Rats with congenital hydronephrosis show increased susceptibility to renal ischemia-reperfusion injury.

Many drug candidates have shown significant renoprotective effects in preclinical models; however, there is no clinically used effective pharmacotherapy for acute kidney injury. The failure to translate from bench to bedside could be due to misleading results from experimental animals with undetected congenital kidney defects. This study was performed to assess the effects of congenital hydronephrosis on the functional capacity of tubular renal transporters as well as kidney sensitivity to ischemia-reperfusion (I-R)-induced injury in male Wistar rats. Ultrasonography was used to distinguish healthy control rats from rats with hydronephrosis. L-carnitine or furosemide was administered, and serial blood samples were collected and analyzed to assess the effects of hydronephrosis on the pharmacokinetic parameters. Renal injury was induced by clamping the renal pedicles of both kidneys for 30 min with subsequent 24 hr reperfusion. The prevalence of hydronephrosis reached ~30%. The plasma concentrations after administration of L-carnitine or furosemide were similar in both groups. I-R induced more pronounced renal injury in the hydronephrotic rats than the control rats, which was evident by a significantly higher kidney injury molecule-1 concentration and lower creatinine concentration in the urine of the hydronephrotic rats than the control rats. After I-R, the gene expression levels of renal injury markers were significantly higher in the hydronephrotic kidneys than in the kidneys of control group animals. In conclusion, our results demonstrate that hydronephrotic kidneys are more susceptible to I-R-induced damage than healthy kidneys. Unilateral hydronephrosis does not affect the pharmacokinetics of substances secreted or absorbed in the renal tubules.

Physiologically Based Pharmacokinetic Models of Probenecid and Furosemide to Predict Transporter Mediated Drug-Drug Interactions.

PURPOSE: To provide whole-body physiologically based pharmacokinetic (PBPK) models of the potent clinical organic anion transporter (OAT) inhibitor probenecid and the clinical OAT victim drug furosemide for their application in transporter-based drug-drug interaction (DDI) modeling. METHODS: PBPK models of probenecid and furosemide were developed in PK-Sim®. Drug-dependent parameters and plasma concentration-time profiles following intravenous and oral probenecid and furosemide administration were gathered from literature and used for model development. For model evaluation, plasma concentration-time profiles, areas under the plasma concentration-time curve (AUC) and peak plasma concentrations (Cmax) were predicted and compared to observed data. In addition, the models were applied to predict the outcome of clinical DDI studies. RESULTS: The developed models accurately describe the reported plasma concentrations of 27 clinical probenecid studies and of 42 studies using furosemide. Furthermore, application of these models to predict the probenecid-furosemide and probenecid-rifampicin DDIs demonstrates their good performance, with 6/7 of the predicted DDI AUC ratios and 4/5 of the predicted DDI Cmax ratios within 1.25-fold of the observed values, and all predicted DDI AUC and Cmax ratios within 2.0-fold. CONCLUSIONS: Whole-body PBPK models of probenecid and furosemide were built and evaluated, providing useful tools to support the investigation of transporter mediated DDIs.

A Pilot Study of the Maternal-Fetal Pharmacokinetics of Furosemide in Plasma, Urine, and Amniotic Fluid of Hypertensive Parturient Women Under Cesarean Section.

