested, in humans, whether MBG levels are related to ambulatory 24-h BP (ABP), controlled long-term increase of salt-intake induces changes in MBG and any salt-induced change in MBG is related to salt sensitivity. METHODS: Thirty-nine healthy individuals (53 ±â€Š11 years old; 20 men and 19 women) had a total daily NaCl intake of 50 mmol (low-salt) and 150 mmol (high-salt) for 4 weeks each, in a random order. ABP and MBG in plasma and urine were measured at baseline (unstandardized salt intake) and after high and low-salt intake. RESULTS: At baseline, plasma MBG (P-MBG) was related to 24-h SBP (r = 0.43, P = 0.007) and DBP (r = 0.32, P = 0.047), whereas 24-h urinary excretion of MBG (UE-MBG) was related to 24-h DBP only (r = 0.42, P = 0.008). Sex-specific analyses revealed that these relationships were significant in men only. Compared with low-salt, high-salt diet increased P-MBG (P = 0.029), mainly driven by results in men. Male P-MBG responders vs. nonresponders (above vs. below median of high-salt induced P-MBG increase) had markedly enhanced SBP (10.4 ±â€Š6.4 vs. 1.0 ±â€Š6.0 mmHg; P = 0.003) and DBP (6.7 ±â€Š5.0 vs. -0.6 ±â€Š3.6 mmHg; P = 0.001) salt sensitivity. CONCLUSION: In men, MBG increases with 24-h ABP, and similar to Dahl salt-sensitive rats, 4 weeks of high-salt induced MBG response is accompanied by marked salt sensitivity. However, these patterns seem to be sex-specific and are not observed in women.

Simultaneous determination of seven bufadienolides in rat plasma after oral administration of Shexiang Baoxin Pill by liquid chromatography-electrospray ionization-tandem mass spectrometry: application to a pharmacokinetic study.

A liquid chromatography-electrospray ionization-tandem mass spectrometry method was described for the simultaneous determination of resibufogenin, bufalin, gamabufotalin, telibufagin, arenobufagin, cinobufagin and bufotalin in rat plasma. Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate. Chromatographic separation was carried out on an ACQUITY HSS T3 column with gradient elution using mobile phase consisting of acetonitrile-0.1% formic acid in water at a flow rate of 0.3 mL/min. All analytes showed good linearity over a wide concentration range (r>0.99). The lower limit of quantification was in the range of 0.5-10 ng/mL for seven bufadienolides. The mean recovery of the analytes ranged from 94.36 to 104.18%. The intra- and inter-day precisions were in the range of 1.74-13.78% and the accuracies were between 89.37 and 101.38%. The validated method was successfully applied to a pharmacokinetic (PK) study of the seven bufadienolides in rat plasma after oral administration of Shexiang Baoxin Pill (SBP). The selected PK marker compounds with typical efficacy/toxicity may provide a practical solution for marker compound selection and dosage design for the therapeutic drug monitoring and PK study of SBP in its clinical applications.

Quantitative determination of arenobufagin in rat plasma by ultra fast liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study.

A rapid, sensitive, and selective ultra fast liquid chromatography-tandem mass spectrometry method was developed for quantitative determination of arenobufagin in rat plasma. Sample pretreatment involved a one-step protein precipitation with methanol using 0.1mL rat plasma. The separation was carried out on a Shim-pack XR-ODS II (75mm×2.0mm, i.d. 2.1µm) column with gradient elution at a flow rate of 0.30mLmin(-1). The mobile phase was acetonitrile and 0.1% formic acid in water. A post-column switching valve was applied to reduce the matrix effect. The detection was performed on a triple-quadruple tandem mass spectrometer in the multiple reaction monitoring mode after electrospray ionization. Linear calibration curves for arenobufagin were obtained over the concentration range 1.056-1056ngmL(-1), with a lower limit of quantification of 1.056ngmL(-1). The intra-day and inter-day precision values were lower than 15% and the accuracy ranged from 5.4% to 9.8% at all quality control levels. The method was successfully applied to the determination and pharmacokinetic study of arenobufagin in rat plasma following intraperitoneal administration.