The third trimester of pregnancy is related to physiological changes that can modify the process of absorption, distribution, metabolism, and excretion and, consequently, the efficacy and toxicity of drugs. However, little is known about furosemide pharmacokinetics and placental transfer in pregnancy. This study evaluated the maternal-fetal pharmacokinetics and distribution to amniotic fluid of furosemide in hypertensive parturient women under cesarean section. Twelve hypertensive parturient women under methyldopa (250 mg/8 h) and/or pindolol (10 mg/12 h) treatment received a 40-mg single oral dose of furosemide 1 to 10 hours before delivery by cesarean section. Blood and urine samples were collected for 12 hours after furosemide administration. At delivery, samples were obtained from maternal and umbilical cord blood (n = 8) to assess the transplacental transfer. Amniotic fluid (n = 4) was collected at the time of delivery. The following furosemide pharmacokinetic parameters were obtained as median (interquartile range): Cmax , 403 ng/mL (229 to 715 ng/mL); Tmax , 2.00 hours (1.50 to 4.83 hours); elimination half-life (t1/2 ), 2.50 hours (1.77 to 2.97 hours); AUC0-12 h , 1366 ng⋅h/mL (927 to 2531 ng⋅h/mL); AUC0-∞ , 1580 ng⋅h/mL (1270 to 2881 ng⋅h/mL); CL/F 25.3 L/h (13.8 to 31.4 L/h); CLR, 2.50 L/h (1.77 to 2.97 L/h); CLNR, 22.7 L/h (12.1 to 25.6 L/h); and Vd /F 82.8 L (34.4 to 173 L). The transplacental transfer of furosemide was 0.43 (0.10 to 0.73), and the amniotic fluid concentration was 11.0 ng/mL (5.51 to 14.6 ng/mL). From a clinical point of view, these results suggest that substrates of uridine diphosphate-glucuronosyltransferase isoenzymes such as furosemide may have increased clearance during pregnancy and could require dose adjustment in this population.

Applications of shun shell column and nanocomposite sorbent for analysis of eleven anti-hypertensive in human plasma.

High-performance liquid chromatography (HPLC) and solid phase micro membrane tip extraction (SPMMTE) methods are developed for the simultaneous analysis of eleven cardiovascular drugs in human plasma. Iron nanoparticles were obtained by the green method, characterized by XRD, FT-IR, TEM, and EDS and utilized in SPMMTE for sample preparation. The mobile phase used was ammonium acetate buffer-methanol-acetonitrile (65:18:17) with a 1.0 mL/min flow rate at 260 nm detection. Column used was Sunshell C18 150 × 4.6 mm, 2.6 µm. The values of k, α, and Rs were ranged from 040 to109.22, 1.20 to 2.67 and 1.0 to 26.18. SPMMTE and HPLC methods were fast, reproducible, precise, robust, economic and rugged for analysis of methyldopa, hydrochlorothiazide, prazosin hydrochloride, furosemide, labetalol, propranolol, valsartan, losartan potassium, diltiazem, irbesartan and spironolactone in human plasma. The recoveries (%) of methyldopa, hydrochlorothiazide, prazosin hydrochloride, furosemide, labetalol, propranolol, valsartan, losartan potassium, diltiazem, irbesartan, and spironolactone were 91.0, 85.2, 92.3, 90.4, 90.1, 85.6, 86.6, 86.2, 85.1, 86.6, and 85.7, respectively. These results showed that SPMMTE and HPLC methods can be applied to test the described drugs in several matrices.

Pharmacokinetics of furosemide in thoroughbred horses subjected to supramaximal treadmill exercise with and without controlled access to water.

BACKGROUND: The primary objective of this study was to assess the disposition of furosemide in Thoroughbred horses treated intravenously with 1 mg/kg of furosemide 4 and 24 h before supramaximal treadmill exercise without and with controlled access to water, respectively. Another objective was to determine whether furosemide was detectable in the plasma of horses after exposure to supramaximal treadmill exercise. Thoroughbred horses (n = 4-6) were administered single intravenous doses of 1 mg/kg of furosemide at 4 and 24 h before supramaximal exercise on a high-speed treadmill, with controlled and free access to water, respectively. Plasma furosemide concentrations were determined using liquid chromatography. RESULTS: Furosemide was detected in all the horses, regardless of whether they were treated 24 h or 4 h before excersice. In both treatment sequence groups of 2 horses, the concentration time profiles of furosemide during the first 4 h after its administration were relatively similar. The average maximum observed concentrations, AUC0-1.5h, and AUC0-3h, of both groups of horses were not different (p > 0.05). There were no significant differences in systemic clearance based on the geometric mean (95% confidence interval) (409 (347-482) mL/h/kg) for 4 h and 320 (177-580) mL/h/kg) for 24 h) between horses that were exercised 4- and 24-h post-furosemide administration. The plasma concentration of furosemide in all the horses fell below the limit of quantification (25 ng/mL) within 12 h after drug administration. In the group treated 24 h before exercise, none of the horses had detectable furosemide at the time of supramaximal treadmill exercise. In the group treated 4 h before exercise, furosemide was detected 1 h before and 2 h after supramaximal treadmill exercise in 4/4 and 3/4 horses, respectively. The mean AUC3-last h of both groups of horses were not different (p > 0.05). CONCLUSIONS: Water restriction did not exert any apparent effect on the disposition of furosemide. It remains to be determined, however, whether the attained plasma concentration of furosemide in combination with other controlled water access protocols have any direct or indirect pharmacological effect that may affect the athletic performance of the horse.

Novel Strategy for the Systemic Delivery of Furosemide Based on a New Drug Transport Mechanism.

We reported a novel transport mechanism of curcumin, independent of improved solubility, which involved direct contact of amorphous solid particles with the cell membrane. This mechanism has potential as a novel systemic delivery system of poorly water-soluble drugs. In this study, the transport mechanism of furosemide (FUR), which is transported by the same novel mechanism, was examined. In vitro cell permeation studies under air-interface conditions (AICs) revealed that the permeation from powders sprayed on cell monolayers was significantly higher than that under liquid-covered conditions (LCCs) from their solutions. The permeation from amorphous solid particles was faster than that from crystals. Similar results were derived from in vitro studies using an artificial membrane, with which the permeation of FUR could be examined without water. These findings clearly indicated that the transport mechanism of FUR is the same as that of curcumin. For the application of this new transport mechanism, the in vivo absorption of FUR was examined after pulmonary insufflation, which allows the solid particles to make direct contact with the epithelial cells. Pulmonary absorption of FUR from the amorphous powder was almost complete and was faster than that after intragastric administration of the solution, suggesting that FUR was absorbed from the lung by the same mechanism as the in vitro study. This new transport mechanism, which is independent of water dissolution, could be exploited to develop a novel delivery system for poorly water-soluble drugs, using pulmonary powder inhalation.

Urine volume as a predicting factor for furosemide clearance during continuous infusion in AKI septic shock patients on hemodiafiltration.

BACKGROUND: This study assessed the contribution of intracorporeal (IC) and extracorporeal clearance (EC) of furosemide in patients with septic acute kidney injury (AKI), and the relationship between plasma concentrations and urine volume. METHODS: Prospective cohort observational study of 15 patients with septic AKI undergoing continuous veno-venous hemodiafiltration (CVVHDF) divided according to urine volume (< 500 ml/12 h, Oliguria group, n = 5; > 500 ml/12 h, Diuresis group, n = 10) during continuous infusion of furosemide (120 mg/12 h) at steady-state condition. Plasma and effluent furosemide concentrations were determined by high-performance liquid chromatography (HPLC)-mass spectrometry every 12 h for 48 h. RESULTS: Furosemide plasma concentrations and total body clearance (TBC) were 6.14 mg/l and 22.1 ml/min for the Oliguria group, and 2.63 mg/l and 54.4 ml/min for the Diuresis group, respectively (p < 0.05). When urine volume was < 500 ml/24 h, the furosemide plasma concentrations peaked at the potentially toxic value of 13.0 mg/l. Furosemide EC was not relevant for the Diuresis group, but it represented 18% of TBC for the Oliguria group. Furosemide plasma concentrations correlated positively with dose infusion for both groups (r = 0.728 and 0.685, p < 0.05), and negatively with urine volume only for the Diuresis (r = - 0.578, p < 0.01) but not for the Oliguria group (r = - 0.089, p = 0.715). CONCLUSIONS: For patients with urine volume > 500 ml/12 h continuous infusion of furosemide up to 480 mg/24 h leads to increasing urine volume, which can predict furosemide plasma levels within its safety range. When the urine volume is lower, the furosemide plasma levels are increased beyond any further diuretic efficacy.