Magnesium sulfate potentiates effect of DigiFab on marinobufagenin-induced Na/K-ATPase inhibition.

BACKGROUND: Immunoneutralization of elevated circulating levels of endogenous digitalis-like Na/K-ATPase inhibitors (i.e. cardiotonic steroids (CTS)) represents a novel approach in the treatment of preeclampsia (PE). Recently we demonstrated that DigiFab (Fab fragments of affinity-purified ovine digoxin antibody) restores PE-induced inhibition of Na/K-ATPase in erythrocytes ex vivo. Previously magnesium ions were shown to antagonize digitalis-induced toxicity, which is mediated by Na/K-ATPase inhibition. We hypothesized that magnesium sulfate would potentiate the effect of DigiFab in the reversal of CTS-induced Na/K-ATPase inhibition. METHODS: To test this hypothesis, we studied the ex vivo effect of DigiFab on Na/K-ATPase activity in erythrocytes from patients with PE in the absence and in the presence of 3 mmol/L magnesium sulfate. RESULTS: Compared with 11 normotensive pregnant subjects (29 ± 1 years; gestational age = 39.0 ± 0.2 weeks; blood pressure = 111 ± 2/73 ± 2 mm Hg), the 12 patients with PE (30 ± 1 years; gestational age = 37.9 ± 0.3 weeks; blood pressure = 159 ± 5/99 ± 3 mm Hg) had plasma levels of marino-bufagenin increased 3-fold (1.38 ± 0.40 vs. 0.38 ± 0.10 nmol/L; P < 0.01) and activity of Na/K-ATPase in erythrocytes was inhibited (1.16 ± 0.11 vs. 2.80 ± 0.20 µmol Pi/ml/h; P < 0.01). Ex vivo, DigiFab (1 µg/ml) restored erythrocyte Na/K-ATPase activity (1.72 ± 0.13 µmol Pi/ml/h; P < 0.01), and 3 mmol magnesium sulfate potentiated the effect of DigiFab (2.30 ± 0.20 µmol Pi/ml/h; P < 0.01). CONCLUSIONS: Magnesium is capable of increasing the efficacy of immunoneutralization of marinobufagenin-induced Na/K-ATPase inhibition.

Enhancement of skin permeation of bufalin by limonene via reservoir type transdermal patch: formulation design and biopharmaceutical evaluation.

A reservoir-type transdermal delivery system (TDS) of bufalin was designed and evaluated for various formulation variables like different penetration enhancers, formulation matrix, rate controlling membranes as well as biopharmaceutical characteristics. Hairless mouse skin was used in permeation experiments with Franz diffusion cells. In vitro skin permeation study showed that terpenes, especially d-limonene was the most effective enhancer when ethanol and PG were used as the vehicle with a synergistic effect. Among different rate controlling membranes, ethylene vinyl acetate (EVA) membrane containing 19% vinyl acetate demonstrated a more suitable release rate for bufalin than the other membranes. In vivo pharmacokinetic study of the bufalin patch in rat showed steady-state of bufalin from 3h to 12 h. In vivo release rate and cumulative amount analyzed by deconvolution method demonstrated the sustained release of bufalin as long as the patch remained on the animal for at least 12 h. The MRT increased from 1h of IV administration to 9h of transdermal administration. In vitro permeation across mouse skin was found to have biphasic correlation with plasma AUC in the in vivo pharmacokinetic study. Current in vitro-in vivo correlation (IVIVC) enabled the prediction of pharmacokinetic profile of bufalin from in vitro permeation results. In conclusion, current reservoir transdermal patch containing 10% D-limonene as a permeation enhancer, 40% ethanol, 30% PG and 15% carbopol-water gel complex provided an improved sustained release of bufalin through transdermal administration. The bufalin patch was successfully applied to biopharmaceutical study in rats and demonstrated the feasibility of this transdermal formulation for future development and clinical trials.