Development and validation of HPTLC and green HPLC methods for determination of furosemide, spironolactone and canrenone, in pure forms, tablets and spiked human plasma.

Two selective and accurate chromatographic methods are presented for simultaneous quantitation of spironolactone (SP) and furosemide (FR) and canrenone (CN), the main degradation product and the main active metabolite of SP. Method A was HPTLC, where separation was completed on silica gel HPTLC F254 plates using ethyl acetate-triethylamine-acetic acid (9:0.7:0.5, by volume) as a developing system and UV detection at 254 nm. Method B was a green isocratic RP-HPLC utilizing a C18 (4.6 × 100 mm) column, the mobile phase consisting of ethanol-deionized water (45: 55, v/v) and UV estimation at 254 nm. Adjustment of flow rate at 1 mL/min and pH at 3.5 with glacial acetic acid was done. Regarding the greenness profile, the proposed RP-HPLC method is greener than the reported one. ICH guidelines were followed to validate the developed methods. Successful applications of the developed methods were revealed by simultaneous determination of FR, SP and CN in pure forms and plasma samples in the ranges of 0.2-2, 0.05-2.6 and 0.05-2 µg/band for method A and 5-60, 2-60 and 2-60 µg/mL for method B for FR, SP and CN, respectively.

Drug-drug interaction of cefiderocol, a siderophore cephalosporin, via human drug transporters.

PURPOSE: Cefiderocol, a siderophore cephalosporin, will be used concomitantly with other medications for treatment of bacterial infections. In vitro studies demonstrated inhibition potential of cefiderocol on organic anion transporter (OAT) 1, OAT3, organic cation transporter (OCT) 1, OCT2, multidrug and toxin extrusion (MATE) 2-K, and organic anion transporting polypeptide (OATP) 1B3. The aim of this study was to assess in vivo drug-drug interaction (DDI) potential of cefiderocol using probe substrates for these transporters. METHODS: DDI potentials of cefiderocol as inhibitors were assessed in a clinical study consisting of 3 cohorts. Twelve or 13 healthy adult subjects per cohort orally received a single dose of furosemide 20 mg (for OAT1/3), metformin 1000 mg (for OCT1/2 and MATE2-K), or rosuvastatin 10 mg (for OATP1B3) with or without co-administration with cefiderocol 2 g every 8 h with 3-h infusion (a total of 3, 6, and 9 doses of cefiderocol with furosemide, metformin, and rosuvastatin, respectively). DDI potentials were assessed based on the pharmacokinetics of the substrates. RESULTS: Ratios (90% confidence intervals) of maximum plasma concentration and area under the plasma concentration-time curve were 1.00 (0.71-1.42) and 0.92 (0.73-1.16) for furosemide, 1.09 (0.92-1.28) and 1.03 (0.93-1.15) for metformin, and 1.28 (1.12-1.46) and 1.21 (1.08-1.35) for rosuvastatin, respectively. Exposures to furosemide or metformin did not change when co-administered with cefiderocol. Slight increase in rosuvastatin exposure was observed with co-administered with cefiderocol, which was not considered to be clinically significant. Each treatment was well tolerated. CONCLUSIONS: Cefiderocol has no clinically significant DDI potential via drug transporters.

Pharmacodynamic assessment of diuretic efficacy and braking in a furosemide continuous infusion model.

INTRODUCTION: Diuretic failure is a potential life-ending event but is unpredictable and poorly understood. The objectives of this study were to evaluate pharmacodynamic markers of furosemide-induced diuresis and to investigate mechanisms of diuretic braking in dogs receiving constant rate infusion (CRI) of furosemide. ANIMALS: Six healthy male dogs. METHODS: Raw data and stored samples from one arm of a previously published study were further analyzed to mechanistically investigate causes of diuretic braking in these dogs. Urine volume was recorded hourly during a 5-h furosemide CRI. Urine and blood samples were collected hourly to measure serum and urine electrolytes, urine aldosterone, and plasma and urine furosemide. Serum electrolyte fractional excretion was calculated. Urine sodium concentration was indexed to urine potassium (uNa:uK) and urine furosemide (uNa:uFur) concentrations, plasma furosemide concentration was indexed to urine furosemide concentration (pFur:uFur), and urine aldosterone was indexed to urine creatinine (UAldo:C). Temporal change and the relationship to urine volume were evaluated for these measured and calculated variables. RESULTS: Urine volume was significantly correlated with urine electrolyte amounts and with uNa:uK. The ratio of pFur:uFur decreased during the infusion, whereas furosemide excretion was unchanged. CONCLUSIONS: There was a strong relationship between urine volume and absolute urine electrolyte excretion. Urine volume was strongly correlated to uNa:uK, giving it potential as a spot indicator of urine production during diuresis. The decrease in uNa:uK over time during the infusion is consistent with mineralocorticoid modification of urinary electrolyte excretion, supporting renin-angiotensin-aldosterone activation as a cause of diuretic braking in this model.

Effect of the angiotensin receptor-neprilysin inhibitor sacubitril/valsartan on the pharmacokinetics and pharmacodynamics of a single dose of furosemide.

AIMS: Sacubitril/valsartan is indicated for the treatment of heart failure and reduced ejection fraction (HFrEF). Furosemide, a loop diuretic commonly used for the treatment of HFrEF, may be coadministered with sacubitril/valsartan in clinical practice. The effect of sacubitril/valsartan on the pharmacokinetics and pharmacodynamics of furosemide was evaluated in this open label, two-period, single-sequence study in healthy subjects. METHODS: All subjects (n = 28) received 40 mg oral single-dose furosemide during period 1, followed by a washout of 2 days. In period 2, sacubitril/valsartan 200 mg (97/103 mg) was administered twice daily for 5 days and a single dose of 40 mg furosemide was coadministered on day 6. Serial plasma and urine samples were collected to determine the pharmacokinetics of furosemide and sacubitril/valsartan and the pharmacodynamics of furosemide. The point estimates and the associated 90% confidence intervals for pharmacokinetic parameters were evaluated. RESULTS: Coadministration of furosemide with sacubitril/valsartan decreased the maximum observed plasma concentration (Cmax ) [estimated geometric mean ratio (90% confidence interval): 0.50 (0.44, 0.56)], area under the plasma concentration-time curve (AUC) from time 0 to infinity [0.72 (0.67, 0.77)] and 24-h urinary excretion of furosemide [0.74 (0.69, 0.79)]. When coadministered with sacubitril/valsartan, 0-4-h, 4-8-h and 0-24-h diuresis in response to furosemide was reduced by ~7%, 21% and 0.2%, respectively, while natriuresis was reduced by ~ 28.5%, 7% and 15%, respectively. Post hoc analysis of the pivotal phase III Prospective comparison of ARNI with ACEI to Determine Impact on Global Mortality and morbidity in Heart Failure trial (PARADIGM-HF) indicated that the median furosemide dose was similar at baseline and at the end of the study in the sacubitril/valsartan group. CONCLUSIONS: Sacubitril/valsartan reduced plasma Cmax and AUC and 24-h urinary excretion of furosemide, while not significantly affecting its pharmacodynamic effects in healthy subjects.

Pharmacokinetics of furosemide administered 4 and 24 hours prior to high-speed exercise in horses.

Furosemide is a diuretic agent used commonly in racehorses to attenuate the bleeding associated with exercise-induced pulmonary hemorrhage (EIPH). The current study describes serum and urine concentrations and the pharmacokinetics of furosemide following administration at 4 and 24 hrs prior to maximal exercise. Eight exercised adult Thoroughbred horses received a single IV administration of 250 mg of furosemide at 4 and 24 hrs prior to maximal exercise on a high-speed treadmill. Blood and urine samples were collected at time 0 and at various times for up to 72 hrs and furosemide concentrations determined using liquid chromatography-tandem mass spectrometry. Serum furosemide concentrations remained above the LOQ (0.05 ng/ml) for 36 hrs in 3/8 and 1/8 horses in the 4- and 24-hrs groups, respectively. Serum concentration data were best fit by a two-compartment model. There was not a significant difference in the volume of distribution at steady-state (0.594 ± 0.178 [4 hrs] and 0.648 ± 0.147 [24 hrs] L/kg) or systemic clearance (0.541 ± 0.094 [4 hrs] and 0.617 ± 0.114 [24 hrs] L/hrs/kg) between horses that were exercised at 4- and 24 hrs postdrug administration. The mean ± SD elimination half-life was 3.12 ± 0.387 and 3.23 ± 0.407 hrs following administration at 4 and 24 hrs prior to exercise, respectively.

Model based population PK-PD analysis of furosemide for BP lowering effect: A comparative study in primary and secondary hypertension.

Though numerous reports have demonstrated multiple mechanisms by which furosemide can exert its anti-hypertensive response. However, lack of studies describing PK-PD relationship for furosemide featuring its anti-hypertensive property has limited its usage as a blood pressure (BP) lowering agent. Serum concentrations and mean arterial BP were monitored following 40 and 80mgkg-1 multiple oral dose of furosemide in spontaneously hypertensive rats (SHR) and DOCA-salt induced hypertensive (DOCA-salt) rats. A simultaneous population PK-PD relationship using Emax model with effect compartment was developed to compare the anti-hypertensive efficacy of furosemide in these rat models. A two-compartment PK model with Weibull-type absorption and first-order elimination best described the serum concentration-time profile of furosemide. In the present study, post dose serum concentrations of furosemide were found to be lower than the EC50. The EC50 predicted in DOCA-salt rats was found to be lower (4.5-fold), whereas the tolerance development was higher than that in SHR model. The PK-PD parameter estimates, particularly lower values of EC50, Ke and Q in DOCA-salt rats as compared to SHR, pinpointed the higher BP lowering efficacy of furosemide in volume overload induced hypertensive conditions. Insignificantly altered serum creatinine and electrolyte levels indicated a favorable side effect profile of furosemide. In conclusion, the final PK-PD model described the data well and provides detailed insights into the use of furosemide as an anti-hypertensive agent.

Quantification of furosemide in camel plasma by high resolution mass spectrometry, application on pharmacokinetics.

We developed and validated a high-resolution liquid chromatography mass spectrometry method for the quantification of furosemide in camel plasma which was used for a pharmacokinetic study in camels. Plasma samples were extracted by supported liquid extraction and furosemide and internal standard (furosemide-D5) were separated on a an Agilent Zorbax XDB C18 column (50 × 2.1 mm i.d., 3.5 µm). Data was acquired in full-scan mode over a mass range of 200-400 Da in negative electrospray mode at a resolution of 70,000. Linear calibration curves were obtained over the concentration ranges of 1.0-10,000 ng/mL. The validated method was then successfully applied in evaluating the pharmacokinetics and metabolites of furosemide in six camels (Camelus dromedarus) and we were able to advice on a withdrawal time of furosemide treatment before racing.

Limitations of the Anticholinergic Activity Assay and Assay-Based Anticholinergic Drug Scales.

OBJECTIVE: The anticholinergic activity (AA) assay is a common method to determine a patient's anticholinergic load. Several limitations, however, are expected when applying the AA assay to patients or using drug scales to estimate anticholinergic burden based on AA levels. This study aims to demonstrate common pitfalls in an experimental setting and outline their clinical consequences. METHODS: The AA was analyzed for five drugs with reported interaction with muscarinic receptors. Concentration-response curves were constructed for furosemide (weak anticholinergic), diphenhydramine (moderate anticholinergic), the strong anticholinergic amitriptyline and its metabolite nortriptyline, and the cholinergic pilocarpine. The Combination Index (CI) was used to assess the interaction of three drug combinations with amitriptyline. RESULTS: All compounds displaced the radioactive tracer from its receptor binding site in a concentration-dependent manner, and full displacement was reached for all compounds except furosemide (Emax 16%). The CI indicated that amitriptyline and thioridazine have antagonistic effects (CI = 1.46) at low and synergistic effects (CI = 0.88) at higher concentrations (p < 0.0001), whereas synergistic effects (CI = 0.47-0.48) were observed for amitriptyline in any concentration combined with pilocarpine (p < 0.001). CONCLUSION: When the patient's anticholinergic load is estimated using AA levels, the actual exposure, combination of anticholinergic drugs, their active metabolites, and also drugs with an opposite pharmacologic action will contribute to AA levels, whereas weak anticholinergic drugs in therapeutic concentrations are rather negligible.

Determination of chloramphenicol in biological matrices by solid-phase membrane micro-tip extraction and capillary electrophoresis.

Solid-phase membrane micro-tip extraction (SPMMTE) and capillary electrophoresis (CE) methods were developed and validated for analysis of chloramphenicol in human plasma and urine samples. Iron composite nanoparticles were prepared using green technology. CE was carried out using a silica capillary (60 cm × 50 µm i.d.), phosphate buffer (50 mm, 8.0 pH)-acetonitrile (95:5, v/v) as the background electrolyte, 10 kV voltage, 280 nm detection, 20 s injection time and 27 ± 1°C temperature. Frusemide was used as an internal standard. The values of migration time, electrophoretic mobility, electrophoretic velocity and theoretical plates of chloramphenicol were 12.254 min, 4.44 × 10, 7.41 × 10 and 11,227. The limits of detection and quantitation of chloramphenicol were 0.1 and 1.0 µg/mL. Recovery of chloramphenicol in the standard solution was 95%. Solid-phase membrane micro-tip extraction and capillary electrophoresis methods may be used to analyze chloramphenicol in human plasma and urine samples of any patient.

Nephron-Specific Deletion of Circadian Clock Gene Bmal1 Alters the Plasma and Renal Metabolome and Impairs Drug Disposition.

The circadian clock controls a wide variety of metabolic and homeostatic processes in a number of tissues, including the kidney. However, the role of the renal circadian clocks remains largely unknown. To address this question, we performed a combined functional, transcriptomic, and metabolomic analysis in mice with inducible conditional knockout (cKO) of BMAL1, which is critically involved in the circadian clock system, in renal tubular cells (Bmal1lox/lox/Pax8-rtTA/LC1 mice). Induction of cKO in adult mice did not produce obvious abnormalities in renal sodium, potassium, or water handling. Deep sequencing of the renal transcriptome revealed significant changes in the expression of genes related to metabolic pathways and organic anion transport in cKO mice compared with control littermates. Furthermore, kidneys from cKO mice exhibited a significant decrease in the NAD+-to-NADH ratio, which reflects the oxidative phosphorylation-to-glycolysis ratio and/or the status of mitochondrial function. Metabolome profiling showed significant changes in plasma levels of amino acids, biogenic amines, acylcarnitines, and lipids. In-depth analysis of two selected pathways revealed a significant increase in plasma urea level correlating with increased renal Arginase II activity, hyperargininemia, and increased kidney arginine content as well as a significant increase in plasma creatinine concentration and a reduced capacity of the kidney to secrete anionic drugs (furosemide) paralleled by an approximate 80% decrease in the expression level of organic anion transporter 3 (SLC22a8). Collectively, these results indicate that the renal circadian clocks control a variety of metabolic/homeostatic processes at the intrarenal and systemic levels and are involved in drug disposition.

Polymeric microcontainers improve oral bioavailability of furosemide.

Microcontainers with an inner diameter of 223 µm are fabricated using the polymer SU-8, and evaluated in vitro, in situ and in vivo for their application as an advanced oral drug delivery system for the poorly water soluble drug furosemide. An amorphous sodium salt of furosemide (ASSF) is filled into the microcontainers followed by applying a lid using Eudragit L100. It is possible to control the drug release in vitro, and in vitro absorption studies show that the microcontainers are not a hindrance for absorption of ASSF. In situ perfusion studies in rats are performed with ASSF-filled microcontainers coated with Eudragit and compared to a furosemide solution. The absorption rate constant of ASSF confined in microcontainers is found to be significantly different from the solution, and by light microscopy, it is observed that the microcontainers are engulfed by the intestinal mucus. An oral bioavailability study in rats is performed with ASSF confined in microcontainers coated with Eudragit and a control group with ASSF in Eudragit-coated capsules. A relative bioavailability of 220% for the ASSF in microcontainers compared to ASSF in capsules is found. These studies indicate that the microcontainers could serve as a promising oral drug delivery system.

Rapid determination of furosemide in drug and blood plasma of wrestlers by a carboxyl-MWCNT sensor.

A novel method is developed for the quantification of furosemide in biological fluids. The method is based on the electro-reduction of Zn(II)-furosemide complex at carboxyl-MWCNT modified glassy carbon electrode. It is shown that, in Britton-Robinson buffer (pH5.7) the reduction peak of Zn(II)-furosemide complex formed at -1.0 V (versus, Ag/AgCl). The increment of current signal obtained from the reduction peak current of the Zn(II)-furosemide complex was rectilinear with furosemide concentration in the range of 0.03 to 140.0 µg ml(-1), with a detection limit of 0.007 µg ml(-1). The drug recovery ranged between 97.8% and 100.8% and the mean drug recovery was 98.89%. The accuracies (relative error% and RSD%) were less than 15% and are acceptable according to the US FDA guideline for bioanalytical method validation. The sensor was used for quantification of furosemide in drug and biological fluid samples. The data of drug analysis were compared with the standard method.

[Detection of plasma and urine furosemide based on online enrichment and restricted- access media coupled with high-performance liquid chromatography].

OBJECTIVE: To establish a method based on restricted access media-high performance liquid chromatography for direct online sample injection and detection of plasma and urine furosemide in rats. METHODS: The column of restricted access media was used as the pre-treatment column and a C18 column as the analytical column. The mobile phase of the pre- treatment column was water-methanol (95:5, V/V) with a volume percentage of formic acid of 0.1%. The mobile phase of the analytical column was methanol-water (65:35, V/V) for plasma and methanol-water (55:45, V/V) for urine samples, all containing a volume percentage of formic acid of 0.1% with a flow rate of 1 ml/min. The detection wavelength was 274 nm and the column temperature was maintained at 25 degrees celsius. RESULTS: The calibration curve showed an excellent linear relationship in rat plasma furosemide concentration range of 0.1-3.2 µg/ml (r=0.9995) and in urine concentration range of 0.5-32 µg/ml (r=0.9991). The average recoveries of furosemide at 3 spiked levels ranged from 101.82% to 113.36% for plasma and from 98.75% to 112.27% for urine samples. The detection showed good intra- and inter-day assay precisions and accuracies with the relative standard deviations all below 5%. The pharmacokinetic parameters AUC(0→24) was 6.265 g/(ml·h) with a t(1/2) of 2.447 h and a C(Max) of 1.414 g/ml. The mean cumulative excretory rate of furosemide in the urine of rats over 24 h was 32.50%-39.08%. CONCLUSION: Detection of furosemide in plasma and urine samples using restricted access media-high performance liquid chromatography is simple and efficient and allows direct online injection of the samples.